Marker Proteins in Inflammation: Volume 2 Proceedings of the Second Symposium Lyon, France, June 27–30, 1983 [Reprint 2020 ed.] 9783112328262, 9783112328255

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Marker Proteins in Inflammation Volume 2

Marker Proteins in Inflammation Volume 2 Proceedings of the Second Symposium Lyon, France, June 27 - 30,1983 Editors P Arnaud • J. Bienvenu • P Laurent

W G DE

Walter de Gruyter • Berlin • New York 1984

Editors Philippe Arnaud, Ph. D., M. D. Department of Basic and Clinical Immunology and Microbiology Medical University of South Carolina 171 Ashley Road Charleston, South Carolina 29403 U.S.A. Jacques Bienvenu, M. D. Biochemical Laboratory Hôpital Jules Courmont F - 6 9 3 1 0 Pierre Bénite France Philippe Laurent, M. D. Department of Immunology Institut Pasteur de Lyon 77, Rue Pasteur F - 69365 Lyon Cedex 2 France CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Marker proteins in inflammation: proceedings of t h e . . . symposium. - Berlin ; New York : de Gruyter Vol. 2. Proceedings of the second symposium, Lyon, France, June 2 7 - 3 0 , 1 9 8 3 . - 1 9 8 4 . ISBN 3-11-009872-5

Library of Congress Cataloging in Publication Data Main entry under title: Marker proteins in inflammation, volume 2. Proceedings of the "Second Symposium sur les marqueurs de l'inflammation," sponsored by the Groupe d'étude et de recherche sur les marqueurs de l'inflammation (GERMI) Bibliography: p. Includes indexes. 1. Inflammation—Congresses. 2. Proteins—Congresses. I. Arnaud, P (Philippe) II. Bienvenu, J. (Jacques), 1950. III. Laurent, P (Philippe), 1949. IV Symposium sur les marqueurs de l'inflammation (2nd : 1983 : Lyon, France) V. Groupe d'étude et de recherche sur les marqueurs de l'inflammation (France) [DNLM: 1. Blood Proteins—diagnostic use—congresses. 2. Blood Proteinsimmunology—congresses. 3. Inflammation—congresses. QZ 150 M3451983] RB131.M353 1984 616'.0473 84-9462 ISBN 3-11-009872-5

Copyright © 1984 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

PREFACE

In the l a s t w e e k

in J u n e

1983, m o r e t h a n 2 5 0 r e s e a r c h s c i e n t i s t s

c l i n i c i a n s m e t at the F a c u l t é A l e x i s C a r r e l , (University, Claude Bernard, participants

Lyon-I)

a School

to p a r t i c i p a t e

sur les M a r q u e u r s de l ' I n f l a m m a t i o n "

s p o n s o r s h i p o f the " G r o u p e d ' E t u d e e t de R e c h e r c h e l'Inflammation" c h o s e n by the provide

a comprehensive

under

S c i e n t i f i c C o m m i t t e e of G E R M I

in I n f l a m m a t i o n a n d t h e i r c l i n i c a l

in h o s t d e f e n s e s ,

main theme running

had

a s u b j e c t of p e r e n n i a l

to the p r o t e a s e

the

proteins

in

the i m p o r t a n c e o f A c u t e P h a s e P r o t e i n s m e a s u r e m e n t

in

zinc, protein deprivation,

and malnutrition

the l a s t s e c t i o n of the book

biological

the r o l e

in h o s t d e f e n s e s . T h e models of

r e p o r t s on c o n f e r e n c e s

t h e i r use as a tool

their

lectins:

biological

to P r o f e s s o r F r a n k W.

of the d e p a r t m e n t of M o l e c u l a r B i o l o g y a n d B i o c h e m i s t r y , (Bloomington,

et

specificity.

W e w i s h to e x p r e s s o u r d e e p e s t g r a t i t u d e

University

fifth

held Caron

aspects of

for the s t u d y o f g l y c o c o n j u g a t e s ,

of

inflammation.

l e c t i n s w o r k s h o p a r r a n g e d by Drs. M i c h e l

and their a f f i n i t y a n d

are

central

A l a i n F a u r e , w h i c h w a s d e v o t e d to t h r e e c o m p l e m e n t a r y

functions,

the

lung

a n d d i s e a s e s o f the

s e c t i o n o f the book d e a l s w i t h

s e c t i o n f e a t u r e s a g r o u p of p a p e r s on animal

a satellite

field

mechanism

interplay

system. The fourth

to

implications.

interest which was

anti-protease

de

been

in o r d e r

a n d the r o l e s s u c h

f l u i d s f o r the d i a g n o s i s o f j o i n t d i s e a s e s ,

during

the

t h r o u g h o u t the m e e t i n g . T h e n e x t t h r e e s e c t i o n s

devoted respectively

Finally,

the

v i e w o f the d i f f e r e n t a s p e c t s o f the w i d e

of Acute Phase R e a c t a n t Proteins synthesis

nervous

in

sur les M a r q u e u r s

The p r o c e e d i n g s begins with a series of papers concerning

diseases,

The

(GERMI). The topics for this second symposium

International

of M a r k e r P r o t e i n s

play

Medicine

l o c a t e d in L y o n , F r a n c e .

had come from fifteen countries

"Second Symposium

of

and

Putnam

Indiana

I n d i a n a , USA) w h o w a s the o u t s t a n d i n g

president

VI

of this Symposium. We also want to extend our thanks to Professor Henry Gewürz, department of Immunology/Microbiology, Rush Medical College (Chicago, USA), Professor Robert Engler, department of Biochemistry, UER des Saints Peres (Paris, France) and Professor Roger Creyssel, department of Biochemistry UER Grange Blanche (Lyon, France), who acted as Symposium Co-Presidents and whose knowledge and understanding in the field made the program and its conferences

possible.

We wish to thank the members of the Organizing Committee for their support and help. This includes Helene Bernon, Jacques

Bienvenu,

Philippe Laurent, Abel Roullet, and J.L. Touraine. The following institutions provided financial

and practical

support: GERMI,

Institut

Pasteur de Lyon et du Sud Est, Fondation Merieux, Société Française d'Immunologie, Association des Anciens Internes et Internes en Pharmacie de Lyon, Commission Médicale Consultative de Hospices Civils de Lyon, BioMerieux, Travenol France, Du Pont de Nemours, HoechstBehring, Boehringer Mannheim, Technicon, Roger Bellon. We would also like to express our sincere gratitude to Professor Maurice Carraz, Director of the Pasteur Institute of Lyon

for his important help, and

Dr. Charles Merieux, who hosted the International

Scientific Advisory

Committee of GERMI and organized the social events. We would like to express our thanks to all other individuals, companies and who contributed to the success of the

institutions

symposium.

The fact that expert researchers agreed to write papers on this rather new and important subject has made our job a considerable pleasure. We also extend our gratitude to the staff of Walter de Gruyter, Berlin, whose labors have led to the rapid publication of this second volume on Marker Proteins in Inflammation.

February

1984

P. Arnaud J. Bienvenu P. Laurent

CONTENTS

SECTION I

THE INFLAMMATORY RESPONSE AND ACUTE PHASE REACTANT PROTEINS

Induction and Control of Acute Phase Reactant Synthesis I. Kushner

3

Use of Immunomorphological Methods and Hemolytic Plaque Tests to Investigate the Synthesis and Secretion of Plasma Proteins by the Liver Cells During the Acute Inflammatory Reaction G. Feldmann

15

Progress in Plasma Proteins: Recent Findings on the Structure of Ceruloplasmin F.W. Putnam

25

Recent Findings on the Isoelectric Points of Human C-Reactive Protein P. Laurent, D.L. Emerson, R.C. Allen, P. Arnaud

43

SAA is a more Sensitive Marker for Chronic Inflammatory Disease than C-Reactive Protein or ct-^-Acid Glycoprotein R.E. Chambers, J.T. Whicher

47

Elastase-a^ Proteinase Inhibitor Complex (E-ct^PI) and C-Reactive Protein (CRP): Early Indicators of Inflammatory Processes M. Jochum, K.H. Duswald, H. Dittmer, S. Neumann

51

Human Kallikrein-Kininogen-Kinins (KKK) System and AcutePhase Reactant Proteins (APRP) in Humans Undergoing Extracorporeal Circulation (ECC) M.E. Faymonville, A. Adam, P. Malengreaux, J. Damas, P. Ers, M. Hulot, M. Lamy, J. Lecomte

57

Vili Effect of Non Sterodial Anti-Inflammatory Drug Acide) on Acute Phase Protein Response.

(Niflumique

P. Laurent, A. Delvil, J. Bienvenu, C. Lahet, B. Colin, J.P. Rebattu, L.F. Perrin

71

Transcortin Activities, Cortisol and Progesterone in Pathological Sera: Inflammation, Septic Shock, Renal and Cardiac Diseases H. Zouaghi, L. Savu, E.A. Nunez, A. Carli, D. Kleinknecht, C. Guerot

75

Measurement of Human High Molecular Weight Kininogen (HMW-K) by Laser Nephelometry A. Adam, G. Calay, P. Ers, H. De Hertogh

SECTION

II

BIOLOGICAL REACTANT

ROLES

OF

ACUTE

79

PHASE

PROTEINS

Functions of Acute Phase Proteins in the Inflammatory Response J.T. Whicher

89

Interaction of the Acute Phase Reactants a^-Acid Glycoprotein, C-Reactive Protein and Serum Amyloid P-Component with Platelets and the Coagulation System B.A. Fiedel

99

Monoclonal Antibodies as Probes of Complement Function S. Ruddy

125

Modulation of Formation of the C3 Amplification Convertase of Human Complement by Acute Phase Proteins E. Fischer, M. Pontet, F. Delers, N. Tran Quang, M. Kazatchkine, R. Enler

135

Materno-Trophoblastic Relationship in Normal Human Pregnancy and Recurrent Spontaneous Abortion W.P. Faulk, C.-J. Yeh, B.-L. Hsi

139

IX

Chemotactic Factors in the Airspaces that may Modulate Inflammation H. Reynolds, C. Roth-Fouret

SECTION

III

PROTEASE

ANTI-PROTEASE

AND L U N G

DISEASES

153

INTERACTIONS

Human a-^-Antichymotrypsin: Purification, Properties and Reactions with some Serine Proteases A. Laine, A. Hayem, M. Davril

171

Alpha,-Antitrypsin-Protease Interactions: The Biological Aspect P. Arnaud, E. Gianazza

181

On the Interaction Between Human Liver Cathepsin L and the two Cysteine Proteinases Inhibitors Present in Human Serum M. Pagano, R. Engler, F. Esnard, F. Gauthier

203

Modulation of Human Lymphocyte Responses by «^-Antitrypsin and o^-Macroglobulin J.-P. Revi1lard, G. Cordier, J. Bata, M. Latour

207

Mediators of Inflammation in Inflammatory Alveolitis R.G. Crystal, J.F. Cordier

219

Idiophathic Pulmonary Fibrosis. Multiparametric Evaluation of Alveolar Lymphocyte and Macrophage Activation J.F. Mornex, G. Cordier, B. Lahneche, J. Pages, P. Chauvot, J.M. Vergnon, T. Wiesendanger, R. Loire, M. Vincent, J.F. Cordier, F. Mornex, J.-P.Revillard, J. Brune

223

The Alpha, Proteinase Inhibitor of the Lower Respiratory Tract. Effect of Cigarette Smoking A. Pelletier, C. Boudier, G. Pauli, J.G. Bieth

227

X Collagenolytic Enzymes and Fibrosis of the Lung J.F. Cordier, 0. Benzerara, Y. Lasne, J. Brune, R. Touraine

231

Elastolytic Activities and Proteinase Inhibitors in Sputum from Patients with Cystic Fibrosis J.M. Tournier, J. Jacquot, E. Puchelle, P. Sadoul, J.-P. Chazalette

235

Serum Derived Proteins and Antibacterial Proteins in Cystic Fibrosis Sputum J. Jacquot, J.M. Tournier, E. Puchelle, P. Sadoul, J.-P. Chazalette

239

Alveolar Fibronectin and Interstitial Lung Diseases N. Biot, D. Gindre, R. Harf, F. Maréchal, B. Vercoustre, M. Perrin-Fayol le

243

Sarcoidosis: IgG, B2 Microglobulin and Evolutivity. D. Duchassaing, P. Leclerc, M. Cambillau, 0. de Fenoyl, M. Paris

247

a^-Proteinase Inhibitor in Meconium from Healthy Infants and Infants with Cystic Fibrosis P. Lafont, Y. Barbier, M. Barbier, J. Mathian, R.C. Boisson, I. Garcia, R. Gilly

251

Qualitative Analysis of Proteins of Alveolar Fluid: A Study by Ultra-Thin Layer Immunoelectrophoresis and Electrofocusing C. Chapuis-Cellier, J.-F. Cordier, J.-M. Vergnon, A. Penet, J. Brune

255

Protease-Ant.iprotease Imbalance in Lung Diseases J.F. Mornex, R.G. Crystal

261

XI SECTION

IV

INFLAMMATION

AND A R T I C U L A R

DISEASES

The Acute Phase Response in Connective Tissue Disease I. Rowe

273

The GLA-Containing Protein öf Bone P.D. Delmas

281

Role of an Acute Phase Reactant, Retroviral gp70, in Murine SLE S. Izui, I. Hara

295

Study of 8 Marker Proteins of Inflammation in Synovial Fluid and Serum of Patients with Joint Diseases P. Bourg, B. Cartier, M. Mathieu, C. Collombel

305

Fibronectin in Arthropathies J. Lecocq, N.Y. Lecocq, L. Asch, M. Roth, M.L. North

SECTION

V

INFLAMMATORY

DISEASES

OF

NERVOUS

311

SYSTEM

Multiple Sclerosis: Clues for a Pathogenesis C. Confavreux, P. Arnaud

319

Immunoglobulins in Neuropathology A. Lowenthal

339

Cellular Inflammatory Response in Cerebrospinal Fluids C. Caudie, 0. Freney, F. Touraine

345

Evaluation of CSF Lymphocyte Subsets Using the OKT Series of Monoclonal Antibodies (OKT 3+ , 0 K T 4 + , 0KTg + ) in Various Neurological Diseases G. Servoz, F. Touraine, C. Quincy

355

XII Cytological Study and Protein Profile in Cerebrospinal Fluid, Before and During Treatment of Neurosyphilis 0. Freney, C. Caudie, S. Gas, C. Quincy

359

Cerebrospinal Fluid Cytology and Protein Parameters in 219 Multiple Sclerosis S. Gas, C. Caudie, C. Quincy

365

Interpretation of the Protein Profile of Cerebrospinal Fluid in Children L. Gerbaut, G. Ponsot

369

Cerebrospinal Fluid Ferritin: Interest as Biological Marker of Central Nervous System Tumors P. Antoine, R. Later, J.C. Eynard, F. Durand, B. Marechal, C. Quincy

375

Clinical Relevance of Beta 2 Microglobulin Levels of the Cerebrospinal Fluid in Children with Acut.e Encephalitis E.J. Raynaud, F. Demeocq, P. Vanlieferinghen, G. Malpuech, G. Gaillard

379

Electrophoretical Patterns of Acetylcholinesterase in Cerebrospinal Fluid J.C. Frot, F. Muller, P. Giraudet

383

Cholinesterase Isoenzymes of Cerebrospinal Fluid and Guillain-Barre Syndrome A. Simplot, S. Guibaud, A. Mercatello, D. Robert, 0. Robert

387

Lymphocytotoxic and Monocytotoxic Antibodies in Multiple Sclerosis L. Rumbach, M.M. Tongio, J.M. Warter, C. Marescaux, M. Col lard, S. Mayer

391

Interest of a High Resolution Agarose Gel Electrophoresis System (Panagel with a Silver Staining Revelation in the Study of Cerebrospinal Fluid (CSF) Immunoglobulin G E. Plan, J.B. Seneterre, C. Quincy

395

XIII Two Dimensional Electrophoresis of CSF Proteins: Electrophoresis Quality Control S. Gehamy, S. Reynier, C. Caudie, C. Quincy

SECTION VI

INFLAMMATION AND

401

MALNUTRITION

Anthropometric and Biological Characterization of Malnutrition States in Man C. André, S. Daniére

409

New Findings on the Role of Zinc as a Biological Response Modifier S. Cunningham-Rundles

421

Immunodeficiency in Protein or Vitamin A Deprived Mice P. Binder, B. Gassies, R. Fontanges

429

Immunological Consequences of Protein Deprivation in Primates S. Qazza

439

Serum Thymic Factor (FTS) Contents of the Thymus in Undernourished Senegalese Children B. Jambon, 0. Ziegler, B. Maire, G. Parent, M.C. Bene, G. Faure, A. Patris, J. Duheille

453

The Clinico-Pathological Consequences of Severe ProteinEnergy Malnutrition in Deceased Senegalese Children O. Ziegler, B. Jambon, G. Parent, M. Maire, R. Dardelin, M. Fall, J. Duheille

457

Protein Profile and Thymic Function in Protein-Energy Malnutrition without Surimposed Infections: Anorexia Nervosa F. Bleiberg, S. Wade, D. Lemonnier, M. Dardenne, H. Flavigny, A. Mosse, J. Lubetzki, P. Chapuis, D. Roche

461

XIV

Malnutrition in Cancer and Cachectic Patients F. Bozzetti

465

Marker Proteins of Inflammation and Nutrition in Advanced Cancer L. Deneux, M. Magalhaes, H. Magdelenat, P. Pouillart

477

Nutritional and Inflammatory Status in 26 Cases of Malignant Hemopathies of the Infancy F. Bienvenu, A. Rochette, G. Souillet

481

Comparison between CRP Serum Values and Hematological Parameters for Diagnosis and Therapeutic Surveillance of Neonatal Infections C. Benattar, D. Vauzelle, A. Lindenbaum, J.F. Magny

485

Contribution of Principal Components Analysis to the Study of Inflammatory and Nutritional Status on Intensive Care Patients J.P. Clavel, D. Gerbet, M.C. Diemert, J. Galli, A. Galli, P. Glaser

489

Effects of Nutrition on the Post-Operative Serum Levels of Prealbumin (PA), Transferrin (T) and Fibronectin (FN) J.F. Zazzo, B. Millat, D. Vauzelle, A. Abella

493

Surgery Induced Modifications of Plasma Protein Levels A. Lepape, J.P. Perdrix, B. Rondelet, V. Banssillon, J. Bienvenu, H. Bernon, A. Roullet, P. Laurent

497

Study of 8 Plasma Proteins during a Case of Severe Heat Stroke J.P. Perdrix, A. Lepape, J.M. Grozel, V. Banssillon, J. Bienvenu, M. Manchon, H. Bernon, A. Roullet

503

Serum Protein Profile Evolution after Sub-Total Hepatectomy M. David, A. Lobera, D. Marée, E. Legrand

507

XV

Malnutrition and Immunity in Chronic Heavy Drinkers with and without Cirrhosis D. Vuitton, P. Rollin, P. Jeambrun, H. Allemand, M.T. Henriet, P. Herve, J.P. Miguet

513

Biological Nutritional Markers which Poorly Reflect the Activity of Crohn's Disease L. Descos, C. André, F. André, P. Landais, J. Fermanian .. 519 Prediction of Success or Failure of Parenteral Alimentation in the Treatment of Severe Forms of Crohn's Disease and Rectocolitis C. André, L. Descos, N. Stremsdorfer 523 Albumin Assay with Astra 8 by a Technique Using Bromocresol Green: Quality Criteria, Comparison with Nephelometry J. Oglobine, B. Bouchet, Z. Pourshaidi, A. Desplaces 527 Quantitative Estimate of the Nutrional Status Through the Measurement of the Electrical Body Impedence A.L. Thomasset, C. Roullet, J. Lenoir

SECTION

VII

MARKER OF

PROTEINS

IN

EXPERIMENTAL

531

MODELS

INFLAMMATION

Characterization of Circulating Alpha-l-Acid-Glycoprotein in Normal and Inflammatory Mice P.M.H. Heegaard, J. Bienvenu, I. Sabbagh, A. Delvil, P. Laurent, T.C. BszSg-Hansen

539

The Inflammatory Reaction of the Developing Rat: Responses of Serum Steroid Hormones and Proteins H. Zouaghi, L.Savu, R. Vranckx, M.E. Martin, E.A. Nunez .. 549 The Effects of Ethinyl-Oestradiol and of Acute Inflammation on the Plasma Level of Rat Alpha 1-Acid Glycoprotein and on the Cell-Free Translation of its Hepatic mRNA M. Diarry-Mehrpour, J. Bourguignon, I. Leroux-Nicollet, D. Vercaigne, D. Biou, M. Hiron, J.P. Lebreton

553

XVI

Evidence for the Identy Between Alpha 1-CysteineProteinase-Inhibitor and Alpha 1-Acute-Phase Globulin in Rat Serum F. Esnard, L. Wibissono, C. Lombart, F. Gauthier

557

Changes in Concentration of Five Rat Serum ProteinaseInhibitors During Experimental Diabetes. Possible Relevance to Non Enzymatic Glycosylation F. Esnard, A. Lepape, W.T. Stauber, J.D. Guitton, F. Gauthier

561

Acute Phase Reactants, Thromboxane and Prostacyclin During Calcium Pyrophosphate-Induced Pleurisy in the Rat M. Tissot, M. Solier, J.P. Giroud, M. D'Asniere, R. Engler

565

Study of the Glycosylated Variants of Chicken Haptoglobin F. Delers, M. Domingo, Y. Rondeau, R. Engler Evidence for the Presence in Chicken Plasma of a Ligand for Haptoglobin which Differs from Hemoglobin F. Delers, M. Domingo, Y. Rondeau, R. Engler

569

575

Effect of an Acute Inflammatory Reaction on the Haptoglobin, Albumin, Cytochrome P-450 Contents and Glucose6-Phosphatase Activity in Hepatic Microsomal Fractions in the Rat J.L. Mahu, G. Feldmann, R. Engler

581

Circadian Variation in PMN-Migration Induced by Live BCG in Swiss Mice J.P. Bureau, L. Garrelly, M. Coupé, G. Labrecque

SECTION VIII

585

LECTINS

Introductive Notes M. Caron, A. Faure

591

XVII

Part Is Lectins as Tools The Use of Lectins to Study Changes in Rat Membrane Glycoproteins Induced by Oral Contraceptives B. Toor, L. McGregor, J. McGregor, S. Renaud, K.J. Clemetson

597

Identification of Cytoplasmic and Membrane Platelet Glycoproteins Using a Combination of SDS-Polyacrylamide Gel Electrophoresis and 125-j.-Labelled Lectins P. Clezardin, J.L. McGregor, E. James, M. Dechavanne, K.J. Clemetson

601

Characterization of Human Epidermal Glycoproteins Recognized by the Lectins Con A and PNA P.W. Ledger, A. Réano, J.Y. Bonnefoy, J. Thivollet

605

Fluorescent Lectins and Quantitative Microscopy M.A. Deugnier, X. Albe, J.C. Bisconte

609

Effects of Serum on Lectin-Induced Lymphocyte Proliferation P.J. Neveu, D. Perdoux 613 Concanavalin A and Prothrombin Complex M. Coulet, M.J. Bezou, P. Travade

615

Modification of Thymidine Transport in LectinStimulated Lymphocytes P. Beaudry, A. Sharif, Y. Goussault

619

Induction of a Thymidine Metabolisme Enzyme in Human Lymphocytes Stimulated by the Robinia pseudoacacia Lectin A. Sharif, P. Beaudry, Y. Goussault

621

Lectins of Robinia pseudoacacia J. Wantyghem, C. Goulut, J.P. Frénoy, Y. Goussault

625

XVIII

Specificity of Human Plasma Fibronectin Towards Biomaterials M. Caron, A. Faure, D. Gulino, C. Boisson, J. Jozefonvicz

627

Study of the Glycosylated Populations of Plasma Alpha 1-Acid Glycoprotein in Human Breast Cancer Patients Treated with Tamoxifen I. Leroux-Nicollet, J.P. Lebreton, M. Hiron, F. Sauger, C. Veyret, P. Bastit

631

Physico-Chemical and Biochemical Characteristics of the Con-A Reactive and Non Reactive Molecular Variants of Human Alpha 1-Acid Glycoprotein I. Leroux-Nicollet, M. Hiron, M. Fontaine, J.P. Lebreton, J.P. Kerckaert, B. Bayard

635

Part 2: Biological Functions of Lectins Biological Functions of Lectins M. Caron, M.A. Deugnier, R. Joubert, P.P. Rouge

641

Localization and Biosynthesis of Lathyrus ochrus Seeds Lectin C. Boisseau, H. Causse, A. Moisand, D. Pere, B. Sousa-Cavada, P. Rouge

651

Surface and Total Receptors for Asialoglycoproteins in Hepatocytes from Diabetic Rats G. Durand, M. Dodeur, J.P.Dumont, S. Coumoul, J. Agneray, J. Feger

655

Part 3: Affinity and Specificity of Lectins Lectin(s) - Ligand(s) Interactions J.P. Frenoy

661

XIX

A Comparative Study of Affinity Constants Measured by Different Methods, Between Peanut Lectin and Sugars with Galactosyl Modified on C(6) or C(l) J. Ohanessian, M. Caron

665

Effect of pH on Binding Properties of Peanut Agglutinin (PNA) M. Decastel, J.P. Frenoy

671

Use of Lectins to Examine the Microheterogeneity of Rat Hemopexin N. Bernard, R. Vranckx

673

AUTHOR INDEX

679

SUBJECT INDEX

683

INDUCTION AND CONTROL OF ACUTE PHASE REACTANT SYNTHESIS

Irving Kushner Department of Medicine, Case Western Reserve University Metropolitan General Hospital, Cleveland, Ohio

I

44109, U.S.A.

The Acute Phase Response

There are both local and systemic responses to infection or tissue injury. The local response to stimulation, inflammation, i s characterized by the consequences of vascular dilatation, increased capillary permeability and leukocyte migration, and i s the subject of intense investigation. Ordinarily, this process leads to repair at the involved s i t e . addition, however, there i s a systemic response (1).

In

Some of the elements

of this response are fever, leukocytosis and a vast variety of hormonal and metabolic changes. The l i v e r plays a major role in this systemic response.

In addition to

many morphologic and enzymatic changes, there i s an increase in synthesis of a number of plasma proteins, the acute phase proteins (or acute phase reactants).

I have a r b i t r a r i l y defined acute phase proteins as those

plasma proteins whose concentrations increase by 25% or more f a i r l y regularly at some point during the f i r s t 5-7 days after stimulus (1). Such changes may not f u l l y reflect changes in synthesis, since increased catabolism may also occur during the acute phase.

I t may be that in the

future a definition based on rate of synthesis would be preferable. TABLE I i s a partial l i s t of the human acute phase proteins, divided into three groups based on the usual magnitude of the plasma changes observed. The most s t r i k i n g changes are seen with C-reactive protein (CRP), the f i r s t acute phase protein recognized, and serum amyloid A protein (SAA), recently discovered.

Neither i s glycosylated.

SAA i s the only acute

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

4

TABLE I

BEST STUDIED HUMAN ACUTE PHASE PROTEINS

Group I - about 50% increase

Group I I - about 2-4 X increase

Ceruloplasmin C3, C4

cq acid glycoprotein a-] anti-trypsin a] anti-chymotrypsin Haptoglobin Fibrinogen

Group I I I - up to 1000 X increase C-reactive protein (CRP) Serum amyloid A (SAA)

phase protein thus far identified which manifests a plasma response comparable to that seen with CRP.

I t i s undoubtedly the precursor of the

major f i b r i l of secondary amyloid tissue.

Much of this presentation will

deal with CRP, which has been our major research focus over the years. Human CRP, with a molecular weight of 105,000, i s a pentraxin; i t i s composed of identical subunits of 21 ,000 daltons, arranged in c y c l i c symmetry.

The related molecule, SAP, another pentraxin, i s a plasma

protein o r i g i n a l l y discovered because of i t s occurrence in all types of amyloid tissue.

This protein shows over 60% amino acid homology with

CRP. I t i s an acute phase protein in some species, such as the mouse, but not in others, such as man. TABLE I I summarizes some of the s i m i l a r i t i e s and differences between three of the plasma proteins which have j u s t been referred to.

SAP i s

glycosylated and makes up about 15% of all types of amyloid t i s s u e , while SAA i s found only i n , and characterizes, the amyloidosis seen in individuals with chronic inflammatory states. One of the s t r i k i n g observations regarding the acute phase response i s the great v a r i a b i l i t y in acute phase behavior from one species to another. Some of these differences, in a few of the proteins, in a few species, are tabulated in TABLE I I I .

The major acute phase proteins in man are

5

TABLE II CRP

SAP

SAA

Acute phase protein in nan

+

-

+

Amino acid homology

+

+

Ca ++ -dependent binding

+

+

Amyloid-associated

+

+

CRP and SAA, in the mouse SAA alone (2), and in the rat a^ acid glycoprotein and "major acute phase a-| protein" described by Schreiber's group (3) and not indicated on this figure.

SAA apparently does not

In each of these species other plasma proteins

occur in the rat.

manifest acute phase behavior, but not of a magnitude comparable to the proteins tabulated.

TABLE III

ACUTE PHASE PROTEINS

SOME MAMMALIAN INTERSPECIES DIFFERENCES CRP

SAA

++++

MOUSE RAT

MAN

SAP

c*2 macro

cq acid GP

++++

0

0

++

+

++++

++

?

++

+

-

0

+++

++++

6 II

Biosynthesis

A.

Site

For many of the studies of C-reactive protein, the rabbit has been used as a model because the serum response appears to be identical to that seen in man.

The major, if not the only, site of CRP synthesis is the

liver, and specifically, the hepatocyte (4).

Following stimulus, the

first cells which can be shown to be synthesizing CRP are located in periportal areas and around the periphery of the hepatic lobules.

With

time, more and more cells can be found to be committed to CRP synthesis; at the peak of the response, virtually all hepatocytes are found to be synthesizing CRP.

Therefore the mechanism of increased synthesis appears

to be progressive recruitment of cells to CRP synthesis.

The sequence is

centripetal, proceeding inward within lobules, suggesting entry of mediator via nerves or blood.

This increased synthesis of plasma proteins is not global.

Some proteins

decrease their plasma concentrations and rates of synthesis during the acute phase. man (5).

Among these are albumin, prealbumin and transferrin in

The term "negative acute phase reactants" has been employed by

some to describe these changes. Two additional points about biosynthesis need to be made: a) on balance, there is a mild but significant increase in total plasma protein synthesis by the liver during the acute phase (6), despite decreased albumin synthesis,

b) The liver is not the only site of synthesis of

acute phase proteins.

For example, complement components and

aj-antitrypsin are also made in monocytes (7, 8); SAA is made to some extent in polymorphonuclear cells (9). synthesized in other sites.

It is not known whether CRP is

Overall, however, it is clear that the liver

is the major site of acute phase protein synthesis.

7 B.

Serum Kinetics

Following stimulus there is a tremendous difference in the rapidity of rise in serum concentrations between CRP and SAA, on the one hand, and the other acute phase proteins. a-j-antichynotrypsin.

The next most rapid rise is shown by

Rate of rise in concentration of other proteins

is even slower than a^-antichymotrypsin, as shown by Laurell's group over a decade ago (5).

These types of observations suggest the

possibility of different mechanisms controlling the synthesis of different acute phase proteins.

In a detailed study of change in serum CRP levels following acute myocardial infarction, an apparently exponential increase in CRP levels was found (10).

The average doubling time was about 8 hours; peak levels

occurred at about 3 days in patients with severe infarction. studies a sort of dose response relationship was observed.

In these In patients

with mild infarction lower CRP levels were achieved than in severe infarction, and they were achieved earlier.

The difference in the

magnitude of response was largely due to the difference in duration of continuing rise in concentration, and, inferentially, to a difference in continuing high rates of hepatic CRP synthesis. C.

Studies of Biosynthesis:

Liver Perfusion and Hepatocyte Culture

We directly measured CRP synthesis by isolated perfused rabbit livers obtained at intervals after intramuscular typhoid vaccine injection (11) A constant linear rate of CRP accumulation was found in each study, which we showed by cycloheximide inhibition studies to be synthesis and not mere secretion.

Rates of synthesis clearly increased with time in vivo

after stimulus.

But synthesis rate did not increase during the course of

each individual perfusion.

Therefore, the increase in rate of CRP

synthesis which occurs in vivo must depend on extrahepatic factors. In comparable studies in which we measured CRP synthesis by primary hepatocyte cultures from rabbits manifesting differing degrees of CRP response, we found rate of CRP synthesis and secretion to correlate with the serum CRP levels at the time of isolation of the hepatocytes (6).

8 Similar results were found in the perfusion studies summarized above. These observations suggested that rates of synthesis observed in these in vitro studies reflected in vivo rates. [1.

Serum Turnover Studies

It was possible to calculate approximate in vivo CRP synthetic rates from serum turnover studies in rabbits. In unstimulated rabbits, the serun half-life of CRP generally lay between 4 and 6 hours, remarkably rapid compared to most plasma proteins.

Fractional catabolic rates derived

from such studies lay in the range of about 15% per hour and did not appear to vary significantly regardless of serum CRP levels (12).

It is

possible that studies of larger numbers of animals or animals with even higher CRP levels might reveal smaller fractional catabolic rates in strongly responding rabbits.

Nonetheless, using these values, we were able to calculate approximate rates of CRP synthesis in some of the rabbits we studied (TABLE IV). Substantial differences between unstimulated and moderately stimulated rabbits were observed (12).

In addition, assuming a fractional

cata-

bolic rate of 15% per hour, we calculated theoretical rates of CRP synthesis in rabbits with low normal serum levels and in rabbits with a marked CP.P response.

These studies showed that synthesis rates in the

latter group might be 4000 times as great as in the former, and would exceed 25% of normal albumin secretion rate in the rabbit.

Thus, in the

rabbit, and presumably in man, there is a major commitment to CRP synthesis in the presence of severe tissue injury or infection.

Major changes in acute phase protein synthesis have been demonstrated in vivo in another species, the rat, by Schreiber, et al (13). synthesis decreased to 1/3 of normal rates.

Albumin

There was a 20-fold

increase, or more, in the two major acute phase proteins in this species.

9 TABLE IV

CALCULATION OF RABBIT CRP SYNTHESIS RATE (yg/Kg/hr) From direct measurement Liver Hepatocyte perfusion culture

From turnover in vivo

Experimental animals: Unstimulated: CRP conc = 0.9 yg/ml 24 h p stimulus; CRP conc = 65-70 ug/ml

6.7 560

410

475

36 h p stimulus; CRP conc = 104 yg/ml

560

Theoretical: for serum CRP conc = 0.1 yg/ml

ca. 0.7

for serum CRP conc = 400 pg/ml

ca. 2,700

For comparison, normally rabbit albumin = ca. 10,600 ug/Kg/hr

III

Molecular Biology

Concentrations of mRMA for rat a-j acid glycoprotein were demonstrated to be increased in livers from stimulated animals compared to normal animals (14). for albumin.

At the same time there was a marked decrease in the mRNA Similar findings have been reported for mouse SAA (15) and

hemopexin (16). CRP (17).

Harvey Colten's group has now found the same for human

Therefore, in all instances studied, evidence of increased

amounts of mRNA during the acute phase has been found, presumably on the basis of increased transcription.

In the case of SAA, increased

transcription itself has been shown (18).

10

IV

Induction

A wide variety of diverse stimuli lead to the acute phase response in vivo (1). induction?

Are there common mechanisms by which all of these lead to APP One set of candidates in which there i s a great deal of

interest i s the products of activated macrophages.

The greatest interest

has focused on a molecule or family of molecules with a molecular weight of about 15,000 , whose a c t i v i t i e s have led to i t s being identified over the years as Endogenous Pyrogen, Leukocytic Pyrogen, Leukocytic Endogenous Mediator and now, Interleukin 1 (19). In in vivo studies, monokines have produced many acute phase phenomena, including increased levels of CRP and other acute phase proteins (20, 21). However, there are reasons to believe that the effect of monokines on the acute phase response i s not entirely straightforward.

For example, there

are questions regarding whether the effect on the l i v e r i s direct or mediated, at least to some extent, via the central nervous system.

In

studies by Turchik and Bornstein in the rabbit, (TABLE V), doses of Leukocytic Pyrogen which failed to produce a CRP response when administered intravenously produced a marked response when injected into the cerebral ventricles (22).

The effects of various doses and routes of

administration paralleled the effect on fever, known to be mediated'via the central nervous system.

TABLE V

ROLE OF CENTRAL NERVOUS SYSTEM

IN CRP RESPONSE TO LEUKOCYTIC PYROGEN from Turchik & Bornstein (ref. 22) Route of administration

24 hr CRP r i s e

T: r i s e in °C

0.1

cerebral ventricle

25 yg/ml

1.9

1.0

cerebral ventricle

50 yg/ml

2.6

1.0

intravenous

1 yg/ml

0.7

10.0

intravenous

10 yg/ml

1.3

Dose of LP

11

In addition, studies of the molecular size of active monokines indicate that more than one molecular species may directly influence acute phase protein synthesis, and that factors other than monokines may be important in this process.

TABLE VI l i s t s reports of acute phase protein induction

in hepatocyte cultures.

Several of these deserve comment:

in molecular sizes of active monokines have been shown.

Differences

A requirement

for dexamethasone i s reported in one instance, rat fibrinogen (23). While SAP induction by Interleukin 1 preparations i s reported by one group (24), an increase of only about 10", hardly persuasive, i s reported by another (25).

There are differences regarding the effect of normal

serum on SAA synthesis.

TABLE VI

FACTORS INDUCING ACUTE PHASE

PROTEIN SYNTHESIS IN HEPATOCYTE CULTURES I MONOKINES MW

Species & Protein

Reference

15,000 10,000 - 50,000 not 10,- 50,000

murine SAA murine a] AGP, SAA, hemopexin murine haptoglobin

not determined

murine SAP (difference of opinion)

25,000 - 30,000 not determined not determined

rat fibrinogen (requires dex) rat a2 macro, ai AT, C3 rat haptoglobin

(23) (27) (28)

rat ai AGP, a 2 macro

(29)

murine SAA but not SAP

(25)

murine SAA (not induced by normal serum)

(30)

I I CORTICOSTEROIDS ALONE I I I NORMAL SFRUM (fetal c a l f , human, mouse) IV LATENT PHASE SERUM

(mouse)

(26) (2) (2) (24, 25)

12

In our own attempts to stimulate CRP synthesis in rabbit primary hepatocyte cultures, we have tested a number of monokine preparations obtained from different investigators and different species, including rabbit, man, and mouse. induction.

We have never been able to show reproducible CRP

Recently we have used an anti-rabbit SAA antiserum (31)

obtained from Drs. Skogen and Natvig of Norway to study whether SAA induction occurred in any of these experiments.

Ue've been able to

employ this only in the absence of serum and have found, in one experiment, a 50% increase in secretion of an antigen reacting with this antiserum.

We have not as yet been able to confirm the s p e c i f i c i t y of

this antiserum.

V Summary Acute phase reactants i s a generic term for the individual plasma proteins whose synthesis increases at variable rates, to variable extents during the f i r s t few days after tissue injury or infection.

During this

period, synthesis of certain other plasma proteins decreases, suggesting complex integration of the l i v e r ' s protein synthetic system. There i s great v a r i a b i l i t y between mammalian species in the spectrum of acute phase reactants. The acute phase response probably results largely from increased transcription of acute phase proteins.

In every instance thus far

studied, increased accumulation of specific mRNA has been shown. Induction of acute phase proteins i s probably an intricate and complex process.

I t can be caused by monokines of varying molecular sizes.

It

i s l i k e l y that other mechanisms, perhaps involving the central nervous system, may participate.

13 References

1.

Kushner, I.: Ann. N.Y. Acad. Sei. 389, 39-48

(1982).

2.

Baumann, H., Jahreis, G.P., Gaines, K.C.: J. Cell. Biol. 97: 866-876 (1983).

3.

Urban, J., Chan, D., Schreiber, G.: J. Biol. Chem. 254, (1979).

10565-10568

4.

Kushner, I., Feldmann, G.: J. Exp. Med. 801, 466-477

5.

Aronsen, K.F., Ekelund, G., Kindmark, C.O., Laurel 1, C.B.: Scand. J. Clin. Lab. Invest. 29, 127-136 (1972).

(1978).

6.

Macintyre, S.S., Schultz, D., Kushner, I.: Biochem. J. 2K), 707-715 (1983).

7.

Col ten, H.R., Ooi, Y.M., Edelson, P.J.: Ann. N.Y. Acad. Sei. 332, 482-490 (1979).

8.

Isaacson, P., Jones, D.B., Judd, M.A.: Lancet 2, 964-965

9.

Rosenthal, C.J., Sullivan, L.: J. Clin. Invest. 62, 1181-1186

10.

Kushner, I., Broder, M.L., Karp, D.: J. Clin. Invest. 61_, 235-242 (1978).

11.

Kushner, I., Ribich, W.N., Blair, J.B.: J. Lab. Clin. Med. 96, 1037-1045 (1980).

12.

Chelladurai, M., Macintyre, S.S., Kushner, I.: J. Clin. Invest. 71_, 604-610 (1983).

13.

Schreiber, G., Howlett, G., Nagashima, M., Millership, A., Martin, H. Urban, J., Kotler, L.: J. Biol. Chem. 257, 10271-10277 (1982).

14.

Ricca, G.A., Hamilton, R.W., McLean, J.W., Conn, A., Kalinyak, Taylor, J.M.: J. Biol. Chem. 256, 10362-10368 (1981).

15.

Morrow, J.F., Stearman, R.S., Peltzman, C.G., Potter, D.A.: Proc. Natl. Acad. Sei. USA 78, 4718-4722 (1981).

16.

McLean, J.W., Taylor, J.M.: Fed. Proc. 42, 1039

17.

Whitehead, A.S., Bruns, G.A.P., Markham, A.F., Colten, H.R., Woods, D.E.: Science 221, 69-71 (1983).

18.

Stearman, R.S., Lowell, C.A., Pearson, W.R., Morrow, J.F.: N.Y. Acad. Sei. 389, 106-115 (1982).

19.

Dinarello, C.A.: Lymphokines 7, 24-74

(1979). (1978).

J.E.,

(1983).

(1982).

20.

Bornstein, D.L., Walsh, E.C.: J. Lab. Clin. Med. 91_, 236-245

21.

Merriman, C.R., Pulliam, L.A., Kampschmidt, R.F.: J. Lab. Clin. Med. 83, 310-322 (1974).

(1978).

22.

Turchik, J.B., Bornstein, D.L.: Infect. Immun. 30, 439-444

23.

Ritchie, D.G., Fuller, G.M.: Ann. N.Y. Acad. Sei. 408, 490-500 (1983).

(1980).

14

24.

Le, P . T . , M ü l l e r , M.T., Mortensen, R.F.: J . Immun. 129, 665-67? (1982).

25.

Tatsuta, E . , Sipe, J . D . , Shirahama, T., Skinner, M., Cohen, A . S . : J . B i o l . Chem. 258, 5414-5418 (1983).

26.

McAdam, K.P.W.J., L i , J . , Knowles, J . , Fuss, N.T., D i n a r e l l o , C.A., Rosenwasser, L . J . , S e i i n g e r , M.J., Kaplan, M.M., Goodman, R.: Ann. H. Y. Acad. Sei. 389, 126-136 (1982).

27.

Fouad, F.M., Scherer, R., Abd-El-Fattah, M., Ruhenstroth-Bauer, G.: Euro. J. Cell B i o l . 2T_, 175-179 (1980).

28.

Hooper, D.C., Steer, C . J . , D i n a r e l l o , C.A., Peacock, A.C.: Biochimica et Biophysica Acta 653, 118-129 (1981).

29.

Baumann, H., Firestone, G.L., Burgess, T . L . , Gross, K.W., Yamamoto, K.R., Heid, W.A.: J. B i o l . Chen. 258, 563-570 (1983).

30.

Benson, M.D.: Ann. N.Y. Acad. S e i . 389, 116-120 (1982).

31.

Anders, R . F . , Matvig, J . B . , S l e t t e n , K., Husby, G., Nordstoga, K.: J. Immun. 118, 229-234 (1977).

USE OF IMMUNOMORPHOLOGICAL METHODS AND HEMOLYTIC PLAQUE TESTS TO INVESTIGATE THE SYNTHESIS AND SECRETION OF PLASMA PROTEINS BY THE LIVER CELLS DURING THE ACUTE INFLAMMATORY REACTION

Gérard Feldmann Laboratoire de Biologie Cellulaire, Faculté de Médecine XavierBichat, 16 rue Henri-Huchard, 75018 Paris and Unité de Recherches de Physiopathologie Hépatique, INSERM U24, Hôpital Beaujon, Clichy, France.

Acute phase reactant proteins

(APRP) form a heterogeneous

group of at least nine plasma proteins : ai-acid glycoprotein (orosomucoid), ai-antitrypsin, ai-antichymotrypsin, a 2 -macroglobulin, C-reactive protein, ceruloplasmin, fibrinogen, haptoglobin and the recently discovered serum amyloid A-protein (1, 2). Except for ai-antichymotrypsin, for which no convincing arguments as to its origin have yet been forthcoming, the hepatic origin of the APRP listed above has been well demonstrated by the use of biochemical technique

(1, 3, 4),

which, however, only provide overall information on the hepatic formation of these proteins. All the biochemical results are based on the assumption that the liver is composed of only one cell type, acting homogeneously both under normal conditions and during the acute inflammatory reaction. In fact, however, the liver is a complicated organ, which, in addition to the different cells constituting the walls of the vessels and biliary ducts, is formed of four different cell cytes, and three kinds of sinusoidal cells and endothelial cells

-

types-hepatoKupffer, Ito

(5). Hepatocytes represent about 78 % of

the cellular volumetric mass, and the remaining 22 % is unequally divided among the extracellular space and the various sinusoidal cells

(6). The cytoplasm of each of these four cells

contains the organelles responsible for the synthesis and secretion of exported proteins i.e., the endoplasmic

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

reticulum

16

and Golgi apparatus (7). Consequently, each cell could produce the APRP. Moreover, in the liver, the four cells are arranged according to a definite order. The morphofunctional unit of the liver is the hepatic lobule, which is surrounded by several portal spaces at its periphery and centered by a unique centro-lobular vein. Between these structures are the hepatic plates, composed of hepatocytes surrounded by the sinusoids, with the sinusoidal cells located very close to the hepatocytes. Depending on the location of the cells in the lobule, cellular metabolism has been demonstrated to differ, at least for some hepatic functions (8). The cells located around portal spaces are the most active metabolically. The opposite situation is observed in the centro-lobular zone around the central vein and an intermediary situation exists in the medio-lobular zone. It should be remembered that the mediators acting in APRP production reach the liver through the portal vessels, and the first liver cells they encourted are those located around the portal spaces (9, 10). The cellular organization of the liver makes it necessary to use methods other than biochemical techniques to obtain information on the biology of the cells responsible for APRP production. Schematically, these methods can be divided into two groups, according to the investigation : 1) In cases when it is necessary to preserve the hepatic lobules, techniques must be used which are capable of identifying the APRP while maintening the structure of the cells intact and their position in the lobule unchanged : so far, immunomorphological techniques have proved the only ones to meet these requirements. Among, the various techniques available, a distinction must be drawn between those that can be used on electron microscopy and are therefore capable of providing information on APRP synthesis and secretion, and those that can be used on optical microscopy and capable simply to demonstrate that a cell contains APRP with no possibility to distinguish between active synthesis and passive accumulation. In the first category, the leading techniques are immunoenzymatic techniques with their principal

17

variant immunoperoxidase (IPO) while in the second categorythere is immunofluorescence (IF). 2) When the nature of the cells with preservation of their structure but not their location in the hepatic lobule is required, isolated liver cells can be investigated, at least in the animals. The behavior of some APRP has been investigated in isolated hepatocytes (11-13X However, in all these reports, APRP synthesis was measured using biochemical techniques which, as it was pointed out above, did not supply any information on functions at single cell level. Immunomorphological techniques can be used on single cell in the same way as they are employed for the whole liver. However, as illustrated below, they are more appropriated when the information required concerns intracellular synthesis and secretion. When extracellular secretion is investigated, techniques like the hemolytic plaque tests are very advantageous.

Contribution of immunomorphological techniques The principle of these techniques is the following : under certain conditions of cell fixation, intracellular proteins preserve their antigenic determinants instead of being destroyed or transformed. They can be recognized in the cells by the use of specific antibodies labelled with a fluorochrome on with the enzyme horseradish peroxidase. Immunomorphological techniques have been used to locate, ai-acid glycoprotein (10), ai-antitrypsin (14), 012-macroglobulin (10), C-reactive protein (9), ceruloplasmin (15), fibrinogen (10) and serum amyloid Aprotein (16) in the liver cells of man, rat, rabbit or mouse. The cells labelled were generally the hepatocytes. However, some discrepancies have been observed between the results obtained with IF and IPO. For instance, fibrinogen (17) and haptoglobin (18) were reported to be present in both hepatocytes and Kupffer cells when IF was used, whereas with IPO, they were only identified in hepatocytes (10). These differences

18

are probably due to the fact that the IPO is more specific and sensitive that IF. The use in IPO of affinity-purified antibodies instead of immunoglobulins as in IF affords greater specificity in cellular localization

(19), and in particular re-

duces the frequency of artifacts. The possibility provided by IPO of examining the cells by electron microscopy easily accounts for its sensitivity. Moreover, ultrastructural observations can definitively confirm that the cell structure is correctly preserved and that the protein correctly localized

(19,

20) . Another feature demonstrated by IF and/or IPO is that only between 1 and 10 % of normal hepatocytes produce a particular APRP at any given time (10, 14, 15). When the cellular kinetics of the APRP were followed during the acute inflammatory reaction, as we did in the rabbit for C-reactive protein (9) and in the rat for four different APRP (10), we observed that at the beginning of the reaction, APRP-producing hepatocytes were visible first around the portal spaces, then in the medio-lobular zone, and later as the inflammatory reaction extended in the centro-lobular zone. At the maximum of the reaction, almost all the hepatocytes were producing the APRP

(Fig. 1) (9, 10).

On electron microscopy, large amounts of APRP were visible in the endoplasmic reticulum and Golgi apparatus (Fig. 2). Optical (Fig. 1) and ultrastructural observation of the hepatocytes (10) suggested, however, that the activity of each cell was not of the same intensity. Fig. 1. Optical microscopy. Immunoperoxidase. Twenty-four hours after the beginning of the acute inflammatory reaction, almost all the hepatocytes produce haptoglobin, indicated on this figure by black deposits visible in the cytoplasm of the cells. Note that the intensity of the staining is variable from cell to cell (nuclei uncont e s t e d ) (x 400). Fig. 2. Electron microscopy. Immunoperoxidase. Same conditions as in Fig. 1. Large amounts of haptoglobin indicated by electron-dense deposits, are visible in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of this hepatocyte. (N : Nucleus ; M : Mitochondria) (x 22,000).

20

The small percentages of APRP-producing hepatocytes, which was also observed for other non-APRP proteins, like albumin (21), transferrin (22) or prothrombin (23), is connected to the technical conditions employed to prepare the liver cells, as it was observed recently (20). When liver fragments were immersed in the fixative (immersion fixation) as it was done in most investigations (9, 10, 14, 15, 17,20), only a small percentage of hepatocytes was observed to produce APRP. The same results were obtained with this method, for instance for fibrinogen, when a cell membrane permeabilizing agent, like saponin, was added to the fixative (20). In contrast, striking differences were seen when the fixation was conveyed to the liver through the portal vein (perfusion fixation) and saponin added to the fixative : instead of a few labelled hepatocytes, almost all the cells were observed to synthetize and secrete fibrinogen (20). Under these

conditions, the recruitment observed in the

hepatic lobule during the acute inflammatory reaction (9, 10) had to be reinvestigated. Preliminary results showed that there was apparently no difference between controls and experimental animals 24 hours after an inflammatory reaction induced by turpentine administration (24). Quantitative electron microscopy studies are now in progress to ascertain whether there are any difference

between the hepatocytes producing APRP.

Finally, immunomorphological techniques have been used to demonstrate that a single hepatocyte is capable to produce several proteins at the same time. Our experiments with primary cultures of rat hepatocytes suggested that the same hepatocyte was able to synthetize fibrinogen and albumin (25). In vivo, when serial liver sections were each incubated in labelled antibodies against different APRP, we observed that during an experimental acute inflammatory reaction a single hepatocyte was capable of producing four different APRP at a given time (10). Consequently, in seems clear that the hepatocyte is not specialized, at least for this function (10). This does not mean, however, that the hepatocyte can produce all the APRP at the same time, or all the plasma proteins coming from the li-

21 ver. It is well known that the different APRP have different kinetics for instance those of C-reactive protein are not the same as those of haptoglobin

(9, 10). Further, it is reasona-

ble to suppose that the activity of the hepatocytes in protein synthesis is restricted to a certain level. For instance, the capacity of hepatocytes to synthetize albumin cannot exceed two of three times (26). The production capacity of the liver for APRP has not yet been investigated at least to our knowlegde. However, a level in their production might exist. This is suggested for instance, by the finding that when the dose of turpentine administered to the rat was double or tripled

(1 ml or

1.5 ml per 100 g body weight instead of the usual 0.5 ml (10)), the plasma concentration of fibrinogen did not differ from that observed with 0.5 ml (24). When hepatocytes are strongly stimulated, they apparently cannot synthetize all the proteins. This situation is not restricted to the proteins exported in the plasma, as we shown in the next paragraph, but also applies to the non-exported proteins like cytochrome Pi, 50 as we observed with J.L. Mahu and R. Engler (see the communication on this subject in this volume). APRP production by hepatocytes under experimental or pathological conditions has so far been little investigated by immunomorphological techniques. In addition to the works already reported ealier

(9, 10), the acute inflammatory reaction was fol-

lowed in the liver for haptoglobin with IF up to 125 hours. In such late times, Kupffer cell localization was possibly an artifact (see above) but interpreted as a morphological sign of cellular catabolism

(18). We investigated the behavior of some

APRP in the hepatocytes of rats with carbon-tetrachloride-induced cirrhosis

(27) and we observed that, despite changes in

the liver, hepatocytes were still able to produce APRP almost as much as those observed

in

animals with a normal liver.

These results suggest that under certain conditions, the hepatocytes remaining in cirrhosis could compensate for the destruction induced by this disease by increasing their protein synthesizing capacity.

22 Contribution of hemolytic plaque tests The hemolytic plaque tests used to explore plasma protein secretion by hepatocytes originated in immunology, a field in which they are very often proposed to investigate immunoglobulin production by plasma cells. The principle of the technique is the following : isolated liver cells are incubated with sheep red blood cells labelled with specific antibodies against a given plasma protein. In the presence of guinea-pig complement, the red blood cells are destroyed and hemolytic plaques appear around the secreting cells. The technique can be semiquantitative, measuring the diameter of the hemolytic plaques and of the secreting cells. Unlike to immunomorphological techniques, hemolytic plaque tests have only begun to be applied to liver cells. We used them to compare the amounts of albumin and fibrinogen secreted by hepatocytes in normal conditions and during an"acute inflammatory reaction induced in the rat by in vivo turpentine administration (28). Firstly, we observed that there was a direct relationship between the diameters of the secreting hepatocytes and the hemolytic plaques, suggesting that protein production is dependent on the quantities of active ribosomes and of nuclear DNA present. Secondly, we saw that while the percentages of albumin and fibrinogen-secreting hepatocytes were respectively about 7 0 % and 1 % in the normal state, the opposite situation was observed 24 hours after the beginning of the inflammatory reaction, when about 50 % of the hepatocytes were secreting fibrinogen, and only 1 %, albumin. This observation confirms other biochemical findings (29, 30) and provides a strong argument in favour of the possibility that even if a single hepatocyte is potentially able to synthetize all proteins, it cannot synthetize all proteins at any given time. In view of these results, the significance of the so-called acute-phase "negative" reactant proteins (31, 32) remains uncertain. It is possible that the behavior of these proteins can only be explained by some physiological cellular functions of

23 the hepatocytes.

Acknowledgments : The authors wishes to think Mrs Maurice and Malet for their help in the preparation of the manuscript.

References 1.

Koj, A.: In Structure and function of plasma proteins, Allison, A.C. edit., Plenum Press, London, Vol 1, pp 73131, 1974

2.

Kushner, I.: In C-reactive protein and the plasma protein response to tissue injury, Kushner, I., Volanakis, J.E., Gewürz, H. edits, Ann. N.Y., Acad. Sei., 389, 39-48, 1982

3.

Schultze, H.E., Heremans, J.F.: Molecular biology of human proteins with special reference to plasma proteins. Elsevier, Amsterdam, Vol 1, pp 354-355, 1966

4.

Putnam, F.W.: In The plasma proteins. Structure, function, and genetic control, Putnam F.W. edit., Academic Press, New York, Vol 1, Second edition, pp 57-131, 1975

5.

Feldmann, G.: In Physiologie humaine, Meyer, P. edit., Flammarion, Paris, pp 113-119, 1983

6.

Blouin, A., Bolender, R.P., Weibel, E.R.: J. Cell Biol. 72, 441-455 (1977).

7.

Palade, G.E.: Science JJ3J3, 347-358

(1975).

8.

Jungermann, K., Katz, N.: Hepatology 2, 385-395

9.

Kushner, I., Feldmann, G.: J. Exp. Med. 148, 466-477 (1978).

(1982).

10. Courtoy, P.J., Lombart, C., Feldmann, G., Moguilevsky, N., Rogier, E.: Lab. Invest. 44, 105-115 (1981). 11. Crane, L.J., Miller, D.L.: J. Cell Biol. 72, 11-25

(1975).

12. Canonico, P.G., Ayala, E., Rill, W.L., Little, J.S.: Am. J. Clin. Nutr. 30, 1359-1363 (1977). 13. Hooper, D.C., Steer, C.J., Dinarello, C.A., Peacok, A.C.: Biochim. Biophys. Acta 653, 118-129 (1981). 14. Feldmann, G., Guillouzo, A., Maurice, M., Guesnon, J.: In Immunoenzymatic techniques, Feldmann, G., Druet, P., Bignon, J., Avrameas, S. edits, Elsevier, Amsterdam, pp 379394, 1976 15. Feldmann, G.: In Electron microscopy and cytochemistry. Wisse, E., Daems, W.Th., Molenaar, I., Van Duijn, P. edits, Elsevier, Amsterdam, pp 145-149, 1973

24

16. Benson, M., Kleiner, E.: J. Immunol. 124, 495-499 (1980). 17. Hamashima, Y., Harter, J.C., Coons, A.H.: J. Cell Biol. 2_0, 271-279 (1964). 18. Peters, J.H., Alper, G.A.: J. Clin. Invest. 45, 314-320 (1966). 19. Feldmann, G.: J. Microscopie Tt, 293-300 (1974). 20. Pignal, F., Maurice, M., Feldmann, G.: J. Histochem. Cytochem. 3J), 1004-1014 (1982). 21. Feldmann, G., Penaud-Laurencin, J., Crassous, J., Benhamou, J.P.: Gastroenterology 63, 1036-1048 (1972). 22. Lane, R.S. : Nature 215, 161-162 (1967). 23. Barhnart, M.I.: J. Histochem. Cytochem. 13, 740-751 (1965). 24. Feldmann, G.: Unpublished data. 25. Guillouzo, A., Feldmann, G., Boisnard, M., Sapin, C., Benhamou, J.P.: Exp. Cell Res. 96, 239-246 (1975). 26. Katz, J., Bonorris, G., Okuyama, S., Sellers, A.L.: Am. J. Physiol. 212, 1255-1260 (1967). 27. Courtoy, P.J., Feldmann, G., Rogier, E., Moguilevsky, N.: Lab. Invest. 45, 67-76 (1981). 28. Bernuau, D., Rogier, E., Feldmann, G.: Hepatology _3» 29-33 (1983) . 29. Princen, J.M.G., Nieuwenhuizen, W., Mol-Backx, G.P.B.M., Yap, S.H.: Biochem. Biophys. Res. Commun. 102, 717-723 (1981) . 30. Morow, J.F., Stearman, R., Peltzman, C.G., Potter, D.A.: Proc. Natl. Acad. Sei. USA 78, 4718-4722 (1981). 31. Lebreton, J.P., Joisel, F., Raoult, J.P., Lannuzel, B., Roger, J.P., Humbert, G.: J. Clin. Invest. jv4, 1118-1129 (1979). 32. Savu, L., Lombart, C., Nunez, E.A.: Febs Lett. 113, 102106 (1980).

PROGRESS

IN PLASMA PROTEINS:

RECENT F I N D I N G S ON THE STRUCTURE

OF CERULOPLASMIN

Frank

W. Putnam, N o b u h i r o T a k a h a s h i ,

Department of B i o l o g y , Bloomington,

Indiana

Introduction: The

(1)

larly

to

reviewed

as

the

First

progress

reflected about

University

in Plasma

in

Proteins

Symposium

in

plasma

At t h a t

some 50

were

properties

available

that

allowed

quantitative

immunoglobulins

a c u t e phase r e a c t a n t s . family

control,

structure

genetic

biosynthesis

states.

for

Furthermore, or

the

of

the

factors

that

are a

greatly

we know l i t t l e

or cause the c h a r a c t e r i s t i c

in

and in

about

regulate

response

of

especial-

function,

differ

the

principal

the i m m u n o g l o b u l i n s

the acute phase p r o t e i n s

control

other

antisera

changes

T h i s was

r e l a t e d in s t r u c t u r e ,

and f u n c t i o n .

and

measurement

and a l s o

Yet, whereas

of p r o t e i n s c l o s e l y

genetic their

disease

much could

specific

and o f s i g n i f i c a n t

in v a r i o u s

the

reac-

They

mobilities

I n many c a s e s

ly

for

proteins.

(2-4).

the b l o o d l e v e l true

Mar-

particu-

of the acute phase

human p l a s m a

range of c o n c e n t r a t i o n

normal

(2-4),

t i m e t h e r e was a l r e a d y

be i d e n t i f i e d by t h e i r e l e c t r o p h o r e t i c physical

on I n f l a m m a t i o n

proteins

the p r o p e r t i e s

t a n t s o f human p l a s m a . information

Ortel

47405

Progress

introduction

kers

Indiana

and Thomas L .

their

inflamma-

tion. At

the

ture,

time of the F i r s t that

bonding

is,

Symposium we knew the p r i m a r y

the c o m p l e t e

pattern,

location

characteristics

of

immunoglobulins

IgG,

four IgA,

amino of of

acid

sequence,

oligosaccharides the

IgM,

five

and

IgE

classes (4).

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in Germany

struc-

disulfide and

other

of

human

Recently

the

26 primary

structure

of

IgD -

has been r e p o r t e d by our

the

laboratory

human i m m u n o g l o b u l i n c l a s s e s Also,

at

the

properties

of

First the

ing f i b r o n e c t i n ) primary

alpha

complete

Then,

1-antitrypsin

amino

published

in

laboratory

acid

1982

has

by

table

acute

phase

At t h a t

sequence

of

Carrell

e_t

completed

the

Antitrypsin Ceruloplasmin C-reactive protein

of

(exclud-

the

complete

the

proteins:

protein,

haptoglobin,

structure

was known

However,

the

1-antitrypsin

was

(6) acid

table

the

of

and

recently

sequence

of

properties

our

human can

be

1.

Pi

Carbohydrate content

Amount in normal plasma (mg/lOOml)

Sequence

40,000

2.7

41.4

55-140

complete

ajAT

54,000

4.8

12.4

200-400

compiete

Cp

135,000

4.4

8.0

15-60

compiete

0

-0>Ti>T3>-n Î i i i r i i i i ï i i î

CRP

%

Haptoglobin

^

Orosomucoid

•

alpha 2 macroglobulin

•

alpha 1 antitrypsin

65 to 177% (p ^

50 30 M I N U T E I0-

LÜ

cr Figure 5

106 E f f e c t s of A A G and H e p a r i n on P l a t e l e t A g g r e g a t i o n / C l o t F o r m a t i o n in PRP I n d u c e d by A c t i v a t e d T h r o m b o f a x R e a g e n t . We n e x t i n v e s t i g a t e d the a n t i - h e p a r i n e f f e c t of A A G f o l l o w i n g c h a l l e n g e of PRP w i t h A c t i v a t e d T h r o m b o f a x R e a g e n t . In this s y s t e m , p l a t e l e t s are a c t i v a t e d by the i n t r i n s i c g e n e r a t i o n of t h r o m b i n as o p p o s e d to its e x o g e n o u s a d d i t i o n as d e p i c t e d in F i g u r e s 1-3; clot f o r m a t i o n is s i m i l a r l y s t i m u l a t e d . As s h o w n in F i g u r e 4, h e p a r i n p r o l o n g e d the o n s e t of p l a t e l e t a g g r e g a t i o n and clot f o r m a t i o n (curve B). The a d d i t i o n of A A G (2 m g ; curve C) s h o r t e n e d the o n s e t of p l a t e l e t a g g r e g a tion but p r o l o n g e d the o n s e t of clot f o r m a t i o n ; A A G (plus h e p a r i n ) at 4 mg (curve D) had e v e n a m o r e d r a m a t i c and dichotomous effect. In the a b s e n c e of h e p a r i n , A A G (4 m g ; c u r v e E) s h o r t e n e d e a c h the o n s e t of p l a t e l e t a c t i v a t i o n and coagulation. A t no c o n c e n t r a t i o n of h e p a r i n or A A G c o u l d an a n t i - h e p a r i n e f f e c t be d e m o n s t r a t e d . M o r e o v e r , the p r o - a g g r e g a n t a c t i v i t y a s s o c i a t e d w i t h A A G w a s not e v i d e n t u n l e s s the clotting system was initiated. T h i s is d e p i c t e d in F i g u r e 5 w h i c h i l l u s t r a t e s that, i n d e p e n d e n t of h e p a r i n , an a m o u n t of A A G w h i c h acts as a p l a t e l e t p r o - a g g r e g a n t a n d / o r p r o - c o a g u lant f o l l o w i n g t h r o m b i n or T h r o m b o f a x c h a l l e n g e of P R P inhibits platelet aggregation following challenge with ADP. C-REACTIVE

PROTEIN.

The c l a s s i c a l acute p h a s e r e a c t a n t , C - r e a c t i v e p r o t e i n (CRP), d e s c r i b e d by T i l l e t t and F r a n c i s in 1930 (19), a p p e a r s in m a r k e d l y e l e v a t e d c o n c e n t r a t i o n in the sera of i n d i v i d u a l s u n d e r g o i n g r e a c t i o n s of acute i n f l a m m a t i o n and tissue d e g r a d a t i o n and is found d e p o s i t e d at sites of tissue injury ( r e v i e w e d in ref. 20). C R P has an Mr of 1 1 5 , 0 0 0 , c o n s i s t i n g of five i d e n t i c a l , n o n c o v a l e n t l y b o u n d s u b u n i t s , and s h a r e s m u l t i p l e functional similarities with immunoglobulins. T h e s e include the a b i l i t y to i n i t i a t e r e a c t i o n s of c o m p l e m e n t c o n s u m p t i o n and p h a g o c y t o s i s and the c a p a c i t y to bind w i t h m o n o n u c l e a r p e r i p h e r a l blood c e l l s _in v i t r o . T h e s e r e a c t i v i t i e s e a c h require C R P to bind w i t h one of its m u l t i v a l e n t l i g a n d s or to be h e a t m o d i f i e d or c h e m i c a l l y a g g r e g a t e d ( r e v i e w e d in 20). We r e c e n t l y r e p o r t e d (3) that t h e r m a l l y m o d i f i e d CRP (H-CRP) i n d u c e d r e a c tions of a g g r e g a t i o n and s e c r e t i o n from i s o l a t e d p l a t e l e t s in a m a n n e r s i m i l a r to t h e r m a l l y m o d i f i e d h u m a n igG (AHGG) but, on a w e i g h t b a s i s , w a s 10-20 fold more e f f e c t i v e than A H G G . We d e s c r i b e here the e v i d e n c e that H - C R P c a u s e s p l a t e l e t s to release a - g r a n u l e c o n s t i t u e n t s , to p r o d u c e and s e c r e t e a p o t e n t a o r t a - c o n t r a c t i n g s u b s t a n c e w h i c h we b e l i e v e to be p r e d o m i n a n t l y t h r o m b o x a n e A2r and to p o t e n t i a t e the o n s e t of b l o o d c o a g u l a t i o n . A c t i v a t i o n of P l a t e l e t s by H - C R P . Thermally modified CRP a c t i v a t e d i s o l a t e d h u m a n p l a t e l e t s to r e a c t i o n s of a g g r e g a t i o n and s e c r e t i o n (Figure 6). I n c r e a s i n g d o s e s of H - C R P r e s u l t e d in c o n c o m m i t a n t l y g r e a t e r a m o u n t s of a g g r e g a t i o n and the release of s e r o t o n i n from the p l a t e l e t s (a m e a s u r e of d e n s e g r a n u l e constituent release). Using a L u m i - A g g r e g o m e t e r , w h i c h p e r m i t t e d a k i n e t i c a s s e s s m e n t of d e n s e g r a n u l e s e c r e t i o n (as ATP) a n d p l a t e l e t a g g r e g a t i o n , we o b s e r v e d that p l a t e l e t a g g r e g a t i o n

107 H-CRFfyg/ml)

UJ O

80

(68)

80

(43)

40

(25)

20

(19)

10

(10)

5

70

CO a. x c? _J UJ >

60 50 40 3020

LU

cr

10I 1 MIN UTE

Figure

6.

R e p r e s e n t a t i v e e x p e r i m e n t i l l u s t r a t i n g the stimulat i o n of p l a t e l e t a g g r e g a t i o n and s e c r e t i o n of s e r o t o n i n by increasing a m o u n t s of H - C R P . The p e r c e n t s e r o t o n i n release is in p a r e n t h e s e s . F r o m r e f e r e n c e (3).

s t i m u l a t e d by H - C R P o c c u r r e d c o i n c i d e n t l y w i t h the s e c r e t o r y event. H - C R P also s t i m u l a t e d p l a t e l e t s to r e l e a s e 8 - t h r o m b o g l o b u l i n a n t i g e n , indicating a c a p a c i t y to m e d i a t e s e c r e t i o n of c o n s t i t u e n t s from p l a t e l e t a g r a n u l e s (Figure 7). However, the r e l e a s e of the l y s o s o m a l acid h y d r o l a s e s ( m e a s u r e d as 3 - g l u c u r o n i d a s e ) w a s not d e t e c t e d in the s u p e r n a t a n t s of platelet suspensions challenged with H-CRP. G e n e r a t i o n of T X B ? and TXA? A c t i v i t y from P l a t e l e t s S t i m u l a t e d by H - C R P . H - C R P induced p l a t e l e t a c t i v a t i o n is m e d i a t e d , in p a r t , by an i n d o m e t h a c i n s e n s i t i v e p a t h w a y . The next e x p e r i m e n t s s o u g h t to identify the c o n v e r s i o n of p l a t e l e t p h o s p h o l i p i d l ^ C - a r a c h i d o n i c acid into 1 4 C - T X B 2 (the stable h y d r a t e d end p r o d u c t of TXA2) d u r i n g p l a t e l e t a c t i v a t i o n w i t h H - C R P .

108

70-

b

a

LJ

1 8,000n

O

£ THROMBIN

7,000-

6,0005,000-

T

10 r 0

2

3

MINUTES

Figure 7.

4

5

10

20

30

40

50

60

70

80

90

H-CRP ( ^ g / m l )

In (a), the aggregation response of isolated platelets to increasing amounts of H-CRP. In (b), the release of 3-thromboglobulin antigen from platelets challenged in (a) using five different H-CRP concentrations marked A-E and corresponding to the H-CRP concentrations used in (a). B-thromboglobulin antigen was measured using radio-immunoassay with platelet activating conditions as described in Materials and Methods. For comparison, the p-thromboglobulin antigen released following challenge of platelet suspensions with a concentration of thrombin yielding a maximal platelet aggregation event is depicted in (b). Data is given as the mean and range of three independent determinations for each H-CRP or thrombin concentration used From reference (21).

Low concentrations of H-CRP (10-20 ug/ml) did not generate detectable 1 4 C-TXB2; however, challenge of platelets with 50-100 ug/ml H-CRP, which produced maximal amounts of aggregation, generated 14q_txB2 (Figure 8). The % conversion of platelet membrane AA into TXB2 in thrombin and H-CRP activated platelets was 8.2% + 4.5% and 2.8% + 0.9% respectively (nine experiments). The inclusion of indomethacin in these systems inhibited TXB2 generation, and imidazole, an inhibitor of thromboxane synthetase, shunted PGG2/PGH2 into production of the prostaglandins (as measured by PGE2). H-CRP, unlike ADP but like thrombin, did not require platelet aggregation to generate TXA2 since the apparent % conversion of platelet AA was similar in the presence or absence of added calcium.

109

A s a d d i t i o n a l e v i d e n c e that H - C R P s t i m u l a t e d p l a t e l e t s p r o d u c e b i o l o g i c a l l y active m e t a b o l i t e s from a r a c h i d o n a t e , we a s s e s s e d w h e t h e r p l a t e l e t TXA2 a c t i v i t y w a s g e n e r a t e d . As cont r o l s , TXA2 a c t i v i t y g e n e r a t e d by a r a c h i d o n a t e , c o l l a g e n and thrombin! w a s also a s s e s s e d . A s d e p i c t e d in Figure 9, H - C R P s t i m u l a t e d p r o d u c t i o n of TXA2 a c t i v i t y from p l a t e l e t s , as did a r a c h i d o n a t e , c o l l a g e n and t h r o m b i n ; TXA2 p r o d u c t i o n w a s a b s e n t in c h a l l e n g e d p l a t e l e t s u s p e n s i o n s in the p r e s e n c e of indomethacin.

THROMBIN THROMBIN + INDO

H-CRP

H-CRP + INDO H-CRP + IMID

TxBz

Figure

8.

PGE 2

R a d i o - t h i n l a y e r c h r o m a t o g r a p h y of lipid p r o d u c t s g e n e r a t e d from i s o l a t e d p l a t e l e t s i n c o r p o r a t e d w i t h I ^ C - a r a c h i d o n i c acid. P l a t e l e t s s u s p e n d e d in TBS (pH 7.4) w i t h 0.2 m M c a l c i u m w e r e c h a l l e n g e d w i t h t h r o m b i n (10 U/ml) or H - C R P (100 u g / m l ) . Indom e t h a c i n w a s used at 20 uM and i m i d a z o l e at lOmM. M i g r a t i o n of n o n - r a d i o a c t i v e p r o s t a g l a n d i n PGE2 a n d of t h r o m b o x a n e B2 (TXB2) are s h o w n below the r a d i o thinlayer chromatograms. Reaction mixtures were m i x e d w i t h an internal ^H-TXBJ s t a n d a r d , a c i d i f i e d (pH 3.5) and e x t r a c t e d w i t h ethyl a c e t a t e . The o r g a n i c m i x t u r e w a s e v a p o r a t e d u n d e r n i t r o g e n , red i s s o l v e d in m e t h a n o l and a p p l i e d to s i l i c a gel G - 6 0 p l a t e s and c h r o m a t o g r a p h e d in two d i m e n s i o n s F r o m r e f e r e n c e (21).

110

lOug/ml

lOOuM

ARACHIDONIC ACID

THROMBIN

COLLAGEN 30SEC IOU/ml

lU/ml

0.2U/ml

-J 60SEC 80uq/ml 4Quq/ml J^

H-CRP

IQuq/ml

60SEC Figure 9.

A comparison of the ability of arachidonic acid, collagen, thrombin or H-CRP to generate TXA2 activity from platelets. Platelets suspensions were challenged with the concentrations shown and the reaction mixtures assayed. Aortic strips were suspended from transducers at a constant tension of 2 gm/cm and calibrated using a polygraph with recorder From reference (21).

The ability of H-CRP and AHGG to Facilitate Blood Coagulation. In an attempt to ascertain whether aggregate CRP or IgG could facilitate blood coagulation, we performed experiments to simultaneously measure clot formation and platelet aggregation in PRP in the presence of H-CRP or AHGG; results are seen in Figure 10. Each decreased the time of onset for clot formation; H-CRP also markedly enhanced (synergistically) the amount of platelet aggregation (Figure 11). This phenomena required the presence of platelets as the onset of clot formation was not enhanced in platelet-poor-plasma.

111 01 M

3 T) t-1 tí o ta o ^ ^ PH 4-1 cri O c o O 0) i S

> • r-1 •H 00 M 4-1 3 3 n) Otí U o O Ci e r4 o U-1 * — tn o O u e ex 3 O tn •H 1-J 4J 4-1 3 O tí O U QJ O •— — ' P. O tß ai Ol 0) n cri ^ o 3 o < 00 1 4-1 •r-t a: 0¡ Ù0 [n -o e 01 1 IJ en »< > 1-1 3 O

— ,\

1 0) 00 0) ^ 00 00 ta w CJ i—i 0) 4-1 0) T-H P. -o tí ta

tí

o •H 4-1 ra g H O 4-1 4-1 O i—i O M — 1 O

tí

O 4-1 ra 4-1 • r4 CL, — i 1 Cri •H u tj 1 ra Ed [n • I-I

>>

*—

i— CM cri 145%) w a s o b s e r v e d . We b e l i e v e that these d a t a r e f l e c t the p r i m a r y m e c h a n i s m s by w h i c h serum a m y l o i d P - c o m p o n e n t i n f l u e n c e s blood coagulation. In c l o s i n g , I have p r e s e n t e d some of our d a t a c o n c e r n i n g the i n t e r a c t i o n of c e r t a i n acute p h a s e r e a c t a n t s w i t h the hemostatic mechanism. T h e s e data p r e s e n t c l e a r e v i d e n c e for an i n t e r - l o c k i n g s y s t e m of i n d u c t i o n and r e g u l a t o r y c o n t r o l t h a t we s u s p e c t w i l l be found to be the c o r n e r s t o n e " p u r p o s e " of the acute p h a s e r e s p o n s e .

124 ACKNOWLEDGEMENTS The author gratefully acknowledges the insight of Henry Gewürz, MD, Chairm a n of the department of Immunology/Microbiology, R u s h Medical Center, Chicago, Illinois, without whose w i s d o m these projects would not have been undertaken. The technical and developmental expertise of Robert M. Simpson, PhD, Cecilia S.L. Ku, PhD and Joyce M. Izzi, BSc, is also greatly appreciated, as is the excellent secretarial assistance of Ms. Mary A n n Briggs. REFERENCES 1- Fiedel, B.A., Costello, M., Gewürz, H. and Hussissian, E. 1983. H a e m o s tasis 13:89. 2 - Fiedel, B.A., Potempa, L.A., Frenzke, M.E., Simpson, R.M. and Gewürz, H . 1982. Immunology 45:15. 3 - Fiedel, B.A. and Ku, C.S.L. 1983. J. Exp. Med. (in press). 4- Fiedel, B.A., Simpson, R.M. and Gewürz, H. 1982, Immunology 45:439. 5 - Haupt, H., Heimburger, N. and Bauder, S. 1972, III Hoppe-Seyler's Z. Physiol. Chem. 353:1841. 6- Lowry, 0., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. 1951. J. Biol. Chem. 193:265. 7- Osmand, A.P., Friedensen, B., Gewürz, H., Painter, R.H., Hofman, T. and Shelton, E. 1977. Proc. Natl. Acad. Sei. (USA) 74:739. 8 - Laemmli, U.K. 1970. Nature 227:680. 9 - Jeanloz, R.W.: in Glycoproteins (ed. A. Gottschalk) p. 560-585. Elsevier North Holland, New York, 1965. 10- Winzler, R.J.: Glycoproteins in disease. In Glycoproteins of Red Cells and Plasma (eds. G.A. Jamieson and T.J. Greenwalt) p. 204-218. J.B. Lippincott, New York, 1971. 11- Schmid, K.: Alpha 1-acid glycoprotein. In, The Plasma Proteins: Structure Function and Genetic Control (ed. F.W. Putnam) p. 163-228, Academic Press New York, 1975. 12- Schmid, K., Kaufman, H., Isemura, S., Bauer, F., Emura, J., Montoyama, T. Ishigura, M. and Nann, S. 1973. Biochem. 12:2721. 13- Snyder, S. and Coodley, E.I. 1976. Arch. Intern. Med. 136:778. 14- Costello, M., Fiedel, B.A. and Gewürz, H. 1979. Nature 281:667. 15- Andersen, P. and Eika, C. 1979. Thromb. Haemostasis 42:299. 16- Godal, H.C. 1961. Scand. J. Clin, and Laboratory Invest. 13:314. 17- Andersen, P. and Godal, H.C. 1977. Haemostasis 6:339. 18- Andersen, P. 1980. Haemostasis 9:303. 19- Tillett, W.S. and Francis, T. Jr. 1930. J. Exp. Med. 52:561. 20- Gewürz, H., Mold, C., Siegel, J. and Fiedel, B.A. 1982. C-reactive protein and the acute phase response. Adv. Int. Medicine (G.H. Stollerman, ed.) v.27, Y e a r Book Medical Pub., Chicago, pgs. 345-372. 21- Simpson, R.M., Prancan, A., Izzi, J.M. and Fiedel B.A. 1982. Immunology 47:193. 22- Fiedel, B.A., Ku, C.S.L., Izzi, J.M. and Gewürz, H. 1983, J. Immunol, (in press). 23- Holmsen, H. 1977. Thromb. (Haemostasis) 38:1030. 24- Pepys, M.B., Becker, G.J., Dick, R.F., McCraw, A . , Hilgard, P., Rosemary, E. and Thomas, D.P. 1980. Clin. Chim. A c t a 105:83.

MONOCLONAL ANTIBODIES AS PROBES OF COMPLEMENT FUNCTION

Shaun Ruddy Medical College of Virginia, Virginia Commonwealth Richmond, Virginia, U.S.A.

University

Introduction Polyclonal antisera directed against individual plasma proteins have been widely used as tools to study the function of these proteins. of

the

complement

than twenty years the

system

antigenic have

been

peptide

(4).

or

useful

(3) and have the

antisera

Such polyclonal

determinants

determining

such

have

been

available

for

more

(1) and have been prepared to all twenty components

(2).

often

medicine

proteins,

In the case

Because

epitopes

for

of

they

on

measuring

sometimes

function

antisera, which the

are

individual

plasma

a

collection

protein

in inhibition

complement of

of

a variety

concentrations

been valuable

a particular

recognize

molecules, in

clinical

studies

protein or

heterogenous

of

for

cleavage

antibodies

directed against all of the epitopes on an individual protein, such antisera are of no utility

in determining

the fine

specificity

involved

in

the protein-protein interactions characteristic of complement activation.

The successful fusion of B-lymphocytes from an immune animal with a myeloma

line

in continuous

culture

(5) has

afforded

the opportunity of de-

veloping reagents in which all of the antibody molecules a single clone.

are products

of

Such monoclonal reagents are homogenous in their reactiv-

ity with a single antigenic determinant on the immunizing protein, and can therefore

be used

to delineate

the function of the protein. four monoclonal

the

importance

of

individual

This manuscript presents

epitopes

information

to

about

antibodies: two directed against Factor B of the alterna-

tive complement pathway, one of which blocks the sis and monocyte spreading

activity of B in hemoly-

(6) and two others reactive with the C3d frag-

ment of the third component which appear to discriminate between the sites on C3d of most importance in the alternative and classical pathways (7).

M a r k e r Proteins in Inflammation, Vol. 2 © 1 9 8 4 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

126 Materials and Methods Production of monoclonal antibodies from rat X rat hybrids DA rats were immunized with human alternative pathway convertase

prepared

by incubating purified C3b (8), B and D (9) for 30 min at 37° C. two

injections

in

Freund's

complete

or

incomplete

adjuvant,

After

rats

boosted by intravenous injection three days prior to the fusion.

were

Myeloma

lines used for fusion were Y3 Ag 1.2.3 (for the anti-B clones) or 210.RCY0 (for the anti-C3 clones). en

in

(10),

myeloma

but

cells

in

in the presence of

from hybrids selected thymidine

Details of the hybridization procedure are givo 7 brief, 10 spleen cells were fused with 5 X 10 polyethylene

glycol

4000.

Supernates

in medium containing hypoxanthine, aminopterin and

(HAT), were screened for anti-B or anti-C3

in an ELISA or RIA

system using purified B or C3 bound to 96-well microtiter plates. ficity

of

reactivity

for

C3

was

determined

using

sheep

Speci-

erythrocytes

coated with C3b, C3bi or C3d prepared with purified CI, C4, C2, C3, H and I (11) and indirect agglutination using goat the

second

antibody.

Positive

pristane-primed

immunoglobulin

culture wells were cloned

either on soft agar or by limiting dilution. injecting

anti-rat

and

subcloned

Ascites fluid produced

DA rats or nu/nu mice with cloned

as

cell

by

lines

was fractionated by precipitation either in polyethylene glycol or caprylic acid, followed by chromatography on DEAE cellulose to yield homogeneous solutions of the monoclonal

antibodies.

Hemolytic assays Sheep erythrocytes were coated with purified complement proteins to generate EAC14

the 0Xy

23b

intermediates was

EAC14 o x y 23b

accomplished

and

by exposure

to

EAC43b.

source

of

of

a reagent containing

fied human C5 (50 U/ml) and a source of C6-C9. with purified

Lysis

The EAC43Bb were

the purilysed

B and D followed by rat serum diluted in EDTA-buffer as a

C3-C9.

Hemolysis

was

quantitated

spectrophotometrically

and

expressed as the average number of sites (Z) per cells according to the Poisson distribution where Z = -ln(l-y) and y is the decimal fraction of cells

lysed. Reagents were adjusted to yield between 80 and 95 per cent

127 lysis corresponding

to Z values of 1.6 and 3.0.

Inhibition of lysis was

calculated from the decrease in Z and expressed as a per cent. Inhibiton of monocyte spreading Human monocytes

were purified from peripheral

blood by counterflow

elu-

triation as described previously and were the gift of Dr. Bruce Rutherford (8).

The

spreading

assay

was

performed

essentially

as

described

by

Sundsmo and Gotze, except that eight-chambered tissue culture glass slides (Lab Tek Products Division of Miles Laboratories, Napierville,

111) were

used in place of glass cover slips contained in plastic plates. were

adhered

to

the

slides

autologous serum for 16 hr.

in medium

containing

20%

heat

Monocytes inactivated

Serum-containing medium was washed away and

serum-free medium replaced, followed by a 3 day incubation before exposure to the reagents used to induce spreading for 3 hr.

Results Monoclonal anti-B For the inhibition of monocyte clonal antibodies were used.

spreading

induced by Factor B, two mono-

C81 had previously been shown to react with

the Bb moiety of B and to inhibit the hemolytic activities of Factor B either

in

EAC43bBbP.

the

fluid

phase

or

when

bound

to

the

cell

intermediate

D21 reacted with the Ba fragment of B and had no effect on

the hemolytic activity of this component.

Cells were exposed to Factor B

which

at 37° C. for 90 min with an

had been activated by preincubation

equal concentration of C3b and l/20th the concentration of Factor D, or to this activated preparation of B in the presence of either monoclonal antibody.

The final concentration of B in the culture medium was 2 y g / m l ;

monoclonal antibodies were added to a final concentration of l O y g / m l . Table

I gives

spreading

the

in medium

results

of

alone was

induced by activated B in the

three

separate

variable, absence

experiments.

as was the extent of

of monoclonal

antibody.

Background spreading In each

128 experiment,

however,

inhibition

of

B-induced spreading

was observed

with

clone C81, but not with D21.

Table I INHIBITION OF HUMAN MONOCYTE SPREADING BY MONOCLONAL ANTIBODIES TO HUMAN B

Cells Spread (per cent)* Expt I

Expt II

7 ± 3

12 ± 4

4± 3

Medium + B

34 ± 8

47 ± 6

28 ± 5

Medium + B + D21

37 ± 6

42 ± 7

31 ± 6

Medium + B + C81

4 ±2

Condition Medium

6

±

Expt III

3

8 ± °>

* > 2 0 0 cells counted per sample; mean + S.E. of triplicate

Monoclonal

Products

determinations.

anti-C3

of

two

clones,

3C4

and 4D7, were studied for their capacity

to

block the hemolytic activity of C3 in either the classical

or

alternative

pathway.

to

agglutinate

EAC43d that 125

In

preliminary

(trypsin) they

I-C3

cells

reacted

was

studies,

as well

with

reduced

the

with

as C3d

both

had

EAC43b

been

and

fragment

shown

EAC43bi

of

2-mercaptoethanol,

the

cells,

C3

indicating

molecule.

incubated

with

When

the

IgG

fraction of 3C4, and excess goat anti-rat Ig added, the alpha chain of C3 was

preferentially

precipitated,

mide gel electrophoresis and To

assess

used B,

to

D

their

inhibit

and

with

C3-C9

effects the in

in

hemolysis, either

following

both

EAC14

0Xy

monoclonal 23

experiment.

of

either

3C4

or

4D7

at 37° C. for

washed free of the antibodies, suspended excess

C5-C9,

cells yielded with

polyacryla-

antibodies

by C5-C9 or EAC14oxy23

limited

and

94 per C3b

allowed

cent

were

to

lysis.

exposed

to

lyse

30 min.

37°

60 min;

In a similar fashion, the

concentra-

The cells

c . for

two

antibodies,

EAC43b

washed,

by

prepared

in a developing reagent at

were

EAC43bBb

were

limited amount of C3b, washed, and incubated with varying

tions

ing

SDS

radioautography.

lysis of the

as judged by subsequent

were

then

containuntreated prepared incubated

129 with

excess

B

cells yielded

and

D,

85 per

and

lysis

cent

lysis.

developed The

with

extent

the two cell types by the two monoclonal

excess

of

C3-C9;

inhibition

antibodies

untreated

of

lysis

of

is shown in Table II.

It is apparent that 3C4 efficiently inhibits the formation of the alternative pathway convertase

as assessed

by the reaction

between

EAC43b,B

and

D; whereas 4D7 is more potent at interfering with the reaction between C3b and

C5

involved

in

the

formation

of

the

terminal

lytic membrane

attack

complex.

Table II INHIBITION OF C3 HEMOLYTIC ACTIVITY BY MONOCLONAL ANTIBODIES TO C3

Reaction

0

0 ^

1.3

6.4

32

160

3C4 Concentration (ng/ml) C3b + C5-C9

0

17*

C3b + B + D + C3-C9

0

7

18

23

28

100

32

95

100

100

4D7 Concentration (ng/ml) C3b + C5-C9

0

0

2

10

55

92

C3b + B + D + C3-C9

0

3

10

23

25

35

*per cent inhibition of average number of hemolytic sites per cell.

Discussion

The in

experiments defining

presented

the

fine

anti-B monoclonal tic

activity

tained

in

of

the

illustrate

specificity

the

power

involved

of monoclonal

in

biologic

C81 had previously been shown Factor

fluid

B regardless

phase

or

already

of

antibodies

reactions.

(6) to block the

whether

this

incorporated

molecule

into

the

pathway convertase

in the form C3bBb or C3bBbP; the presumption the

with

site of this protein. specifically sized with

binding the

Bb fragment

B on or

Direct radioautography

precipitate site.

of

The

the

Bb

fragment,

inactive

Ba fragment which

has

the

active

D21,

biologic

by

with

con-

was

that

enzymatic

showed that C81 did

consistent

monoclonal

no known

near

hemoly-

was

alternative

C81

combined

The

indeed

the

hypothe-

contrast,

combined

activity.

The

finding

130 that

the

active-site

reagent

spreading of human monocytes

C81

blocked

the capacity of Bb to

induce

indicates that the enzymatic site of Bb is

required for this activity as well.

These data independently confirm the

earlier observations (13, 14) that the active serine esteratic site on Bb was necessary for the spreading phenomenon.

A similar definition of fine specificity with the monoclonal

antibodies

tral role in both classical of

complex

interactions

to C3.

is apparent

in the

experiments

This component, which has a cen-

and alternative pathways, undergoes a number

with

other plasma proteins,

including

C42,

the

enzyme for which it is the substrate; C5, which binds to C3b and thereby becomes a better substrate for initiation of the terminal attack sequence; Factor B, which binds to C3b and thereby becomes susceptible to cleavage by Factor D to form the alternative pathway convertase; Factor H, which binds to C3b and renders it both incapable of interaction with B and susceptible to cleavage by Factor I; and properdin, which is believed to bind to

the

alternative

pathway

convertase

active enzymatic form of this complex.

via

C3b

thereby

Although there

stabilizing

the

is evidence

that

B, P and H may react on or near the same site, the remaining

interactions

clearly

these

occur

at

independent

loci

on

the molecule.

Since

sites

vary in their structure, they may also differ in their antigenic composition, and may display epitopes which may be distinguished by the appropriate monoclonal antibodies. preliminary,

Although the data presented in Table II are

it appears that 3C4 preferentially recognizes the B binding

site and thereby blocks formation of the alternative

pathway

convertase;

the effect on hemolysis of EAC1423b in the presence of C5-C9 observed at high concentrations of this monoclonal remains unexplained at present.

By

contrast, 4D7 appears to interfere with the interaction of C5 with C3b, an effect which is more apparent with EAC1423 where the numbers of C3 molecules are continuously limited than with EAC43b where the numbers of C3b molecules are only initially limited when the alternative pathway convertase is being formed, but are relatively plentiful

during the developing

reaction when diluted whole rat serum is used as a source of C3-C9. Monoclonal

antibodies to C3 have been used to characterize the breakdown

of this molecule into fragments by Factor I, and provided evidence for a

131 new

fragment,

C3dg,

which

cleavage of bound C3bi

is

the

(15).

physiologic

fragment

derived

from

Tamerius described several monoclonal

the

anti-

C3 antibodies which blocked binding of H, and others which blocked both P and H binding; none inhibited the binding of B (16). gant studies with eight different monoclonal

In a series of ele-

antibodies

to guinea pig

(17), Burger and co-workers delineated a number of discrete sites ted with various functions of C3. tive

with

antigenic

They described three monoclonals,

determinants

on C3c, which

inhibited

C3

associareac-

the binding

of

125 I-B from

to

those

cells

bearing

presented

C3b.

here for

yet

These 3C4,

blocks

the

results

which

C3d-coated

cells,

convertase

(Table I) and the uptake of

has

formation 1

are

distinctly

been of

found

the

to

different agglutinate

alternative

I-B (data not shown).

pathway Further

experiments will be required to resolve these differences. Although ficity, nature

monoclonal as

in the

should

antibodies studies

be made

just

may

provide

described,

for workers

are

because

listed

of

the

in Table

very

project utilizing monoclonal

some real

homogeneity

III, and should

of exquisite caveats

of

in the field of acute phase

There are a number of disadvantages, arise

reagents several

be

of

a

specigeneral

proteins.

and some potential,

monoclonal

considered

antibodies.

before

embarking

which These on

a

reagents.

Table

III

POTENTIAL DISADVANTAGES OF MONOCLONAL ANTIBODIES FOR THE STUDY OF ACUTE PHASE REACTANTS

Excessive Relative

lability to pH, temperature or other physical

conditions

insolubility

Poor tolerance of labelling procedures (e.g., tyrosine in combining site may result in loss of activity with oxidative iodination) Low affinity of binding (important in immunoassay) Isotype class or species poorly reactive with staphylococcal

protein A

132 Summary Two sets of monoclonal antibodies directed against proteins of the complement system, Factor B and C3, have been developed. active with

Factor

Of two antibodies re-

B, one which reacts on or near the active

esteratic

site of this enzyme blocks both its activity in immune hemolysis and its capacity to induce the spreading

of human monocytes.

Of two antibodies

reactive with C3, both of which react with an epitope present on the C3d fragment of this molecule, one preferentially blocks the reactivity of C3b with Factor B.

These antibodies are illustrative of the high degrees of

specificity which may be obtained with such reagents.

They do not illus-

trate some of their pitfalls, which need also to be considered.

References 1. Müller-Eberhard, H.J., Nilsson, U.:

J. Exp. Med. Ill, 217-234 (1960).

2. Muller-Eberhard, H.J.: Hos. Pract. 12, 33-43 (1978). 3. Ruddy, S., Carpenter, C.B., Muller-Eberhard, H.J., Austen, K.F.: In Miescher, P.A. and Grabar, P. (eds.), Mechanisms of Inflammation Induced by Immune Reactions, Vth Int. Immunopath. Symposium, Schwabe & Co., Basel (1968). pp. 231-251. 4. Ward, P.A.: In Ingram, D.G., (ed), Biological Activities of Complement, S. Karger, Basel (1972). pp. 108-116. 5. Kohl er, G., Milstein, C.:

Nature 256, 495 (1975).

6. Ruddy, S., Lachmann, P.: Molec. Immun. 19, 1399 (1982). 7. Ruddy, S., (1983).

Moxley,

G.

F.,

8. Rutherford, B., Schenkein, H.:

Purkall,

D.B.:

Immunobiology

Immunology 46, 163-173 (1982).

10. Lachmann, P.J., Oldroyd, R.G., Milstein, C., Wright, B.W.: 41:503-515 (1980) 11. Carlo, J., Conrad, D.H., Ruddy, S.:

Immunology

J. Immunol. 123, 523-528 (1979).

Cell. Immunol. 52, 1-11 (1979).

13. Gotze, 0., Bianco, C., Cohn, A.Z.: 14. Sundsmo, J.S., Gotze, 0.:

291

J. Period. Res. 17, 484-486 (1982).

9. Carlson, P., Ruddy, S., Conrad, D.H.:

12. Sundsmo, J.S., Gotze, 0.:

164,

J. Exp. Med. 149, 372-386 (1979).

J. Exp. Med. 154:763-777 (1981).

15. Lachmann, P.J., Pangburn, M.K., Oldroyd, R.G.: 219 (1982).

J. Exp. Med. 156, 205-

133 16. Tamerius, J.D., Pangburn, M.K., Eberhard, H.J.: 518 (1982).

J. Immunol. 128, 512-

17. Burger, R., Deubel, U., Hadding, U., Bitter-Suermann, D.: 129, 2042-2050 (1982).

J. Immunol.

MODULATION OF FORMATION OF THE C3 AMPLIFICATION CONVERTASE OF HUMAN COMPLEMENT BY ACUTE PHASE PROTEINS

Elizabeth Fischer, Michel Pontet, François Delers, N. Tran Quang, Michel Kazatchkine and Robert Engler Inserm U 28, Hôpital Broussais and UER Biomedicale des Saints Pères, Paris, France

Introduction The plasma concentration of acute phase reactants, C-reactive protein (CRP), orosomucoïd and haptoglobin increase during inflammatory processes. CRP has been shown to interact with the human complement system in several ways. Binding of CRP to Pneumococcal

C polysaccharide, to phosphocholine

containing molecules and to polycationic substances results in complement activation through the classical pathway (1). Complexes between CRP and Pneumococcal

C polysaccharide are solubilized by the classical

complement

pathway (2). Binding of CRP to liposomes and to S. pneumoniae inhibits the ability of the liposomes to activate the alternative pathway (3). The present study investigated the effect of human CRP, orosomucoïd and haptoglobin on the formation of the alternative pathway C3 convertase in vitro (4).

Methods CRP was purified from acute phase sera by chromatography on phosphoethanolamine-sepharose using phosphorylcholine in the eluting buffer. Orosomucoïd was purified by ammonium sulfate precipitation and sequential chromatography on CM cellulose and Sephacryl S 200. Haptoglobin (1-1) was purified as previously described (5). The alternative pathway proteins •C3,B,D,P and H were purified to homogeneity from normal human plasma using the methods quoted in ref 4. In order to examine the effect of acute phase proteins on formation of alternative pathway C3 convertase, sheep erythrocytes bearing C3b, EAC4b,

M a r k e r Proteins in Inflammation, Vol. 2 © 1 9 8 4 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

136 3b, were interacted with various concentrations of B,D and P in the presence of 0.5 mM of M g + + at 30° C so as to generate approximately

1.5 sites/

cell. 0.1 ml of the reaction mixture were added to 0.1 ml of buffer alone ( D G V B + + i.e. low ionic strength buffer containing 0.5 mM M g + + and 0.15 mM C a + + ) or containing increasing amounts of purified acute phase

proteins.

After incubation for 30 min at 30° C, cells were washed once, resuspended in 0.2 ml of D G V B + + and incubated with 0.3 ml of a 1/20 dilution of rat serum in 40 mM EDTA for 60 min at 37° C so as to reveal hemolytic sites that had been formed during the first incubation step.

Results CRP, haptoglobin and orosomucoïd inhibited formation of the C3 amplification convertase of complement in a dose dependent manner. Fifty per cent inhibitory concentrations were approximately 35 pg, 70 pg, 140 pg/1.10^ EAC4b3bBbPin 0.2 ml respectively

(Fig 1). Serum albumin at similar con-

centrations had no effect.

ug protein/10 7 EAC43bBbP/0.2 ml

Fig. 1

- Inhibition of formation of P stabilized C3 convertase

sites,

C3b,Bb,P, in the presence of purified CRP ( A — • ), orosomucoïd ( • — • ), haptoglobin ( • — • ) and BSA ( o — o ).

137 Experiments using orosomucoTd and haptoglobin during formation of the C3 convertase in the absence of D and in the presence or absence of P indicated that these two proteins have a minor inhibitory effect on P and D and that the major site of action is between cell bound C3b and factor B. CRP and orosomuco'Td did not affect the spontaneous decay of a preformed P stabilized C3 convertase

; in contrast, haptoglobin actively

dissocia-

ted Bb from a P stabilized convertase similarly to factor H although on a molar basis it is about 10000 times less effective than H (Fig. 2). Thus, acute phase proteins may modulate the expression of the amplification C3 convertase of complement at concentrations similar to those found in inflammatory

sera.

o

? •

Fig. 2

il

To

ng H I0 7 EAC43bBbP. 0 . 2 ml

- Dose dependent decay (20 min at 30° C) of P stabilized C3 convertase sites, C3b,Bb,P in the presence of haptoglobin ( • — • ), in the presence of H ( • — • ) and in the presence of H and 35 ug haptoglobin ( • — • ).

138 References 1.

Kaplan, M.H., V o l a n a k i s , J . E .

2.

V o l a n a k i s , J.E.

: J . Immunol. U 2 , 2135

3.

Mold, C . , Gewurtz, H. : J . Immunol. J_27, 2029 (1981 ).

(1974).

: Ann. N. Y. Acad. S c i . 389, 235 ( 1 9 8 2 ) .

4.

K a z a t c h k i n e , M.D. and Nydegger, U.E.

5.

D e l e r s , F. et a l .

: Prog. A l l e r g y 30, 193

: A n a l . Biochem. 118, 353 ( 1 9 8 1 ) .

(1982).

MATERNO-TROPHOBLASTIC

RELATIONSHIP

RECURRENT

ABORTION

SPONTANEOUS

W. P a g e F a u l k , C h a n g - 3 i n g

IN NORMAL H U M A N P R E G N A N C Y

Yeh, B.-L.

AND

Hsi

INSERM U n i t U2""0, D e p a r t m e n t of I m m u n o l o g y , F a c u l t y of Ave. V a l l o m b r o s e , 06000 Nice

Medecine

Introduction

It m u s t in the f i r s t i n s t a n c e be said that m o s t m e d i c a l p e o p l e to i m a g i n e

the e x i s t e n c e of a m a t e r n o - f e t a l

relationship

that is

g o v e r n e d p r i m a r i l y by the p l a c e n t a , but both of these c o n c e p t s anatomically incorrect. maternal

T h e r e are no i n t e r f a c e s of c o n t a c t

and e m b r y o n i c or fetal

entirely extra-embryonic, presented

to m a t e r n a l

relationship.

t i s s u e s . The p r i n c i p a l

e x i s t at the e n d o v a s c u l a r the i n t e r s t i t i a l

materno-

The

materno-trophoblastic

c o n t a c t , for there are four d i f f e r e n t i n t e r f a c e s

arteries,

between

maternal

materno-trophoblastic

trophoblast uithin

trophoblast within

the

the p l a c e n t a l

spiral bed,

the

and

the

c y t o t r o p h o b l a s t of the c h o r i o n i c a s p e c t of the a m n i o c h o r i o n , syncytiotrophoblast

of the p l a c e n t a l

will be c o n s i d e r e d in

tropho-

and this p h r a s e s h o u l d be usr-d in p r e -

p l a c e n t a is c l e a r l y not r e s p o n s i b l e for all

interfaces

is

tissue w h i c h is

does not e x i s t in o t h e r

to the l e s s m e a n i n g f u l m a t e r n o - f e t a l

and e x t r a - e m b r y o n i c

contribution

s e n s e , but there is such a thing as the

trophoblastic relationship, ference

relationship

are

betueen

cells at all of these i n t e r f a c e s is the

b l a s t . Thus, the m a t e r n o - f e t a l than a b i o c h e m i c a l

tissue. The fetal

and the e x t r a - e m b r y o n i c

tend

c h o r i o n i c v i l l i . Ef-ch of

turn.

M a r k e r Proteins in Inflammation, Vol. 2 © 1 9 8 4 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

these

140 Endovascular

trophoblast

At an i m p r e c i s e l y

known

time b e f o r e

g r a t i o n of c y t o t r o p h o b l a s t i n t o P a r t of terial

these e n t e r a n d m a k e

lumen where

endothelium, occurs

from

w a v e of

the e i g h t h w e e k ,

human decidua

they c h a l l e n g e

and another the s e r o s a l

trophoblast

their way upstream

and e v e n t u a l l y

trophoblastic surface.

invasion

teries

(Pijnenborg

this is r a t h e r o b s c u r e ,

et al..

into

trate into

the m y o m e t r i a l the s p i r a l

histopathological Robertson gested wave. also

segments

arteries'

Hospital Medical

t h a t p r e - e c l a m p s i a is a d i s e a s e Another

characteristic

to be t e m p o r a l l y

filtration

trophoblast

of

f e a t u r e of s p i r a l

is the l o s s of a d i s c e r n i b l e

lock

the v e s s e l s

taneously

disallowing

materials

in the u t e r u s ,

has b e e n p r e s e n t e d material

(Faulk 4 M c l n t y r e ,

e t al.,

arteries

ara suboptimally

1975), and

t h e s i s of a f a i l u r e if i n d e e d

to p o i n t o u t h e r e

t h a t the

of

Whatever they

functions

certainly

a successful

(uteroplacental)

vasoactive

the

trophoblast

trophoblast

moof

(Robertson hypo-

invasion,

is in s o m e w a y

host-parasite

a r t e r y is a m a t e r n a l

Evidence

the R o b e r t s o n

t h a t is a l m o s t w i t h o u t p r e c e d e n t i n i m m u n o b i o l o g y , spiral

re-

simul-

aspects

by e n d o v a s c u l a r

the e n d o v a s c u l a r

create

to

PAS-reactive

in p r e - e c l a m p s i a

the s e c o n d e n d o v a s c u l a r

artery endothelium

to

c e l l s , b u t for

with

in-

function

dilatation while

the r e p l a c e m e n t of b a s e m e n t m e m b r a n e

r e p l a c e m e n t of s p i r a l

serve,

dilated

seems

due

could

t h a t the d i s t a l

t h a t this is c o m p a t i b l e

sug-

second

the p r o s t a g l a n d i n s .

1983)

in

B.

which

to v a s o c o n s t r i c t in r e s p o n s e

s u c h as s o m e of

is p r o b a b l y m a d e by e x t r a - e m b r y o n i c

m e n t i t is p e r t i n e n t spiral

them

pene-

has

the

basement membrane

in m a x i m u m

of oc-

trophoblastic

p l a c e m e n t by a P A S - p o s i t i v e m a t e r i a l . T h i s s t r u c t u r e to o p e r a t i o n a l l y

found

of

arteries

associated with endovascular

ar-

elongation-and

London,

the f a i l u r e

to to

Indeed, Prof. W. School,

second

significance

the s e c o n d w a v e

e n d s as has b e e n

s t u d i e s of p r e - s c l a m p s i a .

at St. G e o r g e ' s

arteries

and spiral

and with inadeguate

distal

ar-

vascular

t h e r e is a

i 9 B " b ) . The p h y s i o l o g i c a l

c u r is a s s o c i a t e d w i t h a f a i l u r e of e n d o v a s c u l a r

d i l a t i o n of

al.,

the

spiral

the e n d o m e t r i u m

b u t f a i l u r e of

et

in the

replace

Several weeks later

that again invades

all of

t h e r e is a m i -

(Pijnenborg

due

to

trophoblast. subset

might

relationship

for the

mature

v e s s e l w h i c h is

lined,

141 infiltrated

and in part surrounded

gin. This seems sequelae,

for l y m p h o c y t e s

and rejection

reactions

not known, unlike

a n d o t h e r c e l l s r e m i n i s c e n t of

1 9 8 1 ) . The e n d o v a s c u l a r

for m a t e r n a l

stimulation

syncytiotrophoblast,

major histocompatibility 1983).

In l i g h t of

to m o u n t i m m u n e

complex

the p r e s e n c e

trophoblast,

iodide

and p l a s m i n o g e n

(Faulk

& Mclntyre,

(MHC)

1983). More importantly,

1 9 8 0 ) . T h e s e new f i n d i n g s w i l l

the n o t i o n of

the i m m u n o p a t h o l o g y

Interstitial At term cally

the p l a c e n t a l

of h u m a n

antigens so

cells a

certain

pregnancy,

some

as a g r a f t w a s

(Barnstable

but

investigators

& Bodmer,

due 1978).

simple.

bed is f i l l e d w i t h

cytotrophoblast.

trophoblast

cells. Sometimes these

f u s i o n s are

i n the p l a c e n t a l

bed which

heterologous In f a c t ,

or m o n o c l o n a l

antibodies

the r e a c t i v i t i e s of a n t i - T A

to be

cells

cannot

hybridization

to i d e n t i f y m u l t i n u c l e a t e d

react with anti-HLA

and

form

thought

but hybridization with maternal

for we are a b l e

morphologi-

they f u s e to

be r u l e d o u t . The m a t t e r of m a t e r n o - t r o p h o b l a s t i c be k e p t o p e n ,

that

T h e s e are in the e n d o m e t r i u m

g i a n t c e l l s . M o s t of

trophoblast-trophoblast,

(TA).

with

was HLA-negative,

had b e e n s t u d i e d ,

to be q u i t e

in a s e a of m a t e r n a l

multinucleated

either

tropho-

trophoblast;

resembles

survive

necessitate

t h a t the s u c c e s s of p r e g n a n c y

transplantation

Alas, it does not appear

possibility

w h i c h are m a r k e r s of e n d o t h e l i a l

for a f t e r i t w a s s h o w n t h a t s y n c y t i o t r o p h o b l a s t

to the a b s e n c e

was

propidium

the e n d o u a s c u l a r

antibodies

trophoblast subsets

a

sera and does not react

(Matter 4 Faulk,

developed

the

& Mclntyre,

m a r k e r s of

this a n u n l i k e l y

are

subset,

in w h i c h e n d o t h e l i u m

a n t i - F a c t o r VIII

before other

recognition

a n t i g e n s o n this s u b s e t ,

b u t the a d d i t i o n a l

receptors make

about

antigens

trophoblast

(Hsi, 1983; Faulk

of M H C

b l a s t is r e a c t i v e w i t h a n t i - t r o p h o b l a s t

a m o u n t of r e t h i n k i n g

distribu-

d o e s e x p r e s s C l a s s I a n t i g e n s of

c a s e of m i s t a k e n i d e n t i t y m i g h t be a s s u m e d confused with

recognition

trophoblast

b u t i t s h o u l d be p o i n t e d o u t t h a t this

ori-

immunological

h a v e b e e n n o t e d in a p e r i - a r t e r i a l

tion (Robertson et al.. responsible

by t i s s u e s of e x t r a - e m b r y o n i c

to n o t be w i t h o u t a c e r t a i n a m o u n t of

giant

sera but not

to t r o p h o b l a s t

sera with

must cells

with antigens

interstitial

142 t r o p h o b l a s t in g e n e r a l

are v a r i a b l e

t h a t this g r o u p m i g h t be m o r e although it probably waves

that spawned

and u n p r e d i c t a b l e ,

heterogeneous

originated

from

the endov/ascular

suggesting

t h a n the o t h e r

the o r i g i n a l trophoblast

subset,

cytotrophoblast

(Pijnenborg

et

al.,

1981b). Another peculiar

a s p e c t of

s u b s e t is i t s p e r i p a t e t i c commonly wandering being

found

into

(Pijnenborg

this s t r a n g e

a t the m o m e n t no r e a s o n a b l e be s a i d a g a i n

for m e m b r a n e m a r k e r s

extraordinary tial

et a l . ,

of t r o p h o b l a s t ,

t h a t the l a c k

than a subset. Finally,

tissues which simply

within

1954; D a l l e n b a c h - H e l l u i e g ,

according

to its h i s t o c h e m i c a l

of

t h e r e is

a phloxine-tartrazine

characteristics.

T h i s is a

mononuclear

in i t s c y t o p l a s m , w h o s e Not surprisingly,

is n o t

tissues

tissue interfaces

trophoblastic mantle

containing

5-7 cells, although

variations, trophoblast. reactions

is v a r i a b l e

Unlike

(Hsi e t a l . ,

1981; Hsi e t a l . , 1 9 8 2 ) ,

extra-

cytotrophoroughly

physiological

to two or e v e n o n e

positive

cyto-

immunocytological, in a fairly

1982), and monoclonal I antigens

with

thickness,

to h u g e

trophoblast,

s e r a are u s u a l l y

c o m m o n d e t e r m i n a n t of M H C C l a s s

reagents.

thinned

the i n t e r s t i t i a l

with anti-TA

in its

this is s u b j e c t

a n d c a n be a b n o r m a l l y

pattern

of m a t e r n a l

is t h a t b e t w e e n d e c i d u a a n d c h o r i o n i c

b l a s t . The

clonal

domain

function

cytotrophoblast

the m o s t e x t e n s i v e

embryonic

geneous

its

known.

Amniochorionic O n e of

cell

et al. . 1980),

appears

to the u t e r u s .

may

intersti-

leukocyte with eosinophilic granules to be l i m i t e d

only

homogeneity

another

uterine

1971; Pijnenborg

is

however

t h a t the g r o u p i n g

the m a t e r n a l

has been called

(Hamperl,

ask

trophoblast forms a subset and

un-

not

but there

to t h i s q u e r y . I t m u s t

location,

cell i d e n t i f i e d

not

but

1981a). One m u s t

s u c h as HLA a n d TA i n d i c a t e

be m o r e of a h o d g e p o d g e

1974),

e a r l y in g e s t a t i o n

group

response

t h a t the i n t e r s t i t i a l

by v i r t u e of i t s a n a t o m i c a l

cytotrophoblastic

(Robertson & Warner,

the m y o m e t r i u m

t h e r e a t term

a b o u t the f u n c t i o n of

nature

the i n t e r s t i t i a l

antisera

are n e g a t i v e

homoto

(Akle

b u t t h i s is n o t true for all a n t i - H L A

T h i s is t h o u g h t to be d u e

to the s h a r i n g

of a

HLA et

al.,

monocon-

143 s t a n t r e g i o n d o m a i n b e t w e e n HLA a n d c e r t a i n TA, p a n c y in r e a c t i o n p a t t e r n s is n o t s e e n w h e n studied on somatic

tissues

problem,

4 Flclntyre,

see F a u l k

(for a m o r e

for s u c h a

the s a m e a n t i s e r a

technical

interaction

a m n i o c h o r i o n uilxch m i g h t h a v 8 i m m u n o p a t h o l o g i c a l covered while conducting

though ample evidence

immunocytological

metrium

to s o m e t i m e s

nal c e l l s m i g r a t i n g ignored.

It w a s

the p r e s e n c e

into

now g r o w n

harbinger

s t u d i e s of

data. Clearly, with disease

a thinned

4 Hsi

the

migration

of

has b e e n

largely

cells deep w i t h i n 1983),

in normal

the c h o r i o n i c

observed

the

cyto-

b u t we

have

amnio c h o r i o n s .

cytotrophoblastic

b u t t h e r e are so few

this

subset

immunopatho-

that conclusions m u s t a w a i t

as has b e e n d i s c u s s e d

endomater-

that we first

(Faulk 4 H s i ,

Al-

has b e e n by F a u l k

more

associated et al.

(19G3) .

trophoblast

The interface

formed

t h a t of

by the v i l l o u s

the e n d o v a s c u l a r

b a t h e d by m a t e r n a l

The v i l l o u s

blood forms a syncytium,

m o s t s t u d i e d of all

t r o p h o b l a s t s u b s e t is trophoblast inasmuch

blood, but other

subsets differ greatly. maternal

territory

cytotrophoblastic mantle

in the f e t u s ,

(1982) and Faulk

The v i l l o u s

tissues

for

dis-

distribution

c e l l s in the m i d s t of

of d i s e a s e ,

these

was

interface.

the p o s s i b i l i t y

this as a c o m m o n f i n d i n g n u m b e r s of

the

the

through decidua and

interest

TA-negative

the p r e s e n c e of m a t e r n a l

is a d e f i n i t e

ally like

extra-embryonic

of a m n i o c h o r i o n

Thus, neither diminished

logical

the m y o m e t r i u m ,

of H L A - P o s i t i v e ,

to a c c e p t

subset nor

bed,

thus w i t h c o n s i d e r a b l e

trophoblastic mantle

s t u d i e s of

been put forward

the p l a c e n t a l

reach

this

in

significance

a t the a m n i o c h o r i o n i c

has already

of c y t o t r o p h o b l a s t i n t o

are

d i s c u s s i o n of

1983).

A n o t h e r a s p e c t of m a t e r n o - t r o p h o b l a s t i c

of TA a n d C l a s s I M H C a n t i g e n s

discre-

than

this,

the e x t r a - e m b r y o n i c

metabolically

the e v o l u t i o n a r y

events

a t the s y n c y t i o t r o p h o b l a s t ' s

(Boyd 4

advantages,

interface.

in an i m m u n o p a t h o l o g i c a l is the b u d d i n g

of

the

Hamilton,

d e v e l o p m e n t of a s y n c y t i u m

surface

with

this is c e r t a i n l y

tissues

important materno-trophoblastic

most bizarre metabolic

as i t is trophoblast

trophoblast which interfaces and a l t h o u g h

1 9 7 0 ) , i t is n o t a t all c l e a r w h a t b i o l o g i c a l derived from

the two

superfici-

if a n y , at

One of

sense

were

this

to

the occur

microvilli,

144 b i t s a n d p i e c e s of p l a s m a m e m b r a n e trophoblastic and e n t e r maternal

sprouts

the i n f e r i o r

vena cava and enter

heart

remain. Histological of i n f l a m m a t i o n

s t u d i e s of

n o m e n o n of a s e e m i n g by

these

1976)

to the m o t h e r .

recognition

and C l a s s

II

& Hsi,

IgG

s u c h as

(Johnson & Faulk,

for s y n c y t i u m , Fc-receptors thelium, many binds

on p l a c e n t a l

b u t n o t to

different to

are

not normally

thought

cross

the p l a c e n t a

able

to m a k e

spontaneous

a s p e c t of

the

times

syncytio-

have

the

9 8 0 ) . In

i9Ri).

immune

as p r o v i d i n g

1970).

high

reverse bind

newborns

spontaneous

IgG

These

re-

into

for the h i g h

(Wood e t a l . .

obdo

a specific

the

known, levels

1978).

abortion

data, Komlos et al..

conclusion

endo-

complexes

IgG

to

contrast,

I n s o f a r as is

thus a c c o u n t i n g

Fc-

monomeric

that m o n o m e r i c

t r a n s p o r t of m a t e r n a l

and B - typing

the i n s i g h t f u l abortions

shown

1

(Johnson & Brown,

(Brambell,

a n t i g e n s in r e c u r r e n t HLA-A

& Johnson,

have

as well

the r e c e p t o r is s p e c i f i c f o r I g G ,

While only using

anti-

and c h o r i o n i c stem v e s s e l

(Faulk

approaches

of o n l y IgG in c o r d b l o o d of n o r m a l

Trophoblast

interesting

macrophages

for

1976;

19B1,

c o m p l e x e s m a d e in the l a b o r a t o r y

for p r o t e c t i o n at b i r t h

parti-

1977) M H C

et a l . .

been confirmed many

to e x p l a i n w h y m a t e r n a l

ceptor-mediated mechanism fetus

et al..

b u t n o t for u n c o m p l e x e d or

trophoblast

technical

be

phe-

is the p r e s e n c e of r a t h e r u n u s u a l

trophoblast Fc-receptors

servations

(Faulk

(Sunderland

1 9 7 7 ) , b u t this s i t u a t i o n is q u i t e

since immune

The

I (Faulk 4 T e m p l e ,

t h o s e on B - l y m p h o c y t e s and m a c r o p h a g e s , complexes

odd

1983).

t r o p h o b l a s tic p l a s m a m e m b r a n e s

for i m m u n e

can p e r h a p s

they

evidence

tissue

.Another i m m u n o p a t h o l o g i c a l l y

receptors,

to p r e s e n t

is a f o r e i g n

findings which have

(for r e v i e w , see F a u l k

affinity

have failed

in

right

where

1 9 5 9 ) , w h i c h is

syncytiotrophoblast

^983),

the

& Toy,

lack of i m m u n e

villi

through

(Bardawil

the a b s e n c e of b o t h C l a s s

et a l . . normal

they c o u r s e

to f i n a l l y p a s s i n t o h e r lung

or r e j e c t i o n

syncytiotrophoblast

Lala et al..

chorionic

to e n t e r

the m o t h e r ' s

g e n s from

a w a y from

''964). T h e s e are c a r r i e d

from w h e n c e

veins

Goodfellow

break

(ikln,

multi-nucleated

the p l a c e n t a

s i d e of

ally explained

spaces

even

through

uterine

since

that normally

the i n t e r v i l l o u s blood

and s o m e t i m e s

that couples with

shared more histocompatibility

(1977)

were

repeated

antigens

than

145 did fertile firmed

couples,

and extended 1

Beer et al. .

to i n c l u d e o t h e r HLfl loci

98i; Mclntyre 4 Faulk,

Plclntyre 4 F a u l k , all f a i r n e s s

an o b s e r v a t i o n w h i c h has b e e n s e v e r a l

it does

couples

n o t seem

al. . 1983). N o n e t h e l e s s , sharing tive

is no m o r e

be a s s o c i a t e d w i t h

important

of

to s t u d y

in

some

( M o w b r a y e_t t h a t HLA

trophoblast-lymphocyte

significance

cross

reac-

performance

of w h y HLA

abortions, and

the r o l e of HLfl i n m a t i n g

the

should

possible

a n t i g e n s , i t is

couples under less

than r e c u r r e n t

the c o n s i d e r a t i o n s

ajpregnancy,

reproductive

the r e s u l t s

severe

spontaneous

clearly

life, and

show

h a v e b e e n the time

the h i s t o r y of p r e g n a n c i e s

of HLfl-fl or - B b e t w e e n

et al.. 1983). These

results indicate

to r e s u l t i n a s u c c e s s f u l

a b o v e f o r TLX r e s u l t s of

a circumstance antigens.

the H u t t e r i t e

compatibility

This appears patibility c a n be m o r e

clearly

relationship TLX

explained

of HLA w i t h TLX antigens

HLA in s u c h a w a y for the o t h e r

are

of the p r e d i c t e d immunity,

antigens

a brief

(Faulk

recognition.

in

the

e f f e c t s of

HLA

in-

immunity.

e f f e c t t h a t HLA a circumstance

com-

which

c o n s i d e r a t i o n of

the

et al. . 1978).

t h o u g h t to be i n l i n k a g e

that compatibility

HLA

described

as the n u m b e r of p r e g n a n c y

following

(Faulk 4 M c l n t y r e ,

l i t y of m a t e r n a l

to t h a t

the g e n e r a t i o n of m a t e r n a l

to g e n e r a t e m a t e r n a l

(Ober

t h a n is

important finding

s t u d y is t h a t the d e l e t e r i o u s

to be the o p p o s i t e

fails

pregnancy

w h i c h is v e r y s i m i l a r

a role for

and

t h a t HLfl d i v e r g e n c y i n a s e x u a l

The new and p o s s i b l y

becomes more obvious

suggesting

a

husband

p a t t e r n s of r e p r o d u c t i o n

u n i o n is m o r e l i k e l y compatibility,

re-

during

the n u m b e r of c h i l d r e n p r o d u c e d ,

that sharing

a n d w i f e is a s s o c i a t e d w i t h s u b o p t i m a l

creases,

sought

indicates

to the a l l o t y p i c s y s t e m of TLX

couples where

to a c h i e v e

couple's

in

T h i s has b e e n d o n e in a l a r g e p o p u l a t i o n of HLfl m a t c h e d

Hutterite quired

It m u s t

1983).

the b i o l o g i c a l

c o n d i t i o n s of r e p r o d u c t i v e abortion.

of

1981;

"'983;

19R2), although

thinking

recurrent spontaneous

this

1983b).

con-

h a v e f o u n d HLfl s h a r -

to h a v e b e e n d i l i g e n t l y

contemporary

(Faulk & H s i ,

Before examining

relationship

(Rocklin et al.,

than sharing

(TLX) a n t i g e n s

1983a;

t h a t n o t all i n v e s t i g a t o r s

ing b e t w e e n a b o r t i n g instances

(Taylor 4 Faulk,

1982; Unander 4 Dlding,

1933a; Mclntyre 4 Faulk,

be s a i d

times

disequilibrium

for o n e d e t e r m i n e s

1983), both disallowing

with

compatibility the

possibi-

T h i s is b o r n e o u t by p r e l i m i n a r y

HLfl

146 and TLX

typing

data obtained with

aborting

and fertile

tical m e c h a n i s m TLX

serves

couples

to e x p l a i n

as a m i n o r

the l e u k o c y t e s

(Mclntyre 4 Faulk,

this a s s o c i a t i o n is

histocompatibility

are a l l o t y p i c ,

and when appropriately

as b o n e m a r r o w

transplants

compatibility

complex

v/ersus-host d i s e a s e

into

recipients

as c o m p a r e d

only a major histocompatibility

underlying mechanism, clinical marker

it s h o u l d

to h e l p i d e n t i f y

m i g h t h a v e an i m m u n o p a t h o l o g i c a l the TLX

compatibility

pathophysiology. linkage

disequilibrium

indicate

chronic abortion in which partners,

1

(Mclntyre 4 Faulk,

9B2a,

Considerations maternal

be c l e a r

1982b,

s t i m u l a t i o n or the lack

stimulated,

the n a t u r e of

closely

the s a m e m a n )

(i.e., women who

though

cytes

(Faulk

4 Plclntyre,

to i n c r e a s e

rather

activity

the s a m p l e

of

the m a t t e r

titred

cytotoxic

immunologi-

to t h i s l i e s these

n o t f o u n d in

spontaneous

children

primary

reeven

cells,

sera with paternal

thought

no

aborters

enough,

they lympho-

have been

the c o m p l e m e n t - m e d i a t e d

! T h i s is p r e s e n t l y

and

b u t now s u f f e r

the h u s b a n d ' s

in

anti-

abortions

1983), and such absorptions

to

lymphocytes

been

by the s a m e m a n ) . O d d l y

than decrease

of

b u t i t is a l s o i m p o r t a n t

they are u s u a l l y

are c y t o t o x i c for

mating

studied

•'983b).

their husband's

c a n n o t be a b s o r b e d f r o m m a t e r n a l

of of

the

and

b u t are n o t u n c o m m o n in s e c o n d a r y

abortions

these a n t i b o d i e s

underlying

between

In the f i r s t i n s t a n c e ,

have had only

who

a n d i t is

be s o m e c a s e s

sharing

the

useful

the d y n a m i c s

been identified

thereof,

h a v e h a d one or m o r e

current spontaneous

sometimes

of

of

aborters

allied with

that there will

1983a and

was

(Hallo-

their problem,

they a b o r t ? The r e s p o n s e

and

there

1993). Regardless

women have very high

their antibodies.

( i . e . , w o m e n who

c h i l d r e n by

graft-

1981). These women have obviously so w h y do

b o d i e s are n o t a n t i - H L A , aborters

surpress

in which

considerations

a n t i b o d i e s w h i c h are o f t e n s p e c i f i c f o r (Faulk 4 Plclntyre,

histo-

thus f a r h a v e o n l y d e a l t w i t h

point o u t that some aborting

cally

and major

recurrent spontaneous

have already

complexes

t h a t HLfl is o n l y a

b a s i s for

genethat

transferred

inccmp atibility

t h e r e is no HLA

and s u c h c o u p l e s

for such

they s i g n i f i c a n t l y

complex

Another

c e l l s are

to t r a n s p l a n t s

w h i c h is m o r e

Indeed, simple

1983a).

incompatible minor

t9B 1 ; H a y e s & C l a m o n ,

Psnneko 4 Festenstein,

recurrent

the p o s s i b i l i t y

complex,

matched

from

known

cytotoxic

to be due

to

the

147 removal

of h i g h - a f f i n i t y

antibody

by a b s o r p t i o n w h i c h

ted the m o r e e f f i c i e n t c o m p l e m e n t f i x i n g c o m p e t i t i o n for

target sites and

Immunological

evidence

thereby

loui-affinity

anti-HLA

and

data.

t h a t the l y m p h o c y t o t o x i c

As d i s c u s s e d a b o v e ,

they c a n n o t be c o m p l e t e l y

secondary

aborters

tion with

the H L A - n e g a t i v e

can completely m o s t of

with

remove

completely

removed

monstrated

the s a m e

anti-trophoblast John Mclntyre that seminal

at Southern

that these absorptions

material

Ranta et al..

Illinois University

the s e r u m of

one

there

was

the

in S p r i n g f i e l d

again like

The i d e n t i f i c a t i o n of TLX a n t i g e n s

has

to TLX

shown

the

rabbit

reactive as

plasma

that

w i t h PP,_ from b

the

plasma

in s e m i n a l

s o u r c e of =>nt.ioenic e x o o s u r e

that this early s t i m u l a t i o n primes respond

Dr.

a c t i v i t y , anri

trophoblast cross

properties

de-

rabbit

In f a c t ,

a protein in seminal

be the i n i t i a l

ab-

preparations

that her anti-TLX

remove antibody

and immunological

t h a t this m a y

to s u b s e q u e n t l y

that

p l a s m a is n o t w i t h o u t p r e c e d e n t , i n a s m u c h

( 9 8 1 ) have identified

the w o m a n , a n d

amniochorion

indicating

cytotoxic antibodies

are s p n c i e s - s p e c i f i c ,

shares physicochemical

suggests

l a y e r of

activity,

e a r l i e r in this p a p e r .

1

human placenta.

absorp-

indicating

s e r a . The f i n d i n g of

in s e m i n a l

of

type of a l l o t y p i c v a r i a t i o n as d i d

sera discussed

not

the s e r a

a n d o n l y one of

the c y t o t o x i c i t y ,

are

However,

In a d d i t i o n ,

plasma but not sperm will

anti-trophoblast

antibodies

largely

from

cytotrophoblastic

ten d i f f e r e n t c h o r i o n s ,

antibodies comes

lymphocytes.

all l y m p h o c y t o t o x i c

aborter with husband-specific

sorbed with

these

absorbed

their husband's

the a n t i b o d i e s w e r e a n t i - T L X .

secondary

from

complement.

f o u n d i n the b l o o d of s o m e a b o r t i n g w o m e n are a n t i - T L X from s e r o l o g i c a l

libera-

antibody

antigens on

her i m m u n e

for

system

the s u r f a c e

of the

blasto-

regarding

w h y some

cyst. The q u e s t i o n h a s y e t to be a n s w e r e d orting women have antibodies b a s i s of r e p r o d u c t i v e

genic recognition. Whether but within several

to TLX

have been provided

Such antibodies

are usually

information

from c l i n i c a l

found in secondary

t e r s , a n d this c o u l d r e p r e s e n t a c o l l a p s e d

the

immunological

to be a f a i l u r e

this is an a n s w e r a b l e

the c o n t e x t of a v a i l a b l e

clues

antigens when

f a i l u r e is p u r p o r t e d

ab-

of

q u e s t i o n is it would

and basic

antidebatable,

appear

and not primary

or s h o r t e n e d

that

studies. abor-

time f r a m e

of

148 the n e g a t i v e

biological

performance

which

tive h i s t o r y , study

of HLA c o m p a t i b i l i t i e s

19P3).

s h o w n in the r e s u l t s

The o b s e r v a t i o n s

t h a t they are u s u a l l y

t h a t the n a t u r e of

restricted

the w o m e n ' s

t e r m i n e d by the m a n ' s

HLA

immune

tially appropriate

throughout pregnancy

clone

that was

a maternal

trophobias totoxic,

In c o n t r a s t , ternal

stimulation

to r e c o g n i z e

tainly

normal,

for

antigens

active

dren during

they o f t e n

aborters

down

suggests

presented

can c o n c e i v e

to b e g e t p r o g e n y . S u c h c l i n i c a l

to h e r T L X - c o m p a t i b l e from

compatibilities

she w o u l d

The a b o v e Taylor 4 Faulk latter group leukocytes phocytes

to TLX

receive

hypothesis

(19R1)

as w e l l

has e m p l o y e d

from

aborting

has now

unable

women's

HLA

cer-

plus

chil-

are

the bulk

prompted inertia

of

Faulk

to

by i m m u n i z i n g

respond

her

with

incompa-

would

elicit

the a l l o t y p i c

in-

"!981).

been successfully

tested

by

(1981), although

different approach

immunizations

de-

to

second,

they

(and thus T L X )

as by B e e r et a l . .

a conceptually

prob-

and

their

which

histories

by v i r t u e of

to be a s a t i s f a c t o r y

on

ma-

have had normal

(Faulk & M c l n t y r e ,

the f a t h e r . W h e t h e r

is g o i n g

measurable

that such immunizations

antigens

produ-

the c u r t a i n

abort during

c o u l d be o v e r c o m e

the i d e a b e i n g

response

particu-

T h e s e w o m e n are

and a n t i b o d i e s

erythrocyte-compatible,

tible l e u k o c y t e s , a protecting

mate

the

by a n o t h e r p a r t n e r

but only

r e s e a r c h on TLX

leukocytes

was

not without precedent

background

that a primary

HLA

ini-

stimulation

t h a t they are

and r e m a r r y f o l l o w i n g

antigens

that

de-

the

to them by t h e i r m a t e s ,

by m e n and w o m e n w h o

can divorce

to p r o p o s e

is

pregnancy.

again able

(19 01)

antigens

sex l i f e ,

of an i m m u n o l o g i c a l l y

their first m a r r i a g e s

and t h e s e p e o p l e

suggest

d r o v e o u t an a n t i b o d y

i n f a n t at t e r m . It is a l s o

l e a r n of s e c o n d m a r r i a g e s

ex-

aborters

response

antigenic

a b l y o n the b a s i s of a l l o t y p i c c o m p a t i b i l i t i e s .

liver a normal

reproduc-

Hutterite

could have been

bringing

of a s u c c e s s f u l

the lack

in p r i m a r y

the TLX

to TLX

primary

but with continuous

and a subsequently

the f u t u r e p o s s i b i l i t y

the

that such antibodies

aborter

lar c o m b i n a t i o n of HLA a n d TLX a n t i g e n s cing

reproductive

type w i t h w h i c h i t is p r e s e n t e d . T h u s

to p r o d u c e

protective,

of

to s e c o n d a r y

response

type of the m a l e p a r t n e r of a s e c o n d a r y

essentially

on

become more apparent late in a couple's

as f o r e x a m p l e

(Qber e t a l . .

i s t and

effects

by

with paternal

approach awaits

further

the

using lyminves-

149 tigation,

but Taylor 4 Faulk

unreported

groups

term b a b i e s

following

Taylor 4 Faulk

(1983)

as well

h a v e now d e l i v e r e d immunizations

(i903)

as s e v e r a l

with

have delivered

third party

n i n e o u t of

ers,

the o n e u n s u c c e s s f u l

case a b o r t e d a t the

only

appeared

at the

n o n e of bringing

therapy

abortions

or f e t u s

tenth week.

had a n y u n e x p e c t e d

some doubt into

spontaneous embryo

for

these infants

the h i t h e r t o

are d u e

o t h e r as

r e c u r r e n t a b o r t e r s of

leukocytes.

ten r e c u r r e n t

twelfth week,

to u n d e r l y i n g

abort-

having

It is n o t e w o r t h y

chromosomal

broadly

that

aberrations,

accepted idea

genetical

( B o u £ e t a l . . 1975; BouiS 4 B o u S ,

yet

normal,

that

d e f e c t s in

most the

1977).

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(1974).

Robertson, Ul., Brosens, I. & Dixon, G.: European J. Obstet. Gynecol. Reprod. Biol. 5: 47-65 (1975). Robertson, Id., Brosens, I. 4 Dixon, G.: In: van Assche, A. 4 Robertson, Id. (eds): Fetal Growth Retardation, Churchill Livingstone, London, 126-138 ( I98i). Rocklin, R., Kitzmiller, J. 4 Garroy, M.R.: Clin. Immunol. Immunopathol. 22: 305-315 (1982). Sunderland, C.A., Naiem, M., Mason, D.Y., Redman, C. 4 Stirrat, G.: 3. Reprod. Immunol. 3: 323-331 ( 19R1). Taylor, C. 4 Faulk, W.P.s Lancet 2: 6B-70

(19B1).

Taylor, C. 4 Faulk, Id.P.! Recurrent spontaneous abortions can be prevented by leukocyte transfusions.(Submitted for publication)(1983). Unander, 1*1. 4 Olding, L.: Amer. J.Reprod. Immunol, (in press)(19B3). Idood, G., Reynard, J., Knishnan, E. 4 Racela, L.: Cell. Immunol. 35: 191-204 (1978).

CHEMOTACTIC FACTORS

Herbert Reynolds

IN T H E A I R S P A C E S T H A T M A Y M O D U L A T E

INFLAMMATION

M.D.,

Pulmonary sections, Yale University School of 333 Cedar Street P.O. Box 3333 New Haven Connecticut 06510 USA

Catherine Roth-Fouret,

Medicine,

M.D.

Laënnec Hospital, 42, r u e de S è v r e s 75340 PARIS Cedex 07

1.

Introduction.

The c a p a c i t y to d e v e l o p a s e l e c t i v e

i n f l a m m a t o r y r e a c t i o n in l u n g

tissue

is p a r t o f t h e h o s t ' s o v e r a l l r e s p i r a t o r y t r a c t d e f e n s e s y s t e m a n d augment local cellular and humoral factors

in the a i r s p a c e s t h a t

ly c a n c l e a r o r i n a c t i v a t e p a r t i c l e s a n d m i c r o - o r g a n i s m s rated into the lungs

cipally polymorphonuclear neutrophils

i n s p i r e d or

(PMNs), a n d f l u i d c o m p o n e n t s

I n f l a m m a t i o n m a y b e g i n in s e v e r a l d i f f e r e n t w a y s , a s

f r o m t h e r e s p i r a t o r y t r a c t to p r e s e r v e h e a l t h ,

a r e a m o n g the m o s t c o m m o n o f f e n d e r s ,

prin-

from of

illustra-

alveolar-

surface. Many foreign particles and noxious substances must

removed daily

aspi-

is i n i t i a t e d a n d m o d u l a t e d f r o m t h e a i r s i d e

t e d in t h e f i g u r e , b u t u l t i m a t e l y a f f e c t s all l a y e r s o f t h e capillary

ordinari-

(1, 2). T h i s r e a c t i o n in w h i c h p h a g o c y t i c c e l l s ,

plasma enter the airspaces the lungs.

can

and

so t h e y p r o v i d e a c o n v e n i e n t

be

bacteria example.

A c k n o w l e d g e m e n t s . T h i s r e s e a r c h w a s s u p p o r t e d in p a r t b y G R A N T f r o m H L B I , N I H , B e t h e s d a , M a r y l a n d a n d by a r e s e a r c h f e l l o w s h i p f o r D r R e y n o l d s f r o m t h e I n s t i t u t N a t i o n a l de l a S a n t é e t de l a R e c h e r c h e M é d i c a l e , L a ë n n e c Hospital (UNITE 214), PARIS.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • New York - Printed in G e r m a n y

154 Bacteria, gram positive and gram negative species with aerobic or bic metabolism,

reside

in the n a s o - o r o p h a r y n x

anaero-

and can be aspirated

the airways or entrained with inspired air and airbourne

i n t o the

M o s t w i l l a d h e r e o r i m p a c t o n the a i r w a y m u c o s a a n d m u c o - c i l i a r y r e m o v e s t h e m , b u t s o m e r e a c h the a l v e o l a r s u r f a c e w h e r e m o b i l e

lungs. clearance

macrophages

ingest them and presumably kill or contain them. After deposition micro-organisms

or p a r t i c l e s o n t h e a l v e o l a r s u r f a c e ,

c r o p h a g e s p r o c e e d s q u i c k l y a n d is q u a n t i t a t i v e l y iary inflammatory or particularly

r e a c t i o n is u s u a l l y n e e d e d .

virulent,

and a more general

almost complete.

If the inoculum

then local alveolar mechanisms may not

may generate other chemotactic systems

is

inflammatory reaction must be generated. This

factors from macrophages,

of

p h a g o c y t o s i s by

c a n p r o c e e d in a t l e a s t t h r e e w a y s , w h i c h w e w i l l r e v i e w chemotactic

into

No

suffice reaction

: liberation

factors, and mobilization of other

s e c r e t e d by in v i t r o c u l t u r e d r e s p i r a t o r y c e l l s m a y b e c o m e

lung diseases

b r o s i s a n d s a r c o i d o s i s w i l l be

2. A l v e o l a r m a c r o p h a g e

; their status

mediator

important

in

(BAL) to a v a r i e t y

in i d i o p a t h i c p u l m o n a r y

fi-

chemotaxins.

occur after manipulating

in n o r m a l , n o n - i n f e c t e d a i r w a y s

the r e s p i r a t o r y t r a c t b y b r o n c h o s c o p y

(5). In m o n k e y s r e p e a t e d l a v a g e s

(4)

analysis of this fluid, two substances were

caused potent, directed migration of PMNs

area

(5).

From

identified

that

(into a m i c r o p o r e f i l t e r in B o y -

den chambers). One substance was a fragment of the fifth component of plement

(Cca) which was about 15,000 daltons molecular weight and

5 , 0 0 0 d) w a s n o t e d to be s e c r e t e d by n o r m a l m o n k e y a l v e o l a r

stimuli

intratracheal

injection of h e a t killed S t a p h y l o c o c c u s

(about

macrophages,

i n c u l t u r e b u t in e n h a n c e d a m o u n t s a f t e r v a r i o u s

( T a b l e 1). A l v e o l a r m a c r o p h a g e s f r o m g u i n e a p i g s ,

com-

promoted

m i g r a t i o n of m o n o c y t e s as well as PMNs. The other smaller s u b s t a n c e

spontaneously

will

and

in t h e s a m e a n a t o m i c a l

o f the l u n g , c a u s e d a s t r i k i n g r e c o v e r y o f P M N 1 s in l a v a g e f l u i d immunochemical

or

discussed.

An accumulation of inflammatory cells

lung lavage

lavage

of

that

(3). D e t e c t i o n o f p h l o g i s t i c f a c t o r s p r e s e n t in l u n g f l u i d s

of inflammatory

auxil-

large

complement pathway activation

the diagnostic application of broncho-alveolar

ma-

phagocytic

in s i t u a f t e r

aureus or in

an

cultu-

155 re, also secreted a rather similar, small molecular size chemotactic factor (6). This factor when purified and reinstilled into the

airways, pro-

duced a PMN exudate without alveolar hemorrhage. It is important to note that the macrophage derived chemotactic factor elicited a PMN response in the absence of erythrocyte leakage which may distinguish its biologic activity from some other inflammatory mediators, preformed immune complexes or active C,_a, that cause appreciable hemorrhagic injury to the alveolar5 capillary membrane when instilled in the airways.

COMPARISON OF ALVEOLAR MACROPHAGE DERIVED FACTORS

Origin

Monkey

Size

(5)

Stability

5,000 daltons

Characteristics

Heat labile

Attracted PMN3 but not monocytes or eosinophils

Guinea pig (6)

1,500 daltons

Heat stable

Generated after in vivo phagocytosis, had in

(600 d)

vivo chemotactic activity

Human

(7,8,9)

8,500

daltons

Sensitive to

PMNs attracted by PI

trypsin and

5.0 peak

heat stable

C1,000

daltons

(600 d)

Resistant to

PMNs attracted in prefe-

trypsin and

rence to monocytes and

proteases, h e a t

eosinophils

and pH stable

release of elastase and

; promoted

lysozyme from PMNs

Sensitive to serum PI 7.6 and 5.2, in part chemotactic inhibitor

factor lipid, but not HETE includes LTB„ 4

156 Human alveolar macrophages also produce chemotactic t i a l l y a t t r a c t PMfos. M e r r i l l a n d C o l l e a g u e s cells by b r o n c h o a l v e o l a r

(7) o b t a i n e d

( > 9 0 %) o f m a c r o p h a g e s .

preferen-

respiratory

lavage of normal smoker and n o n s m o k e r

which consisted principally in vitro culture,

factors that

volunteers,

After adherence

s u p e r n a t a n t f l u i d f r o m the c e l l s h a d

a c t i v i t y , b u t the l e v e l o f a c t i v i t y w a s v e r y d e p e n d e n t o n c e r t a i n manipulations.

The stimulus of cell adherence

chemotactic activity which was detectable

and

chemoattractant cellular

itself provoked release

in s u p e r n a t a n t f l u i d

specimens

a t 6 a n d 24 h o u r s , w h e r e a s s t i m u l a t i o n w i t h a n I g G o p s o n i n o r i m m u n e plex produced a maximal response within 3 hours. Tumbling the cells prevent adherence greatly

d i m i n i s h e d the o u t p u t o f c h e m o t a c t i c

of

comto

activity

f o r up to 22 h o u r s . C e l l s u p e r n a t a n t f l u i d w a s g e l c h r o m a t o g r a p h i e d to solate active chemotactic macrophage cultures guished by size,

components

for f u r t h e r c h a r a c t e r i z a t i o n .

The

(> 97%) produced two substances w h i c h c o u l d be

about 8,500 d and about 1,000 d on calibrated

G - 5 0 S F g e l c o l u m n s , a n d by p h y s i c a l c h a r a c t e r i s t i c s . p r o v e d to be c o m p l e x w h e n s e p a r a t e d b y i s o e l e c t r i c

distin-

Sephadex

The larger

focusing

substance

into a t

5 p e a k s b u t c h e m o a t t r a c t a n t a c t i v i t y w a s c o n f i n e d to j u s t o n e p e a k a p i o f 5.0. The s m a l l f a c t o r w a s n o t a n a l y z e d in d e t a i l b u t its release

into macrophage

c e l l s u p e r n a t a n t w a s d e c r e a s e d w h e n the

early lipoxyge-

A number of

genase products have chemoattractant activity

12-hydroxy

10, 1 4 - E i c o s t e t r a e n o i c

acid

(HETE) a n d l e u k o t r i e n e

duced by s t i m u l a t e d alveolar m a c r o p h a g e s Hunninghake

and colleagues

LTB^

; b o t h c a n be

(9) a l s o f o u n d t h a t a l v e o l a r m a c r o p h a g e s its

as a small 400-600 daltons sized factor. Although adherent, but

from source

activity stimuli

t h i s a c t i v i t y to be p r e s e n t a f t e r 3 h r . P a r t i c u l a r l y

tent were opsonized bacteria and IgG immune complexes

; selective

tion of c o m p l e m e n t r e c e p t o r s on m a c r o p h a g e s was n o t an effective A c t i v e c e l l c u l t u r e s u p e r n a t a n t s w e r e f r a c t i o n a t e d to i s o l a t e t h e t i c a c t i v i t y w h i c h r e s i d e d in a s m a l l s i z e f a c t o r . A s c o n t r a s t e d 1, it h a s d i f f e r e n t p r o p e r t i e s

po-

stimulamethod. chemotacin

Table

f r o m the l a r g e r m a c r o p h a g e c h e m o t a x i n .

partial extraction with organic solvents,

pro-

otherwise

cells would produce maximal amounts of c h e m o t a c t i c

i n s u p e r n a t a n t f l u i d a f t e r 18 h r o f c u l t u r e , v a r i o u s p h a g o c y t i c would accelerate

lipoxy5, 8,

(8).

nonsmoker normals produced chemotactic activity and identified

unstimulated,

least with

nase pathway of p r o s t a g l a n d i n metabolism was inhibited. including

i-

indicated that this factor

Its was

157 in part lipid and a lipogenase pathway

substance.

AIRWAY-ALVEOLAR SURFACE

CAPILLARY-ENDOTHELIAL SURFACE In s u m m a r y

( F i g u r e 1), v a r i o u s p a r t i c l e s a n d m i c r o - o r g a n i s m s

sited on the alveolar surface where they are scavenged cytic macrophages

can be

depo-

e f f i c i e n t l y by

a n d d e g r a d e d o r c l e a r e d by t h e s e c e l l s . H o w e v e r ,

phago-

macro-

p h a g e s are capable of secreting at least two factors that can produce

di-

rected migration of auxiliary phagocytes, principally PMNs. Although

much

o f the e x p e r i m e n t a l w o r k h a s b e e n d o n e w i t h in v i t r o c u l t u r e d c e l l s ,

the

chemotactic

factors when purified and instilled intratracheally

to h a v e b i o l o g i c a c t i v i t y a n d p r o d u c e a P M N - E x u d a t e

do

seem

without hemorrhage.

s e e m s p l a u s i b l e t h a t a l v e o l a r m a c r o p h a g e s c a n i n i t i a t e the i n f l u x o f into airspaces through liberation As y e t uninvestigated,

(by a c t i v e s e c r e t i o n )

of

chemotaxins.

a r e t h e i r m e c h a n i s m s o f a c t i o n to a l t e r

t y o f the a l v e o l a r e p i t h e l i u m o r i n t r a c e l l u l a r j u n c t i o n s a n d (membrane solubility) barrier.

permeabili-

diffusability

through layers of the a l v e o l a r - c a p i l l a r y

membraneous

A n i m p o r t a n t p o i n t o f a c t i o n w o u l d be o n the c a p i l l a r y

l i u m w h e r e c i r c u l a t i n g P M N s in the v a s c u l a t u r e

It

PMNS

are a t t r a c t e d f o r

endothesticking

158

(margination)

and cell polarization as a prelude for migration into

extravascular

location.

3. C o m p l e m e n t - d e r i v e d

an

chemotaxins.

The r o l e o f the c o m p l e m e n t s y s t e m

in h o s t d e f e n s e o f the a i r w a y s a n d

lar surface remains controversial.

P r o p e r d i n F a c t o r B (10) a n d a

of other complement components have been identified

alveo-

number

in lung lavage

fluids

(11) w h i c h s u p p o r t t h e c o n c e p t t h a t a l t e r n a t e p a t h w a y a c t i v a t i o n m a y i m p o r t a n t in t h e l u n g

( R e v i e w e d in R e f e r e n c e 2). I n t e r e s t f o c u s e s

ly o n C,_a a n d C^_a d e s arg, c o n s i d e r e d to be t h e m o s t p o t e n t mediators and chemotactic molecules

primari-

inflammatory

liberated by the c o m p l e m e n t

cascades.

T h e s e are c a p a b l e by t h e m s e l v e s o f c a u s i n g a c u t e a i r w a y h e m o r r h a g e exudation after of alternative

intratracheal

instillation

(12, 1 3 ) . If d i r e c t

complement pathway actually occurs

be

and

activation

in the a i r w a y s ,

then

i t is r e a s o n a b l e to p o s t u l a t e t h a t a v a r i e t y o f i n h a l e d o r a s p i r a t e d c l e s a n d m i c r o b e s t h a t g a i n a c c e s s to the r e s p i r a t o r y

tree can

parti-

trigger

the complement cascade

(C„ a n d C,_ p l u s o t h e r i n t e r m e d i a t e c o m p o n e n t s like o 5 t h e t r i m o l e c u l a r c o m p l e x C,._„ c o u l d b e l i b e r a t e d a n d p r o d u c e a n u m b e r o f 567 f r a g m e n t s w i t h c h e m o a t t r a c t a n t properties). Q u a n t i t a t i v e l y , derivatives 5 s e e m to b e the m o s t e f f e c t i v e a n d p o t e n t o f the c o m p l e m e n t g r o u p . A potential

complement derived chemotactic

factor,

s u c h a s C a, m i g h t 5

be

g e n e r a t e d through a c t i v a t i o n of the c o m p l e m e n t system or e x i s t as a

prefor-

med component

for

in l u n g f l u i d . T h e s i t u a t i o n

is n o t c l e a r a t p r e s e n t ,

s o m e o f the c o n f l i c t i n g r e s u l t s h a v e b e e n p r e s e n t e d o n l y form.

In n o n h u m a n p r i m a t e s

identified whereas,

in

preliminary

( b a b o o n s ) , C,_ w i t h f u n c t i o n a l a c t i v i t y b

in g e n e r o u s a m o u n t s

in c o n c e n t r a t e d l u n g l a v a g e f l u i d

in l a v a g e f l u i d f r o m n o r m a l n o n s m o k e r s ,

c a n be (14) ;

it is n o t p r o m i n e n t

(15).

M a c r o p h a g e s can secrete a number of c o m p l e m e n t components w h i c h c o u l d

pro-

v i d e a l o c a l s u p p l y w i t h i n the a i r s p a c e s .

com-

Two s t u d i e s t h a t i d e n t i f i e d

p l e m e n t c o m p o n e n t s s e c r e t e d by h u m a n alveolar m a c r o p h a g e s found different components

in c u l t u r e

: in o n e C„ a n d C w e r e d e t e c t e d 3 b

o t h e r n o t e d C^ a n d F a c t o r B a n d o c c a s i o n a l l y

C^

(16)

fluid

; the

(17).

T h e n e c e s s i t y o f h a v i n g a n i n t a c t c o m p l e m e n t s y s t e m to c l e a r b a c t e r i a

depo-

s i t e d in t h e a i r s p a c e s

of

is n o t w e l l e s t a b l i s h e d e i t h e r . W h e n a v a r i e t y

159 gram positive and gram negative bacteria were aerosolized

i n t o the

lungs

of mice, previously d e p l e t e d of c o m p l e m e n t activity by COBRA Venom ment or intact normal controls,

the c l e a r a n c e o f

was impaired about 25 per cent versus controls, proliferation quently,

Streptococcus and Pseudomonas

i n t h i s l u n g m o d e l w a s l e s s t h a n in c o n t r o l s

treat-

pneumoniae aeruginosa

(18).

Subse-

stronger evidence has been provided that Pseudomonas clearance

better w i t h an intact complement system

(19). O b v i o u s l y , m o r e r e s e a r c h

is is

n e e d e d to u n d e r s t a n d the r e l a t i o n o f the c o m p l e m e n t s y s t e m a n d l u n g h o s t d e f e n s e , b e f o r e a t t e m p t s a r e m a d e to m a n i p u l a t e c o m p l e m e n t - m e d i a t e d mation or opsonic phagocytosis

w a y s o r i n h i b i t i o n o f the a c t i v i t y o f i n d i v i d u a l

4. O t h e r i n f l a m m a t o r y o r c h e m o t a c t i c

As i l l u s t r a t e d

in F i g u r e

(20),

mediators.

triggered

i n c l u d i n g the c l a s s i c a l p a t h w a y

intrinsic coagulation,

fibrinolysis and kinin

It is n o t e s t a b l i s h e d t h a t H a g e m a n F a c t o r c o m p o n e n t o f the a i r s p a c e

(Factor XII)

is a n

lining fluid, nor that potential

these enzymatic systems must depend on this molecule.

important

is e s t a b l i s h e d

in the a i r s p a c e s ,

b e the r o l e o f m a c r o p h a g e c h e m o t a c t i c f a c t o r s o r c o m p l e m e n t .

may

perhaps

it a s m a y Protease

m e s t h a t c a n be r e l e a s e d by P M N s a n d f r o m p u r i f i e d l u n g m a s t c e l l s

the dependent systems.

enzy-

can

for triggering some

of

T h i s s e e m s to be t h e c a s e in a d u l t r e s p i r a t o r y

tress syndrome where a PMN inflammatory reaction can develop p a n i e d by p r o t e o l y t i c a c t i v i t y

in l u n g s e c r e t i o n s

in lung

(21) a n d l u n g

fluid that can cleave Hageman Factor, prekallikrein,

plasminogen,

(22). R e l e v a n t

our review are the various intermediate

factors that may have

include plasminogen activator and kallikrein,

kinin which has vasopermeability

activity

and could contribute

disaccom-

lavage

molecular weight kininogen and complement components

activity. These

a

contribute

to p e r p e t u a t e o r a u g m e n t the r e a c t i o n r a t h e r t h a n to i n i t i a t e

a c t i v a t e H a g e m a n F a c t o r a n d c o u l d be a m e c h a n i s m

of

It is, i n s t e a d ,

p r o d u c t s to the i n f l a m m a t o r y r e a c t i o n . A c t i v a t i o n o f t h e s e s y s t e m s inflammation

of

formation.

activation

c o n v e n i e n t w a y to g r o u p a n u m b e r o f r e l a t e d s y s t e m s t h a t m a y

be important once

path-

components.

1, a n u m b e r o f i m p o r t a n t s y s t e m s c a n b e

by the a c t i v a t i o n of H a g e m a n Factor complement

inflam-

through suppression of the complement

high to

chemotactic and

brady

to the

lea-

160 k a g e o f p l a s m a a c r o s s the c a p i l l a r y - a l v e o l a r t o r is o f s p e c i a l

interest because

membrane. Plasminogen

it c a n be g e n e r a t e d by

o r c a n be s e c r e t e d by a l v e o l a r m a c r o p h a g e s a n d , t h e r e f o r e , different

activa-

fibrinolysis could arise

in

ways.

The interaction between various Hageman Factor dependent pathways and p l e m e n t i n the a i r w a y s

is l a r g e l y a n u n e x p l o r e d n e t w o r k

i m p o r t a n t in i n i t i a t i n g o r e s t a b l i s h i n g

t h a t is

involved,

s i u m , a n d the o v e r a l l b i o l o g i c s i g n i f i c a n c e o f

in w h i c h c h e m o t a c t i c

fluids or cell culture

in t h i s

fibrosis

tic factor might participate

f a c t o r s m a y b e f o u n d in l u n g

(IPF).

in the i n f l a m m a t o r y r e a c t i o n

and colleagues

chemotac-

in h u m a n

(23) s o u g h t e v i d e n c e o f

lung

chemotaxin

s e c r e t i o n by a l v e o l a r c e l l s l a v a g e d f r o m p a t i e n t s w i t h I P F . T h i s w a s r e l e v a n t d i s e a s e to s t u d y b e c a u s e a l o w - g r a d e a l v e o l a r often present, especially sis of bronchoalveolar among the respiratory

Sympo-

them.

the concept that an alveolar m a c r o p h a g e - d e r i v e d

diseases, Hunninghake

re-

of

media.

a. I d i o p a t h i c p u l m o n a r y To s u b s t a n t i a t e

more

the i m p o r t a n c e

o t h e r a c u t e p h a s e r e a c t a n t s , d i s c u s s e d by o t h e r p a r t i c i p a n t s

5. E x a m p l e s o f D i s e a s e s

probably

inflammation. Obviously,

s e a r c h is n e e d e d to u n r a v e l t h e c o m p o n e n t s

com-

inflammation

in a n e a r l y , c e l l u l a r s t a g e o f d i s e a s e .

lavage fluid from IPF patients

that

PMNs

( m e a n a b o u t 30 %, 5 to 8 0 r a n g e

o f the c e l l s . A s m a l l p e r c e n t a g e o f e o s i n o p h i l s

is

Analy-

(11) r e v e a l e d

cells retrieved from untreated patients,

ted for a consistent percentage

a

accoun-

observed)

( a b o u t 4 %) a l s o w a s

cha-

racteristic . A g r o u p o f 1 5 p a t i e n t s w i t h IPF, c o n s i d e r e d to be in m i d s t a g e o f disease, were investigated derived chemotactic

for p o s s i b l e secretion of a l v e o l a r

macrophage-

factor and compared with 8 nonsmoker normal

(23). A f t e r B A L to r e t r i e v e r e s p i r a t o r y c e l l s f r o m b o t h g r o u p s , w e r e s u b m i t t e d to H y p a q u e - F i c o l l

their

volunteers the

the purified mononuclear cells, principally alveolar macrophages, cultured for 3 h after w h i c h supernatant fluids were h a r v e s t e d a n d ted for chemotactic cultures

cells

d e n s i t y c e n t r i f u g a t i o n to r e m o v e P M N s ;

a c t i v i t y . V a r i o u s s t i m u l i w e r e g i v e n to the

were evalua-

macrophage

in the f o r m o f c o n c e n t r a t e d B A L f l u i d f r o m I P F p a t i e n t s ,

Sepharo-

161 se 4B b e a d s , a n d I g G c o a t e d ox e r y t h r o c y t e s . R e s u l t s o f r e s p i r a t o r y

diffe-

r e n t i a l c e l l c o u n t s in t h e p a t i e n t s s h o w e d t h a t 9 o f 15 h a d g r e a t e r

than

10 % P M N s a m o n g t h e c e l l s

(approximately

t i e n t s h a d l e s s t h a n 10 %

; the n o r m a l n o n s m o k e r s d i d n o t h a v e P M N s .

in culture,

3 5 % ) , a n d t h a t the o t h e r

pa-

m a c r o p h a g e s f r o m I P F p a t i e n t s w i t h > 1 0 % P M N s in t h e i r

nal m i x t u r e of lavage cells spontaneously

released chemotactic

Once

origi-

activity

i n t o t h e c u l t u r e m e d i u m , w h e r e a s c e l l s f r o m t h o s e w i t h f e w e r t h a n 10 % PMNs generally

did not release activity, nor did the cells from

nonsmo-

kers. The IPF macrophages generated large amounts of chemotactic quickly

(by 3 h )

; when this activity was localized

( S e p h a d e x G - 2 5 g e l ) o f the s u p e r n a t a n t f l u i d ,

in the

activity

chromatogram

it w a s f o u n d in the 4 0 0

6 0 0 m o l e c u l a r w e i g h t r a n g e f r a c t i o n s e l u t e d f r o m the c a l i b r a t e d

to

column.

Further analysis of this material showed preferential chemoattractant vity for PMNs compared w i t h b l o o d monocytes, and this activity was extractable with organic solvents, n e n t to b e p r e s e n t .

largely

indicating a significant lipid

compo-

I g G i m m u n e c o m p l e x e s w e r e c o n s i d e r e d to be a n

impor-

t a n t stimulus for p r o d u c t i o n of this c h e m o t a c t i c ke a n d c o l l e a g u e s s u c c e e d e d lar weight, partially

factor. Thus,

Hunningha-

i n i d e n t i f y i n g the s a m e k i n d o f s m a l l

lipid-containing chemotactic

viously found from normal alveolar macrophages that a chemotactic

acti-

molecu-

factor they had

(9). T h i s s t u d y

pre-

indicated

factor was released from alveolar m a c r o p h a g e s of

pa-

t i e n t s w i t h I P F w h o w e r e in a n a c t i v e p h a s e o f a l v e o l i t i s w i t h P M N s

pre-

s e n t in b r o n c h o a l v e o l a r

l a v a g e f l u i d . I g G i m m u n e c o m p l e x e s a p p e a r to be

an important macrophage

stimulus.

Thus an endogenous,

airside

c a n b e r e l e a s e d q u i c k l y w i t h i n a few h o u r s a n d t h i s c a p a b i l i t y w i t h an e l e v a t e d PMN count

b.

( > 1 0 % respiratory

correlates

cells).

Sarcoidosis.

Sarcoidosis teristically

is a c o m m o n , m u l t i s y s t e m d i s e a s e o f u n k n o w n c a u s e t h a t

charac-

produces non-caseating epithelial-giant cell granulomas

a f f e c t e d t i s s u e . A l t h o u g h the r e s p i r a t o r y t r a c t is i n v o l v e d tients,

chemotaxin

pulmonary sarcoidosis

majority of them. However, is p r o g r e s s i v e

in m o s t

is u s u a l l y s e l f - l i m i t e d o r r e s o l v e s

i n a b o u t 2 0 p e r c e n t , the p u l m o n a r y

a n d c a n l e a d to a c h r o n i c s t a g e o f i n t e r s t i t i a l

and cystic airspace degeneration.

in pa-

in t h e

disease fibrosis

In recent onset or intermediate

stages

162 of lung disease,

the histologic response

lar i n f i l t r a t i o n w i t h l y m p h o c y t e s , in interstitial

tissue, bronchial

in t i s s u e

is a m i x t u r e

primarily, and granuloma and vascular structures.

of

alveo-

distributed Sampling

the

airspaces w i t h lung lavage yields an increased n u m b e r of r e s p i r a t o r y with many macrophages and a striking increase, usually,

in

(24). T h i s s a m p l e o f a i r s p a c e o r a l v e o l a r l u m i n a l c o n t e n t s a preponderance

of T-lymphocytes

is n o t a b l e

in w h i c h the s u b p o p u l a t i o n o f

c e l l s c a n b e i n c r e a s e d in v e r y a c t i v e p h a s e s o f a l v e o l i t i s not prominent

cells

lymphocytes for

T-Helper

(25). P M N s

are

in l a v a g e c e l l s r e t r i e v e d f r o m e a r l y a n d a c t i v e p h a s e s ,

but

w i t h p r o l o n g e d d i s e a s e a s s o c i a t e d w i t h f i b r o s i s , P M N s c a n be i n c r e a s e d l a v a g e f l u i d in s o m e p a t i e n t s

(26). C l i n i c a l l y ,

it w o u l d be d e s i r a b l e

have some marker that might distinguish those patients who will have gressive disease. Moreover, excessive,

P M N s in t h e a i r w a y s c o u l d be a s s o c i a t e d

local concentrations

of p r o t e o l y t i c enzymes and

which could foster the fibrotic process. tic substances litis,

in a m a n n e r a n a l o g o u s to I P F p a t i e n t s

From the clinical

s i s as p a r t o f d i a g n o s t i c including

evaluation

inflammatory

(n = 5), a n d l o n g d u r a t i o n d i s e a s e

sarcoi-

and lung lavage for cellular

(n = 5), r e c e n t o n s e t

(n = 4 ) , r e c e n t o n s e t b u t o n t r e a t m e n t w i t h

fluid and respiratory

(i* 2 y r )

disease

corticosteroids

(n = 5). T h e a c e l l u l a r

lavage (direc-

ted m i g r a t i o n of b l o o d PMNs) u s i n g a leading front m i g r a t i o n assay (7). C e l l s w e r e f r a c t i o n a t e d b y

tion o n discontinuous albumin-density tes from macrophages

density

to s e p a r a t e

( w i t h a b o u t 90 p e r c e n t e f f e c t i v e n e s s )

te a l v e o l a r m a c r o p h a g e s dient

gradients

in

centrifuga-

lung

lymphocy-

and to

separa-

i n t o s e v e r a l p o p u l a t i o n s a c c o r d i n g to s i z e a n d g r a -

(28). M a c r o p h a g e s w e r e e s t a b l i s h e d

t e d f o r 6 a n d 16 h o u r s to o b s e r v e tances into cell supernatant, lenge with zymosan.

analy-

defined

cells were assessed for chemotactic activity

blind well diffusion chambers

alveo-

(23).

(24, 2 7 ) . S e v e r a l g r o u p s w e r e

: normal non smoker volunteers

(«5;6 m o n t h s )

chemotac-

service at Laennec Hospital, patients w i t h lung

dosis underwent fiberoptic bronchoscopy

prowith

inflammation

T h i s l e d u s to l o o k f o r

in l u n g f l u i d t h a t m i g h t be s t i m u l a t i n g

in c u l t u r e a n d

incuba-

s p o n t a n e o u s release of c h e m o t a c t i c

subs-

and t h e r e a f t e r s t i m u l a t e d by p h a g o c y t i c

Lung lavage fluid and cell culture supernatants

(organic solvent)

Results are summarized.

elution

chal-

were

gel filtered through calibrated columns or chromatographied w i t h high sure liquid

in to

pres-

gradients.

Bronchoalveolar

lavage fluids, with cells

removed

163 by centrifugation,

and concentrated at 4° C with an Amicon UM-05

h a d no detectable chemotactic activity for PMNs, except several

membrane, specimens

from long duration sarcoidosis patients. W h e n these active BAL fluid ples were chromatographied vity monitored

sam-

through Sephacryl G-300 SF and chemotactic

in t h e e l u a n t f r a c t i o n s ,

it w a s o n l y d e t e c t a b l e

tions that eluted at about 95 per cent of gel bed volume,

in

acti-

frac-

corresponding

to a s m a l l m o l e c u l e o f s e v e r a l t h o u s a n d d a l t o n s s i z e . T h i s r e a s o n a b l y cluded several other possible chemotactic C^a 5

(15,000 d), neutrophil chemotactic

or lipopolysaccharide

(endotoxin)

factors of larger size

factor of asthma

percentages patients

flect an elevated For macrophages

However,

the

perPMNs

so t h e p r e s e n c e o f c h e m o t a c t i c a c t i v i t y d i d n o t

in c u l t u r e , n o r m a l c e l l s d i d n o t s p o n t a n e o u s l y Macrophages

secrete from

onset sarcoid patients and those receiving corticosteroid therapy did not spontaneously

lation w i t h zymosan did n o t induce activity either.

recent

also

make chemotactic activity, but, surprisingly,

these cells were unresponsive,

It is u n c e r t a i n

stimuwhy

f o r s a r c o i d m a c r o p h a g e s are g e n e r a l l y

In c o n t r a s t m a c r o p h a g e s f r o m l o n g d u r a t i o n p a t i e n t s

were spontaneously

re-

count.

c h e m o t a c t i c a c t i v i t y , b u t d i d so w i t h s t i m u l a t i o n .

make

(29)

in t h e r e s p i r a t o r y c e l l c o u n t s w e r e n o t v e r y e l e v a t e d in t h e s e

(up to 4 % ) ,

vated cells.

including

( 6 0 0 , 0 0 0 d)

from contaminating bacteria added

h a p s f r o m the t r a n s n a s a l p a s s a g e o f the b r o n c h o s c o p e .

ex-

secreting activity or could

acti-

either

be readily stimulated

to

it.

Cell supernatants were collected from active macrophage cultures, w i t h o u t c o n c e n t r a t i o n o r e x p o s u r e to f r e e z i n g t e m p e r a t u r e w e r e to a calibrated Sephadex G - 2 5 SF column and gel filtered with buffered saline, pH 7.2. Chemotactic activity

and

applied phosphate

f o r P M N s w a s a s s a y e d in the

e f f l u e n t c o l u m n fractions and two peak areas of activity were one centered about 50-60 per cent of gel bed elution volume,

identified corresponding

to a 5,000 daltons marker protein, and another peak about 90 per cent

elu-

tion volume which was less than 1,000 daltons size. This pattern of

chemo-

tactic activity

cells

in m a c r o p h a g e

that were spontaneously

supernatants was the same for sarcoid

s e c r e t i n g a n d f o r t h o s e s t i m u l a t e d to s e c r e t e

m o t a c t i c a c t i v i t y . A n a l y s i s o f the a c t i v e c o m p o n e n t s ve p e a k s

in t h e s e t w o

respecti-

is in p r o g r e s s a n d r e q u i r i n g u s e o f u l t r a s e p a r a t i o n m e t h o d s .

expect the larger size chemotactic

che-

f a c t o r to be a c o m p l e x m i x t u r e o f

We pro-

164

t e i n s as w a s f o u n d by M e r r i l l a n d c o l l e a g u e s

(7) w h e n a s i m i l a r s i z e

fac-

t o r w a s s u b m i t t e d to i s o e l e c t r i c f o c u s i n g s e p a r a t i o n . W i t h t h e r e c e n t

inte-

r e s t in a r a c h i d o n i c a c i d m e t a b o l i t e s p r o d u c e d by a l v e o l a r m a c r o p h a g e s

(7,

8, 9, 30, 3 1 ) , a n a l y s i s o f t h e s m a l l e r c h e m o t a c t i c

factor has

t a k e n t h i s d i r e c t i o n , w i t h t h e c o l l a b o r a t i o n o f B. A r n o u x

ry b i o l o g i c a l c h a r a c t e r i z a t i o n h a s n o t detected any p l a t e l e t factor or slow reacting substance of anaphylaxis. been extracted

Prelimina-

aggregation

When the substance

u s i n g a m e t h a n o l gradient a c o m p l e x e l u t i o n

15 d i s t i n c t p e a k s e m e r g e s

; chemotactic

is p r e s e n t i n a t l e a s t t h r e e o f the peaks. L e u k o t r i e n e one, but other leukotrienes,

activity L T B ^ is

for

from the

active sarcoid macrophages than was found with calcium tion of ostensibly normal alveolar macrophages

PMNs

probably

stimula-

(8). small

factor correlated amazingly well w i t h the degree of

t r o p h i l i a in l u n g l a v a g e c e l l s

(23) a n d s e e m e d to r e l a t e

stimulation. We have not found such a relationship

spaces

are

spontaneously

ionophore

T h e c a p a c i t y o f a l v e o l a r m a c r o p h a g e s f r o m I P F p a t i e n t s to s e c r e t e a size chemotactic

prespat-

LTC^, LTD^ and LTE^ which comprise SRS,

n o t p r e s e n t . T h e p a t t e r n a p p e a r s to be m o r e c o m p l e x

dosis patients,

has

in e t h a n o l a n d t h e n f r a c t i o n a t e d by r e v e r s e p h a s e h i g h

sure liquid chromatography tern with some

naturally

(31).

to i m m u n e

neu-

complex

in long d u r a t i o n

sarcoi-

f o r m o s t p a t i e n t s do n o t h a v e e x c e s s i v e P M N s in t h e i r

; p r o g r e s s i o n to a f i b r o t i c s t a g e o f l u n g d i s e a s e

is t h e

air-

outcome

in a m i n o r i t y o f p a t i e n t s a n d m a y n o t be i n d u c e d in a n a n a l o g o u s w a y .

Yet,

alveolar macrophages from these sarcoid patients can p r o d u c e

chemotactic

s u b s t a n c e s w h i c h m i g h t r e l a t e to a g e n e r a l s t a t e o f e n h a n c e d

macrophage

a c t i v a t i o n a n d t h u s b e a p h e n o m e n o n t h a t is m e a s u r a b l e f e s t e d by a n in s i t u c o r r e l a t i o n . solvent soluble components

although not

The secretion profile of p r o t e i n

is c o m p l e x a n d c h e m o t a x i s as a s s e s s e d

maniand

with

P M N s a n d o t h e r i n f l a m m a t o r y c e l l s m i g h t n o t b e the c o r r e c t b i o l o g i c to c h a r a c t e r i z e

6•

these macrophage

assay

products.

Summary.

A selective

i n f l a m m a t o r y r e a c t i o n in l u n g t i s s u e

tory host defense that can augment local cellular and humoral defense against micro-organisms

is a m e c h a n i s m o f (alveolar

and particles

respira-

macrophages)

that reach

the

165 alveolar surface.

T h i s r e a c t i o n in w h i c h p h a g o c y t i c c e l l s ,

polymorphonuclear neutrophils,

principally

immune effector cells, and fluid

components

f r o m p l a s m a a r e a t t r a c t e d to t h e a l v e o l i a p p e a r s to be i n i t i a t e d a n d trolled from the airside chemotactic

of the lung. In the early phase of

inflammation,

f a c t o r s m a y b e g i n the i n f l u x o f a s s e s s o r y p h a g o c y t e s .

factors can be l i b e r a t e d by alveolar m a c r o p h a g e s teins

con-

Such

in the f o r m o f s m a l l

(about 5,000 d mole, wt. ) and lipogenase pathway derived

o f a r a c h i d o n i c a c i d o f w h i c h l e u k o t r i e n e B ^ is a n e x a m p l e o r c o u l d be plement components. alternative

complement pathway

(C,_a) or, p o s s i b l y , s e c r e t e d l o c a l l y 5

Other mediators with chemotactic and/or

from bacteria must be considered,

by

vaso-

a c t i o n s m a y a r i s e f r o m the f i b r i n o l y t i c o r k i n i n

Soluble exo-products

systems.

too. When

inflamma-

t i o n is e s t a b l i s h e d a n d e x u d a t i v e f l u i d f i l l s t h e a i r s p a c e s , c e l l u l a r zymes released from injured or dying cells may help perpetuate tion. Thus, factors from several sources can individually or be involved in modulating lung inflammation. feature

interstitial and alveolar

the

s u c h as i d i o p a t h i c macrophages

a source of c h e m o t a c t i c activity that may r e f l e c t p a r t of a poorly reaction.

en-

reac-

collectively

In c e r t a i n l u n g d i s e a s e s

inflammation,

ry fibrosis and s a r c o i d o s i s of long d u r a t i o n , a c t i v a t e d

ted inflammatory

com-

A c t i v a t e d complement fragments are g e n e r a t e d by the

alveolar macrophages. permeability

pro-

metabolites

that

pulmonaare regula-

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H.R., Clin.Res.29,

18. G r o s s , G . N . , R e h m , S . R . , P i e r c e ,

R.G.

(1982). R.M.

(1980).

G.E., Matthews, W.G., Rössing,

F.S., Colten,

Am.

(1981).

15. R o b b i n s , R . A . , G a d e k , J . E . , R e n n a r d , S . I . , C h e n , Y . F . , C r y s t a l ,

(1978).

Z.A.,

(1980).

11. R e y n o l d s , H . Y . , F u l m e r , J . D . , K a z m i e r o w s k i ,

547-548

Clin.

T.K.C., Cohn,

10. R o b e r t s o n , J . , C a l d w e l l , G . R . , C a s t l e , G . , W a l d m a n , R . H . , J.

Frank

117,

(1980).

Scott, W.A., Proc. Natl. Acad. Sei, U.S.A., 79, 7866-7870 9.

59,

(1978).

Invest. 65, 268-276 8.

(1980).

J . A . , G a l l i n , J . I . , R e y n o l d s , H . Y . J. C l i n . I n v e s t .

(1977).

Hunninghake, 15-23

Simmons,

(1980).

4.

273-281

(Editor

450

T.H., Gash, D.J.,

(abstr)

Cole,

(1981).

A . K . , J. C l i n . I n v e s t . 6 2 ,

373-378

167 19. Larsen, G.L., Mitchell, B.C., Harper, T.B., Henson, P.M., Am. Rev. Respir. Dis.126, 306-311

(1982).

20. Ghebrehiwet, B., Randazzo, B.P., Dunn, J.T., Silverberg, M., Kaplan, A.P. J. Clin. Invest. 71, 1450-1456

(1983).

21. Lee, C.T., Fein, A.M., Lippmann, M., Hottzman, H., Kimberly, P., Weinbaum, G., N. Engl.J. Med. 304, 192-196

(1981).

22. Mc Guire, W.W., Spragg, R.G., Cohen, A.B., Cochrane, C.G. J. Clin. Invest. 69, 543-553

(1982).

23. Hunninghake, G.W., Gadek, G.E., Lawley, T.G., Crystal, R.G., J. Clin. Invest.68, 259-269

(1981).

24. Reynolds, H.Y., Lung 155, 225-242,

(1978).

25. Hunninghake, G.W., Crystal, R.G., N. Engl J Med. 305, 429-434

(1981).

26. Roth, C., Huchon, G.J., Arnoux, A., Stanislas-Leguern, G., Marsac, G., Chretien, J., Am. Rev. Respir Dis 124, 9-12

(1981).

27. Roth-Fouret, C., Reynolds, H.Y., Laval, A-M., Sandron, D., Huchon, G., Chretien, J., Rev. Fr. Mai. Respir 11, 347 (Abstr)

(1983).

28. Sandron, D., Reynolds, H.Y., Laval, A-M., Venet, A., Roth-Fouret, C., Huchon, G.J., Chretien, J. Rev. Fr. Mai. Respir. 11, 348 (abstr) 29. Nagy, L., Lee, T.K., Kay, A.B., N. Engl. J. Med. 306, 497-501 30. Rankin, J.A., Hitchock, M., Merrill, W., Bach, M.K., Brashler, Askenase, P.W., Nature 297, 329-331

(1982).

31. Joseph, M., Tonnel, A-B., Torpier, G., Capron, A., Arnoux, B., Benveniste, J., J. Clin. Invest. 71, 221-230

(1983).

(1983)

(1982). G.R.,

SECTION PROTEASE

III ANTI-PROTEASE

INTERACTIONS AND LUNG DISEASES

HUMAN

cyANTICHYMOTRYPSIN

SOME SERINE

: PURIFICATION,

PROPERTIES

AND

REACTIONS

WITH

PROTEASES

Anne Laine, Annette Hayem, Monique Davril Unité INSERM N°16, Place de Verdun 59045 Lille Cédex, France

Introduction

Q-l-antichymotrypsin fically

(a^Achy)

is a plasma protease

inactivates chymotrypsin-like

enzymes

inhibitor which

(1,2).

It represents one of

the major acute phase proteins (3). It appeared that a-|Achy was ly concentrated tis

in the bronchial

lumen

speci-

selective-

in patients with chronic

(4). On the other hand, a^Achy which has been shown to be

bronchiidentical

to the protein termed 64 DP (5) has a high affinity for DNA-cel1ulose and it

has

role

been

in

diagnosing

antibody

response

inhibitory and till

suggested

its now

that

malignant and

capacity

reaction

(6)

this

(7).

little

diseases.

with

the inflammatory nule

contents

medium

; thus

Moreover

seems

biological

the

serine

between

it

is

elastase

of

interest

a-|Achy to

be

We present

enhances

related

significance

proteases

prominent

it

of

its

Q-jAchy

inhibits

here a simpler

the

to

have

purifica-

one we previously published (8) and some a^Achy

and leukocyte enzymes

process, polymorphonuclear

including

not

the

attention.

tion procedure than the original studies about the reaction

64 DP would have a quite

property

In fact

mechanism

received

this

and to

leukocytes release their gra-

cathepsin know

: during

how

G into the a^Achy

extracellular

reacts

with

these

enzymes, or how these enzymes react upon a^Achy. Immunochemical led

us

to

studies

conclude

we

that

carried

out

quantitation

using three different of

such

a component

procedures

which

under different molecular forms must be very carefully exploited.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany

can

be

172 Results 1)

Purification

procedure.

a^Achy was prepared

the procedure p r e v i o u s l y graphy

steps

were not

equilibrated NaN 3

0.02%

loaded

an

described glycine 7.4

by d i a l y s i s .

(8).

The

0.5

concentrated present

tion

Sephacryl

on

a

After

centrifugation,

by

M NaCl

S

fraction

200

column.

The s l i g h t

pure

inhibitor

Purity.

In SDS-PAGE

(Fig.

with

:

25

Diaflo

apparatus.

remaining

filtracontami-

to

PI

Sepharose

30

mg of

columns.

a-jAchy

as s i n g l e

are

procedure.

1) p u r i f i e d a^Achy g i v e s a s i n g l e band of

M^ : 58,000 with or w i t h o u t p r i o r r e d u c t i o n . A s p a r a g i n e was fied

0.2 M

a d j u s t e d t o pH

(a^ P i ) , were removed

and a n t i - a - |

a^Achy

o b t a i n e d from 100 ml normal serum u s i n g t h i s

2)

a

previously

f r a c t i o n were e l i m i n a t e d by gel

anti-albumin is

in

as

eluted

immediately

ultrafiltration

in t h i s

successively

Unbound

chromato-

the serum was

prepared

fraction

was

serumalbumin and a ^ - p r o t e a s e

using

of

human pooled serum was

column

containing

c^Achy

pH 2 . 8 ,

Aggregates nants,

: the two c o v a l e n t

anymore. Normal

anti - a ^ A c h y - S e p h a r o s e

buffer

and

(8)

a modification

i n 0.01 M sodium phosphate b u f f e r pH 7 . 4 , 0 . 3 M NaCl, (PBS)

on

described

used

by

amino a c i d i n N - t e r m i n a l

position

identi-

by Edman d e g r a d a -

tion. 3)

Properties. (8),

Chemical

further

tryptophan,

composition

studies both

c o n t e n t , a^Achy

of

is

indicate them

a very

has that

represent labile

been

previously

concerning about

1%

determined

half-cystine of

the

and

amino-acid

component which has t o be kept

in

PBS pH 7.4 at 4°C or f r o z e n i f s t o r e d f o r a long p e r i o d . 4)

Interactions purulent formation and

with c a t h e p s i n G. C a t h e p s i n G was i s o l a t e d from human

sputum of

cathepsin

(9).

In

an equimolar G

occurred c^Achy

modified

form

of

Moreover

when

active

fragment,

migrating

as

a

previous complex

paper

cathepsin a peptide

we

(M^ near 78,000)

concurrently which

(10),

had G of

no

with

inhibitory

reacted M

the

near

with

noticed

between formation capacity a^Achy

5-6,000

was

a

that

a^Achy of

a

left. small

released

173

and

observed

in

SDS-PAGE.

More r e c e n t l y

complex was not s t a b l e with time Spectrophotometry (12)

using

amounts of amount

of

enzymatic

enzyme

10 ^M), a q u i t e

(final

as

enzyme

performed

substrate.

equimolar

according

When

to

increasing

5 min at 25°C w i t h a f i x e d

concentration

inhibition

in

the

assays

: 3 x

curve was o b t a i n e d and e x t r a p o l a -

p a r t gave an I/E molar r a t i o equal t o 1.76/1 f o r

inhibition.

A time c o u r s e study i s a-jAchy with various

were

were incubated f o r

linear

t i o n of the l i n e a r complete

assays

Suc-Ala2-Pro-Phe-NA inhibitor

we showed t h a t

(11).

cathepsin

times.

performed on s e v e r a l G (molar

ratio

Spectrophotometric

I/E

identical = 2/1)

mixtures

kept at 25°C

enzymatic a s s a y s on a l i q u o t s

of for sho-

wed no reappearance of a c t i v i t y when the i n c u b a t i o n time i n c r e a s e d . After

PMSF

addition

to

each

s t u d i e d by e l e c t r o p h o r e t i c that

the

increases are

the

equimolar

methods.

complex

: transient

incubation

mixture

the

samples

In SDS-PAGE ( F i g . 1) we observe

dissociates

when

the

incubation

components appear then and the f i n a l

m o d i f i e d a^Achy having a M

were

equal

fragment ( i n d i c a t e d by the arrow on F i q .

time

products

t o 55,000 and the

small

1). i

94,000 67,000 43.000

+

30 000

20,100

^

14,400

M

I

E

C

1 10 min min

1h

3h

6h 24h 48h

8 days

I

F i g . 1. SDS-PAGE on a g r a d i e n t gel (5 to 30% acrylamide) of samples withdrawn from i d e n t i c a l m i x t u r e s of a , A c h y with c a t h e p s i n G (molar r a t i o I/E = 2/1) kept f o r v a r i o u s i n c u b a t i o n times at 25°C. M : M markers ; I = n a t i v e a-,Achy ; E = c a t h e p s i n G ; C = c o n t r o l m i x t u r e , i n which c a t h e p s i n G has been p r e v i o u s l y i n a c t i v a t e d w i t h PMSF.

174

F i g . 2. Characterization of chymotrypsin inhibit o r s (13) after a l k a l i n e PAGE (10% aery1 amide at pH 8.3) of samples w i t h drawn from identical mixtures of a,Achy with cathepsin G (molar ratio 2/1) kept for various i n cubation times at 25°C. I = native a,Achy 1/2 = half d i l u t i o n of I .


+6 (1983).

81. Putnam, C.W., Porter, K.A., Peters, R.L., Ashcavai, M., Redeker, A.G., Starzl, T.E.: Surgery 8^, 258-261 (1977). 82. Hood, J.M., Koep, L.J., Peters, R.L.: (1980).

N. Engl. J. Med. 302, 272-275

ON THE INTERACTION BETWEEN HUMAN LIVER CATHEPSIN L AND THE TWO CYSTEINE PROTEINASES INHIBITORS PRESENT IN HUMAN SERUM.

Maurice Pagano, Robert Engler L a b o r a t o i r e des Protéines de la Réaction Inflammatoire Faculté de Médecine - 45 rue des S a i n t s - P è r e s - F 75270 P a r i s Cédex 06 F r é d é r i c Esnard, and F r a n c i s

Gauthier

L a b o r a t o i r e de B i o c h i m i e , Faculté de Médecine 2, b i s Boulevard Tonnellé - F 37032 Tours Cédex

Introduction Human serum contains two d i f f e r e n t c y s t e i n e - p r o t e i n a s e s - i n h i b i t o r s

: a

high molecular weight i n h i b i t o r named aCPI which appears under two d i f f e r e n t molecular forms a f t e r p u r i f i c a t i o n (HMr aCPIl molecular weight 90.000 and HMr aCPI2 molecular weight 175.000) ( 1 - 3 ) , and a low molecular weight one (LMr CPI molecular weight 12.000) ( 4 - 5 ) . The former

inhibits

s t r o n g l y papain and f i c i n but i n h i b i t s Cathepsin B only weakly ( 1 - 3 ) . The l a t t e r e x h i b i t s a low d i s s o c i a t i o n constant ( K i ) a g a i n s t Cathepsin H and B and p l a y s probably a p h y s i o l o g i c a l proteinases

r o l e in the control of these

(5).

I n a previous paper (6) we have described the i n h i b i t i o n of human l i v e r Cathepsin L (E.C. 3 . 4 . 2 2 . 1 5 ) by HMr C P U and 2. A q u a n t i t a t i v e study of the i n t e r a c t i o n of t h i s enzyme with the two s p e c i f i c i n h i b i t o r s i s reported here.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

204 Material and methods The assay of human liver Cathepsin L was carried out by using Benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methyl-7-coumarylamide

(Zphe-Arg-N-

Mec) as a substrate. Active site titration of cysteine proteinases was made up with L trans epoxy succinyl-leucyl amino (4 guanidino) butane (E 64). All proteinase and inhibitor concentrations given in this paper refer to "active" concentrations.

Results and discussion Table 1 Kinetic constants for the interaction of the specific cysteine proteinases inhibitors with Cathepsin L.

Inhibitor Ki M

k : M- 1 S- 1

Delay time Sec.

k

l

s-i

Stabi1i ly time min. 4

5

3

1.3

HMr CPI

3.0I0- 9

S.OIO- 4

12.5

6 8IO-

LMr CPI

1.510- 8

7.010- 4

70

2 .410-

Io/ Ki

I 600 66

In this study, we have found that the two varieties of cysteine proteinases inhibitors can play a physiological function in the regulation of Cathepsin L activity. This enzyme has a high proteolytic activity (7), degrades collagen (8) and would be identical with the "col.lagenolytic" Cathepsin (9). The high molecular weight inhibitor seems to be the most efficient : it has the lowest Ki value and there is three order of magnitude between the plasma concentration of this inhibitor and the Ki value. For the low molecular weight inhibitor, the kinetic data are more unfavorable than for

205 the high molecular weight one. By electroimmuno assay, we have also investigated the plasma concentration of HMr CPI : it ranged between 0.4 g/1 - 0.8 g/1 (5.0 I0- s M - I.I0- 5 M on the basis of a molecular weight of 90.000 which corresponds to HMr aCPIl, the only molecular form present "in vivo"). These assays were carried out with thirty sera of known haptoglobin content (0.50 to 6.50 g/1) : the differences observed in the plasma concentration of HMr aCPI are not related to the inflammatory state estimated by the haptoglobin concentration. On the contrary, in the rat, this inhibitor is identical with the al acute phase globulin (10). On the other hand, with both inhibitors, a2 Macroglobulin was able to remove the free enzyme from the enzyme-inhibitor equilibrium. This latter inhibitor would play a role in the plasma elimination of Cathepsin L.

References 1. Sasaki, M., Minakata, K., Yamamoto, H., Niwa, M., Kato, I., and Ito,N.: Biochem. Biophys. Res. Commun. 76, 917-924 (1977). 2. Ryley, H.C.: Biochem. Biophys. Res. Commun. 89, 871-878 (1979). 3. Jarvinen, M.: FEBS Letters J08, 461-464 (1979). 4. Lenney, J.F., Liao, J.R., Sugg, S.L., Gopalakrishnan, V., Wong, H.C.H., Ouye, K.H., and Chan, P.W.H.: Biochem. Biophys. Res. Comun. J08, 15811587 (1982). 5. Gauthier, F., Pagano, M., Esnard, F., Mouray, H., and Engler, R.: Biochem. Biophys. Res. Commun. jJO, 449-455 (1983). 6. Pagano, M., and Engler, R.: FEBS Lettets J38, 307-310 (1982). 7. Barrett, A.J., and Kirschke, H.: In methods in enzymology vol.80 (Lorand L. ed.) 535-561. Academic Press. London (1982). 8. Kirschke, H., Kembhavi, A.A., Bohley, P., and Barrett, A.J.: Biochem.J. 201, 367-372 (1972). 9. Mason, R.W., Taylor, M.A.J.,and Etherington, D.J.: FEBS Letters 146, 33-36 (1982). 10. Esnard, F., and Gauthier, F.: accepted for publication.

MODULATION OF HUMAN LYMPHOCYTE RESPONSES BY GLOBULIN

ai

ANTITRYPSIN AND a 2 MACRO-

Jean-Pierre Revillard, Geneviève Cordier, Jacqueline Bata, Mireille Latour Laboratoire d'Immunologie, F- 69374 Lyon Cedex 08.

INSERM U.80 CNRS-ERA 782, Hôpital E. Herriot

Summary Several lines of evidence indicate the role of proteinases and of their natural inhibitors in the regulation of lymphocyte responses. A proteolytic activity can be demonstrated at the surface of intact living cells, including lymphocytes. Two major serum anti-proteinases, ajAT and a 2 m were shown to bind to lymphocyte subpopulations and to modulate proliferative or cytotoxic lymphocyte responses. 012m may serve as carrier molecule for various lymphokines. Therefore proteinase-antiproteinase systems are likely to contribute to the regulation of the immune response, especially at the site of inflammatory reactions.

Since the work of Grassmann and Dyckerhoff in 1928 (1) peptide hydrolases or proteases have been classified in two distinct groups: the exopeptida^ ses (aminopeptidases, carboxypeptidases and dipeptidylpeptidases) and the endopeptidases or proteinases. The latter comprise four main groups defined according to the chemical nature of the groups responsible,for catalytic activity : serine proteinases, thiol or cysteine proteinases, carboxyl or aspartic proteinases and metalloproteinases (2, 3). The activities of the proteinases in vivo are controlled by two systems. One is represented by the fact that most of the serine proteinases are synthetized as inactive precursors that require limited proteolysis to activate them. This provides a convenient inactive form of the enzyme for storage, but also allows complex control system with positive feedback, such as activation of trypsinogen by trypsin, or cascades of enzymes each activating the next, a system most highly developped amongst the blood coagulation factors. The

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

208 second form of control of the serine proteinases activity is the system of protein inhibitors. About 10 % of the human plasma protein consists of serine proteinase inhibitors, the most abundant beinga2-macroglobulin (012M) and aj-proteinase inhibitor or aj,-antitrypsin (ctjAT), on a weight basis.

1)

Proteinase activity of lymphocyte surface.

Many inactive zymogens

for serine proteinases can be found in plasma, including enzymes of the blood coagulation and fibrinolysis cascades, the complement system and the interconnected kinin-generation system.Other proteinases are located in cells and tissues, among which two cell types were shown to contain large amounts of active enzymes : the polymorphonuclear leukocytes and the mast cells. The enzymes are located in the cytoplasmic granules. The presence of proteinase on the cell surface has not been extensively studied so far. Hatcher and coworkers (4) extracted a proteinase from human lymphocytes, the cytotoxicity of which was demonstrated at neutral pH using human bladder carcinoma target cells. This protease may be involved in lymphocyte mediated cytotoxicity but its precise localization in the cytoplasm or on the membrane has not been investigated. Similarly a cytotoxic substance was isolated from human lymphocyte membrane

though

its possible protease nature has not been investigated (5). Tokes and Kiefer have designed a method allowing the demonstration of proteolytic activity at the surface of lymphocytes (6). Radioiodinated casein covalently linked to polystyrene beads was found to be hydrolyzed when put in contact with mouse viable lymphocytes in the absence of serum. Proteolytic activity was higher in spleen than in lymph nodes. No difference was found between preparations of thymus and bone-marrow lymphocytes (7). Using the same technique we were able to demonstrate a proteolytic activity on blood and tonsillar human lymphocytes. Also, a significant proteolytic activity was observed dn erythrocytes, but little or no protease was released from these cells. No major difference in surface protease activity appeared between T-enriched and T-depleted cell suspensions (8). A nearly complete inhibition of protease surface activity was achieved by addition of purified ct j AT at a concentration of 0.2 mg/ml. Proteolytic activity of lymphocyte surface has also been demonstrated by Grayzel et al. (9). More recently Zucker-Franklin and coworkers studied the bin-

209 ding and hydrolysis of serum amyloid A by leukocytes (10). By using specific inhibitors, they could demonstrate a-naphtyl-acetate-esterase activity on the surface of monocytes and a subpopulation of 8-15 % lymphocytes. 2)

Binding of proteinase inhibitors to lymphocyte surface.

The binding

of proteinase inhibitors provides indirect evidence for the presence of a proteinase activity of the cell cytoplasmic outer membrane, assuming that the binding is specific. Trasylol, a low molecular weight polypeptide obtained from bovine lung, is a broad spectrum antiproteinase. It was shown to bind to human peripheral blood lymphocytes and polymorphonuclear neutrophils (11). Four independent studies have shown the binding of ajAT to human lymphocytes. Incubation of highly purified ajAT with human tonsil cells in serum-free medium resulted in a transient decrease of ajAT activity in the supernatant (12). More direct evidence of the binding of ajAT was brought by Moreau et al. (13) and by our own studies (14) using iodinated ajAT. We reported that the binding was fast and reversible, that the cells could be saturated. Each lymphocyte can bind a maximum of approximately 1.2 x 10^ molecules of ajAr with an association constant of 0.7 x 10^ M

1. The binding was inhibited by addition of cold ai AT » Soybean

Trypsin inhibitor, and partially by ci2M- lodination by a ] A T which results in the oxidation of the methionine group of the active site with a loss of functional activity, did not allow to demonstrate any binding of a jAT, suggesting that the integrity of the functional site of the inhibitor was necessary. Finally the addition of cell-free supernatants from lymphocytes incubated at 37°C was foumd to decrease the binding of ajAT, suggesting that the proteinase receptor was released from the cell surface. The radiolabelling method, however, brings no information on the possible heterogeneity of a^AT distribution among the cell populations nor does it indicate the presence of ajAT on untreated lymphocytes. We therefore investigated these two points using indirect fluorescence and flow cytometry analysis (15). The presence of a^AT was revealed on a variable percentage of untreated peripheral blood and tonsillar lymphocytes. The percentage of labelled lymphocytes was increased by preincubation with purified ajAT and inhibited by TPCK and by EDTA (3 mM) which could also displace OjAT initially bound to the lymphocyte surface (15). Two other studies have shown

210 the presence of ajAT on the surface of RAJI cells (16) and on concanavalin A stimulated lymphocytes whereas no such binding was found with unstimulated lymphocytes cultured for 72 hrs (17). These experiments does not demonstrate that the antiprotease is actually synthetized by the cells rather than taken up from the serum containing culture medium, through a proteinase receptor at the cell surface. The association of a 2 M with 'lymphocyte surface was first demonstrated by Mc Cormick et al. (18) and confirmed by other reports (19, 20, 21). By indirect fluorescence, a 2 M was detected mostly on a subpopulation of lymphocytes bearing surface Ig. Since a 2 M i s synthetized by monocytes but not by T lymphocytes and quite unlikely by B lymphocytes (22), the most likely explanation for its presence on lymphocyte surface is its binding to a surface receptor. In conclusion, proteinases associated with the external surface of lymphocyte cytoplasmic membrane can be demonstrated by a variety of techniques. Such enzymes are responsible for the binding of antiproteinases like cijAT and a 2 M - Proteinase- ajAT complexes are released in the extracellular environment whereas new enzymatic sites are expressed. The binding of ajAT, although specific and saturable, is characterized by a low affinity and a great capacity. The distribution of proteinases and antiproteinases on the cell surface may depend greatly on the mode of separation of lymphocytes and the concentration of serum and plasma in the medium. 3)

Effect of proteolytic enzymes on lymphocyte responses.

The role of

proteolytic enzymes in the triggering and regulation of lymphocyte activation was suggested by experiments showing that lymphocyte transformation could be inhibited by antiproteases and triggered by neutral proteases (table 1). Trypsin, thrombin and pronase were shown to stimulate DNA synthesis in mouse B cells in the absence of other stimulating agents (23, 24, 25). The stimulating effect was usually not demonstrable on T cells, except with pronase (24). Similar results were reported in other species including hamster (26), rabbit (27) and man (28, 29). Human lymphocytes however, seem less susceptible to activation by proteases than those of other species (30, 31). Human B lymphocytes were slightly more activated than T cells (24, 32). In addition to its own mitogenic effect, pronase,

211 Table 1.

Effect of neutral proteases on lymphocyte transformation.

Species

Proteases

Mouse

Trypsin Thrombin Pronase

Hamster Rabbit

Trypsin, chymotrypsin

Man

Trypsin, chymotrypsin Elastase,, cathepsin G Trypsin, pronase Trypsin Elastase,, chymotrypsin

Lymphocyte transformation Ref. + + +

0 0 + + (Í)

26 27

+ +

++

+ 0 0

23, 24 25 24

28 29 24, 32 30 31

but not trypsin, was shown to enhance the proliferative response to PPD (32). The potentiation of the response to phytohemagglutinin (PHA) was less readily demonstrable (27, 32). However, Havemann and coworkers (31) clearly demonstrated a marked potentiation of PHA-induced.lymphocyte stimulation by two granulocyte neutral proteases, elastase and chymotrypsin, especially if low and medium protease concentrations, short time incubation with the enzymes, and sub-optimal amounts of mitogen were employed. This potentiating effect was no longer demonstrable after removal of monocytes from the culture. These results suggested that potentiation of lymphocyte responses was mediated by an indirect effect via activation of monocytes. Such activation was paralleled by changes in other functional parameters such as inhibition of monocyte migration and chemotaxis, and increase of phagocytosis (31). Several hypotheses has been put forward postulating a role for proteases in lymphocyte triggering by antigens or mitogens. Vitteta and Uhr proposed that binding of multivalent antigens to specific IgD receptors on B cells could expose a critical site in the hinge region of the molecule to the proteolytic activity in the membrane of B cells or adjacent accessory cells (33) . A second signal might be mediated by proteolytic conversion of macrophage derived B lymphocyte bound C3 to C3b (23, 26,34) . These two mechanisms account for a preferential effect of proteases on B cells. However it should be stressed that lymphokines as well as a number

212 of factors of inflammation known to modulate the immune response, are dependent upon protease activity for their synthesis, release and/or activity (35). Therefore the modulating effect of proteases may well be not restricted to certain forms of immune responses, thus making the effects of natural antiproteinases rather unpredictable. 4)

Immunomodulatinq effect of otiflT.

Although Vischer et al. (36) could

not demonstrate a significant inhibitory effect of aj AT on the proliferative response of Balb/c spleen cells to various polyclonal activators, Arora et al. showed that the primary in vitro antibody response to sheep erythrocytes was inhibited by addition of ajAT at concentration of 0.1 to 1 mg/ml (37). Inhibition was achieved by pre-incubation of ajAT with B cells but not with adherent nor with T cells. Injection of ajAT in vivo also depressed the primary response and appropriate controls showed that inhibition could not be attributed to antigenic competition, despite the fact that human ajAT was used in these experiments (37). Using human and mouse lymphocytes, we have found that ajAT inhibits DNA synthesis triggered by several mitogens or by allogeneic cells, and that the effect could be demonstrated with T cells as well as B cells (38, 39, 40). The inhibitory activity of cijAT appeared to be related to its protease inhibitory capacity since ajAT preparations which had lost their anti-trypsin activity during purification were not inhibitory (39). Inhibition of PHA response byajAT was also reported by Baranova et al. (41) . Finally, ctjAT was shown to decrease antibody-dependent cell cytotoxicity and natural Killer activity in a dose-dependent pattern (42). The demonstration of various immunosuppressive activities of ajAT at concentrations below those found in normal plasma is of great importance in view of the possible therapeutic use of this molecule (see Crystal, this volume). It would be of great interest to monitor primary antibody responses, natural killer activity and antibody-dependent cell cytotoxicity in patients treated with ajAT as well as in those with deficient ajAT phenotypes.

213 5)

Modulation of lymphocyte responses by a-,H.

The inhibitory effect of

a 2 M on PHA-induced proliferation of homologous lymphocytes has been observed in man (43) and hamster. Hamster a 2 M

was

reported to depress the pro-

liferation of a B lymphoma cell line as shown on morphological or isotopic grounds (44, 45). a 2 M prepared from human plasma inhibits the proliferative responses of human lymphocytes to PHA and Con A at concentrations of 1 mg/ml (46). In contrast to these observations, Vischer was unable to observe an inhibitory effect of a 2 M »

at a

concentration of 1.4 mg/ml, on

the proliferative response of mouse lymphocytes cultured in a serum-free medj-um (36) . The effect of additional a 2 M present in fetal calf serum may have contributed to some extent to the inhibition reported by other authors (47) . The inhibitory effect of ct2M

was

extended to other mitogens

including LPS on hamster lymphocytes (45) and PWM on human lymphocytes (43). The possibility for inhibition to be due to a direct interaction between mitogen and Q 2 M that prevents the binding of the mitogen to the lymphocyte surface was discussed for PHA (48, 49). The inhibitory effect of a 2 M on proliferative responses is not restricted to mitogen-induced responses but has been observed in mixed lymphocyte culture as well. Doses showing inhibitory effect were 1,000 to 10,000 times lower than those required to prevent mitogen-induced proliferation (46). Moreover, a 2 M exerted an inhibitory effect only when added during the early phase of the reaction, suggesting an effect at the recognition stage (50). This was further supported by experiments showing the decreased production of macrophage slowing factor by lymphocytes incubated with antigen and a 2 M (51). None of these observations considered the relation between the effects of

&2M

preparations and their biological activity. Some discrepan-

cies between different authors might be due to variations in biological activity. However, it was reported that denaturation of a 2 M by ammonium sulfate with a loss in the esterase-inhibitor activity resulted in increasing the inhibitory effect of the a 2 M on proliferative response in mixed lymphocyte cultures (46). Finally the regulatory role of fr2M itself remains questionable since other factors carried by a 2 M might account for some of the reported results. Hence small inhibitory peptides (2,000 to 5,000) daltons) were extracted from ct2M by acid dialysis. Other

aglobu-

lins with documented immunosuppressive effect, such as the pregnancy zone protein, might be antigenically related to a 2 M.

214 The immunomodulating effects of a 2 M may be mediated by the many substances that can be carried by a 2 M (52) . For instance, a 2 M from plasma of patients with rheumatoid arthritis was shown to induce polyclonal activation of B cells (53) . This effect was recently attributed to an a 2 M bound lymphokine produced by T cells in crowded cultures. The lymphokine is associated with a protease activity (54). a 2 M is also a potent inhibitor of aarious cytotoxic reactions including antibody dependent cell cytotoxicity mediated by neutrophils (55) or by lymphocytes (38, 42, 56). The a 2 M subunits were shown to be more potent inhibitors than native a 2 M (56) .

Conclusion Proteolysis is part of many biological reactions especially in inflammation and the immune response. Proteolytic activity can be demonstrated on the outer membrane of lymphocytes, but the enzymes have not been characterized as yet. Natural antiproteinases including ajAT and

a 2 M are

therefore involved in the regulation of lymphocyte responses. Since these antiproteinases are produced by monocytes and macrophages, proteinaseantiproteinase systems may represent one of the regulatory pathway of interaction between lymphocytes and accessory cells.

215 References 1.

Grassmann, W., Dyckerhoff, H: Hoppe-Seyler1s, Z. 41 (1928).

2.

Barrett, A.J.: Proteolysis in health and disease. Ciba Foundation symposium N° 75. Evered, D. and Whelan, J., eds. Excerpta Medica, Amsterdam.

3.

Barrett, A. J. : Fed. Proc. 39^ 9 (1980).

4.

Hatcher, V.B., Oberman, M.S., Lazarus, G.S., Grayzel, A.I.: J. Immunol 120, 665 (1978).

5.

Kahn-Perles, B., Golstein, P.: Eur. J. Immunol. 8, 71 (1978).

6.

Tökes, Z.A., Kiefer, H.: J. Supramol. Struct. 4, 507 (1976).

7.

Tökes, Z.A.: Fed. Proc. 3£, 533 (1975).

8.

Bata, J., Martin, J.P., Revillard, J.P.: Experientia 37, 518 (1981).

9.

Graizel, A., Hatcher, V.B., Lazarus, G.L.: Cell. Immunol. JJ3, 210 (1975) .

Physiol. Chem. 179,

10.

Zucker-Franklin, D., Lavie, G., Franklin, E.: J. Histochem. Cytochem. 29_, 451 (1981) .

11.

Thomson, A.W., Pugh-Humphreys, R.G.P., Tweedie, D.J., Hörne, C.H.W.: Experientia 34, 528 (1978).

12.

Bata, J., Deviller, P., Colobert, 1499 (1977).

13.

Moreau, P., Dornand, S., Kaplan, J.G. : Can. J. Biochem. (1975) .

14.

Bata, J., Deviller, P., Revillard, J.P.: Biochem. Biophys. Res. Com. 98, 709 (1981).

15.

Bata, J., Cordier, G.: Ann. Immunol. 133D, 263 (1982).

16.

Dierich, M.P., Landen, B., Schmitt, M.: Z. Immun. Fursch. 156, 153 (1979) .

17.

Lipsky, J.J., Berninger, R.W., Hyman, L.R., Talamo, R.C.: J. Immunol. 122, 24 (1979) .

18.

Mc Cormick, J.N., Tunstall, A.M., James, K. : Nature New Biol. 146, 78 (1973) .

19.

Gelder, F., Hurtubise, P., Scillian, J., Murphy, S.: Clin. Res. 23, 291A (1975).

20.

James, K., Tunstall, A.M., Parker, A., Mc Cormick, J.N.: Clin. Exp. Immunol. _19, 237 (1975).

21.

Jakab, L., Pozsonyi, T,, Feher, J., Papp, G.: Protides of the Biological Fluids. Peeters, H. Ed. Pergamon Press, Oxford, p. 767 (1977).

22.

Hovi, T., Mosher, D., Vaheri, A.: J. Exp. Med. 145, 1580 (1977).

23.

Vischer, T.L.: J. Immunol. 113, 58 (1974).

L.: C.R. Acad. Sei. Paris 285D, 1337

216

24.

Kaplan, J.G., Bona, C.: Exp. Cell. Res. 88, 388 (1974).

25.

Chen,L.B., Teng, N.N.H., Buchanan, J.M.: Exp. Cell. 101, 41 (1976).

26.

Hart, D.A., Streilen, J.S.: Exp. Cell. Res. 102, 246 (1976).

27.

Goodall, D.M., Pardoe, G.I., Gregory, J.K. : Clin. Exp. Immunol.9^, 645 (1971) .

28.

Mazzei, D., Novi, C., Bazzi, C.: Lancet 2, 232 (1966).

29.

Vischer, T.L., Bretz, U. , Baggiolini, M. : J. Exp. Med. 144, 863 (1976)

30.

Algom, D. , Graham, B., Fyson, R. , Richter, M.: Immunol. Coram. 5_, 145 (1976).

31.

Haveman, K., Schmidt, W., Bogdahn, U., Gramse, M.: Neutral proteases of human polymorphonuclear leukocytes. Haveman, K., Janoff, A., eds. p. 306 (1978).

32.

Girard, J.P. , Fernandes, B.: Europ. J. Clin. Invest. 6_, 347 (1976).

33.

Vitetta, E.S., Uhr, J.W.: Science 189, 964 (1975).

34.

Gisler, R.H., Vischer, T.L., Dukor, P.: J. Immunol. 116, 1354 (1976).

35.

Becker, E.L., Herrson, P.M.: Adv. Immunol. YJ_, 94 (1973).

36.

Vischer, T.L.: Immunology 36, 811 (1979).

37.

Arora, P.K., Miller, H.C., Aronson, L.D.: Nature 274, 589 (1978).

38.

Bata, J., Cordier, G., Kevillard, J.P., Bonneau, M., Latour, M.: Transplantation and Clinical Immunology 59 (1981).

39.

Bata, J., Deviller, P., Vallier, P., Revillard, J.P.: Ann. Immunol. 132C, 275 (1981) .

40.

Bata, J., Revillard, J.P. : Agents and Action

41.

Baranova, F.S., Bermann, A.A., Zarestskaya, Y.M.: Bull. Exp. Biol. Med. 8£, 44 (1981).

42.

Ades, E.W., Hinson, A., Chapuis-Cellier, C., Arnaud, P.: Scand. J. ^5, 105 (1982).

43.

Goutner, S., Simmler, J.T. , Rosenfeld, C. : Differentiation (1976).

44.

Streilein, J.S.: Ph.D. Thesis, University of Texas, Health service Center, Dallas (1976).

614 (1981).

171

45.

Stein-Streilein, J., Hart, D.A. : Fed. Proc. y]_, 2042 (1978).

46.

Johannsen, R., Haupt, H., Bohn, H., Heide, K., Seiler, F.R., Schwick, H.G.: Z. Immunit. Forsch. 152, 280 (1976).

47.

Streilein, J.S., Hart, D.A.: Infect. Immun. J.4, 463 (1976).

48.

Chase, P.S.: Cellul. Immunol. 5_, 544 (1972).

49.

Amlot, P.L., Unger, A.: Clin. Exp. Immunol. 25, 520 (1976).

50.

Johannsen, R., Carlsson, A.B., Haupt, H., Heide, K.: Behring. Inst. Mitt. 54, 33 (1974).

217

51.

Ford, W.H. , Caspary, E.A. , Shenton, B.: Clin. Exp. Immunol. _15, 169 (1973).

52.

James, K. ; TIBS JJ5, 43 (1980).

53.

Teodorescu, M., Chang, J.L., Skosey, J.L.: Int. Arch. Allergy Appl. Immunol. 66, 1 (1981).

54.

Chang, J.L., Ganea, D., Dray, S., Teodorescu, M.: J. Immunol. 130, 267 (1983).

55.

Cordier, G., Revillard, J.P.: Experientia 36, 603 (1980).

56.

Gravagna, P., Gianazza, E., Arnaud, P., Neels, M., Ades, E.W.: Scand. J. Immunol. 15, 115 (1982).

M E D I A T O R S OF

INFLAMMATION

Moderators

: R.G.CRYSTAL

Th-is Aound

table, pAovided

between gists, most

clinicians, since The

AeseaAch. paAt

hoA

next

symposium.

summaAy

discussion and

alveolitis oh the

paAticipants

The

AeadeA

texts

will

in the

oh R.G. CRVSTAL

lung

interstitial

structures,

includes a c c u m u l a t i o n

chemotactic

and

H.

REyfJOLPS

the p r o c e s s of a l v e o l i t i s . the major They

factors

origin

consist

of immune and

is

hoA

of such

effector

O n e or more

can

release injury.

of cells

alveolar

Alveolar

factors

of small p r o t e i n s

in

these cells

into a i r s p a c e s .

take a part spaces

Other

to the increase

: plasminogen

(at least

or

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

is

side of

2 diffe-

chemotactic

in cellular

activator,

will

macrophage

in the air

have been i d e n t i f i e d ) p r o d u c i n g an influx

polymorphonuclears

of

to alveolar

factors a t t r a c t i n g

initiate

of a l v e o l a r

gAeateA

bAieh

they

contributing

probably

will a l s o

a

in the.

a

structures.

types are a c t i v a t e d , and of i n f l a m m a t i o n

Since a l v e o l i t i s

rent

that

discussion,

and

is defined a s a c c u m u l a t i o n

t h e s e cell

uAge

the

diseases

be AeseAved

^-¿nd theAeahteA

appAoache.d

one. oh

oh pulmonary

should

moApholo-

data.

cells w i t h i n

the lung.

i.6 pAobably

inteAest

to the

complementaAy

mediators

constAuctive

iield

themes

(Lyon)

immunologists,

in the

inhlammation

ALVEOLITIS

J.F.CORDÏER

subjects

oh the

Alveolitis

a veAy

biochemists,

pulmonaAy

AeheAAed

( B e t h e s d a ) and

inhlammatoAy

exciting

IN INFLAMMATORY

of

factors

populations

fragments

220 derived

from

diseases are

the

or i m m u n e

examples

tactic

of

substances,

Oxydant s are which are vation,

highly

cellular

of

membranes. of

lung

direct

infectious

pulmonary of

fibrosis

these

chemo-

extracellular

Oxygen

by

nization,

will

deficiency),

region part

injury

mechanism

of

still

of

and

taken the

lung

the lungs

no-

inacti-

is

of an

There are

enzymes.

enzy-

: superoxyde Apart

susceptible structures

to

from lower

can derange

if a n t i p r o t e a s e

inactivated

or w h e n

collagen

proteases,

oxydants is s t i l l

of

and

activity are

I),

collagen

is

and

im-

elastase, the

orga-

types,

connective

proteases

debated,

in

the

also

for a b o u t

4 0 X of

turns the

to

the

cells

f i b r o b l a st. of

lung

This

cell

inter stitium,

and

matrix.

intersmajor

underterminated.

Attention

is

absent

release

will alter

to t h e

the

(functional

proteases

attachment

by

enzyme

peroxydation

of o x y g e n

distribution

cell

of

metabolism

when antiproteases

or

neutral

source

oxygen

such as

(directed against

activator,

relative

occur

both

antiproteases.

in a l v e o l a r

deficiency)

repartition

the

oxydants are

This

Collagenase

plasminogen

of

for

radicals

the lung,

of

structures.

to f i b r o s i s .

inactivating

(which

to i n j u r y

first

lipid

and glutathione

of

"CI a n t i t r y p s i n portant.

derange

free

matrix

antitrypsin

and

toxicity

leading

present

not s u f f i c i e n t .

the

cells

proteases,

toxic activities

catalase,

when

as

products

with DNA,

injury

defences

Proteases

contribute in e x c h a n g e

considered

toxic activity,

alveolar

titial

in

alveolitis

oxydants and

defences against

dismutase,

will

matrix

reactive

interactions

example

The

are i.e.

capable

spaces,

state)

extracellular

Polymorphonuclears

(s^l

in i d i o p a t h i c the p r o d u c t i o n

in a l v e o l a r

in an a c t i v a t e d

matic

for

Bacteria

factors.

cells and xious

system.

complexes

stimuli

Whai a c c u m u l a t e d are

complement

account and

it

221 produces

most

Alveolar

macrophage

alveolar

fibroblasts

factor

for

part

of

connective

may

modulate

by

fibroblasts.

which

blasts.

The

of

bolic

colJagen After

takes a part

functions and

of

its

reviewing

the

discussion

focused

in c l i n i c a l

predicting

those

is d i f f i c u l t

cells

on the

lable. Actually,

both

gallium

together

and

¿7

scan,

pathologic

evaluating

the

risk

The

with

findings,

are of

of

enzyme

for

fibro-

as

the

meta-

both

collagenase).

is

the

data

X-ray,

the m o s t a c c u r a t e

that

fibrosis

indicator

lavage

alveolitis.

wall

inflammatory

of d e v e l o p i n g

clinical,

of growth

research,

conclusion

no d e f i n i t e

bronchoalveolar

intensity

as well

of l u n g

evaluation at

a

macrophages

of p r o d u c i n g

degrading

subjects

since

rate

in t h e a l v e o l a r

(capable

practice.

patients

to a s s e s s

alveolar

in f i b r o g e n e s i s ,

specifically

alveolitis

replication

is a c h e m o a t t r a c t a n t

fibroblasts

the a b o v e

matrix.

(when activated)

Furthermore,

fibronectin,

probably

the

releasing

produce

increase

extracellular

is

avai-

and

functional methods

for

222

IDIOPATHIC PULMONARY FIBROSIS. MULTIPARAMETRIC EVALUATION OF ALVEOLAR LYMPHOCYTE AND MACROPHAGE ACTIVATION.

JF MORNEX, G CORDIER, B LAHNECHE, J PAGES, P CHAUVOT, JM VERGNON, T WIESENDANGER, R LOIRE, M VINCENT, JF CORDIER, F MORNEX, JP REVILLARD, and J BRUNE.

Centre de Cytofluorométrie, UER de Biologie Humaine & INSERM U 80, ERA CNRS 782, Pavillon P, Hôpital Edouard Herriot, 69374 Lyon Cedex 08,France Service de Mëdecine Nucléaire, Centre Léon Bérard. Laboratoire d'hématologie, d 1 anatomo-pathologie & d'exploration fonctionnelle respiratoire, and service de Pneumologie, Hôpital Louis Pradel.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung of unknown etiology. It includes inflammation of the alveolar structure, and interstitial fibrosis. The alveolitis of IPF is characterized by a combination of mononuclear phagocytes, lymphocytes and neutrophils (1). The neutrophil component of the alveolitis can mediate the alveolar derangement of IPF and is considered to play a major role in the pathogenesis of the disease. But some IPF patients have a predominantly

lympho-

cytic alveolitis and a better response to corticosteroid treatment (2). As it is the case in sarcoidosis and hypersensitivity pneumonitis, IPF lymphocytic alveolitis can be critical in the development of lung fibrosis. In order to estimate the intensity of alveolitis in IPF patients we studied in parallel lung lymphocyte and macrophage activation.

Patients and methods

Eleven patients (5 women, 6 men, mean age 61.9 /_ 37 - 76 _ / ) were studied. The diagnostic criteria of IPF (2) were : breathlesness, widespread end respiratory rales, diffuse reticular or reticulo-nodular pattern on the chest Xray and consistent physiologic features with a low diffusing capacity and exercice hypoxia (FVC = 92.5 + 22.8 % predicted, FEV 1 =

M a r k e r Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

224 89.6 + 25.0, DLCO/AV = 84.3 + 20.7, exercise Pa 02 = 7.8. + 1.3) and a decrease compliance in 3/8 patients. Other known causes of chronic lung shadowing, such as inhaled inorganic or organic dusts, sarcoidosis or medications mere excluded. Histological confirmation was obtained by transbronchial lung biopsy from 5 patients (including the 3 patients with normal Xray). The follow up of the patients revealed reversal of the disease and improvement under corticosteroid treatment in 5 of them. 2) Lung lymphocyte activation was measured on cells from bronchoalveolar lavage as previously described (3). The criteria (4) were

of T cell activation

presence of large T cells, expression of HLA DR antigens on

T lymphocytes, labelling by monoclonal antibodies directed against in vitro activated lymphocytes (MLR 1-3), the study was performed by flow cytometry using immunofluorescence labelling with monoclonal antibodies (5). Various cell types were identified by their light scattering properties. Simultaneous measurement of DNA and RNA content by acridine orange staining was used for cell cycle analysis. Lymphocyte proliferation was evidenced by an increased percentage of cells in S + G2 + M phases of the cell cycle (6). 3) Lung macrophage activation was assessed by in vivo Ga 67 uptake (1,7). Each patient received 2 m Ci of Ga 67 citrate intravenously 48 or 72 H prior to scanning. A whole body imaging was obtained with an Angers' tomoscintigraphic camera. A semi-quantitative index was computed according to Line and Crystal (1).

Results. 1) Patients with IPF were characterized by an increase percentage of BAL lymphocytes (50.6 + 24.7) that were mainly T cells (38.9 + 10.4 % of total cells). Blood lymphocytes showed a decreased percentage of cells identified byt 0KT4 and 0KT8 markers, respectively 36.4 + 16.7 and 20.0 + 15.3, US 54.6 +.9.6 and 30.9 + 7.8 in 34 healthy controls, and an increased 0KT4/0KT8 ratio(3.9 + 4.3, US 1.9 + 0.7). Conversely

225 these values in BAL lymphocytes were not significantly different

from

control blood (0KT4 : 45.6 + 12.3, 0KT8 : 30.0 + 9.2, 0KT4/0KT8 : 1.6 + 0.5).

2) Activated T lymphocytes mere identified in the BAL of 3 patients by the presence of large T cells (1 case), MLR positive cells (2 cases) and HLA DR positive T cells (1 case). Lymphocyte proliferation was observed in two of these three patients

: up to 10 % of the lymphocytes were in

the S + G2 + M phases. In one patient lymphocyte proliferation was no longer detectable after 6 months of corticosteroid

treatment.

3) Macrophage activation was demonstrated in 10/11 patients by an index over 50 (117.3 + 6 3 . 9 / 5

investigated

- 230_/ . Comparison between lym-

phocyte and macrophage activation revealed no discrepancy within IPF patients.

Comments

From a series of 16 IPF patients (excluding collagen vascular

disease

associated IPF) admitted in a two year period, 11 had a lymphocytic alveolitis. Lung gallium scanning demonstrated macrophage activation in all cases but one. Activation of alveolar T cells was demonstrated in all cases available for bronchoalveolar lavage lymphocyte studies. It was associated with lymphocyte proliferation in two patients. These investigations are presently extended to a larger group of patients. The preliminary results reported here suggest that macrophage

activa-

tion may be associated with T lymphocyte activation. Whatever the yet unknown triggering signal, interactions between these two cell types are likely to play a critical role in the development of the disease. Measurement of lymphocyte and macrophage activation allows to monitor the alveolitis of patients. Since assessing the effect of treatment according to physiologic data is difficult over a short period of time the monitoring of the alveolitis would have to be useful (8). A combination of lymphocyte and macrophage activation measurements can be per-

226 formed routinely and sequentially in the course of the disease.

Aknowledgments

: we thank G. Corte, L. Moretta and J. Brochier for mono-

clonal antibodies and C. Quintin for secretarial

assistance.

References

1. Crystal,R.G, Gadek, J.E., Ferrans, V.J., et al : Am. J. Med. 70 542-568 (1980) 2. Rudd, R.M., Haslam, P.L., Turner-Warwick, M. : Am. Rev. Respir. Dis. 124, 1-8 (1981) 3. Mornex, J.F., Cordier, G., Pages, J. et al : Monographs in allergy in press 1983 4. Mornex, J.F., Cordier, G., Revillard, J.P. 11, 293-300 (1983)

: Rev. fr. Mai. Resp.

5. Cordier, G., Mornex, J.F., Lefebvre, R. et al : in Poncelet Ed. Cytofluorometrie et anticorps monoclonaux dans le suivi des therapeutiques. In press 1983 6. Pacheco, Y., Cordier, G., Perrin-Fayolle, M., et al : Am. J. Med. 73, 82-82 (1982) 7. Mornex, J.F., Lahneche, B., Mary, P., et al : Lyon Med. in press 1983 8. Keogh , B.A., Bernardo, J., Hunninghake, G.W., et al : Am. Rev. Respir. Dis. 127, 18-22 (1983)

THE ALPHA1] PROTEINASE INHIBITOR OF THE LOWER RESPIRATORY TRACT. EFFECT OF CIGARETTE SMOKING

A.Pelletier, M.B., C.Boudier PH.D., G.Pauli M.D., J.G.Bieth PH.D. Service de Pneumologie (Prof.E.ROEGEL), C.H.U., B.P. 426 67091 STRASBOURG and INSERM Unité 237, Faculté de Pharmacie, U.L.P., B.P.10,67048 STRASBOURG CEDEX (France)

Cigarette smoking is an important risk factor of pulmonary emphysema. In vitro experiments document on cigarette smoke-induced inactivation of alpha-] proteinase inhibitor (djiPl) (1), a protein which is thought to protect the Lung against neutrophil elastase as shown by the association between emphysema and inherited ^1PI deficiency (2). Neutrophil elastase itself is able to induce pulmonary emphysema in animals models (3). We studied the in vivo effect of cigarette smoking ont^-|PI activity of the lower respiratory tract fluid collected by bronchoalveolar lavage (BAL).

Methods The studiedpopulation consisted of twenty healthy volunteers divided in two equal groups : ten smokers (8 ± 5 pack years) and ten non smokers (mean age 28 ± 9 years). To the best of our knowledge, the smokers did not smoke for at least 24 hours before BAL. BAL was performed as usually described using five 60 ml aliquots of sterile 0.9 X saline for each BAL. The fluid recovered was centrifuged and the supernatant frozen at -30°C until concentration ca five-times by Amicon UM2 ultrafiltration at +4°C. In 17 volunteers, serum was obtained by venous punction just before BAL. Porcine pancreatic elastase isolated by us (4) was 95-98% active as assessed by active site titration (5,6). The elastase inhibitory capacity of serum and lavage fluid was measured using the kinetic method described in the figure Legend.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

228 Serum and Lavage fluid levels ofc^iPI and albumin were determined by radial

immunodiffusion.

Results

Reaction of increasing amounts of serum with constant amounts of porcine pancreatic elastase yields a straight inhibition curve up to at least 80% inhibition (see the figure). Since oj-]PI is the only porcine pancreatic elastase inhibitor present in human serum (7), elastase can be used to titrate serum ¿ ^ P I , if the association betweenojiPI and elastaseiias gone to completion i.e. if serum + elastase mixtures have been preincubated long enough prior to addition of substrate (8). The preincubation time we used is 90 minutes. For the sake of clarity, we have expressed the serum and lavage fluid concentration in reaction medium in mole of immunoreactiveC^iPI per mole of elastase

(see the figure). As expected for a 1 : 1 c ^ P I

: elastase

stoichio-

metry (8), the serum inhibition yields an equivalence point of 1.03. By contrast the lavage fluid curve yields an equivalence point of 2.33. This indicates that 58% of the immunoreactiveo'ilPI

is inactive on elastase.

The table lists the results obtained for the twenty healthy

subjects.

We use albumin as reference protein. There is no difference in immunoreactivec\lPI between smokers and non smokers. The last column lists the number of moles of elastase inhibited per mole of

of the table immunoreactive

0^-|PI. This is the fraction of functionally activedy|PI. It can be seen that the lavage fluid non smokers and smokers contains 38% and 54% of activeC^qPI respectively

(this«, f i gures are not significantly

different).

Discussion

Our data are at variance with those published by GADEK et al (9) and CARP et al (10) who claim that non smokers have fully activeC^iPI in their vage fluids whereas smokers have significantly reduced levels of

la-

functio-

nal(^iPI. The use of completely different methodologies may account for the

229 FIGURE

MOLEodPl/MOLE

ELASTASE

Inhibition of porcine pancreatic elastase by the serum and the BAL fluid from a subject. Increasing amounts of fluid were added to constant amounts of elastase contained in a spectrophotometer cuvette. After 90 minutes, the time required for complete association of elastase w i t h d ^ P I ,

constant

amount of succinyl-Ala3-p-nitroanilide was added to measure the activity of non inhibited elastase

(12).

TABLE IMMUNOREACTIVE ALBUMIN A N D ^ P I AND ELASTASE INHIBITORY CAPACITY OF BAL FLUIDS AND SERUM (MEAN ± SD) .

MOLE cV]PI/M0LE ,M0LE OF ELASTASE ALBUMIN x 1 0 2 INHIBITED PER MOLE OF «t-jPI

ALBUMIN (pM)

LAVAGE FLUID

0.64 ± 0.28

9.3 ± 3.1

0.38 Í 0.14

SERUM

590

i

30

5.9 Í 0.76

0.94

0.45

t

0.16

9.3 ± 2.6

0.54 ± 0.15

600

t

40

6.0 Í 0.9

0.98 ± 0.08

NON S M 0 K E R S

LAVAGE

FLUID

SMOKERS SERUM

t

0.08

230 divergent results. Another explanation may be that our smokers did not smoke 24 hours before lunglavage whereas in one of the reports

(10)they

were "permitted to smoke up to the time of the Lavage". It would suggest that oxydi zed 0I1PI is rapidly regenerated by in vivo reducing systems (11) We do not know yet why, in our hands, 50% of immunoreactive

has

its functional activity and why other investigators find100%

lost

active

This work was supported by grants from INSERM (CRL 815021, CRL 815030 and CP 810121) and Fondation pour La Recherche

Médicale.

References

1. CARP H., JAN0FF A. : Amer. Rev. Resp. Pis.,118, 617-621

(1978)

2. LAURELL C.B., ERIKSSON S. : Scand. J. Clin. Lab. Invest.,J_5, (1963)

132-140

3. JAN0FF A., SLOAN B., WEINBAUM G., DAMINA0 V., SANDHAUS R.A., ELIAS J., KIMBEL P. : Amer. Rev. Resp. Dis., 115, 461-478 (1977) 4. SH0TT0N D.M. : Methods. Enzymol., _19, 113-140

(1970)

5. WINNINGER C., LESTIENNE P., DIMIC0LI J.L., BIETH J.G. : Biochim. Biophys. Acta, 526, 227-234 (1978) 6. POWERS J.C., GUPT0N B.F. : Methods. Enzymol., 46, 208-216,

(1977)

7. MEYER J.F., BIETH J.G., METAIS P. : Clin. Chim. Acta, 62, 43-53

(1975)

8. BEATTY K., BIETH J.G., TRAVIS J. : J. Biol. Chem., 2^5, 3931-3934 9. GADEK J.E., FELLS G.A., CRYSTAL R.G. : Science, 206, 1315-1316

(1980)

(1979)

10. CARP H., MILLER F., HQIDAL J.R., JAN0FF A. : Proc. Natl. Acad. Sci . (USA) 79, 2041-2045 (1982) 11. ABRAMS W.R.,. WEINBAUM G., WEISSBACH L., WEISSBACH H., BROT N. : Proc. Natl. Acad. Sci. (USA), 78, 7483-7486 (1982) 12. BIETH J.G., SPIESS B., WERMUTH C.G. : Biochem. Med., 11, 350-357

(1974)

COLLAGENOLYTIC

Jean Jean

ENZYMES AND

FIBROSIS

François CORDIER, Odette BRUNE, Roger TOURAINE

Centre

Hospitalier

OF

THE

BENZERARA,

Universitaire

LUNG

Yves

de Lyon,

LASNE,

France

Introduction Interstitial the

pulmonary

heterogeneous

Alveolitis,

group

cells

event

sequence

of

least

the

clear,

mic processes GADEK

et al

of

thic pulmonary this

found

but

of

they

lavage

fibrosis,

fluid

But of

respiratory

to

lung

fibrosis.

and

collagenase of

postulated

distress

of

was

patients syndrome

release and

not

in the

dyna-

collagens. present with

it c o u l d activity with

initial

cells

still

patients

collagenolytic

certain

are

the

and

; proteases

disturbances

degradation

fluid

is

for

(1).

immune

These

injury

fibrogenesis

and

diseases of

structures,

tissue

include

(BAL)

denominator

and activation

early

synthesis

condition.

in BAL

and acute

of

(2) d e m o n s t r a t e d

bronehoalveolar for

interstitial

leading

The m e c h a n i s m s

completely

is a c o m m o n

in the a l v e o l a r

two m e d i a t o r s

oxydants.

of

i.e. a c c u m u l a t i o n

inflammatory at

fibrosis

be

in

specific

(CA) w a s

sarcoidosis

(4).

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

the

idiopaalso (3)

232 Patients and

methods

We various usual

studied 40

origin

and

people

(table

lavage

was

and

(1).

; the cell

BME

collagen

supernatant

labelled by

gradient

gel

agarose.

CA

was

was

done

of

I).

carried

out

fluid was frozen

after

diseases using

the

immediately

for C A

suspension

analysis of

the

pellet

medium.

6 0 jil of

analyzed

The a s p i r a t e d

count

Analysis

with

pulmonary

normal

centrifuged in E a g l e

with

Bronchoalveolar procedure

later,

patients

of CA in B A L 1 25 with

I were

unconcentrated

SDS

electrophoresis, was detected

of

tracings,

and measured

collagen

by

: 700

gel and

fluid.

human

37°C The

for

isoelectric

visualization

of

TLC linear

type I 2

samples

electrophoresis,

on a u t o r a d i o g r a p h i e s by

ng of

i n c u b a t e d at

alveolar

polyacrylamide

fractions

fluid

hours were

SDS

focusing

in

degradation

of

analyzer

the LB

gel 2832

Berthold.

Results 1) C A

was

present

2) C A

was

complex.

since a p

to

collagenase ments

at

in

o( f r a c t i o n s

the

weight

fragments

was

with

not

by

shown

at l e a s t

conversion

times

of

was

other with

differential

t h a n 5 X of

is

in

neutral was

characteristic

collagen

correlated

in BAL

more

f l u i d ).

the

initial

Finally,

clears

It i n c l u d e d

(giving

tion). 3) C A

17 p a t i e n t s a s

kinetic degraded

table

I.

proteases,

present, 3/4 a n d

studies

and

1/4 of

in small

frag-

degrada-

molecular

proteases. the cell

presence count

of

(11

polymorphonuclears

polymorphonuof

had

17

patients

no CA

in

BAL

233

P a t i e n t s w i t h CA i n BAL

fluid

P a t i e n t s w i t h o u t CA i n BAL

fluid

R e s u l t s e x p r e s s e d i n jjg c o l l a g e n d e g r a d e d / h o u r / m g a l b u m i n i n BAL f l u i d Interstitial

fibrosis

:

I d i o p a t h i c pulmonary

(7/7)

Control healthy patients

fibrosis

76.1 11.3 486

A s s o c i a t e d with scleroderma

21.6

Associated with hypersensitivity pneumonitis

10.1 1.3

Apical

55.9

fibrosis

Radiation pneumonitis

Sarcoidosis

: (4/4)

4.2 5.0 3.2 2.0

: (4/15)

Eosinophilic

Sarcoidosis

(11/15)

C h r o n i c pneumonia

(3/3)

V a r i o u s pulmonary d i s e a s e s

(6/6)

neoplastic lymphangitis, a s b e s t o s i s , emphysema, mycobacterial infection, bronchiectasis, i a t r o g e n i c cyclophosphamide pneumonitis.

6.3 20.5 5.8 5.8

pneumonia

:

10.9 13.6

Table I - C o l l a g e n o l y t i c a c t i v i t y

D

(3/3)

i n BAL

fluid

iscussion

CA

was

present

in

diseases

of

dered

specific

tures, is

as

exceeding

probably

although the In

which

of

for the

one

of

CA

to

of

patients

and

aetiology.

of

of

interstitial

was its

always

radiation

CA

in

not

alveolar

matrix

fibrosis

fibrosis and

in

proteases

and

interstitial

should

antiproteases

present

origin,

sarcoidosis

of

connective

part

with

therefore

capacities

mechanism

whatever

leads

cases

fluid

origin

respective

study,

fibrosis

few

a

the

development our

BAL

various

in ;

in

is

oxydants

of

radiation it

was

struc-

protection,

still

also

eosinophilic

consi-

derangement,

and

cases

be

in

debated

(5).

pulmonary pneumonitis present

pneumonia,

in

a

those

234 diseases and

being

sometimes

Thus occur

capable

of d e r a n g i n g

including

fibrosis.

collagenolytic in v a r i o u s

specific

mentaux

pulmonary

mediator

(Supported

enzymes of

by a g r a n t

contre

la

interstitial

release

in a l v e o l a r

diseases,

inflammation of C o m i t é

tuberculose

et

in

and

they

structures structures,

are a

non-

alveolitis.

National

et C o m i t é s

les m a l a d i e s

Départe-

respiratoires).

References 1. C r y s t a l RC, Hunninghake

G a d e k JE, F e r r a n s V J , F u l m e r JD, L i n e G W : A m . J . M e d , 70, 5 4 2 - 5 6 8 ( 1 9 8 1 )

BR,

2. G a d e k JE, K e l m a n JA, F e l l s G, W e i n b e r g e r SE, H o r w i t z R e y n o l d s H Y , F u l m e r JD, C r y s t a l RG : N . E n g l . J . M e d , 301, 7 3 7 - 7 4 2 ( 1 9 7 ? )

AL,

3. C o r d i e r J F , L a s n e Y, G r i m a u d JA, T o u r a i n e R : In C h r e t i e n M a r s a c J, S a l t i e l JC, Eds, I X t h I n t e r n a t i o n a l C o n f e r e n c e on S a r c o i d o s i s a n d O t h e r G r a n u l o m a t o u s D i s e a s e s P a r i s , P e r g a m o n Press, 204-208 (1982) 4.

F e i n A M , C h r i s t n e r P, G o l d b e r g SK, L i p p m a n M, A b r a h m s W R , W e i n h a u n G : A m . R e v . R e s p i r . D i s . J_27 ( s u p p l ) , 95 ( 1 9 8 3 )

5.

Snider

GL

: Am.

Rev.

Respir.

D i s . J_27, 5 3 5 - 5 3 9

( 1 983 )

J

ELASTOLYTIC ACTIVITIES AND PROTEINASE INHIBITORS IN SPUTUM FROM PATIENTS WITH CYSTIC FIBROSIS

Jean-Marie Tournier, Jacky Jacquot, Edith Puchelle, Paul Sadoul INSERM Unité 14, Plateau de Brabois, F-54511 Vandoeuvre-1es-Nancy Jean-Pierre Chazalette Hôpital Renée Sabran, Bd Edouard Herriot, F-83406 Giens

Introduction Cystic fibrosis (CF) is characterized by progressive pulmonary impairment associated with purulent sputum, very often contaminated by aeruginosa

Pseudomonas

(Ps aer). As leukocytes and Ps aer are important sources of

proteinases (1, 2), an imbalance between proteinases and their inhibitors in sputum may be an important factor in the pulmonary pathogenesis of this disease. In this study, we attempted to analyse in CF sputum 1) the main elastolytic activities : human leukocyte elastase (HLE) and Ps aer elastase and 2) the main proteinase inhibitors : c^-antitrypsin (aiAT^ derived from serum, and the locally produced bronchial inhibitor (brl).

Materials and methods Patients and bronchial secretions : Bronchial secretions were collected from 8.30 to 9.00 am in 15 CF patients aged 8 to 18, using a method avoiding salivary contamination (3). All subjects were in-patients hospitalized in hôpital Renée Sabran, Giens. The leukocytic and bacteria content of sputum was performed as previously described (4). The remaining sputum was homogenized (5) and a protein extract was obtained using the method of Harbitz et al. (6). Elastases and proteinase inhibitors assays : The total elastolytic

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

236

a c t i v i t y was measured using an elastin agarose diffusion plate (7) containing human lung elastin. Succinyl-trialanine-pNA and elastin-remazol were used as substrates of HLE (8) and Ps aer elastase respectively. For the l a t t e r , the complete inhibition of HLE and cathepsin G was previously obtained by an excess of aiAT. aiAT concentration was measured by rocketimmunoelectrophoresis (9). The electrophoretic mobility of aiAT was compared to a standard serum aiAT using crossed Immunoelectrophoresis. Immunoreactive brl was assessed using an ELISA test (10). The active fraction of brl was determined after dissociation of brl-proteinase complexes by acidification and was expressed as the ratio of brl which inhibited HLE to the immunoreactive brl.

Results and discussion Principal origins of proteinases in sputum are from leukocytes and bacteria such as Ps aer. In our study, leukocyte and Ps aer numerations ranged respectively from 0.4 x 10® to 31.2 x 10^/ml and from 0 to 6.2 x 109/ml. In 8/15 cases, leukocyte numeration was higher than 5 x 106/ml. Ps aer was present in 11/15 sputa and sputum culture yielded Ps aer as the sole isolate from 9 patients. All sputum samples showed a free e l a s t o l y t i c a c t i v i t y on human lung e l a s t i n which was closely correlated to the HLE a c t i v i t y (r = .85, p < .001). That means that the elastase was i n s u f f i c i e n t l y inhibited and that leukocytes played an important part in the local secretion of elastase in CF sputum.Ps aer elastase was detected in 9/11 sputa contaminated by Ps aer. The higher the Ps aer numeration, the higher the a c t i v i t y of Ps aer elastase (r = .85, p < .001). In spite of the generally high numeration of Ps aer, the concentration of active Ps aer elastase was always lower than the concentration of active HLE (0.14 + 0.15 yM and 11.9 + 9.9 yM, respectively). aiAT concentration ranged from 0.037 to 14.8 pM and was closely correlated with the leukocyte numeration (r = .83, p < .001) confirming that a ^ T w a s a

good marker of bronchial inflammation. In all sputum samples, aiAT

was observed as a multiform arc of precipitation and i t s electrophoretic mobility was always less than that of standard serum aiAT, indicating that

237

all aiAT was present either in complex form with different proteinases or in an inactive form, brl i s the main proteinase inhibitor in bronchial lavage f l u i d s obtained from non-infected individuals (12). I t s concentration in CF sputum ranged from 0.76 to 3.47 yM and was not s i g n i f i c a n t l y correlated to leukocyte or Ps aer numeration. The active fraction of brl was very high (124 + 17 %) and did not depend on Ps aer elastase concentration. These results mean that, although in v i t r o experiments demonstrated that brl i s inactivated by Ps aer elastase (13), this inactivation does not necessarily occur in vivo. In summary our results show that, in CF sputum : 1) there i s a marked free e l a s t o l y t i c a c t i v i t y , mainly due to leukocyte elastase. 2) aiAT i s present in complex

or inactive forms. 3) The presence of Ps aer

elastase does not necessarily imply, in vivo, a loss of brl a c t i v i t y . (Supported by grant from AFLM 2.81/3).

References 1.

Morihara, K., Tsuzuki, H.: Infect. Immun. 15, 679-685 (1977).

2.

Ohlsson, K.: B u l l . Europ. Physiopath. resp. 16 (suppl.), 209-222 (1980).

3.

Beck, G., Puchelle, E., Laroche, D., Mougel, D., Sadoul, P.: Bull. Europ. Physiopath. resp. 18, 885-892 (1982).

4.

Puchelle, E., Beck, G., Thevenin, F.: Respiration, 34, 220-231 (1974).

5.

Girard, F., Tournier, J.M., Polu, J.M., Puchelle, E., Beck, G., Sadoul, P.: Clin. Chim. Acta 113, 105-109 (1981).

6.

Harbitz, 0 . , Jenssen, A.O., Smidsrod, 0.: Europ. J. resp. Dis. 6^, 84-94 (1980).

7.

McGi11ivray, D., Burnett, D., Afford, S.C., Stockley, R.A.: Clin. Chim. Acta, 111, 289-294 (1981).

8.

Bieth, J.G., Spiess, B., Wermuth, C.G.: Bioch. Med.lU 350-357 (1974).

9.

Laurel 1, C.B.: Analyt. Biochem. 10, 21-37 (1965).

10.

Tournier, J.M., Jacquot, J . , Sadoul, P., Bieth, J.G.: Anal.Bioch. 131, 345-350 (1983).

11.

Tegner, H.: Acta Otolaryngol. 85, 282-289 (1978).

12.

Johnson, D.A., Carter-Hamm, B., Dralle, W.M.: Am. Rev. respir. Dis. 126, 1070-1073 (1982).

SERUM DERIVED PROTEINS AND ANTIBACTERIAL PROTEINS IN CYSTIC FIBROSIS SPUTUM

Jacky Jacquot, Jean-Marie Tournier, Edith Puchelle, Paul Sadoul Unité de Physiopathologie Respiratoire INSERM U. 14 54511 Vandoeuvre-les-Nancy, France Jean-Pierre Chazalette Hôpital

Renée Sabran, Bd Edouard Herriot

83406 Giens, France

Introduction In cystic f i b r o s i s (CF), the exact factors responsible for the frequent bronchial superinfections are unknown. They could be related to either the rheological abnormalities of sputum due to an abnormal protein content, or to the local

d e f i c i t of antibacterial a c t i v i t y proteins in the

bronchial lumen. The purpose of this study was to investigate the protein composition of sputum in CF patients. The total protein content, the concentration of main proteins with antibacterial

activity

(immunoglobulins A, G, M, bronchial lysozyme and lactoferrin) and serum derived proteins (serumalbumi n and aj antitrypsin) were measured in CF sputum. Results were analyzed taking into account 1) the degree of bronchial superinfection estimated by a quantitative cytobacterial study and 2) the severity of the disease assessed by the Shwachman score.

Materials and Methods 1) Patients : In 17 CF patients aged 8 to 18, sputa were collected from 8.30 to 9.00 a.m. by a protected method avoiding the salivary contamination (1). The c l i n i c a l status of each patient was estimated by the Shwachman score (2).

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

240 2) Technics Cytobacteriological

analysis

: Immediately after their collection, sputum

samples were homogenized with a sterile solution of 2.3 di hydro 1.4 dithiobutane. Bacteria and leukocytes were identified and numerated (3). Protein

analysis

: The protein extract was obtained by centrifugation

(17000 x g, 30 min) of sputum sample previously homogenized by ultra sonic treatment (4) and solubilized by shaking at +4° C for 12h in 0.5 M Nacl in a phosphate buffer (0.07 M pH = 6.0) (5). The total protein content in each extract sputum was determined according to Lowry et al. (6). The concentrations of IgA, lysozyme (LZM), lactoferrin (LF), serumalbumin (SA) and aj antitrypsin (aiAT) were measured by rocket immunoelectrophoresis (7) and IgG, IgM by radial immunodiffusion (8).

Results 1)

Cytobacteriology : All 17 CF sputum samples were found infected. A high bacterial numeration (3.1 + 3.4 x 10^/ml) was generally associated with a high leukocyte numeration (9.2 + 9.0 x 10 3 /yl). aeruginosa,

Pseudomonas

identified in 14 cases out of 17 (82 %) was in mucoid form

in 10 cases (71 %). 11 CF sputa out of 17 were severely superinfected as judged by a leukocyte numeration > 5000/ul associated with a bacterial numeration > 10 8 /ml. 2)

Proteins : In the group of the 11 CF superinfected sputa, the average concentration of total proteins (30.0 + 13.6 g/1) was significantly greater (p < 0.01) than in the group of the CF infected sputa (15.9 + 5.3 g/1). No significant difference was found between the infected and superinfected CF sputa for the main proteins with antibacterial activity except for IgG as shown in fig. 1,(a).

241

Fig. 1 . — Antibacterial activity proteins @ and serum derived proteins in CF infected sputa (I) and CF superinfected sputa (SI) **p < 0.01, NS : not significant.

(b)

On the other hand, the average concentration of serum derived proteins used as markers of bronchial

inflammation (SA and c^AT) in the SI group

was significantly higher (p < 0.01) than that of the group I (fig. 1, High molecular weight proteins such as IgM (PM = 900 000) were found in all CF sputa.

9 /l

Fig.2.- Relationship between the Shwachman score and the concentration of aiAT in CF sputum.

242

The Shwachman scores ranged from 40-76 (mean + SD, 62 + 18). A s i g n i f i c a n t negative c o r r e l a t i o n (r = -0.56 p < 0.01) was observed ( f i g . 2) between the Shwachman score and the concentration of axAT i n CF sputum.

Discussion Our r e s u l t s show that in CF, the bronchial i n f e c t i o n i s not associated i n sputum with marked changes in the concentration of proteins with a n t i bacterial a c t i v i t y . O n the other hand, the bronchial i n f e c t i o n i s accompanied by a severe local inflammation reflected by very high concent r a t i o n of serum derived proteins present i n sputa. The p o s i t i v e c o r r e l a t i o n between the sputum protein content and the s e v e r i t y of the disease suggests that t h i s marked bronchial inflammation may be of c r i t i c a l importance in the pathogenesis of c y s t i c

fibrosis.

References 1. Beck, G., Puchelle, E . , Laroche, D., Mougel, D., Sadoul, P.: B u l l . Europ. Physiopath. resp. 18, 885-892 (1982). 2. Shwachman, H., Kulczychi, L . L . : Am. J. D i s . C h i l d . 96, 6-15 (1958). 3. Beck, G., Puchelle, E . , Polu, J.M.: Rev. frang. Mai. Resp. 8, 357-366 (1980). 4. G i r a r d , F . , Tournier, J.M., Polu, J . M . , Puchelle, E . , Beck, G., Sadoul, P.: C l i n . Chim. Acta 1_13, 105-109 (1981). 5. Harbitz, 0 . , Jenssen, A.O., SmidsriSd, 0 . : Europ. J. Resp. D i s . 61, 84-94 (1980). 6. Lowry, O.H., Rosebrough, N . J . , F a r r , A . L . , Randall, R.J.: J. B i o l . Chem. 193, 265-275 (1951). 7. Laurel 1, G.B.: Analyt. Biochem. 10, 21-37 (1965). 8. Mancini, G., Carbonara, A.O., Heremans, J . F . : Immunochem. 2, 235-254 (1965). 9. Brogan, T . D . , Ryley, H.C., Lynne, N., Janet, Y.: Thorax 30, 72-79 (1975).

ALVEOLAR FIBRONECTIN AND INTERSTITIAL LUNG DISEASES

N. Biot, D. Gindre, R. Harf, F. Maréchal, B. Vercoustre, M. Perrin-Fayolle Service de Pneumologie, C.H.U. Lyon-Sud, F-69230 Saint-Genis-Laval

Introduction Fibronectin (FN), a high molecular weight glyco-protein, plays a major role in cellular homeostasis. FN i s present as well in the extracellular compartment in the plasma (where i t i s also called the cold insoluble protein CIG) as in the c e l l u l a r compartment where i t can be found at the surface or included in the basal membrane of most i n t e r s t i t i a l c e l l s . The question to know i f CIG derives from c e l l u l a r FN is controversory. FN i s synthetized by various types of c e l l s but principally by f i b r o b l a s t s , vascular endothelial c e l l s and macrophages (1). Numerous biological functions have been described : 1) In the i n t e r s t i t i a l t i s s u e , FN represents mainly the f i b r i l l a r matrix between the c e l l s and the connective tissue (specific binding structures to collagen, fibrinogen and c e l l s - especially to macrophages, have been described) (2). Animal experiments have shown that paraquat was able to increase the amount of FN within the alveolar structures and i t s production by the alveolar macrophage. Cancerous cell membranes exhibit a loss in FN, which could explain reduced adhesivity and thus metastatic a b i l i t y FN at least seems necessary in fibroblast replication. 2) Circulating FN acts as a non specific opsonic signal for macrophages by increasing their phagocytic capacity (3). From these data showing that FN was essentially implicated in non specific host defence mechanisms and f i b r o t i c processes, we investigated the possible

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

244

v a r i a t i o n s of FN concentrations within the a l v e o l a r structures during various i n t e r s t i t i a l

lung disturbances ( a l v e o l i t i s and f i b r o s i s ) .

Material and Methods Investigated populations were the following : - 20 young (mean age = 24 years) healthy volonteers, 40 % (8) of which were smokers. - 33 patients with h i s t o l o g i c a l l y proven s a r c o i d o s i s , mean aged of 40 years including 7 smokers. - 14 e x t r i n s i c a l l e r g i c a l v e o l i t i s patients (mean age = 46 y e a r s , 5 smokers). - 15 pneumoconiosis (mean age = 50 y e a r s , 8 smokers). - 8 women undergoing thoracic radiotherapy following breast cancer exerese (mean age = 46 y e a r s , 2 smokers). FN was determined in both serum and broncho-alveolar lavage (BAL) supernatant. BAL was performed as u s u a l l y by f i b r o p t i c method. All

sarcoidosis

patients - except 2 ) underwent a double lavage procedure i . e . a total of 64 supernatants. These 64 samples were divided in two groups : - 24 corresponding to absent a l v e o l i t i s were characterized by a low proportion of lymphocytes ( hydroxyapatite metamorphosis in vitro (11), Because of these features, it is tempting to speculate that BGP plays a role in mineralization of bone. Such a role, however, needs to be demonstrated. Experimental models in vivo have used vitamin K antagonists that induce the production of decarboxylated BGP. Thus, dicumarol treatment decreases the level of BGP in chicken bone, but without alteration of the calcium and phosphorus content of the bone ash (30), Warfarin, an other vitamin K antagonist, can reduce the BGP content of rat bone to 2% of control values, probably because the decarboxylated BGP

285 cannot bind to the mineral phase of bone (31). When rats were maintained from birth to two months of age on this protocol, no impairment of growth, bone structure and mineralization was found (31). More recently however, excessive mineralization characterized by complete fusion of the tibial growth plate and cessation of longitudinal growth have been reported in rats maintained on warfarin from birth to eight months of age (32). A new exciting aspect of the putative function of BGP has been provided by the demonstration of the role of vitamin D in BGP secretion. Where osteosarcoma cells are cultured with physiologic doses of l,25-(OH)2D2 - the most active metabolite of vitamin D - both intracellular and secreted levels of BGP are elevated 6 - to 10 - fold (33) ; this response is due to specific regulation of BGP synthesis since there is no general increase in protein synthesis. Also, the level of serum BGP increases 3 - to 4 - fold following intravenous administration of 1,25(OH)„D- to either young or old rats (34). Rats weaned onto a Ca 2+ - deficient diet exhibit marked increases in serum BGP and in serum 1,25 (OH)^D, while serum BGP is not elevated if the 2+ diet is deficient in vitamin D as well as Ca (35). BGP concentrations in bones from vitamin D - deficient rachitic chickens is decreased (50%) (36), These results emphasize the relationship between vitamin D and BGP in vivo. Finally, BGP has a chemotactic activity. BGP evokes a dose-dependent chemotactic response in human monocytes (37). Osteoclasts derive most likely from circulating monocytes, and there is growing evidence that the recruitment of monocytes for osteo clast development occurs by chemotaxis ; because BGP is directly produced by the osteoblasts, this chemotactic activity of BGP shed a new light on the coupling between bone resorption and bone formation. More recently it has been shown that BGP is also chemotactic for breast cancer cells as well as osteoblast like osteogenic sarcoma cells and causes their unidirectional migration (38).

286

IV. BGP circulates in serum, a) Radioimmunoassay for BGP, Sensitive and specific radioimmunoassays have been developed for bovine (7, 39) and rat (25, 39) BGP. In these animals, serum BGP levels range from 25 ng/ml in the adult cow to 200 ng/ml in the fetal calf and in rats. Because antibody directed against calf BGP crossreacts with purified human BGP (7), measurement of serum BGP in human uses a radioimmunoassay system developed with bovine BGP (7, 40, 41). Price has shown that the molecular weight of plasma BGP is identical to that of BGP extracted from bone, and that both plasma and bone BGP bind to hydroxyapatite with the same affinity, indicating that plasma BGP is fully carboxylated (7, 39, 42). A bovine antibody has been shown to cross react with human, horse, monkey, baboon and cat BGP, but not with dog and mouse BGP (39). We have developed a radioimmunoassay system for measuring serum BGP (40). BGP was extracted from bovine bone in 0.5 M EDTA and purified by gel filtration over Sephadex G-100 and subsequent gradient elution from DEAE-Sephadex A-25. This purified bovine BGP was used for standard and tracer. Tracer was prepared by chloramine-T radioiodination and antiserum to bovine BGP was raised in rabbits. Rabbit antiserum was precipitated by adding goat antiserum to rabbit -globulin. Previous studies have shown that purified bovine BGP and human BGP displace BGP tracer from antibody with equal effectiveness (7). We verified that the presence of BGP in human plasma causes the displacement of tracer from our antibody by demonstrating that standard curves obtained with bovine BGP and with dilutions of human serum were strictly parallel (Fig.l). We also verified that gel filtration of human serum showed a single immunoreactive peak that coeluted with pure bovine BGP (40). With our system, the intra- and inter-assay variations are less than 10% and 7% respectively, the sensitivity of the assay is 0.1 to 0.2 ng of BGP and the concentration of BGP can be determined in > 99% of normal subjects (40).

287

Plasma (ml/tube) 1

5

10

25

5 0 100

200

300

10 08

B/B 0 06 04

0 2

0 Bovine BGP (ng/tube) o-o

Fig, 1 - Standard curve for BGP radioimmunoassay. Dilutions of human plasma and bovine BGP standard show similar reactivity , b) Serum BGP levels in normal subjects. In adults, circulating BGP is lower in females than in males, with mean concentrations ranging from 5 to 10 ng/ml (7,39,30). Values are higher in children (10-30 ng/ml), reach a peak at puberty (up to 40 ng/ml), and then decline to the adult levels (39) . We measured serum BGP in 174 women, 30-94 yr of age, who were randomly selected from the population. We found that serum BGP increased linearly with aging (p 0.75 or it can be normal. The albumin ratio is < 0.65%. In addition to bacterial meningitis, this pattern is found most often in subacute sclerosing panencephalitis, encephalitis, foetal diseases, and multiple sclerosis. An interesting profile which combines the features of the transudative and IgG profiles is observed frequently in Herpes encephalitis. Fig. it:

Degenerative profile [ 31 •

A: serum; B: CSF

This profile, which is often found in children, has not yet been assigned to a specific syndrome [5]. Three major causes can lead to an erroneous interpretation: - The presence in the gamma globulin region of beta traces or of post gamma bands due to a degenerative profile. - The contamination of CSF by blood which will lead to the presence of bands corresponding to hemoglobin A2 and carbonic anhydrase. - A monoclonal or oligoclonal pattern due to serum IgG. Doubtful patterns can be solved by performing an immunofixation with an anti IgG antiserum.

373 In conclusion: In the IgG profiles, the high resolution electrophoresis is more sensitive than the IgG index. In the transudative profiles, the albumin ratio is more significant than the electrophoretic pattern. The simultaneous analysis of the serum is absolutely necessary. In doubtful cases, an immunofixation needs to be performed.

CEREBROSPINAL FLUID FERRITIN : INTEREST AS BIOLOGICAL MARKER OF CENTRAL NERVOUS SYSTEM TUMORS.

Pierre ANTOINE, Roger LATER, Jean-Christophe EYNARD, Françoise DURAND, Bruno MARECHAL, Claude QUINCY Laboratoire de biochimie, Hôpital Neurologique 6 9 003 - Lyon - France

Introduction Many compounds have been proposed as biochemical markers of central nervous system (CNS) tumors and measured in cerebrospinal fluid (CSF) (1, 2, 3). They have been widely investigated and their utility in the diagnosis of tumors and in the monitoring of the treatment is well defined. More recently, ferritin has been investigated in CSF of patients with cerebral infarction or haemorrhage (4), in various neurological disorders with only a few cases of tumors (5) and in lymphoproliferative disorders with CSF infiltration (6). In this study, we have determined blood and CSF ferritin concentration, of control subjects, patients having various neurological disorders and patients having CNS tumors. Then, we have compared ferritin with four other biological markers.

Material and methods Cerebrospinal fluid is obtained by lumbar puncture, blood is collected on the same day. After centrifugation, aliquots are kept frozen at -20°C until analysed. Patients are assigned to one of these three groups : group I is constituted from 22 patients having various minor

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

376 neurological disorders and normal CSF regarding total protein content (< 0,40 g/1) determined by a turbidimetric method with benzethonium chlorid (7), number of cells per liter (< 2.106/1), glucose (3 to 4 mmol/1) and qualitative cytology. They are regarded as control subjects. Group IX is constitued from 98 patients having various neurological disorders and abnormal cerebrospinal fluid. Five classes are distinguished : multiple sclerosis (21), nerve diseases (26), subarachnoidal haemorrhage and haematoma (14), degenerative disorders (8) and other neurological diseases (29). Group III included 44 patients having central nervous system tumors. Ferritin is determined before any surgery or treatment. Four classes are distinguished : primary leptomeningeal tumors (12), primary parenchymal tumors (14), other primary tumors (3) and metastases of carcinoma (15). The assay of ferritin is performed with a sandwich-type enzyme immunoassay method. We have used NEIA Ferritin Kit from New England Immunology Associates^ and each determination has been done in duplicate, including standards and controls. Individual values in each group are shown in figure 1. A 300 »UO

H0"

m;2s

OOOO^AA o O O à&AAA • f l o o o o o AAAAA 0 0 •••••(OOOOOAAAAA

o o o ODO

Figure

.V W

1

Control subjects

2

Neurological disorders :

. multiple sclerosis ( ° ) . nerve diseases (» ) . subarachnoidal haemorrages and hematoma ( » ) . degenerative disorders ( °) . others ( ' ) 3 CNS tumors : . parenchymal Primary tumors ( » ) . Leptomeningeal Primary tumors ( ' ) . Other primary tumors ( • ) . metastases of carcinoma ( • )

377

Results For ferritin, we have determined the mean (4,5 jag/1) and the standard deviation (3,2 /ig/1) for control subjects. Assuming the upper normal limit is mean + 2 SD (10,9 yug/1) ; specificity (fraction of group II patients giving negative results), sensibility (fraction of group III patients giving positive results) and efficiency (fraction of all results that are correct) are calculated. Values are given in table I. Then, we have made a comparison

between ferritin and four

other biological markers determined in our laboratory : Beta glucuronidase (Fluorimetry (8), carcinoembryonic antigen p (CEA - EIA from Abbott ) and polyamines : putrescine and spermidine (HPLC (9)). But for these four markers, the populations are subgroups of those analysed for ferritin (group II : 65 amongst 98 ; group III : 32 amongst 44). Results are given in table I. Specificity

Sensibility

Efficiency

Ferritin

79,6

45,5

69

Putrescine

68,9

60,7

65,8

Spermidine

84,4

55,6

72,6

Beta glucuronidase

85,9

37,8

68,3

C.E.A.

90,7

21,6

65,4

In no case blood levels could explain CSF levels. TABLE I

Discussion Ferritin in CSF seems to be a new biological marker of CNS tumors. Specificity is good except in the case of haemorrhage but we found no correlation between cells count, CSF total proteins content and CSF ferritin concentration ; Halgreen describes the same phenomena. In order to precise our study, specificity should be determined for subjects with bacterial

378 or viral infection of CNS. Sensitivity is weak but better for primary tumors considered as a whole. Performances of ferritin and all of the other markers do not make it possible to recommend any one of them in particular as an absolute tumor marker ; but it seems presently we must use all markers together for the greatest aid in the diagnosis of CNS tumors.

Acknowledgement The authors wish to thank J. SALANDRE and S. DUNAND for their excellent technical assistance.

References 1.

Seidenfeld, J., Marton, L. J. : J.N.C.I. 63, 919-931 (1979)

2.

Schold, S. C., Bullard, D. E. : J. H. Woods : Neurobiology of cerebrospinal fluid. Plenum Press, 539-559 (1980)

3.

Wasserstrom, W. R., Schwartz, M. K., Fleisher, M., Posner, J. B. : Am. Clin. Lab. Sei. 11, 239-251 (1981) Hällgren, R., Terent, A., Wide, L., Bergström, K., Birgegard, G. : Acta Neurol. Scandinav. 61, 384-392 (1980)

4. 5.

Syndic, C. J. M., Collet-Cassard, D., Cambiaso, C. L., Masson, P. L., Laterre, E. C. : J. Neurol. Neurosurg. psychiatry 44, 329-333 (1981)

6.

Vincente, V., Gonzalez, M., Lopez Borrasca, A. : Acta Paediatr. Scand. 71, 325-326 (1982)

7.

Iwata, J., Nishikaze, 0. : Clin. Chem. 25, 1317-1319 (1979)

8.

Gehler, J., Cantz, M., Tolksdorf, L., Spranger, J. : Human Genetik 23, 149-158 (1974)

9.

Seiler, N., Knodgen, B. : J. Chromatogr. 221, 227-235 (1980)

CLINICAL RELEVANCE OF BETA 2 MICROGLOBULIN LEVELS OF THE CEREBROSPINAL FLUID IN CHILDREN WITH ACUTE ENCEPHALITIS Elie Jean Raynaud, François Deméocq, Philippe Vanlieferinghen, Georges Malpuech, Ginette Gaillard. Clinique médicale infantile - Hôtel-Dieu - C.H.U. and Centre Jean Perrin 63000 - Clermont-Ferrand, France

Introduction In acute brain disorders of childhood, clinical, biological and electroencephalographic data do not always allow one to distinguish between non inflammatory encephalopathy which requires a symptomatic treatment and encephalitis which some times requires an antiviral therapy. In a prospective study in children who had miscellaneous neurological disorders, we have found that the cerebroscpinal fluid (CSF) Beta 2 microglobulin (/32m) was significantly increased in acute encephalitis (1). Moreover we have observed an increase in the /32m levels in CSF after prophylactic cranial irradiation in children with hemopathies. This increase of the /32m is correlated to the outbreak of the early post irradiation encephalopathy (3). These findings have led us to study the CSF /32m in children with either encephalitis or non inflammatory encephalopathy in order to differentiate between these two kinds of cerebral disorder. The other biological markers of the cerebral disorder (CSF enzymology and cytomorphology) were also recorded (4, 5). Material and methods The study involved 22 children. 11 of whom (7 males, 4 females average ages 6.5 years) had biologically proved acute inflammatory disorders of the CNS : measles (3), herpetic (2), indetermined viral (1), eosinophilic (2), legionella pneumoniae (1), chiken-pox (2) encephalitis. The other 11 patients (6 males, 5 females average age 3.5 years) had acute ischemic encephalopathy : status epilepticus (4), long lasting severe seizures with transient hemiplegia (7). There were 10 controls, matched for age, admitted for diseases without any neurolo- gical disturbance. The CSF and serum samples were collected once during the periods, between D1-D3, again between D5-D8, and lastly between D12-D15. The /32m was measured using a radio-immunologic assay (phadebas /32 microtest). Lacticodeshydrogenase (LDH) and aspartate amino transferase (ASAT) activities determined by ultra violet spectrophotometry. A cytomorphologic study was performed in the CSF after

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in Germany

380

1000 r.p.m. cytocentrifuging (Shandon centrifuge) by May Grumwald Giemsa staining. Results In the group of patients with encephalitis, we have observed (fig) a clear rise of the CSF 02m at the onset of the disease (3.20 nq/ml ± 0.96 ; p < 0.001 ; controls mean : 0.81 ± 0.35). The levels of the CSF /32m slowly returned to the normal range (D5-D8 : 2.40 ± 0.77 ; D12-D15 : 1.29 ± 0.46). These variations of the CSF /32m levels were not related to the blood levels which remained normal. The LDH and ASAT activities in the CSF were slightly disturbed. For the first three days, the levels increased marginally : LDH : 51.6 ± 15.6 UW (normal mean : 26 ± 5) ; ASAT : 18.1 ± 5 mUI/ml (normal mean : 12 ± 5). They decreased thereafter to their normal levels. These variations were not related to the blood levels which were nomal. The CSF cytology showed a statistically significant cellular reaction which progressively dicreased from D1-D3 to D12-D15, with the lymphocytosis remaining greater than 50 % of the sample during the whole follow up period.

Table : Qualitative cytology < the CSF Encephalitis Encephalopathies D1 to 3 D5 to 8 D12 to 15 D1 to 3 D5 to 8 D12 to 15 Leukocytes count (mm3) Lymphoid cells % Histiomonocytoid cells %

72

34

16

9

14

3

56 27

54 16

50 42

30 52

30 60

20 75

In the group of patients with encephalopathy, we have noticed (fig) no significant changes of the CSF and blood /32m levels. On the other hand, a clear increase of the enzymatic activities was statiscally significant, especially at D5-D8 (LDH : 68.% ± 16.5 ; ASAT : 26.8 ± 5.4 ; p < 0.05). This increase of enzymatic activities was not related to the blood levels. The cellular reaction in the CSF was mild and mainly composed of histiomonocytic cells whose relative percentage was progressively raised from 52 % at D1-D3 to 75 %at D12D15. In both groups, there was a statistically significant linear correlation between the number of leucocytes per mm3 and the level of the CSF (32m (R = 0.64 ; p < 0.0001). Comments These results indicate that the CSF /32m is significantly increased in children with ence-

381

LEUKO/ ß 2M MM 3

L

LDH

4

ENCEPHALITIS

U.W.

60 50

75 40 50

25

30

l

20

10

60 50 75 40 50

30 20

25 10

CONTROL '

DAY I to 3

' DAY 5 to 8

' DAY 12 to 15

Variations of p 2m ( T), LDH (•) and leukocytes count (•) in CSF during encephalitis and encephalopathies.

382 phalitis. The increase of the CSF (32m could be used to diagnose true encephalitic states in the child. In children with encephalopathy no noticable change of the CSF /32m levels was observed during the follow up period. The increase of the CSF /32m probably represents a type of cerebral disturbance different from the one involved in the increase of the enzymatic activities. The increase of the/32m should beattribued to a localized synthesis which is supported by to arguments : first, the CSF /32m rise is not related to the blood levels ; on the other hand, there is a correlation between the levels of the /32m and the cell reaction. Thus, the localized synthesis could have a lymphocytic source. The increase of the CSF enzymatic activities in encephalopathy reinforces the already known concept that is represents a cellular release of enzymes due to a plasma membran injury or due to a cell necrosis, whatever the site of the cell disorder. The /32m molecule seems to be a marker protein, in inflammation of the CNS, useful for the diagnosis of true encephalitis in the large group of acute cerebral disorders in children. References 1 - Deméocq, F., Debost, M., Malpuech, G. and Al. (Abstracts) Archives F. Pédiatrie, 901, 38,1981 2 - Deméocq, F., Malpuech, G., Raynaud, E.J. and Al. N. Engl. Jour, of Medicine, 304, 1366,1981 3 - Deméocq, F., Malpuech, G., Raynaud, E.J. and Al. In pediatric oncology X I I I ^ meeting of the international society of pediatric oncology, Marseille, C. Raybaud. Editors Excepta Medica, 3 5 5 - 3 5 6 , 1 9 8 2 4 - Viallard, J.L., Gaulme, J., Dalens, B. and Al. Clin. Chim. Acta, 89, 4 0 5 4 0 9 , 1978 5- Dalens, B., Bezou, M.J., Raynaud, E.J. and Al. Acta pediatr. Scand., 70, 161-166, 1981

ELECTROPHORETICAL PATTERNS CEREBROSPINAL FLUID.

J.C.

FROT,

F . MÜLLER,

HopiLal Ambroise 9 Avenue Charles

P.

OF A C E T Y L C H O L I N E S T E R A S E

IN

GIRAUDET.

P a r é , L a b o r a t o i r e C e n t r a l de B i o c h i m i e , de G a u l l e , 9 2 1 0 0 B o u l o g n e , F r a n c e .

Introduction. In previous work, we studied the different forms of cholinesterases in human brain during the development of embryo and in adults (acetylcholinesterase, AChE, E.C. 3.1.1.7. and butyrylcholinesterase, BChE, E.C. 3.1.1.8.) The

distribution of molecular forms of enzymes in brain areas was signifi-

c a n t l y different between adults (1,2). One of the factors which could be involved is a specific neurological pathology, and, in this work, we try to demonstrate that a relationship exists between electrophoretical pattern of cholinesterases in cerebrospinal fluid (CSF) and some specific neurolo-gical disorders.

Material and 1)

Methods.

One hundred CSF samples, obtained

by lumbar puncture were collected

from ninety nine patients, and stored at -20°C. Patients' ages start from one day old to 91 years old with 4 newly borns, 31 children and 64 adults. Diagnosis was unquestionably assessed in 70 patients. 2)

Qualitative assay of CSF cholinesterases. Polyacrylamide gel electro-phoresis was carried out using B.5% gels in a LKB Multiphor apparatus with a Tris-glycine pH 8.9 buffer. Gels were preelectrophoresed at 50 mA for 30 minutes. Five ^il CSF were layered in each slit and runs were carried out at 15 V/cm for three hours. Gels were pre-incubated at room temperature in a phosphate buffer pH 7j after 15 minutes they

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in Germany

384 were stained according to the method of Karnovsky and Roots (3),modi-fied as follows: gels were immersed in a phosphate buffer pH 7 contain i n g 0.005 mol/L potassium ferrycyanure, 0.003 mol/1 copper sulphate, and 0.007 mol/1 sodium citrate. After 30 minutes, 0.003 mol/1 of either acetylthiocholine iodine or butyrylthiocholine iodine were added and gels were left overnight. To inhibit BChE activity, tetra isopropyl-pyrophosphoramide (iso 0MPA) was included in the incubation mixture at a concentration of 0.001 mol/1. To inhibit AChE activity, 1.5 bis (4 allyldimethylammonium) pentan 3 one bromide (BW 284C51) was includ e d in the incubation mixture at 0.01 mol/1. The gels u/ere dried and passed through a densitometer to determine the number and density of each band.

3)

Quantitative assay of CSF cholinesterases. Activity of cholinesterases u/as estimated by the photometric method of Ellman et al. (4) applied to the V.P. Abbott apparatus. Inhibitors u/ere used at the same concent r a t i o n . In each sample, proteins were dosed by the method of Lowry and cells counted as to remove hemorragic CSF.

Results. (Figure 1) In general we found two bands: one slow migrating band, not very dense and a second faster moving band which was more dense. But above all we noted a great variability from one band to four bands with large differences in density. The density of bands correlated well with the activity, but corre-lation is weak with total CSF proteins. This variability has no relationship with either age and sex of the patient or any disorder, neurological and non neurological. In one case we found no band at all, but a sample of this patient's serum showed an absence of two bands in comparison to normal serum. Activity in the presence of inhi-bitors confirmed the specificity of CSF

with AChE since bands are totally

inhibited by BW 284C51 and activity remained identical with or without iso 0MPA added to the solution.

385

Figure 1. This picture clearly poi nts out the great variability of CSF cholinesterases ; from one band to three bands at the far right.

Discussion. Despite a relatively large population of common neurological disorders, we are not able to establish a relationship between electrophoretical patterns of cholinesterases and a specific neurological disorder. Numerous facts

for this result have to be noted: the collection of samples by

lumbar puncture could be a source of error if not performed by the same person at the same time on a patient at rest for hours; another difficulty is to define a "normal" CSf. If this is eventually possible for children since a lumbar puncture is easily performed even in the absence of neurological signs, it is quite impossible in a population of adults. Besides these problems, we must also point out that the population studied came from a general hospital and that none of the patient had an illness of the cholinergic pathways, such as Huntington disease. Finally it is known that the brain contains several iso-enzymes of acetylcholinesterase (1,5) and are the methods used in this work precise enough to detect these different forms7 It would be therefore more appropriate to find out if the great variability in our results were not due to the presence of such isoenzymes and we are now continuing our work in that direction. To conclude, and despite of other work in which

electrophoresis of CSF

cholinesterases exhibits a large, unique and diffuse AChE band (6), we found in general, two AChE bands completly inhibited by BW 284C51. Whatever the neurological disorders are, we cannot find any relationship between the illness and AChE bands. We think that CSF obtained by lumbar puncture represents a mixture of AChE secreted by the nervous system

386 from different areas and the only way to demonstrate a specific pattern, if any, is to study the different iso-enzymes.

References. 1. MULLER, F. : Cholinesterases et développement chez 1'homme.Thèse Paris Uli, 1982. 2. VALETTE, F.M., MARSH, D.J., MULLER, F., MASSOULIE, 3. :Comparative affinity chromatography of acetylcholinesterases from five different species. J. Chromatogrphy 257,285-296 (1983). 3. KARNOVSKY, M.J., ROOTS, L. : A direct coloring thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, 219-221 (1964). 4. ELLMAN, G.L., COURTNEY, K.D., ANDREAS, \l., FEATHERSONE, R.M. : A new rapid colorimetric method of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95 (1961). 5. CHUBB, I.W., GOODMAN, S., SMITH, A.D.,: Is acetylcholinesterase secreted from central neurons into the cerebrospinal fluid? Neuroscience 1, 57-62 (1976). 6. GUIBAUD, S., SIMPLOT, A., MERCATELLO, A. : CSF Cholinesterase and Guillain-Barré syndrome. Lancet, _2, 1456 (1982).

CHOLINESTERASE ISOENZYMES OF CEREBROSPINAL FLUID AND GUILLAIN-BARRE SYNDROME

A. S i m p l o t , S . Guibaud L a b o r a t o i r e de Biochimie ( S c e Pr Reboud) Hôpital de l a C r o i x Rousse 69317 Lyon France A. M e r c a t e l l o , D. Robert S e r v i c e des I n s u f f i s a n t s R e s p i r a t o i r e s , H ô p i t a l de l a C r o i x Rousse 69317 Lyon France 0. Robert S e r v i c e de N e u r o l o g i e , Hôpital Neurologique Lyon

France

Introduction The d i a g n o s i s o f Gui 11 a i n - B a r r e syndrome r e s t s on c l i n i c a l electromyography and a l b u m i n o - c y t o l o g i c a l

dissociation in

observations, cerebrospinal

f l u i d (CSF) but the causes of t h i s syndrome, the c e l l u l a r and molecular mechanisms i n v o l v e d , remain unknown and the d i a g n o s i s i s not easy. Working on c h o l i n e s t e r a s e isoenzymes, we have observed a s p e c i a l

pattern

i n the cerebrospi nal f l u i d o f these p a t i e n t s .

Material and methods Cerebrospinal

f l u i d specimens - 181 - are obtained by standard lumbar

puncture techniques from 152 p a t i e n t s the r e p a r t i t i o n o f which i s : . m e n i n g i t i e s - 25 . v a r i e d n e u r o l o g i c desorders - 48 (cerebral

v a s c u l a r deseases 5, tetanus 4 , m u l t i p l e s c l e r o s i s 8, me-

t a s t a t i c tumors 3, H o r t o n ' s desease 1, cerebral a t r o p h i e s 3, s e i z u r e s 7, metabolic encephalopathies 3, coma a f t e r cardiac a r r e s t 6, s e p t i c

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

388 encephalopathies

2, P a r k i n s o n ' s desease 1, t r i c y l c i c

cephalopathy 1, p o s t s u r g e r y 1, v i r a l . peripheral

encephalities

antidepressants

en-

3)

n e u r o p a t h i e s - 19 -

( s p i n a l c o r d compression 1, m y e l i t i s suspected p o l y r a d i c u l o n e u r o p a t h i e s

1, p o l y r a d i c u l o n e u r o p a t h i e s

2, p o l y n e u r o p a t h i e s

13,

2)

. o t h e r s - 60(fewer o f undetermined o r i g i n ) Only u n c o l o r e d specimens w i t h o u t b l o o d c o n t a m i n a t i o n are

selected.

C h o l i n e s t e r a s e isoenzymes are s e p a r a t e d by e l e c t r o p h o r e s i s

on 6 p e r cent

p o l y a c r y l a m i d e g e l s by a m o d i f i c a t i o n o f the method o f C l a r k e (1)

: 40 ul

o f CSF mixed w i t h 10 ul o f 40 p e r c e n t s u c r o s e s o l u t i o n are a p p l i e d to each tube which i s run a t 1 mA u n t i l

the albumin-dye marker

(bromophenol

blue) band has m i g r a t e d towards the anode. A f t e r s e p a r a t i o n , the bands o f c h o l i n e s t e r a s e a c t i v i t y appear on the e x t r u d e d g e l s as dense t a t e s a f t e r i n c u b a t i o n o v e r n i g h t i n a medium c o n t a i n i n g iodide

, sodium

precipi-

acetylthiocholine

m a l e a t e , sodium c i t r a t e , s u l f a t e copper and p o t a s s i u m

f e r r i c y a n i d e as p r e c e d e n t l y d e s c r i b e d ( 2 ) . Two i n h i b i t o r s are used w i t h a good degree o f s p e c i f i c i t y cholinesterase

: L y s i v a n e (ethopropazine)

(nS Ch) ( E C . 3 . 1 . 1 . 8 )

methy 1 ammonium p h e n y l - p e n t a n - 3 - o n e )

f o r non s p e c i f i c

and BW 284 C 51 (1-5 b i s - 4 a l l y l d i for acetylcholinesterase

(A ChE)

(EC 3 . 1 . 1 . 7 )

Results On 162 specimens - see Table I - we o n l y observed one l a r g e , d i f f u s e band w i t h a c h a r a c t e r i s t i c m o b i l i t y . but L y s i vane r e s i s t a n t ,

side

T h i s band i n h i b i t e d by BW 284C51

corresponds to the A ChE a c t i v i t y .

On 19 specimens - see Table II - we observed w i t h the A ChE band, an additional

slow band i n h i b i t e d by L y s i v a n e but BW 284C51 r e s i s t a n t

ponding to the nSCh a c t i v i t y .

the Gui 1 l a i n - B a r r e syndrome ( p o l y r a d i c u l o n e u r o p a t h y w i t h gical

dissociation).

corres-

These specimens c o r r e s p o n d to p a t i e n t s

with

albumino-cytolo-

389 Table I : Patient r e p a r t i t i o n and CSF isoenzyme pattern.

Pathologies

n . . Protein (5; - g . r i )

CSF

Patients

Bands » rKr

A

ChE

nSCh

Meningitis

25

33

0,67

+

0

Varied neurologic disorders

48

56

0,54

+

0

Peripheral

19

30

4,25

+

See Table

60

62

0,41

+

0

neuropathies

Others

Table I I

: Peripheral neuropathies and CSF isoenzyme pattern

Patients

CSF

Spinal cord compression

1

3

55

10,03

+

0

Myelitis

1

1

2

0,14

+

0

Polyradiculoneuropathies (with albumino-cytological d i s s o c i a t i o n )

10

18

4

2,38

+

+

Polyradiculoneuropathies (with low increased CSF proteins)

2

3

2

0,44

+

0

2

2

12

1,5

+

1+ lo

Polyradicul oneuropathy (4 monthes later)

1

1

2

0,53

+

0

Polyneuropathies

2

2

2

+

0

Pathologies

Suspected p o l y r a d i c u l o neuropathies ?

Leuk. Protein (Mega T 1 ) ( x - g l " l )

25,6

Bands A ChE nSCh

II

390

An uninterpretable pattern i s obtained in case of blood contamination see photo 1. Photo 1 Cholinesterase

isoen-

zyme pattern i n CSF. From l e f t to r i g h t : .two "standard" CSF .CSF from Guillain-Barre .blood contaminated CSF

We have observed the nSCh band only in the cases of polyradiculoneuropathy with high level of CSF proteins and albumino-cytological

dissociation.

This band does not appear neither for the unfortunately alone patient punctured 4 monthes l a t e r when the c l i n i c a l

symptoms are normalized,

nor for the polyradiculoneuropathies with low increased CSF p r o t e i n s . This band i s not only related to high level of CSF proteins since i t i s not observed in the cases of polyneuropathies and spinal

compression

with very high level of CSF p r o t e i n s . Now, no mechanism explanation may be proposed but A ChE isoenzyme separation might be a help in d i f f i c u l t c l i n i c a l

context.

References 1. Clarke, J.T. : Ann. N. Y. Acad. S c i . , 121, 428-436

(1964).

2. Guibaud, S . , Simplot, A . , Bonnet, M., Thoulon, J.M., Guibaud, P . , Robert, J.M. : J. Genet, hum., 30, 119-134 (1982).

LYMPHOCYTOTOXIC AND MONOCYTOTOXIC ANTIBODIES IN MULTIPLE SCLEROSIS

1 2 1 Lucien Rumbach , Marie-Marthe Tongio , Jean-Marie Warter , 1 1 2 Christian Marescaux , Maurice Collard , Simone Mayer 'clinique Neurologique, Hospices Civils de Strasbourg 2

Institut d'Hematologie, Hospices Civils de Strasbourg et Centre de Transfusion Sanguine, 67091 Strasbourg Cédex,France

Introduction Terasaki et al (3) demonstrated cold lymphocytotoxic antibodies (LCA) in the serum at 15°C of patients suffering from disseminated lupus erythematosis. This cytotoxic activity was different from that of the HL-A allo-antibodies ; since then it has been reported in many conditions which have often as a common factor a disturbance of the immune system (1). We have recently studied warm and cold lymphocytotoxic and monocytotoxic antibodies in the serum and cerebrospinal fluid (CSF) in multiple sclerosis (MS) (2).

Materials and methods This study comprised 21 patients with "clinically definite" form of MS. For controls, serum of 32 healthy blood donors, and serum and CSF of 6 patients suffering from diseases other than MS were studied. Detection of antibodies in serum and CSF was done by a modified microlymphocytotoxic technique of Terasaki and Mc Clelland (2). Serum and CSF of each MS and

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

392 non-MS patient were tested at 15 and 37°C with their total lymphocytes (TL), B lymphocytes (BL), monocytes (M) autocells and then with fresh TL, BL, M alio cells of healthy blood donors. Tests were performed before and after absorption on platelets. If more than 20 % of the cells were stained with eosin, the reaction was considered to be positive.

Results 1) patients with MS. - serum : results are summarized in Table 1. Table 1 Cytotoxic

activity

In the. ie.Jiu.rn o{

MS

tested in the presence of following allocells 15°C

sera TL

The

37 °C M

BL

TL

BL

M

9 cases

-

-

-

-

-

-

4 cases

+

+

+

-

-

-

3 cases 4 cases 1 case

+

+

+

-

-

+

+

+

+

-

+

+

+

+

+

+

+

+

itiength

+ positive

ofi the reaction,-

reaction negative

Ii

not

Indicated

In thli

table.

reaction.

The search for cytotoxic antibodies was negative in 9 cases, at 15 and 37°C. The sera of the other 12 MS reacted positively at 15°C but there were differences at 37°C : 4 patients reacted negatively to TL, BL and M, 3 reacted negatively to TL, BL but positively against M, 4 negatively to TL and positively against BL, M and one serum reacted positively against TL, BL and M. After absorption on platelets, the number of positive reactions was reduced. Only 6 of the 21 MS

393 sera contained autoantibodies. - CSF : results were very different. The reactions were positive against autocells and allocells in all the samples tested, at 15 and 37°C. 2) patients with other diseases : no cytotoxic activity was detected in 5 sera. However the CSF cytotoxic activity of all 6 patients was strongly positive to alio- and autocells. In the absence of complement, the reaction against M was positive at 37°C, the tests against TL and BL were negative. 3) healthy controls : 5 of the 32 sera tested were positive. No auto antibodies were found in the serum. 4) clinical and biological parameters : no relation was established between cytotoxic activity and various clinical parameters. We noted no correlation between CSF albumin, CSF immunoglogulin and HL-A typing and the cytotoxic activity.

Discussion Cold lymphocytotoxic antibodies are found mainly when there is some alteration of the immun system, but their role is still unknown and their site of cellular action is also controversial. They have been observed in the serum of 30-67 % of MS patients ; our work confirmed this occurence. However this study demonstrated that : 1) the cytotoxic activity was directed not only against total lymphocytes but also, and predominantly, against monocytes ; 2) some of these antibodies were detected not only at 15°C but also at 37°C ; 3) tests were positive with allocells but in certain patients with autocells ; 4) reactions were strongly positive in all

394 CSF studied. As this activity is directed against 3 cellular populations, it involves probably not one type of antibody. These antibodies may play a role in immunomodulation ; further investigation of antibody effects on MS lymphocyte subpopulations is warranted.

References 1. Mayer, S., Falkenrodt, A., Tongio, M.M. : Ann. Immunol., 126 C, 621-627 (1975) . 2. Rumbach, L., Tongio, M.M., Warter, J.M., Marescaux, C., Mayer, S., Rohmer, F. : J. Neuroimmunol., 3, 263-273. 3. Terasaki, P.I., Mottironi, V.D., Barnett, E.V. : N. Engl. J. Med., 283, 724-728 (1970).

INTEREST OF A HIGH RESOLUTION AGAROSE GEL ELECTROPHORESIS SYSTEM (PANAGELR) WITH A SILVER STAINING REVELATION IN THE STUDY OF CEREBROSPINAL FLUID (C. S. F.) IMMUNOGLOBULIN G

Elisabeth Plan, Jean-Bernard Seneterre, Claude Quincy Laboratoire de biologie - Hôpital Neurologique - 59,boulevard Pinel - 69 003 LYON

Introduction Demonstration of oligoclonal bands by CSF proteins

electro-

phoresis (1) is an important aid in establishing the diagnosis of chronic inflammatory diseases of central nervous system (C.N.S.). In this report, we compare two systems : - high resolution agarose gel system (Panagel ) with silver staining - polyacrylamide disc gel electrophoresis.

Material 150 CSF samples were studied from : - 85 patients with multiple sclerosis (M.S.) 5 patients with subacute sclerosing panencephalitis (S.S.P.E.) 3 patients with neurosyphilis -

and 57 reference patients with various non inflammatory

other neurological diseases. CSF were stored at + 4°C ; it is known that keeping samples longer than one week may alter oligoclonal pattern : generally the bands become diffuse or disappear.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

396

Methods . Polyacrylamide (P.A.A.) disc gel electrophoresis : Electrophoresis, using a discontinuous system, is carried out in vertical gels according to the method described by Ornstein (2) and Davis (3). It requires 0,6 ml of unconcentrated C.S.F., concentration occuring in situ during electrophoresis. Because of the high power of resolution of P.A.A., electrophoretic patterns, stained with amido black, give many informations. But their interpretation is difficult due to many bands which are not immunoglobulins but migrate in the gamma zone (haptoglobin polymers ; carbonic anhydrase). . High resolution agarose gel electrophoresis : Agarose gel electrophoresis using Panagel out as described by Johanson

system is carried

(4).

Due to the high sensitivity of the silver staining (5) , this method requires only 7 ul of unconcentrated C.S.F. This technic presents several advantages versus P.A.A. : - easiness of handling - speed of realisation - smaller initial volume of sample - high resolution power in the gamma globulin area which makes agarose a useful supporting medium to demonstrate gamma bands. Electrophoretic patterns, obtained by the two methods, were examined in simple blind to search the presence of oligoclonal bands without knowledge of the patients' identity or the result given by the other system. For each system, we calculated sensitivity and specificity (6): . Sensitivity : was defined at 100 times the number of patients with M.S. who had positive tests (presence of oligoclonal bands) divided by the sum of this number plus the number of patients who had negative results (absence

397

FIGURE

:

-C.S.F.

electrophoretic

-Multiple

-Non

Sclerosis

inflammatory

patterns :

from

A

Agarose

C

P.A.A.

other

patients gel gel

neurological

B

Agarose

D

P.A.A.

with

. diseases

gel gel

.

:

:

398

of oligoclonal bands). . Specificity : was defined at 100 times the number of patients having no M.S. who had negative tests divided by the sum of this number plus the number of patients without M.S. who had positive tests .

Results High resolution agarose gel system revealed oligoclonal bands for 57 of the 85 patients with M.S. (sensitivity : 67 %) ; P.A.A. gel revealed bands only for 43 of them (sensitivity : 50,5 %) . For the 57 reference patients, agarose gel demonstrated extra bands in the gamma region for 9 of them (specificity : 77 %) ; with P.A.A. gel in 13 cases (specificity : 73 %). We did not include in our calculations cases of S.S.P.E. and neurosyphilis which are known for their local production of IgG. Oligoclonal aspect was found in all the C.S.F. from S.S.P.E. (5 cases) and neurosyphilis (3 cases) when agarose gel was used. P.A.A. showed oligoclonal bands in all the cases of neurosyphilis, but only in 3 out of 5 cases of CSF from S.S.P.E. patients.

Discussion We evaluated the performance of 2 electrophoretic methods to reveal the presence of oligoclonal bands in demyelinating diseases as M.S. The high resolution agarose system is more sensitive : 67 % (P.A.A. : 50,5 %) and more specific : 77 % (P.A.A. : 73 %).

399

The apparent "false positives" should not be viewed as diminishing the value of the test. Oligoclonal bands represent locally produced immunoglobulins of restricted heterogeneity which are known to occur in the electrophoretic patterns from CSF of patients with bacterial and viral encephalitis, Guillain Barré syndrome, etc... (7,8,9,10) P.A.A. is not as sensitive as agarose gel to reveal the oligoclonal aspect, particularly in the study of C.S.F. from S.S.P.E. patients. In this case, the absence of information results in the loss of gammaglobulins which migrate in very cathodical position.

Conclusion Comparison of two electrophoretic methods using different support mediums permitted to confirm the superiority of high resolution agarose gel for routinely qualitative study of CSF gamma globulins in clinical laboratories. High resolution agarose gel system with silver staining is more pratical and more performant than P.A.A. gel to demonstrate oligoclonal IgG locally produced in chronic inflammatory C.N.S. disorders.

References 1.

Gerson, B.,Krolikowski, F. I., Gerson, I. M. : Clin. Chem. 26/2 , 343-345 (1980)

2.

Ornstein, L. : Ann. N. Y. Acad. Sci. 121 , 321-349 (1964)

3. 4.

Davis, B. J. : Ann. N. Y. Acad. Sci. 121 , 404-427 (1964) Johansson, B. G. : Scand. J. Clin. lab. Invest. 29^ , suppl. 124, 7-19 (1972)

5.

Kerenyi, L., Gallyas, F. : Clin. Chim. Acta 3¡8 , 465-467 (1972)

400 6.

G e r s o n , B., C o h e n , S. R., G e r s o n , 2. M . , G u e s t , G. H. : C l i n . C h e m . 2 7 / 1 2 , 1 9 7 4 - 1 9 7 7 (1981)

7.

L a t e r r e , E. C . , C a l l e w a e r t , A . , H e r e m a n s , J. S. : N e u r o l . 20 , 9 8 2 - 9 9 0 (1970)

8.

L i n k , H . , M u e l l e r , R.

9. 10.

: A r c h . N e u r o l . 25^ , 326-344

(1971)

: Ann. Neurol. 2 , 425-431

(1977)

J o h n s o n , K . , N e l s o n , B.

J o h n s o n , K., A r r i g o , S. C . , N e l s o n , B . J. e t al. : N e u r o l o g y 27 , 2 7 3 - 2 7 7 (1977)

TWO DIMENSIONAL ELECTROPHORESIS OF CSF PROTEINS : ELECTROPHORESIS QUALITY CONTROL

Salma Gehamy, Sylvie Reynier, Christiane Caudie, Claude Quincy Laboratoire de biologie (Professeur Quincy) - Hôpital Neurologique, 59, boulevard Pinel - 69 003 - LYON

Introduction A high resolution two dimensional gel electrophoresis procedure has been developed for the analysis of complex mixtures of proteins (O'Farrell 1975). By combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate (SDS) gel electrophoresis in the second dimension, proteins are separated according to isoelectric point and molecular weight- This technique is an extremely powerful investigative tool for analysis of CSF proteins. We report electrophoresis quality control data for our two dimensional gel electrophoresis system to allow the routine examination of CSF specimens.

Material and methods . First dimension Apparatus :

:

The first dimensional separation or isofocali-

sation in glass tubes is performed in the Pharmacia gel electrophoresis apparatus GE-4 : (glass tubes 150 X 1,2 mm in size, gel rods Reagents :

T = 4 % , C = 5 % ) .

Ampholytes are purchased from LKB Instruments,

Inc. (pH : 3,5 - 9,5 78 %, pH : 2,5 - 4 11 %, pH : 9 - 11 11 %) and all other reagents and solvents used are of the highest purity available.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

402

Sample preparations

:

We use fresh sera and CSF and Rabbit

muscle Powder (Sigma) to study the electrophoresis quality control of our system (repeatability and reproducibility). Each serum or CSF, and rabbit muscle powder specimen are prepared by specific methods, respectively Tracy's (4) and Anderson's (1). . SDS electrophoresis Dimension Apparatus

:

:

For the second dimension, the vertical

polyacrylamide gel electrophoresis apparatus built by Mula , France, is used, (gel concentration 12 % or 14 % and gel slab dimension 140 X 140 X 1 mm). Reagents

:

Calibration kit Pharmacia

(lmw), running

gel buffer based composed of tris-glycin-SDS, pH : 8,3-8,5 (2) Staining

:

silver staining is performed as described

by Oakley et al. (3). Background staining is eliminated by a longer washing after the silver diammine treatment (2 hours instead of 2 mn in Oakley's technique with agitation and eight changes of glass-distilled water).

SDS electrophoresis dimension 94000

1-2-3-4

67000 5

43000

30000

20100

J

2

3

,4

: different concentrations of rabbit muscle : calibration kit

403 Results . A typical 2D gel pattern of CSF is shown in the following figure. It demontrates the capability of this gel electrophoresis system.

. Electrophoresis quality control : First dimension

:

The curves for pH gradients are plotted

routinely according to Tracy (4) and we calculate the slope and intercept of the regression line. The gels used as Iso dimension quality control gels for pH gradients do not contain samples. The following table lists repeatability and reproducibility CVs for the slope and intercept of the regression line of our system. The values are 5,5 % or less as Tracy's (4).

404 i i

"o 1 rH II G

Repeatability

1

i •H 0 • "3 tJ >i ! II 0 -P ^ -RH 1 G OJH a) -H 1

Pi X)

PH Slope

i i

x

=

0,354

i

SD

=

0,0195

i

cv

5,5%

i

x

=

4,629

i ,

SD

=

0,255 5,5 %

, ,

i

cv

I

=

0,370

I

X

=

4,423

,

=

0,020 5,4 %

I

SD

=

,

CV

0,178 4,02 %

, , ;

CV

I

Second dimension

i ,

1

Intercept

i

I

i x , SD I

1

gradient i

1 :

The curves for molecular mass are

plotted routinely with rabbit muscle powder and Pharmacia calibration kit (lmw). The following table lists CVs values for the slopes of the molecular mass curves (log Mr Vs relative mobility). The values are 5,9 or less, close to Tracy's values (4). I

Log Mw curves

I 1 •H

o 1—1 +I || 0 -P U -H FTH Q) -H A .Q

I I

CV

= —

Intercept

I

0,376

X

" SD !

I

Slope

I

0,012

"

3,1 %

1

!

X

=

5,2

SD

=

0,018

!

3,3 %

!

CV

=

I

_

0,385

!

x

=

5,213

!

! SD

=

0,023

!

SD

=

0,025

!

! CV

=

5,9 %

!

CV

:

4,7 %

!

!

x

I

2D electrophoresis

I

:

An important cause of poor reproduci-

bility are losses of proteins during manipulation and particulary during equilibration. To measure these losses,

405

we introduced in each batch a sample of rabbit muscle proteins. In view of results, we progressively decrease the duration from 30 mn to 5 mn and finally we eliminated this step without effect on the quality of the rabbit muscle pattern (no more streaks). The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation.

Conclusion We examined the various steps of the 2D electrophoresis for repeatability and reproducibility. The quality control of

our simple 2D electrophoresis system seems sufficient

to allow us to begin developing methods for CSF protein study. The rabbit muscle powder, easy to use and of little cost in 2D electrophoresis routine technique, can be utilised for electrophoresis quality control.

References 1.

Giometti, C. S., Anderson, N. G. and Anderson, N. L. : Clin. Chem. 25 , 11 , 1877 - 1884 (1979)

2.

Johnson,B. J. : Analytical Biochem. 127 , 235-246 (1982)

3. 4.

Oakley, R. B. : Anal. Biochem. 105 , 361-363 (1980) Tracy, R. P., Currie, R. M., Young, D. S. : Clin. Chem. 28 , 4 , 908 - 914 (1982)

ANTHROPOMETRIC AND BIOLOGICAL CHARACTERIZATION STATES IN MAN Claude André and Simone

OF MALNUTRITION

Danière

1

Groupe d I m m u n o p a t h o l o g i e digestive, INSERM U 45, Pavillon Centre Hospitalier Lyon Sud, 69310 PIERRE BENITE, FRANCE

1G,

Introduction Defining

the

normal

human

nutritional

state

There are n e v e r t h e l e s s several

situations

sification

instance

is

necessary,

compare

populations

quiring

medical have

we must

because

they

that

subjects had

time

identify

body

in

problem.

this we

clas-

wish

individuals

a

higher could

or shorter

be

life

Underweight

individuals

spans, but to a lesser

extent.

The

of

nutritional

comparison to

state

anthropometric

criterion(a)

does not

an

prevent

has(have) us

from

also

individual

can

or biological not

using

yet

have

been

currently

be

standards. employed

to determine

indispensable therapeutic

complements.

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

to

expectanand

life-

determined

predict the chances of success of failure of a given

or

com-

used

shorter

defined,

to re-

death

American insurance

weights

longer

a

There is in fact a clear association with mortality

overweight.

fect

each

are

In this context,

established

determine which cies.

or when

care

illness risk group. panies

for

is

in which

in

A

per-

but

this

methods

to

treatment

410

Anthropometric

Standards

1) Mean weight - Ideal weight The simplest measurement

is that of body weight.

that this parameter is height- and sex-related. studies This

have shown

"normal"

that mean

weight

life expectancy.

gain

weight

We admit Population

increases with

is undesirable

if

we

aging.

refer

to

This idea has led to the definition of an

ideal weight, constant in an adult regardless of age. A

number

of

formulas

has been

proposed

ideal weight. The most classical of Lorenz and Vague,

to

calculate

where the ideal theoretical weight is

equal to height expressed as cm above one m, subtract

a number

this

formula for adults is that

equal

to

1/4

of the

from which we

difference

between

150 and height expressed in cm: Ideal weight

(kg) = (height cm-100) - (height cm-150)/4-

There are two major

criticisms to

be leveled

against

this

formula. It neglects sex and, more importantly, it neglects individual

morphology.

among 4 compartments:

Body weight

is in fact

the skeleton, adipose tissue, extra-

cellular liquid and the cellular mass, cles, tions

and blood and visceral proteins. in

relation

to

distributed

a standards

may

which includes musBody weight variabe

the

result

of

a

large skeleton or large muscle mass as much as of large fat reserves.

The size of the skeleton may be estimated by the

width

of

rence

of the

measured

the pelvis wrist.

between

the

or the shoulders and by the The

slight

anterior

type and

circumfe-

has a pelvic

superior

iliac

width, spines

which is less than 28 cm and a wrist circumference which is less than 16 cm. The heavy skeletal type has an iliac width

411

greater than 20 cm.

29 cm and a wrist circumference greater than

The mean type has intermediate values.

account for this skeletal parameter,

In order to

Monnerot-Dumaine

(1)

proposed another formula in which ideal weight is given by the equation: W = (H - 100 + 4C)/2, most widespread

where C is wrist circumference.

current us involves tables

(2).

The

Neverthe-

less, the comparison of data must also include the clinical examination. dromes,

Edema

accompanying certain

nutritional

syn-

for example in children with kwashiorkor, causes a

weight increase which may

cause the degree of

denutrition

to be underestimated. 2) Importance of the lipid compartment Virtually all human energy reserves are in adipose tissue. In

a

normal

weight,

male,

it

accounts

for

10 to

15%

of

total

while the corresponding figure in normal women is

around 20 to 25%.

Half this fat is subdermal with varying

distributions.

The value of determining the adipose tissue

content

organism

of

the

is obvious.

Obesity

in

fact

is

physiologically defined as an excessive proportion of this tissue.

On the contrary,

and

a bit too excessive,

the

Littre dictionary defines the state emaciated as an individual whose subcutaneous tissue contains little or no fat. On the

practical

level,

there

is no

means

for

directly

measuring the mass of adipose tissue.

It is calculated by

subtracting the non-fatty

extracellular

and support tissue

cell

from total

mass,

body weight.

water

The non-fatty

cell mass is determined isotopically as the dilution space

412

of

potassium-42;

measuring

inulin

extracellular or

mannitol

exchangeable sodium chloride. tissues

is

determined

function of height. difference

between

The weight

of an

by

water

is

dilution

or

determined by

by

measuring

Finally, the mass of support

estimations

from

tables

as

a

An alternative method uses the density adipose

tissue

individual

is

and the

expressed

non-fatty on the

mass.

basis

of

his volume measured in a bathtub and reading off a nomogram gives the proportion of adipose tissue. baum et al.

however

(3),

the exact determination

As noted by Apfel-

the difficulty of this method is

of respiratory

volumes.

Two

indirect methods which are easier to use are more employed. with

an

g/mm

,

regularly

One is the measurement of skin folds, determined appropriate

compass

exerting

a

pressure

sub-scapular, regions.

pectoro-axi11ary,

supra-iliac and abdominal

The approximate weight of the adipose

ness of the skin fold in women

and

on the posterior

5-7 mm

in men.

compartment

The normal

tion indices have also been described

These

data

vary

however,

is

as a

correla-

between per cent body

(or skin fold) and the weight/size ratio consensus,

thick-

face of the arm

function of age and also of racial type. Excellent

is no

10

usually the tricipital, bicipital,

can be deduced from these measurements.

fat

of

regardless of the spread. The measurement should be

done at several points,

15 mm

other

concerning

the

(W/S). ideal

There

formula:

W / S ^ or W/S"^, or even an exponent W/S^, where P accounts for age, sex and a racial factor (4, 5).

413

3) Importance of the non-fatty mass The proportion composition

of proteins,

of

the

organism

especially muscles, can

be

determined

in the by

easy

measurements or may require special equipment. The simplest method is arm circumference,

measured halfway between the

olecranon and the acromion. Muscular diameter in the middle of the arm can be calculated by deducting the contribution of skin and sub-cutaneous fat with the formula: muscle diameter = (arm circumference/3•14) - tricipital skin fold,

where values are in mm.

Similarly,

the

muscle surface in the middle of the arm is calculated with: 2 muscle surface (mm ) = 3-14/4 x (diameter of arm 2

muscles) . The protein content of children and adults is well shown by the muscle surface of the arm, and the diameter or circumference of its muscles (6, 7). Some authors perform these measurements with the thigh. A more sophisticated method for determining total protein in the organism involves measuring body nitrogen (8), total cell

mass by measuring exchangeable

potassium

and (9),

the latter excluding bone cells.

Biological Standards 1 ) Laboratory data Anthropometric obesity however,

as

tests

much

as

are for

used

for

the

dénutrition.

determination Laboratory

of

tests,

are primarily concerned with the study of malnu-

trition. The significance of certain tests is mainly at the level of general status, while others are more specific.

Urea nitrogen tein supply infectious tion.

may be

low if there

in the diet. state

which

is an insufficient

Standard blood tests indicate an

is

often

This is as important

associated

in the

case of

are the determinations of cholesterol an

obese

have

subject.

long

general

included

decrease

number

of

proposed

plasma

proteins as

an

as in

the

of

leaves

synthesized

index,

of

denutrition

proteins;

immunoglobulins by

including

denutri-

denutrition

criteria

levels

with

and triglycerides

The biological usually

pro-

the

liver

albumin,

the

intact. have

A

been

prealbu min,

siderophi1 in and retinol binding protein. The determination of these parameters must not neglect the fact that such

as

albumin,

concentration

of

are

large

retinol

reserves,

binding

while

protein or

plasma

prealbumin

much lower. In addition, these proteins have very half lives:

certain,

the

20 days for albumin, 8 days for

is

different

siderophilin,

48 hours for prealbumin and 12 hours for the retinol

bind-

ing protein.

reac-

These data explain the highly different

tivities of these biological

Serum crease

complement in

and

protein

indices.

hemoglobin

and

calorie

concentrations malnutrition.

also

de-

Creatinine

clearance is also an excellent, indication of muscle mass in adult, as in children ed

for

diet.

72

hours

in

(10). Creatinine clearance is measurthe

absence

of

meat

and

fish

in

the

The index is determined as the ratio between urinary

creatinine

per

24 hours

and

the

same

parameter

healthy size-paired subjects. A number of clinical tions sometimes lead to the search for more local

in

normal

observaassociat-

ed deficiencies, for example vitamins, folic acid, elements such as zinc.

415

Malnutrition caused

by

is

important

sign

lymphocytes. mined

often

a decreased of

denutrition,

by

as

On a more functional

if the immune

nutritional presenting

accompanied

insufficiency

hypersensitivity

to

infections

Lymphopenia

is

the

level,

is

decrease

an

of

T

it can be deter-

system is or- is not compromised by the

a positive

tetanus anatoxin,

frequent

immune function.

by

studying

anamnestic

contact

the

response

antigens

of

such

capacity delayed

as

of skin

tuberculin,

Candida, diphtheria anatoxin,

Trichophy-

ton , varidase, Proteus or Streptococcus. Usefulness of markers of nutritional

state

It is a fact that severe malnutrition, such as observed in the third world or in concentration camp victims, causes irreversible sequelae intractable to any therapy. A lowered to trauma subjects 20%.

in the form of major surgery who

pre-operative

body

weight

decreases

These situations are unfortunately

no less maximalist.

The

problem posed

that of selecting high

risk subjects,

tients

would

whose

prognosis

be

resistance

is also established more

too frequent

in

than

and are

in Western nations of defining

improved

by

is

those

pa-

nutritional

and

immune therapy. A large number of studies has been inspired by this idea.

In addition, there is an upsurge of in interest in

nutrition today. this key

word

There are in fact about 20,000 entries under

in the last two years of MEDLINE.

sions of this work are far from unanimous, case

of

plasma

concentration vital

albumin

lower than

prognosis

in

levels.

Some

25 g/l

is an

subjects

with

The

for example in the

workers

believe

unfavorable

severe

in the

nephrology

department

(12).

that

sign

diseases

predicts a longer hospitalization arid more frequent in patients

conclua

for a

(11)

or

infections

Treatment

of

416

surgical patients by enteral or parenteral nutrition,

accord-

ing to whether it

concen-

succeeds in increasing the

tration over 35 mg/1

or fails to do so,

element in survival or death Other authors,

albumin

is also a predictive

(13).

however, believe that post-operative complica-

tions can be better predicted by the dynamometric

measurement

of hand clenching force than by albumin assays (14,

15)- This

questioning of the value of albumin assays is consistent

with

other workers who believe that albumin is a correct reflection of the less

nutritional

reliable

on

state an

on an epidemiological

individual

basis

(16).

level

but

Similarly,

is in

cancer patients on parenteral alimentation, increased survival is

better

(17)-

predicted

by

siderophilin

levels

than

Plasma proteins with short half lives,

protein

and

excellent

prealbumin,

indices

of

are

also

nutritional

by

albumin

retinol

binding

considered

status

(18)

to

be

or

unreliable

markers as a result of excessively rapid fluctuations There are also supporters hypersensitivity test.

and detractors

(19).

delayed

skin

The former admit a predictive capacity

for the course of the disease property

of the

either-

(20),

the latter

denying this

(21).

Conclusions The critical analysis of these data, tive prognosis, gence

of

the

especially in pre-opera-

is deceiving as long as there exists a diver-

results

obtained.

The

has

caused

well

nutrition workers to emit defeatist considerations (22).

known They

believe that a careful clinical examination contributes information

at

least

sophisticated

identical

anthropometric

to

that

arid

furnished

biological

by

the

most

determinations.

417

This hard line opinion may nevertheless contain nuances. authors

ask

if

a clinical

spirit

as precise

as this

Some is

not

specific to several specialists in nutritional problems and if their

experience

can

not

have

been

partially

acquired

as a

result of practicing those very same anthropometric and biological indices.

Also, although biological assays would seem to

be superfluous on a practical level in cases of extreme clinical situations, they are useful in equivocal cases Finally,

(23).

a review of the literature gives the impression that

defiance of anthropometric and biological determinations could very

well

studies. analysis during

be

related

For

example,

of

several

the

last

inconclusive

12

the treatment context,

immune

hundred only

given was

of

skin

articles

explains

tests,

in

why

3 studies used

taken

into

a

English the

of

patients It

in

is

the

critical appearing

results

age-paired

remain

controls,

in the disease

account..

In

this

nor

special con-

although it is the rreasui ement of an anamnes-

response.

whom

also

We

thus

understand

what

when

they

are

the search for an anergic state in patient

undergoing digestive (25).

in terms

inadaptation

neither the differences

homogenous series, of

frequent

the effect of repeating the skin tests was not

sidered either , tic

a

years

(24):

and in most cases

to

tract operations a

clear

leads

hyperalimentation that

the

malnutrition would be beneficial

to

the

selection

reduces

mortality

correction

in the

of

treatment

a of

state

of

a large

number of diseases.

The use be

of anthropometric

encouraged,

providing

greater strictness effectiveness.

and biological that

this

use

in its application,

indices should is

accompanied

thus by

the only guarantee

a of

418 References 1.

Monnerot-Dumaine, M.: Presse Med. 6_3, 1037-1038

2.

Geigy, Bale

3.

J.R.:

Scientific

edition

Geigy

SA,

(1963)

Apfelbaum,

M.

Vigy,

Paris 10506 B10-9 4.

Tables,

( 1955 )

Cronk.,

M.,

Igoin, L.:

Encycl. Med.

Chir.

(1973)

C.E., Roche, A.F.: Am. J. Clin. Nutr. 35,

347-354

(1982)

5.

Frisancho, A.R., Flegel, P.N.: 697-699

Am. J. Clin. Nutr. ¿ 6 ,

(1982)

6.

Durnin,

J.V.G.A.,

7.

(1974) Cureton, K.J., Boileau, R.A., Lohman, T.G.: Hum. Biol. 321-326

8.

J.: Br. J. Nutr. ¿2,

77-97 47,

(1975)

McNeill, S.L.,

Womersley,

K.G.,

Memagh,

Harrison,

J.E.:

J.R., Am.

J e e j e e b h o y , K.N., Wolman,

J. Clin. Nutr. _32, 1955-1961

(1979) 9.

Talso,

P.J.,

Metabolism

Miller,

9., 456-471

10. Trowbridge,

F.L.,

Clin. Nutr. 3 6 ,

C.E.

Carballo, A.J., Vasquez,

I.:

(I960)

Hiner,

69I-696

C.D.,

Robertson, A.D.: Am. J.

(1982)

11. Apelgren, K.N., Rombeau, J.L., Twomey, P.L., M i l l e r , R.A.: Crit. Care Med. J_0, 305-307

( 1982)

12. Anderson, C.F., Wochos, D.N.: Mayo Clin. Proc.

57., 181 - 1 8 4

(1982) 13- Ching, Mills,

N.,

Grossi,

C.B.,

C.E.,

Nealon,

T.F.:

Angers, J., Zurawinsky, Surg. Gynecol. Onstet.

H.S., 151,

199-202 (1980) 14- Klidjian, A.M., Foster, K.J., K a m m e r l i n g , R.M., Cooper, A, Karrah, S.J.: Br. Med. J. 281, 899-901 15-

(1980)

Klidjian, A.M., Archer, T.L., Foster, K.J., Karran, J.P.E.N. 6, 119-121

(1882)

S.J.:

419 16. Forse, R.A., Shizgal, H.M.: J.P.E.N. 4, 450-454 17. Eriksson,

B.,

Douglass,

H.O.:

J.A.M.A.

(1980)

243,

2049-2052

( 1980) 18. Cals,

M.J.,

Delacoux,

Drevillon,

C.,

Raffray,

Y.,

Miocque, M.: Sem. Hop. Paris ¿ 8 , 2833-2837 19- Farthing, M.J.G.: Nutr. Res. 2, 561-568 20. Tasseau,

Succari, M.,

E., Evstigneeff, T., Desmoulins, D., Arsac, M.,

F.

(1982)

(1982)

Gaucher, L. Nicolas, F.: Sem. Hop. Paris

58,

7 8 1 - 7 8 4 (1982) 21. Brown, C.A.,

R.,

Bancewicz,

Farrand,

R.J., Pumphrey, R.S.H., Irving, M.:

Med. J. 284, 851-853 22. Baker,

J.P.,

Stewart,

S.,

J., Hamid, J . , Patel, N.J., W a r d , Brit.

(1982)

Cetsky,

A.S.,

Wesson, D.E., W o l m a n ,

S.L.,

Whitewell, J., Langer, B. J e e j e e b h o y ,

K.N.:

N. Eng. J. Med. 396, 969-972 (1982) 23. Collins, J.A.: N. Eng. J. Med. 306, 987-988 24. Twomey,

P.,

Ziegler,

(1982)

D., Rombeau, J.: J.P.E.N.

6^, 50-58

(1982) 25- Champault, G., Fabre, F.,Patel, J.C.: Nouv. Presse Méd. 9_, 1559-1563

(1980)

NEW FINDINGS ON THE ROLE OF ZINC AS A BIOLOGICAL RESPONSE MODIFIER

Susanna Cunningham-Rundles, Ph.D. L a b o r a t o r i e s o f C l i n i c a l Immunology and Human Immunogenetics Memorial S l o a n - K e t t e r i n g Cancer Center 1275 York Avenue, New York, NY 10021

Introduction Several l i n e s of i n v e s t i g a t i o n have suggested that the c e l l u l a r immune response may be modulated

both in v i t r o and i n v i v o by network

i n t e r a c t i o n s i n which z i n c may act as a p o t e n t i a t i n g agent ( 1 - 4 ) .

We and

others have p r e v i o u s l y demonstrated that zinc i s a monocyte dependent T lymphocyte mitogen i n v i t r o ( 5 , 6 ) and that l o s s of t h i s

function

c o r r e l a t e s with advance of malignancy in cancer p a t i e n t s .

Furthermore,

we have found that z i n c can a c t i v a t e human B lymphocytes to d i f f e r e n t i a t e i n v i t r o i n t o antibody s e c r e t i n g c e l l s

(7) and to act s y n e r g i s t i c a l l y

t h i s system with other B c e l l s t i m u l a t o r s .

in

We and others have found that

zinc may improve immune response i n zinc d e f i c i e n t persons (8) i n some cases.

In a d d i t i o n , we have found a c l o s e l i n k between zinc and thymic

hormone, as reported a l s o by Bach ( 9 ) . In the s t u d i e s described here, p o s s i b l e r e g u l a t o r y i n t e r a c t i o n s with the natural k i l l e r , NK, system were explored using ZnCl, Z n + + as a source of zinc.

Methods Peripheral blood mononuclear c e l l s , PBM, were i s o l a t e d from normal, healthy v o l u n t e e r s by p r e v i o u s l y described methods ( 1 0 ) , washed and resuspended i n RPMI 1640 supplemented with p e n i c i l l i n , glutamine, and 10% pooled normal human serum.

The 4 hr

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

streptomycin, 51

C r r e l e a s e assay

422 was used to assess natural killer, NK, function against the K562 tumor target cell as previously described (11).

Details of the longer term

activated NK assay have been described (12).

The Z n + + used was freshly

diluted from a 30 mM stock solution of ZnCl.

Results and Discussion In view of previous findings that zinc has the potentiality to enhance immune response in vitro and in some individuals in vivo in situations reflecting enhanced proliferation, studies were undertaken to examine the effect of zinc on natural killer activation in vitro. natural killer, NK, function were assessed.

Two types of

The first, endogenous NK

activity, was studied using freshly isolated peripheral blood mononuclear cells, PBM, in a 4 hr

51

C r release assay against the K562 target.

The

second, activated NK function, was examined following culture of PBM for six days with and without NK activators followed by exposure of these prestimulated cells to labeled K562 target cells in the 4 hr assay.

51

C r release

Concurrent addition of NK augmenting agents, specifically a or y

interferon, IFN, was carried out in the 4 hr assay in both procedures in some cases to assess pre-NK activity and residual potential pre-NK activity.

All studies were conducted in pooled normal human serum to

avoid possible stimulating effects of fetal calf serum.

Addition of Z n + + to freshly isolated PBM in a 4 hr assay of cytotoxic activity against the K562 tumor target was observed to produce a sharply inhibitory effect on lytic activity when present in the assay period only. As shown in Table I, this effect was highly dependent upon concentration and was rapidly diluted out so that at a [Zn + + ] of 4.0 X 10" 5 M, this was no longer observed.

One day incubation without Z n + + followed by exposure

to the labeled tumor cell target produced loss of NK activity.

This

spontaneous loss of activity is usually observed in studies of normal human peripheral blood lymphocytes and has been widely reported. here, Z n

++

further depressed lysis.

was seen at 4 days.

As shown

Additional diminution of NK activity

Similarly, NK augmentation by alFN was apparently

423 i n h i b i t e d by the presence of Z n + + in the absence of preculture and again t h i s effect was r a p i d l y l o s t with lower [ Z n + + ] , Table I .

Effect of Zn + + on NK A c t i v i t y 1 Preculture Peri od 4 Days 1 Day

Augmenting Agent

None

0 7,.5 X l O " 4 M 2..0 X 10" 4 M 4..0 X 10" 5 M

None None None None

21.8 4.6 6.7 21.2

10.6 2.8 5.8 N.D.

0 7..5 X 10" 4 M 2.,0 X 10"' + M 4..0 X 10" 5 M

alFN alFN alFN alFN

39.9 10.6 18.1 39.2

39.2 19.2 46.4 47.9

Zn

J 2

i

4 hr Final

i 9

51

C r release assay against K562; E:T r a t i o , concentrations.

5.5 1.0 N.D. N.D. 7.4 21.0 N.D. N.D.

100:1.

In c o n t r a s t , enhancement of ciIFN mediated augmentation was observed following 1 day preculture with both alFN and Z n + + together at concentrations of Z n + + l e s s than 5 X 10" 4 M and furthermore, by day 4, a concentration of 7.5 X 10" 4 was associated with a 3 - f o l d increase i n NK a c t i v i t y over cultures with alFN alone. Experiments designed to test for p o s s i b l e effects of Z n + + on the target c e l l s revealed that Z n + + i n h i b i t s both spontaneous and detergent induced lysis.

As shown in Table I I , the i n h i b i t o r y effect was most marked at the

higher concentration.

Since spontaneous release was equally affected, the

S/T r a t i o did not change.

The net r e s u l t was to reduce effector c e l l

lysis. Thus, the r e s u l t s shown i n Table I considered in l i g h t of Table suggest that Z n + + may act on both target and effector c e l l s .

II

In

experiments where Zn + + was present in a preculture phase with NK augmenting agents p r i o r to target cell a d d i t i o n , effector cell enhancement was predominant.

Z n + + was a l s o able to prevent l o s s of alFN induced

a c t i v i t y over time, not by d i r e c t l y a c t i v a t i n g NK c e l l s but perhaps by prolonging the l i f e of newly matured NK c e l l s recruited by alFN.

424 Table I I . Zn + +

Z n + + Associated Target Protection 1

Total Release 2

% Inhibition Total Release

Spontaneous Release 3

S/T

3134 2545 2764 2939

0 62.0 11.8 6.0

344 268 292 308

0.11 0.11 0.11 0.10

0 7..5 X K T 4 M 2..0 X I O - 4 M 4..0 X I O ' 5 M

*4 hr assay. cpm, detergent induced l y s i s . cpm, target c e l l s alone.

2

3

P o s s i b l e s t a b i l i z i n g e f f e c t s of Z n + + on NK function during a c t i v a t i o n by alFN and ylFN were studied in further experiments c a r r i e d out over 6 days. In these experiments, PBM were cultured at a density of 1.5 to 2.5 X 10 6 lymphocytes per ml and then recollected and plated with targets.

As shown i n Table I I I , Z n

most normal donor lymphocytes.

++

51

Cr labeled

did not a c t i v a t e NK function of

( I n a few cases, 2 of 10 persons'

lymphocytes tested, augmentation by Z n + + alone was found.

This may

suggest that the NK system was being stimulated in vivo and that Z n + + acted as a cofactor to promote s u r v i v a l of newly emerging mature NK cells.) Table I I I .

Z n + + Potentiated Induction of Activated NK C y t o t o x i c i t y 1 No Addition

100: 1 50: 1 25:: 1

1.9 1.3 1.2

Zn++

cJFN

4.6 1.5 1.3

16.4 9.8 5.1

alFN + Z n + + 37.1 35.1 24.5

% A

126.2 258.2 380.3

day culture system: data are shown as % 5 1 C r release at 3 effector target r a t i o s in 4 hr assay against K562. The presence of alFN during the culture period was associated with 6 - f o l d augmentation of l y t i c a c t i v i t y compared to unstimulated NK function at an E:T r a t i o of 100:1.

In t h i s culture system, Z n + + s t r o n g l y enhanced NK

induced target l y s i s and t h i s e f f e c t was stronger proportionately at lower E:T r a t i o r e l a t i v e to cultures with alFN alone as shown by the increased net change i n Table

III.

The a b i l i t y of Z n + + to modulate NK function was a c o n s i s t e n t f i n d i n g in a l l normal donors tested.

There was v a r i a t i o n in the magnitude o f the

425

boosting e f f e c t that appeared to be proportional to response to a or ylFN in the absence of Z n + + .

As shown in Figure 1, enhancement of Z n + + was

quite concentration dependent and in t h i s longer period of c u l t u r e , of 2.5 X 10

-lt

[Zn++]

M (corresponding to Zn 1:5 in Figure 1) was the most

e f f e c t i v e concentration.

The enhancement was r a p i d l y d i l u t e d out and did

not change the slope of the curve at concentrations where no augmentation was seen. E5 E0 55 50 45 U1 >- H0 35 UJ

30

a:

25 20

15 I0 5

ISEY B=STFS »-«IFN+ZN *-°f IFN+ZN I;20 FN+ZN I;5

0

I 00 : I

50 : I

E :T

25 :

RHTI•

Figure 1 Concentration dependent enhancement of alFN induced NK a c t i v a t i o n by Z n + + . Data are shown as percent l y s i s at d i f f e r e n t e f f e c t o r target r a t i o s . [alFN] was 800 U/10 5 lymphocytes. [ Z n + + ] were as follows: 1:5 = 2.5 X I O " 4 M; 1:20 = 8.0 X I O " 5 M; and 1:100 = 6.0 X I O " 5 M. Additional studies were conducted using ylFN as an NK augmenting agent in the 6 day activated NK assay.

In studies with normal donors,

enhancement of ylFN mediated NK augmentation was c o n s i s t e n t l y observed with considerable v a r i a t i o n in magnitude and d i f f e r e n t i a l e f f e c t on a range of E:T r a t i o s .

Typical data are shown in Figure 2.

426

75 70 E5 B0 55 50 Ln H5 H0 35 tr 30 25 20

_KEY_ • -"ÏTFN *- Ï1FN+ZN I¡0E *-ÏIFN+ZN I 2H +-JIFN+ZN I

I5 I0 5 0

I 00 : I E•T

50 : I

25 : I

RBTI•

Figure 2 Zn++

Effect of on y l F N augmented NK a c t i v i t y . Concentrations of Zn++ given in legend to Figure 1. [ y l F N ] was 64 U / 1 0 6 lymphocytes. The concentration dependence of Zn++ enhancement in experiments with yIFN was less restricted.

As for alFN at ineffective concentrations of Zn++,

no significant deviation from the slope of the curve observed with ylFN alone was seen. In summary, the results described here demonstrate a s i g n i f i c a n t enhancement of NK a c t i v i t y by Zn++.

The studies suggest that the

mechanism may a f f e c t the binding reaction or s t a b i l i z e NK c e l l maturation induced by NK augmenting agents in short term culture.

The time

dependence of the results presented may r e f l e c t either a requirement for metabolism of Zn++ or reduced a v a i l a b i l i t y of Zn++ as a target protecting agent.

In longer term cultures, Zn++ was found to exert a strongly

enhancing e f f e c t on augmented NK a c t i v i t y while being i n s u f f i c i e n t alone to have any potentiating impact on the NK system.

Since the time

427

required for maximum expression of enhancement coincided exactly with the peak of Z n + + induced lymphocyte p r o l i f e r a t i o n as shown previously ( 5 ) ,

it

i s possible that precursor NK c e l l s might be within the expanded c e l l u l a r compartment and thus Z n + + would provide a larger pool of pre-NK c e l l s IFN recruitment.

for

The magnitude of Z n + + enhancement was s t r i k i n g ,

p a r t i c u l a r l y at lower E:T r a t i o s .

Since in v i v o , lower r a t i o s to

putative targets might be more r e a d i l y achieved, one may speculate that Z n + + could act as a cofactor in mediating mechanisms of immune surveillance.

Acknowledgments The e x c e l l e n t technical assistance of Mrs. K.M. Smith i s

gratefully

acknowledged.

NIH NCI CB

These studies were supported in part by:

08748-17, The Richard Molin Memorial Foundation for Cancer Research, and NIH POI CA 29502-04.

References 1.

Brummerstadt, E . , Flagstad, T., Basse, A., Anderson, E.: Acta Pathol. M i c r o b i o l . Scand. (Sect. A) 79, 686 (1971).

2.

Iwata, T . , Incefy, G.S., Tanaka, T., Fernandes, G., Menendez-Botet, C . J . , Pih, K., Good, R.A.: Fed. Proc. 37, 1827 (1978).

3.

Fraker, P . J . , De-Pasquale-Jardun, Zwickl, C.R., Luecke, B.W.: Proc. Nat. Acad. S c i . U.S.A. 76, 457 (1979).

4.

Bendtzen, K.: Scand. J. Immunol. 12, 489 (1980).

5.

Garafalo, J . A . , Cunningham-Rundles, S . , Braun, D.W., Good, R.A.: J. Immunopharmacol. 1, 60 (1980).

6.

Ruhl, H., Kirchner, H., Bochert, G.: Proc. Soc. Exp. B i o l . Med. 137, 1089 (1971).

7.

Cunningham-Rundles, S . , Cunningham-Rundles, C., Dupont, B . , Good, R.A.: C l i n . Immunol. Immunopathol. 16, 115 (1980).

Int.

428 8.

Cunningham-Rundles, C., Cunningham-Rundles, S . , Iwata, T., Incefy, G., Garafalo, J . A . , Menendez-Botet, C., Lewis, V., Twomey, J . J . , Good, R.A.: C l i n . Immunol. Immunopathol. 21, 387 (1981).

9.

Bach, J . F . : Immunology Today, 1981.

10.

Cunningham-Rundles, S . , Hansen, J . A . , Dupont, B. in C l i n i c a l Immunobiology Vol. 3, ed. F. Bach, R.A. Good. Academic P r e s s , New York, p. 151, 1976.

11.

Cunningham-Rundles, S . , F i l i p p a , D.A., Braun, D.W., A n t o n e l l i , P., A s h i k a r i , J . : J. Nat. Cancer I n s t . 67, 585 (19 ).

12.

Cunningham-Rundles, S. in NK C e l l s and Other Natural Effector C e l l s , ed. R.B. Herberman, Academic Press, New York, p. 1133, 1982.

IMMUNODEFICIENCY IN PROTEIN OR VITAMIN

A

DEPRIVED MICE

Studies of immunological responses in immediate hypersensitivity with egg albumine or vaccination with ribomunyl

Patrice Binder, Bernard Gassies and Robert Fontanges Centre de Recherches du Service de Santé des Armées Division de Microbiologie 108, boulevard Pinel 69275 Lyon Cedex 03 France

Introduction

It is generally accepted that protein malnutrition and certain specific nutrient deficiences have an adverse effect on the immune system (1,2,3). Investigations on the interactions of protein or vitamin malnutrition and various immune parameters have led to some understanding of the etiology of the increased susceptibility to infectious diseases (4,5,6). Effects of nutrition on atopic reactions are the less known facts. Magnesium deficiencies may induce a temporary allergy like crisis (7) and eicosapentanoic acid enriched diet increases synthesis of IgE and IgG (8). Abassy (9) described a less sensibility to atopy in protein deprived children although IgE level was increased, probably because these children were infected with many parasitic agents. A recent work studies atopic children who developped hypervitaminasis A (10). The vaccination study on fed deprived people is an important problem and the hazards may be increased : atopy, immune complexe diseases, infections with living vaccines. The level and duration of protection have never been evaluted. In a previous work on S. typhimurium used as a living vaccine we observed that this germ became pathogenic in protein deprived Balb/c mice (11). In the present work, two types of responses were described after Normal, Protein deficient or Vitamin A deprived mice immunization. In the first work Balb/c mice, nourished with 19 % protein feed (Normal), 4 % protein feed (P deprived) or 19 % protein feed without vitamin A (Vit. A deprived),

M a r k e r Proteins in Inflammation, Vol. 2 © 1 9 8 4 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

430 were immunized with egg-albumin (E.A.). Immediate hypersensitivity (I.H.) and various related parameters were studied. In the second work, vaccination with Ribomunyl of Normal or P. deprived mice was studied. This ribosomial vaccine is a protective agent against K. pneumoniae infection (12)

Material and Methods

E®®d_and_Animals *

Feeds. Feeds were made by UAR . Their composition is given in table I. Animals. Male and female Balb/c mice were bred in our laboratory 4-5 weeks old mice were weaned and the diet was only started when mice were 6 weeks aged. Animals were weighted weekly.

Feed percent of total wei ght Composition Casein D.L. Methionin

Normal 20 0,3

Protein deprived 4 0,3

Vit. A deprived 20

**

0,3 **

Cornstarch

15

20

15

Dextrose

50

61

55

5

5

5

Cornoil

5

5

-

Linoleic acid

-

-

0,5

Cellulose

Linolenic acid

-

-

1

Mineral mix

3,5

3,5

4,5

Vitamin mix

1

1

4,5

Cholin butyrate

0,2

0,2

0,2

** Vitamine A control = No Vit. A Tableau I : Composition of feeds

* U.A.R. Villemoisson 91360 Epinay sur Orge France

431

- MojrtjiljXy jstudiej^. I.H. w i t h E.A. was studied previously

(13). In this

work w e used this antigen at various doses after various duration of feed control. The best moment for immunization had been observed to be started 3 weeks after feed control. Balb/c were randomised in 3 groups for feed control. Every group was divided in 7 subgroups for each sex. After 3 weeks of dietary, any subgroups was simultaneously immunized w i t h 9 killed B. pertussis, 6 x 10

germs intraperitonealy

doses of E.A. subcutaneously

(S.C.). 2 weeks later any mouse had received

2 mg of E.A. intravenously

(I.P.) and various

(I.V.). I.H. shock was quick and mortality

data were observed in any subgroup. -

of Serotonin. Three identical dietary groups were studied w i t h

female Balb/c mice. Seven doses were used in any group of 10 mice. These doses were 0,100,150,200,250,300 and 400 mg/kg. Serotonin was I.V. injected. Number of death was computed for 30 m n and we had used these date for L.D. 50 determinations. - E.A. specific antibodies. W e used the passive hemagglutination

test.

E.A. was adsorbed on sheep red blood cells (S.R.B.C.). Balb/c were

ran-

domised and any group was divided in 4 subgroups of feed control duration 1,2,3 or 4 weeks before immunization. Mice were immunized w i t h 2 mg/mouse of E.A. and B. pertussis, as previously described. In any subgroups bloods of 5 mice were collected and pooled at 1,2,3,4 and 5 weeks after immunization. Sera were

frozen.

Vacc ination_with_Ribomun^l * - MJjomuriyl . It is a ribosomal complex w i t h ribosomes of K. pneumoniae, S. pyogenes, S. pneumoniae and H. influenzae

(14). These ribosomes are

mixed w i t h proteoglycan of K. pneumoniae membranes 25 |Jg of this product are equivalent to a vaccinal dose. - Dietary. Mice received a Normal or P. deprived feed as previously described. - VaccimitjLon. M i c e were randomised in every feed groups and distributed in 2 subgroups

: vaccinated and no vaccinated animals. The vaccinated

* Laboratoires Pierre Fabre, Castres 81100 France

432 subgroups were immunized S.C. with 2 doses (25 pg x 2) of Ribomunyl. The first dose was injected after 4 weeks and the second dose after 5 weeks of feed control. - Challenge^ Two weeks after the last injection, 20 mice of the vaccinated and the no vaccinated subgroups were challenged with K. pneumoniae (2 x 3 10

germs/mouse I.P.). Mortality data was computed over 21 days and these

data were evoluted by a statistical method previously described

(15).

- Specific antibodies. Ribosomial and proteoglycan specific antibody titers were estimated in 5 pooled sera 1,2 and 3 weeks after the first immunization in both vaccinated and no vaccinated subgroups. E.L.I.S.A. technic was used for titration. Related data are the differences between vaccinated and no vaccinated subgroups. - Di£ect_PFC_test_. Direct H?C testfor SRBC were done 1,2 and 3 weeks after the first injection of vaccine. For any assay 8 mice of every subgroup g were immunized IP with 10

SRBC, 4 days before splenectomy. We used the

method of Cunningham (16). The numbers of spleen cells and PFC/10^ cells were computed. - Inmunoglobulij^dos^ges. Different classes of immunoglobulins were estimated by the single radial immunodiffusion method. 5 sera of any subgroups were pooled 1 week after the first vaccination.

Results

Immediate_HY2ersensitivity -

reality. We observed (table IX) a major decrease of mortality

average in P. deprived mice. The sex factor was also very important, while E.A. dose was a minor factor. Bfelow 1 mg/mouse the mortality was minimal. The most significative differences between any group were observed with 2 or 3 mg/mouse. The group of Vit. A deprived mice gave comparable results to controls. - L^D._50 of serotonin^ L^D. 50 was normal or decreased in P. deprived or Vit. A deprived mice (table III).

433

Anima1s sex Normal

Vit. A Deprived

Normal

Vit. A Deprived

alive

dead

dead

alive

dead

alive

dead

alive

1

9

1

9

2

8

4

6

2

8

3

7

1

1

9

1

9

0

10

4

6

0

10

6

4

2

2

8

1

9

1

9

8

2

1

9

5

5

3

3

7

1

9

3

7

7

3

4

6

10

0

4

3

7

1

9

5

5

6

4

3

7

7

3

10

5

5

1

9

3

7

5

5

4

6

6

4

alive

dead

P.Deprivec

alive

P.Deprived

0,4

Number

Mg/animal

Female

dead

Feed

0. A.

Male

Table II : Mortality after E.A.

shock

i n t h e d i f f e r e n t g r o u p s of B a l b / c

Feed control

in weeks

1

2

Normal Feed

mice

3

4

262

P.Deprived Vit.A Deprived

223

196

229

191

203

240

231

196

T a b l e I I I : R e s u l t s of L . D . 5 0 of s e r o t o n i n (doses i n m g / k g )

434 ~ iL-Ar_S2.ecif_i.c_ antibodies. In Normal mice the E.A. specific antobodies showed a steady rise untill the 5th week (table IV). In P. deprived or Vit.A deprived mice the levels of antibodies were always lower when duration of dietary was 3 or A weeks before immunization. The differences between Normal and deprived mice were very important 4 and 5 weeks after immunization. At this moment, antibody titers in Vit. A deprived mice were very low.

Feed

Norma!

Feed control before immunization (weeks) 1 Antibodies titer for every weeks

16

Vit.A deprived

P. Deprived

0

1

2

3

4

0

1

2

3

4

64

8

4

16

64

2

2

8

32

64

2

128

128

64

64

128

128

32

16

32

64

64

3

512

256

256

256

256

256 128

64

64

128

64

4

1024

512

512

512

512

512 256

256

256

256

32

5

2048

024

1024 1024

512

128

2048

512

256

32

69

Table IV : Titers of E.A. antibodies during different periods of immunization.

Y§££.i2§£i°D_with_Ribomunyl - Chall_en_ge_. Vaccination with Ribomunyl gave a good protection in Normal mice (table V). P. deprived mice were not protected, but showed a longer mean survival time. P . deprived but not vaccinated mice had a fulminant disease. - Specif^c_a^tjibodies_. Proteoglycan antobodies were higher than ribosomes antibodies in every feed and vaccinated group. At any time the titers of these antibodies were lower in P. deprived than in controls (fig. 1). -

A*- a n y time the numbers of spleen cells were dramati-

cally decreased in P. deprived mice. The rate of S.R.B.C. antobodies bearing cells were the same in any feed groups. These rates were

435 Normal

Feed Vaccination Mortality 21th day after challenge Daily mortality Survival average (days)

P. deprived

Yes

No

Yes

No

5 %

90 %

100 %

100 %

0,05

0,39

0,47

2,99

10,93

6,45

2,68

1,54

Table V : Survival curves parameters increased when Normal or P. deprived mice were just vaccinated (table VI)

Feed

Normal Yes

Vaccination 1 WEEKS AFTER

Cells spleen 223 ± 40 x 106 b PFC/10 1099 ± 145 cells Cells/spleen x 106

2

VACCINATION 3

P. Deprived no

Yes

No

214 ± 42

54 1 10

704 ± 227

1080 ± 498

337 + 129

56 t 9

61 ± 15

39 - 9

173 1 27

152+50

1119 1 50

1285 Ì 774

404 ± 111

458 ± 269

Cells spleen

181 ± 31

195 Ì 19

42+11

47 + 11

PFC/10b cells

495 ± 109

507 ± 243

355 ± 247

326 + 229

PFC/10b cells

Table VI : Results of direct P.FoC. test

- Immunoglobulin classes. IgM were increased after vaccination. The level of these antibodies was lower in P. deprived than in Normal mice. Only IgA were higher in P. deprived than in Normal mice (table VII).

436 A

O.D.

1000

ANTIBODIES LEVEL IN VACCINATED GROUP MICE

500 450 400 350 300 250 200 150 100 50

1

2

3

WEEKS AFTER VACCINATION —

Ab a proteoglycan K.d., Normal feed,

---

Ab a proteoglycan K.p., P.Deprived feed, Ab a ribosomes K:p., Normal feed, Ab â ribosomes K.p., P.Deprived feed

Figure 1 : Specific antibodies in vaccinated mice

437

Feed

Normal Yes

No

IgG1

5,83

6,91

5,«

IgG2a

3,22

3,39

2,20

IgG2b

1,18

0,86

1 ,58

Ig M

0,93

0,32

0,49

Ig A

1 ,05

1,10

2,30

Vaccination

Ig Class

P. Deprived Yes

Table VII : Results of estimations of Immunoglobulin class

Discussion and Conclusions

Malnutritions are impairment factors for immunological responses. Observed modifications may be very important and opposed : protein deficiency may be a good thing for atopy and very bad thing for vaccinal protection ! Systems for immunological responses have regulation steps. Nutritional disorders can be induced on increase or decrease of atopy, vaccinal protection and immunological memory. Fundamental studies on AMPc (17), histamin release (18) or IgE receptors of mast cells are very important for understanding the role of unbalanced nutrition in atopy. Likewise antibodies affinity and cellular cooperations are essential factors. Their modifications may be cause an infectious disease in spite of vaccination or immunostimulation (19).

Acknowledgment. P. Binder thanks Pierre Fabre Laboratories for the free gift of Ribomunyl.

438 References 1.

Chandra, R.K., Newberne, P.M. : Nutrition Immunity and Infection, mechanism of interactions, Plenum Press, New York, London 1979.

2.

Bounous, G. , Kongshawn, P.A.L. : J. Nutr. JJ_2, 1 /47-1755 (1982).

3.

Watson, R.R. , Mc Murray, D.N. : CRC Critical Reviews in Food Sciences and Nutrition, _1_2, 1 13-150 (1979).

4.

Shaedler, R.W., Dubos, R.J. : J. Exp. Med. J_K), 921-934 (1959).

5.

Scrimshaw, N.S., Taylor, C.E., Gordon, J.E. : WHO, Monograph, serie 57,(1968).

6.

Newberne, P.M., Hunt, C.E., Young, V.R. : Brit. J. Exp. Rathol. 4_9, 448 (1968).

7.

Claverie-Benureau, S., Lebel, B., Gaudin-Harding, F. : Reprod. Nutr. Developp. 2J_, 591-600 (1981).

8.

Prickett, J.D., Robinson, D.R., Black, K.J. : Immunol. 46, 819-826 (1982).

9.

Abassy, A.S., El Din, M.K., Hassan, A.I., Aref, G.H., Hammad, S.A., El Araby, I.I., El Din, A.A. : J. Trop. Med. Hyg. 77, 18 (1974).

10. Silverman, S.H., Leks, H.I. : Clin Pediatr. 2_j_, 172-174 (1982). 11. Binder, P., Fontanges, R. : Personnal Communication SO1"*1 Reunion of Association of Physiologist (France), Toulouse 14-17 Juin 1982. 12. Fontanges, R., Robert, D., Content, Y., Nis, G. : Arzneim. Forsh./ Drug Res. 30, 142-172 (1980). 13. Fournier, J.M., Accominotti, J.C., Leclerc, J.L., Fontanges, R. : Rev. Franç. Allergol., 135-141 (1976). 14. Fontanges, R., Robert, D., Content, U., Nils, G. : Rev. Franc. Allergol _T7, 35-81 (1977). 15. Binder, P., Epinat, R., Eprinchard, Y., Fontanges, R. : C.R. Soc. Biol. 176, 345-351 (1982). 16. Cunningham, A.J. , Szenberg, A. : Immunol., U\_, 599-600 (1968). 17. Spach, C., Askhenasy, A. : J. Nutr. _109, 1265-1273 (1979). 18. Gaudin-Harding, F., Claverie-Benureau, S., Armier, J., Davy, J., Lebel, B. : Internat. J. Vit. Nutr. Res. 50, 185-192 (1980). 19. Lambert, R. : Cahier de Nutrition et de Diététique, 1, 53-57 (1976).

IMMUNOLOGICAL CONSEQUENCES OF PROTEIN DEPRIVATION IN PRIMATES

Saman Qazzaz Department of Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT.

Introduction

The association of malnutrition and infectious disease is the most common problem of disease in tropical and sub-tropical countries. disease nearly always worsens co-existing malnutrition.

Infectious

Furthermore, the

consequences of infection are likely to be more serious in a malnourished host than in a well nourished one.

When infection aggravates malnutri-

tion or when malnutrition weakens resistance to infection, the term "synergism of infection and malnutrition" has been applied to this association (Scrimshaw, Taylor and Girdon, 1959).

Protein-energy mal-

nutrition is usually accompanied by suppression of the immune response as well as by severe infections.

Reports of field studies in children with

severe malnutrition have documented the suppressed immune response (McFarlane et al, 1970).

Nevertheless, because of the frequent episodes

of infection in these children, it is still not clear whether it is the infection in these children or the malnutrition, or both, that are responsible for the immune suppression.

The present study was therefore

designed to investigate this problem.

Materials and Methods

Establishment of malnutrition in primates

Three baboons and one cynomolgus monkey, mean age 2h years, were used. The primates were offered a Mazuri primate diet which was obtained from British Petroleum (BP) Nutrition, Essex, England.

This diet consisted of

217. protein, 427. carbohydrate and 4.27„ digestible crude oil containing

M a r k e r Proteins in Inflammation, Vol. 2 © 1 9 8 4 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

440 both saturated and unsaturated fatty acids. 3851 calories. vitamins.

One kg of the diet contained

The rest of the diet consisted of trace minerals and

The control baboon received 220 g of this diet as established

in preliminary studies and the remaining 2 baboons and the m o n k e y received 44, 11 and 22 g/day respectively. 5 and 107, of the control diet.

The animals were fed twice daily on 20,

In addition to their respective diets each

primate received 4 g of a soluble multivitamin mixture containing all the known vitamins, calcium pantothenate and choline daily in their drinking water, as well as 130 g/day of fresh vegetables and fruits comprising apples, carrots, grapes, cauliflower and bananas.

Prior attempts to feed

these primates w i t h any other type of food were unsuccessful and hence this factor had to be taken into account in the design of the study.

After 4 weeks on their respective diets attempts were made to achieve a steady metabolic state by increasing the amount of food for all the m a l nourished animals to 357„ of the Mazuri primate diet and maintaining it at this level.

Assessment of the nutritional

status

General appearance. The animals were examined daily for any changes in appearance, behaviour and signs of infection.

Body weight. The animals were weighed twice weekly after being anaesthetised.

Plasma protein quantitation. The serum albumin, transferrin and C3 concentrations were measured by the specific single radial method (Mancini, Carbonara and Heremans,

immunodiffusion

1965).

Assessment of the immune response

Delayed hypersensitivity. This was performed according to the m e t h o d of Catalona et al, 1972.

In preliminary experiments w i t h different

ing doses of dinitrofluorobenzene

sensitiz-

(DNFB) in 0.1 ml acetone on the shaved

forearm all the primates produced a distinct inflammatory response w i t h i n

441 2 days regardless

of their n u t r i t i o n a l

sensitizing dose a challenging w a s a p p l i e d to the o p p o s i t e

status.

Fourteen days after

the

d o s e of D N F B of 100, 50 a n d 25 jUg i n 0 . 1 m l

f o r e a r m w h i c h w a s r e - e x a m i n e d a t 24 a n d 48 h

for e v i d e n c e of d e l a y e d c u t a n e o u s h y p e r s e n s i t i v i t y .

A positive

response

w a s i n t e r p r e t e d as a n a r e a h a v i n g d i s t i n c t e r y t h e m a ,

induration

and

vesiculation.

T h e s i z e s of s u c h a r e a s w e r e m e a s u r e d w i t h a W e l l c o t e s t 2 the a r e a of w h i c h w a s c a l i b r a t e d i n c m .

skin reaction gauge

Skin transplantation. performed according grafts, examined

First and second set skin transplantations

to the m e t h o d d e s c r i b e d b y M e d a w a r

were Auto-

allografts and x e n o g r a f t s were done, and all skin grafts

were

daily.

Passive cutaneous anaphylaxis out in primate kwashiorkor levels.

(1944).

(PCA). S t a n d a r d P C A r e a c t i o n s w e r e

skin with serum specimens

f r o m (i) c h i l d r e n

carried

with

a n d (ii) c h i l d r e n w i t h a s t h m a k n o w n to h a v e r a i s e d s e r u m

Serum specimens with

n o r m a l c o n c e n t r a t i o n s of I g E f r o m

m a t c h e d h e a l t h y c h i l d r e n s e r v e d as c o n t r o l s .

A positive PCA reaction was

i n t e r p r e t e d as a d i s t i n c t b l u e a r e a , w h i c h a p p e a r e d o n the b a b o o n s ' within 5 minutes after

the a l l e r g e n c h a l l e n g e .

T h e s i z e s of the

reactions were m e a s u r e d w i t h a ruler calibrated in cm and w i t h Wellcotest

skin reaction

skin

PCA

a

gauge.

Lymphocyte activation by mitogen. The method used was m o d i f i e d from of J a n o s s y a n d G r e a v e s , haemagglutinin

1971, a m i c r o - m e t h o d being employed.

DNA synthesis

(PHA) s t i m u l a t i o n of D N A s y n t h e s i s b y p e r i p h e r a l

blood

primates.

(E) a n d to

1 9 7 5 ) , i n w h i c h the p e r c e n t a g e of r o s e t t e s a m o n g

lymphocytes was estimated. established by counting duplicate.

to s h e e p e r y t h r o c y t e s

antibody

(EAC) r o s e t t e s w e r e p e r f o r m e d as p r e v i o u s l y d e s c r i b e d

and McFarlane,

after

t h a t 40 j/Ug of P H A p e r 1 m i l l i o n c e l l s c a u s e d m a x i m u m

i n the n o r m a l

Rosetting. Spontaneous rosettes complement

that

Phyto-

l y m p h o c y t e s w a s p e r f o r m e d a t l e a s t t w i c e p e r w e e k for e a c h p r i m a t e , having established

IgE

age-

The reproducibility

in t r i p l i c a t e a s a m p l e

(Jones 200

of the c e l l c o u n t s t h a t w a s s e t up

in

was

442 Immunoglobulin G (IrG) measurements. This was quantitated by the single radial immunodiffusion assay (SRID). Test for infection Microbiological methods. Throat and conjunctival, swabs taken at least twice weekly from each primate into Stuart's transport medium were examined by means of Gram-stained films, cultures on blood agar, chocolate agar and MacConkey agar, and anaerobic cultures carried out on brain-heart infusion agar with 107o blood.

All colony types were Gram-

stained then subcultured to appropriate media for further examination. Organisms were identified by the method of Cowan and Steel (1974). Peripheral blood from each primate was also inoculated into brain-heart infusion broth and Brewer's thioglycollate.

Subcultures were made on to

blood agar and chocolate agar plates at 24 and 48 h and at 7 days.

At the end of the experiment the animals were killed and specimens of lungs, liver, spleen, kidneys and lymph nodes were cultured as above.

Results Changes in behaviour By the end of the first week on the restricted diets the animals appeared more aggressive and by the fourth week they had become withdrawn and lethargic. Body weight Changes in total body weight are summarized in figure 1.

When body

weight was expressed as a percentage of initial weight, a significant change (p t > I-

5

S

4

! D

3

£ X

2

H

1 0

controls

anorexia nervosa

Figure 1. Thymulin activity in anorexia nervosa

464 References 1.

Herbeth, B., Henny, J. and Siest, G. : Ann. Biol. Clin. 41, 23-32 (1983).

2.

Pertschuk, M.J., Crosby, L.O., Barot, L. and Mullen, J.L. : Am. J. Clin. Nutr. 35., 968-972 (1982).

3.

Richard, J.L., Rodier, M., Bringer, J., Bellet, M.H., Mirouze, J. : Nouv. Presse Med. 11, 3327-3330 (1982).

4.

Ingenbleek, Y. : In The Thyroid Gland (M. de Visscher, ed.) 499-527, Raven Press, New-York (1980).

MALNUTRITION IN CANCER AND CACHECTIC PATIENTS Federico Bozzetti, M.D. Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan.

Cancer cachexia is a metabolic syndrome characterized by weakness, anorexia, depletion and translocation of host components and loss of immunocompatence which often occurs in patients with cancer independently from the tumor burden. It is related to the type and site of origin of tumor, stage and duration of the disease (5). Also therapy (chemotherapy, radiation and surgery) may occasionally precipitate a cachectic state in patients with borderline malnutrition. From a clinical point of view, cachexia manifests as asthenia, anorexia, significant loss of body fat, protein and other components, anemia, electrolyte abnormalities, hormonal aberrations and sometimes fever. The patients appear chronically ill and emaciated, the skin is atrophic and shiny, with loose folds. The temporal fossae and cheeks are sunken leaving prominent zygomatic protuberances and old denture plates become large. Depletion of fat tissue is revealed with prominent borders of the sternocleidomastoid muscles, larynx, clavicles and vertebral spines. The sternum and xyphoid and ribs are often well visible, the interspaces are sunken and the breast sag and are shrivelled. The abdomen is protuberant or scaphoid and pubis is prominent. The genitalia are atrophic, and sexual interest and arousability almost absent. The elbows and knees look large compared to atrophic muscle masses. The sacrum and the greater trochanters and sometimes the scapulae, lateral malleoli and heels are prominent; they

may present erythematous

and bullous lesions which subsequently may evolve into decubitus ulceration with dry eschar or undermining necrosis of the entire skin. Fluid may accumulate in the bulbar conjunctiva and along the chest and abdominal walls posterolaterlly as an eccentric bulging or may occupate the entire low back and occur also in the scrotum and prepuce and in the lower legs. Bizzarre patterns of fluid accumulation occur in patients who assume unusual constant postures, usually to avoid pain. Fluid accumulation may contribute to mantain a falsely normal or subnormal weight. The loss of body weight consists primarily of the loss of

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

466 muscle mass and secondly of body fat, while the visceral life-supporting system is, to a considerable extent, spared (11) Pathogenesis of cachexia is multifactorial. It includes an increased energy requirements despite a decrease in the caloric intake, and alterations in protein, carbohydrate and lipid metabolism. Increased energy requirements The energy requirements in patients affected by tumor include the energy required by the tumor for active transport of nutrients and ions and synthesis of cellular materials and the caloric requirements imposed on host (Cori cycle activity, assimilation and transport of nutrients to tumor and synthesis of host tissues in response to tumor).

In tumor cells, glucose

is metabolized via glycolysis to lactate (18, 60). The lactate which is formed is returned to the host liver, where it is resynthesized to glucose (29). This cyclic metabolic pathway, involving metabolism of glucose to lactate and the conversion of lactate to glucose, is termed the Cori cycle. Utilization of lactate and gluconeogenic aminoacids for glucose synthesis requires energy within the host liver and kidney and an increased rate of gluconeogenesis has been proposed a possible mechanism for increased energy expenditure in the cancer patients (18, 21). Holroyde et al. (25) have recently reported studies of Cori cycle activity in cancer patients. Patients who were not losing weight had normal levels of Cori cycle activity, but patients with progressive weight loss had increased Cori cycle activity, showing that lactate production was higher in these patients. In a critical appraisal of data of Holroyde and other authors (46, 63, 71) Young (71) has estimated that less than 10 percent of daily total expenditure in the cancer patients can be accounted by the cost of lactate recycling. Furthermore it can be estimated that, if 15% of total lactate production is oxidized to CO^ and 1^0, and 85% of lactate is converted to glucose, there will be a maintenance of high-energy phosphate balance. It is difficult to accept therefore, that changes in the Cori cycle activity are a significant cause of the marked body wasting in patients with progressive neoplasia. Nevertheless, it is of interest that Holroyde et al. ( 2 5 ) observed highest Cori cycle activity in patients with greatest total

467 energy expenditure. Additional causes for the increased energy expenditure include activation of futile cycles. These cycles occur when there are two opposing metabolic pathways in the cell in which the reactions in the forward and reverse directions are catalyzed by separate enzymes. For example three futile cycles in carbohydrate metabolism are: the glucose 6-phosphate, fructose 6-phosphate and phosphoenolpiruvate cycles. In one direction the reaction requires the participation of a high energy compound, such as ATP, while the reaction in the opposite direction is energetically spontaneous. Although there is no net flux of reactants in these cycles, the cycling causes ATP hydrolysis and thus an energy wastage. The increased energy expenditure could be the result of some alteration in the energy yielding reaction and the coupling mechanism of

oxidative phosfhorylation. In fact

there is some documentation in vitro (15, 41, 42) of alteration in the oxidative phosphorylation in the tumor bearing animal, but convincing evidence is still lacking in vivo (14, 24). Finally the protein turnover (synthesis and degradation) accounts for a significant proportion of basal energy expenditure (about

507c).

An incre-

ase in protein turnover, either in synthesis or breakdown could explain the alteration of resting metabolic expenditure. Alterations in protein metabolism can occur in malignant tissue (21, 22, 23, 32) and recently Carmichael et al. (10) have demonstrated

a correlation between rates of

protein synthesis and degradation and extent of tumor in patients with colorectal carcinoma and have suggested a translocation of protein stores from muscle to areas of more rapid protein synthesis such as tumor. Finally it is of particular interest the observation of changes which inevitably occur in various body organs in malignant disease. The liver has a high oxygen consumption and therefore contributes relatively more per unit weight to energy expenditure than other tissues. Although liver size is often diminished in malnutrition (27), may be increased in patients with cancer independently from presence of hepatic metastases. An alteration in the liver function cannot be ruled out as contributory factor to the change in the resting metabolic expenditure.

468 In humans, when energy expenditure has been estimated as basal metabolic rate, this rate frequently has been found elevated and there are very few reports in the 1 terature of a low basal metabolic rate in patients with metastatic cancer (58). Some variation in reported basal metabolic rates among and within the various studies probably reflects differences in methodological and biological variables, as well as possibly effects of different cancers and severity and stage of disease. If we accept +15-20% as the upper limit of the normal basal metabolic rate on the basis of the date of Boothby and Sandiford (3), than the frequency of abnormal BMR pooled from several studies (6, 8, 37, 39, 68, 49, 57) (Table l) ranges from 35% for patients with carcinoma to 74% for patients with leukemia (4). Table 1 INCIDENCE AND RATE OF INCREASED BMR IN PATIENTS WITH MALIGNANCY Type Leukemia

No. pts. 133

% pts. with high BMR

Mean value

74.4

+35

Plasmacytoma Lymphoma

4

50

+28

Miscellanea

31

35

+38

INT series

65

59

+29

64.8

+32

Total

233

469 Despite the extra energy requirement is relatively small, its cumulative effect may be significant over a long period. In fact a correlation between increased energy expenditure and malnutrition evaluated as weight loss and decrease in serum transferrin has been reported by Bozzetti and Co. (6). From a speculative point of view it is intriguing the fact that although healthy subjects with decreased alimentation usually demonstrate decreased metabolic rate, in contrast, patients with cancer may show increased metabolic rate despite their falling caloric intake. Starvation causes a decrease in oxygen consumption, resulting in diminished ATP requirements since the formation of glycogen, fatty acids and triglycerides that follows the transient excess of foodstuffs is diminished. Waterhouse (64), however, demonstrated that in patients with cancer long after the postabsortive state, the oxidative metabolism persists at increased rates. It appears therefore that the energy expenditure is inappropriately high in the semistarved patient with cancer. Decreased caloric intake The second major factor in negative energy balance in cancer patients is decreased caloric intake. The insufficient food consumption may be consequence of (1) localized disease-related effects (lesions localized to the upper digestive tract), or of (2) systemic disease-related effects. These include anorexia, changes in taste and small sensation, increased sense of fullness interfering with eating. Finally (3) treatment-related effects include for instance undernutrition following total gastrectomy, malabsorption following pancreatectomy, and nutritional complications of radiotherapy and chemotherapy. A special problem for both radiation and chemotherapy is the development of conditioned aversions. If a food is eaten shortly before the patient receives therapy, the acute malaise resulting from therapy may be psychologically associated with the food eaten before therapy. The patient may, thereafter, get a conditioned aversion for that food. Malabsorption Idiopathic steatorrhea or villous atrophy has been associated with carcinoma of the head and neck, bronchus, gastrointestinal tract, skin and ova-

470 ry (7). The presence in cancer patients of mucosal atrophy accompanied by weight loss and steatorrhea led Creamer (12) to suggest the term "cancer enteropathy" to indicate that some of the ill h e a l t h and loss of weight in advancing cancer is caused by abnormal small intestinal mucosa. H o w e ver subsequent studies (2, 19, 28) failed to confirm the findings of Creamer and others (13, 17, 20). Moreover, it has b e e n demonstrated (2) that the mucosal changes observed in cancer patients are quite similar to those in patients died from wasting disease other than cancer or gastrointestinal disease. Although these mucosal changes occur in cancer patients, they are not specific for cancer and the concept of "cancer

enteropathy"

is no more tenable. It seems probable that the mucosal changes are not the cause but the effect of cachexia, even if they m a y perpetuate to p r o g r e s sive malnutrition in a vicious circle. Energy balance A study which attempts to put the relative roles of increased caloric d e mand and decreased caloric intake into quantitative perspective h a s b e e n reported by Warnold et al. (62). In a series of

10 cancer patients, e n e r -

gy expenditure per 24h was calculated from oxygen consumption and heart rate data, and energy intake was calculated from records of food intake and standard food composition tables. These data were compared to the findings in control patients of similar age w i t h diseases affecting

physical

activities to about the same extent. Energy intake (mean 1270 Kcal/day) although varying greatly, w a s not significantly different from the intake in the controls (mean 1470 Kcal/day). Both the daily energy expenditure (mean 2020 Kcal/day) and the resting metabolic rate (mean 1630 Kcal/day) were significantly greater in the cancer patients than they were in the controls (mean, 1420 and 1170 Kcal/day, respectively). Thus the cancer patients has an average caloric intake that was nearly sufficient for a r e sting state, while their measured energy expenditure was comparable to that expected w i t h moderate activity.In the experience of Warnold (62) and of Bozzetti (6) treatment of tumor resulted in a drop of energy expenditure. Protein metabolism

471 Three separate studies have demonstrated that some cancer patients have anomalously h i g h protein turnover rates (10, 53, 4 5 , 9). Carmichael et al. (10) studied 11 patients w i t h colorectal cancer. W h e n synthesis was p l o t ted against stage of disease, synthesis and breakdown (ie. protein turnover) were found to increase as the severity of the tumor burden increased. A parallel increase in the apparent synthesis rate of the mixed plasma proteins w i t h increasing tumor burden was also found. Stein et al. (53) found that relative to normal controls (healthy young adults) those cancer patients who were o n TPN had a higher m e a n protein synthesis rate. Also Norton et al. (45) w h o studied patients w i t h various intestinal tumor found that three patients had a marked elevation of their whole body p r o tein synthesis rate. The reason w h y protein turnover is increased in some cancer patients is unclear. It is possible that this increase is not real but merely reflects the fact that the pool of faster turning over visceral proteins is r e l a t i vely expanded due to the depletion of the slower turning over muscle p r o teins (11, 69). Alternatively, the increase is real and probably it d e r i v e s from the host's tissue rather than from tumor. In fact a study on operable gastrointestinal malignancies, Stein and C o . determined the tissue fractional protein synthesis rate and concluded that "the tumor did not have an exceptionally h i g h fractional protein sinthesis rate relative to other visceral tissues, but it did sequester a disproportionate amount of parenterally administered aminoacids" (40, 54). Another possible

explana-

tion correlates the elevated protein synthesis rate to liver dysfunction. According to this hypothesis (52) the elevated protein synthesis rate would not be a direct consequence of the tumor on the host's protein m e t a bolism, but rather and indirect effect secondary to the metabolic load placed on the host's metabolic capacity by the tumor. The synthesis of v i s c e ral proteins is variously effected by the presence of cancer. Synthesis of secretary proteins such as acute-phase reactants (43), immunoglobulin (64) fibrinogen m a y be increased while the synthesis of liver structural p r o teins is normal. The liver seems to be protected from catabolic protein balance in the tumor-bearing host (34, 3 5 , 55) and liver size remains n o r m a l or increases possibly because of increased adrenocortical activity (21). The liver normally accounts for 20-25% of total oxygen consumption,

472 therefore, continued hepatic protein synthesis m a y be an important factor in energy balance in cancer patients (71). In contrast synthesis of album i n is less than normal (56) and there is some evidence that a factor in the blood of the tumor-bearing host m a y suppress albumin synthesis

(59).

Synthesis of muscle proteins is generally depressed and breakdown increased (21, 22, 23, 31, 32, 33). Reduction in protein synthesis is comparable to that of pair-fed healthy controls who showed however a decreased breakdown of muscle protein. W h e n protein synthesis is measured in cultures of tissues , the depressed synthesis is only partially corrected by an addition of an excess of aminoacids (32) or insulin (36). Enzyme activities in muscle tissue of patients w i t h cancer show depression of key enzymes of anabolism and increases activity of catabolic enzymes such as Cathepsin D w h i c h correlates w i t h the increased protein degradation. Aminoacids released from skeletal muscle are used for tumor protein synthesis and for gluconeogenesis to provide glucose for tumor metabolism (44, 18). Carbohydrate metabolism Carbohydrates provides a major source of the energy requirements of tumor tissue which metabolizes glucose predominantly v i a anaerobic glycolysis. The uptake of glucose is proportionate to tumor size and is approximately 1.4 g/24h/g of tumor according to perfusion experiments by Norton (45).This level of glucose uptake(230g/24h) by tumor agrees w i t h the increased g l u cose turnover of 170 g/24h reported by Holroyde (25). One consequence of anaerobic metabolism is the release into circulation of lactate by tumor cells. The recycling of lactate v i a the Cori cycle requires an energy e x penditure of 6 moles of ATP per mole of glucose synthesized. The anaerobic breakdown of glucose to lactic acid by the tumor yields 2 moles of ATP for a potential net loss of 8 moles of high-energy phosphate by normal tissue. If production of lactate exceeds the ability of liver or kidney to m e t a b o lize it, lactic acidosis m a y develop. Excessive lactate production m a y have a role in producing anorexia. A n interesting feature of carbohydrate metabolism w h i c h m a y be relevant to cachexia is abnormality of glucose

in-

tolerance (38, 5 1 , 66, 70). Various authors have demonstrated that cancer patients have a higher m e a n blood glucose, w i t h a slower insulin response,

473 lower insulin/glucose ratio and insulin resistance when compared to controls (48, 50, 51). Lipid metabolism The cachectic patients demonstrate an overt wasting of body fat. Free fatty acids serve as an important metabolic full for the liver, the cortex of the kidney, and cardiac and skeletal muscle. Under conditions of long-term fasting the latter three tissues, as well as the brain, can obtain energy from the combustion of acetoacetate and ^-hydroxybutyrrate, and the ketone bodies which are products of fatty acid oxidation. Fasting free fatty acid levels are usually normal (25, 48) in the cancer patients and they fall appropriately in response to insulin (36, 47). However, free fatty acid oxidation was suppressed to a lesser extent by glucose loading of cancer patients than that observed in normal controls (66) in contrast to recent observations by Axelrod and Costa. The decreased suppression of free fatty acid oxidation probably reflects utilization of glycerol for the accelerated gluconeogenesis (47). CONCLUSION Interest in cancer cachexia is not merely speculative. It has been demonstrated that 10-23% of terminal cancer patients ultimately die exclusively because of cachexia (26, 61). More recently DeWys et al. (16) using data from 3047 patients enrolled in 12 protocols of the Eastern Cooperative Oncology Group, definitely showed that median survival was significantly shorter in nine protocols for the patients with weight loss compared with the patients with no weight loss, and chemotherapy response rate were lower in patients with weight loss. Decreasing weight and frequency of weight loss were generally correlated with decreasing performance status and with increasing number of anatomic sites involved with metastases, but within homogeneous performance status categories and categories of anatomic involvement, weight loss was associated with decreased median survival. As it always occurs when people get interested in some kind of clinical nutrition which is not directly related to starvation, as cancer cachexia or infection or injury, the boundaries between alterations due to uncomplicated undernutrition and dismetabolic malnutrition are not clear. Even if we define the nutritional status, according to an old definition as "the who-

474 le of physical and biochemical parameters which are altered by a poor or unbalanced nutrition and are reversed to normal by an adeguate intake of nutrients" (F. Black, personal communication), we frequently observe that some "nutritional" parameters are altered in these diseases independently from the nutritional state and it is therefore unlikely that they may be corrected by a conventional nutritional support. Knowledge of the metabolic alterations of cancer is therefore essential to the clinicians involved to the nutritional support of patients with cancer and to the researchers who are interested to the nutritional manipulation of cancer.

References 1.

Axelrod L., Costa G.: Cancer 2: 81-83, 1980.

2.

Barry R.E. : Gut 15: 562

3.

Boothbay W.M., Sandiford I.: J. Biol. Chem. 54: 783

, 1974.

4.

Bozzetti F.: Argom. Oncol, (in stampa).

5.

Bozzetti F., Migliavacca S., Scotti A., Bonalumi M.G., Scarpa D., Ba-

, 1922.

ticci F., Ammatuna M., Pupa A., Terno G., Sequeira C., Masserini M., Emanuelli H.: Ann. Surg. 196: 170-179, 1982. 6.

Bozzetti F., Pagnoni A.M., Del Vecchio M.: Surg. Gyn. Obstet. 150:

7.

Brezechwa-Ajdukiewicz A., McCarthy C.F., Austad W., Cornes J., Harri-

8.

Burke M., Bryson E.I., Kart E.A. Br. Med. J.

9.

Burt M.E., Stein T.P., Schwabe J.G. et al.: Am. J. Clin. Nutr. 34:

229-234, 1980. son W.J., and Read AEA. Gut 7: 572

, 1966. 211-215, 1980.

628, 1981. 10.

Carmichael M.J., Clagne M.B., Keir M.J., Johnston I.D.A.: Br. J. Surg.

11.

Cohn S.H., Gartenhaus A., Sawitsky A., Rai K., Zanzi I., Vaswani A.,

67: 736-739, 1980. Ellis K.J., Yasumara S., Cortes E., Vantsky D.: Metabolism 30: 222229, 1981. 12.

Creamer B.: Br. Med. J. 2: 1435, 1964.

475 13. Deller D., Murrell T.G.C., Blowes R.: Austral Ann. Med. 16: 236 1967. 14. Devlin T.K., Costa G.: Proc. Soc. Exp. Biol. Med., 116: 1095, 1964. 15. Devlin T.M., Pruss M.P.: Fed. Proc. 17: 211

, 1958.

16. DeWys W.D., Begg C., Layin P.T. et al.s Am. J. Med. 69: 491-497, 198a 17.

Dymock J.W., Mackay N., Miller V., Thomson T.J., Gray B., Kennedy E.H., Adams J.F.: Br. J. Cancer 21: 505

, 1967.

18. Fenninger L.D., Mider G.B.: Adv. Cancer Res. 2: 229 19.

H.M.: Cancer 18: 1278 20.

, 1954.

Fischer R.A., Rosoff B.M., Altshuler J.J., Thayer W.R. Jr., Spiro , 1965.

Gilat T., Fischel B., Dammon J., Loenthal M.: Digestion 7: 147

,

1972. 21.

Gold J.: Ann. N.Y. Acad. Sei 230: 103

22.

Goodlad G.A.J., Clark C.M.: Eur. J. Cancer 16: 1153-1162, 1980.

, 1974.

23.

Goodlad G.A.J., Tee M.K., Clark C.M.: Biochem. Med. 26: 143-147, 1981.

24.

Green A.A.: Cancer Res. 20: 233

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Holroyde C.P., Gaburda T.G., Putnam R.C., Paul P., Reichard G.A.:

, 1960.

Cancer Res. 35: 3710-3714, 1975. 26.

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27.

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28.

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ty of Minnesota. Press. 1950. , 1969.

29.

Krebs H.A.: Adv. Enzyme Regul. 10: 397

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Loehry C.A., Creamer B.: Br. Med. J. 1: 827

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Lundholm K., Bemegard K., Eden E., Svaninger G., Emery P.W., Remie

, 1966.

M.J.: Cancer Res. 42: 4807-4811, 1982. 32.

Lundholm K., Bylund A.C., Holm J., Scherstein T.: Eur. J. Cancer 12: 465

, 1976.

33.

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34.

Lundholm K., Ekman L., Edstrom S., Karlberg I., Jagenburg R., Schersten T.: Cancer Res. 39: 4657-4661, 1979.

35.

Lundholm K., Ekman L., Karlberg I., Edstrom S., Schersten T.: Cancer Res., 40: 1680-1685, 1980.

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Terepka A . R . , Waterhouse C . : A m . J . Med. 20: 225

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Theologides A . : A n n . N Y . Acad. Sci 230: 14-22,

59.

Toporek M . : Cancer Res. 31: 1962,

, 1956

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Warburg 0 . : L o n d o n , Constable,

61.

Warnold I., Lundholm K . , and Schersten T.: Cancer Res. 38:

62.

W a r r e n S.: A m . J. M e d . Sci 184: 610-615,

63.

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64.

Waterhouse C.: Ann. NY Acad. Sci 230: 86

65.

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66.

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67.

W a t k i n D.M.: A m . J . Clin. Nutr. 9: 446

68.

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MARKER PROTEINS OF INFLAMMATION AND NUTRITION IN ADVANCED CANCER

Laurent Deneux, Marco Magalhaesf Henri Magdelenat, Pierre Pouillart Institut Curie - 75231 Paris Cédex - France * Universidade Fédéral da Paraiba - Brésil

Introduction The host-tumor relashionship is more and more frequently evoked as a control mechanism of tumor progression (1,2). Among the parameters easily attainable in the investigation of the host response to a tumor burden are the marker proteins of inflammation and nutrition. Some of these proteins, particularly those with a rapid metabolic turnover or a sensitive "switchon - switch-off" synthesis may prove to be usefull for the follow-up of the disease and the efficiency of the therapy. Three marker proteins of nutrition, the retinol binding protein (RBP), prealbumin (PA) and transferrin

(TF) and a marker protein of inflammation,

C reactive protein (CRP) were assayed in ^2 patients with advanced cancers before and during the course of chemotherapy. The initial values were compared to those of healthy donors and cancer patients in clinical remission.

Patients and Methods Cancer bearing patients : 4-2 patients admitted at the Institut Curie for chemotherapy of advanced cancer (CA).

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

478

Table 1 : Cancer patients (Age : 21-65 ; Mean : 47) Primary site

Number

With metastasis

With liver metastasis

Breast

7

6

ORL

5

4

Ovary

4

2

Cervix

4

4

Lung

3

2

Testis

3

3

Sarcoma

3

2

Kidney

2

2

Uterus

2

1

Melanoma

2

1

Other

7

4

4 1 1

Normal values were obtained from 100 healthy blood donors (N) 50 patients in complete remission were also assayed (REM). Serum proteins were assayed by immunonephelemetry using Atlantic Antibodies and a Hyland Disc 120 Nephelometer. Carcinoembryonic antigen (ACE) was assayed by radioimmunoassay

(CIS).

Results As seen in Figure 1, initial CRP values are generally elevated in cancer patients (22/42 patients above the upper normal limit) in contrast with REM patients who do not differ from normals. The difference is statistically significant (p 0,30

"lower

upper limit

0,10 limit

Figure 1 : orosomucoid and prealbumin serum levels at the onset (O) and at the recovery ( R )

References

1. Reid M.M., Craft A.W. and Cox J.R. : J. Clin. Pathol. 34, 479-482 (1981). 2. Koller M.E., Romslo I., Finne P.H. and Haneberg B. : Acta Paediatr. Scand. 68, 93-96 (1979). 3. Schofield K.P., Voulgari F., Gozzard D.I., Leyland M.J., Beeching N.J. and Stuart J. : J. Clin. Pathol. 35, 866-869 (1982). 4. Caldani C., Milano G., Thyss A., Lesbats,G. and Schneider M. : Nouv. Presse Med. U , 3576 (1982).

COMPARISON BETWEEN CRP SERUM VALUES AND HEMATOLOGICAL PARAMETERS FOR DIAGNOSIS AND THERAPEUTIC SURVEILLANCE OF NEONATAL

INFECTION

C. Benattar, D. Vauzelle and A . Lindenbaum Laboratoire de Biochimie (R. Leluc), Hopital A Beclere, 157 Rue de la porte de Trivaux, 921^1 Clamart (France)

J.F. Magny See de Reanimation neonatale (J.C. Gabilan), Hopital A Beclere, 157 Rue de la porte de Trivaux, 921^1 Clamart (France)

The severity of neonatal infections and the scarcity of their clinical symptoms at an early stage has promoted the research for biological parameters of infection which would be both reliable and precocious. a i m of this work was to compare the value of CRP a n d hematologic for an early diagnosis of neonatal

The

criteria

infection.

The clinical samples included 36 newborn hospitalized during the first 3 days of life which were suspected of maternofetal infection. lated C R P values measured by radial immunodiffusion Behring) w i t h hematologic parameters of infection

We c o r r e -

(Partigen LC plates,

(hyperfibrinogenemia,

neutrophilia or neutropenia, myelemia, thrombopenia) a n d w i t h the bacteriological tests.

The latter allowed to differentiate

retrospectively three groups of children.

1.

Eleven newborns presented w i t h septicemia and CRP levels were above

18 mg/1 (19-122 mg/1, m e a n 68.5) at the first examination performed between 2 and 31* hours

(except for 3 patients admitted at 72 hours).

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

486 Initial hematological examinations, performed at the same time as CRP measurement, showed significant abnormalities due to infection in only 27% of the patients.

At U8 hours, 73% of these exhibited clear-cut

hematological abnormalities. regular

The follow-up of CRP levels showed a

decrease of its values, with normal values between 2 and 10 days

following efficient antibiotic treatment. 2.

Nine children had at least 2 bacteriological examinations positive

for the same infectious agent.

One of them had CRP values at 6 mg/1 and

in two of them CRP could not be detected. present any hematological abnormalities.

These three newborns did not The remaining six had increased

CRP levels (l8 - 100 mg/1, mean 20.6) of which only one presented hematological abnormalities following admission, and three after H8 hours. In three cases, the clinical symptoms favored a real infection, but for the other two, it was not possible to confirm the infection; however, in view of the high CRP levels, they were considered as infected and treated accordingly. 3.

Sixteen children had negative bacteriological examinations.

Only one

of them had a high CRP value (21 mg/l) at 15 hours without hematological abnormalities.

CRP levels were not detectable in the 15 other patients,

6 of which had only one, and two had two hematological abnormalities. In conclusion, CRP levels appear to be a valuable parameter: - for a precocious diagnosis of neonatal infection; - for the separation of the children with infection from those which are settled; - and to follow the efficacy of the treatment.

487 Therefore, the determination of CRP values should allow an early diagnosis and a more efficient use of antibiotic treatment in newborns in w h i c h an infection is questioned.

C O N T R I B U T I O N O F P R I N C I P A L C O M P O N E N T S A N A L Y S I S TO T H E S T U D Y O F FLAMMATORY

A N D N U T R I T I O N A L S T A T U S ON I N T E N S I V E C A R E

•Jfr

-3-

-K-

C L A V E L J . P . , G E R B E T D., D I E M E R T M . C . , G A L L I J . , G A L L I

-ft

IN-

PATIENTS

A.,

L a b o r a t o i r e de B i o c h i m i e de l a S a l p é t r i è r e 4 7 , B o u l e v a r d de l ' H ô p i t a l 7 5 0 1 3 P A R I S .

GLASER**P. S e r v i c e d ' A n e s t h é s i e R é a n i m a t i o n de l a P I T I E

INTRODUCTION

The

biological

status The

survey

interpretation

difficult and often The

application

variables

of

principal data

of

in p o s t - o p e r a t i v e

and

of

data

to

multiple

entire

MATERIALS -

METHODS

We

a

by

the

biologist

non

statistical

automated

level serum acid

of

analysis)

allows

and concrete

multiple

of

seven

analysis

protein,

of

laser

albumin,

glycoprotein

Multidimensionnal analysis,

the

physician

view

indépendant

a simple of

patients

who

instrument

evolution

were

was

each

of in

(like complex

relation

IgG,

to

IgM,

and

to

serum

determine

ceruloplasmin, atomic

absorption

levels.

t r e a t m e n t of these data, p e r f o r m e d by principal a method

every

determine

method

transferrin, IgA,

surveyed

surgery.

used

immunonephelemetric

prealbumin,

(orosomucoid),

is

is

biological

procedure

description

patient

spectrometric m e t h o d for zinc and copper p l a s m a

nents

or

tests.

tions.

sample

total

levels

immunological

laboratory

analysis

t h r e e o r f o u r d a y s d u r i n g a 3 - 4 weeks p e r i o d a f t e r An

and

a lot of

quantitative

multidimensionnal

to t h e t e s t e d f u n c -

present

these

nutritional

requires

incomplete.

components an

inflammatory, patients,

to p r e s e n t p a t i e n t s a n d t e s t s

Marker Proteins in Inflammation, Vol. 2 © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

compo-

in a t w o

axis

490

AXE "Inflammatoire

«.V

(•Il

Éi)

a

trf.

Cu " cerul.

li.

prealb. album. Zn ai.-macro. prot.

.s r « AXE "Nutritionnel"

I9G I9M I9A

il

ïl

i i l " of romcuter

rtonnnmO

ë

491 system

in

analyses

relation

to

correlations

which

they are most discriminate.

between

variables

in

order

to

This method

reveal

masked

informations in univariate analysis, and to observe groupings of variables raising various hypothesis. This multidimensionnal analysis method can be used on most microcomputers .

RESULTS

We observed

the usual correlations between Zinc/Prealbumin,

Zinc/Albu-

min, Copper/Ceruleoplasmin, Ceruloplasmin/®