Marker Proteins in Inflammation: Volume 2 Proceedings of the Second Symposium Lyon, France, June 27–30, 1983 [Reprint 2020 ed.] 9783112314883, 9783112303696

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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 27 - 30,1983. - 1984. 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. QZ150 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 250 r e s e a r c h

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 , a School (University, Claude Bernard, participants

Lyon-I)

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

" S e c o n d S y m p o s i u m 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 " sponsorship

of 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

provide a comprehensive of Marker Proteins

under

the

sur les M a r q u e u r s

Scientific Committee of GERMI

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

and their clinical

in o r d e r

the

mechanism

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

p l a y in h o s t d e f e n s e s , a s u b j e c t o f p e r e n n i a l t h r o u g h o u t the m e e t i n g .

proteins

interest which was

the

T h e n e x t three s e c t i o n s

anti-protease

interplay

in

lung

in

biological

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 ,

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

central

n e r v o u s s y s t e m . T h e f o u r t h s e c t i o n o f the book d e a l s w i t h the role zinc, protein deprivation, section features Finally, during

and malnutrition

a g r o u p of p a p e r s on animal

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

a satellite

in h o s t d e f e n s e s . m o d e l s of

The

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

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. Michel

held Caron

aspects of

t h e i r use as a tool

their

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

and their a f f i n i t y and

lectins:

specificity.

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

biological

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

University

of fifth

inflammation.

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,

are

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

devoted respectively

to the p r o t e a s e

to field

implications.

begins with a series of papers concerning

main theme running

de

been

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

of A c u t e P h a s e R e a c t a n t P r o t e i n s

diseases,

The

in the

( G E R M I ) . T h e t o p i c s f o r this s e c o n d s y m p o s i u m had

c h o s e n by the I n t e r n a t i o n a l

The proceedings

and

Medicine

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

had come from fifteen countries

l'Inflammation"

scientists of

Putnam

Indiana

I n d i a n a , U S A ) 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 w a n t 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

institutions

who contributed to the success of the 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

REACTANT

RESPONSE

AND A C U T E

PHASE

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 a^-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

VIII 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. Zouaghl, 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 LUNG

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 a -Antitrypsin and i^-Macroglobulin J.-P. Revi 1 lard, 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-Fayolle

243

Sarcoidosis: IgG, 82 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 of 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, O. Freney, F. Touraine

345

Evaluation of CSF Lymphocyte Subsets Using the OKT Series of Monoclonal Antibodies (OKT 3+ , OKT 4 + , OKT g + ) 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 Acute 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

401

INFLAMMATION AND 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 0. 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. Hervé, 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

531

MARKER PROTEINS IN EXPERIMENTAL MODELS OF 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. B(zSg-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

569

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

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. Coupe, G. Labrecque

SECTION VIII

585

LECTINS

Introductive Notes M. Caron, A. Faure

591

XVII P a r t 1: L e c t i n s

as

Tools

T h e U s e o f L e c t i n s to S t u d y C h a n g e s i n R a t M e m b r a n e Glycoproteins Induced by Oral Contraceptives B. Toor, L. M c G r e g o r , K.J. Clemetson

J. McGregor,

S.

Renaud, 597

I d e n t i f i c a t i o n of C y t o p l a s m i c a n d M e m b r a n e P l a t e l e t G l y c o p r o t e i n s U s i n g a C o m b i n a t i o n of S D S - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s and 125].-Labelled L e c t i n s P. C l e z a r d i n , J.L. M c G r e g o r , K.J. Clemetson

E. James, M.

Dechavanne, 601

C h a r a c t e r i z a t i o n of Human Epidermal R e c o g n i z e d b y the L e c t i n s C o n A a n d

Glycoproteins PNA

P.W. Ledger, A. Réano, J.Y. Bonnefoy, J. Thivollet Fluorescent Lectins M.A. Deugnier,

and Quantitative

Microscopy

X. A l b e , J.C. B i s c o n t e

609

Effects of Serum o n L e c t i n - I n d u c e d Lymphocyte P.J. Neveu,

Proliferation

D. P e r d o u x

613

Concanavalin A and Prothrombin

Complex

M. C o u l e t , M . J . Bezou, P. T r a v a d e M o d i f i c a t i o n of T h y m i d i n e T r a n s p o r t in Stimulated Lymphocytes P. B e a u d r y , A. Sharif,

605

615 Lectin-

Y. G o u s s a u l t

619

I n d u c t i o n of a T h y m i d i n e M e t a b o l i s m e E n z y m e i n H u m a n Lymphocytes S t i m u l a t e d by the R o b i n i a p s e u d o a c a c i a L e c t i n A. Sharif,

P. B e a u d r y ,

Lectins of R o b i n i a J. W a n t y g h e m ,

Y. G o u s s a u l t

621

pseudoacacia

C. G o u l u t ,

J.P. Frénoy,

Y. G o u s s a u l t

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

SECTION I THE INFLAMMATORY RESPONSE AND ACUTE PHASE REACTANT PROTEINS

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 v/hose 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 catabolisn 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 II - about 2-4 X increase

Ceruloplasmin C3, C4

a-) acid glycoprotein oq anti-trypsin a] anti-chymotrypsin Haptoglobin Fibrinogen

Group III - 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.

It is undoubtedly the precursor of the

major fibril 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, is a pentraxin; it is composed of identical subunits of 21 ,000 daltons, arranged in cyclic symmetry.

The related molecule, SAP, another pentraxin, is a plasma

protein originally discovered because of its occurrence in all types of amyloid tissue.

This protein shows over 60% amino acid homology with

CRP. It is an acute phase protein in some species, such as the mouse, but not in others, such as man.

TABLE II summarizes some of the similarities and differences between three of the plasma proteins which have just been referred to.

SAP is

glycosylated and makes up about 15% of all types of amyloid tissue, while SAA is found only in, and characterizes, the amyloidosis seen in individuals with chronic inflammatory states. One of the striking observations regarding the acute phase response is the great variability 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 III.

The major acute phase proteins in man are

5

TABLE II CRP

SAP

SAA

Acute phase protein in man

+

-

+

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. occur in the rat.

SAA apparently does not

In each of these species other plasma proteins

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

et] 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

(^-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 i s a tremendous difference in the rapidity of r i s e in serum concentrations between CRP and SAA, on the one hand, and the other acute phase proteins. a^-antichyniotrypsin.

The next most rapid r i s e i s shown by

Rate of r i s e in concentration of other proteins

i s even slower than a^-antichymotrypsin, as shown by L a u r e l l ' s group over a decade ago (5).

These types of observations suggest the

p o s s i b i l i t y 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 e a r l i e r .

The difference in the

magnitude of response was largely due to the difference in duration of continuing r i s e in concentration, and, i n f e r e n t i a l l y , 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 l i v e r s 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. D.

Serum Turnover Studies

It was possible to calculate approximate in vivo CRP synthetic rates from serum turnover studies in rabbits. In unstimulated rabbits, the serum 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 narked 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 yg/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 yg/ml

ca. 2,700

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

III

Molecular Biology

Concentrations of mRNA for rat a-| 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 Col ten'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 (IP).

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 ug/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 lists 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 is reported in one instance, rat fibrinogen (23). While SAP induction by Interleukin 1 preparations is reported by one group (24), an increase of only about 10", hardly persuasive, is reported by another (25).

There are differences regarding the effect of normal

serum on SAA synthesis.

TABLE VI

FACTORS INDUCING ACUTE PHASE

PP.OTEIN 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, al AT, C3 rat haptoglobin

(23) (27) (28)

rat ai AGP, a2 macro

(29)

murine SAA but not SAP

(25)

murine SAA (not induced by normal serum)

(30)

II CORTICOSTEROIDS ALONE III NORMAL SFRUM (fetal calf, human, mouse) IV LATENT PHASE

SERUM

(mouse)

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

12

In our own attenpts 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 t h i s 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 t h i s

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, 10565-10568 (1979).

4.

Kushner, I., Feldmann, G.: J. Exp. Med. 801_, 466-477 (1978).

5.

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

6.

Macintyre, S.S., Schultz, D., Kushner, I.: Biochem. J. 2J0, 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 (1979).

9.

Rosenthal, C.J., Sullivan, L.: J. Clin. Invest. 62, 1181-1186 (1978).

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. 7_1_, 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, J.E., 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 (1983).

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.

Dinarel lo, C.A.: Lymphokines ]_, 24-74 (1982).

20.

Bornstein, O.L., Walsh, E.C.: J. Lab. Clin. Med. 91_, 236-245 (1978).

21.

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

22.

Turchik, J.B., Bornstein, D.L.: Infect. Immun. 30, 439-444 (1980).

23.

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

14

24.

Le, P . T . , Müller, M.T., Mortensen, R.F.: J . Immun. 129, 665-672 (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. M. 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 . 21_, 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 . Chem. 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., Hordstoga, 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, a2-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

-

types-hepatoKupffer, Ito

and endothelial cells (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 reticulum

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

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 raaintening 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 category there 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 (11-13). 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), a2-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 in the rat for four different APRP

(9) and

(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). Opti-

cal (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 reasonable 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» so 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 followed 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 Mirs Maurice a.nd Malet for their help in the preparation of the manuscript.

References 1.

Koj, A.: In Structure and function of plasma proteins, All ison, A.C. edit., Plenum Press, London, Vol 1, pp 73 — 131, 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 189, 347-358

8.

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

9.

Kushner, I., Feldmann, G. : J. Exp. Med. .U8, 466-477 (1978).

(1975). (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. 12, 11-25

(1975).

12. Canonlco, 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. 2J3, 271-279 (1964). 18. Peters, J.H., Alper, G.A. : J. Clin. Invest. 4_5, 314-320 (1966) . 19. Feldmann, G. : J. Microscopie 2_1, 293-300 (1974). 20. Pignal, F., Maurice, M., Feldmann, G.: J. Histochem. Cytochem. 1004-1014 (1982). 21. Feldmann, G., Penaud-Laurencin, J., Crassous, J., Benhamou, J.P.: Gastroenterology 6j3, 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., Eeldmann, G., Rogier, E., Moguilevsky, N.: Lab. Invest. 45, 67-76 (1981). 28. Bernuau, D., Rogier, E., Feldmann, G.: Hepatology 2 2 9 - 3 3 (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 7_8, 4718-4722 (1981). 31. Lebreton, J.P., Joisel, F., Raoult, J.P., Lannuzel, B., Roger, J.P., Humbert, G.: J. Clin. Invest. 64, 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

in Plasma

the

First

progress

reflected

in

about

Symposium

in

plasma

At t h a t

some 50

physical

properties

available

normal

that

I n many c a s e s quantitative

disease states.

immunoglobulins

structure their

genetic

specific

antisera

for

of

changes

T h i s was the

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

the acute phase p r o t e i n s

and f u n c t i o n .

biosynthesis

could other

control

Furthermore, or

the

factors

that

are a

greatly

we know l i t t l e

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

of

principal

function,

differ

the

especial-

Y e t , whereas the i m m u n o g l o b u l i n s

f a m i l y of p r o t e i n s c l o s e l y genetic control,

and a l s o

much

and

measurement

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

ly

the

reac-

They

mobilities

(2-4).

in various

for

proteins.

allowed

acute phase r e a c t a n t s .

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

(2-4),

Mar-

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

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

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

proteins

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 were

Ortel

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

and Thomas L .

University

47405

Progress

introduction

kers

Indiana

in

about

regulate

response in

and

their

inflamma-

tion.

At

the time of the F i r s t

ture,

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

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 ing

of

First the

primary

principal

had been

for

alpha

complete

Then,

published

in

laboratory

acid

1982

has

table

acute

phase

of

Carrell

completed

the

shown here as T a b l e

Antitrypsin Cerulopl asmin C-reactive protein

of

the

haptoglobin,

structure

(6)

acid

table

of

proteins: was known

However,

the

1-antitrypsin

was

and

recently

sequence

of

properties

our human

can

be

1.

Pi

Sequence

40,000

2.7

41.4

55-140

complete

^AT

54,000

4.8

12.4

200-400

complete

Cp

135,000

4.4

8.0

15-60

complete

0

en

20 40 60 SAP ( ug/ml) Figure 16: v Effect of SAP on clot times (±sd) generated by addition of Russmll's Viper Venon-cephalin (RVV) in the presence or absence of heparin (see Figure 14). From reference (4).

NO HEPARIN

20

30

40

SAP (¿¿g/ml)

116 The p o t e n t a n t i c o a g u l a n t a c t i v i t y of v a r i o u s c o n c e n t r a tions of SAP in the R W assay in the p r e s e n c e of h e p a r i n is s h o w n in Figure 16. A s R W assay initiates the c o m m o n c o a g u l a t i o n p a t h w a y at the level of F a c t o r X, this r e s u l t , t o g e t h e r w i t h r e s u l t s from F i g u r e s 14 and 15, show that in the p r e s e n c e of h e p a r i n e a c h the i n t r i n s i c and e x t r i n s i c p a t h w a y s w e r e a f f e c t e d by small a m o u n t s of SAP ( 9 0 %) of macrophages.

preferen-

respiratory

lavage of normal smoker and nonsmoker

which consisted principally in vitro culture,

factors that

(7) o b t a i n e d

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 p l e x p r o d u c e d a m a x i m a l r e s p o n s e w i t h i n 3 h o u r s . T u m b l i n g the c e l l s prevent adherence greatly diminished the output of 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 components macrophage cultures

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 which could be

g u i s h e d by s i z e , a b o u t 8 , 5 0 0 d a n d a b o u t 1 , 0 0 0 d o n c a l i b r a t e d 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 .

distin-

Sephadex

The larger

substance

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 by i s o e l e c t r i c f o c u s i n g i n t o 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 one p e a k a p i o f 5.0. T h e 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 m a c r o p h a g e 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 t h e

least with

early lipoxyge-

nase pathway of prostaglandin metabolism was inhibited. A number of genase products have chemoattractant activity 10, 1 4 - E i c o s t e t r a e n o i c a c i d

duced by s t i m u l a t e d alveolar macrophages Hunninghake

and colleagues

including 12-hydroxy

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

LTB^

lipoxy5, 8,

; b o t h c a n be

its

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

in 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

from source

otherwise

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

activity stimuli

w o u l d a c c e l e r a t e 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 complement receptors 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 the tic activity which resided

in a s m a l l s i z e f a c t o r . As 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 T a b l e

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-

(8).

(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

nonsmoker normals produced chemotactic activity and identified

unstimulated,

i-

indicated that this factor

Its was

157 in part lipid and a lipogenase pathway

substance.

AIRWAY-ALVEOLAR SURFACE

CAPILLARY-ENDOTHELIAL SURFACE In summary

( 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

s i t e d o n t h e a l v e o l a r s u r f a c e w h e r e t h e y are s c a v e n g e d

c a n be

depo-

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

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

di-

rected migration of auxiliary phagocytes, principally PMNs. Although

much

o f t h e 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 have biologic activity and produce a PMN-Exudate

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 u n i n v e s t i g a t e d ,

(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

ty o f the a l v e o l a r e p i t h e l i u m or 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)

PMN

It S

permeabili-

diffusability

t h r o u g h l a y e r s o f the a l v e o l a r - c a p i l l a r y

membraneous

b a r r i e r . 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 t h e 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 a r e a t t r a c t e d f o r

endothesticking

158 (margination) and cell polarization as a prelude for migration into an extravascular

location.

3. Complement-derived

chemotaxins.

The role of the complement system in host defense of the airways and alveolar surface remains controversial. Properdin Factor B (10) and a number of other complement components have been identified in lung lavage fluids (11) which support the concept that alternate pathway activation may be important in the lung (Reviewed in Reference 2). Interest focuses primarily on C^a and C,_a des arg, considered to be the most potent 5

inflammatory

b

mediators and chemotactic molecules liberated by the complement cascades. These are capable by themselves of causing acute airway hemorrhage and exudation after intratracheal instillation (12, 13). If direct activation of alternative complement pathway actually occurs in the airways, then it is reasonable to postulate that a variety of inhaled or aspirated particles and microbes that gain access to the respiratory tree can trigger the complement cascade (C„3 and C,. b plus other intermediate components like the trimolecular complex C „ „ could be liberated and produce a number of 567 fragments with chemoattractant properties). Quantitatively, C,. derivatives 5 seem to be the most effective and potent of the complement group. A potential complement derived chemotactic factor, such as C,_a, might be 5 generated through activation of the complement system or exist as a preformed component in lung fluid. The situation is not clear at present, for some of the conflicting results have been presented only in preliminary form. In nonhuman primates (baboons), C,_ 5 with functional activity can be identified in generous amounts in concentrated lung lavage fluid (14) ; whereas, in lavage fluid from normal nonsmokers, it is not prominent

(15).

Macrophages can secrete a number of complement components which could provide a local supply within the airspaces. Two studies that identified complement components secreted by human alveolar macrophages in culture fluid found different components

: in one C„ and C,_ were detected (16) ; the 3 5

other noted C^ and Factor B and occasionally C^ (17). The necessity of having an intact complement system to clear bacteria deposited in the airspaces is not well established either. When a variety of

159

gram positive and gram negative bacteria were aerosolized

i n t o the

lungs

o f m i c e , p r e v i o u s l y d e p l e t e d o f c o m p l e m e n t a c t i v i t y by C O B R A V e n o m 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

Streptococcus

pneumoniae

and Pseudomonas

in 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-

aeruginosa

(18).

Subse-

quently, stronger evidence has been provided that Pseudomonas clearance better with 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 t h e 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

host

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

inflam-

m a t i o n o r o p s o n i c p h a g o c y t o s i s t h r o u g h s u p p r e s s i o n o f the c o m p l e m e n t ways or inhibition of the activity of individual

4. O t h e r

inflammatory or chemotactic

As i l l u s t r a t e d in F i g u r e

components.

mediators.

1, a n u m b e r of i m p o r t a n t s y s t e m s c a n be

triggered

b y t h e a c t i v a t i o n o f H a g e m a n F a c t o r 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 complement

(20),

intrinsic coagulation,

It is n o t e s t a b l i s h e d

fibrinolysis and kinin

that Hageman Factor

( F a c t o r X I I ) is a n

c o m p o n e n t o f the a i r s p a c e l i n i n g f l u i d , n o r t h a t p o t e n t i a l these enzymatic systems must depend on this molecule.

formation.

important

is e s t a b l i s h e d

in the a i r s p a c e s ,

factors or complement.

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 b y 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 activate Hageman Factor and could be a mechanism the d e p e n d e n t s y s t e m s .

enzy-

can

for triggering some

of

T h i s s e e m s to be the 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

plasminogen,

molecular weight kininogen and complement components

(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

fluid that can cleave Hageman Factor, prekallikrein,

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 be 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

of

It is, i n s t e a d ,

p r o d u c t s to t h e 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

activation

convenient way to group a number of related systems that may

be important once

path-

high to

chemotactic and brady to the

lea-

160 kage of plasma across the capillary-alveolar membrane. Plasminogen activator is of special interest because it can be generated by fibrinolysis or can be secreted by alveolar macrophages and,therefore, could arise in different ways. The interaction between various Hageman Factor dependent pathways and complement in the airways is largely an unexplored network that is probably important in initiating or establishing inflammation. Obviously, more research is needed to unravel the components involved, the importance of other acute phase reactants, discussed by other participants in this Symposium, and the overall biologic significance of them.

5. Examples of Diseases in which chemotactic factors may be found in lung fluids or cell culture media.

a. Idiopathic pulmonary fibrosis

(IPF).

To substantiate the concept that an alveolar macrophage-derived

chemotac-

tic factor might participate in the inflammatory reaction in human lung diseases, Hunninghake and colleagues (23) sought evidence of chemotaxin secretion by alveolar cells lavaged from patients with IPF. This was a relevant disease to study because a low-grade alveolar inflammation is often present, especially in an early, cellular stage of disease. Analysis of bronchoalveolar lavage fluid from IPF patients (11) revealed that among the respiratory cells retrieved from untreated patients, PMNs accounted for a consistent percentage (mean about 30 %, 5 to 80 range observed) of the cells. A small percentage of eosinophils

(about 4 %) also was cha-

racteristic . A group of 15 patients with IPF, considered to be in midstage of their disease, were investigated for possible secretion of alveolar

macrophage-

derived chemotactic factor and compared with 8 nonsmoker normal

volunteers

(23). After BAL to retrieve respiratory cells from both groups, the cells were submitted to Hypaque-Ficoll density centrifugation to remove PMNs ; the purified mononuclear cells, principally alveolar macrophages,

were

cultured for 3 h after which supernatant fluids were harvested and evaluated for chemotactic activity. Various stimuli were given to the macrophage cultures in the form of concentrated BAL fluid from IPF patients,

Sepharo-

161 se 4B beads, and IgG coated ox erythrocytes. Results of respiratory

diffe-

rential cell counts in the patients showed that 9 of 15 had greater than 10 % PMNs among the cells (approximately 35 %), and that the other p a tients had less than 10 % ; the normal nonsmokers did not have PMNs. Once in culture, macrophages from IPF patients with > 1 0 % PMNs in their original mixture of lavage cells spontaneously released chemotactic activity into the culture medium, whereas cells from those with fewer than 10 % PMNs generally did not release activity, nor did the cells from nonsmokers. The IPF macrophages generated large amounts of chemotactic activity quickly (by 3 h)

; when this activity was localized in the chromatogram

(Sephadex G-25 gel) of the supernatant fluid, it was found in the 400 to 600 molecular weight range fractions eluted from the calibrated column. Further analysis of this material showed preferential chemoattractant activity for PMNs compared with blood monocytes, and this activity was largely extractable with organic solvents, indicating a significant lipid component to be present. IgG immune complexes were considered to be an important stimulus for production of this chemotactic factor. Thus,

Hunningha-

ke and colleagues succeeded in identifying the same kind of small molecular weight, partially lipid-containing chemotactic factor they had previously found from normal alveolar macrophages

(9). This study

indicated

that a chemotactic factor was released from alveolar macrophages of patients with IPF who were in an active phase of alveolitis with PMNs present in bronchoalveolar lavage fluid. IgG immune complexes appear to be an important macrophage stimulus. Thus an endogenous, airside can be released quickly within a few hours and this capability with an elevated PMN count ( > 1 0 % respiratory

chemotaxin correlates

cells).

b. Sarcoidosis. Sarcoidosis is a common, multisystem disease of unknown cause that characteristically produces non-caseating epithelial-giant cell granulomas in affected tissue. Although the respiratory tract is involved in most p a tients, pulmonary sarcoidosis is usually self-limited or resolves in the majority of them. However, in about 20 per cent, the pulmonary disease is progressive and can lead to a chronic stage of interstitial

fibrosis

and cystic airspace degeneration. In recent onset or intermediate stages

162 of lung disease,

the h i s t o l o g i c r e s p o n s e

lar infiltration w i t h lymphocytes, in interstitial

in t i s s u e

primarily,

is a m i x t u r e o f

and granuloma

tissue, bronchial and vascular structures.

alveo-

distributed Sampling

the

a i r s p a c e s w i t h l u n g l a v a g e y i e l d s a n i n c r e a s e d 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

c e l l s c a n be i n c r e a s e d not prominent

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

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

cells

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

with prolonged disease associated with fibrosis, PMNs can be increased 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

h a v e some m a r k e r t h a t m i g h t d i s t i n g u i s h t h o s e p a t i e n t s w h o w i l l h a v e gressive disease. Moreover, excessive,

P M N s in the 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 proteolytic enzymes and

w h i c h c o u l d f o s t e r the f i b r o t i c p r o c e s s . tic substances litis,

with

chemotac-

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 i n f l a m m a t o r y

alveo-

(23).

From the clinical service at Laennec Hospital, patients with lung

sarcoi-

dosis underwent fiberoptic bronchoscopy and lung lavage for cellular sis as part of diagnostic evaluation including

(24, 27). S e v e r a l g r o u p s w e r e

: normal non smoker volunteers

("¡^6 m o n t h s )

(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

(n = 5), a n d l o n g d u r a t i o n d i s e a s e fluid and respiratory

(i»2 y r )

tion o n discontinuous

disease

corticosteroids

(n = 5). The a c e l l u l a r

lavage

cells were assessed for chemotactic activity

tes from macrophages

density

g r a d i e n t s 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

(7). C e l l s w e r e f r a c t i o n a t e d by

albumin-density

analy-

defined

(direc-

ted migration of blood PMNs) using a leading front migration assay blind well diffusion chambers

in

centrifuga-

lung

lymphocy-

a n d 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

in c u l t u r e a n d

incuba-

t e d f o r 6 a n d 16 h o u r s to o b s e r v e s p o n t a n e o u s r e l e a s e o f c h e m o t a c t i c tances

into c e l l s u p e r n a t a n t ,

and thereafter

subs-

s t i m u l a t e d by p h a g o c y t i c

lenge with zymosan. Lung lavage fluid and cell culture supernatants

(organic solvent) elution

chal-

were

g e l f i l t e r e d t h r o u g h c a l i b r a t e d c o l u m n s or c h r o m a t o g r a p h i e d w i t h h i g h sure liquid

to pro-

inflammation

T h i s l e d u s to look f o r

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

in

pres-

gradients.

R e s u l t s are s u m m a r i z e d . B r o n c h o a l v e o l a r

lavage fluids, with cells

removed

163 by centrifugation, and concentrated at 4° C with an Amicon UM-05 membrane, had no detectable chemotactic activity for PMNs, except several

specimens

from long duration sarcoidosis patients. W h e n these active BAL fluid samples were chromatographied through Sephacryl G-300 SF and chemotactic activity monitored in the eluant fractions, it was only detectable in fractions that eluted at about 95 per cent of gel bed volume,

corresponding

to a small molecule of several thousand daltons size. This reasonably cluded several other possible chemotactic factors of larger size

ex-

including

C^a (15,000 d), neutrophil chemotactic factor of asthma (600,000 d) (29) b or lipopolysaccharide

(endotoxin) from contaminating bacteria added per-

haps from the transnasal passage of the bronchoscope. However, the PMNs percentages in the respiratory cell counts were not very elevated in these patients (up to 4 St) , so the presence of chemotactic activity did not reflect an elevated count. For macrophages in culture, normal cells did not spontaneously

secrete

chemotactic activity, but did so with stimulation. Macrophages from recent onset sarcoid patients and those receiving corticosteroid therapy also did not spontaneously make chemotactic activity, but, surprisingly,

stimu-

lation with zymosan did not induce activity either. It is uncertain why these cells were unresponsive, for sarcoid macrophages are generally activated cells. In contrast macrophages from long duration patients either were spontaneously secreting activity or could be readily stimulated to make

it.

Cell supernatants were collected from active macrophage cultures, and without concentration or exposure to freezing temperature were applied to a calibrated Sephadex G-25 SF column and gel filtered with phosphate buffered saline, pH 7.2. Chemotactic activity for PMNs was assayed in the effluent column 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 elution volume which was less than 1,000 daltons size. This pattern of chemotactic activity in macrophage supernatants was the same for sarcoid cells that were spontaneously secreting and for those stimulated to secrete chemotactic activity. Analysis of the active components in these two respective peaks is in progress and requiring use of ultraseparation methods. We expect the larger size chemotactic factor to be a complex mixture of p r o -

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 the 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 b y 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 biological

c h a r a c t e r i z a t i o n has n o t detected any p l a t e l e t

f a c t o r or s l o w r e a c t i n g s u b s t a n c e o f a n a p h y l a x i s . been extracted

; chemotactic

is p r e s e n t in 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

has

active sarcoid macrophages

L T B ^ is

from the

than was found with calcium

tion of ostensibly normal alveolar macrophages The 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 size chemotactic

activity for

prespat-

PMNs

probably

L T C „ , LTD„ a n d L T E , w h i c h c o m p r i s e S R S , 4' 4 4 f >

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

are

spontaneously

ionophore

stimula-

(8).

f r o m I P F p a t i e n t s to s e c r e t e a s m a l l

f a c t o r c o r r e l a t e d a m a z i n g l y w e l l w i t h the d e g r e e o f n e u -

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 to i m m u n e

stimulation. We have not found such a relationship

spaces

aggregation

W h e n the s u b s t a n c e

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

one, but other leukotrienes,

dosis patients,

Prelimina-

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 b y r e v e r s e p h a s e h i g h

sure liquid chromatography tern with some

naturally

(31).

complex

in l o n g 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

i n 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 be 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 a l t h o u g h n o t 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 PMNs and other to c h a r a c t e r i z e

6-

inflammatory

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 a s a s s e s s e d

maniand

with

c e l l s m i g h t n o t be t h e c o r r e c t b i o l o g i c

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. This reaction in which phagocytic cells, principally polymorphonuclear neutrophils, immune effector cells, and fluid components from plasma are attracted to the alveoli appears to be initiated and controlled from the airside of the lung. In the early phase of inflammation, chemotactic factors may begin the influx of assessory phagocytes. Such factors can be liberated by alveolar macrophages in the form of small proteins (about 5,000 d mole, wt.) and lipogenase pathway derived metabolites of arachidonic acid of which leukotriene B^ is an example or could be complement components. Activated complement fragments are generated by the alternative complement pathway (C,_a) or, possibly, secreted locally by alveolar macrophages. Other mediators with chemotactic and/or vasopermeability actions may arise from the fibrinolytic or kinin systems. Soluble exo-products from bacteria must be considered, too. W h e n inflammation is established and exudative fluid fills the airspaces, cellular enzymes released from injured or dying cells may help perpetuate the reaction. Thus, factors from several sources can individually or collectively be involved in modulating lung inflammation. In certain lung diseases that feature interstitial and alveolar inflammation, such as idiopathic pulmonary fibrosis and sarcoidosis of long duration,activated macrophages are a source of chemotactic activity that may reflect part of a poorly regulated inflammatory

reaction.

166 REFERENCES. 1.

Reynolds, H.Y., Chest 75 (suppl.), 239-242

2.

Reynolds, H.Y., Merrill W.W.

: iri Current Pulmonology

D.H.) Houghton Mifflin, Boston, 2, 299-323 3.

(1979). (Editor Simmons,

(1980).

Reynolds, H.Y., Am. Rev. Respir. Dis. 127, 516-25,

1983.

4.

Cohen, A., Batra, G. Am.Rev. Respir. Dis. 122, 239-247

5.

Kazmierowski, J.A., Gallin, J.I., Reynolds, H.Y. J. Clin. Invest. 59,

6.

Hunninghake, G.W., Gallin J.I., Fauci, A.S., Am Rev Respir Dis 117,

273-281

15-23 7.

(1977).

(1978).

Merrill, W.W., Naegel, G.P., Matthay, R.A., Reynolds, H.Y., J. Clin. Invest. 65, 268-276

8.

(1980).

(1980).

Fels, A.O.S., Pawlowski, N.A., Cramer, E.B., King, T.K.C., Cohn, Z.A., Scott, W.A., Proc. Natl. Acad. Sei, U.S.A., 79, 7866-7870

9.

(1982).

Hunninghake, G.W., Gadek, J.E., Fales, H.M., Crystal, R.G., J. Clin. Invest. 66, 473-483

(1980).

10. Robertson, J., Caldwell, G.R., Castle, G., Waldman, R.H., J. Immunol. 117, 900-903

(1976).

11. Reynolds, H.Y., Fulmer, J.D., Kazmierowski, J.A., Roberts, W.C., Frank , M.M., Crystal, R.G., J. Clin. Invest., 59, 165-175

(1977).

12. Desai, U., Kreutzer, D.L., Showeil, B.S., Arroyave, C.V., Ward, P.A., Amer. J. Pathol. 96, 71-83

(1979).

13. Shaw, J.O., Henson, P.M., Henson, G., Webster, R.O., Lab. Invest. 42, 547-548

(1980).

14. Kolb, W.P., Kolb, L.M., Wetsel, R.A., Rogers, W.R., Shaw, J.O., Am. Rev. Respir. Dis. 123, 226-231

(1981).

15. Robbins, R.A., Gadek, J.E., Rennard, S.I., Chen, Y.F., Crystal, R.G. Am Rev. Respir. Dis 125, 53 (Abstr)

(1982).

16. Scherzer, H.H., Kreutzer, D.L., Varani, J., Desai, U., Senior, R.M. Am Rev. Respir Dis 121, 92 (abstr)

(1980).

17. Pennington, G.E., Matthews, W.G., Rössing, T.H., Gash, D.J., Cole, F.S., Colten, H.R., Clin.Res.29, 450 (abstr)

(1981).

18. Gross, G.N., Rehm, S.R., Pierce, A.K., J. Clin. Invest. 62, 373-378 (1978).

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

(1983)

(1982).

30. Rankin, J.A., Hitchock, M., Merrill, W., Bach, M.K., Brashler, G.R., 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).

SECTION PROTEASE

III ANTI-PROTEASE

INTERACTIONS AND LUNG DISEASES

HUMAN a.|-ANTICHYMOTRYPSIN

: PURIFICATION,

PROPERTIES AND REACTIONS WITH

SOME SERINE PROTEASES Anne Laine, Annette Hayem, Monique Davril Unité INSERM N°16, Place de Verdun 59045 L i l l e Cédex, France

Introduction a^-antichymotrypsin

(a^Achy) i s a plasma protease i n h i b i t o r which s p e c i -

f i c a l l y i n a c t i v a t e s chymotrypsin-like enzymes ( 1 , 2 ) .

I t represents one of

the major acute phase proteins ( 3 ) . I t appeared that a^Achy was s e l e c t i v e l y concentrated in the bronchial tis

lumen i n patients with chronic

bronchi-

(4). On the other hand, a^Achy which has been shown to be i d e n t i c a l

to the protein termed 64 DP (5) has a high a f f i n i t y for DNA-cel1ulose and it

has been suggested

role

in

diagnosing

antibody

response

inhibitory and till

its

reaction

that t h i s

malignant and

capacity

now received

(6) this

(7).

diseases. property

Moreover

seems

not

In fact the b i o l o g i c a l

mechanism with little

64 DP would have a quite prominent enhances

be related

significance

the serine proteases

attention.

t i o n procedure than the o r i g i n a l

a^Achy to

it

of

its

a^Achy

inhibits

We present here a simpler

the

to

have

purifica-

one we p r e v i o u s l y published (8) and some

studies about the reaction between a^Achy and leukocyte enzymes : during the inflammatory process, polymorphonuclear leukocytes release t h e i r g r a nule contents medium

including

; thus i t

is

elastase

of

interest

and cathepsin G into the e x t r a c e l l u l a r to know how

a^Achy reacts

with

these

enzymes, or how these enzymes react upon c^Achy. Immunochemical led

studies

we c a r r i e d

us to conclude that

out

quantitation

using three d i f f e r e n t of

procedures

such a component which can be

under d i f f e r e n t molecular forms must be very c a r e f u l l y exploited.

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

172 Results

1)

Purification

procedure.

a^Achy

the procedure p r e v i o u s l y graphy

steps

were

equilibrated NaN 3

0.02%

loaded

7.4

(8).

by

a^Achy

pH 2 . 8 ,

present

tion

Sephacryl

on

a

After

by

S

column.

fraction

anti-albumin is

in

2)

Purity. M

In SDS-PAGE

pure

(Fig.

a

c^Achy

:

25

previously with

Diaflo

apparatus.

by gel

remaining

filtracontami-

to

PI

Sepharose

30

mg

columns.

a^Achy

of

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

: 58,000 w i t h 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

anti-cL|

o b t a i n e d from 100 ml normal serum u s i n g t h i s

as

eluted

slight

inhibitor

and

the serum was

prepared

immediately

The

chromato-

pH 7 . 4 , 0 . 3 M N a C l ,

f r a c t i o n were e l i m i n a t e d

200

of

human p o o l e d serum was

fraction

was

ultrafiltration

in this

successively

Unbound

: the two c o v a l e n t

column

M NaCl

a modification

centrifugation,

serumalbumin and « ^ - p r o t e a s e

using

by

Normal

containing

0.5

concentrated

Aggregates

nants,

dialysis.

The

buffer

and

(8)

anymore.

an a n t i - Q^Achy- S e p h a r o s e

described glycine

described

used

prepared

i n 0 . 0 1 M sodium phosphate b u f f e r (PBS)

on

not

was

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

content.a^Achy

of

is

indicate them

a very

represent labile

PBS pH 7.4 at 4°C or f r o z e n i f

4)

Interactions purulent formation and

with cathepsin

sputum of

cathepsin

(9).

In

an equimolar G

occurred c^Achy

modified

form

of

Moreover

when

active

fragment,

migrating

as

a

been

has that

previously

concerning about

1%

G. C a t h e p s i n previous complex

a peptide

the

amino-acid

(M^ near

had G of

no

(10),

the

inhibitory

near

we

78,000)

with

reacted M

in

period.

G was i s o l a t e d from human

paper

concurrently which

of

and

component which has t o be kept

stored f o r a long

cathepsin

determined

half-cystine

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 recently we showed that

equimolar

complex was not stable with time (11). Spectrophotometric (12)

using

enzymatic

assays

Suc-Ala 2 -Pro-Phe-NA

as

were

performed

substrate.

according

When

to

increasing

amounts of inhibitor were incubated for 5 min at 25°C with a fixed amount of enzyme

(final

enzyme concentration

in the assays

: 3 x

10 ^M), a quite linear inhibition curve was obtained and extrapolation of the linear part gave an I/E molar ratio equal to 1.76/1 for complete inhibition. A time course study is performed on several a^Achy with cathepsin

G

(molar ratio

identical mixtures of

I/E = 2/1) kept at 25°C for

various times. Spectrophotometric enzymatic assays on aliquots showed no reappearance of activity when the incubation time increased. After

PMSF

addition

to

each

incubation

mixture

the samples

were

studied by electrophoretic methods. In SDS-PAGE (Fig. 1) we observe that

the

increases

equimolar

complex

dissociates

when

the

incubation

time

: transient components appear then and the final products

are the modified o^Achy having a M r equal to 55,000 and the small fragment (indicated by the arrow on Fig. 1).

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

Fig. 1. SDS-PAGE on a gradient gel (5 to 30% acrylamide) of samples withdrawn from identical mixtures of a.Achy with cathepsin G (molar ratio I/E = 2/1) kept for various incubation times at 25°C. M : M markers ; I = native a,Achy ; E = cathepsin G ; C = control mixture, in which cathepsin G has been previously inactivated with PMSF.

174

Fig. 2. Characterization of chymotrypsin inhibit o r s (13) a f t e r a l k a l i n e PAGE (10% aery1 amide at pH 8 . 3 ) of samples w i t h drawn from i d e n t i c a l mixt u r e s of a,Achy with c a thepsin G (molar ratio 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. I = n a t i v e a-,Achy 1/2 = h a l f d i l u t i o n of I .

IL I 1 5 10 30 60 90 2h /i min min min min min min In

Fig.

shows the

2,

that

the

characterization

active

reaction

is

chymotrypsin

inhibitors

(13)

beginning

of

up between 30 min and 1 h of i n c u b a t i o n .

In

inhibitor

used

of

which

is

present

at

the

t h i s e l e c t r o p h o r e t i c system without d e n a t u r i n g agent o n l y the c o l o u red m a t e r i a l min is

which does not enter the gel

and 10 min of i n c u b a t i o n , noticeable

that

the

Fig.

intensity

of

the

which m i g r a t e s f a s t e r

than a c t i v e

of i n c u b a t i o n

the o n l y v i s i b l e

; it

is

(visible

3) r e p r e s e n t s band of

f o r the times 1 the complex modified

;

it

a^Achy

i n h i b i t o r i n c r e a s e s with the time component at the end of the

reaction.

ts*

v

+

I

C

1 ID min min

1h 3h 6h 24h 48 h 8 I days

F i g . 3. A n a l y s i s by a l k a line-PAGE of samples w i t h drawn from i d e n t i c a l m i x t u r e s of a,Achy with c a thepsin G (molar ratio 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. I = n a t i v e a.|Achy. C = c o n t r o l mixture (as in Fig. 1).

175 Isoelectrofocusing sion)

containing

was a

carried out in Isogel

mixture

of

Pharmalytes

(Marine Colloid (Pharmacia)

Divi-

(Fig.

Native a^Achy gives 5 bands with pis between 3.9 and 4.35

4).

(results

close to (14)).

a . Achy/Cath G

2/1 f i g . 4. Isoelectrofocusing pattern in Isogel containing Pharmalytes to obtain a pH gradient between 2.5 and 10. Cathepsin (near

G

10.0

precipitates

in

this

(15)). A component

medium

with

a pi

before

a

mixture

reaching

its

components,

min

has disappeared after 90 min of incubation,

coloured the than

zone

reaction the

antiserum

; it

is then

native against

a^Achy. a^Achy

the obtained patterns reacting

form

seen

only modified

of

A

pi

near 6.5, which does not

exist in the initial incubation

of

is seen in the mixture after a 10 a

in the range of pi 5.2. At the end of a^Achy

is observed,

second

dimension

in

having Isogel

a pi

lower

containing

was carried out for the three mixtures ;

(data not shown) proved that an antigenically

a Achy was

present

in the components of pis 6.5

176 and

5.2

as

in

the

modified

c^Achy

obtained

at

the

end

of

the

reaction. In

the

tion

presence

is

of

faster

an excess of c a t h e p s i n G the complex

and we do not

observe

dissocia-

a protein degradation

going

f u r t h e r than the p r o d u c t i o n of the m o d i f i e d a^Achy of M^ = 55,000. Thus,

the

ciates

equimolar

under

these

activity

is

chymotrypsin may e x p l a i n the

it

and

between

the

conditions

is

recovered,

unlike

(16). the

complete

when

complex

spontaneously

molecule

the

with

by

that

a^Achy

modified results

;

is

generated

no

enzymatic

obtained

with

bovine

of the a ^ A c h y - c a t h e p s i n G system

high molar r a t i o

inhibition

reacts

the

The u n s t a b i l i t y

very

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 LettersJ38, 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 a j ANTITRYPSIN AND a 2 MACROGLOBULIN

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. a 2 m 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 too 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 de Gruyter & Co., Berlin • New York - Printed in Germany

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 being 012-macroglobulin (ct2M) and aj-proteinase inhibitor or a ^antitrypsin (ajAT) , 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 a^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 cijAT with human tonsil cells in serum-free medium resulted in a transient decrease of aiAT 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 a^AT with an association constant of 0.7 x 10^ M *x 1. The binding was inhibited by addition of cold ai AT # Soybean Trypsin inhibitor, and partially by ci2M- lodination by ajAT 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 ai AT > 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 found to decrease the binding of ctjAT, suggesting that the proteinase receptor was released from the cell surface. The radiolabelling method, however, brings no information on the possible heterogeneity of ajAT distribution among the cell populations nor does it indicate the presence of a j AT 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 cijAT and inhibited by TPCK and by EDTA (3 mM) which could also displace a^AT initially bound to the lymphocyte surface (15). Two other studies have shown

210 the presence of a^AT 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 ct2M 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 is 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 ajAT 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

+ (Í)

Man

Trypsin, chymo tryps in Elastase, cathepsin G Trypsin, pronase Trypsin Elastase, chymotrypsin

+ +

Lymphocyte transformation B

T

+ + +

0 0 +

0 0

23, 24 25 24 26 27

+

++

Ref.

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 ths 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 ctiAT.

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 cqAT 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 ctjAT 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 ajAT appeared to be related to its protease inhibitory capacity since cqAT 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, a^AT 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 g?M.

The inhibitory effect of

a 2 M on PHA-induced proliferation of homologous lymphocytes has been observed in man (43) and hamster. Hamster

CI2M

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 a2M#

at a

concentration of 1.4 mg/ml, on

the proliferative response of mouse lymphocytes cultured in a serum-free medium (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 a 2 M 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 a 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 Q2M preparations and their biological activity. Some discrepancies 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 a 2 M 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 a 2 M by acid dialysis. Other

aglobu-

lins with documented immunosuppressive effect, such as the pregnancy zone protein, might be antigenically related to ct2M.

214 The immunomodulating effects of 012M may be mediated by the many substances that can be carried by CI2M (52). For instance, ei2M 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). CI2M 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 ct2M (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 cqAT 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-Seyler's, Z. 41 (1928).

Physiol. Chem. 179,

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.

6.

Tökes, Z.A., Kiefer, H.: J. Supramol. Struct. 4, 507 (1976).

71 (1978).

7.

Tökes, Z.A.: Fed. Proc.

8.

Bata, J., Martin, J.P. , Revillard, J.P. : Experiential*

9.

Graizel, A., Hatcher, V.B., Lazarus, G.L.: Cell. Immunol. (1975) .

533 (1975). 518

(1981). 210

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. 53^ 1337 (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. 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,

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. Comm. 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. V7_, 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-, Revillard, J.P., Bonneau, M., Latour, M.: Transplantation and Clinical Immunology _1J_, 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 U_, 614 (1981).

41.

Baranova, F.S., Bermann, A.A., Zarestskaya, Y.M.: Bull. Exp. Biol. Med. 89_, 44 (1981) .

42.

Ades, E.W., Hinson, A., Chapuis-Cellier, C-, Arnaud, P.: Scand. J. _15_, 105 (1982).

43.

Goutner, S., Simmler, J.T. , Rosenfeld, C. : Differentiation 5_, 171 (1976).

44.

Streilein, J.S.: Ph.D. Thesis, University of Texas, Health service Center, Dallas (1976).

45.

Stein-Streilein, J. , Hart, D.A. : Fed. Proc. 37_, 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. 14, 463 (1976).

48.

Chase, P.S.: Cellul. Immunol. 5_, 544 (1972).

49.

Amlot, P.L., Unger, A.: Clin. Exp. Immunol. 26, 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. (1973) .

169

52.

James, K. ; TIBS

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).

43 (1980).

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).

MEDIATORS OF INFLAMMATION

Moderators

: R.G.CRYSTAL

Thi-i round

table. provided

between gists,

clinicians, since

IN INFLAMMATORY

(Bethesda) and J.F.CORDIER

a very

biochemists,

inflammatory

ALVEOLITIS

constructive

discussion

immunologists,

and

alveolitis

i-4 probably

mo-it exciting

¿abject*

in the

{¡¿eld of pulmonary

research.

interest

of the

participant*

The

part

for pulmonary

next

symposium.

nummary referred

of the to the

complementary

inflammation

The

reader

theme* texts

will

approached

(Lyon)

should find

urge

one. of

in the

of R.G. CRYSTAL

a

¿n

greater the

a

brief

discussion,

and

and

the

diseases that

be reserved thereafter

morphoto-

is

H. R E V N O L V S for

data.

A l v e o l i t i s is defined as a c c u m u l a t i o n of immune a n d cells w i t h i n lung interstitial

effector

structures. One or more of

these cell types are a c t i v a t e d , a n d they can mediators of i n f l a m m a t i o n contributing

release

to alveolar

injury.

Since a l v e o l i t i s includes a c c u m u l a t i o n of cells in alveolar structures,

c h e m o t a c t i c factors attracting

these cells will

initiate the process of alveolitis. Alveolar macrophage probably

is

the major origin of such factors in the air side of

the lung. They consist of small proteins rent factors have been identified) polymorphonuclears

(at least 2 d i f f e -

producing an influx of

into airspaces. Other chemotactic

will also take a part to the increase in cellular of alveolar

spaces

: p l a s m i n o g e n activator,

or

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

factors

populations

fragments

220 derived

from

diseases are

the

examples

tactic

of

stimuli

cells and

substances,

Oxydants

are

which are vation, example

highly

in

infectious

pulmonary

the p r o d u c t i o n

of

lung

of

these

fibrosis chemo-

catalase,

when

extracellular

matrix

1 antitrypsin

nization,

relative

part

injury

the lung,

of

mechanism

still

Attention

also

occur

of

the lung

enzy-

: superoxyde Apart

susceptible structures

to

from lower

can d e r a n g e

if a n t i p r o t e a s e

inactivated

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

turns the

to

the

cells

fibroblast. of

lung

This

cell

inter stitium,

and

matrix.

intersmajor

underterminated.

f o r a b o u t 4 0 % of

is

absent

release

will alter

to t h e

the

(functional

proteases

distribution

by

of an

antiproteases.

cell attachment

taken

is

There are

enzymes.

or w h e n

neutral and

no-

inacti-

peroxydation the lungs

when antiproteases

or

of

metabolism

enzyme

of o x y g e n

(directed against

activator, region

are

in a l v e o l a r

deficiency),

repartition

the

oxydants

of

such as

to f i b r o s i s .

inactivating

source

of o x y g e n

for

radicals

deficiency)

Collagenase

plasminogen derange

free

both

structures.

first

lipid

and glutathione

This will

1 antitrypsin

portant.

by

and

(which of

proteases,

toxicity

leading

present

not s u f f i c i e n t .

the

products

with DNA, Oxygen

toxic activity,

Proteases

as

cells

to i n j u r y

in e x c h a n g e

toxic activities

injury

defences

matrix

alveolitis

contribute

oxydants and

defences against

direct

spaces, will

reactive

of

membranes.

alveolar

titial

Bacteria

in i d i o p a t h i c

considered

i.e.

capable

dismutase,

The

are

interactions

cellular

(

state)

extracellular

Polymorphonuclears

matic

for

in a l v e o l a r

in an a c t i v a t e d

xious

system.

complexes

factors.

When a c c u m u l a t e d are

complement

or i m m u n e

account and

it

221 produces

most

Alveolar

macrophage

alveolar

fibroblasts

factor

for

part

of

connective

may

modulate

by

fibroblasts.

which

blasts.

The

of

bolic

increase

takes a part

functions

colJagen and

After

of

its

reviewing

the

the a b o v e

discussion

focused

in c l i n i c a l

predicting

those

is difficult

fibroblasts

(capable

on the

at

since

risk

together

scan,

evaluating

the

with

findings,

are

intensity

of

fibrowall

the

meta-

both

collagenase).

is

the

data

X-ray,

that

fibrosis

indicator

the m o s t a c c u r a t e alveolitis.

as

inflammatory

lovage

clinical,

growth

research,

of d e v e l o p i n g

gallium

67

of

no d e f i n i t e

both bronchoalveolar

pathologic

enzyme

The conclusion

lable. Actually, and

for

producing

of l u n g

evaluation

practice.

patients

to a s s e s s

of

of

macrophages

as well

degrading

subjects

a

in t h e a l v e o l a r

in f i b r o g e n e s i s ,

specifically

alveolitis

alveolar

is a c h e m o a t t r a c t a n t

cells

rate

(when a c t i v a t e d )

Furthermore,

fibronectin,

matrix.

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

releasing

produce probably

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 Médecine Nucléaire, Centre Léon Bérard. Laboratoire d'hématologie, d'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 lymphocytic 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 =

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

224 89.6 + 25.0, DLCO/AU = 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 were 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-guantitative 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 mere 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 were 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 investigated by an index over 50 (117.3 + 63.9 J_ 5 - 230_/ . Comparison between lymphocyte 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 activation 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. : Rev. fr. Mai. Resp. 11, 293-300 (1983) 5. Cordier, G., Mornex, J.F., Lefebvre, R. et al : in Poncelet Ed. Cytofluorometrie et anticorps monoclonaux dans le suivi des thérapeutiques. In press 1983 6. Pacheco, Y., Cordier, G., Perri n-Fayolle, M., ©t 3l i Am. J. Msd. 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 ALPHA-) 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.

S e r v i c e de Pneumologie ( P r o f . E . R O E G E L ) , C . H . U . , B . P . 426 67091 STRASBOURG and INSERM U n i t é 2 3 7 , F a c u l t é de P h a r m a c i e , U . L . P . , B . P . 1 0 , 6 7 0 4 8 STRASBOURG CEDEX ( F r a n c e )

C i g a r e t t e smoking i s an i m p o r t a n t r i s k

f a c t o r o f pulmonary emphysema.

v i t r o e x p e r i m e n t s document on c i g a r e t t e smoke-induced i n a c t i v a t i o n

of

alpha-] p r o t e i n a s e i n h i b i t o r

to

(dj-jPI) ( 1 ) , a p r o t e i n which i s thought

p r o t e c t the lung a g a i n s t n e u t r o p h i l e l a s t a s e as shown by the between emphysema and i n h e r i t e d < ^ | P I d e f i c i e n c y itself

In

association

(2). Neutrophil

i s a b l e to induce pulmonary emphysema i n a n i m a l s models

elastase (3).

We s t u d i e d the i n v i v o e f f e c t of c i g a r e t t e smoking on 10 pg/ml) such as NZB, BXSB, MRL/1, NZW and 129, while strains such as C57BL/6, BALB/c and C3H mice, whose gp70 levels are lower ( < 5 ug/ml), make little or no response. Although LPS has been shown to be able to activate and release endogenous retrovirus in cultured spleen cells of various strains of mice including BALB/c mice (8), the induction of high serum levels of gp70 by LPS is not a result of the production of endogenous retrovirus. This conclusion was reached following our demonstration that neither retrovirus nor retroviral antigens other than gp70 are detectable, after injection of LPS, in the circulating blood of LPS-responding strains such as 129, BXSB and MRL/1 mice (6). Clearly, the expression of serum retroviral gp70 does not require the production of complete virus particles and serum gp70 is probably a partial expression of a single provirus. The serum gp70 is apparently synthesized by hepatic parenchymal cells and released into the circulating blood. Serological and immunohistochemical studies have demonstrated that (a) the liver is the only organ whose gp70 content increases after injection of LPS with a parallel increase in serum gp70, and (b) the subsequent injection of colchicine, an inhibitor of protein release from hepatic cells, suppresses the rise in serum levels of gp70 but increases amounts of gp70 in liver extracts (7). In all probability, hepatic cells are the major source of serum gp70. This is compatible with the fact that serum levels of gp70 are generally higher in male mice than in females and are dependent on testosterones

298

(9), since the liver is a known source of several proteins whose serum concentrations are markedly influenced by sex hormones (10). A particularly significant observation is that inducers of acute phase reactants (APR) such as turpentine oil, casein or polyriboinosinic-polyribocytidylic acid (poly I - poly C) are able to induce the production of gp70 in kinetics identical to those induced by LPS (Table I). Furthermore, these gp70 responses are kinetically identical to those of APR such as haptoglobin or serum amyloid A protein (7, 11). Consequently, serum gp70 has the quality of an APR, and its expression is controlled by a mechanism similar to that for other APR. Supporting evidence is that restriction of caloric intake in mice lowers serum levels of one of the APR, haptoglobin, as well as gp70 to less than half of those in control mice, while albumin levels are not affected (12). Table I. Induction of gp70 by various APR Inducers Inducers

gp70 (yg/ml)

Haptoglobin (%)

LPS

(25 jag)

590 + 69

421 + 52

Turpentine oil

(0,05 ml)

413 + 38

533 + 183

Casein

(200 mg)

575 + 84

513 + 107

543 + 122

438 + 29

Poly I - poly C (500 jag) Saline

63+6

119 + 32

Serum levels of gp70 and haptoglobin were determined 1 day after intraperitoneal injection of APR inducers into 2 mo old (NZBxNZW) F1 male mice. Concentrations of haptoglobin are expressed as a percentage of values from the pooled sera of 3 mo old C57BL/6 male mice.

299 In view of (a) the similarity in expression of serum gp70 and a number of other proteins that are products of the host genome, and (b) the failure of other retroviral gene products to be expressed along with serum gp70, it is concluded that serum gp70 behaves as a host gene product under the same regulatory influences as other normal host constituents. Thus, serum gp70 should be considered as an autologous protein. Clearly, the endogenous retrovirus by itself does not directly cause the development of autoimmunity, but contributes to murine SLE by providing a potential autoantigen which is significantly involved in the pathogenesis of murine SLE. Pathogenic Significance of gp70-anti-gp70 IC in Murine SLE It is now clear that only murine strains with SLE respond immunologically to their own serum retroviral gp70 by forming antibodies. Because of the large excess of retroviral gp70 in sera from these strains, antibodies reacting with serum gp70 are not detectable in a free form; instead, one can find antibody-bound gp70 IC in sera from all the SLE-prone mice. Sera from all older SLE-prone mice contain, in addition to a free form of 5S gp70, a rapidly sedimenting heavy form of gp70 that is specifically absorbed with anti-IgG antibodies (13). More directly, immunoglobulins are isolated from the heavy form of gp70 and are shown to have an antibody activity reacting with serum gp70 (14). Immunoprecipitation analysis with anti-IgG antibodies indicates approximately 20 - 50% of serum gp70 from older SLE mice is associated with anti-gp70 antibodies. In contrast, immunologically normal strains of mice, even those with comparable levels of serum gp70, do not develop such a heavy form of antibody-complexed gp70.

300 Several lines of evidence suggest that gp70-anti-gp70 IC (gp70 IC) play a role in the pathogenesis of glomerulonephritis that accompanies murine SLE. The gp70 antigen is heavily deposited in a granular pattern in glomerular lesions of SLE mice and distributed in the peripheral capillary loops as well as in the mesangia, similar to bound immunoglobulins and complement (2, 3). Its glomerular deposition usually appears to coincide with the manifestation of significant proteinuria and renal disease. The appearance of gp7 0 IC in the circulating blood varies with age among the SLE susceptible strains, but parallels the onset of renal disease and persists throughout its course (13). Generally, circulating gp70 IC becomes detectable several months before the mice develop fatal renal disease and increases numerically as the disease worsens. Thus, one can predict the course of renal disease by determining serum levels of gp70 IC. Genetic studies on (NZBxNZW)xF2 and (NZBxNZW)xNZW backcross mice in which the presence of such IC is significantly associated with the development of fatal glomerulonephritis (15, 16), further underline gp70 IC as a potential source of renal injury. We have recently demonstrated that one of the SLE-prone mice, male BXSB, has a significant incidence of abnormally elevated levels of gp70 in sera during the course of their SLE (17). Such increased levels of serum gp70 are highly significantly associated with the presence of hepatic sinusoidal lymphocytosis. Hepatic sinusoidal lymphocytosis is characterized by the infiltration of mononuclear leukocytes within sinusoids of liver and Kuppfer cell proliferation. It is often seen in human patients with viral infection such as infectious mononucleosis or with chronic systemic infection such as malaria

301

(18). This suggests that infection of unknown origin occurring uniquely in male BXSB mice might be responsible for the enhanced expression of serum gp70, because of its nature as an APR. Strikingly, the enhanced expression of serum gp70 in adult male BXSB mice is associated with increased formation of circulating gp70 IC, presumably related to increased antigp70 antibody production, and with accelerated progression of renal disease. This further constitutes good evidence that gp70 IC is prominently involved in the renal disease of murine SLE. Furthermore, the amount of autoantigens as a source for antigenic stimulation and for the formation of IC, is notably one of the important factors which control the development of SLE. In this context, it is noteworthy that the expression of serum gp70 is genetically controlled by several genes (6, 9, 19) and its production is modified by environmental factors such as hormones (9), diet (12) as well as non-specific inflammatory stimuli (6, 7). Finally, an association of hepatic inflammation, increased production of an autoantigen and increased formation of IC with accelerated SLE suggest that a similar situation may be partly responsible for the exacerbation of SLE often observed in patients during microbial infection.

Conclusion Serum retroviral gp70 which is the envelope glycoprotein of endogenous retrovirus, is expressed in a selective manner without the production of virus particles or any other viral gene products. The serum gp70 molecule, like many other serum proteins, is synthesized in hepatic parenchymal cells and re-

302 leased into the circulating blood. It is striking that nonspecific inflammatory stimuli, which are inducers of APR, greatly increase serum concentrations of gp70 in kinetics identical to those of APR. This indicates that serum gp70 is a gene product of endogenous retrovirus, but it behaves essentially as a normal host constituent and is controlled under the regulatory mechanisms similar to those for host gene products, such as APR. Thus, the gp70 is considered as an autoantigen, and the anti-gp70 antibody an autoantibody. Serum retroviral gp70 complexed to anti-gp70 antibody is prominently involved in the renal disease of SLE mice. Not only SLE-prone mice but also many immunologically normal mice have relatively high concentrations of gp70 in their sera; however, only SLE mice develop antibodies directed against serum retroviral gp70 as a result of their immunologic abnormalities. Subsequently, these antibodies combine with circulating gp70 and form IC that are deposited in the renal glomeruli where they produce glomerulonephritis. A significant association of circulating gp70-anti-gp70 IC with the development of glomerulonephritis has been demonstrated in SLEprone mice under several experimental conditions. These combined results strongly suggest that gp70-anti-gp70 IC play a significant role in the pathogenesis of murine SLE, and are a useful and predictive indicator of murine SLE.

Acknowledgement The author would like to express his sincere appreciation to Dr. Frank J. Dixon, Scripps Clinic and Research Foundation, La Jolly, California, U.S.A. for his invaluable advice and discussion during the course of this work. The excellent

secretarial assistance of Mrs. Ruth Preston is gratefully acknowledged. This work was supported by grant 3.196.0 82 from the Swiss National Foundation for Scientific Research.

References 1.

Lambert, P.H., Dixon, F.J.: J. exp. Med. 127, 507-521 (1968).

2.

Andrews, B.S., Eisenberg, R.A., Theofilopoulos, A.N. , Izui, S., Wilson, C.B., McConahey, P.J., Murphy, E.D., Roths, J.B., Dixon, F.J.: J. exp. Med. 148, 1198-1215 (1978).

3.

Yoshiki, T. , Mellors, R.C., Strand, M. , August, J.T.: J. exp. Med. 140, 1011-1027 (1974).

4.

Lerner, R.A., Wilson, C.B., Del Villano, B.C., McConahey, P.J., Dixon, F.J.: J. exp. Med. 143, 151-166 (1976).

5.

Izui, S.: Folia allergol. immunol. clin. 30, 32-54 (1983).

6.

Hara, I., Izui, S., McConahey, P.J., Elder, J.H., Jensen, F.C., Dixon, F.J.: Proc. natl. Acad. Sci. U.S.A 78, 4397-4401 (1981).

7.

Hara, I., Izui, S., Dixon, F.J.: J. exp. Med. 155, 345357 (1982).

8.

Moroni, C., Schuman, G., Robert-Guroff, M., Suter, E.R. Martin, D.: Proc. natl. Acad. Sci. U.S.A. 72, 535-538 (1975).

9.

Obata, Y., Stockert, E., Yamaguchi, M., Boyse, E.A.: J. exp. Med. 148, 793-798 (1978).

10.

Gustaffson, J.A., Eneroth, P., Pousette, A., Skett, P., Sonnenschein, C., Stenberg, A., Ahlen, A.: J. Steroid Biochem. 8, 429-435 (1977).

11.

McAdam, K.P.W.J., Sipe, J.D.: J. exp. Med. 144, 11211127 (1976).

12.

Izui, S., Fernandes, G., Hara, I., McConahey, P.J., Jensen, F.C., Dixon, F.J., Good, R.A.: J. exp. Med. 154 1116-1124 (1981).

304

13.

Izui, S. , McConahey, P.J., Theofilopoulos, A.N., Dixon, F.J.: J. exp. Med. 149, 1099-1116 (1979).

14.

Izui, S., Elder, J.H., McConahey, P.J., Dixon, F.J.: J. exp. Med. 153, 1151-1160 (1981).

15.

Izui, S., McConahey, P.J., Clark, J.P., Hang, L.M., Hara, I., Dixon, F.J.: J. exp. Med. 154, 517-528 (1981).

16.

Nakai, Y., Maruyama, N., Ohta, K., Yoshida, H., Hirose, S., Shirai, T.: Immunol. Lett. 2, 53-58 (1980).

17.

Izui, S., Hara, I., Hang, L.M., Elder, J.H., McConahey, P.J., Dixon, F.J.: Submitted for publication.

18.

Marsden, P.D., Hutt, M.S.R., Wilks, N.E., Valler, A., Blackman, V., Shah, K.K., Connor, D.H., Hamilton, P.J.S., Banwell, J.G., Lunn, H.F.: Br. med. J., 1, 89-91 (1965).

19.

Maruyama, N., Lindstrom, C.O.: Immunogenetics, in press (1983) .

STUDY OF 8 MARKER PROTEINS OF INFLAMMATION IN SYNOVIAL FLUID AND SERUM OF PATIENTS WITH JOINT DISEASES.

Pascal Bourg, Brigitte Cartier, Monique Mathieu Laboratoire de Biochimie - Hôpital des Charpennes 69603 Villeurbanne Cedex - France

Christian Collombel Laboratoire de Biochimie C - Hôpital E. Herriot 69374 Lyon Cedex 08 France

Introduction

More and more the plasma proteins are studied as markers of inflammatory

process.

interesting

Then quantitative

for

diagnosis.

The

most informative marker proteins

and

qualitative

aim

of

this work

contents seem to be was

to

select

the

in different cases of joint diseases

and to compare their content in synovial fluid (SF) and in plasma.

Material

29 patients have been studied, corresponding to 4 clinical classes : -

group

1

(10

cases)

is

a

control

one

of

adults

suffering

from

mechanical arthrosis without any inflammatory process, - group 2 concerns 4 patients with chondrocalcinosis, -

group

3 comprises

6 cases

of rheumatoid

arthritis,

patients have rheumatoid factor in synovial fluid, - group 4 concerns 5 cases of septic arthritis.

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

among them, 5

306 The marker proteins we studied are the following : haptoglobin

(Hp),C reactive protein

alpha-l-antitrypsin plement C

(A^AT),

alpha

(CRP),orosomucoid,

2 macroglobulin

ceruloplasmin,

(alpha

2 M) , com-

and C .

Methods :

-

Serum

samples

separate,

then

were after

obtained

from

centrifugation

veinous serum

puncture,

were

allowed

analyzed

to

within

4

hours, or stored frozen at - 25°C before analysis. -

Synovial

then

fluids

treated

with

(SF)

were

collected

hyaluronidase

in heparinized

(5 ^ig/ml

of

SF)

plastic

tube,

during 1 hour

at

37°C. -

Specific

proteins

immuno-diffusion Behring

determinations

(IDR)

reagents.IDR

and by and

laser

were

both

nephelometry

by

radial

(INL)

using

INL were compared and exhibited a

tion coefficient above 0.91, except for per and lower

performed

immuno

CRP.

In

this

correla-

case, both

up-

values of CRP were found with INL.

Results

The

next

figures

orosomucoid,

gives

the

ceruloplasmin,

values alpha

of

proteins

1 antitrypsin,

: haptoglobin,

CRP,

alpha 2 macroglobu-

lin, C^ and C^ complement.

The

values

are

indicated

in g/liter of serum and synovial

the 4 groups of joint diseases.

fluid,

in

307

sA

Haptoglobin'

CRP

g/1

0, 3

0, 2 * *

+

t *

?

0, 1

0

Ceruloplasmin

% *

i»

group

Legendes :

group

+ serum . synovial fluid

1

F

( Q SF with seronegative RF)

308

Alpha 2 m a c r o g l o b u l i n

Alpha 1 Antitrypsin

gA

g/1

8 + •

7 6 5 4 3 2

group

2

3

group

4

T

2

Complement C4

C o m p l e m e n t C3

gA

g/1

2

2

1,5

1,5

+ •f

9 0,5

*

group

Legendes :

3~"

•

0,5

group

+ serum . synovial fluid

t *

?

• s

I

1

( o SF with seronegative RF)

309 Conclusion

In the present limit of our experience we can conclude the following : 1 - concerning good

correlation

although

SF

the

between

seems

to

be

interest serum

of

biological

and

a better

synovial medium

samples,

fluid

there

marker

for alpha 2

is

a

proteins,

macroglobulin

and complement C^-C^. 2 - Haptoglobin

seems the most sensitive

marker

(no overlap

in

controls and patients). 3 - CRP and orosomucoid are, as usually known elevated in septic arthritis. 4

-

Alpha

1 AT

and

ceruloplasmin

are

not

sensitive

markers

place

marker

in

any clinical groups. 5

-

synovial

Alpha

2

membrane

macroglobulin lesions

with

has

a

special

a markedly

of

for

elevated content in S.F.,

this fact may be related to the high molecular weight of this protein which

normally

does

not

allow

it

to

diffuse

across

the

synovial

membrane. 6

- Cg

and

C^

complement

cases of rheumatoid arthritis

seem

to be of interest mainly

in the

: they are diminished in SF of seroposi-

tive and increased in SF of seronegative R.A.

As a general conclusion of our still limited experience we can assume that

in

the

case

of

joint

diseases,

inflammation the most interesting are C „. Besides, their 4 synovial fluid.

determinations

is

among

8

marker

proteins

in

: Hp, CRP and complement C^ and valuable

both

in

serum

and

310 References.

1. Chales G., Panlotsky Y., Le Treut A., Catheline H., Le Gall J., Cloarec L. : Revue du Rhumatisme 47, 7-9, 493-499 (1980). 2. Gobelet C., Gerster J.C., Saudan Y : Med. Hyg. 37, 4526-4530 (1979). 3. Muller W. : Medical Laboratory 3, 21-30 4. Niedermaier W., Cross R., Beetham W.P. 355-359 (1965)

(1977) : Arth. and Rheum. 8,

5. Takahiro 0., Kunio Y., Keiro 0. : Annals of rheumatic diseases 235-240 (1980).

FIBRONECTIN

IN

ARTHROPATHIES

3. LECOCQ, M.A. LECOCQ, L. ASCH Service de Rhumatologie, C.H.U. 67200 STRASBOURG-HAUTEPIERRE M. ROTH et M.L. NORTH Centre de Transfusion Sanguine, 10 rue Spielmann, 67000 STRASBOURG

Introduction Fibronectin, a high molecular weight glycoprotein, is secreted by cells of connective tissue and exists in synovial fluid and plasma. After others (1, 2, 3, 5, 6), we now report an investigation of plasma and synovial fluid fibronectin level in arthropathies, the diagnostic usefulness of these measurements and the place of synovial fibronectin (S Fn) beneath other synovial fluid parameters. The concentration of Fn in plasma (4) and in hyaluronidase (25 U/ml - pH 6,9) treated synovial fluid was measured by laser nephelometry. 104 patients with various joint disorders have been investigated : - 44 non inflammatory arthropathies formed of osteoarthritis and post traumatic arthropathies, - 26 rheumatoid arthritis (RA), - 34 non rheumatoid inflammatory arthritis.

Results 1) Plasmatic Fibronectin (p Fn) concentrations are scattered, but stay within normal range (408 mg/1 ± 224 : Mean ± 2 SD). There are no statistically significant difference between the three

groups of diseases.

2) Synovial fluid fibronectin levels are similar in patients with non inflammatory arthropathies (374 mg/1 ± 116) and with non rheumatoid

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

312 inflammatory arthritis (381 mg/1 ± 158). On the other hand, patients with RA (583 mg/1 ± 196) have significantly higher levels (p < 0,001) (figure 1). k6 percents of RA have S Fn concentration higher than 575 mg/1 while only four non rheumatoid arthritis (5 %) have such levels : one osteoarthritis, one articular chondrocalcinosis, one gout, one ankylosing spondylitis. The S Fn of seropositive RA is significantly higher in comparison with seronegative RA (p = 0,035). 3) Ability of S Fn to differenciate RA of other inflammatory arthritis has been compared with others synovial fluid proteins by stepwise discriminant analysis ; decreasing classification is CH 50, Fn, (3 2 microglobulin, C^, C^, total proteins. k) Among non rheumatoid inflammatory arthritis (figure 2) the mean of crystal-induced arthritis (401 mg/1 ± 193), ankylosing spondylitis and reactive arthritis (419 mg/1 ± 157), infectious arthritis (220 mg/1 ± 180) and non defined and miscellaneous arthritis (366 mg/1 ± 65) are not significantly different. Yet S Fn level in infectious arthritis is low but the sample size is too small to be statistically significant. 5) There is no correlation between S Fn or p Fn and acute phase proteins (ESR, fibrin, haptoglobin, orosomucoid, CRP, C9). 6) No correlation has been found between S Fn and inflammatory synovial parameter, but a positive correlation exists between S Fn and synovial lymphocyte count (r = 0,4-0 ; p < 0,001) or its markers

(gammaglobulins,

r = 0,34- ; p < 0,001, 0 2 microglobulin, r = 0,37 ; p < 0,001). S Fn is unrelated to polymorphonuclear leucocytes count, macrophagic cells count. In more than half of the patients with non inflammatory or inflammatory arthritis (in 88 percents of RA ; in RA the mean ratio S Fn/p Fn = 2) S Fn is higher than p Fn. This finding suggests local secretion of S Fn by synovium cells.

S Fn could well be a local marker of diverse connective synovial tissue modifications rather than an inflammatory protein or a specific pathologic protein. It could be a diagnostic help in RA. Its usefulness in infectious arthritis needs to be confirmed in later studies.

313 O O

900

O

O O O

600

O

O

O

O

O

'

O O O

S Fn

O

p < 0,001

O

500

o oo

mg/1

o o oo o

300 "

o 200

non inflammatory arthropathies

rhumatold arthritis

FIGURE 1

non rhumatold inflammatory arthritis

314

NON

RHEUMATOID

INFLAMMATORY

ARTHRITIS

500

• o • •

•

•

300 •

D

100

crystal induced arthritis

ankylosing spondylitis + reactive arthritis

infectious arthritis

FIGURE 2

non defined and miscellaneous arthritis

315 References 1. Carsons, S., Mossesson, M.W., Diamond, H.S. : Arthritis and Rheumatism , 2k, 10, 1261-1267 (1981). 2. Clemmesen, I., Andersen, R.B. : Scand. 3. Rheumatol, suppl., 33, 127 (1980). 3. Lu Steffes, M., Iammartino, A.O., Schmid, F.R., Castor, C.W., Davis, L., Entwistle, R., Anderson, B. : Annals of clinical and laboratory science, 12, 3, 178-185 (1982). k. Pott, G., Meyering, M. : 3. Clin. Chem. Biochem., 18, 893-895 (1980). 5. Scott, D.L., Farr, M., Crockson, A.P., Walton, K.W. : Clin. Sci., 62 (2), 71-76 (1982). 6. Vartio, T., Vaheri, A., Von Essen, R., Isomaki, H., Stenman, S. : Eur. 0. Clin. Invest., 11 (3), 207-212 (1981).

SECTION V INFLAMMATORY OF NERVOUS

DISEASES

SYSTEM

MULTIPLE SCLEROSIS:

CLUES FOR ITS PATHOGENESIS

Christian Confavreux Hopital Neurologique, Service de Neurologie, 59 Bd Pinel, 69003 Lyon (France)

Philippe Arnaud Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, 1J1 Ashley Avenue, Charleston, South Carolina, 29U25 (U.S.A.)

"To study the phenomena of disease without books is to sail an uncharted sea, while to study books without patients is not to go to sea at all." Sir William Osier

(1895)

Introduction

To know Multiple Sclerosis (MS) is, as sometimes said, to know Neurology. How modest might be the neurologists as some "franc-tireurs" use to consider MS as a disease with an unknown etiology, an unknown prognosis and an unknown treatment.

In fact, definite data are not lacking. in young adulthood.

MS is the most frequent disease

Its prevalence, i.e. the number of cases encountered

at a given time in a given population, is about U0/100,000 persons. Therefore, 25,000 French people are affected with MS.

Three masterwords mark the MS pathology: and sclerosis.

demyelination, inflammation

The lesions appear as focus or "plaques" with inflam-

matory denyelination and axonal preservation disseminated in the white

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

320 matter of the central nervous system (CNS). different stages:

the perivascular inflammatory cuffs, the centrifugal

denyelination and the sclerosis.

There is a continuous appearance of new

plaques as shown by sequential CT scans. course.

Each lesion goes through

Each plaque follows its own

A given brain contains lesions of different ages.

Lower and/or upper limbs dysfunction, paresthesia, sphincteric disturbances, optic neuritis, diplopia, dizziness, . . . are usual clinical manifestations.

At a first glance, they would seem proteiform but they

reflect the elective CNS white matter tropism of the disease. The disease course is governed by two mechanisms:

the "relapses", i.e.

exacerbations of the neurological symptoms followed by remissions, and the "progression" with a steady and continuous worsening of the neurological condition. plaques?

Are relapses reflecting the course of one or several

This is possible but a number of these plaques have no clinical

correspondence and the usual duration of a relapse is to too short for a demyelination-remyelination mechanism being the single interpretation. And what reflects the progression?

Probably, an extensive and progres-

sive gliosis of the CNS which, finally, overcomes the expression of the relapses.

Consequently, the course of MS is marked by two phases:

remittent one defined by the relapses, and a progressive.

a

This is the

unique sequence if these two phases are featured in a given patient. Eighty per cent of the cases present a remittent onset of the disease, and twenty per cent a progressive onset. this profile of the MS course?

In a recent work [l], we tried, after

many others, to answer this question. be decisive:

Is there any interpretation for

The age of the patient seemed to

the remittent cases start at 30 years of age as a mean, the

directly progressive cases at 38.

More thoroughly, the course is marked

"by relapses without sequelae since 29 years of age at mean, relapses with sequelae since 3^ y e a r s , and the progressive phase since 38 y e a r s .

This

is only a scheme, with large individual variations possible but correlation between course profile and age are tight.

Other explanations must

of course be sought.

On the whole, MS is infamous and this is fully justified.

With our

cohorts, we have estimated using the actuarial technique that, at mean, M S patients were disabled after a 6 y e a r duration of the disease, homeconfined after 18 years and dead after 30 years.

Cases do exist however

without any disability after 15 years of disease duration:

these benign

cases represent 10-15 per cent of the MS population.

Is a prognosis feasible?

For us, three factors were decisive.

the age at the onset of the disease: faster the disability.

First,

the later the disease starts, the

This is not to say that, statistically, the

y o u n g e r patients are not disabled at an earlier age than the senior patients.

Second, the interval between the first two relapses in the

remittent forms: the disease.

the shorter this is, the faster will be the course of

Third, the onset of the progressive phase, which, on the

w h o l e , is of pejorative significance. may be of importance: immunoglobulin levels. still unpredictable.

For other authors, other factors

symptomatology at onset, HLA typing,

intrathecal

In fact, the precise course of a given patient is

322 As for the course and the prognosis, the pathogeny of MS is still an enigma.

The efforts of epidemiologists, geneticists, microbiologists and

immunologists result in a very rough description.

A.

Exogenous and endogenous factors act jointly to induce MS.

These

are the lessons of epidemiology and genetics.

1.

Epidemiology [4,5,10]

There is a precise planetary geographical distribution of MS prevalence in the 2 hemispheres: grows.

it increases as the distance from the Equator

This is an argument for the role of an ENVIRONMENTAL FACTOR.

Nevertheless, great variations in prevalence have been noted in very close regions such as the Finnoscandian districts; Scotland and the surrounding islands are even a better example:

the MS prevalence is at

its highest world-wide level in Scotland (1^4/100,000) and in OrkneysShetland Islands (203/100,000) while it is very low in the FaroB Islands. The understanding of such disparities in very close areas appear to be critical.

The analysis of migrations in the U.S.A., Israel, and South Africa shows that the migrant keeps a risk of developing MS corresponding to the area in which he spent his first fifteen years.

The exogenous factor to MS

susceptibility would be ACQUIRED during infancy.

MS would be a disease

with a long incubation period.

The study of foci with a naturally low MS prevalence is meaningful: is then easy to detect epidemies. was known there before I9U0.

See the FaroB Islands:

it

no MS case

Twenty four cases were diagnosed from 19^3

323 to i960 and only one since then. Iceland:

The same observation was made in

the two world wars were followed by a clear increase in the MS

incidence.

According to these "epidemies", the MS exogenous factor

would be TRANSMISSIBLE. While the role of the foreign troops occupation was questioned in these epidemies, suspicion was aroused about house pets.

According to Cook and

Dowling, MS patients were more often than controls in contact with dogs during their childhood.

This was not confirmed by others.

A temporal

relationship between the canine distemper encephalitis epidemics and the MS "epidemies" has been unsuccessfully sought.

House pets then appear to

be cleared.

2.

Genetics [5,9,10]

The racial influence is strong. twice less common in Blacks.

MS is the "privilege" of Whites.

It is

It is even n»re infrequent in Orientals and

this is particularly clear in a country with a temperate climate such as Japan.

For a given race, the former geographical distribution still pre-

vails, showing the superimposition of endogenous and exogenous factors.

Familial cases are about 5% in the different series in the literature. For first-degree relatives of an MS patient, the prevalence of MS is estimated to 0.6/100 for the parents, 1.2/100 for brothers and sisters, 0.6/100 for the children.

This is twenty times the prevalence observed

in the general population (1+0/100,000) but this is too low for an MS genetic transmission governed by a simple Mendelian procedure with an only gene and a complete penetrance.

324 For twin studies, it appears that the concordance of MS is 15% for dizygotic twins, and 30% for monozygotic twins.

When the whole genome is

in common "between two individuals, one of whom is affected with MS, the risk for the other to be also affected does not exceed 30%.

This clearly

shows the limitation of the genetic factors in MS susceptibility.

The results of familial and twin studies are debatable: nity or environmental community?

genetic commu-

This is why the detection of a linkage

between particular phenotypes and the transmission of MS has been of the utmost importance, showing unequivocally the influence of genetic factors.

This demonstration came from HLA studies.

sian population, the prevalence of the HLA - A3 -

In a normal CaucaB7 and DR2, is, in

each case, about 20%; for MS Caucasians, it is about 30%, 1*0% and 50%, respectively.

The appearance of certain HLA groups increases the risk of

developing MS. fold.

For HLA-DR2 the increase in relative risk is about three-

Note, however, that these "predilection" groups are not found in

some MS populations, e.g. Israelian and Japanese.

Furthermore, the study

of HLA haplotypes sharing among affected sib-pairs with MS shows that they are HLA identical in 37% of the cases, i.e. more often than expected by chance (25%).

The concept of an MS susceptibility gene, HLA linked

and in linkage disequilibrium with HLA-DR2 may therefore be proposed. Similarly, another linkage between MS transmission and a highly polymorphic genetic system, the Gm allotypes of Immunoglobulins G (igG) heavy chains, has recently been described.

If this result is confirmed, the

existence of other MS susceptibility genes, not linked to the Major Histocompatibility Complex (MHC) may be postulated.

325 B.

The Immunological Mediation

[2,3,5,6,7,8,10,11,121

Exogenous factors acting in susceptible individuals are needed for MS to develop.

Abnormal immunological reactions are also operating:

in the

plaques of CNS white matter demyelination, there is a perivascular cuffing with lymphocytes, plasma cells and monocytes.

The presence of

immunoglobulins (Ig), complement and immune-complexes can be shown; in the cerebro spinal fluid (CSF), there is often a moderate pleiocytosis with mononuclear cells, and an increase of the IgG levels, with an intrathecal synthesis and oligoclonal distribution after electrophoresis; in the peripheral blood, (although we are here far from the primary lesion) it was thought that clear-cut abnormalities were absent, until the recent observation of a defect of the T suppressor lymphocytes.

Thorough details must be sought about these immunological perturbations. 1.

The Abnormalities of Humoral Immunity [2,3,6,7,8]

a.

The most classical one is the increase of the CSF IgG level first

noted by Kabat in the forties and fully confirmed since then. concerns IgG but also, in some cases, IgM and IgA.

It

Three interpretations

can be discussed: - an increase in the serum IgG levels is unlikely.

Indeed, only a major

increase in plasma IgG can influence CSF IgG levels and serum IgG levels are usually normal in MS; - an alteration in the Blood-Brain-Barrier (BBB):

the CSF is a biologi-

cal fluid which contains 200 times less proteins than serum.

It is even

relatively poorer in IgG, as the IgG/total proteins quotient is physiologically 8 to 10 percent in CSF versus 15 to 18 percent in serum.

An

326 alteration in the BBB results in an increase in CSF IgG. transudated IgG.

This is

A BBB alteration is detected by the presence in CSF of

increased levels of proteins with an exclusive systemic synthesis, such as albumin.

The total amount of transudated IgG can be estimated from

the level in the CSF increase in the albumin.

Note, however, that BBB is

altered in only 20 to 1*0 per cent of the cases according to the different series in the literature and that this alteration is only minimal to moderate:

a total protein amount higher than 0.80 g/1 in CSF is strong

evidence against the diagnosis of MS; -An intrathecal synthesis can be assumed when CSF IgG level is increased if the CSF albumin level is normal.

It can be detected in the case of a

BBB alteration when the calculated amount of transudated IgG is not sufficient to account for the total measured CSF IgG level. modes of expression can be used:

Different

the result of the dosage of IgG

(normal < 60 mg/1.), the IgG/Total Protein ratio and the IgG/Albumin ratio are the most classical; other calculations take into account serum IgG and albumin levels:

IgG index (ratio between the CSF IgG/Albumin

quotient and the serum IgG/Albumin quotient) and Tourtelotte's formula which is rather complex but gives an estimate of the daily CNS IgG synthesis.

Finally, intrathecal synthesis is considered as present in at

least one case out of two in MS.

Evidence for an intrathecal synthesis of IgG given by these calculations is only indirect.

More direct arguments are available:

the Ig amounts

are increased in cerebral MS plaques; intracytoplasmic Ig can be detected by immunofluorescence in lymphoid cells from MS CSF and brain; plaqueforming cells are present in MS CSF; there is a close relationship

327 between the presence of plasma cells in CSF and the increase in the intrathecal IgG levels (personal data, work in preparation). Is there any parallel between an increase in the CSF IgG levels and the relapses, a fastly deteriorating course of the progression, in the course of disease activity?

No coherent results have emerged from the different

series in the literature and each neurologist has observed malignant cases with normal CSF IgG levels and, conversely, benign cases with high CSF IgG levels. b.

The CSF IgG modifications are quantitative but also qualitative with

an oligoclonal distribution on electrophoresis, as shown by Lowenthal and Laterre in the sixties [referenced in 13]. The electrophoresis pattern can be visualized on various migration supports (agar, agarose, cellulose acetate, polyacrylamide) and using concentrated or, unconcentrated CSF.

Oligoclonal Ig appears as well

defined bands of restricted number in the immunoglobulin zone. shown with immunofixation that these bands are mainly IgG.

It can be

The band

pattern varies from patient to patient and is relatively stable for a given patient.

Some bands can disappear following intensive cortico-

therapy. Upon electrophoresis, the oligoclonal pattern is present in 50 to 95 per cent of MS cases, depending on the technique used and the cases studied (definite MS cases or not). CSF IgG level.

It is more common than the increase in the

It has a strong diagnostic value, as it is infrequent in

other neurological diseases, with the exception of other inflammatory CNS diseases but which can rarely be confused with MS such as neurosyphilis,

328 subacute sclerosing panencephalitis (SSPE) and trypanosomiasis.

With

isoelectrofocusing, the presence of CSF IgG bands is the rule in MS.

It

seems to be also frequent in other neurological diseases, inflammatory or not.

The improvement in sensitivity is partly lost in specificity and

these techniques seem to have less value for MS diagnosis than the conventional electrophoresis. This oligoclonal pattern of IgG is also present in MS cerebral plaques, but the band position may vary from plaque to plaque, data which is puzzling from a pathogenetic viewpoint [13]Formerly, it was thought that each band of oligoclonal IgG reflected one antibody synthesized by one clone of plasma cells.

This represents an

over simplification but it can still be considered that oligoclonal IgG has a restricted heterogeneity reflecting the synthesis of selective antibodies possibly directed against a definite target, the nature of which is still unknown. c.

Other alterations in humoral immunity have been reported.

It is

difficult, at the moment, to integrate them into a pathogenetic scheme: - The Kappa/Lambda ratio to IgG light chains in CSF is physiologically equal to one.

In MS CSF, it can increase to 2,3.

The same trend has

been noted for other inflammatory diseases of the CNS. -immune complexes:

according to different workers using various

techniques, they can be detected in serum, but also in CSF in about 50 per cent of the MS patients.

Their concentration is low when compared to

active systemic lupus erythematosus patients.

Similar results have been

reported in diseases without known immunological mediation such as

329 Amyotrophic Lateral Sclerosis.

A relationship could exist b e t w e e n the

presence of immune complexes and relapses.

The nature of the antigen and

the antibody in the complexes is unknown; - complement:

an increase in CU, C3 Proactivator and, more generally,

the acute phase reactants of inflammation such as C-Reactive Protein and orosomucoid has b e e n reported, mainly during relapses.

Hypocomplemen-

t e m i a has also b e e n reported to b e more common in MS patients than in controls.

2.

The Abnormalities of Cellular Immunity

a.

Until recently, the moderate pleiocytosis in CSF was the only known

common alteration.

[2,3]

It concerns mononuclear cells.

It is usually

moderate, i.e. 5 to 10 cells per mm3; w h e n it is higher than 50 cells, the diagnosis of MS must b e reevaluated. of MS cases.

b.

It is seen in about one third

The relationship with relapses is not constant.

The cytomorphological study of CSF is now commonly performed.

made after sedimentation, or better, after centrifugation.

It is

A normal CSF

contains about 75 per cent of lymphocytic cells and 25 per cent of histiocytic cells.

In our experience with lUj M S patients (work in

preparation), the cellular distribution is not modified, b u t , in more t h a n one case out of two, lymphocytic and/or histiocytic cells tend to b e c o m e basophilic and enlarged, and in about every other case typical plasma cells are present in small amounts, even when pleiocytosis is lacking.

As already indicated there is a clear relationship between

increased CSF IgG levels and the presence of plasma cells.

330 c.

lymphocyte populations and subpopulations

Total T-lymphocytes and B-lymphocytes counts, studied by the sheep red blood cells rosetting and surface Ig detection, respectively, show variations in blood and CSF but they are not consistent in the different studies published. According to different studies using various techniques (percentage of avid T cells; study of the non specific suppressor activity of T cells after stimulation by Concanavalin A; percentage of T cells with a receptor for the Fc fragment of IgG; percentage of T cells reacting with OKT5 and OKTg monoclonal antibodies) the systemic suppressor T cells are decreased.

This is clear mostly during the relapses, even during the

week preceeding the relapse. of the diseases.

It returns to normal during the remissions

Are these cells sequestered in the CNS?

This does not

seem to be the case, as no increase in 0KT8 subpopulations of lymphocytes is found in the CSF.

Similarly, in the brain, their distribution is

restricted to the margin of the lesion and the perivascular region.

How-

ever, from these data, a derepression leading to an abnormal immunological reaction can be hypothesized and a new therapeutic approach can be explored using manipulation of suppressor T cells.

d.

Other alterations worthy of mention:

- nonspecific stimulation of lymphocytes by polyclonal activators such as Pokeweed Mitogen, Phytohemagglutinin and Concanavalin A shows a very weak response in CSF, and an almost normal one in blood; - there is also a low proliferative response of MS lymphocytes in allogenic mixed lymphocytic cultures;

331 - the production of leucocytic Interferon and the NK cells activity are usually decreased. 3.

Immunological disturbances were the basic justification for immuno-

suppressive trials.

The efficiency of these protocols on the disease

course would have been a reciprocal evidence for the immunological mediation in the MS process.

In fact, this efficiency is still debated.

From several long term studies with Azathioprine it seems to be moderate or minimal although effective at the remittent phase of the disease. C.

Is there any target for these immunological reactions?

There does exist evidence for an antiviral and for an antinervous tissue process in MS patients.

Here are the still current pathogenetic theories

which have prevailed since the beginning of the century. 1.

The infectious theory [3,5,10]

The role of bacteria and parasites, suspected as soon as 188U by Pierre Marie, has been ruled out for some time.

Suspicion concerns viruses and

indirect evidence comes from the epidemiology and the comparison with various models: - the slow virus diseases, marked by a long incubation phase in the target organ of a nonconventional infectious agent; - the post vaccinal and post infectious encephalomyelitis; - some animal diseases:

Theiler's virus infection in the mouse, canine

distemper encephalitis, . . . - in other infectious diseases of the CNS with increased levels and oligoclonal patterns of CSF IgG, most of these IgG can be absorbed with

332 the etiological agent, for example, measles virus in SSPE, and Treponema pallidum in neurosyphilis. Other evidence comes directly from the study of MS patients.

It is known

that antimeasles antibody levels are increased in the serum and, even more, in the CSF of MS patients compared to controls.

An intrathecal

synthesis of these antibodies can be calculated in 50 percent of MS patients.

MS lymphocyte reactivity is abnormal in the presence of the

measles virus as shown by the lymphoblastic transformation test and the peripheral T lymphocyte adherence on measles infected cells.

Curiously,

in the leucocytic migration inhibition test, there is a defect of MIF production in the presence of measles virus. But the arguments against a virus action in MS, notably the measles virus, are several: - the increased antibody levels, their intrathecal synthesis, the abnormal lymphocyte reactvity have been described for measles virus but also for a number of other viruses:

rubella, mumps, vaccinia,

parainfluenza, herpes simplex, . . . - the increased antiviral antibody levels are noted, although to minor degree, in the sibs of the MS patients.

It could be explained by a

genetic contribution; - its

quantitative importance is modest:

the absorption experiments on

viral antigens can subtract only a small fraction (5 to 10 percent) of the CSF IgG. - virus isolation, viral particles detection by electron microscopy, inoculation in to primate hosts with a more than ten year follow-up in some cases, cocultures techniques, in situ hybridation techniques with

333 viral nucleic acid complementary probes are all experiments which have failed to give consistent results; - finally, an overt measles infection has been reported in only one case of a MS patient. Consequently, nonspecific defective immunoregulation instead of a specific and antiviral activity is currently hypothesized.

Another

possibility is that the actual "casual" virus in MS still needs to be discovered. If it really operates in the MS process, several ways are offered to the virus:

a direct cytopathogenetic effect on oligodendroglia; the induc-

tion of immunological cross reaction between this virus and an antigenic determinant in the nervous tissues; the induction of immunological reactions against the oligodendroglia or the DQrelin sheets from virusinduced neoantigens; the activation of macrophages which would become able to phagocytize the myelin or to release toxic factors for myelin, such as proteases ("bystander" effect).

2.

The autoimmune theory [3,5,10]

This stems from the comparison with experimental allergic encephalitis (EAE), of which chronic relapsing forms can now be produced. Other evidence emerges from the study of MS patients: - MS serum added to myelinated nervous tissue in culture induces a myelin destruction, a nyelination inhibition and a blockade of the neuroelectric activity.

The responsible factors can be antibodies or enzymes;

- myelin fragments can be observed by electron microscopy and Myelin

334 Basic Protein (MBP) can be assayed by radioimmunological techniques in MS CSF.

The MBP levels give a good idea of the disease activity.

and anti brain antibodies are present in MS CSF.

Anti MBP

Similarly, antioligo-

dendrocyte antibodies have been reported in sera of 90 percent of MS versus 30 percent of controls; - a lymphoblastic transformation of MS CSF lymphocytes is observed in the presence of MBP, mainly during disease relapses.

The same observation

can be made with peripheral blood lymphocytes or with other brain antigens.

The leukocytic migration inhibition test is usually positive

with MBP for MS patients. The real significance of the data can be questioned, as there is also evidence against the autoimmune theory: - the _iii vitro nyelinotoxic activity of serum is found in 50 percent of the cases, whether MS or not . . .; - the absorption experiments on MBP or brain extracts concerns only a small proportion of the CSF IgG from MS patients; - the MBP release in CSF may be only the indication of the nervous tissue damage.

Its presence has been reported in patients with stroke,

encephalitis and leukodystrophy.

The anti MBP immunization could be only

the result of the MBP release and not the cause of myelin destruction; - the anti-oligodendrocyte antibodies could be nonspecific as oligodendrocyte membranes possess receptors for the Fc fragment of IgG; - the leucocytic migration inhibition test in the presence of MBP can give positive results in patients with stroke or alcoholic neuropathy, i.e. with diseases for which no immunological factor is thought to play a role;

335 - the cutaneous delayed hypersensitivity reactions with MBP and lyophilized human cerebral white matter are negative in MS patients. It is possible to sensitize MS patients against these antigens by repeated subcutaneous injections, but with theoretical risk of inducing an EAE.

Conclusion Any coherent conclusion is still premature.

Clearly, exogenous factors

and genetic susceptibility act in concert to induce MS.

The genetic

part, as shown by twin studies, is of minor importance.

The immunologi-

cal abnormalities are puzzling.

It is tempting to hypothesize a

derepression of the antibody production in the CNS and to attribute it to a defect of the suppressor T lymphocutes.

This scheme is oversimplified

and the possibility that the immunological abnormalities reflect only the genetic predisposition cannot be dismissed.

For many years, it seemed

that a virus could be responsible for MS but the number one suspect, i.e. measles virus, can be probably dismissed.

The autoimmune theory is still

attractive but, despite many reports, the present target antigen is unknown.

Moreover, it cannot be assumed that the autoimmunity signs

present in MS reflect the cause of the disease and not only the consequence of the destruction of the nervous tissue.

For a better understanding of the pathogenesis of MS several questions, among others, need to be answered: - Do the oligoclonal IgG produced inside the CNS have any pathological action?

They have been recently shown to possess a demyelinating effect

on the tadpole optic nerve.

336 - Are they directed against a unique antigen?

If this is the case, the

viral or tissue target still needs to be unravelled.

The genetic back-

ground may be critical for the recognition of oligoclonal band specificities.

As for murine monoclonal antibodies directed against influenza

virus, it is possible that the antibody specificity could be identified only when the viral antigen is presented in combination with the host MHC antigens. - Conversely, do they only reflect a nonspecific derepression of antibody production in the CNS?

This may be the case as idiotypes defined by

anti-idiotype heteroantisera prepared from the CSF IgG of MS cases are not present in the CSF IgG from other MS cases. - Do the immunocompetent cells of MS patients react against a precise viral or tissue target?

In this field, the syngenic restriction

phenomena mast be taken into account for the cytotoxicity experiments. This has not yet been the case and may explain the negative results so far observed. Undoubtedly, data concerning Ms pathogenesis have accumulated to induce confusion with only minor progress in our understanding of the disease. The knowledge of these data is however crucial for further research. classical sentence of Sir William Osier is still valid.

Acknowledgements This work was supported by a grant from the "Association pour la Recherche sur la Sclerose en Plaques" (A.R.S.E.P.).

The

337 References Only recent general reviews have been selected. Brain 103, 281-300 ( 1 9 8 0 ) .

1.

Confavreux, C., Aimard, G., Devic, M.:

2.

Fishman, R.A. ( 1 9 8 0 ) in Cerebrospinal Fluid in Diseases of the Nervous System pp. 1-381+, W.B. Saunders, Philadelphia.

3.

Iivanainen, M.F.:

1+.

Kurtzke, J.F.:

5.

McFarlin, D.E., McFarland, H.F.:

J. Neuroimmunol. _1, 11+1-172 ( 1 9 8 1 ) .

Trends Neurosci. 6, 75-80 ( 1 9 8 3 ) . N. Engl. J. Med. 307, 1183-1188;

121*6-1251 (1982). 6.

Schuller, E.:

7. 8.

Schuller, E.: Nouv. Presse Med. 8, 1+27-1+32 (1979). Schuller, E. ( 1 9 8 1 ) in Les Proteines du Liquide Cephalo-rachidien et Les Maladies Immunitaires du Systeme Nerveux pp. 1-116, J.B. Bailiiere, Paris.

9.

Spielman, R.S., Nathanson, N. ( 1 9 8 2 ) in Epidemiologie reviews, Vol. 1+, pp. I+5-65, Johns Hopkins University.

10. Waksman, B.H.:

Nouv. Presse Med. 8^, 351-357 (1979).

Immunol. Today 2, 87-93 ( 1 9 8 1 ) .

11. Weiner, H.L., Hauser, S.L.:

Ann. Neurol. 11, 1+37-1+1+9 (1982).

12. Weiner, H.L., Hauser, S.L.:

Ann. Neurol. 12, 1+99-509

13. Lhermitte, F., lyon-Caen, 0.:

395-1+06 (1983).

(1982).

Rev. Neurol. (Paris) 1 3 9 , 323-333;

IMMUNOGLOBULINS

Armand

IN N E U R O P A T H O L O G Y

Lowenthal

Laboratory of Neurochemistry, Born-Bunge Foundation, Universitaire Instellinq Antwerpen, B-2610 Antwerp, Belgium

The e x i s t e n c e o f a h u m o r a l

immune r e a c t i o n p u z z l e s

neurolo-

g i s t s s i n c e t h e y d i s c o v e r e d t h a t the c o l l o i d a l r e a c t i o n the r a t i o a l b u m i n / q l o b u l i n s are m o d i f i e d

in the c e r e b r o s p i n a l

in m u l t i p l e s c l e r o s i s

(MS), w i t h o u t

fluid

(CSF)

modifica-

t i o n o f the t o t a l p r o t e i n c o n t e n t . For a long t i m e ,

these

reactions remained empiric, reflecting a qualitative c a t i o n o f the p r o t e i n d i s t r i b u t i o n .

and

modifi-

It is o n l y w i t h the use

o f m o r e s o f i s t i c a t e d t e c h n i q u e s such as e l e c t r o p h o r e s i s i s o e l e c t r i c f o c u s i n q t h a t in the C S F of p a t i e n t s

and

affected

w i t h M S , t h e s e m o d i f i c a t i o n s w e r e l o c a t e d at the level o f the g a m m a g l o b u l i n s and m o r e p r e c i s e l y at that of the

IgGs.

W e w e r e the first to d r a w a t t e n t i o n t o t h i s p e c u l i a r i t y the g a m m a g l o b u l i n s ^ .

Instead o f s h o w i n g an h o m o q e n e o u s

pattern, they presented a succession of protein (Fiq.1).

bands

It took m a n y y e a r s to u n d e r s t a n d that t h i s

n o n c o r r e s p o n d s to t h a t o b t a i n e d e x p e r i m e n t a l l y i m m u n i z e d a n i m a l s . As a m a t t e r o f fact the

phenome-

in h y p e r -

experimental

h y p e r i m m u n i z a t i o n w a s o n l y s e e n in the s e r u m . T h i s non, o b s e r v e d in the IqG r e q i o n , w a s n a m e d IqG w i t h ted

of

phenomerestric-

heterogeneity.

A f t e r o u r first d e s c r i p t i o n

in M S , we o b s e r v e d the

same

p h e n o m e n o n n o t o n l y in c h r o n i c d i s e a s e s such as S S P E ,

trypa-

nosimiasis, syphilis, filariosis, cysticercosis etc.,

but

a l s o in a c u t e d i s e a s e s such as h e r p e t i c e n c e p h a l i t i s .

It can

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

340

I I 1 I XI Fiq. 1. CSF protein aqar qel electrophoresis with restricted heteroqenity of the qlobulins

be observed also in animal diseases such as Icelandic Visna, chronic relapsinq experimental allerqic encephalomyelitis (EAE), etc. In MS the fractionation of the qamma qlobulins is hardly ever observed in the serum. On the other hand, in SSPE, trypanosomiasis, cysticercosis, chronic relapsinq EAE, this phenomenon is observed in the serum and in the CSF (Fiq.2).

Ill f I I • Fiq. 2. CSF and serum protein aqar qel electrophoresis with restricted heteroqeneity of the qamma qlobulins.

341 S i n c e the first d e s c r i p t i o n of t h i s p h e n o m e n o n ,

many

q u e s t i o n s h a v e b e e n r a i s e d w i t h the h o p e that the

answers

m i q h t lead s o o n e r or l a t e r to the u n d e r s t a n d i n q of

the

physiopatholoqy of MS. T h r e e o f t h e m we w i s h to d i s c u s s as w e l l as the there

answers

upon.

W h a t is the o r i g i n of the i m m u n o g l o b u l i n s w i t h

restricted

h e t e r o g e n e i t y or f r a c t i o n s ? M a n y a u t h o r s are in f a v o u r o f a t h e o r y a t t r i b u t i n g o r i g i n to an i n t r a t h e c a l

s y n t h e s i s . At the start there w a s a

certain confusion between intrathecal

s y n t h e s i s and

t i s s u e s y n t h e s i s . At p r e s e n t the p r e v a i l i n q

theory

t h e s y n t h e s i s o c c u r s in c e l l s , l y m p h o c y t e s or h a v i n q e n t e r e d the c e n t r a l n e r v o u s s y s t e m

nervous is that

plasmocytes,

(CNS) and

n a t i n g f r o m the b l o o d . An i n t r a t h e c a l p h e n o m e n o n e x p l a i n w h a t is o b s e r v e d

their

oriqi-

could

in M S , but it c o u l d less

easily

e x p l a i n w h a t is o b s e r v e d in o t h e r d i s e a s e s w h e r e the m e n o n is g e n e r a l i z e d and is a l s o o b s e r v e d

in the b l o o d .

t h e s e c a s e s , p e r h a p s a l s o in M S , we are p r o b a b l y m e n i n q e s as w e l l as i n t r a t h e c a l l y

the

and w h i c h w o u l d be m o r e

i n t r a t h e c a l l y , than n o r m a l l y could be

f r o m the o b s e r v a t i o n s m a d e o u t s i d e the A c c o r d i n q to t h e s e o b s e r v a t i o n s ,

expected

CNS.

it can be a s s u m e d that M S

is p r i m a r i l y an i m m u n o l o q i c a l d i s e a s e and that the

anti-

b o d i e s t h u s f o r m e d w o u l d a t t a c k the C N S and the m y e l i n t h u s p r o d u c e the c h a r a c t e r i s t i c s e m i o l o q y of the Numerous publications have been discussing this however

In

dealinq

with a generalized phenomenon which occurs outside pronounced

pheno-

and

disease. hypothesis,

it m a y not y e t be c o n s i d e r e d p r o v e n . As a m a t t e r

fact w h e n q a m m a q l o b u l i n s w i t h r e s t r i c t e d h e t e r o q e n e i t y

of are

342 o b s e r v e d , we c a n a s s u m e a q e n e r a l i z e d

immune p h e n o m e n o n

at l e a s t , a p h e n o m e n o n w h i c h e v o l v e s t h e r e w h e r e b l o o d miqrated

or, cells

into the C N S .

W h a t are the s p e c i f i c i t y of the a n t i b o d i e s w i t h heterogeneity

restricted

?

In e x p e r i m e n t a l h y p e r i m m u n i z a t i o n ,

it seems that t h o s e

t i o n s are a c t i v e a q a i n s t the a n t i q e n i n j e c t e d .

frac-

It is n o t

c e r t a i n b u t we m a y a s s u m e that in S S P E t h e s e f r a c t i o n s

have

an a n t i b o d y a c t i v i t y d i r e c t e d a q a i n s t the S S P E v i r u s ^ .

May

we r e m i n d t h a t this v i r u s is c l o s e l y r e l a t e d to the m e a s l e s v i r u s . The real a c t i v i t y of these f r a c t i o n s r e l a p s i n q E A E is u n d e r d i s c u s s i o n .

in

For some a u t h o r s ,

f r a c t i o n s are a n t i b o d i e s d i r e c t e d a q a i n s t

mycobacterium

t u b e r c u l o s i s w h i c h is a d d e d to the i n j e c t e d a n t i q e n . s h o u l d be c a r e f u l

One

in d r a w i n q c o n c l u s i o n s b e c a u s e h i q h

of anti-myelin basic protein

(anti-MBP) are m e a s u r e d

b l o o d of q u i n e a p i q s a f f e c t e d w i t h c h r o n i c r e l a p s i n q A n t i b o d i e s to MBP h a v e not y e t b e e n l o c a l i z e d electrophoresis

the

in the q a m m a q l o b u l i n

titers in the EAE.

after

reqion.

The p r o b l e m is m o r e c o m p l e x for M S . Some a u t h o r s q o so far as to d e c l a r e that the a n t i b o d i e s w i t h r e s t r i c t e d

heteroqe-

n e i t y are a n o n s e n s e r e a c t i o n . The s p e c i f i c a n t i b o d y

activi-

t i e s r e v e a l e d till n o w , r e p r e s e n t o n l y a small p e r c e n t a q e the IqGs c o n c e r n e d . N o n e o f the h y p o t h e s e s p u t f o r w a r d be c o n s i d e r e d W e still

can

acceptable.

iqnore the s p e c i f i c i t y of the a n t i b o d y

activity

r e v e a l e d by e l e c t r o p h o r e s i s or i s o e l e c t r i c f o c u s i n q in the C S F o r in the s e r u m o f p a t i e n t s a f f e c t e d w i t h diseases,

in a n y case in M S .

neuroloqical

of

343 The third question is to know how this hyperimmunization occurs. One knows that in experimental hyperimmunization, the phenomenon persists as lonq as the animal is iniected with the qiven antiqen. We indeed should admit that in SSPE, the antiqen persists in the orqanism durinq the whole development of the disease. The same is true for chronic relapsinq EAE. Nothinq can be said reqardinq MS. If, nevertheless, we accept that we are dealinq here with a phenomenon of hyperimmunization similar to that described experimentally, we should also accept the idea that in all those diseases, MS included, an antiqen persists in the presence of antibodies, iust like in SSPE. Consequently it may be asked how it is possible to find both hiqh measles antibody titers in SSPE serum and the correspondinq virus in the tissues of these patients. Several explanations are suqqested: 1. the virus would be deficient. It would probably lack one of its polypeptides (the M-protein). This would explain the resistence of the virus to the specific antibodies. We wish to emphasize the fact that, amonq the measles antibodies found in the blood and the CSF of SSPE patients, antibodies directed aqainst M-protein are also found^. 2. another explanation is that an inhibitinq factor would prevent the normal antiqen/antibody reaction, more precisely, that this inhibitinq factor would help to reach extremely hiqh titers of hyperimmunization. The action of an inhibitinq factor could eventually be one of the clues to the "network theory"^.

344 3. Finally these two hypotheses should not be considered completely independent from one another. They could be partially or totally associated.

Conclusion We wish to say that in a number of neuroloqical diseases an extremely important humoral immune phenomenon occurs. This phenomenon enables to assume, in these diseases, an hyperimmunization due to the persistence of an antiqen. Different hypotheses have been put forward restinq on these bases. None of them can be retained with certainty. We express the hope that a better definition of the physicochemical and immunoloqical characteristics of the CSF and the serum immunoqlobulins will lead to more precise conclusions.

References 1. Lowenthal, A., Van Sande, M., Karcher, D.: J. Neurochem. 6, 51-56 (1960). 2. Karcher, D., Matthyssens, G., Lowenthal, A.: Immunol. 23, 93-99 (1972). 3. Karcher, D., Thormar, H., Lowenthal, A., Noppe, M.: J. Neurol. 227, 29-34 (1982). 4. Karcher, D.: In "Humoral Immunity in Neuroloqical Diseases", Ed. by Karcher, D., Lowenthal, A., Strosberq, A.D., Plenum Press, New York, 1979, pp. 541-551.

CELLULAR INFLAMMATORY RESPONSE IN CEREBROSPINAL FLUID

Christiane Caudie, Odile Freney, Françoise Touraine Laboratoire de biologie, service du Professeur Quincy Hôpital Neurologique - 59, boulevard Pinel - 69 003 - LYON

Introduction The study of cellular inflammatory response has often been considered as of little clinical value because of poor slide preparations and mechanical alterations in the cells. For the last ten years, thanks to the development of new slide-preparation methods better suited to the special properties of the CSF, the cytology of CSF has become an integral part of the complete biological study of CSF. Its essential interest is to define a diagnosis or to eliminate a pathology. 3 routine enrichment methods are used : sedimentation in special chambers, membrane filtration and cytocentrifugation. They all have advantages and disadvantages. Some modifications are sometimes necessary in order to obtain good slide preparations (precentrifugation and addition of proteins). Statistics in our laboratory from 3 194 fluids show by cytocentrifuge method (Shandon-Elliot centrifuge) that 10 % of the preparations are acellular or paucicellular whatever the number of their leukocytes (34 % of them have 2 or less than 2 leukocytes per mm3)(Statistics only from 1982). Cytological studies should be done soon after puncture for accuracy.

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

346 1

-

Cytogram of a normal CSF

In the last years, various terms designated CSF different cellular types according to their origins. Now it is generally agreed that the various leukocytes in CSF are derived chiefly from the circulating blood (1,2). A normal CSF is a paucicellular fluid most often containing 2 or less than 2 leukocytes and less than 10 erythrocytes per 3 mm in lumbar puncture only for the first cubic centimeters. It is composed of 60 to 95 % lymphocytes and 5 to 40 % monocytes. On rare occasions elements which exfoliated from the arachnoid, from the choroidal epithelium and from the ependyma may be found in CSF particularly in infants when there is a hydrocephalus. It is important to be able to recognize them in order not to confuse with tumor cells because as tumor cells they may be in clusters. A normal cell count does not mean a normal cytogram. It is important to examine slide preparations from cell fluids even if they are paucicellular because in this case, cytology may provide the clue for the diagnosis, for example in multiple sclerosis, in old hemorrhages or in tumors.

II

-

Cytogram during inflammatory processes

4 000 CSF with inflammatory reactions have been analysed, and the most diverse cellular reactions were observed, following septic, traumatic, tumorous, chemical, physical or unknown agressions. These cellular reactions are rarely isolated, they are often intricated and of rapid evolution. 4 different types of cytological inflammatory reactions can be distinguished. We present the cases where inflammatory responses are of interest.

347 1.

Cellular inflammatory reactions associated with a majority of neutrophils.

This pattern is always the result of a leptomeninges inflammation. It is the primary cellular reaction and does not depend on the causative agent. It may be of short duration. 1.1.

In bacterial meningitis the cell count reaches the

highest level during the first few days (more than 10 000 per mm^). In neurosurgical wards, meningitis are essentially with "hospital" pathogenic organisms after a trauma, a neurosurgical intervention or a shunt derivation. In infants, the early diagnosis of purulent meningitis is often difficult. Repeated lumbar punctures serve as a guide to the clinical course and management. In a retrospective study in our laboratory on 115 children (600 samples) who had had shunts inserted for congenital or acquired hydrocephalus, we found complications caused by staphilococci (62 %), enterobacteria (14 %), streptococci (11 %). The intensity of the response was greatest in the case of staphilococci epidermidis complications. Early diagnosis is primordial. A quick reply from the cytologist is a great help in diagnosis and for the first application of antibiotics. We usually distinguish 3 phases which are well known (3). . Acute exsudation phase dominated by altered neutrophils which constitute over 90 % of the cells. . Proliferative phase with a rapid decrease in number of cells ; in this phase macrophages, transformed lymphocytes and plasma cells are present. . Repair phase with the almost complete disappearance of granulocytes. The stages of bacterial meningitis may last some days or some weeks. It depends on the virulence of the pathogenic organism, the effectiveness of the antibiotic and the

348 resistance of the patient. Normalization of CSF with therapy is usually prompt and parallel to the overall clinical improvment. For correct interpretation of inflammatory cellular cytology, it is required the knowlegde of the other biological parameters of the CSF and the clinical history because of the non specific sign of an inflammatory disorder during proliferative and repair phases. 1.2.

Aseptic meningeal reactions is the term given to

meningeal fever signs in addition to an increase in polymorphomononuclear leukocytes. CSF is sterile in cultures. This term represents different clinical situations, so it is necessary to determine the cause of the reactions and to follow the evolution. These reactions are often observed in : - neurosurgery wards after intervention without infectious complications. The problem is to distinguish an early bacterial meningitis from a simple inflammatory response. Our study of 55 inflammatory responses after surgery without infectious complications has shown that the cell count varies 3 (mean : 2 50). Cytogram from 2 5 to 3 050 leukocytes per mm results : 60 to 80 % non altered neutrophils 5 to 40 % very often transformed lymphocytes 5 to 40 % transformed monocytes and macrophages. - partially treated bacterial meningitis - meningitis revealing a spreading intracranial process whether infectious or not (brain abscess, empyema, extradural sepsis, tumor, hematoma, cerebral thrombosis...).

2.

Cellular inflammatory reactions with eosinophils

This kind of reaction is relatively less frequent and of weak intensity in our country. Any increase in the number of

349

eosinophils demands special attention although these cells are easily recognized. This reaction is of special significance in allergic processes. Statistics in our laboratory from 3 194 CSF show that 6 % of them have eosinophils. 2.1.

Eosinophils are present in any infectious pleocytosis

The cell count is about 1 % of the total cell number. In our experience as in KBlmel's, eosinophils are relatively more numerous in meningoencephalitis and lymphocytic meningitis than in bacterial meningitis. We generally find eosinophils, in CSF of children with a ventricular shunt and of patients with a tumor of the central nervous system, after a cerebral hemorrhage. 2.2.

On rare occasions it is a true eosinophilic meningitis

As an example, we present 3 cases : - A_garasitic_menin2itis (hypoderma bovis) that was followed for one month. Eosinophils and basophils were at significative high levels (table I).

! Chamber ! Proteins % E. ! % B. , count ^ i cells/mm ! g/i i ,

542

576

, 1 056 , 480

,3'4

, , ,

% L. ! % M.

15

,

5

60

, 15

2,7 4 , 60 2

40

,

5

45

, 10

50 50

,

5

35

,

1

40

, 10 , 10

,

5

60

,

% P.

5

,

240

,

1,86

30

,

124

,

1,41

30

,

5

50

, 10

5

i ,

74

,

1,90

30

,

5

50

40

50

,

767

45

, 20

,

240

! i ,

2,7

, 10 , 10

5

960

35

3

40

1, 15

! very i ' few i

TABLE E

:

eosinophils

M

: monocytes

45

5

5

!

glucose ! mmol/1 ! 1,4 2

, ,

2

,

1,8 2

, ,

2,1 2,4

, ,

1,6 2,4

, ,

I

-

B

: basophils

-

-

P

: plasma cells

L

:

lymphocytes

350

- An_aller2ic reaction developed in a 3 months old baby submitted to ventriculoperitoneal shunt for an hydrocephalus (spina bifida). The eosinophilic reaction stopped when an external ventricular shunt was performed (table II).

!Proteins ! chamber ^ , count cells/mm !p g/i

% E.

! % L. i i

1

80

!

1,50

30

i "

i

30

,

1,41

20

!

150

!

1,48

20

150

!

3,70

25

,

170

,

1,65

25

!

256

! i

1, 30

E

:

eosinophils

M

: monocytes

P

:

% M. ! % N. ! % P.

55

15

i

30

15

i

35

! i

30

15

!

35

15

!

60

15

,

60

i ! i

60

30

!

10

!

!

TABLE II L : lymphocytes -

N

: neutrophils

plasma cells

- An_asegtic_eosinophilic_menin2itis in an adult, 2 days after an intervention for a meningioma. 55 % of cells were eosinophils.

3.

Cellular inflammatory reactions with monocytes and macrophages.

An extraordinary cell reaction takes place if blood infiltrates the CSF. This is the case after an hemorrhage, a cerebral contusion, brain surgery or a traumatic lumbar puncture.

351

3.1.

The cellular reaction is typical during the days and

the weeks after the bleeding. - The first reaction is the appearance of all hematologic cell forms ; some of them transformed, with a majority of granulocytes. CSF may contain several thousands to many 3 hundreds of thousands of red cells per mm . -Then phagocytosis takes place for the elimination of the blood as foreign material. The monocytes are transformed into macrophages. The days after the hemorrhage, we observed erythrophages (they are able to absorb

one to more than a dozen of red

cells), then pigmentophages(containing hemosiderin or hematoldin) and leukophages (containing phagocytosed leukocytes) if the phagocytic reaction is intense. 3.2.

Monocytic and macrophagic reactions may be observed

when there is an irritation in leptomeninges by physical, mechanical or chemical agents : intrathecal therapy, pneumo encephalography, presence of a shunt or a tumor (4). 3.3.

Basophilic and macrophagic multinuclear giant cells can

be seen in different pathologic processes. We observed them after subarachnoid hemorrhages in children and in the ventricular puncture of a young man with a ventricular block one year after a tuberculous meningitidis (the pleocytosis was 430 per mm^). These giant cells are rarely observed in tuberculous meningitis and have little pronostic significance.

4.

Cellular inflammatory reactions with predominance of

lymphocytes. We often observe these reactions in various inflammatory diseases. An increase in the number of lymphocytes may be an indication of a local cell-mediated or humoral antigenantibody reaction or it may occur as a non specific response to tissue injury.

352

4.1.

Intense cellular response is generally observed in

viral meningitis and meningo-encephalitis. They are caused by heterogeneous viruses. Cellular response is greatest in patients with predominant signs of meningeal irritation. The cell count may be increased to levels of 1 000 leukocytes per mn\3 ¿ u r ing the acute phase. The cytogram shows a variety of transformed cells (lymphocytes and monocytes). Plasma cells are also numerous. Some eosinophils and basophils are very often associated in cases of coxsackie meningo-encephalitis. Normalisation of cell count and cellularity often requires several weeks. 4.2. Moderate pleocytosis is observed in tuberculous meningitis, cerebral and spinal syphilis, brucellosis. There are no characteristic differences in the cellularity. The changes in CSF are quite variable reflecting the stage of the disease. 4.3.

Low pleocytosis and normal cell count

- The increased number of mononuclear leukocytes of patients with multiple sclerosis could reflect cellular infiltrates found in the regions of demyelinating lesions. But data obtained from several large groups of patients reveal a normal cell count or a low level of pleocytosis. In our experience in 219 multiple sclerosis, leukocytes count was less than 2 leukocytes per mm^ in 38 % of patients, between 2 to 5 in 12 %, between 6 to 10 in 28 % upper than 10 in 22 %. The cytogram is presented in table III.

353 1 ! ! M. S.

1 Cell count! cells/mm3 ( 1

! %. M. i,

o .P "6

! P ! presenqe

! 84 + 15 ! 10 + 7 i ! 83 + 17 " 10 + 8 i

4 + 3 ! 52 % ! i i 5 + 3 • 56 % • i i

! 87 + 13 ! 9 + 5

5 + 4 ! 40 % !

7 + 25

80 + 19 " 10 + 4 !

2 + 1 " 15 % " i I

9 + 12

! 65 + 28 ! 11 + 7 i i

3 + 4 ! 10 % ! i i i I

n = 219! 1 2 + 1 8

! confirmed M.S.! n = 95 , i probable M.S. . n = 64 i i possible M.S. * ! n = 60 ! , non M.S. , groups n = 198" ! ! i i

% L.

9+12 7 + 6

TABLE III Immature and mature plasma cells such as lymphocytes with only some of them transformed, are characteristic of multiple sclerosis, the CSF total cell count being normal or nearnormal . Plasma cells in our study are present in 56 % of confirmed multiple slcerosis. There is a correlation between the degree of cell count elevation and the presence of plasma cells (p as the increase in gammaglobulin content (p

1%0) as well l%o), but they

are some patients with plasma cells and a normal cell count (11 cases in 95 confirmed multiple sclerosis in our study) . We found no correlation between the biological parameters and the stage of the disease (lumbar punctures have most often been performed during a period of exacerbation). - brain tumors naybe associated with a leukocytic pleocytosis composed of lymphocytes and monocytes as well as with malignant cells in fluid. Tumor cells may be found in the CSF with the gamut of neoplasms that affect the brain or meninges. Their identification is a valuable diagnostic aid particularly with primary and secondary tumors that infiltrate the leptomeninges and also in the management of meningeal leukemia (5).

354

Conclusion The study of CSF cellular inflammatory response, routinely performed with lumbar, cisternal or ventricular punctures i of great importance for diagnosis of neurological diseases. This is particularly true with septic and aseptic meningiti cerebral hemorrhage, multiple sclerosis and with primary and secondary tumors in the CNS.

References 1.

Oehmichel, M., Griininger, H. : J. Neurol. Sci. 22_ , 165-176 (1974)

2.

Oehmichel, M. : Acta cytol. 20, 548-552 (1976)

3.

Kolmel, H. W. : Atlas of cerebral spinal fluid cells, second enlarged edition, Heidelberg . New York (1977)

4.

Morantz, R. A., Wood, G. W. , Foster, M., Clark, M. , Gollahon, K. : J. Neurosurg., 50 , 305 - 311 (1979)

5.

Balhuizen, J. C., Bots, G. T. A. M., Schaberg, A., Bosman, F. T. : J. Neurosurg., 48^, 747-753 (1978)

Acknowledgements We are grateful to Dr Suzane GUIBAUD for many helpful discussions.

EVALUATION OF CSF LYMPHOCYTE SUBSETS USING THE OKT SERIES OF MONOCLONAL ANTIBODIES (OKT3+ , OKT 4 +, OKTg+) IN VARIOUS NEUROLOGICAL DISEASES

Gilles Servoz, Françoise Touraine, Claude Quincy Laboratoire de biologie - Hôpital Neurologique 59, boulevard Pinel - 69 003 - LYON

Introduction In various neurological diseases, study of iiranunoregulation, and more precisely, studies of lymphocyte subsets in CSF may be a useful tool for their diagnosis and their prognosis. These immunological studies become more valuable if biochemical and viral determinations are simultaneously tested. CSF lymphocyte subset with phenotype 0KT 3 +, 0KT 4 +, 0KT g + determinations have been performed in patients with multiple sclerosis (M.S.) or other neurological diseases (polynevritis, Behget syndrome, Guillain Barré syndrome,...)(O. N. D.). Our purpose was to modify the usual technology for the phenotyping so that only an aliquot of 5 to 7 ml of CSF would be required. On this same sample, routine biochemical and immunochemical studies are performed.

Material and methods Material 2 5 CSF samples have been studied (obtained by lumbar puncture) subdivided in 2 groups : group 1 : 16 CSF from patients with severe progressive M.S. group 2 :

9 CSF from patients with O.N.D.

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

356

Method The total number of white cells in the CSF is counted ; 5 to 7 ml of CSF are centrifuged in a conical tube for 30mn at 350 g, at 10°C. The supernatant is then nearly totally discarded and will be used for biochemical and immunochemical studies. The pellet, divided in 3 aliguots is distributed in 3 Eppendorf conical tubes of 1,5 ml, approximatly 30 % of AB serum is added to the aliquots and respectively 5 Ail of antiserum 0KT 3 , 0KT 4 , 0KT g

(Orthoclone ). The tubes are

incubated 30 mn at 4°C, then the cells are washed with 500 jul of Hanks balanced salt solution (HBSS) and centrifuged 10 mn at 400 g. 20 jul of the appropriate dilution of fluorescein conjugated goat anti mouse IgG antiserum and 100 ill of HBSS with 30 % of bovine serum albumin (HBSS/BSA) are respectively added to each tube. After a 30 mn incubation at 4°C, the cells are washed with 500 jul of HBSS/BSA and centrifuged 10 mn at 4 50 g. The supernatant is discarded and the pellet is placed between slide and coverslip with a special modification so that the cells are concentrated and this allows the count of at least 100 cells, on a microscope with an epifluorescence (Zeiss).

Results Preparations with no more than 100 cells counted are not included in the results. The results are on table I.

357

Group I !

0KT 3 +

, 0KT 4 +

: S.E• P .

Group II : O.N D.

i

i

i

I

!

n=l 1

80,1 % + 7,8

n=9

56,2 % + 18,4

n=l 1

57,8 % + 12,5

i I n=7 , N.S. ,

I

N.S.

t !

0KTg+

! T

4 /T8

!

85,6 % + 7,4

i

i

34,6 % + 6,7

n=12

39,3 % + 14,7

n=9 ! 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. U:

Degenerative profile [ 31 -

A: serum; B: CSF

This profile, which is often found in children, has not yet been assigned to a specific syndrome [5lThree 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 69 003 - 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 II 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 Associates1* and each determination has been done in duplicate, including standards and controls. Individual values in each group are shown in figure 1. HO"

Control subjects

2

Neurological disorders :

. multiple sclerosis ( ° ) . nerve diseases (« ) . subarachnoidal haemorrages and hematoma ( • ) . degenerative disorders (0 ) . others ( " )

mSs1 ">000 0 ••• •««loo 0ooO « o aA 0 0 ODD »f ' -1 1 1 r "i—i—r 1

1

2

Figure

3

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 yug/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 (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 Ferritin

79,6

Sensibility 45,5

Efficiency 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) Syndic, C. J. M., Collet-Cassard, D., Cambiaso, C. L., Masson, P. L., Laterre, E. C. : J. Neurol. Neurosurg. psychiatry 44, 329-333 (1981)

4. 5. 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 de 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 y.q/m\ ± 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 02m 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 of the CSF Encephalitis

Encephalopathies

D1 to 3

D5 to 8

D12 to 15

72

34

16

9

Lymphoid cells %

56

54

50

Histiomonocytoi'd

27

16

42

Leukocytes count

Dlto3

D5 to 8

D12 to 15

14

3

30

30

20

52

60

75

(mm3)

cells % In the group of patients with encephalopathy, we have noticed (fig) no significant changes of the CSF and blood 02m levels. On the other hand, a clear increase of the enzymatic activities was statiscally significant, especially at D5-D8 (LDH : 68.% ± 16.5 ; A S A T : 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 02m is significantly increased in children with ence-

381 LEUKO/ ß 2M 4 ß g/l MM*

LDH ENCEPHALITIS

U.W.

60 50

75 40 50

25

1

T

\

30 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 /} 2m ( • ) , 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 be attribued 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, 355-356,1982 4 - Viallard, J.L., Gaulme, J., Dalens, B. and Al. Clin. Chim. Acta, 89, 405409, 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. M Ü L L E R ,

Hôpital 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 k 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 8.5% gels in a LKB Multiphor apparatus with a Tris-glycine pH 8.9 buffer. Gels were preelectrophoresed at 50 mA for 30 minutes. Five (jl 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 de Gruyter & Co., Berlin • New 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 were dried and passed through a densitometer to determine the number and density of each band.

3)

Quantitative assay of CSF cholinesterases. Activity of cholinesterases was estimated by the photometric method of Ellman et al. (4) applied to the V.P. Abbott apparatus. Inhibitors were 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 points 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 forms? 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 VII, 1982. 2. VALETTE, F.M., MARSH, D.J., MULLER, F., MASSOULIE, J. ¡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. 3. Histochem. Cytochem. 12, 219-221 (1964). 4. ELLMAN, G.L., COURTNEY, K.D., ANDREAS, V., 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. Simplot, S. Guibaud Laboratoire de Biochimie ( S c e Pr Reboud) Hôpital de la Croix Rousse 69317 Lyon France A. Mercatello, D. Robert Service 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 , Hôpital de l a Croix Rousse 69317 Lyon France 0. Robert Service de Neurologie, Hôpital Neurologique Lyon

France

Introduction The diagnosis o f G u i l l a i n - B a r r e syndrome rests on c l i n i c a l

observations,

electromyography and albumino-cytological d i s s o c i a t i o n in 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 involved, remain unknown and the diagnosis i s not easy. Working on cholinesterase isoenzymes, we have observed a special i n the cerebrospinal

pattern

f l u i d of 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 patients the r e p a r t i t i o n of which i s : . meningities - 25 . varied neurologic desorders - 48 (cerebral vascular deseases 5, tetanus 4 , multiple s c l e r o s i s 8, met a s t a t i c tumors 3, Horton's desease 1, cerebral atrophies 3, seizures 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 e n c e p h a l o p a t h i e s 2, P a r k i n s o n ' s desease 1, t r i c y l c i c a n t i d e p r e s s a n t s cephalopathy 1, p o s t s u r g e r y 1, v i r a l . peripheral

encephalities

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 i 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 per c e n t 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 per c e n t sucrose 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

b l u e ) 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 ,

(bromophenol 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 p r e c i p i 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

m a l e a t e , sodium c i t r a t e ,

acetylthiocholine

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 a good degree o f s p e c i f i c i t y

: L y s i v a n e (ethopropazine)

are used w i t h

f o r non s p e c i f i c

chol i n e s t e r a s e (nS Ch) ( E C . 3 . 1 . 1 . 8 )

and BW 284 C 51 (1-5 b i s - 4 a l l y l d i -

methylammonium p h e n y l - p e n t a n - 3 - o n e )

for acetylcholinesterase

(A ChE)

(EC 3 . 1 . 1 . 7 )

Resul ts 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 .

side

This band i n h i b i t e d by BW 284C51

but L y s i v a n e r e s i s t a n t , 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 G u i l 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-

Table I : Patient r e p a r t i t i o n and CSF isoenzyme pattern.

Pathologies

CSF

Patients

-

0

. . Protein -|-l)

Bands . ,r „cr, A rChE nSCh

n

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

Pathologies

Patients

CSF

Spinal cord compression

1

3

55

10,03

+

0

Myelitis

1

1

2

0,14

+

0

Polyradi culoneuropathies (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

Polyradicul oneuropathy (4 monthes later)

1

1

2

0,53

Polyneuropathies

2

2

2

Suspected p o l y r a d i c u l o neuropathies ?

Leuk. Protein (Mega l" 1 ) ( x - g l " 1 )

25,6

Bands A! ChE nSCh

1+ lo +

0

+

0

II

390

An üninterpretable pattern is obtained in case of blood contamination see photo 1.

r?

Photo 1 Cholinesterase isoenzyme pattern in CSF. From l e f t to right : .two "standard" CSF .CSF from GuiHain-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 later when the c l i n i c a l symptoms are normalized, nor for the polyradiculoneuropathies with low increased CSF proteins. 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 proteins. 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 2 1 Christian Marescaux , Maurice Collard , Simone Mayer 'clinique Neurologique, Hospices Civils de Strasbourg 2

Institut d'Hématologie, 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 Germany

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 ¿zAum

o 6 MS

tested in the presence of following allocells 15°C

sera TL

37 °C

BL

M

TL

BL

M

9 cases 4 cases

-

-

-

-

-

+

+

+

-

-

3 cases

+

+

+

-

4 cases

+

+

+

1 case

+

+

+

The ¿tA.ength 0(J the. reaction + poiitive

reaction,-

+

i& not indicated

negative

+ +

+

+

+

in thii> 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., 263-273. 3. Terasaki, P.I., Mottironi, V.D., Barnett, E.V. : N. Engl. J. Med., 28j3, 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 • N e w 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

i^ FIGURE

m

:

-C.S.F.

electrophoretic

-Multiple

Sclerosis

patterns : A C

-Non

inflammatory

other

from

Agarose P.A.A.

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. 2_9 , suppl. 124, 7-19 (1972)

5.

Kerenyi, L., Gallyas, F. : Clin. Chim. Acta 38 , 465-467 (1972)

400 6.

Gerson, B., Cohen, S. R., Gerson, J. M., Guest, G. H. : Clin. Chem. 27/12, 1974-1977 (1981)

7.

Laterre, E. C., Callewaert, A., Heremans, J. S. : Neurol. 20 , 982-990 (1970)

8. 9.

Link, H., Mueller, R. : Arch. Neurol. 25_ , 326-344 (1971) Johnson, K., Nelson, B. : Ann. Neurol. 2 , 425-431 (1977)

10.

Johnson, K., Arrigo, S. C., Nelson, B. J. et al. : Neurology 27 , 273-277 (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 T = 4 Reagents :

% , 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

JL.

3

4

5

: 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

1

PH

"o 1 ^

II c

Repeatability

1

! II

!

c

!

T) >i 0 -P M -H ftnH i 0

=

i !

0

X

" SD !

; i

Slope

2D electrophoresis

_ -

=

0,385

J

0,023 5,9 %

!

SD

= =

!

CV

:

X

t

: 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)

SECTION VI INFLAMMATION AND MALNUTRITION

ANTHROPOMETRIC AND BIOLOGICAL CHARACTERIZATION OF MALNUTRITION STATES IN MAN Claude André and Simone Danière Groupe d 1 Immunopathologie digestive, INSERM U 45, Pavillon 1G, Centre Hospitalier Lyon Sud, 69310 PIERRE BENITE, FRANCE

Introduction Defining

the

normal

human

nutritional

state

is

a

problem.

There are nevertheless several

situations in which this clas-

sification

instance

is necessary,

compare populations quiring medical

care

illness risk group. panies

have

for

or when we must because

they

are

In this context,

established

that

each time we wish

identify in

individuals

a higher

death

or

American insurance com-

body weights

could

be

determine which subjects had longer or shorter life cies.

to re-

used

to

expectan-

There is in fact a clear association with mortality and

overweight.

Underweight

individuals

also have

shorter

life-

spans, but to a lesser extent. The

nutritional

state

of an

comparison to anthropometric fect

individual

criterion(a) has(have) not yet

does not

prevent us

can be determined

or biological

from using

been

currently

standards. defined,

in

A perbut

this

employed methods to

predict the chances of success of failure of a given treatment to determine indispensable therapeutic

complements.

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

410

Anthropometric

Standards

1) Mean weight - Ideal The simplest

weight

measurement

is that of body weight.

that this parameter

is height- and sex-related.

studies

that mean

This

have

"normal"

shown

weight

life expectancy.

gain

number

of

increases

undesirable

formulas

has

of Lorenz and Vague,

been

proposed

number

There

aging.

refer

to

calculate

to

equal

to

1/4

of

the

is

from which we

difference

between

in cm:

(kg) = (height cm-100) - (height

are two major

this

where the ideal theoretical weight

150 and height expressed Ideal weight

we

formula for adults is that

equal to height expressed as cm above one m, a

with

if

in an adult regardless of age.

ideal weight. The most classical

subtract

Population

This idea has led to the definition of an

ideal weight, constant A

weight

is

We admit

criticisms

to

be

cm-150)/4-

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,

cellular liquid and the cellular mass, cles, tions

and blood in

and visceral

relation

to

distributed

a

proteins.

standards

may

extra-

which includes musBody weight be

the

result

variaof

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

the pelvis

rence

of

the

measured which

wrist.

between

the

or the The

shoulders

slight

anterior

type and

and by the

circumfe-

has a

pelvic

width,

superior

iliac

spines

is less than 28 cm and a wrist circumference which

less than 16 cm. The heavy skeletal

is

type has an iliac width

411

greater than 20 cm.

29 cm

and a wrist circumference

greater

The mean type has intermediate values.

account for this skeletal proposed

another

formula

parameter,

than

In order to

Monnerot-Dumaine

(1)

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, weight

Edema

accompanying

certain

nutritional

syn-

for example in children with kwashiorkor, causes a increase

cause the

which may

degree of

denutrition

to be underestimated. 2) Importance of the lipid Virtually In

a

all human energy

normal

weight,

compartment

male,

it

reserves are in adipose tissue.

accounts

while the corresponding

around 20 to 25%.

for

10

figure

to

15%

of

total

in normal women is

Half this fat is subdermal

with

varying

distributions.

The value of determining the adipose tissue

content

organism

of

the

physiologically tissue.

defined

On the

is obvious.

Obesity

in

fact

is

as an excessive proportion of this

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

extracellular

and

the

non-fatty

support tissue

cell mass

cell

from total

is determined

mass,

body weight.

isotopically

The

water'

non-fatty

as the dilution

space

412 of

potassium-42;

measuring

extracellular

inulin

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

and the

is 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 ej; al .

however

(3),

the exact determination

As noted by Apfel-

the difficulty of this method is

of respiratory

volumes.

Two

other

indirect methods which are easier to use are more regularly employed. with

an

g/mm ,

One is the measurement of skin folds, determined appropriate

compass

exerting

pressure

sub-scapular, regions.

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

W/S

consensus,

or W/S

however,

is

as a

correla-

between per cent body

(or skin fold) and the weight/size ratio L

thick-

face of the arm

function of age and also of racial type. Excellent

is no

10

usually the tricipital, bicipital,

pectoro-axi1lary,

can be deduced from these measurements.

fat

of

regardless of the spread. The measurement should be

done at several points,

15 mm

a

concerning 3

the

(W/s). ideal

There

formula:

, 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

especially

organism

can

be

muscles,

in

determined

by

the 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, meter or circumference of its muscles (6,

and the dia-

7). Some authors

perform these measurements with the thigh. A more sophisticated method

for determining total

in the organism involves measuring body nitrogen total

cell

mass by

measuring

exchangeable

protein

(8),

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. are an

is

of

proposed

siderophilin these

much

lower.

and

retinol

half

lives:

48

hours

ing

In

Serum

of

These these

in

data

and

is

also

adult,

in

children

ed

as

for

diet.

72

hours

The

index

creatinine healthy tions ed

per

sometimes

as

in is

24

hours

zinc.

for

the

the

denutrition

have

min,

determination that

while or

have

A

been

prealbu

The

certain,

the

plasma

prealbumin very

for

is

different

siderophil i n ,

the

highly

the

intact.

liver

fact

for

retinol

bind-

different

reac-

malnutrition.

indication

Creatinine

and

example

of

concentrations

absence as the

subjects.

leac' t o

in

indices.

calorie

(10).

triglycerides

proteins;

protein

hours

explain

excellent

the

the

proteins

12

denutrias

albuirin,

reserves,

determined

size-paired

deficiencies,

such

an

the

protein.

hemoglobin

and

clearance

by

an

denutrition

immunoglobulins

al buiri ri, 8 d a y s

and

biological

protein

plasma

neglect

these

for

and

pro-

indicate

with

of

criteria

of

binding

addition,

complement

crease

not large

prealbumin

protein.

tivities

are

days

case

cholesterol

binding

insufficient tests

associated

including

must

an

blood

the

leaves

retinol

20

for

in

synthesized

index,

of

often

levels

usually

albumin,

is

biological

the

parameters

as

of

an

concentration

there

Standard is

The

proteins as

if

important

included

decrease

number

such

which

as

subject.

long

general

of

low

diet.

determinations

obese

have

be

the

state

This

the

may

in

of

same

A number

search

clearance

for

vitamins,

and

ratio

is

folic

local acid,

in

measurin

between

clinical

more

mass

fish

parameter of

de-

Creatinine

of m u s c l e

meat

the

also

the

urinary

in

normal

observaassociatelements

415 Malnutrition

is

often

caused by

a decreased

important

sign

lymphocytes. mined

of

accompanied

dénutrition,

as

On a more functional

if the immune

nutritional

a positive

hypersensitivity tetanus anatoxin,

to

frequent

infections

Lymphopenia

is

the

level,

is

decrease

an

of T

it can be deter-

system is or- is not, compromised by the

insufficiency

presenting

by

immune function.

by

studying

anamnestic

contact

the

response of

antigens

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

These

pre-operative

body

weight

decreases

situations are unfortunately

no less maximalist.

The

problem

posed

that of selecting high

risk subjects,

tients whose

would

prognosis

be

resistance

is also established more

in

than

too frequent 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/1 is an

subjects

with

The

workers

believe

unfavorable

severe

in the

nephrology

department

(12).

that

sign

diseases

predicts a longer hospitalization arid more frequent in patients

conclu-

for example in the a

for a

(11)

or

infections

Treatment

of

416 surgical patients by enteral or parenteral nutrition,

accord-

ing to whether

concen-

it

succeeds in increasing the

tration over 35 mg/l 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

(18)

to

be

or

unreliable

markers as a result of excessively rapid fluctuations There are also supporters hypersensitivity test.

and detractors

of the

either

(19).

delayed

skin

The former admit a predictive capacity

for the course of the disease property

albumin

retino! binding

considered

status

by

(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 diverresults

obtained.

The

has

caused

nutrition workers to emit defeatist considerations

well (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

be superfluous on a practical

level

cal situations, they are useful Finally,

assays would seem to

in cases of extreme cliri.i-

in equivocal cases

(22).

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,

only

of

inadaptation

skin

articles

explains

tests,

in

why

3 studies used

taken

into

a

English the

of

appearing

results

age-paired

remain

controls,

in the disease

acc ount.

Tn

this

special

in

is

con-

whom

also

We

thus

understand

what

when

they

are

the search for an anergic state in patient

undergoing digestive It

nor

although it is the measurement of an anamnes-

response.

patients

the

critical

the effect of repeating the skin tests was not

immune

(25).

frequent

neither the differences

homogenous series, of

hundred

given was

sidered either, tic

a

hi terms

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. 63., 1037-1038 ( 1955)

2.

Geigy, Bale

J.R.:

Scientific

Tables,

ö^*1 edition

Geigy

SA,

(1963)

3.

Apfelbaum,

M.

Vigy,

4.

Cronk.,

5.

Frisancho, A.R., Flegel, P.N. :

Paris 10506 B10-9

M.,

Igoin, L.:

Encycl. Med. Chir.

(1973)

C.E., Roche, A.F.: Am. J. Clin. Nutr. 35, 347-354

(1982) 697-699 6.

Am. J. Clin. Nutr. 36^,

(1982)

Durnin,

J.V.G.A.,

Womersley,

J.: Br. J. Nutr. 32, 77-97

(1974) 7.

Cureton, K.J., Boileau, R.A., Lohman, T.G.: Hum. Biol. 47,

8.

McNeill,

321-326 S.L.,

(1975) K.G.,

Memagh,

Harrison,

J.E.:

J.R.,

Jeejeebhoy, K.N., Wolman,

Am.

J. Clin. Nutr. _32, 1955-1961

C.E.

Carballo, A.J., Vasquez, I.:

(1979) 9.

Talso,

P.J.,

Miller,

Metabolism 9, 456-471 10. Trowbridge,

F.L.,

(I960)

Hiner,

C.D.,

Robertson, A.D.: Am. J.

Clin. Nutr. ¿6, 691-696 (1982) 11. Apelgren, K.N., Rombeau, J.L., Twomey, P.L., Miller, R.A.: Crit. Care Med. ^0, 305-307 (1982) 12. Anderson, C.F., Wochos, D.N.: Mayo Clin. Proc. 57, I8I-I84 (1982) 13. Ching,

N.,

Grossi,

Mills,

C.B.,

199-202

(1980)

C.E.,

Nealon,

T.F.:

Angers, J., Zurawinsky, H.S., Surg. Gynecol. Onstet. 151,

14. Klidjian, A.M., Foster, K.J., Kammerling, R.M., Cooper, A, Karrah, S.J.: Br. Med. J. 28l, 899-901 15.

(1980)

Klidjian, A.M., Archer, T.L., Foster, K.J., Karran, S.J.: J.P.E.N. 6, 119-121

(1882)

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. Cais,

M.J.,

Delacoux,

Drevi11 on,

C.,

Raffray,

Y.,

Miocque, M.: Sem. Hop. Paris 58, 2833-2837 19. Farthing, M.J.G.: Nutr. Res. 2, 561-568 20. Tasseau,

781-784

F.

(1982)

(1982)

Gaucher, L. Nicolas, F.: Sem. Hop. Paris 58,

(19o2)

21. Brown, C.A.,

Succari, M.,

E., Evstigneeff, T., Desmoulins, D., Arsac, M.,

R.,

Bancewicz,

Farrand,

R.J., Pumphrey, R.S.H., Irving, M.: Brit.

Med. J. 284-, 851-853 22. Baker,

J.P.,

Stewart,

S.,

J., Hamid, J., Patel, N.J., Ward,

(1982)

Cetsky,

A.S.,

Wesson, D.E., Wolman, S.L.,

Whitewell, J., Langer, B. Jeejeebhoy, 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., Fahre, F.,Patel, J.C.: Nouv. Presse Méd. 9_, 1559-1563

(1980)

NEW FINDINGS ON T H E R O L E OF ZINC AS A BIOLOGICAL R E S P O N S E

Susanna C u n n i n g h a m - R u n d l e s ,

MODIFIER

Ph.D.

L a b o r a t o r i e s o f Clinical Immunology a n d Human Memorial S l o a n - K e t t e r i n g C a n c e r C e n t e r 1275 York A v e n u e , New York, NY 10021

Immunogenetics

Introducti on

Several

lines of i n v e s t i g a t i o n have s u g g e s t e d t h a t the c e l l u l a r

r e s p o n s e m a y be interactions

modulated

immune

b o t h in v i t r o and in vivo by network

in which zinc m a y a c t as a p o t e n t i a t i n g a g e n t (1-4).

We and

others have p r e v i o u s l y d e m o n s t r a t e d that zinc is a m o n o c y t e d e p e n d e n t T lymphocyte m i t o g e n in vitro (5,6) and t h a t loss of this c o r r e l a t e s w i t h a d v a n c e of m a l i g n a n c y

function

in cancer p a t i e n t s .

Furthermore,

w e have found that zinc can a c t i v a t e human B lymphocytes to in vitro into a n t i b o d y s e c r e t i n g cells

(7) and to a c t s y n e r g i s t i c a l l y

this s y s t e m w i t h o t h e r B cell s t i m u l a t o r s .

We and o t h e r s have found

zinc m a y improve immune r e s p o n s e in zinc d e f i c i e n t persons cases.

differentiate in that

(8) in some

In a d d i t i o n , w e h a v e found a c l o s e link b e t w e e n zinc and thymic

h o r m o n e , as r e p o r t e d also by Bach

(9).

In the studies d e s c r i b e d h e r e , possible r e g u l a t o r y i n t e r a c t i o n s w i t h the natural

k i l l e r , NK, s y s t e m w e r e e x p l o r e d using Z n C l , Z n + + as a source o f

zinc.

Methods

Peripheral

b l o o d m o n o n u c l e a r cells, PBM, w e r e isolated from n o r m a l ,

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

(10), w a s h e d and

r e s u s p e n d e d in RPMI 1640 s u p p l e m e n t e d w i t h p e n i c i l l i n , g l u t a m i n e , a n d 10% pooled normal human serum.

T h e 4 hr

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

streptomycin, 51

C r release

assay

422 was used to assess natural k i l l e r , NK, function against the K562 tumor target cell as previously described (11).

D e t a i l s of the longer term

activated NK assay have been described (12).

The Z n + + used was f r e s h l y

diluted from a 30 mM stock s o l u t i o n of ZnCl.

Results and D i s c u s s i o n In view of previous f i n d i n g s that zinc has the p o t e n t i a l i t y to enhance immune response in v i t r o and in some i n d i v i d u a l s in vivo in s i t u a t i o n s r e f l e c t i n g enhanced p r o l i f e r a t i o n , studies were undertaken to examine the effect of zinc on natural k i l l e r a c t i v a t i o n in v i t r o . natural k i l l e r , NK, function were assessed.

Two types of

The f i r s t , endogenous NK

a c t i v i t y , was studied using f r e s h l y i s o l a t e d peripheral blood mononuclear c e l l s , PBM, in a 4 hr

51

Cr release assay against the K562 target.

The

second, activated NK f u n c t i o n , was examined following culture of PBM for s i x days with and without NK a c t i v a t o r s followed by exposure of these prestimulated c e l l s to labeled K562 target c e l l s i n the 4 hr assay.

51

Cr release

Concurrent addition of NK augmenting agents, s p e c i f i c a l l y a or y

i n t e r f e r o n , IFN, was carried out in the 4 hr assay in both procedures in some cases to assess pre-NK a c t i v i t y and residual potential activity.

pre-NK

A l l studies were conducted in pooled normal human serum to

avoid possible stimulating effects of fetal c a l f serum. Addition of Z n + + to f r e s h l y i s o l a t e d PBM in a 4 hr assay of cytotoxic a c t i v i t y against the K562 tumor target was observed to produce a sharply i n h i b i t o r y effect on l y t i c a c t i v i t y when present in the assay period only. As shown in Table I , t h i s effect was h i g h l y dependent upon concentration and was r a p i d l y diluted out so that at a [ Z n + + ] of 4.0 X 10" 5 M, t h i s was no longer observed.

One day incubation without Z n + + followed by exposure

to the labeled tumor c e l l target produced loss of NK a c t i v i t y .

This

spontaneous l o s s of a c t i v i t y i s u s u a l l y observed in studies of normal human peripheral blood lymphocytes and has been widely reported. here, Zn + + further depressed l y s i s . was seen at 4 days.

As shown

Additional diminution of NK a c t i v i t y

S i m i l a r l y , NK augmentation by alFN was apparently

423 inhibited by the presence of Z n + + in the absence of preculture and again this effect was rapidly lost with lower [Zn + + ]. Table I.

Effect of Z n + + on NK Activity

Augmenting Agent

None

0 7.5 X lO"4 M 2.0 X lO" 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_lt M 2.0 X 10"'4 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„ + +

2

x 2

4 hr 5 1 Cr release assay agai Final concentrations.

Preculture Period 1 Day 4 Days 5.5 1.0 N.D. N.D. 7.4 21.0 N.D. N.D.

K562; E:T ratio, 100:1.

In contrast, enhancement of alFN mediated augmentation was observed following 1 day preculture with both alFN and Z n + + together at concentrations of Z n + + less than 5 X 10"4 M and furthermore, by day 4, a concentration of 7.5 X 10" 4 was associated with a 3-fold increase in NK activity over cultures with alFN alone. Experiments designed to test for possible effects of Z n + + on the target cells revealed that Z n + + inhibits both spontaneous and detergent induced lysis.

As shown in Table II, the inhibitory effect was most marked at the

higher concentration.

Since spontaneous release was equally affected, the

S/T ratio did not change.

The net result was to reduce effector cell

lysis. Thus, the results shown in Table I considered in light of Table II suggest that Z n + + may act on both target and effector cells. experiments where Z n

++

In

was present in a preculture phase with NK

augmenting agents prior to target cell addition, effector cell enhancement was predominant.

Z n + + was also able to prevent loss of alFN induced

activity over time, not by directly activating NK cells but perhaps by prolonging the life of newly matured NK cells recruited by alFN.

424 Table II. Zn++

Z n + + Associated Target Protection1

Total Release 2

% Inhibition Total Release

Spontaneous Release 3

3134 2545 2764 2939

0 62.0 11.8 6.0

344 268 292 308

0 7..5 X 10"4 M 2 .0 X 10"4 M 4..0 X 10" 5 M

S/T 0.11 0.11 0.11 0.10

hr assay. cpm, detergent induced lysis. 3 cpm, target cells alone.

2

Possible stabilizing effects of Z n + + on NK function during activation by a I FN and ylFN were studied in further experiments carried 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 in Table III, Z n

most normal donor lymphocytes.

++

51

Cr labeled

did not activate NK function of

(In 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 survival of newly emerging mature NK cells. Table III. 100:1 50:1 25:1 1

Z n + + Potentiated Induction of Activated NK Cytotoxicity 1 No Addition

Zn++

1.9 1.3 1.2

4.6 1.5 1.3

a IFN

16.4 9.8 5.1

alFN + Z n + +

% A

37.1 35.1 24.5

126.2 258.2 380.3

6 day culture system: data are shown as % 5 1 Cr release at 3 effector target ratios in 4 hr assay against K562.

The presence of aIFN during the culture period was associated with 6-fold augmentation of lytic activity compared to unstimulated NK function at an E:T ratio of 100:1.

In this culture system, Z n + + strongly enhanced NK

induced target lysis and this effect was stronger proportionately at lower E:T ratio relative to cultures with a I FN alone as shown by the increased net change in Table III. The ability of Z n + + to modulate NK function was a consistent finding in all normal donors tested.

There was variation in the magnitude of the

425

boosting e f f e c t that appeared to be proportional to response to a or ylFN in the absence of Zn ++ .

As shown in Figure 1, enhancement of Zn ++ was

quite concentration dependent and in this longer period of c u l t u r e , [Zn + + ] of 2.5 X 10"14 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 rapidly d i l u t e d out and did

not change the slope of the curve at concentrations where no augmentation was seen. ES sia 50 in m

H5 40 35

Ld ^ 30 CE 25 Ld CL

20

I5 I0 5

KEY D=«TFT i •-«I FN+ZN FN+ZN I; 20 +-«1 FN+ZN I IS

0

I 00 : 1

50 : I

25 : I

:

E T RHTI• Figure 1 Concentration dependent enhancement of alFN induced NK a c t i v a t i o n by Zn ++ . 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/106 lymphocytes. [Zn + + ] were as follows: 1:5 = 2.5 X IO' 4 M; 1:20 = 8.0 X IO" 5 M; and 1:100 = 6.0 X IO" 6 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 consistently 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

LTl Ln >_1 I— z; LJ

Ql LJ

I 00: I

50: I

25: I

E : T RflT I 0 Figure 2 ++

E f f e c t of Z n on ylFN augmented NK a c t i v i t y . Concentrations of Z n + + given in legend to Figure 1. [ylFN] was 64 U/106 lymphocytes. The concentration dependence of Z n + + enhancement in experiments with ylFN was l e s s r e s t r i c t e d .

As for alFN at i n e f f e c t i v e concentrations of Z n + + ,

no s i g n i f i c a n t deviation from the slope of the curve observed with ylFN alone was seen. In summary, the r e s u l t s 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 Z n + + .

The studies suggest that the

mechanism may affect the binding reaction or s t a b i l i z e NK cell maturation induced by NK augmenting agents in short term culture.

The time

dependence of the r e s u l t s presented may r e f l e c t e i t h e r a requirement for metabolism of Z n + + or reduced a v a i l a b i l i t y of Z n + + as a target protecting agent.

In longer term c u l t u r e s , Z n + + was found to exert a s t r o n g l y

enhancing effect 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 proliferation as shown previously (5), it is possible that precursor NK cells might be within the expanded cellular compartment and thus Z n + + would provide a larger pool of pre-NK cells for IFN recruitment.

The magnitude of Z n + + enhancement was striking,

particularly at lower E:T ratios.

Since in vivo, lower ratios to

putative targets might be more readily achieved, one may speculate that Z n + + could act as a cofactor in mediating mechanisms of immune surveillance.

Acknowledgments The excellent technical assistance of Mrs. K.M. Smith is gratefully acknowledged.

These studies were supported in part by:

NIH NCI CB

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. Microbiol. 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. Sci. 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.: Int. J. Immunopharmacol. 1, 60 (1980).

6.

Ruhl, H., Kirchner, H., Bochert, G.: Proc. Soc. Exp. Biol. Med. 137, 1089 (1971).

7.

Cunningham-Rundles, S., Cunningham-Rundles, C., Dupont, B., Good, R.A.: Clin. Immunol. Immunopathol. 16, 115 (1980).

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 . Inmunol. Immunopathol. 21, 387 (1981).

9.

Bach, J . F . : Immunology Today, 1981.

10.

Cunningham-Rundles, S . , Hansen, J . A . , Dupont, B. i n 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 © 1984 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

lËËÉ_5Hd_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 weight 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

Cellulose

5

5

5

**

Cornoil

5

5

-

Linoleic acid

-

-

0,5

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 Immediate^^gersensitivit^^I^H^ - M o r t a l i t y .studies. I.H. with 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 2 mg of E.A. intravenously

(I.P.) and various

(S.C.). 2 weeks later any mouse had received

(I.V.). I.H. shock was quick and mortality

data were observed in any subgroup. - L.D. 50 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 domised and any group was divided in 4 subgroups of feed control

ran-

duration

1,2,3 or 4 weeks before immunization. Mice were immunized with 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.

Vaccination with_Ribomun2l * - JRit)omuiiyl . 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 pg of this product are equivalent to a vaccinal dose. - Dietary. Mice received a Normal or P. deprived feed as previously described. - Vaccination. Mice 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. - Chall_enge_. 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. - Direct_PFC_test^. Direct IFC 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. - Immuno|;l^b^lin_dosa.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 -

I_»H*_mor_ta_lity. We observed (table II) a major decrease of mortality

average in

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 \fit. 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

Animals sex Normal

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

Vit. A Deprived

alive

P.Deprivec

dead

Normal

alive

Mg/animal

Vit. A Deprived

0,4

Number

O.A.

P.Deprived

dead

Feed

Female

Male

Table II : Mortality after E.A. shock in the different groups of Balb/c mice

Feed control in weeks

1

2

Normal Feed

3

4

262

P.Deprived Vit.A Deprived

223

196

229

191

203

240

231

196

Table III : Results of L.D.50 of serotonin (doses in mg/kg)

434 ~ E_.A._sge^ific_ ant_ibodies. 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)

0

1 Antibodies titer for every weeks

P. Deprived

16

Vit.A deprived

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

512

512

512

512 256

1024 1024

512

128

4

1024

512

5

2048

024

69

256

256

256

32

2048

512

256

32

Table IV : Titers of E.A. antibodies during different periods of immunization .

Ribomun^l - Chall_enge_. 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. - Specific_antibodies. 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). - Dir^ec_t_PFC_t£st_. At any time the numbers of spleen cells were dramatically 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

2

VACCINATION 3

no 214 ± 42

Yes 54 1 10

No 39-9

704 ± 227

1080 ± 498

337 ± 129

1/3 Î 27

152 ± 50

56 t 9

61 ± 15

1119 1 50

1285 i 774

404 ± 227

458 ± 269

Cells spleen

181 ± 31

195 Î 19

42+11

47 + 11

PFC/10b cells

495 ± 109

507 Ì 243

355 Î 247

326 + 229

Cells/spleen x 106

WEEKS AFTER

Cells spleen 223 ± 40 x 106 b PFC/10 1099 ± 145 cells

P. Deprived

PFC/10b cells

Table VI : Results of direct P.F„C. 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 4

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.p., Normal feed,

---

Ab à 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

Vaccination

Ig Class

P. Deprived

Yes

No

Yes

IgG1

5,83

b, 91

5,43

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

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. 2.

Chandra, R.K., Newberne, P.M. : Nutrition Immunity and Infection, mechanism of interactions, Plenum Press, New York, London 1979. Bounous, G. , Kongshawn, P.A.L. : J. Nutr. U 2 , 1 /47-1755 (1982). Watson, R.R., Mc Murray, D.N. : CRC Critical Reviews in Food Sciences and Nutrition, J_2, 113-150 (1979).

4.

Shaedler, R.W. , Dubos, R.J. : J. Exp. Med. _n0, 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. 49, 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. Tl_, 18 (1974).

10. Silverman, S.H., Leks, H.I. : Clin Pediatr. 2_1_, 172-174 (1982). 11. Binder, P., Fontanges, R. : Personnal Communication 50*"^ 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., J_6, 135-141 (1976). 14. Fontanges, R., Robert, D., Content, U., Nils, G. : Rev. Franç. Allergol J_7, 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.,

, 599-600 (1968).

17. Spach, C., Askhenasy, A. : J. Nutr. J09, 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 © 1984 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 monkey 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 malnourished 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

immunodiffusion

method (Mancini, Carbonara and Heremans, 1965).

Assessment of the immune response

Delayed hypersensitivity. This was performed according to the method 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 within

441 2 days regardless of their nutritional status.

Fourteen days after the

sensitizing dose a challenging dose of DNFB of 100, 50 and 25 JUg in 0.1 ml was applied to the opposite forearm which was re-examined at 24 and 48 h for evidence of delayed cutaneous hypersensitivity.

A positive response

was interpreted as an area having distinct erythema, induration and vesiculation.

The sizes of such areas were measured with a Wellcotest 2

skin reaction gauge the area of which was calibrated in cm .

Skin transplantation. First and second set skin transplantations were performed according to the method described by Medawar (1944).

Auto-

grafts, allografts and xenografts were done, and all skin grafts were examined daily.

Passive cutaneous anaphylaxis (PCA). Standard PCA reactions were carried out in primate skin with serum specimens from (i) children with kwashiorkor and (ii) children with asthma known to have raised serum IgE levels.

Serum specimens with normal concentrations of IgE from age-

matched healthy children served as controls.

A positive PCA reaction was

interpreted as a distinct blue area, which appeared on the baboons' skin within 5 minutes after the allergen challenge.

The sizes of the PCA

reactions were measured with a ruler calibrated in cm and with a Wellcotest skin reaction gauge.

Lymphocyte activation by mitogen. The method used was modified from that of Janossy and Greaves, 1971, a micro-method being employed.

Phyto-

haemagglutinin (PHA) stimulation of DNA synthesis by peripheral blood lymphocytes was performed at least twice per week for each primate, after having established that 40^jg of PHA per 1 million cells caused maximum DNA synthesis in the normal primates.

Rosetting. Spontaneous rosettes to sheep erythrocytes (E) and to antibody complement (EAC) rosettes were performed as previously described

(Jones

and McFarlane, 1975), in which the percentage of rosettes among 200 lymphocytes was estimated.

The reproducibility of the cell counts was

established by counting in triplicate a sample that was set up in duplicate.

442 Immunoglobulin G (IgG) 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 m e d i u m 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 w i t h 107. blood.

All colony types were Gram-

stained then subcultured to appropriate m e d i a for further examination. Organisms were identified by the m e t h o d of Cowan and Steel

(1974).

Peripheral blood from each primate was also inoculated into brain-heart infusion broth and Brewer's thioglycollate.

Subcultures were m a d e 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.

W h e n body

weight was expressed as a percentage of initial weight, a significant change (p

0.01) in the body weight of all malnourished primates occurred

only after day 14 of protein-energy deprivation.

After 14 days of

placing the primates on the protein-energy deprived diet, they continued to lose weight and by the end of 28 days, the primates which were fed 20,

443 10 and 5 per cent of the control diet, had lost 237., 207. and 257. of their initial weights respectively.

However, when the animals were placed on

357. diet, there was a corresponding increase in body weight, which remained at a steady state until there were further increases in the amount of food offered to the animals.

Serum proteins

The first significant decrease (P

0.001) in the concentration of the

serum transferrin and complement C3 occurred as early as the second and third day of the protein-energy deprivation in the baboons receiving the 5 and 107. diets.

The serum from the primates on the 207. Mazuri diet

showed such changes one day later.

As shown in Table I, comparable

significant changes in the serum albumin concentration did not occur until 21, 28 and 35 days.

There was a greater increase in the concentra-

tions of serum transferrin and C3 than of albumin when the diets were increased to 357..

140'

> a o

BODY WEIGHT

60' — C O N T R O L PERCENT OF CONTROL 20

40'

10 5

0

3

7

10

14

17

21

24

28

31

35

38

42

63

70

77

FEEOING TO 35 per cant OF CONTROL

Fig. 1. Comparative changes in total body weight of the different primates during the feeding programme

444 Table I.

The first day on w h i c h a significant reduc-

tion (P 0.05) in lymphocyte

function in the one on 207. diet, during the same period.

Furthermore,

the

initial increase in lymphocyte transformation response in all malnourished animals, was followed by no further increase and remained steady for a long period of time.

E-rosettes. The m e a n number of E rosettes in the normal primates ranged from 30 to 407., m e a n 367«.

In the human the corresponding values were 50

to 607., with a m e a n of 527. (Table III).

The first significant reduction

of the proportion of E rosettes occurred on Day 7 in the baboon with the severest malnutrition and on Day 14 in the other 2 malnourished primates Thereafter there was a consistent reduction in the total number of E

446 rosettes, the lowest value being found on Day 21.

During the steady meta-

bolic state the number of E rosettes remained remarkably constant (Fig. 2).

EAC rosettes. As shown in Table III, the normal values for EAC rosettes in primates ranged from 17.0 to 237», mean 201, and was significantly lower than in healthy human beings.

Nevertheless, the pattern of reduction of

EAC rosettes in the malnourished baboons was similar to that observed for the E rosettes, the lowest number of EAC rosettes occurring on Day 35 of the feeding programme.

It was surprising to observe such a marked and

sustained decrease of the EAC rosettes in the malnourished primates. After 14 days of protein-energy deprivation there was a significant (P

0.001)

reduction in the number of B cells in all the malnourished baboons, although this reduction occurred about 4 days earlier in the baboon receiving the 5% diet.

Null cells. In all primates there was a comparatively large proportion of lymphocytes which did not react with E or EAC erythrocytes.

E and EAC rosettes in peripheral blood, spleen and bone marrow post mortem. Table IV summarizes the comparative values of E and EAC rosettes in the peripheral blood, spleen and bone marrow from primates immediately after they were killed.

It should be noted that the malnourished animals had

been in a steady metabolic state for 4 weeks before being killed.

E and

EAC rosettes were significantly more numerous in the peripheral blood than in the corresponding spleen and bone marrow of all the primates, regardless of their nutritional status.

Furthermore, the number of null cells in the

spleen and bone marrow were also proportionately lower in the peripheral blood than in either the spleen or bone marrow of the primates.

Serum IgG. There was no significant change in the IgG concentration in any of the malnourished primates although the values were slightly higher in both primates on the lower percentage diet.

447 Table III.

Comparative values of E and EAC rosettes and null

cells (7.) in healthy and malnourished primates after the latter had been on their restricted diets for 4 weeks.

The

corresponding values are given for healthy human beings. E rosettes

EAC rosettes Null

rt Range

Mean

Range

Mean

cells

Primates

34-40

36

17-23

20

40

Human

50-60

51

25-38

32

17

Normals Subj ects

Malnourished primates % diet 20

16

11

73

10

15

9

76

5

13

7

80

Table IV.

E and EAC rosettes (7„) in peripheral blood, spleen

and bone m a r r o w immediately after the primates were killed Percent- Peripheral blood age *

Spleen

Bone m a r r o w

A

E

EAC

E

EAC

E

Control

39

22

13

10

7

5

20

19

13

6

5

2

3

10

16

12

8

4

9

3

5

20

15

3

12

4

5

of diet

Microbiological

EAC

findings

Conjunctival swab. None of the experimental animals showed any clinical signs of eye inflammation although scanty pus cells were seen in the Gramstained films from a few of the swabs.

Staph, pyogenes was the predominant

pathogen isolated from the conjunctiva from both the control and m a l nourished primates.

448 Throat swab. There was no macroscopic or microscopic evidence of inflammatory disease.

Streptococcus viridans group, Haemophilus spp. and staphylo-

coccus comprised the major resident flora whilst the Neisseria spp. comprised a portion of the transient microflora of the throat.

There was no

significant difference in the type and numbers of organisms isolated from the throat swabs of the control and malnourished primates.

Blood culture. After 4 weeks on the protein-energy deficient diet, coagulase-negative staphylococcus was isolated more frequently from the blood culture of the malnourished primates than from the controls.

Organ culture. The cultures of lungs, liver, spleen, kidneys and inguinal lymph nodes collected post mortem from the malnourished and the control primates remained sterile.

Histopathological light microscope findings

There was a mild nonspecific mononuclear-cell infiltration of hepatic portal tracts and parenchyma of all the primates.

The spleen and lymph

nodes of the malnourished as well as the control primates showed follicular hyperplasia and other nonspecific inflammatory changes. some polymorph infiltration in the spleen.

There was also

Active germinal centres were

prominent in the spleens of the malnourished animals.

Discussion

Changes in the serum transferrin and C3 concentrations were observed ve*y early, being significantly reduced after 2-3 days on the restricted diet, unlike the serum albumin concentration which remained static for at least 21 days.

On refeeding the primates the rate of return towards normal concen-

trations of these 3 plasma proteins showed a similar pattern.

These results

suggest that the reduction in the serum albumin is a relatively late event in the malnourished individual, and that both the serum transferrin and C3 concentrations provide much more sensitive indices of nutritional status than either the anthropometric measurements or albumin serum concentrations.

449 T - CELLS ROSETTES IN NON - HUMAN

PRIMATES

Fig.2. Pattern of E rosettes (T cells) in the peripheral blood of the various baboons on theis restricted diets compared to the controls.

CONTROL

—

PERCENT OF CONTROL

z 40 i I 30 V)

>v

E 1 20'

3

7

10

14

21

28

I

35

13 (1974).

2.

Bang, B.G., Foard, M.A., Bang, F.B„: Proc. Soc. exp. Biol. Med. 143, 1140 (1973).

3.

Catalona, W.J., Taylor, P.T., Rabson, A.S., Chretien, P.B.: N. Engl. J. Med. ¿86, 399 (1972).

4.

Cowan, S.T., Steel, S.: Manual for the Identification of Medical Bacteria, Second edition, Cambridge University Press, London, p.42 (1974).

5.

Edelman, R., Suskind, R., Olson, R.E., Sirisinha, S.: Lancet, i, 506 (1973).

6.

Harland, P.S„G.E., Brown, R.E.: E. Afr. Med. J. 42, 233 (1965).

7.

Jones, S.V., McFarlane, H. : Br. J. Haematol. _31> 545 (1975).

8.

McFarlane, H., Reddy, S., Adcock, K.J., Adeshina, H., Cooke, A.R., Akene, J.: Br. med. J. 4, 268 (1970).

9.

McFarlane, H., Hamid, J.: Clin. exp. Immunol. _13, 153 (1973).

10. McFarlane, H., Olusi, S.0„, Ade Serrano, M.A., Osunkoya, B.O.: Swedish Nutrition Foundation Symposium XIII. Ed. L.Hambaeus, L.A.Hanson, H.McFarlane. Stockholm: Almqvist and Wiksell. p.23 (1977). 11. Mancini, G., Corbonara, A.O., Heremans, J.F.: Immunochemistry 2, 235 (1965). 12. Medawar, P.B.: J. Anat. (Lond.) 78, 176 (1944).

452 13. Ruch, T.C.: Diseases of Laboratory Primates, W.B.Saunders, Philadelphia, p.42 (1959). 14. Scrimshaw, N.A., Taylor, C.E., Girdon, J.E.: Am. J. med. Sei. 237, 367 (1959). 15. Smythe, P.M., Schonland, M.M., Brereton, G.G., Goovadia, H.M., Grace, H.J., Loening, W.E.K., Mafoyane, A., Parent, M.A., Trace, Vos G.H.: Lancet 2, 939 (1971). 16. Uphill, P.F.: J. appl. Bact. 36, 501 (1973). 17. Uphill, P.F., Wilde, J.K.H., Berger, J.: J. appl. Bact. 37, 309 (1974). 18. Woodroffe, R.C.S., Shaw, D.A.: In The Normal Microbial Flora of Man. Ed. F.A.Skinner and J.G.Carr. Academic Press, London, p.13 (1974).

S E R U M THYMIC FACTOR SENEGALESE CHILDREN

(FTS)

CONTENTS

"A p o s t - m o r t e m i m m u n o - h i s t o l o g i c a l

B.JAMBON1,

O.ZIEGLER2,

A.PATRIS4,

J.DUHEILLE4.

1

Correspondence

:

THE

G.PARENT3,

; 30RST0M,SENEGAL

Laboratoire

THYMUS

IN

UNDERNOURISHED

study"

B.MAIRE3,

; 2CHU,NANCY

ORSTOM,NANCY

OF

M.C.BENE4,

G.FAURE4,

; 4 F A C U L T Y OF M E D I C I N E , N A N C Y .

d'Immunologie,

Faculté

de

Médecine

de NANCY, B.P. 184, 54505 V A N D O E U V R E CEDEX, F R A N C E .

INTRODUCTION

Protein-energy

malnutrition

of cellular

immunity

of

lymphocyte

thymic

(PEM)

(1,2,3).

in

children

The study,

differentiation

results

in the

in

blood,

(circulating

a

deficiency

o f the

F.T.S.

in vitro l y m p h o c y t e m a t u r a t i o n by thymic factors), s u g g e s t s a n of

hormonal

secretion

hypothesis

by

a

involution

and

the

by

the

post-mortem F.T.S.

thymus

(4,5,6) We

immunohistological

contents

have study

o f the t h y m u s

function activity, impairment

confirmed of

the

among y o u n g

this thymic

children

p r e s e n t i n g w i t h P.E.M. a t the time o f death.

M A T E R I A L S ANS M E T H O D S

Fifty stages

eight c h i l d r e n a g e d from of

nutritional

1 day to 4 y e a r s ,

dificiency,

were studied.

who d i e d a t d i f f e r e n t This study c o n s i s t s o f

clinical e x a m i n a t i o n s , a n t h r o p o m e t r i c m e a s u r e m e n t s and h i s t o p a t h o l o g i c a l s t u d i e s o f the v i s c e r a l o r g a n s for the evidence o f

infection.

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

454 The thymus in each case was rapidly but carefully dissected out, weighed and

deep-frozen

in

liquid

nitrogen.

Cryostat

sections

were

then

made for histopathological studies of macrophages and immunofluorescence of F.T.S. and fibronectin . The F.T.S. contents of the thymus was evaluated considering the number and the immunofluorescent intensity of the cellular elements stained by the anti-serum. Finally

a statistical

analysis of the different

parameters was done

making it possible to draw conclusions.

RESULTS AND CONCLUSIONS

Forty severe forms of P.E.M. (i.e. marasmus, Kwashiorkor and marasmicKwashiorkor), out of the 58 undernourished children studied, assumed an identical picture. There was an involution of the thymus, always marked, and

associated

with

the

disappearance

(sometimes

completely) of the

F.T.S. contents of the thymus. This disappearance of F.T.S. from the thymus (Fig.l) follows a double process : 1) A diminution of the F.T.S. concentration and therefore possibly its intracellular synthesis in the epithelial cell network and in the Hassall's bodies, which seems to act as a function of nutritional deficiency . 2) In extreme cases a total disappearance of these epithelial elements, which appears linked with the necrotic process of involution. 3) The thymic involution is associated with an inflammatory reaction as expressed by the double invasion of the parenchyme by the macrophages and by cellular elements which stain for fibronectine (fibroblasts and newly formed capillaries). In our study we have not formed a relationship between infection and other

parameters

studied

inflammatory reaction.

except

those

which

are

essentially

due to

455

100

Score in case of normal thymus moderate malnutrition

marasmus

N 5

50

kwashiorkor

I

00

17

15

marasmic-kwashiorkor

11

Fig.1) EVALUATION OF THE INTRA-THYMIC CONTENTS OF F.T.S. EXPRESSED AS THE PERCENTAGE OF NORMAL (3T+s)

REFERENCES 1) CHANDRA (R.K.), Br. Med. J. 3, 608-609, 1974 2)

SMYTHE GRACE

(P.), (H.),

SCHONLAND

LOENING

(M.),

BRERETON-STILES,

(W.), MAFOYANE

(A.), PARENT

COOVADIA

(H. ) ,

(M.), VOS

(G.),

LANCET, 2, 939, 1971 3) SUSKIND

(R.M.), Malnutrition and the immune response, Raven press,

New-York 1977 4) CHANDRA (R.K.), Clin. Exp. Immunol. 38, 228-230, 1979 5) OLUSI (S.O.), THURMAN (G.B.), GOLDSTEIN (A.L.), Clin. Immunol. Immunopath. 15, 687-691, 1980 6) JACKSON (T.M.), SHAMIWA (N.), ZANAN, Clin. Exp. Immunol. 39, 717-721, 1980

THE CLINICO-PATHOLOGICAL CONSEQUENCES OF SEVERE PROTEIN-ENERGY MALNUTRITION IN DECEASED SENEGALESE CHILDREN. O.ZIEGLER 1 ,

B.JAMBON 2 ,

G.PARENT 3 ,

B.MAIRE 3 ,

R.DARDELIN 4 ,

M.FALL 4 ,

5

J.DUHEILLE .

1

CHU,NANCY

5

; 2 0RST0M,NANCY ; 3 0RST0M,SENEGAL ; 4 H0PITAL LE DANTEC,DAKAR ;

FACULTY OF MEDICINE,NANCY.

Correspondence

: Laboratoire d'Immunologie, Faculté de Médecine de NANCY,

B.P. 184, 54505 VANDOEUVRE CEDEX, FRANCE.

INTRODUCTION

The

clinico-pathological

consequences

of

the severe

forms of

protein-

energy malnutrition (P.E.M.) were studied in 40 children who died between the ages of 8 days and 4 years. We have compared, on the one hand, the thymic

involution

pancreas)

and,

the

severe

three

with

on

that

the

of

other

the

hand,

forms of P.E.M.

visceral the

organs

respective

(marasmus,

(liver,

spleen,

consequences

Kwashiorkor

and

of

marasmic-

kwashiorkor).

MATERIALS AND METHODS

The

nutritional

wasting of height The

ratio,

thymus,

relation (using

status

was

assessed

height/age

liver,

ratio),

table

of

expressed

STOWENS),

(presence

allowed

(liver

accurately

as a percentage for

of

edema,

(weight, height, weight/

and histologically

spleen and pancreas were

to the boody height, the

clinically

fat and muscle), anthropometrically

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

Their

of the normal

comparisons,

independent of age, to be made.

steatosis).

weighed.

which

were

458 All

in

all

58

children

of both

sexes

were

studied,

40 of

these

were

severely malnourished. Their mean age was 12 months (+ 2.7). In

another

work

by

Jambon

et al

(also reported

in this

symposium) an

immunohistological study of the thymus was carried out. The

different

parameters

collected

were

analyzed

various

degrees

statistically

by the

method of data processing.

RESULTS AND CONCLUSIONS

The

children

studied

weight/height (+ 4.5)

ratio

had of

and height/age

69,6

%

ratio

(+

of

2.9),

95,9 %

of malnutrition a

weight/age

with

ratio

a

of

mean

65,0 %

(+ 1.8). These ar shown in the

table below :

Nutritional status Marasmic

moderate Marasmus

Kwashiorkor Kwashiorkor

Malnutrition

1)

Number of cases

18

15

11

14

Weight/age ratio (%)

78 %

52 %

74 %

55 %

The

thymic

malnutrition

involution

is

always

marked

in

the

severe

forms

of

(Fig.l)

2) This involution of the thymus is more important than those of other organs 3)

(Figs.1,2)

There

studied

are

no

significant

differences

in the

among the severe forms of malnutrition

pancreas)

weights

of the organs

(thymus, liver,

; the same was observed with the serum thymic factor

and fibronectine contents of the thymus.

spleen, (F.T.S.)

459 The

different

nutritional parameters studied normally

(weight/age

ratio,

ratio)

fatty

and

weight/height infiltration

ratio) of

the

stature liver

: weight

deficit

(a

deficit

(height/age

reflection

of

the

visceral protein pool) have different significances. The

independent

behaviours,

vis

à

vis the totality of the

nutritional

criteria and histological and immunological parameters that were studied, suggest course

the

existence

of

many

physiopathological

same

thymus agree

in

the

of P.E.M., the syndromes of marasmus, Kwashiorkor and marasmic-

Kwashiorkor demonstrate these. However, the

machanisms

ultimate

phase

and

these three clinical forms have

consequences

i.e. a severe atrophy of the

and a disappearance of the F.T.S. secretion. These with

the

modern

concept

which

pathological continuity

(1,2,3).

100

normal ratio

describes

P.E.M.

observations

as

a

clinico-

] moderate malnutritic

50

T

X 00

Fig.1)

m

N 5

18 WEIGHT/HEIGHT

15

11

RATIO

OF

OF NORMAL (3T+s)

marasmus kwashiorkor marasmic kwashiorkor

THE

THYMUS

EXPRESSED

AS

THE

PERCENTAGE

460

normal ratio

100

N ~ 5 moderate malnutrition

marasmus

50

3 8 1 7 - 3 8 2 9 (1972).

4.

F o u r n e t , B., M o n t r e u i l , J., S t r e c k e r , G., D o r l a n d , L., H a v e r k a m p , J., V l i e g e n t h a r t , J., B i n e t t e , P., S c h m i d , K. : B i o c h e m . 17, 5 2 0 6 - 5 2 1 4 (1978).

5.

Brig-Hansen, T . C . , P r a h l , P., L o w e n s t e i n , H. 2 9 3 - 3 0 7 (1978).

6.

W e l l s , C., BeSg-Hansen, T . C . , C o o p e r , E.A., G l a s s , M . R . A c t a 109, 5 9 - 6 9 (1981 ) .

7.

N i c o l l e t , I., L e b r e t o n , J . P . , F o n t a i n e , M . , H i r o n , M. B i o p h y s . A c t a 668, 2 3 5 - 2 4 5 (1981).

8.

Laemmli, U.K.

9.

0'Farrell,

: Nature 277, 680-685

P.H.

: J. I m m u n o l . M e t h .

Chim.

: Biochim.

(1970).

: J. B i o l . C h e m . 250, 4 0 0 7 - 4 0 2 1

10. B a y a r d , B., K e r c k a e r t , J . P .

: Clin.

22,

: E u r . J. B i o c h e m .

(1975). 113, 4 0 5 - 4 1 4

(1981).

PART 2 BIOLOGICAL FUNCTIONS OF LECTINS

BIOLOGICAL FUNCTIONS OF LECTINS

Michel CARON; Marie-Ange DEUGSIIER, and Raymonde JOUBERT UER Bicmédicale, 93012 Bobigny Cedex, France Pierre-Paul ROUGE Faculté des Sciénces Pharmaceutiques, 31062 Toulouse Cedex, France

The occurence of lectins in a wide variety of organisms supports the idea of their important role in various functions of living systems. The main concepts about the function of lectins concern défense against bacterial invasion in plants, unicellular interaction and recognition irtechanians, elimination of degraded glycoproteins and cells,... In the present paper several examples of lectin activities are reviewed : I. LECTIN ACTIVITIES IN PLANTS II. LECTIN" "ACTIVITIES IN ANIMALS III. LECTIN ACTIVITIES IN MAN

LECTIN ACTIVITIES IN PLANTS

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

642 Evidence of a biological role(s) of lectins in plants is suggested by the fact that Leguminosae lectins appear as veil conserved and evolutionarily related proteins. Despite our increasing knowledge on the distribution and the physicochemical properties of plant lectins, little is known about their physiological functions. Since the discovery of lectins, many hypothesis concerning their possible roles have been postulated but none of them has not been yet consistently demonstrated. Among the postulated hypothesis, those concerned with the involvement of lectins in recognition mechanisms have been well documented : - experiments conducted by DAZZO and coworkers on the Tri folium repensRhizobium trifolii interaction have shown that the lectin trifoliin could play a role in the recognition and subsequent fixation of the symbiont. However, many contradictory results have been obtained about the specificity of the interaction between various legume lectins and infective Rhizobium strains.

- the work of MIEELMAN and coworkers which shows that WGA from Triticum vulgare inhibits the hyphal growth and conidiation of both pathogenic and saprophytic fungi argues for the fact that lectins could act in the defense of plants. A similar functicn has been recently postulated for PHA from Phaseolus vulgaris. However, we lack informations about the occurrence and the specificity of such a defensive mechanism in the plant itself. Recently, other attractive hypothesis have been proposed in relation with the localization of lectins in plants and their main physicochemical properties, namely their ability to bind various glycoconjugates via specific or hydrophobic interactions : - the ability of lectins to bind specifically glycoconjugates together with their localization in the protein bodies has led BASHA to postulate that lectins could be involved in cross-linking the storage proteins into aggregates within the protein bodies. However, this hypothesis should be regarded with caution since only a very small amount of seed proteins react with lectins in vitro. - experiments on the possible interaction between lectins and plant hormones have been conducted by WANG and coworkers using Con-A and ji -indole acetic acid and by ROBERTS with lima bean (Phaseolus lunatus) lectin and cytokinins. Although these interactions are weak and of hydrophobic

643 nature, their possible involvement in the plant hormones pathway cannot be excluded. - PEUMANS and coworkers have postulated that endogenous plant receptors could bind lectins and that the resulting lectin-receptor complexes could play a biological role, e.g. by triggering the cell membranes in order to pass messages to the nucleus. The nature of receptors still remains to be established. Another interesting question about the possible role of lectins in plants arises from the work of PUEPPKE and coworkers which shows that seeds of several cultivated soybean (Glycine max) lines are devoid of lectin. Similar findings have been reported in Phaseolus species. Plants lacking lectins develop in the usual way, thus suggesting that lectins are not necessary for infection and nodulation by Rhizobium since these plants nodulate equally well. Hovever, new lectins different from the "classic" seeds lectins have been recently discovered in Phaseolus lacking lectins by PUSZTAI, which could play a similar role in the specific interaction with Rhizobium. Whatever the hypothesis, the biological functions of the plant lectins remain cbscure. To answer this question it seems necessary to have a better picture on the localization and changes of lectins in the different parts of plants during their life cycle since many reported results have been only obtained in vitro. Another hypothesis, concerning lectins found in .pollens, is that they may act as allergens (G. PELTKE, unpublished results). t

If lectinic activities have mainly been studied for a hundred years in a wide variety of plants, they are now found to take place in several mechanians of biological importance, in animal and man. LECTIN ACTIVITIES IN ANIMALS Lectins have been described in various animals organs for approximately ten years (1, 2, 3). The following examples concern lectinic activities detected in the nervous systan. It has been reported that brain extracts of different species prcmote in vitro proliferation of nervous ceils (4). Adult rat brain extract added

644

to the culture medium seans to increase the reassociation and clustering of mouse embryonic cells. These observations led to search a lectin-mediated activity (5). A galactosamine and lactose-inhibitable hemagglutinating activity was found in this rat brain extract. No inhibition was detected with heparin and no fibronectin traces measured. The lactose-specificity permitted the preparation of an extract specifically depleted in this activity. This preparation was obtained by adsorption on lactose linked to agarose beads ( Lactogel, E.Y. ). The lectin-depleted extract was found to have lost its hemagglutinating properties. Then, the effects of both native and lectin-depleted extracts were investigated on dissociated mouse embryonic cells in a low density suspension. In such conditions the spontaneous reassociation of nerve cells was strongly limited. The lectin-depleted extract, unlike total brain extract, was not found to be a pranotingfactor of cell reassociation. Assays performed with glutaraldehyde fixed cells support the hypothesis that cell aggregation obtained with total brain extract is not secretion dependent. The direct or indirect interaction of lactose-inhibitable lectin with appropriate binding sites of brain cell membranes is believed to be responsible for this aggregation. In a second example, preliminary results about the rosette-forming ability of mouse neuroblastoma cells were reported ( M.A. DEUGNIER, unpublished results). Neuroblastoma cells constitute a model system of great interest since they can be stimulated in vitro to morphologically and/or biochemically differentiate. When grown in supplemented rredium these cells rapidly proliferate and form large cohesive aggregates. In a previous vrork, TEICHBERG et al. (2) reported that extracts of mouse neuroblastoma cells contain a lactose-inhibitable lectin, which agglutinates trypsin-treated rabbit erythrocytes. To determine whether neuroblastoma cells express lectinic activity (ies) on their surface, the rosette formation of "undifferentiated" neuroblastoma cells (NS20 clone) with trypsinized glutaraldehyde-fixed rabbit erythrocytes (TGRs) was tested. It appeared that 80 to 90 % of the neuroblastoma cells formed rosettes, provided that they were allowed to react w ith TGRs in Eagle's minimum essential medium (MEM, pH 2+

8.0). In phosphate buffered saline (PBS) supplemented with Ca

2+

and Mg ,

TGRs failed to bind to the surface of neuroblastoma cells. The addition of glucose (l~mg/ml) as wall as adjustment of pH to 8.5 did not restore a

645

positive response. The rosette formation in MEM was totally inhibited by low concentrations of D-galactosami ne (10 mM). A 50 % inhibition was obtained in the presence of 100 mM lactose but it was also pointed out that in such experimental conditions, this saccharide induced the self-agglutination of TGRs. The tested saccharides which did not lead to a significant inhibition at concentrations up to 100 mM, include galactose, thiodigalactoside, N-acetylD-galactosamine, N-acetyl-D-glucosamine and D-mannopyranose. The effect of other saccharides are now planed to be studied. Indeed, the inhibition obtained with D-galactosamine but not with the N-acetyl derivatives suggests that other osamines could also inhibit the rosette formation. Anyway these preliminary data indicate, i) that the rosette-forming ability of neuroblastoma cells strongly depends on the reactional medium, ii) and that in well-defined experimental conditions, neuroblastoma cells are able to specifically bind 1GRs. This latter result suggests that neuroblastoma cells display lectinic activity(ies) on their surface. LECTINIC ACTIVITIES IN MAN Many lectinic activities have been described in man, but very few is known about their biological significance. A number of them is discussed below. 1) Hepatic lectin. This protein is related to the animal hepatic binding protein (1, 6) . it recognizes molecules with free Gal or GalNac (V). 2) Leucocytes lectins. Several lectinic activities have been described in lymphocytes. The E-rosette formation of T-lymphocytes with sheep erythrocytes has been interpreted as the interaction between a lectin and a glycolipid (8). Among these rosette-forming-cells, a subset agglutinated by a complex polysaccharide (PVCH) and freed by the addition of a-methylmannoside is largely enriched in T suppressor (Ts) cells and produces a 6-desoxymannose inhibitable suppressor factor (9) (Fig 1). These results can be related to previous description of selective inhibition of Ts function by a-methyl-mannoside (10). On another hand, it has been observed that a number of sugars ( N-acetyl-D-galactosamine, lactose,...) are capa-

646

LEUCOCYTES PWCH PWCH

PWCH

c+ "i U'

|PWCH' E

¥

Ts

tPWCH 1

T

Monocytes

\PWCH (

T1. jPWCH

E

f T

I

* : inhibitable by e