Marker Proteins in Inflammation: Volume 1 Proceedings of the Symposium Lyon, France, April 22–25, 1981 [Reprint 2019 ed.] 9783110837643, 9783110086256

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Marker Proteins in Inflammation

Marker Proteins in Inflammation Proceedings of the Symposium Lyon, France, April 22-25,1981 Editors Robert C. Allen • Jacques Bienvenu Philippe Laurent • Robert M.Suskind

W DE

G Walterde Gruyter • Berlin • New York 1982

Editors Robert C. Allen, Ph. D., Professor of Biochemistry Department of Laboratory Animal Medicine and Pathology Medical University of South Carolina 171 Ashley Avenue, Charleston, South Carolina 29403, USA Jacques Bienvenu, M. D. Biochemical Laboratory, Hôpital Jules Courmont 69310 Pierre Bénite, France Philippe Laurent, M. D. Department of Immunology, Institut Pasteur de Lyon 55 Rue Pasteur Robert M.Suskind, M. D. College of Medicine, Department of Pediatrics University of South Alabama

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Bibliothek

Marker proteins in inflammation: proceedings of the symposium Lyon, France, April 22-25,1981/ed. Robert C.Allen... - Berlin; New York: de Gruyter, 1982. ISBN 3-11-008225-5 NE: Allen, Robert C. [Hrsg.]

Library of Congress Cataloging in Publication

Data

Main entry under title: Marker proteins in Inflammation. Proceedings of the First International Symposium on the Marker Protein of Inflammation sponsored by the Group d'étude et de recherche sur les marqueurs de l'inflammation (G.E.R.M.I.) Bibliography: p. Includes index. 1. Inflammation-Congresses. 2. Blood proteins-Diagnostic use-Congresses. I. Allen, R. C. (Robert Chadbourne), 1924. II. International Symposium on the Marker Protein of Inflammation (1st: 1981: Lyon, France) III. Groupe d'étude et de recherche sur les marqueurs de l'inflammation (France) [DNLM: 1. InflammationPhysiopathology-Congresses. 2. Proteins-Physiology-Congresses. QZ150 M3451981] RB131.M351982 616'.0473 82-9939 ISBN 3-110-08625-5 (Germany) AACR2

Copyright © 1982 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: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany

Preface

The First International Symposium on the Marker Protein of Inflammation was held in Lyon, France from April 22 to April 25, 1981, under the sponsorship of the "Groupe d'Etude et de Recherche sur les Marqueurs de 1'Inflammation (G.E.R.M.I.)" (President: J.L. Touraine, Lyon) with Professor Frank W. Putman, distinguished Professor of Molecular Biology and Biochemistry, Indiana University as Symposium President. Some 320 participants from 14 different nations attended this initial Congress. 46 papers and 92 posters were presented, the majority of which appear in this volume. This volume is divided into four sections and contains presentations made either as plenary lectures or posters. The manuscripts have been divided into four major sections entitled (I) The Inflammatory Response, (II) Acute Phase Reactants, (III) Malnutrition and the Immune Response and (IV) Posters, which are subdivided further by category. Production of these proceedings has been made possible through the cooperation of the many authors, and we wish to thank all of them for their efforts. We also appreciate, in no small way, the efforts of the staff of Walter de Gruyter, Berlin, which led to the rapid publication of this volume. The editors would also like to express their profound gratitude to Mrs. Sharon Fields, Mrs. Mae Jean Reeves and Miss Patricia A. Corcoran of the Department of Pediatrics, University of South Alabama, Miss Brenda Altman of the Department of Laboratory Animal Medicine, Medical University of South Carolina, and Mrs. Dominique Karsenty, Laboratory of Biochemistry, Hopital Jules Courmont, Lyon, for their outstanding efforts in manuscript-retyping and secretarial assistance.

VI The Convenors would also like to express their sincere appreciation for the outstanding efforts of Mr. A. Roullet who acted as Secretary-General of the meeting and was always present to help the speakers and attendees, no matter what the problem or request, and also for those of his Assistant, Miss Helene Bernon, We wish also to express our appreciation to Miss Daniele Ferreboeuf, Miss Maris-Alix Fournier and Mrs. Odile Damour for their help in the smooth running of the Symposium and in hosting the social events. We would like to thank Miss Debbie Impastato as well, particularly for her aid as an interpreter to those whose French was lacking. Also, her efforts as a tour and shopping guide in Lyon were much appreciated. We would also like to express our sincere gratitude to the Mayor of Lyon, Dr. Maurice Carraz - Director, Institute Pasteur,Lyon - and Dr. Charles Merieux as hosts of the splendid social events, and to Monsieur Jacques Barrot - Minister of Health - for governmental support. We also wish to thank the following exhibitors who helped make this Symposium possible: Beckman Instruments (France), Biolyon, Biomerieux, Boehringer-Mannheim (France), Laboratoires Fumouze, Helena (France), Hoechst-Behring, Hyland-Travenol, Immuno (France), Sebia, Compagnie Technicon.

R.C. Allen Charleston, SC

P. Laurent Lyon, France

J. Bienvenu Lyon, France

R.M. Suskind Mobile, Alabama

Contents

Section I The Inflammatory Response Progress in Plasma Proteins: Hallmarks of Health and Markers of Disease F.W. Putnam Plasma Proteins Implicated in the Inflammatory Respons L.M. Killingsworth Acute Inflammatory Process P. Laurent, J. Bienvenu The Inflammatory Response D.A. Willoughby, A. Sedgwick, J. Edwards Relationships between Acute Non-Specific Inf lamination and Non-Specific Defense Mechanisms of the Host M. Pelletier, I. Florentin, D. Nolibe, M. RochArveiller, J.M. Jadin, J.P. Giroud The Role of Thrombocytes in Inflammation J. Benveniste

Section II Acute Phase Reactants Clinical Usefulness of C Reactive Protein Measurement P. Laurent Control Proteins of the Alternative Complement Pathway In Vivo Correlates of In Vitro Function S. Ruddy

VIII Plasma Fibronectin, its Biochemical and Biological Properties and its Pathological Changes in Man I. Millet, P. Laurent

99

Interaction between C-Reactive Protein (CRP) and Platelets B.A. Fiedel, R.M. Simpsop, H. Gewürz 111 Interactions between CRP and Mononuclear Cells K.K. James, C. Mold, H. Gewürz

131

Clinical Usefulness of Orosomucoid Determination J. Bienvenu

13 9

Alpha^ Acid Glycoprotein - Structure, Genetics and Biological Significance P. Arnaud, E. Gianazza

159

Alpha^-Antitrypsin Structure and Genetics P. Arnaud, C. Chapuis-Cellier, E. Gianazza

171

Affinity-Immunodeletion (AID) Isoelectric Focusing on Ultra-Thin Gels and Staining with Silver Diamine Applied to the Marker Proteins of Inflammation R.C. Allen, P. Arnaud

185

A Survey of the Measurement, Distribution of Values and Phenotypes of the Haptoglobins 0. Hever

197

Recent Findings on the Biological Role of Haptoglobin in Rats R. Engler

209

Biochemical Aspects of Complement Activation M.G. Colomb, J.C. Bensa, A. Reboul

215

The Acquired Abnormalities of the Complement System in the Acute or Chronic Inflammatory Response F. Berthoux, B. Laurent, C. Genin, J.-C. Sabatier, R. Gonthier Acute Phase Reactant Protein Profiles in Cancer: An Approach to Deciphering their Message E.H. Cooper, J. O'Quigley Plasma Protein Profile: A Model of Data for Interpreta tion with Special Reference to Inflammatory Conditions J.C. Frot, P. Giraudet, H. Hofmann, F. Muller

Section III Malnutrition and the Immune Response Protein-Calorie Malnutrition-Clinical Biochemical and Immunological R.M. Suskind Complement, Orosomucoid, Transferrin, Retinol Binding Protein and Prealbumin in Malnutrition F. Touraine, G. Gay, J.L. Touraine Cellular and Humoral Immunity in Malnutrition R.K. Chandra, S. Sahni, S. Chandra Immune Deficit in Kwashiorkor J.-L. Touraine, G. Gay Inhibition of Cell-Mediated Immunity by Serum Abnormal ities in Protein-Calorie Malnutrition G. Gay, J.-L. Touraine Host Resistance in Malnutrition W.P. Faulk, V. Wallis, A.J.S. Davies

X

Usefulness of Prealbumin as Nutritional Indicator Y. Ingenbleek

405

Section IV Posters A. Acute Phase Reactants in Neonates A Comparative Study of the Sequential and Coupled Determination of CRP and Orosomucoid during Neonatal Infections F. Bienvenu, C. Chantin, L. Sann

417

CRP and Neonatal Sepsis R. Alt, D. Willard, J. Messer, P. Metais, C. Goester

421

Characterization of a C2 Derived Oligopeptide with Biologic Activity M. Lopez-Trascasa, M. Moisy, E. Pirotzky, Y. Blouquit, C. Blanc, A. Sobel

423

Direct Quantitation of the Complement C3 Split Product C3d in Plasma in Immunopathological Disorders I. Brandslund, B. Teisner, S.-E. Svehag

424

Variations of Orosomucoid Levels in New-Born Twins B. Capolaghi, O. Baubin, C. Grasmick, C. Marchal, P. Roos

427

Complement Levels in Infants of Diabetic Mothers over the First Month of Life A.-M. Freyria, A. El Mohandes, T. Raffin, J.-P. Lamelin, B. Salle, J.-L. Touraine

429

B. Protein Status in Clinical States Protein-Status and Malnutrition, Preliminary Results of a Prospective Study of 177 Patients J. Jouquan, M. Garre, J.F. Menez, P. Youinou, J.F. Morin, J.M. Boles, Y. Pennec, G. Le Menn

437

XI Research of Relationship between Protein and Folic Acid Deficiencies J.M. Boles, P. Youinou, M. Garre, D. Lhostis, J. Morin, J. Jouquan, G. Le Menn, C.H.U. Morvan

438

The Measurements of Plasma Proteins in Patients with nutritional Deficiency G. Levy, G. Francois, M.-C. Liechtensteger, J.-P.Ardissone, J. Romette, J. di Costanzo, B. Mallet 439 Value of Plasma Prealbumin in the Early Recognition of Protein-Energy Malnutrition in Sahelian Children A. Froment, L. Monjour, F. Bourdillon, A. Fribourg-Blanc, J.M. Kyelem, E. Gouba, M. Gentilini

440

Visceral and Muscular Proteins in Anorexia Nervosa: An Example of Severe Malnutrition in Industrialized Countries. Effects of Renutrition J.L. Richard, J. Bringer, L. Monnier, J. Mirouze

442

C. Protein-Calorie Malnutrition and the Immune Response Effects of Protein-Calorie Malnutrition on the CellMediated Immune Response in Mice G.-A. Conge, P. Gouache, P.-H. Langrange, D. Lemonnier

447

Effects of Early Under- and Over-Nutrition on the Immune Response in Mice S. Wade, D. Lemonnier, F. Bleiberg, J. Delorme, P. Chappuis

450

D. Inflammatory Proteins in Patients with Cancer Plasma Transferrin Level: A Preoperative Prognosis Indicator in Esophageal Cancer Patients J.-P. Alibeu, C. Arvieux, C. Alibeu, V. Danel, R.Sarrazin

455

Usefulness of a Protein Profile as an Indicator for a Gastrostomy in Patients with Advanced Head and Neck Cancer J.-F. Gillette, J. Susini, J. Oglobine, R.T. Saracino 457

XII Acute Phase Reactant Proteins and Cancer L. Israel, R. Edelstein, R. Samak, F. Pontet, F. Rousselet, P. Giraudet

460

Plasmapheresis in Cancer Patients and Acute Phase Reactant Proteins L. Israel, R. Samak, R. Edelstein, J. Baudelot, F. Pontet, F. Rousselet, P. Giraudet

462

Prognostic Value of the Macrophage Component of an Inflammatory Reaction Induced by Skin Abrasion in Cancer Patients L. Israel, R. Samak, M. Samak, R. Edelstein

465

Nutritional Markers in Advanced Large Bowel Cancer J.C. Hall, J. O'Quigley, G.R. Giles

467

Effects of Acute Phase Reactant Proteins on PHA-Induced Lymphocyte Blastogenesis R. Samak, L. Israel, R. Edelstein

469

Effects of Acute Phase Reactant Proteins on Monocyte Chemotaxis in Vitro R. Samak, L. Israel, R. Edelstein, M. Samak

471

E. Protein Changes in Other Clinical States Evaluation of Inflammation Proteins, Cardiac Enzymes and Myoglobin in Myocardial Infarcts C. Maurice, 0. Vernet, J.C. Rymer, M. Roncato, M. Paris, M. Leclerc

477

Human Serum Perchlorosoluble Glycoproteins in Myocardial Infarct M. Succari, M.-J. Foglietti, F, Percheron

480

Elevated C-Reactive Protein Levels in Sarcoidosis Patients J.-F. Mornex, J. Brune, J.-P. Revillard, C. Vincent

483

XIII Determination of Plasma Fibronectin Concentration in Pulmonary and Circulatory Failure J. Labrousse, J.M. Coulaud, J.P. Salmona, A. Tenaillon, J. Lissac, P. Beyne, J. Rapin, A. Jacqueson, D. Allard, H. Jerome

486

Protein, Alpha^ Antitrypsin, Lysozyme in Bronchoalveolar Lavage Liquid in Case of Sarcoidosis J.-J. Buneaux, H. Gosselin, F. Buneaux, P. Leclerc

489

Macrophage Phagocytic Activity and Lysozyme Concentration in Bronchoalveolar Lavages S. Guibaud, A. Simplot, A. Plumet, J.C. Guerin

491

Determination of Human Serum Complement Using the Hemolytic Radial Immunodiffusion Micromethod daring Ventilatory Tests of Allergenic Provocation in Asthmatics F. de Massia-Castex, M. Bastide, J.-M. Bastide, Ch. Seignalet, F.-B. Michel

493

Inflammation Markers in Patients with Pulmonary Pathology N. Biot, A. Revol, R. Harf, Y. Pacheco, M. PerrinFayolle

496

Usefulness of C-R P /C4 - 1 Index of Detection and Prognosis of Septic States in the Burn Patients P. Laurent, J. Marichy

497

Assessment of Appropriate Laboratory Measurements to Measure Crohn's Disease Activity C. André, L. Descos, F. André, F. Paille, S. Perrin

503

Assessment of Appropriate Laboratory Measurement to Measure Ulcerative Colitis Activity L. Descos, C. André, F. André, F. Paille, S. Perrin

505

Diagnostic and Prognostic Value of the Plasma Variations of Prealbumin and Transferrin in the Post-Operative Period of Gastrointestinal Tract Surgery - Preliminary Results J.F. Zazzo, D. Vauzelle, A. Abella

507

XIV Serum C-Reactive Protein Assay in Renal Transplantation J.-P. Revillard, M. Laville, C. Vincent, J. Traeger

509

Relation between the Quantity of Serum Lysozyme and the Amount of Succinatedehydrogenase (SDH) and Alpha-Glycerophosphate-Dehydrogenase (alpha-GDPH) on Activated Lymphocytes Membranes as a Possible Index of the Immune Response after Human Renal Transplantation K. Metodiev

511

Study of Some Markers of Inflammation (C Reactive Protein, Haptoglobin, Orosomucoid) in Chronic Inflammatory Articular Rheumatic Diseases F. Bertrand, X. Herbeuval, P. Paysant, G. Faure, A. Gaucher

513

Levels of Serum C9 Complement Factor in Various Chronic Inflammatory Articular Rheumatic Diseases P. Montagne, G. Faure, M.-Ch. Bene, A. Gaucher, J. Duheille

516

Modifications of "Inflammatory Proteins" (Transferrin, Orosomucoid, Coeruloplasmin, Alpha-l-Antitrypsin in Human Gonadotropin Induced Ovulation S. Ferrand, F. Touraine, H. Dechaud, B. Claustrat, C. Quincy, J.M. Thoulon

518

Changes of Distribution of Glycosylated Forms of Human Orosomucoid and Alpha-2-HS Glycoprotein during Inflammation and Pregnancy: Study by Isoelectric Focusing and Crossed Immuno-Affinity Electrophoresis I. Nicollet, J.-P. Lebreton, M. Fontaine, M. Hiron

520

Variation of C Reactive Protein in Serum Following Severe Skull Trauma C. Goester, G. Ferard, A. Bourguignat

524

Studies on some Blood Proteins in Medullary Patients B. Sallier, G. Pilonchery, J. Depassio, P. Minaire, A. Revol

528

XV The Prostaglandin Induced Acute Phase Response and its Failure in Scleroderma J.T. Whicher, M.F.R. Martin, P.A. Dieppe, L. Marshall

535

F. Acute Phase Reactants in Rodents Serum Amyloid P Component in the Rat: Isolation, Characterization and Original Features M. Pontet, M. d'Asnieres, R. Engler, J. Escaig

541

Transcortin (CBG) Actitivities, Endogenous Corticosterone Levels and other Serum Parameters in the Course of the Acute Inflammatory Reaction of the Rat L. Savu, H. Zouaghi, Ch. Lombard, E.A. Nunez

544

Secretion of 3 Proteins during Experimental Inflammation in Rats F.N. Dinh, J. Davy, M. Appel, G. Durand, J. Feger, J. Agneray

547

Identification of Intracellular Acute Phase Proteins in the Liver of Mice by Crossed Immunoelectrophoresis H.-B. Oh, T.C. B(Zig-Hansen

550

Studies on the Mechanism of the Prostaglandin Induced Acute Phase Response; its Failure in Endotoxin Tolerant Animals J.T. Whicher, J. Unwin, A. Bell, P. Southall

552

G. Proteinase Inhibitors Inhibition of Formation of the Human Amplification C3 Convertase by Fluid Phase Polycations F. Maillet, M.D. Kazatchkine

557

Determination of the Degree of Desialylation for Orosomucoid and Alpha, Antitrypsin. Application to Rheumatoid Polyarthritic Sera M. Bordas, N. Serbource-Gogue1, D. Biou, J. Féger, G. Durand

558

XVI Proteinase Inhibitors Controlling Biologie Activity of Lymphoid Tissue Cells F. Baranova, A. Berman, L. Popova, Y. Zaretskaya

561

Binding of Alpha-l-Antitrypsin Specific Antibodies to Human Lymphocytes: A Flow Cell Microfluorometry Study J. Bata, G. Cordier, J.-P. Revillard

563

Suppression of Antibody-Dependent Lymphocyte Mediated Cytotoxicity by Human Alpha-2 Macrog&obulin G. Cordier, J P. Revillard. M. Latour

565

H. Methodology Protein Evaluation of Populations in Intertropical Regions A. Fribourg-Blanc, P. Druilhe, L. Monjour, B. Carme, D. Richard-Lenoble, M. Gentilini, P. Carnevale, J.F. Trape, J.F. Molez, E. Bois 569 Immunophelometric Quantitation of C.R.P. - Importance of Samples Storage H. Bernon, J. Bienvenu, M. Perouse de Montclos, A. Roullet, P. Laurent

573

Pre Albumin Titration Using a Rate Nephelometer. Critical Study. Applications in Nutrition anfl Oncology J. Oglobine, M. Meslin, C. Bouchet, R.T. Saracino, J.-F. Gillette, J. Susini

575

Plasma Fibronectin: Its Dosage in Clinical Biology M.-L. North, Y. Dudt, M. Schwartz, J.-P. Cazenave, S. Rabinovitch, B. Audhui, P. Ciret

579

Quantitative Immunoelectrophoretic Methods R. Guinet, S. Gabriel

582

Protein Profile in Surgical Intensive Care M.C. Diemert, J. Galli, A. Galli, F. Baud, F. Rouillon, P. Glaser

586

Section I The I n f l a m m a t o r y

Response

PROGRESS IN PLASMA PROTEINS:

HALLMARKS OF HEALTH AND MARKERS

OF DISEASE

Frank W. Putnam Department of Biology, Indiana University Bloomington, Indiana 4740 5, USA

Introduction In recent years great progress has been made in knowledge of the plasma proteins and of their role in health and disease (1-4). The advances include better methods for identification and isolation, ultrasensitive and precise techniques for quantitation, better knowledge of their metabolism and of their interaction with each other and with cell surfaces. Complex cascades of enzymatic reactions such as blood coagulation, complement action, fibrinolysis, and kinin formation are being dissected and reassembled on a molecular basis. The intricate interactions of these cascades are being delineated, and their myriad pharmacologically active products— both proteins and peptides—are being discovered. The periodic table of the immunoglobulins is now probably complete with the recent determination of the structure of human IgD in my laboratory (5). The complete amino acid sequences and in a few cases even the three-dimensional structures of a number of human plasma proteins are already known, and much more is on the way. Gene sequencing is revealing startling new discoveries such as the phenomenon of skipping genes and may be on the verge of deciphering the mechanism of genetic control of expression and regulation (6-8). Many of you on the symposium committee, speakers on the program and others in the audience have been participants in

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1 9 8 2 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

2

this parade of progress. Some in basic research, others in laboratory medicine and clinical research. Our program will assemble and integrate the many diverse themes I have referred to into a holistic view through our three topic sessions: 1) Protein Profile in Malnutrition; 2) Protein Profile in Inflammation, and 3) Future Trends on Acute Phase Proteins. My purpose today is to give you an overview of recent progress in plasma proteins, to relate this progress briefly to the three major themes of the Symposium, and then to illustrate the highlights of some of our current work on human IgD as it relates to the themes of the Symposium. Progress in Plasma Proteins According to an exhaustive survey made in 1975 (1, 2), about 86 human plasma proteins had been described, but the function was known for only about a dozen (see Table I, Ch. 2 of Ref. 1). Today, about 100 have been purified and the function is known for about 40 including many trace proteins. In 1975 there was a scarcity of structural data on human plasma proteins (see Table II, Ch. 2 of Ref. 1); in 1981 there are probably more amino acid sequence data on human plasma proteins in press or ready for publication than were extant in 1975. To use immunoglobulins as an example: In March 1965, when we reported our first sequence data for a Bence Jones protein (9), the world's literature then contained a total of only about 50 amino acid residues of immunoglobulin sequence. By 1979 even before DNA sequencing began, about 47,500 residues had been published. The rate has been increasing exponentially since the advent of the commercial automatic protein sequencer and is doubling every three years, and DNA sequencing will soon shorten the doubling time. However, it is not the rate of accumulation of data that I want so much to emphasize but rather the pace of increase of molecular understanding of antibody specificity, structure, and genetic control.

3

Nonetheless, as I will outline later, we still have much to learn about the biological effector functions of the Fc domain, such as the site and mechanism of complement fixation, cytotropic reactions, and binding to cells. And these are the antibody functions that are critical to many inflammatory processes. Whole systems of plasma.proteins besides the immunoglobulins have been thoroughly investigated. Much progress has been made with the coagulation proteins especially the serine proteases, the antiproteases, and the complement system. The mechanisms of transport of metal ions, heme, drugs, and organic ligands are being elucidated, and the importance for human health of the'interaction and competition of these processes is being clarified. As we shall see in this Symposium, inflammation is involved in many of these processes. Despite the parade of progress in plasma proteins much remains to be learned. As yet, we know the function of less than half of the 100 or more readily detectable plasma proteins, and we are hardly aware of the hundreds of lesser components, some of which may have important physiological roles. C-reactive protein is the prototype of the acute phase reactant. Yet serendipity led to its discovery 50 years ago through the lucky accident that it forms a specific precipitate with the C polysaccharide of pneumococcus, and this was observed in the leading laboratory for study of pneumococcal polysaccharides. Still today we are only beginning to learn the biological function of CRP. How many such marker proteins remain to be discovered? In previous reviews I have listed a dozen alpha and beta glycoproteins in search of a function (1, 4). Conversely, I could name a dozen other proteins that were isolated, characterized, and named before their function was discovered and then found to be already known under another name related to their function. One example is fibronectin, the plasma form of which was long known as "cold-insoluble globulin". The role of this cell surface glycoprotein in cellular interactions remains to be clarified, and we look forward to

4 Dr. Laurent's discussion of fibronectin. Theme 1 - Protein Profile in Malnutrition Although plasma protein represents only about 2% of the tissue protein mass, the ease of sampling and of analysis early led to many studies of changes in total plasma proteins in severe human malnutrition or as the result of protein depletion in experimental animals. This early work has been rather critically reviewed by Garrow (10) who emphasized three aspects: 1) the interaction of plasma protein levels and tissue protein stores, where the greatest effect is on liver, 2) the diagnostic value of plasma proteins as a measure of nutritional stress, and 3) dynamic changes in plasma protein metabolism. However, in recent years the emphasis has focused on two more significant phenomena that are the subject of the first theme of this Symposium, i.e. 1) general and specific effects of malnutrition and concurrent infection on humoral and cellular immunity, and 2) specific effects on the plasma protein profile such as characteristic decreases in certain proteins other than the general indicator, serum albumin. An important question to ask is whether the effect on the plasma protein profile caused by malnutrition or marasmus, or more generally by protein-calorie malnutrition, is mainly a general change in profile or whether there is some specific and significant effect, for example on the immune response. Last year this question was the subject of an entire symposium on nutritional deficiency, immune responses, and infectious illness that was chaired by Dr. Chandra (11). At our Symposium, he, together with Drs. Page Faulk, Touraine, and Gay will lead the discussion on our first theme—Protein Profile in Malnutrition. In this matter it is difficult to separate the individual roles of malnutrition, infection, and socio-cultural status. We will look forward to their efforts to dissect the effects of these conditions both on cell-mediated immunity

5

(CMI) and on humoral immunity and to seek to identify the ways in which specific nutritional deficiencies may alter CMI responses. In contrast to the current indication of possible specific effects of nutritional deficiencies on the CMI response, is the apparently benign effect on humoral immunity. In the absence of severe infection, serum IgG, IgA, and IgM levels are usually normal or somewhat increased; though, to be sure, serum IgE may be elevated and complement levels decreased (12). Thus, current interest centers on specific effects on the plasma protein profile, notably the profound deficit of transferrin in kwashiorkor, as well as the decline in ceruloplasmin, B-lipoproteins, and in other proteins to be discussed by Touraine. Theme 2 - Protein Profile in Inflammation The terms "acute-phase proteins," "acute-phase reactants," and AP-proteins or AP-reactants have traditionally been used to denote plasma proteins whose concentration increases significantly in the acute phase of inflammatory processes, and often in pregnancy, cancer, and various diseases (13). Originally the AP-proteins represented an uncharacterized group consisting mainly of ct-glycoproteins with two characteristics in common: the presence of carbohydrate and synthesis in the parenchymal cells of the liver. These are not very clear distinguishing features because all typical plasma proteins except immunoglobulins are synthesized in the liver, and virtually all plasma proteins are glycoproteins except for albumin, retinol-binding protein, and notably C-reactive protein - the archtype of AP-reactants. Much of the early literature dealt with seromucoid, an a-glycoprotein fraction of plasma which contains ai-glycoprotein (orosomucoid) as its major component. As summarized in Table I, in recent years the principal APreactants have been isolated and well characterized. In most

6 TABLE 1.

Properties of Acute-phase Reactants of Human Plasma Amount in normal Sequence plasma (mg/100 ml)

Symbol

Molecular Weight

Pi

Carbohydrate content {%)

ai-Acid glycoprotein (orosomucoid)

ajS

40,000

2.7

41.4

55-140

complete

ai-Antitrypsin

en AT

54,000

4.8

12.4

200-400

advanced

Cp

135,000

4.4

8.0

15-60

half-done

?

0

Protein

Ceruloplasmin C-reactive protein Haptoglobin (type 1-1) Fibrinogen

CRP Hp -

(21,500)5-6

iinitial rise in C-R.P. level until

reaching a maximum in 48 to 72 hours, in uncomplicated cases, C-R.P. returns to normal values -within a few days ( 14 ) . In cases of postsurgery complications, the persisting h i g h values after 72 hours, or a new rise, must immediately lead the clinician to look for infection and/or necrosis

...

The everyday determination of C-R.P. levels is an excellent indicator of postoperative infection . In the same way, an increase of C-R.P. level after renal transplantation should start infection or

serum sickness

a search for a rejection episode, an

( 32, 44 ) .

The patients w i t h active Systemic Lupus Erythematosus high

( SLE ) do not have

levels C-R.P. in their sera ( 21 ) . Thus the C-R.P. changes

distinguish the patients w i t h SLE from those w i t h active Rhumatoid

should Arthri-

tis which disease is generally marked w i t h a high levels of C-R.P. during acute phase . On the other hand, an increase of C-R.P. concentration in patients w i t h SLE is a satisfactory and reliable

index of a

superimposed

infection ( 2J ) . Many other pathological states such as b u r n trauma, bacterial

infections

in a non—surgical setting, myocardial infarction, and rheumatic diseases are the areas w h e r e C-R.P. has b e e n found useful

. But, the use of C-R.P.

changes in a diagnostic w a y m u s t be limited to some definite cases w h i c h may

b e analysed v e r y well . We must not forget that C-R.P. is a n o n -

specific Inflammatory Marker . The m o s t frequent illustration of prognostic value of C-R.P. changes is in the rheumatic diseases in which it is often difficult to assess the course of disease activity . In acute rheumatic fever or rheumatoid arthritis or rheumatic heart disease the C-R.P. levels are typically elevated and usually in proportion to the activity and seriousness of the disease process ( 2, 47 ) , The decrease of C-R.P. concentration is a good prog-

85

nostic sign for the termination of a rheumatic attack but does not preclude the possibility of a future polycyclic recurrence

( 56 ) .

The use of the C-R.P. and ESR for detecting inflammation has led to several comparaisons of the advantages and disadvantages of these tests . The ESR is more frequently abnormal in negative cases than C-R.P., and is influ enced b y b o t h anaemia and polycythaenia ( 8 ) . I n one study, the ESR was consistently normal where active rheumatic heart disease was accompanied by congestive heart failure ( 56 ) . A

recent study suggests that elevated

ESR in patients is not caused or influenced by C-R.P. production although b o t h phenomena may share in many cases common triggering processes ( 1 ) .

4) E F F E C T S ON

OF ANTI-MICROBIAL

C-R.P.

AND

ANTI-INFLAMMATORY

TREATMENT

LEVELS

C-R.P. level is a reliable indicator to determine the effectiveness of anti microbial therapy . A quick fall of C-R.P. concentration following is observed w h e n bacteria are fully sensitant ve

therapy

. In contrast an ineffecti -

anti-microbial treatment shows a persisting high CRP level ( 22 ) .

Cortisone, A C T H or Predinsone treatment in cases of rheumatoid arthritis (RA) cause

a fall in C-R.P. concentration ( 13, 18, AO ) . The effect of trea-

tement w i t h lod and dapsone in patients w i t h in C-R.P. levels

RA

are similar in changes

( 40 ) . These changes in C-R.P. reflect the efficacy of

the drugs, and complete normalization of C-R.P. levels have b e e n found to correlate w i t h suppression of disease activity . W i t h the cessation of antiinflammatory therapy, clinical and biological manifestation of

inflammation

including C-R.P., usually return and mark the recurrent inflammation ( 13 ). The disappearance of C-R.P. from human blood during anti-inflammatory

the-

rapy is probably secondary to suppression of the process rather than a primary effect of these agents upon the metabolism of C-R.P. ( 17 ) . The experimental studies established that it was not possible to suppress the production of A P R induced by different

inflammatory agents after pretrea-

tment w i t h anti—inflammatory drugs ( 17 )

86

CONCLUSION C-R.P.

is a p a r t i c u l a r l y interesting n o n - s p e c i f i c m a r k e r of

b e c a u s e of its i m m u n o r e g u l a t o r y role in h o s t r e s p o n s e

inflammation

. In clinical

it is a r e l i a b l e and easily a c c e s s i b l e indicator of a c t i v i t y

of

practice

inflamma-

tory p r o c e s s and u s e f u l in d e t e c t i o n of these i n f l a m m a t o r y c o m p l i c a t i o n s . The Immuno N e p h e l o m e t r i c A s s a y is the b e s t a d a p t e d technique of its q u a n t i f i c a t i o n b e c a u s e of its s e n s i v i t y and r a p i d i t y b y C - R . P . changes a true c o u r s e of the complications

patient

. T h e r e f o r e it m a y e x p o s e d to

show

inflammatory

( m a i n l y n e c r o s i s and b a c t e r i a l i n f e c t i o n s ) . In this p e r s -

p e c t i v e the C - R . P . c a n b e c o n s i d e r e d a

BIOLOGICAL ALARM .

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CONTROL PROTEINS OF THE ALTERNATIVE COMPLEMENT PATHWAY: IN VIVO CORRELATES OF

IN VITRO FUNCTION.

Shaun Ruddy Medical College of Virginia, Richmond, Virginia, U.S.A. (Currently at: Mechanisms in Tumour Immunity Unit, MRC Centre, Hills Road, Cambridge, England)

Introduction The alternative pathway for complement activation constitutes a means by which the inflammatory effects of the complement system may be recruited independent of specific antibody and the classical early complement components (C1,C4- and C2) {reviewed in l). Interaction of native C3 or its major cleavage fragment, C3b, with factor B (B) in the presence of magnesium ions renders B susceptible to cleavage by the serine protease, factor D (Figure 1).

The complex of C3 or C3b with the major

fragment of B, (Bb), is itself a protease, C3bBb, whose substrate is C3.

The instability of C3bBb at 3 7°C. is counter-

acted by properdin (P), which prolongs the half-life of decay of C3bBb by physically binding to it to form C3bBbP.

Both the

unstable and the properdin-stabilized alternative pathway C3 convertases catalyze the same reaction, the cleavage of C3 at arginine 7 7 to release the anaphylatoxin, C3a,and produce the major fragment, C3b. Since C3b is very much more efficient than native C3 in its interaction with B to form additional convertase, a positive feedback loop is inherent in the system, in which the product of a reaction, C3b, participates in the formation of additional enzyme capable of catalyzing the reaction.

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1982 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

The activity of C3b

90

Figure 1: The alternative pathway for complement activation, (reproduced with permission from 1) in this feedback is damped by two control proteins, C3b Inactivator (I) and glH Globulin (H).

The former is an endopepti-

dase which cleaves the »-chain of C3b,producing the inactive form, C3bi. H physically binds to C3b, making it susceptible to proteolysis by I, preventing its interaction with B to form C3bBb, and accelerating the decay of preformed C3bBb or C3bBbP. In normal plasma, continuous low grade generation of C3b by the interaction of native C3, B and D occurs, but the activity of the regulatory proteins, I and H, serves to control the cycling of this feedback loop.

The function of some activating sur-

faces, such as the yeast cell wall polysaccharide zymosan or rabbit erythrocytes,is to provide a microenvironment in which C3b and the properdin-stabilized convertase, C3bBbP, are protected from the action of these regulatory proteins.

The assem-

bly of a surface-associated alternative pathway convertase, resistant to inactivation by the control proteins, leads to an amplification phase which activates the terminal complement components and deposits additional C3b on the activating surfaces

91

This is a report of measurements of serum concentrations of I and H in normal individuals and in patients with arthritis. The strong correlations observed between levels of these control proteins and of other constituents of the alternative pathway suggest that they may regulate the normal cycling of the feedback loop in vivo in the same way that they have previously been shown to function in vitro.

Materials and Methods Serum specimens were obtained from 50 normal individuals who had been interviewed to exclude individuals with a recent history of respiratory infections or other illnesses or who were taking any medication, including oral contraceptives. All 29 patients with rheumatoid arthritis had definite or classical disease as defined by the American Rheumatism Association criteria (2); they were divided into seropositive (RA pos) or seronegative (RA neg) groups depending upon the results of a latex fixation test for rheumatoid factor. Serum was also obtained from seven patients with degenerative joint disease of the knees or hips. Measurements of serum concentrations of C4, B, P, I and H were made by single radial immunodiffusion against antisera as previously described (3).

Results The normal range (mean jf 2 SD) for serum I concentrations was 5 7.9 - 116.5% of a standard serum pool. For H the corresponding limits were 196 - 400 ug/ml. Both I and H concentrations in the normal sera were strongly correlated with each other (r = 0.66) and with C3, B and P, but not C4. The data for I vs C3 are shown in Figure 2 and for H vs C3 in Figure 3.

92

140

120 • • .

100 C3blNA

(%STD)

i•

80

60 40

r = 0.4050 p < 0.005

20 1000

J 2000

l

C3 ( ^ g / m l )

Figure 2.

Correlation of serum concentrations of Inactivator and complement component 3.

93

400

• • 300

BIH (>ig/ml) 200

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INTERACTIONS BETWEEN CRP AND MONONUCLEAR CELLS

Karen K. James, Carolyn Mold, and Henry Gewürz Department of Immunology/Microbiology, Rush Medical Center Chicago, I l l i n o i s 60612, USA

Itymphocytes-Revlew

A p o s s i b l e i n t e r a c t i o n between C - r e a c t l v e p r o t e i n (CRP) and lymphoc y t e s was s u g g e s t e d i n 1937 when Abernethy and F r a n c i s (1) r e p o r t e d a d e l a y e d " t u b e r c u l i n - t y p e " s k i n r e a c t i o n t o t h e C - s u b s t a n c e of pneumococcus which c o r r e l a t e d w i t h t h e p r e s e n c e of CRP.

Hornung (2)

in-

j e c t e d complexed CRP i n t o a human v o l u n t e e r and observed a delayed s k i n r e a c t i o n ; b i o p s y of t h e l e s i o n a t 42 h r showed a mononuclear

cell

i n f i l t r a t e with p e r i v a s c u l a r c u f f i n g , histology similar to delayedt y p e h y p e r s e n s i t i v i t y (DTH).

Hornung showed t h a t low l e v e l s

(10

ug/ml) of p u r i f i e d CRP s t i m u l a t e d lymphocyte b l a s t o g e n e s i s (3) and t h a t CRP-stimulated normal lymphocytes could d e s t r o y human melanoma c e l l s i n c u l t u r e ( 4 ) , s u g g e s t i n g t h a t lymphocyte a c t i v a t i o n i s a n o r mal p h y s i o l o g i c f u n c t i o n of CRP.

Hokama e t a l . ( 5 , 6 ) , n o t i n g t h a t CRP

i s e l e v a t e d i n c a n c e r and t h a t s e r a from c a n c e r p a t i e n t s i n h i b i t

the

3 H- t h y m i d i n e i n c o r p o r a t i o n i n l e u k o c y t e c u l t u r e s , used b o t h p u r i f i e d CRP and CRP-containing s e r a and found i n h i b i t i o n of

phytohemagglutin

( P H A ) - s t i m u l a t e d b l a s t o g e n e s i s which was r e v e r s i b l e by phosphocholine (PC).

Additionally,

they r e p o r t e d t h a t f l u o r e s c e i n - l a b e l e d CRP bound

t o p e r i p h e r a l blood lymphocytes.

This b i n d i n g could be i n h i b i t e d by

c h o l i n e phosphate o r by p r e t r e a t m e n t of t h e c e l l s w i t h p h o s p h o l i p a s e C (5).

These a u t h o r s concluded t h a t t h e CRP e f f e c t on lymphocytes was

I n i t i a t e d through b i n d i n g t o phosphocholine r e s i d u e s on t h e phosphol i p i d membranes.

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1982 by W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k

132

In 1974, this laboratory set out to investigate the effects of CRP on lymphocyte functions in vitro (7-10).

Mortensen, et al.

(7) observed

that purified CRP would selectively bind jji vitro to 50% of the T lymphocytes, inhibit their ability to form rosettes with sheep erythrocytes (ShE), and inhibit their proliferative response to allogeneic cells in a one-way mixed lymphocyte culture (MLC).

CRP was not inhibi-

tory to mitogen-stimulated cultures (PHA and Concanavalin A); did not affect the detection of B lymphocytes by surface immunoglobulin (S-Ig), by complement receptors (CR) or by Fc receptor (FcR) reactivity as detected by aggregated human gamma globulin (AHGG); did not inhibit antibody-dependent cellular cytotoxicity reactions (ADCC); and did not exert any effect on the stimulator cells in a MLC response (7).

Sub-

sequently human CRP was demonstrated to bind to more mature murine T cells, i.e., only 6% of thymocytes bound CRP compared to 40% of spleen cells and 50% of lymph node cells (8).

Similar to the effects

on human cells, CRP inhibited the murine MLC but had no effect on the mitogen responsiveness of murine lymphocytes.

In the mouse system,

CRP inhibited the in vitro generation of cytolytic T cells (8).

Further experiments demonstrated selective binding of CRP to antigenstimulated (but not mitogen-stimulated) lymphocytes (9).

Cells cul-

tured in the presence of CRP synthesized decreased quantities of migration inhibitory factor (MIF) and macrophage chemotactic factor from both antigen-stimulated and mitogen-stimulated lymphocytes (10). More recently, Mortensen reported that human CRP, added to murine cultures, inhibited the in vitro induction of T-dependent IgM and IgG antibody formation (11).

This inhibition appeared to be effected by

the CRP-mediated generation of a suppressive T cell population. Collectively, these findings suggested that CRP reacted selectively with a subpopulation of T lymphocytes and influenced certain T cell-dependent functions.

In support of a reactivity with T cells, Bieber, et al. reported that CRP was the critical component in an ShE rosette inhibitory factor

133 which has been isolated from spleens of patients with Hodgkin's Disease (12).

This inhibitory factor was a complex containing CRP,

Clq and low-density lipoprotein; inhibition was reversible by CPS, but not by chloroform-methanol extraction.

Isolated CRP could also

inhibit ShE rosette formation, thus implicating CRP as the mediating factor. Lymphocytes-Current Studies More recently our laboratory has re-investigated the CRP-lymphoid interaction and found that highly purified CRP did not bind to mononuclear cells and did not inhibit reactivity of any of the functional assays we had previously reported.

Thus, we decided to direct our

efforts toward the binding reaction at the membrane level, both to define the conditions required to achieve CRP-binding and to study the nature of the reactivity at the cell surface.

These investigations

have shown that CRP binding to mononuclear cells requires complex formation of CRP with a multivalent binding specificity, achievable with either C-polysaccharide (CPS) or with PC coupled to a carrier molecule (13).

This reactivity, demonstrable by immunofluorescence

and by radioassay, was influenced by the purity and state of the CRP, the ratio of CRP:ligand, incubation conditions, and calcium concentration.

CRP has many effector functions analogous to antibody

(agglutination, precipitation and complement activation), which led us to establish methods for detecting CRP-binding cells analogous to those used to detect FcR; i.e., soluble complexes, heat modification, and rosette formation with E-CPS-CRP.

In normal individuals, these

three methods detected equivalent small percentages of lymphocytes (3.0 + 1.7%) as well as approximately half of the phagocytic mononuclear cells.

Characterization by double marker studies indicated that the lymphocytes capable of binding complexed CRP were not restricted to T cells, but instead overlapped the T, B, and "null" cell categories in a 2:1:1

134

ratio (14).

Preferential overlap was seen with IgG FcR-bearing cells

since 70% of the CRP-binding cells formed EA rosettes; however, only 12% of the EA rosetting cells bound CRP, suggesting that CRP detected a subset of cells bearing the IgG FcR.

This is in agreement with the

investigations of Williams, et al. (15) which detected CRP bound in vivo to lymphocytes from patients with rheumatic fever (16 + 8% in patients compared to 3 + 3% in normals).

No apparent correlation was

observed between serum CRP levels and the percentage or absolute numbers of CRP bearing lymphocytes.

Williams et al. used multiple

marker systems to characterize the CRP binding lymphocyte subpopulations and found a 50% overlap of CRP binding and IgG FcR bearing lymphocytes (using either AHGG or IgG EA), but no overlap with lymphocyte bearing IgM FcR.

They further defined the CRP bearing

population as 40% T lymphocytes (based on subsequent binding of ShE) and 40% B lymphocytes (by detection of S-Ig).

The preferential binding of complexed CRP to IgG FcR bearing cells prompted us to study several cultured cell lines for CRP-binding. CRP-CPS bound to both human and murine cultured lines which bound aggregated human gamma globulin (AHGG), but lesser or no binding of CRP-CPS was detectable with cell lines negative for the IgG FcR. These cultured cell lines have provided a consistent source of cells for use in studying the interactions of CRP at the cell-surface level, including the possibility that CRP may bind to the IgG FcR.

However,

preliminary investigations with both cultured cells and peripheral blood mononuclear cells have shown that CRP-CPS does not block AHGG binding or vice versa, suggesting that different receptors or expressions of the receptor are involved.

The E-CPS-CRP rosette method of detecting CRP-binding cells facilitated study of the morphologic appearance of these cells and the majority were large, granular lymphocytes.

This information, coupled

with the small percentage of CRP-binding cells and some overlap with

135 ShE rosetting cells, prompted us to investigate the effect of CRP on natural killer cells which have been reported to have similar char*acteristics.

Baum, et al. (16) has shown that purified CRP, either

native or complexed, had no effects on NK activity.

F(ab')2 frag-

ments of antl-CRP, however, enhanced NK activity and whole molecule anti-CRP and complement substantially depleted this effector function.

These effects, demonstrable with the same antibody used in

the binding studies, and the demonstration by Williams, et al. (15) of CRP-bearing cells, prompted us to look for CRP-bearlng cells using a more sensitive indirect immunofluorescent assay.

Preliminary

investigations indicate that CRP-bearing cells are present in equivalent percentages to CRP-binding cells.

In certain rheumatic

fever patients, Williams et al. found increased percentages of CRP-bearing cells after 18 hr in culture (15).

Whether this mem-

brane-bound CRP is cytophillically attached from serum CRP or is produced by the cell remains to be determined.

In any case, CRP

in the presence of a multivalent ligand binds preferentially to a subset of lymphocytes bearing the IgG FcR, and cell-bound CRP may influence certain of the functions of these cells.

Monocy t e s-Ma c rophage s The reactivity of CRP with Clq to initiate activation of the clasical complement pathway suggested a functional analogy between CRP and the Fc region of Ig (17-19).

This led to investigations of CRP-mediated

phagocytosis and immune adherence to monocytes/macrophages.

Mortensen

et al. (20) reported that CRP could effectively replace Ig in an EAC system (i.e., E-CPS-C was equivalent to E-CPS-anti CPS-C) to initiate not only the attachment but also the ingestion of these coated erythrocytes.

This suggested an interaction of CRP with a membrane

receptor, analogous to the binding of IgG to the FcR.

CRP was

required to activate complement initially (for the deposition of C3b

136

and C4b), but CRP deposition was also necessary to promote phagocytosis of these coated red cells.

Subsequently, Mortensen, et

al. demonstrated that immobilized CPS-CRP complexes could inhibit the ingestion of either EA or EAC cellular complexes (21).

This

inhibition of phagocytosis was directly proportional to the concentration of CRP, in a manner analogous to the inhibition mediated by immobilized Ab-Ag complexes.

It was also shown that the reversible

inhibitor of carbohydrate metabolism, 2-deoxyglucose (2-dG), blocked the effects of CRP-CPS complexes on macrophages, analogous to 2-dG inhibition of both IgG and C3b mediated ingestion by macrophages.

Recently, the opsonic effects of CRP in vivo were investigated in mice by Nakayama et al. (22) using human CRP complexed to murine E coated with CPS.

The presence of CRP altered the site of sequestration of

^^Cr-labeled E-CPS from the liver to the spleen, an effect which required thé participation of complement as well.

Thus, the in vitro

and in vivo data provide support for a functional role for CRP as an opsonin.

Studies with both lymphocytes and monocytes-macrophages

indicate reactivity of CRP with a receptor analogous to or identical with the FcR, and suggest functional consequences for this interaction. (Supported by NIH grant AX-12870.)

137 References

1. Abernethy, T. and Francis, T.: J. Exp. Med. ^5:59-73 (1937). 2. Hornung, M: in "Non-Specific Factors Influencing Host Resistance", Braun, W. and Ungar, J., eds. , Karger Publ. Co., Basel, pp. 354-357 (1973). 3. Hornung, M. and Fritchi, S.: Nature New Biol. (USA) 230:84-85 (1971). 4. Hornung, M.: Proc. Soc. Exp. Biol. Med. 239:1166-1169 (1972). 5. Hokama, Y., Paik, Y. , Yanagihara, E. and Kumura, L.: Reticuloendothel. Soc. 13:111-121 (1973). 6. Oishi, N., Toukawa, C., Ochiai, H. and Hokama, Y.: J. Retlculoentothel. Soc. =242-249 (1973). 7. Mortensen, R. , Osmand, A. and Gewürz, H; : J. Exp. Med. 141 :821839 (1975). 8. Mortensen, R. and Gewürz, H.: J. Immunol. 116:1244-1250 (1976). 9. Croft, S., Mortensen, R. and Gewürz, H. : Science 193:685-687 (1976). 10. Mortensen, R. , Braun, D. and Gewürz, H. : Cell. Immunol. 28:59-68 (1977). 11. Mortensen, R. : Cell. Immunol. 44:270-282. 12. Bieber, M., Fuks, Z. and Kaplan. H. : Clin. Exp. Immunol. 29:369375 (1977). 13. James, K. , Hansen, B. and Gewürz. H. : Fed. Proc. J38:14 3 5 (19 79). 14. James, K., Hansen, B. and Gewürz. H.: Fed. Proc. J39 : 941 (19 80). 15. Williams, R., Kilpatrick, K., Kassaby, M. and Abdin, Z. : J. Clin. Invest. jy.:1384-193 (1978). 16. Baum, L. , Glaviano, R. , James, K. and Gewürz, H. : Fed. Proc. 40: 1045 (1981). 17. Volanakis, J. and Kaplan, M. : J. Immunol. JJ_3 :9-17 (1974). 18. Osmand, A.P., Mortensen, R.F., Siegel, J. and Gewürz, H. : J. Exp. Med. _142:1065-1077 (1975). 19. Claus, D., Siegel, J., Petras, K. , Osmand, A. and H. Gewürz: J. Immunol. 219:187-192 (1977). 20. Mortensen, R. , Osmand, A., Lint, T. and Gewürz, H. : J. Immunol. J_17 :774-781 (1976). 21. Mortensen, R. and Duszekiewicz, J.: J. Immunol. 119:1611—1616 (1977). 22. Nakayama, S., Mold, C., Gewürz, H. and DuClos, T.: Fed. Proc. 40:1046 (1981).

CLINICAL USEFULNESS O F OROSOMUCOID DETERMINATION

Jacques Bienvenu Laboratoire de Biochimie - Est Hôpital Jules Courmont - Sainte Eugénie 69310 Pierre Bénite, France

Introduction

Orosoraucoid

or

c(>

-

acid

studied plasma proteins. reactants

glycoprotein

a

better

extensively

It belongs to the so-called typical acute phase

(APR). Orosomucoid

is the APR with the lowest molecular weight

(40.000). Some of its physico-chemical for

is one of the most

understanding

of

the

properties are to be pointed

following.

These

noteworthy

out

charac-

teristics are (1) (2) : -

a very

high

sialyl residues orosomucoid

carbohydrate

content

(45 %)

with

(12 %) which play an important role

(5,5 days

for

the native

form,

a

large

number

of

in the half-life of

2 minutes

for the

desialy-

lated form). - a very acidic isoelectric point (2,7 in phosphate buffer). - the microheterogeneity of the glycoprotein is now clearly

demonstr-

ated (cf P. ARNAUD). - an important degree of homology w i t h immunoglobulins

After its

a rapid

survey

of

the

(3).

different methods used

determination, we shall summarize the clinical usefulness of

for

oroso-

m u c o i d measurement.

Methods for orosomucoid measurement :

non-immunological methods : The addition of perchloric acid to plasma leads to the historical

seromucoid soluble fraction

(4). Due to the simplicity of its

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1982 by W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k

140

isolation,

the

seromucoid

fraction

has

been

investigated

by

many

workers. This fraction can also be obtained by the use of sulphosalicylic acid or trichloracetic acid and by boiling. The amount of the seromucoid

is

content

determined

from

protein

(with Folin-Ciocalteau

orcinol).

The mean normal

content

reagent) or

values

(biuret),

from

from

hexose

tyrosine

content

(with

for seromucoid level in human serum

given by WINZLER are 0.612 (as protein), 0.124 g/1 (as hexose) and 0.034 (as tyrosine). Orosomucoid

constitutes

seromucoid fraction which also contains

more

than

90 per

cent of

the

^ ^-glycoprotein, haemopexin,

C^-antitrypsin and Cl^-antichymotrypsin. The solubility of orosomucoid

in these acidic media is

probably due to its high carbohydrate content which possesses a protective

effect against

acidic

denaturation

(cleavage

of the 0-glycosidic

linkage of the sialyl residues). As recently shown by THAW and ALBUT (5), the seromucoid assays are difficult to reproduce and depend a lot on working conditions (timing, mixing, filtering...). Nevertheless, methods

show

a good

the

correlation

results

given

by

these

chemical

(r = 0.98) with the data of immuno-

chemical methods for orosomucoid determination. THAW and ALBUT conclude that

immunochemical

methods which

are

more

precise

and specific must

replace the seromucoid assay for orosomucoid determination.

- Immunochemical analyses : Radial Immunodiffusion (RID) based on the MANCINI'S method (6)

is

the

first

immunochemical

applied for orosomucoid analysis

technique

to

have

been

intensively

(7). Due to its low molecular weight

the diffusion of the protein in the gel is rapid. Precision of the assay is satisfactory

for clinical practise with a between-run precision of

less than 5 % (5) and a detection limit of about 5 mg/1 for commercially available kits. An important fact is that RID is not influenced by the degree of desialylation of orosomucoid (8).

141

Electroimmunoassay

(EIA) according

to

LAURELL'S

proce-

dure (9)has been also frequently used for orosomucoid determination (10) (11). The performances of EIA are quite similar to those obtained by RID with a slight preference for EIA concerning sensitivity and accuracy and for RID concerning precision. The time needed

for EIA is shorter (about

4 hours). Usually, correlation

between

EIA

and RID

is excellent

but sometimes, as it has been stressed by BI0U and al. (8), a discrepancy between the two techniques may occur. When it is the case, results given

by

EIA

are

lower

than

those

obtained

by RID.

This

difference

between the two techniques is related to the presence of desialylated orosomucoid

which

presents

mobility compared to

a

pronounced

decrease

in

electrophoretic

native orosomucoid. So, RID appears more adequate

for estimation of both native and desialylated orosomucoid since diffusion and antigenic properties are not related to the sialic acid content. Modifications

of the classical EIA have been described

and for example a simultaneous determination of orosomucoid and ceruloplasmin has been performed (11). In that case, the antiserum mixture is usually prepared in such a way that the two proteins cause precipitation peak

at different

average peak height

; orosomucoid gives the higher

peaks and the precipitates of ceruloplasmin are stained by using oxidasic

properties

of

the protein. For

screening

purpose, three height

levels of precipitates derived from three antigen-antibody

systems may

be used. In the recent years, immunonephelometric

methods

have

been largely developped for specific proteins analysis including orosomucoid

measurement. Different commercially available systems have been

proposed using continuous flow analysis (AIP Technicon) or laser nephelometry (with Polyethylene glycol : PDQ Hyland or without : LN Behring) or

rate

nephelometry

sensitive and rapid

(ICS Beckman).

These

methods

are

more

precise,

(less than 1 hour) than RID. Besides,automation is

possible. Precision data we obtained by such a technique are shown in table I.

142

Table I : Precision of the orosomucoid immunonephelometric assay (n = 30 for each concentration, results were obtained using the LN - BEHRING nephelometer, module I)

Within-Run

Concentration

Low

Medium

Between-Run

High

Low

Medium

High

Mean g/1

0.35

0.82

1.68

0.35

0.80

1.66

SD g / 1

0.015

0.019

0.036

0.018

0.028

0.049

CV %

4.2

2.4

2.1

5.2

3.5

2.9

Correlation of laser nephelometry (y) with RID (x) is excellent (r = 0.989 ; y (g/1) = x (g/l)-0.013).

Immunoturbidimetric been

adapted

Rotochem

to

centrifugal

quantitation

analysers

such

as

(13). Advantages of these techniques

of

orosomucoid

Centrifichem

lie

in a good

has

(12) and precision

(within-run precision = 2 % (12) ), low cost and the ability to process large numbers of assays in short periods of time.

Some measurement gastric

in

techniques

medium

where

have

its

been

introduced

concentration

is

for

orosomucoid

usually

low

(i-e

juice, ascitic or pleural fluid...). A

solid-phase

enzyme

linked

immunosorbent

described by WANG and CHU (14) allows the detection of 4

assay

yig of orosomu-

coid per liter. a Radioimmunoassays

using

a

H

coid are able to detect 0,3 ng of orosomucoid

125 or

I-labelled orosomu-

(15) (16). LUDWIG and al.

(17) developped a very sensitive electroradioimmuno assay for measuring acid glycoprotein in gastric juice.

143

In conclusion, methods for orosomucoid determination are numerous ; so, the biologist can find a technique adapted to the problem he has to face (measurement of low concentration or desialylated forms, need for a quick method. ..). It is important to keep in mind that an orosomucoid measurement has a clinical usefulness only if results can be obtained

rapidly.

allow one

to

Immunonephelometric

obtain the

methods which

are

sensitive and

data within one hour seem to us particularly

well-suited.

Normal range and physiological variations of serum orosomucoid : orosomucoid in infants : In

newborns,

orosomucoid

levels

are

low

compared

to

adult levels

(18) (19) (20). The mean value of orosomucoid reported by

BIENVENU

al.

thereafter

et

(20)

in

full-term

infants

at

birth

the protein follows a rapid post-natal

is 0,18 g/1 ;

increase during the

first week of life (figure 1). • • • • • * 26-32 WEEKS

Figure 1 : Evolution of serum orosomucoid levels with postnatal and gestational age in normal infants (results obtained using LN-Behring).

144 At one month, orosomucoid levels are close to adult ones. Prematurity influences

orosomucoid

concentrations

since

values

are

significantly

lower at day 1 and 2 in the 26-32 weeks group than in full-term infants. These results are in agreement with the fact that despite of the very low

molecular

weight

of orosomucoid,

there

is no

transfer

of this

protein from mother to the foetus. (21) - orosomucoid in adults : * Biological variations of orosomucoid : The

biological

variations

of

orosomucoid

have

been

investigated by WINKEL et al. (22) in 12 healthy male volunteers. The average coefficients of variation for the within-day intra-subject variation,

for

the

day-to-day

intra-subject variation and for the inter-

subject variation were respectively 1.37 %, 11.1 % and 17.1 % (22) (23). No seasonal variation of orosomucoid has been found by LYNGBYE and KR0LL (24) who compared the levels of the protein in three winter and three summer months. * Sex and age influence : By use of a quantitative immunoelectrophoretic method, LYNGBYE and KR0LL (24) have evaluated serum orosomucoid in 260 normal subjects, 8 to 95 years old. The behavior of orosomucoid in males and females is significantly different

: the protein shows a tendency to

increasing values until the age of 30 in the male and after 40 in the female (figure 2). In the period 30-40 years, the levels are significantly higher in men than in women.

Figure 2 : Evolution of serum orosomucoid in adults from LYNGBYE and KR0LL (24)

145 These sex-associated differences are to relate to hormonal influence : * Hormonal influence : Cortisol (25,26) and anabolic hormones stimulate o r o s o mucoid

synthesis

(27).

orosomucoid

levels

about

(18).

20

steroid

%

On

(28). This

metabolism

the

other

During variation

during

hand,

pregnancy

this

has

decrease

serum

is

decreased

attributed

to

the

been

state,

oestrogens

orosomucoid

but

other

by

modified

alterations

such

as

variation in body fluids may infuence the homeostasis of this protein. Even

if the

alone

(29).

decrease It or

(30-33).

that

6 months

In

since after

progestogens gens

achieved more

+

case,

the et

of

cannot

be

explained by

that the administration of oral

LAURELL

a

oestrogens

(32).

after

level

oestrogens

use

to

alone

its

is known

(oestrogens

pregnancy

of

progestogens)

fall al.

induces

of orosomucoid

contraceptives

similar

changes

is greater than

(30) report a 36 % orosomucoid

megestrol-mestranol has no

Maximum

hemodilution

association.

effect on changes

influence

a 4 months therapy

the

Addition

induced by

contraceptive

of

oestro-

therapy

is

(33). The decrease of orosomucoid

is

important under ethinylestradiol

of

during

decrease

than under mestranol, w h i c h paral-

lels the estrogenic activity of these two compounds.

* Normal values in adults : The

following

reference

values

can

be

used

for

serum

orosomucoid levels : males : 0.40 - 0.90 g/1 females (without contraceptives)

: 0.35 - 0.80 g/1

Pathological variations of serum orosomucoid :

Diminution of serum orosomucoid : Circumstances

in

which

orosomucoid

is

decreased

are

rare : * liver

cancer,

synthesis

is

Severe

liver

disease

(decompensated

fulminans hepatitis). The decrease late

in

these

severe

liver

cirrhosis,

in orosomucoid

disorders

compared

hepatic

to

biological parameters (clotting factors, prealbumin for example).

other

146

* drug administration : Oestrogen administration (diethyl stilboestrol) induces an

orosomucoid

diminution

in

patients

with

prostatic

cancer.

In

addition, in these patients, the concanavalin A crossed immuno-affinoelectrophoresis shows a typical pattern associated to hormonal effect (34). This, prolonged oestrogen administration modifies the carbohydrate content of orosomucoid, and may alter the function of this protein. * nephrotic syndrome

:

Due to the low molecular weight of orosomucoid, there is an important urinary loss of this protein during nephrotic syndrome. Recently, STRAPRANS et al. (35) suggested that the defect in lipoprotein-lipase

which

induces

elevated

plasma

triglycerides

in

nephrotic

patients was related to the loss of a glycosamino glycan-0(^acid glycoprotein

complex.

The

glycosaminoglycan

associated

with

the

Ct^ acid

glycoprotein in urine appears to possess a cofactor function and can restore lipoprotein lipase activity in presence of C II apoprotein.

Elevation of serum orosomucoid : Clinical

studies reporting

orosomucoid

elevations

are

numerous ; they show that levels of this plasma protein are remarkably increased in patients with inflammatory or infectious diseases. The

surgical

trauma

provides

an

excellent

model

to

define the response times of orosomucoid comparatively to the other APR (36-39). As outlined by FISCHER et al. (39), during a non-complicated postsurgical response two sets of acute phase proteins can be defined : 1)

the

first

set

contains

CRP, oC^antichymotrypsin,

these

proteins are detectable within 6 hours after surgical trauma with a peak at 48 hours. 2) orosomucoid belongs to the second set of proteins with antitrypsin, haptoglobin, and ceruloplasmin.

147

They are detected 12-24 h after tissue injury with a maximum between

72-96 h.

The normalization

occurs

18-21

days after surgery

Figure 3 : Changes in plasma concentration of haptoglobin, orosomucoid and C^ - antitrypsin after cholecystectomy, adapted from ARONSEN et al. (37).

Orosomucoid is increased in various diseases : In malignant diseases, OC^ - acid-glycoprotein has been studied extensively cancer

(40,41),

(43),

large

especially bowel

in breast

cancer

cancer

(42),

(44), neuroblastoma

in cervical

(45), Hodgkin's

disease (46). Due to its non specificity, the interest of orosomucoid elevation in cancer diagnosis is poor. Nevertheless, in liver diseases it can be helpful since an important

serum orosomucoid increase is

highly suggestive of hepatoma (figure 4). The great value of orosomucoid lies in its sequential determination for the monitoring of cancer patients : it can help to define the stage of the malignancy, to appreciate a remission by its normalization, to

early

detect

a

recurrent

or metastatic

state

by

its

elevation

(figure 5) and to distinguish between infective and neoplastic state.

148 300 • A • •

250

A A 200-

A A

150-

i

• l

A f p

Hi* 100

so

A. A A A A A A

• •• • •

•

Lg....

••• •

• v : ••••

HEPATOMA C I R R H O S I S (36) (30)

•

• •. •

HEPATITIS (35)

Figure 4 : Orosomucoid in patients with hepatoma, cirrhosis and hepatitis. indicates reference range, from CHIO and OON (47).

Figure 5 : Disturbance of orosomucoid profile caused by recurrent tumour of the pelvis, from MILFORD WARD et al. (44).

149 It is important to know that a normal value for orosomucoid in a patient with cancer is of more clinical value than an elevated level. In Hodgkin's and non Hodgkin's lymphoma, CHILD et al. (46) conclude that a stable orosomucoid profile is characteristic of remission, that APR increase coincides with relapsed disease and that an instable profile is observed during relapsing disease or inadequate treatment. Some variants of

acid-glycoprotein have been described in

malignant diseases. ABEL and GOOD

(48) isolated from the urine of a

patient with Hodgkin's disease an orosomucoid with a lower sialic acid and a higher

fucose content than normal. RUDMAN et al. (49,50) showed

that orosomucoid

from

neoplastic

sera or

effusions

differ

from the

normal protein in its carbohydrate content.In 1977, BACCHUS (51) using polyacrylamide gel electrophoresis reported a typical profile in sera from patients with cancer : in these patients there is an increase of fractions with a medium mobility while in patients with inflammation an elevation of bands with slow mobility is observed (figure 6). ACRYIAMIDE Oil IlICTIOPHOtlSIS fATTlINJ MOlfCUlAlt WEIGHT COMPONENTS

M|

%

ruui cil- amtauaiK uum CIIMU

IMUMNMU

«TU UXIU «Util

Figure 6 : Acrylamide gel disc electrophoresis of seromucoid of normal subjects and of patients with neoplastic and non neoplastic diseases, from BACCHUS (51).

In

1978, PAPSIDERO

associated orosomucoid

et al.

(52) identified a breast tumor-

by use of concanavalin A - affinity chromato-

graphy ; this abnormal orosomucoid is tightly bound to Con A - Sepharose.

150

Orosomucoid than

serum

the

during

has

been

neoplastic

determination

of o(

-

investigated

states acid

in other

(17,53).

For

glycoprotein

is

biological

BARBOTTE more

detection of gastric cancers than the CEA measurement

fluids

et al.

useful

(15)

for

the

; the same authors

found a significant correlation between the two proteins. Orosomucoid is elevated

in

related

to

(54,55),

cancerous change

in

pleural

fluids

vascular

(53),

permeability

this

increase

(54).

For

could

several

be

authors

it seems difficult to distinguish a cancerous process from

inflammatory

one

by

orosomucoid

measurement

in

ascites

and

an

pleural

effusions.

Inflammatory Bowel diseases Inflammatory ulcerative

bowel

diseases

especially

Crohn's

diseases

colitis are characterized by a fluctuating course. So,

and there

is a need for objective determinations to follow the evolution of these diseases. authors reliable the

In

this

(56-58).

purpose, It

serum

is generally

orosomucoid has b e e n measured by concluded

many

that orosomucoid provides

indicator of disease evolution, this protein being elevated

beginning

of

the

disease

and

its normalization

occuring

with

a at

the

treatment efficiency.

Liver diseases

case

of

Orosomucoid

is generally normal

steatosis),

contrasting

(59-61). KINDMARK and LAURELL proteins

during

inoculation

with

in liver diseases

high

level

of

(except

in

O l ^ - antitrypsin

(62) described a typical profile of plasma hepatitis

with

increased Ol^- antitrypsin,

normal orosomucoid and decreased haptoglobin.

Rhumatoid disease Rhumatoid Serum

protein

KILLINGSWORTH orosomucoid the

arthritis

profile (65)

(RA)

is

is characterized

considered

that,

a

typical by

inflammatory

a rise

of nine

of

plasma

the

APR

proteins

of

drugs

in

the

treatment

of

the

RA

has

(63,64). measured,

was the most sensitive marker of disease activity.

efficiency

disease.

Besides,

often

been

151

supported studies

by

normalization

of

orosomucoid

levels

(66,67).

Experimental

in the r a t have shown that phenylbutazone and salicylate

a reduction of orosomucoid synthesis and KUTRYK presence

(68)

of

observed

that

concentrations

; using r a t liver slices,

synthesis

of

0,5

mM

was significantly

or greater

of

induced JAMIESON

decreased

in

phenylbutazone

or

concentrations of 2 mM or greater of salicylate.

Myocardial infarction : The cardial

sequential

infarction

are

changes

quite

of

orosomucoid

similar

observed

after

to the variations of this

myo-

protein

after surgical trauma. The maximal

increase is observed by about 5 days

and the

days

found

normalization

a

by

quantitative

about

21

relationship

infarct size. These modifications but

they have

no

creatine kinase

(69).

between

SMITH

and coworkers

orosomucoid

and

(70)

enzymatic

in orosomucoid levels are to be known

real diagnostic interest since enzyme studies such as - MB

isoenzyme are a m u c h more sensitive

indicator.

Infectious diseases : GANROT infectious

(71)

diseases

reported

in adults

an

important rise

(about

200

%

of

in orosomucoid

during

the normal value) and a

normal concentration after recovery. Increased orosomucoid values in newborns w i t h bacterial tions,

has

sensitive

been

indicator

in the neonatal orosomucoid of

148

found for

period

by the

G0T0H

et

al.

diagnosis

(19).

and

A need

for

infec-

a quick

the monitoring of

and

infections

is obvious. For this purpose, we measured

serum

in infants who were thought to have an infection. In a group

infants

with

confirmed bacterial

serum orosomucoid values

infections 1 1 8 , ( 8 0 %) had h i g h

(20).

Figure 7 shows the results obtained in full-term infants :

152

2,5-

1,5-

1•

0,5-

Day*

1 2

3-4

5-6

7-9

10-15

-fh 16-32

Figure 7 : Serum orosomucoid in bacterial septicemia meningitis and peritonitis (full term infants). O staphylococcus, A streptococcus, • E. Coli, • Proteus, • Klebsiella, Salmonella, • Listeria, 9 others, upper limit of reference values, from BIENVENU et al. (20) infants who died.

In our experience fibrinogen and IgM are less sensitive than orosomucoid

in early detection of bacterial

parasitological

infections. In viral and

infections serum orosomucoid did not prove to be very

useful since, in only 16 of the 30 cases studied, elevated values were observed.

153

Evolution of serum orosomucoid in eight cases of septicemia is presented in figure 8.

Figure 8 : Evolution of orosomucoid in 8 cases of neonatal from BIENVENU et al. (20)

septicemia,

Increase of the protein is very rapid after the beginning of with

infection

a maximum b e t w e e n 3 and 6 days after the onset of infection and a

normalization

after

about

7

to

10

days

since

onset.

In

complicated

cases, this normalization could need up to three weeks.

Several usefulness

peculiarities

must

of orosomucoid measurement

neonatal period :

be

pointed

in bacterial

out

to

explain

the

infection during the

154

In

the

neonatal

period,

the

increase

of

this

APR

is

generally related to an infectious process, which gives a certain degree of specificity to orosoraucoid increase.

The capacity to synthetize orosomucoid occurs early and elevation

of orosomucoid

is a very newborns

sensitive (so

it

is

which

can b e as h i g h as in adult

parameter

since

not

to

rare

normal

observe

values

values

the

inflammation

are very low in the

15

times

higher

than

normal).

The variation of orosomucoid level can help in the treatment of the infection by controlling its efficiency.

In our experience four infected infants with normal oid

values

died.

protective

role

synthetize

These

results

during

seem

infection

to

and

show

that

that

the

orosomuc-

orosomucoid

ability

of

has

liver

a to

large quantity of orosomucoid is an indicator of the capacity

of the newborn to fight infections.

Yore time.

Due

(increase

recently,

to of

their

CRP

is

we

measured

different more

CRP

and

response

rapid),

these

orosomucoid

times two

at

during

proteins

the

same

inflammation

allow

a

precise

monitoring of bacterial infection (72).

Conclusion

Orosomucoid acute

phase

usefulness perfectly must

be

contribute

can

be

considered

proteins particularly in detection

illustrates measured to

and

the

by

precise

a

in chronic

follow

clinical rapid

the

as

up

of

the

most

reliable

inflammatory diseases.

of neonatal bacterial

utilization

technique.

exact

one

Such

biological

especially the interaction with the immune system.

of

this

role

infections

protein

clinical of

Its

studies

which may

orosomucoid,

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37. Aronsen, K.F., Ekelund, G., Kindmark, C.O., Laurell C.B. : Scand. J. clin. Lab. Invest., 29, suppl 124, 127-136 (1972) 38. Wandall, J.H.

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Simonsen, E.

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58. Buckell, N.A., Lennard-Jones, J.E., Hernandez, M.A., Kohn, J., Riches, P.G., Wadsworth, J. : Gut, 20, 22-27 (1979) 59. Hallen, J., Laureil, C.B. : Scand. J. clin. Lab. Invest., 29, suppl. 124, 97-103 (1972) 60. Agostini, A., Marasini, B., Stabilini, R., Del Ninno, E. Pontello, M. : Clin. Chem., 20, 428-429 (1974) 61. Carlson, J., Eriksson, S. : Acta Med. Scand., 207, 79-83

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72. Bienvenu, F., Bienvenu, J., Chantin, Ch., Sann, L.: A comparative study of the sequential and coupled determination of CRP and orosomucoid during neonatal bacterial infections. Poster session "Symposium on inflammation Markers" Lyon 22-25 april, 1981.

ALPHAi ACID GLYCOPROTEIN - STRUCTURE, GENETICS AND BIOLOGICAL SIGNIFICANCE

Philippe Arnaud and Elisabetta Gianazza Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29I+25

Introduction

Alphas - acid glycoprotein is present in plasma and several biological fluids.

The state of the art on this protein has been recently reviewed by

K. Schmid in 1975 (l)*

The aim of the present review is to discuss some

new data concerning the structure and genetics of A]_AGP and to extend them to the possible role of this protein as a modulator of immunological parameter^. Structure of A]_AGP The structure of A]_AGP has been extensively studied by K. Schmid and coworkers.

The complete amino acid sequence was determined in 1973, after

isolation and purification of the protein from Cohn fraction VI of pooled human plasma obtained from 2,500 donors (2).

The purification process

involved a Cohn fractionation followed by ion exchange chromatography (3,1*).

A^AGP structure was characterized by two unusual features:

at

least 21 amino acid substitutions could be detected, and a significant degree of homology with the variable region of the K-type light chain of human immunoglobulins was demonstrated.

Previous work of the same labora-

tory had shown homology between the carboxy-terminal part of the molecule and the constant region of the heavy chain of immunoglobulins (5).

From

these data, the authors postulated a relationships between A^AGP and the ancestral immunoglobulin. Other procedures for the purification of A]_AGP based upon ion exchange chromatography have been reported (6).

Preparative isoelectric focusing

M a r k e r Proteins in Inflammation © 1982 by W a l t e r de Gruyter & Co., Berlin • N e w Y o r k

160 (7) allows for the isolation of the protein from small amounts of plasma, thus permitting to purify A^AGP from single donors (see Fig. l).

Fig. 1. Microheterogenity and polymorphism of A ^ A G P . A ^ A G P was purified from plasma of single individuals according to ref. 7 and analyzed by thinlayer isoelectric focusing in a pH gradient 2.5-5 (anode at the top). Note the microheterogeneity (at least 7 bands in each individual preparation) and the difference in migration of the whole pattern (polymorphism). The sugar content of AqAGP has been extensively studied.

Indeed, this

protein is characterized by an exceptionally high carbohydrate content, which corresponds to 65% of the total weight of the protein. studies [reviewed in (l)] indicated that

A^AGP

Earlier

possessed five carbohydrate

units linked to asparagine residues of the protein moiety.

Recent work

from Schmid's laboratory characterized ten different peptides carrying carbohydrate chains (18).

Five of them corresponded to different parts of

the molecule while five others were due to amino acid substitutions, some of them already reported (2) and some of them new, thus bringing the total amino acid substitutions of

A^AGP

to at least 23.

Taking into account

variations in the protein moiety, 1 6 different glycopeptides could be distinguished

(9)«

By nuclear magnetic resonance spectroscopy, five

classes of heteroglycans were detected:

class A possesses a bi-, class B a

tri- , and class C a tetraantennary structure.

Two other structures were

161 characterized by an additional fucose residue linked to the B(BF) and the C(CF) chains.

Carbohydrate units with different structures can be linked

to the same glycosylation site on the peptide chain.

As an example,

glycosylation site I can accept either A, B or BF carbohydrate units (10). Finally, the ability of the penultimate galactose to accept terminal sialic acids was shown to vary according to its type of linkage to the other components of the sugar chain, indicating a high degree of specificity of the siayltransferases (ll). This raises the question of the importance of sialic acids for the charge of A^AGP molecule.

It was postulated earlier that the large number of

sialyl residues in the molecules was responsible for the acidic isoelectric point of A^AGP.

Sialic acid represent approximately 11% of the molecule,

which should correspond to 16 residues of sialic acid/mole of glycoprotein. AjAGP exhibits a large degree of microheterogeneity, and current hypotheses attribute a major role in the differences in charge exhibited by the different isotypes of several plasma glycoproteins to uneven sialylation. We have studied (j) the role of sialic acid in the microheterogeneity of A]AGP obtained from different individuals.

After complete desialylation,

the microheterogeneity of the protein was still present, indicating that it was not due to differences in sialic acid content of the different protein bands.

These results have been confirmed recently (12).

The heterogeneity of A^AGP with respect to differences in the carbohydrate content has been demonstrated recently indirectly by Wells et al. (13) who studied the interaction of the protein with Concavanalin A (ConA) by crossed immunoaffinoelectrophoresis.

They showed the presence of three

A^AGP fractions, one of which was not retarded by ConA, while the two others were retarded to a different extent.

Another important point of

this study is that quantitative variations in the distribution of the variants were modulated by factors such as pregnancy and estrogen treatment, which increase the overall synthesis of A]AGP.

On the other hand,

Micollet et al. (lU) isolated two populations of A^AGP by chromatography on immobilized ConA.

The first peak does not bind to the lectin column and

represents the major part of the protein.

In addition, a slight charge

difference between the two fractions could be observed by analytical isoelectric focusing.

Crossed immunoaffinoelectrophoresis confirmed the

findings of Wells et al. (13), but, in contrast with their results,

162 Nicollet et al. (l4) found that during inflammation, the percentage of ConA-reactive A]AGP was clearly increased.

Keeping in mind the fact that

both pregnancy" and inflammation increase the plasma concentration and synthesis rate of A]_AGP, these two publications suggest that the glycosidic structure of A]_AGP produced in either condition is different.

In view of

the known requirements for the binding of mannosyl-containing glycans to ConA (15), it is likely that the ConA reactive material should possess relatively simple chains (type A) when the other populations should correspond to more complicated structures (type B and C).

Therefore,

pregnancy and estrogen treatment would result in increased synthesis of A^AGP possessing mostly tri and tetraantennary structures, when, in contrast, acute-phase reaction would increase the synthesis of biantennary AqAGP. The biosynthesis of the sugar moiety of AQAGP has been studied in rats (16-18).

It appears to follow the pattern described for glycoproteins

(19-21).

After release from ribosomes, the polypeptide chain is processed

by the rough endoplasmic reticulum.

A lipid-linked oligosaccharide unit is

transferred from the carrier to the asparagine of the peptide chain. oligosaccharide contains N-acetylglucosamine, mannose and glucose.

This The

glucose and excess mannose are then processed in the smooth endoplasmic reticulum, where additional N-acetylglucosamine and galactose are added. The terminal sialic acids are then added in the smooth endoplasmic reticulum and/or in the Golgi apparatus. The glycosidic moiety of A]AGP plays a major role in its catabolism. Regoeczi et al. (22) have shown in rats that the major catabolic pathway is the one postulated by Ashwell and Morell (23).

Exposure of the penultimate

sugar, galactose (upon removal of the terminal sialic acid) promotes the binding of the protein to a specific membrane-bound hepatic lectin, followed by endocytosis.

The kidney appears also to contribute to the

catabolism of asialo A^AGP (22-24).

Genetics of A]AGP Although the so-called "polymorphism" of A^AGP was demonstrated as early as 1962 (4) by starch gel electrophoresis of pooled plasma, and that

163 differences in the primary or tertiary structure (25), the possibility that different individuals could present individual variants was first proposed in 1964 ( 2 6 ) . this study.

One hundred and twenty seven individuals formed the basis of After purification, A ] A G P was studied by starch gel

electrophoresis.

Results indicated that each sample could be resolved in 5

t o 8 bands.

Family studies suggested that the pattern was genetically

determined.

Further studies indicated that this genetic polymorphism

appeared more clearly after desialylation of the protein (27-28).

Finally,

clear-cut demonstration of the genetic inheritance of the asialo-patterns w a s demonstrated on whole serum by specific immunofixation and crossed Immunoelectrophoresis in 216 individuals.

Studies performed in several

families indicated this mode of inheritance to be autosomal codominant. The two genes, called F and S, produced three phenotypes, two homozygous (FF and SS) and one heterozygous

(FS).

Study of the gene frequencies

in

various ethnic groups suggested significant differences related to the ethnicity, with a relative scarcity of the S gene in Japanese. additional, points of interest came from this study:

Two

the suggestion that

b o t h structural genes could be present in all individuals, and the absence of a genetic control of serum concentrations of A^AGP by the genes W e have also studied A ^ A G P polymorphism by thin-layer isoelectric in polyacrylamide gels followed by print immunofixation.

focusing

The protein shows

b o t h microheterogeneity and polymorphism, the latter being under control.

(29).

genetic

In addition, additional "subtypes" can be recognized, the

existence of which were ascertained by family studies (30) (see Fig. l).

Biological role of AjAGP

A^AGP is an acute-phase reactant protein and as such its plasma levels increase in response to a number of stimuli, including infection,

inflam-

mation, and several disease states such as malignant cell proliferation. For the latter, levels of A^AGP have been proposed to assess and/or monitor development of malignancy, but this test does not appear to be reliable (31).

The mechanism by which A]AGP is increased, during acute-phase

reaction, is still unknown. synthesis by liver cells.

It certainly corresponds to an increased Anti-inflammatory agents appear to possess a

direct negative effect on A ^ A G P synthesis (32).

Interestingly, in rats, it

164 was shown that the half-life of asialo-A]AGP, but not of native A]_AGP, was increased during experimental acute-phase, suggesting that, during this process, there could be production of incompletely sialylated protein which would compete for the membrane receptors responsible for the clearance of asialo-forms (33). The exact biological role of A^AGP is still not fully understood.

In

plasma, this protein binds a number of ligands, including cationic drugs such as propanolol and chlorpromazine (3*0, quinidine (35), imipramine and alprenolol (36), lidocaine (37), perazine (38), methadone (39) > etc.

As

with many drug-binding proteins, the protein-ligand interaction has two consequences:

it modifies the half-life of the drug, through alteration of

its distribution volume, hepatic and renal clearance, and it also decreases the free part of the ligand, normally the only one accessible to tissues, preventing a possible toxic effect.

This represents an important parameter

with respect to drug disposition and pharmacokinetics. hormones bind A^AGP (UO-Ul).

Similarly, steroid

Although this appears of minor physiological

significance for sex steroids [(because of the high affinity of steroidbinding ^-globulin and the high capacity of albumin (^2)] it could affect more significantly other steroids such as progesterone C*l), although its affinity is lower for A]AGP than for corticosteroid-binding globulin.

In

addition, several experiments have shown that A^AGP is able to inactivate heparin (1*3), which can be of significance when anticoagulant treatment is performed during acute-phase reactions.

A]AGP has been found to inhibit

platelet aggregation induced by several agents (1+4-45), but the exact physiological significance of this property remains to be determined.

In

this respect, the asialo-form of A^AGP is more effective than the native molecule (45).

In addition, A]AGP interacts with collagen, and Franzblau

et al. (l) speculated a role for this protein in the regulation of the formation of striated collagen. One of the most promising properties of A]AGP is represented by its possible effect on the immune response.

It must be kept in mind that A^AGP

possesses a significant degree of homology with immunoglobulins (12).

In

addition, A]AGP can be expressed on the surface of human lymphocytes (46). An interaction of A^AGP with the immune system was first reported by Chiu et al. C*T).

The protein was found to markedly inhibit at physiological

concentrations, proliferation of lymphocytes in response to several mito-

165 gens, including Concanavalin A, phytohemagglutinin and pokeweed mitogen, as well as the responding cells in mixed lymphocyte culture.

Together with

these data, inhibition of T- and not B-cell rosette formation suggested that the inhibition by A]_AGP was T-cell mediated.

Further evidence

suggested the effect of A]_AGP to be confined on a T-cell lymphocyte subset. In contrast, AjAGP was shown to promote growth of several cell lines, including fibroblasts, HeLa cells, and lymphoblastoid cell lines (1»8). This effect, which was obtained for low concentrations of A]AGP, was not abolished by sialic acid removal. The role of A]AGP as a regulator of the immune response was recently reexamined by Bennett and Schmid C*9). cells as effectors.

These authors used mouse spleen

In agreement with Chiu et al. (UT) they found that

A^AGP inhibited lymphocyte mitogenesis and mixed lymphocyte reaction. addition, it suppressed the antibody response to sheep erythrocytes. contrast with data reported by Maeda et al.

In

(W3), A3AGP did not affect the

tumor cell proliferation of a lymphoma cell line (EL-I4). did not inhibit natural killing.

In

Finally, A]AGP

We found no effect of human Aj_AGP neither

on natural killing nor in antigen dependent cell-mediated cytotoxicity, using human T lymphocytes as effector cells, therefore confirming and extending the above findings (50).

One of the most original points of

Bennett and Schmid's paper was to study the effects of AjAGP with modified sugar chains.

Often these derivatives were more active as immunoregulatory

agents than the native molecule.

This could be explained on the basis of

specific sugar interactions with the cell membrane, or, alternatively, by a better access of a specific peptide sequence to a membrane receptor. In conclusion, as far as its structure is concerned, A]AGP represents one of the best characterized plasma proteins.

Not only its complete sequence

has been established, including several amino acid substitutions, but its glycosidic moiety has been extensively studied.

Its polymorphism, although

conclusively established, is still not commonly used for genetic studies, and no disease associations have been reported in relation to its genetic variants.

In striking contrast, the exact biological role of this protein

is still not elucidated.

Nevertheless, recent evidence strongly suggests

that AxAGP plays an important role as a modulator of the immune response, both at the level of IgG production and on parameters of cell-cell inter-

166 actions.

The respective importance of the peptide backbone and of the

glycosidic moiety in these interactions remains to be elucidated. Acknowle dgment s Elisabetta Gianazza is a postdoctoral fellow of the College of Graduate Studies, Medical University of South Carolina, on leave of absence from the Department of Biochemistry, University of Milano, Via Celoria 2, Milano 20133, Italy.

Publication no. 451 from the Department of Basic and

Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina.

Research supported in part by USPHS Grants

HD-09938 and CA-2571+6, and by General Medical and Faculty Research Appropriation Grant A-301.

We thank Charles L. Smith for excellent

editorial assistance. References 1.

Schmid, K., in the Plasma Proteins, vol. I (Ed. by F.W. Putnam), Academic Press, New York, 1975, PP- 183-228.

2.

Schmid, K., Kaufmann, H., Isemura, S., Bauer, F., Hnura, J., Motoyama, T., Ishiguro, M., and Nanno, S. , Biochemistry, 12, 2711-2721+ (1973).

3.

Burgi, W. , and Schmid, K. , J. Biol. Chem. , 236, 1066-1071» (l96l).

1». Schmid, K. , Binette, J.P., Kamiyama, S. , Pf ister, V., and Takahashi, S., Biochemistry, 1, 959-966 (1962). 5.

Ikenaka, T., Ishiguro, M. , Emura, J., Kaufmann, H., Isemura, S., Bauer, W., and Schmid, K., Biochemistry, 11, 3817-3829 (1972).

6.

Bezkorovainy, A., Biochim. Biophys. Acta, 101, 336-3^2 (1965).

7.

Arnaud, P., Gianazza, E., Righetti, P.G., and Fudenberg, H.H., in: Electrophoresis 79 (Edited by B.J. Radola) Walter de Gruyter, Berlin, New York, pp. I5I-I63 (1980).

8.

Schmid, K., Nimberg, R.B., Kimura, A., Yamaguchi, H., and Binette, J.P., Biochim. Biophys. Acta, U92, 291-302 (1977).

9.

Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P., and Schmid, K., Biochemistry, 17, 5206-521U (1978).

167 10. Schmid, K., Binette, J.P., Dorland, L., Vliegenthart, J.F.G., Fournet, B., and Montreuil, J., Biochim. Biophys. Acta, 5 8 1 , 356-359 (1979)• 11. Eijnden, D.H., Joziasse, D.H., Dorland, L., Van Halbeek, H. , Vliegenthart, J.F.G., and Schmid, K., Biochem. Biophys. Res. Commun., 92, 839-81+5 (1980). 12. Berger, E.G., ifyss, S.R., Nimberg, R.B., and Schmid, K., Hoppe Seyler's Z. Physiol. Chem., 3 6 1 , 1567-15762 ( 1 9 8 0 ) . 13* Wells, C., Bog-Hansen, T.C. , Cooper, E.H., and Glass, M.R., Clin. Chim. Acta, 109, 59-67 (1981). ll+. Nicollet, I., Lebreton, J.P., Fontaine, M., and Hiron, M. , Biochim. Biophys. Acta, 668, 235-21+5 ( 1 9 8 1 ) . 15. Goldstein, I.J., Hollerman, C.E., and Smith, E.E., Biochemistry, 1+, 876-883 (1965). 16. Jamieson, J.C., Can. J. Biochem., 55, U08-I+IU (1977). 17. Friesen, A.D., and Jamieson, J.C., Can. J. Biochem., 5 8 , 1101-1111 (1980). 18. Nagas-hima, M. , Urban, J., and Schreiber, G. , J. Biol. Chem., 255, 1+951-1+956 (1980). 19. Turco, S.J. , and Robbins, P.W. , J. Biol. Chem., 25I+, 1+560-1+567 (1979). 20. Hubbard, S.C., and Robbins, P.W., J. Biol. Chem., 25l+, I+568-I+576 (1979). 21. Varki, A., and Kornfeld, S. , J. Biol. Chem., 255, 1081+7-10858 ( 1 9 8 0 ) . 22. Regoeczi, E., Debanne, M.T., Hatton, M.W.C., and Koj, A., Biochim. Biophys. Acta, 5I+I, 372-381+ (1978). 23. Ashvell, G. , and Morell, A. G. , Adv. Enzymol. , 1+1, 99-128 (197!+). 2l+. Shibata, K. , Okubo, H. , Ishibashi, H. , Tsuda-Kawamura, T. , and Yanase, T., Brit. J. Exp. Pathol. 59, 601-608 ( 1 9 7 8 ) . 25. Marshall, W.E. , J. Biol. Chem., 2l+l, 1+731-1+737 (19 66). 26. Schmid, K., Binette, J.P., Tokita, K., Moroz, L., and Yoshizaki, H., J. Clin. Invest., 1+3, 231+7-2352 (I96I+). 2 7 . Schmid, K., Tokita, K., and Yoshizaki, H. , J. Clin. Invest., 1+1+, 1391+-11+01 ( 1 9 6 5 ) . 28. Hunziker, K., and Schmid, K. , Anal. Biochem. 20, 1+95-501 ( 1 9 6 7 ) .

168 29» Johnson, A.M., Schmid, K., and Alper, C.A. , J. Clin. Invest., 1*8, 2293-2299 (1969). 30. Arnaud, P., Gianazza, E., and Fudenberg, H.H., Fed. Proc., 39» 3U25A (1980). 31. Chaimoff, C., Shaked, P., and Lubin, E. , Israel J. Med. Sei., 16, 863-861+ (1980). 32. Jamieson, J.C., and Kutrik, M. , Biochem. Med., 23, 293-301 (I98O). 33. Wong, M.W.C. ,• and Jamieson, J.C., Life Sei., 25, 827-831* (1979). 3l+. Piafsky, K.M., Borga, 0., Odar-Cederlof, I., Johansson, C., and Sjoqvist, F., N. Engl. J. Med., 299, l ^ - l ^ (1978). 35* Fremstad, D., Bergervd, K., Haffner, J.F.W., and Lunde, P.K.W., Eur. J. Pharmacol. 10, UUl-UUU (1976). 36. Borga, 0. Piafsky, K.M., and Nilsen, O.G., Clin. Pharmacol Ther. 22, 539.51+1+ (1977). 37* Routledge, P.A., Barchowsky, A. , Bjornsson, T.D., Kitchell, B.B., and Shand, D.G., Clin. Pharmacol. Ther., 271, 31+7-351 (1980). 38. Schley, J., Siegert, M., and Muller-Oerlinghausen, B., Eur. J. Clin. Pharmacol. 18, 501-501+ (1980). 39. Romach, M.K., Piafsky, K.M., Abel, J.G., Khouw, V., and Sellers, E.M., Clin. Pharmacol. Ther. 29, 211-217 (l98l). 1+0. Ganguly, M., Carnighan, R.H., and Westphal, U., Biochemistry, 6, 2803-28114 (1967). 1+1. Kerkay, J., and Westphal, U., Biochim. Biophys. Acta, 170, 321+-333 (1968). 1+2. Abramovich, D.R., and Towler, C.M. , J. Steroid Biochem. 9, 791-791+ (1978). 1+3- Andersen, P., and Godal, H.C. , Haemostasis, 6, 339-31+6 (1977). 1+1». Synder, S., and Coodley, E.L., Arch. Internal. Med., 136, 778-781 (1976). 1*5- Costello, M., Fiedel, B.A. , and Gewürz, H. , Nature 281, 677-678 (1979). 1+6. Gahmberg, C.G., and Andersson, L.C., J. Exp. Med., 148, 507-521 (1978). 1+7* Chiu, K.M., Mortensen, R.F., Osmand, A.P., and Gewürz, H. , Immunology,

32, 997-1005 (1977).

169 U8. Maeda, H., Murakami, 0. , Kann, M., and Yamane, I., Proc. Soc. Exp. Biol. Med., 163, 223-227 (1980). 1»9. Bennett, M. , and Schmid, K. , Proc. Natl. Acad. Sei. USA, 77, 6109-6113 (1980). 50. Ades, E.W., and Arnaud, P., Unpublished data ( 1 9 8 1 ) .

ALPHAi-AliTITRYPSIN S T R U C T U R E A N D

GENETICS

P h i l i p p e A r n a u d , C. C h a p u i s - C e l l i e r , and E.

Gianazza

D e p a r t m e n t of B a s i c a n d C l i n i c a l I m m u n o l o g y and M i c r o b i o l o g y , M e d i c a l U n i v e r s i t y o f S o u t h C a r o l i n a , C h a r l e s t o n , South C a r o l i n a 29k2$.

Introduction

Alpha^-antitrypsin

(A^AT)* r e p r e s e n t s t h e major p r o t e a s e inhibitor

e x t r a c e l l u l a r fluids (l).

in

I n d i v i d u a l s g e n e t i c a l l y deficient in this

pro-

t e i n a r e p r e d i s p o s e d to a series of clinical d i s o r d e r s , of w h i c h the c l a s s i c a l feature is r e p r e s e n t e d b y p r e c o c i o u s p u l m o n a r y e m p h y s e m a K n o w l e d g e of the s e v e r a l d i s e a s e a s s o c i a t i o n s

(2).

recently r e p o r t e d w i t h A ^ A T

d e f i c i e n c y has p r o m p t e d e x t e n s i v e r e s e a r c h in two a r e a s , namely

structural

s t u d i e s to d e t e r m i n e t h e d i f f e r e n c e s b e t w e e n common a n d v a r i a n t

products,

a n d g e n e t i c studies to d e l i n e a t e t h e different alleles and a t t e m p t to c l a r i f y their mode of t r a n s m i s s i o n .

B o t h a s p e c t s of this w o r k are

comple-

m e n t a r y a n d t h e a i m o f t h i s r e v i e w is to summarize recent d a t a in this field.

The c l i n i c a l m a n i f e s t a t i o n s a s s o c i a t e d w i t h A]_AT d e f i c i e n c y

b e e n reviewed elsewhere

have

(3).

S t r u c t u r e o f A-|AT

Since t h e first d e s c r i p t i o n of t h e i s o l a t i o n a n d p u r i f i c a t i o n of A ^ A T

(4),

s e v e r a l d e t a i l e d reports have shed light o n t h e p h y s i c o c h e m i c a l

charac-

t e r i s t i c s of this p r o t e i n .

paralleled

The p r o g r e s s of their k n o w l e d g e has

t h e p r o g r e s s in its p u r i f i c a t i o n .

Indeed, A ^ A T r e p r e s e n t s only 3% o f t h e

p l a s m a p r o t e i n s , and in a d d i t i o n it is d i f f i c u l t to s e p a r a t e from a number

* A l p h a ^ - a n t i t r y p s i n , n a m e d a c c o r d i n g to S c h u l t z e (4), is the t e r m u s e d b y m o s t g e n e t i c i s t s and c l i n i c i a n s . In c o n t r a s t , b a s i c r e s e a r c h e r s appear to p r e f e r , a c c o r d i n g to the s u g g e s t i o n of P a n n e l l et al. (5), the t e r m a l p h a s - p r o t e a s e (or p r o t e i n a s e ) i n h i b i t o r .

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1 9 8 2 b y W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

172 of proteins which share similar size and/or charge. is presented in its sensitivity to denaturation.

An additional problem

Major progress in the

purification procedures has been provided by affinity chromatography, which uses several ligands, either nonspecific [lectins (6), Thiol-disulfide interchange (7), metal chelate (8), dye-ligand (9)1, or specific.

The

latter uses direct binding of A^AT on immobilized antibodies (10), or subtraction of the contaminants by antibodies directed towards them (11,12). The purified protein possesses a molecular weight of approximately 53,000, a sedimentation coefficient of 3.35, and an isoelectric point between lt.lt and It.7 (l). laboratories.

Its amino acid composition has been determined in several A]_AT contains relatively large amounts of glutamic (V2$) and

aspartic (12%) acid; this partly accounts for its mobility as an alphasglobulin, and its acidic isoelectric point.

The content in cysteine is

still a matter of controversy, varying between 1 and U, in different reports with an average value of 2.

This value raised the possibility of

the presence of one disulfide bridge inside the molecule.

This is an

important point, because of the stabilizing role of disulfide bridges on the configuration of proteins.

Jeppson et al. (13) contended that a second

free cysteine could be bound to a single residue of the protein core through formation of a mixed disulfide.

The latter can be removed by

oxidation, but even when using this technique, different laboratories report discrepant data (13-15)In contrast with previous results indicating that the NH2~terminus of A^AT was blocked, more recent investigations have determined the NH2~terminal sequence of A^AT.

The terminal amino acid is a glutamic acid, and the

sequence has been determined up to residue no. It5 (l6).

An important point

is there appears to exist some heterogeneity at the NH2~terminus of A^AT (see Table l).

In particular, in four documented studies (llt,l6-l8), resi-

due no. 10 in the M variant is found to be Glu in two cases, and Lys in two others.

This is important, because the substitution Glu-Lys (see below)

modifies the charge of A]AT significantly and would lead to isoelectric focusing patterns clearly different, which does not appear to be the case. If confirmed, these results would imply the existence of balanced substitution in another domain of the molecule, which is a feature only

173 Table 1. Sequence data on the Ni^-terminal part of the A]AT molecule8-

5 1(b)

N

H

E

2

D

P

Q

10 G

D

A

A

Q

2(c)

E

3(d)

K

Me)

K

a

E

15 T

D

T

S

H

H

D

The one-letter code is used for designation of amino acids.

From references 14(b), 70(c), 16 (d), and 18(e).

reported in the case of hemoglobin variants.

In addition, the sequence of

the 152 COOH-terminal amino acids of A^AT has been determined (see below). A^AT is fi glycoprotein, and its total carbohydrate content reported in the literature varies between 11 and 18%.

It contains N-acetyl glucosamine,

mannose, galactose and N-acetyl neuraminic acid (19).

Each molecule of

A]AT would accept 3 to 4 sugar chains, each linked to an asparagine residue of the protein core.

In addition, one type of chain appears to be

biantennary, and the other triantennary (20,21).

The ratio between these

different chains in the molecule has been found different in different laboratories (20-21+). When studied by electrophoretic systems with high resolution, such as starch gel electrophoresis in discontinuous buffers or isoelectric focusing (26,27), A^AT exhibits microheterogeneity.

Each homozygous phenotype is

composed of up to 8 bands (isotypes) of which the most important are represented by bands 4 and 6, according to the classification of Fagerhol

(28).

It is important to note that the distribution of A]AT microheterogeneity is identical from an individual to another within the same phenotype. Furthermore, the microheterogeneity of different homozygous phenotypes is identical (29), with only two established exception, the I and the P allele products (30-32).

For I, band 6 is weak, and for P, band k is weak.

In

addition, the microheterogeneity varies in two circumstances, acute-phase reaction, and at birth.

In the latter, bands 1+ and 7 are practically

174 absent, and the adult pattern is established only after a few weeks of life (33,34).

Only one case of adult with a "fetal" microheterogeneity has been

reported in the literature (35)» The basis of A]AT microheterogeneity is still a matter of controversy.

A

classical hypothesis to explain the microheterogeneity of glycoproteins is that it is due to uneven sialylation of the different isotypes.

Complete

desialylation of A^AT, which should, in this hypothesis, lead to the disappearance of thèse isotypes, with reversion to a single band, has given contradictory results (13,36-39).

In our laboratory, desialylation of

several homozygous phenotypes has always led to a heterogeneous pattern (40).

Vaughan and Carrell (24) have recently shown that the isotypes of

A]AT can be separated in two groups (I and II) by their binding properties to Concanavalin A.

They concluded, on the basis of structural analysis of

the sugar chains of the two groups, that type I possesses biantennary sugar chains, when type II possesses both bi-and triantennary structure.

This

could explain in part the microheterogeneity of A]AT and also would explain the "fetal" pattern on the basis of a temporary deficiency of tri-antennary side chain branching mechanisms. Other reasons are necessary to explain completely the microheterogeneity of A^AT.

They could involve post-secretory modification such as acetylation

and deamidation (2*0.

They could also be due to amino acid substitutions

in the polypeptide chain of the A]AT molecule (4l). A-|AT polymorphism The existence of a genetic polymorphism of A]_AT was first demonstrated on the basis of a different migration of A]AT from deficient individuals (further recognized as Z A^AT) upon agarose electrophoresis (42). study of

AQAT

But the

polymorphism was really initiated by the demonstration by

Fagerhol and Braend (25) of different migration patterns of A^AT phenotypes following electrophoresis of plasma on acid-starch gel electrophoresis. This polymorphism, referred to by the symbol Pi (for protease inhibitor) could also be demonstrated by agarose-gel electrophoresis followed by immunofixation (43) and by agarose-acrylamide electrophoresis (44).

A major

breakthrough in the study of A]_AT polymorphism was represented by the

175 introduction in 19lb of the analysis of Pi variants by thin-layer isoelectric focusing

(PAGIF) (26,27)-

In a first step, it was shown that

the polymorphism of A]_AT as resolved by PAGIF was identical to that demonstrated by starch gel (U5).

Further developments of the PAGIF

technique, and especially separator isoelectric focusing

(U6), allowed the

demonstration that the most common M phenotype could be subdivided into a series of subtypes.

At least 5 subtypes have been identified so far.

As a

consequence, individuals that possessed a variant phenotype from the common M have increased from less than 10% to up to 50%.

This greatly

increases

the genetic information that can be obtained by Pi typing and has a considerable importance in disputed paternity and forensic medicine.

Table 2

lists the Pi alleles demonstrated so far on the basis of isoelectric focusing and family studies. It indicates that A]_AT is the most polymorphic protein in human plasma.

Finally, it must be pointed out that some

phenotypes present an increased sensitivity to heat and/or pH. case for Msanfrancisco b e identical.

This is the

(kj) a n d Msalla (U8), although the two alleles could

These observations are of great importance, because these

variants cannot be detected by classical techniques based upon immunological or biological determination of A]_AT and would need population screening on a larger scale using changes in temperature and p H variations. It is possible that the instability of these variants affects also their ability to bind proteases "in vitro", therefore opening the field of functional defects of A]_AT. In addition to its role as a genetic marker, A^AT possesses a number of interesting genetic features.

First, some alleles are associated with a

decrease of plasma A]_AT concentration (Table 3). can be distinguished. the S a n d I allele. lower than normal.

Three groups of alleles

Those responsible for mild A^AT deficiency

include

The A]_AT values associated with them are about 205» The basis for this deficiency is still unknown, but, at

least for the S allele, cannot be explained on the basis of an increased catabolism of the protein (49). tion controlled by a "lazy" gene.

It is probably due to a low hepatic

secre-

A second group is responsible for

severe

A]AT deficiency and includes the Z allele, the P allele and the recently reported MMalton and MDuarte alleles. are only 10-30% of normal (50).

Values associated with these alleles

As discussed in the next section, all the

severely deficient alleles appear to be associated with A^AT retention

176 Table 2. Pi alleles classified according to their identification on isoelectric focusing. MUnstable and MSan Francisco (46,1+7) present a thermal instability. Data from refs. 48,82,83 and unpublished observations. Mx M5 m3

Balhambra Bsaskatoon

51

s3

52 Sl4 T V W Wgazak Wtoulouse Wconstantine Wsalerno

M2 Msalla Mchapel Hill Mmalton Mduarte Munstable Msanfrancisco N Nhampton Nlyon Nletrait P Psaintlouis Pbudapest Pkyoto R

D E Elemberg Ecincinnati Etokyo E2 F "F" G Gcler 12 L Lvibeuf

X

Y Z Zcathodal Zpratt Z2

Table 3. Alpha -antitrypsin expression associated with deficient alleles. Mean values for the M allele were considered as 100%. From refs. 31 and 50 and unpublished data. Pi allele

% expression

M S I P Z Mmalton Mduarte Hull

100 72.7 68.3 34.9 26.3 10-20 5-10 0

Serum

AqAT

Normal Moderately deficient Moderately deficient Severely deficient Severely deficient Severely deficient Severely deficient Absent (or trace)

177 within the liver cells.

This retention could be responsible, through a

feedback mechanism, for the decreased synthesis of the protein ( 5 1 ) .

.A

third catergory of A]_AT deficiency occurs in individuals possessing the "null" allele, responsible for levels of A]AT not detectable by usual techniques ( 5 2 ) .

Nevertheless, it has been shown that, by radiolabeled

techniques, traces of about 5 ng/ml of the protein can be demonstrated.

It

is apparently identical to normal A]AT by migration on starch gel ( 5 3 ) -

In

addition, at least in the few cases studied, no liver A]_AT retention appears to be associated with the possession of the "null" gene

(53,5^)-

An additional point of importance in the genetics of AjAT is the study of its linkage relationship.

The only genetic marker shown to be linked to

A^AT is the Gm allotypic system of immunoglobulin heavy chains

(55).

Interest comes from the demonstration that normal alleles (including M subtypes) are linked to the Gm markers in both sexes in an identical fashion, with recombination fractions around 0 . 2 9 in both sexes ( 5 6 ) . the contrary, for the Z and S alleles ( 5 7 , 5 8 ) , according to the sex.

To

the linkage appears to vary

No clear difference is seen in females; in contrast,

in males there is a strong evidence for closer linkage.

This allele-

specific effect on the recombination fractions in males, which represents a unique feature in mammalian genetics, is not yet explained, although several hypotheses have been proposed

(56,57).

Finally, in two laboratories, a preferential transmission of the Z deficient allele from the father to the offspring has been shown (59560). Further work in other laboratories has given various results, some

(61,62)

not showing this trend, in contrast, others being in agreement with this finding ( 5 6 ) .

Apart from biases of selection, these discrepancies could be

explained by ethnic variations.

Indeed, the distribution of Pi phenotypes

varies considerably according to the ethnicity.

For instance, there is

scarcity of Pi variants in individuals of Black or Asian origin (63), and high frequency of the S allele in Spanish and Portugese people ( 6 3 , 6 4 ) .

A

recent study of the epidemidogy of A^AT deficiency in the Netherlands illustrates its clustering in small rural communities, the distribution of the deficiency varying from 15 to 59 for 10,000 from region to region (65).

178 Basis of AiAT polymorphism

Comparison of the amino acid composition of isolated variants is not precise enough to ascertain any significant difference "between them.

The

first report of an amino acid difference related to A]AT genotypic variation was presented by Owen et. al (66).

Comparison of tryptic digest

of M and S proteins indicated the presence of a variant peptide in the latter.

The variation was due to the substitution of a valine residue in S

for a glutamic acid in M.

Similarly, the difference between M and Z was

shown to be due to the substitution of a lysine in Z for a glutamic acid in M (67-69).

Further work showed that these two substitutions were located

in the same large (109 AA) fragment, but far apart, one being localized at residue no. 22 ( s ) and the other at residue 100 (Z) of this peptide (70,71).

In addition, these substitutions were located far apart from the

asparagine which carries the sugar chain of this peptide.

Similar work has

shown that M2 differed from M]_ by a substitution Glu — > Asp (72), and a fast variant, called Balhambra carried two mutations, Glu — > Asp (as M2) and Lys — > M2 — >

Asp, suggesting the following sequence in evolution:

B (73).

M]_ — >

The precise location of the substitutions for the two

latter variant proteins is not yet determined. No other substitutions in relation to variant A^AT has yet been published, but a large part of the basis for A]_AT polymorphism probably will be demonstrated in the next few years.

Through the analysis of the

substitutions, it is likely that a better understanding of the evolution of the A]_AT gene will result.

As an example, comparison of the 153 amino

acids at the COOH-terminus of A^AT showed a significant homology with both ovalbumin and antithrombin III, suggesting that A]_AT, ovalbumin and antithrombin III belong to the same protein superfamily, and have probably evolved from a common ancestor gene (7^,75). As noted above, the severely deficient alleles Z, P, Mduarte and Mmalton are associated with a defect in the secretion of the variant protein.

This

is illustrated by the retention within the hepatocytes of A^AT which is present as a PAS positive material within the cysternae of the rough endoplasmic reticulum.

Identification of this material as A^AT is

performed by immunofluorescence using specific anti A^AT antibodies

(76).

179 The structural relationship between this material and plasma A]AT has been extensively studied.

Matsubara et al. (77) isolated from ZZ liver a

material of M.W. 18,000 which probably corresponded to A-j_AT fragmented by endogeneous proteases.

Eriksson and Larsson (78) extracted from the liver

a protein very similar to plasma A]AT, but completely devoid of sialic acids and without any biological activity.

Further work showed a close

similarity between the polypeptide moieties from liver and plasma.

In

contrast, the carbohydrate portion of the former was markedly altered.

Not

only sialic acids were absent, but other sugars were markedly reduced. This could explain the high tendency of this material to aggregate (79). Finally, recent data have shown that the intrahepatic form possesses an excess mannose (80), which suggests a blockade of the glycoprotein processing at the level of the rough endoplasmic reticulum, although the mechanisms which prevents ZZ A]AT to proceed from the rough to the smooth endo-plasmic reticulum remain unknown.

Enzymatic cleavage of the excess

mannose and addition of the next sugar, N-acetyl glucosamine, was possible in vitrtf

(8l).

The importance of this observation lies in the fact that,

using this experimental model, it is now possible to get a better knowledge of the compartmentation of the glycosylation of glycoproteins in human hepatocytes.

An intriguing fact is that the microheterogeneity of

sequestrated A]_AT is preserved, even in the absence of sialic acid, galactose and terminal N-acetyl glucosamine.

No information is available

concerning liver A^AT associated with other deficient phenotypes. Recently, A]_AT has been extracted from liver of a normal (MM) individual (10,15).

Both cytosolic and microsomal A^AT appear identical to the

circulating protein.

The absence of biological activity of this material

appears to be related to the extraction procedure. In conclusion, studies of A]_AT genetics and structure conducted in parallel in recent years have been especially informative.

A]_AT represents to date

the most polymorphic of the plasma proteins, and the basis of this polymorphism has been established for the common alleles.

Study of the reten-

tion of A]AT within the liver cells associated with the Z allele represents an experimental model for the study of the processing of the carbohydrate moiety of liver glycoproteins.

The existence of genetically determined

A]AT deficiency has helped to understand the pathophysiology of the asso-

180 ciated diseases.

Finally, comparison of A^AT sequence with sequences of

other proteins should help to elucidate the evolution of the A]_AT gene.

Acknowledgments

C. Chapuis-Cellier is a postdoctoral fellow of the College of Graduate Studies of the Medical University of South Carolina, on a leave of absence f r o m the University Claude Bernard, Lyon, France.

E. Gianazza is a post-

doctoral fellow of the College of Graduate Studies of the Medical University of South Carolina, on a leave of absence from the Department of Biochemistry, University of Milano, V i a Celoria 2, Milano 20133, Italy. Publication no. 1*50 from the Department of Basic and Clinical

Immunology

a n d Microbiology, Medical University of South Carolina, Charleston, South Carolina.

Research supported in part by USPHS Grants HD-09938 a n d

CA-257^6, and by General Medical and Faculty Research Appropriation Grant A - 3 0 1 , Medical University of South Carolina.

We thank Charles L. Smith for

excellent editorial assistance.

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183 50. Cox, D.W., Billinsgley, G.D., and Smyth, S., In: Electrophoresis "8l" (Ed. by R.C. Allen and P. Arnaud), Walter de Gruyter, Berlin, New York, pp. 505-510 (19Ö1). 51. Laurell, C.B., Nosslin, B., and Jeppson, J.O., Clin. Sei. Mol. Med., 52: i+5T-^6l (1977). 52. Talamo, R.C., Langley, C.E., Reed, C.E., and Makino, S., Science, l8l: 70-71 (1973). 53* Martin, J.P., Sesboue, R., Charlionet, R., and Ropartz, C., Humangenetik, 30: 121-125 (1975). 5l+. Arnaud, P., Bernheim, J., Chapuis-Cellier, C. ,and Creyssel, R. , In: Alpha^-antitrypsin and Pi system, INSERM Colloq.. 1+0: 109-120 (1975). 55* Gedde-Dahl, T. Jr., Fagerhol, M.K., Cook, P.J.L., and Noades, J., Ann. Hum. Genet. 35: 393-399 (1972). 56. Gedde-Dahl, T. Jr., Frants, R.R., Olaisen, B., Eriksson, A.M., Loghem, Van E., Lamm, E. , Ann. Hum. Genet., 1+5: 11+3-153 (1981). 57« Gedde-Dahl, T. Jr., Fagerhol, M.K., Cook, P.J.L., and Noades, J., Ann. Hum. Genet. 39: 1+3-50 (1975). 58. Chapuis-Cellier, C. , Verdier, M., Lepetit, J.C., Fudenberg, H.H., and Arnaud, P., J. Immunogenetics (in press) (1981). 59. Chapuis-Cellier, C. , and Arnaud, P., Science, 205: 1+07-1+08 (1979). 60. Iammarino, R.M., Wagener, D.K., and Allen, R.C., Amer. J. Hum. Genet., 31: 508-517 (1979). 61. Cox, D.W., Amer. J. Hum. Genet., 32: 1+55-1+57 (1980). 62. Mittman, C., and Madison, R. , Amer. J. Hum. Genet., 32: 1+58-1+59 (I98O). 63. Fagerhol, M.K., and Tenfjord, O.W., Acta. Pathol. Microbiol. Scand., 72: 601-608 (1968). 61+. Martin, J.P., Sesboue, R. , Charlionet, R. , Ropartz, C., and Pereira, M.T., Hum. Hered., 26: 310-311+ (1976). 65. Dijkman, J.N., Penders, T.J., Kramps, J.A., Sonderkamp, H.J.A., Van Den Broek, W.G.M., and Haar, B.G.A. , Hum. Genet., 53: 1+09-1+13 (1980). 66. Owen, M.C., and Carrell, R.W. , FEBS Letters, 79: 21+5-21+7 (1977). 67. Jeppson, J.0., FEBS Letters, 65: 195-196 (1976).

184 68. Yoshida, A., Lieberman, J., Gaidulis, L., and Ewing, C., Proc. Natl. Acad. Sci. U.S.A., 73: 1324-1328 (1976). 69. Owen, M.C., Carrell, R.W., and Brennan, S.O., Biochim. Biophys. Acta, 1+53: 257-261 (1976). 70. Owen, M.C., Lorier, M., and Carrell, R.W., FEBS Letters, 88: 234-236 (1978). 71. Shochat, D., Staples, S., Hargrove, K., Kozel, J.S., and Chan, S.K., J. Biol. Chem., 253: 5630-5634 (1978). 72. Yoshida, A., Taylor, J.C. , and Vanderbrock, W.G.M., Amer. J. Hum. Genet., 31: 564-568 {1919)• 73* Yoshida, A., Chillar, R. , and Taylor, J.C., Amer. J. Hum. Genet., 31: 555-563 (1979). 74. Carrell, R., Owen, M., Brennan, S., and Vaughan, L., Biochem. Biophys. Res. Comm., 91: 1032-1037 (1979). 75« Hunt, L.T., and Dayhoff, M.O., Biochem. Biophys. Res. Comm., 95: 864-871 (1980). 76. Sharp, H.L., Bridges, R.A., Krivit, W., and Freier, E.F., J. Lab. Clin. Med., 73: 934-939 (1969). 77* Matsubara, S., Yoshida, A., and Lieberman, J., Proc. Natl. Acad. Sci. U.S.A., 71: 3333-3337 (1974). 78. Eriksson, S., and Larsson, C., N. Engl. J. Med., 292: 176-180 (1975). 79- Jeppson, J.O., Larsson, C., and Eriksson, S., N. Engl. J. Med., 293: 576-579 (1975). 80. Hercz, A., Katona, E., Cutz, E., Wilson, J.R., and Barton, M., Science, 201: 1230-1232 (1978). 81. Hercz, A., and Harpaz, N., Can. J. Biochem., 58: 644-648 (1980). 82. Charlionet, R., Sesboue, R., Morcamp, C., Le Febvre, F., and Martin, J.P., Hum. Hered., 31: 104-109 (l98l). 83. Cox, D.W., Am. J. Hum. Genet., 33: 354-365 (1981).

AFFINITY-IMMUNODELETION (AID) ISOELECTRIC FOCUSING ON ULTRATHIN GELS AND STAINING WITH SILVER DIAMINE APPLIED TO THE MARKER PROTEINS OF INFLAMMATION

R. C. Allen* and P. Arnaud** Departments of Laboratory Animal Medicine and Pathology* and Basic and Clinical Immunology and Microbiology Medical University of South Carolina Charleston, South Carolina 29425

Introduction A number of recent developments have been made in electrophoretic techniques which allow, not only the detection of polypeptides in sub-nanogram amounts (1,2,3) but also in the resolution where one-thousand or more polypeptides may be resolved on a single gel using two-dimensional techniques. The Iso-Dalt System (4,5) has the theoretical potential of resolving up to ten thousand polypeptides on a single gel. As commonly utilized, two-dimensional PAGIF-SDS PAGE suffers from several deficiencies as a practical application to clinical studies. First, the procedure requires considerable time, effort and precise control to carry out and achieve adequate confidence limits for spot location. This problem is magnified further in comparing data between laboratories, although automated scanners and sophisticated computer programs such as the matching and stretching of two-dimensional gel patterns (6) may eventually solve this problem. Second, SDS denaturation in the second dimension requires the difficult, if possible, renaturation in situ of the proteins for enzyme and immunological identification of individually separated spots. This may be avoided by using pore limiting gradients in the second dimension to achieve size separation (5) on ultrathin

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1 9 8 2 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

186

gels. However, even reagent penetration for enzymological, immunological or enhanced immunoenzyme staining may not have the required sensitivity at higher gel densities of 20 per cent T or more. On the other hand, two-dimensional techniques may be looked on as being simply a deletion system whereby increased information is gained by a method of removing overlying proteins from a given point by charge differences in one dimension and size in the second dimension. Thus, the likelihood that two or more proteins separated by two parameters will occupy the same position in an x,y plane is reduced. An alternative solution is to selectively and or sequentially remove proteins by standard procedures to allow similar resolution in one dimension, namely, by PAGIF. An additional desired capability would be to extend the resolving capacity of the isoelectric focusing procedure itself. As commonly employed, this procedure utilizes voltage gradients of 100-200 volts per centimeter; yet, Swendson (7) has shown that for a four-fold increase in voltage gradient, a two-fold decrease in band width results; thus, effectively increasing theoretical plates and resolution by using higher voltages. Such practical effects have been previously reported by one of us using ultrathin layer isoelectric focusing with an increased cooling capacity system (2). This, in conjunction with selected sequential removal of proteins by Affinity-Immunodeletion (8), has been applied in this report as a clinical approach to the study of the marker proteins of inflammation.

Methods and Materials PAGIF:

Ultrathin-layer

isoelectric focusing was performed

on 250u thick 5 per cent T 3.5 per cent C gels bonded on

187 glass plates with Polyfix 1000 as previously described (2 ) ( 8 ) conventional PAGIF was performed on 0.75 mm gels not bonded to glass as previously described (8 ). In the former, the electrode distance was six cm and in the latter 11 cm. Peak voltage gradients were up to 500 V/cm in the former and 180 V/cm in the latter. Staining procedures used were Coomassie Blue R250 at 55°C and silver diamine staining (2) . Affinity-immunodeletion techniques were carried out as previously described (8) with the exception that protein A (sigma B 20 mg binding 9 mg IgG) was used to remove antibody after reaction with specific antisera. Specific antisera were reacted with serum in a 1:1 ratio for 15 minutes and then centrifuged. The supernatant was then reacted with protein A diluted to a concentration of 5 mg/ml. Trypsin at a concentration of 20 mg/ml and trypsin bound to sepharose were used to determine deletion of A-1AT the former to detect complexes and the latter to provide a complex free pattern for comparison.

Results The affect of voltage gradient on resolution is shown in Figure 1 where it is readily apparent with pH 3-10 Pharmalyte that as the voltage gradient is raised above 4 50 V/cm that much fuller resolution is obtained over the entire pH gradient and that with the times and temperatures used no cathodal drift was apparent.

188

LU —I < o CO z> LL. Q O o

Figure 1. Densitometric traces of 0.8 ul volumes of granulocyte extracts run at various voltages on ultrathin gels with a Pharmalyte pA 3-10 gradient. Staining was with silver diamine and relative distance is shown in the scale insert. In Figure 2, immunodeletion alone illustrates that the marker proteins of inflammation with isoelectric points lying outside the pi range of albumin and the IgG fraction of the specific antisera may be identified readily by immunodeletion followed by PAGIF, and may titered in fact, by varying the amount of antisera. Those marker proteins such as alpha-2-macroglobulin and Gc protein, however, are completely obscured by overlying albumin. In Figure 3, albumin was removed prior to immunodeletion by reacting the serum with Blue Sepharose CL6B and the gel subsequently stained with silver diamine. Here the alpha-2-macroglobulin immunodeletion and that of the Gc protein is clearly evident.

However, the rabbit IgG fraction of anti-Gc globulin

clearly contains some antibody to alpha-l-antitrypsin as well as indicated by its diminution.

Similarly, the IgG fraction

189

Figure 2. Immunodeletion of various serum proteins. A, human serum + anti-A-lAT; B,Serum + saline and serum + anti-alpha 2 macroglobulin; C,serum and saline and serum + anti-haptoglobin; D,serum + saline and serum + anti-Gc; E, serum + saline and serum + anti-transferrin and F,Serum + saline and serum + antibodies used.

A

B

C

D

E

F

G

H

Figure 3. A, Human serum + saline 1:2; B, human serum plus IgG fraction anti A-1AT; C, antibody IgG fraction + saline; D, human serum plus saline 1:2; E. human serum + IgG fraction anti-alpha-2-macroglobulin; F, alpha-2-macroglobulin antisera IgG fraction plus saline 1:2; G, Human serum + saline 1:2; H, Human serum + IgG fraction anti Gc. Ampholine was used at a pH Gradient of 3.5-8 on 250 u thick gel with maximum voltaqe 375 V/cm. of rabbit anti alpha-l-antitrypsin also diminished the Gc globulins, thus, indicating that the monospecificity of the antisera may be suspect.

190 Since the Blue-Sepharose also may remove some antibodies from the serum, an alternative method to study alpha-l-antitrypsin and transferrin deletion was employed in which serum was reacted with concanavalin A covalently linked to sepharose, the unbound fraction discarded and the bound glycoproteins eluted by alpha-D-methyl glucoside. The releasate was then reacted with "specific" antibody and the unbound antibody and complex removed by the addition of Protein-A as shown in Figure 4.

A B C D

E F G H

IJK

L M

Figure 4. A, human serum + saline 1:2} B, releasate from concanavalin A-Sepharose; C, human serum + anti-transferrin IgG fraction, D, anti-transferrin IgG fraction + saline 1:2; E, concanavalin A releasate + anti-transferrin, then reacted with Protein A; F, protein A + saline 1:2; G, human serum + saline 1:2; H, human serum concanavalin A releasate + antialpha-l-antitrypsin IgG fraction; I, human serum concanavalin A releasate ; J, anti-alpha-l-antitrypsin IgG fraction; K, same as H + protein A; L, protein A; M, same as A and G. Ampholine pH 3.5-8 in 0.75 mm gels subsequently stained with Coomassie R250 was employed. A similar system with purified transferrin was compared as

191 shown in Figure 5.

Figure 5. A, Purified transferrin at a concentration of 320 mg/dl; B, A/diluted 1:2 with saline; C, transferrin + anti-transferrin IgG fraction of the + protein A, D, anti-transferrin IgG fraction diluted 1:2 with saline. E, concanavalin A releasate; F, human serum plus saline 1:2; G, same as D. The pH Gradient and stain are the same as in Figure 4.

It was apparent in the anti-transferrin antibody IgG fraction's that there was also rabbit transferrin present as marked by the small arrows. All transferrin fractions were removed and the multiple forms apparent in the purified material probably represent different oxidized states. It was of interest to note that in the concanavalin A - alpha-D-methylglucoside releasate that the M6/A-1AT appears to have different binding characteristics than M4, which is in much heavier concentration than M4 after release. Proteinase binding proteins in both the A-1AT in serum and trypsin binding proteins of human granulocytes and lymphocytes were identified by ligand binding and by affinity deletion techniques. Direct ligand binding of trypsin to A-1AT is compared to affinity binding by trypsin immobilized on Sepharose

192 in Figure 6.

Figure 6. A, Human + serum + saline 1:2; B, Human serum + trypsin 4 ul serum + 2 ul trypsin at a concentration of 20 mg/ml; Human serum + saline 1:2 + immobilized trypsin; D, same as A; E, Non-binding proteins from concanavalin A - Sepharose; F, concanavalin A bound proteins released with alpha-D-methyl glucoside.

Direct ligand binding deletes the A-1AT similarly to specific immunodeletion shown in Figures 2, 3 and 4; however, the two complexes indicated by arrows also have a similar pi to protein in the IgG region shown in A by the small arrow. Removal of the complex by the immobilized trypsin clearly shows the comparative advantages of each method. Additional trypsin binding proteins are shown in both granulocytes and leukocytes in Figure 7, Deleted bands binding trypsin are shown by arrows as are the resulting complexes with altered pis.

Unbound trypsin with a

pi of pH 10 is designated by the small white arrows located at the edge of the cathode wick.

It was not apparent that any

193

Sjfe Mfa

A

B

C

D

E

F G H

I

Figure 7. A, human granulocyte extract A 0.8 ul of 10^ cells/ ml extracted with Triton X100 + freezing and thawing; B, A + 0.2 ul trypsin; C, human lymphocytes of an 0.8 ml sample similarly extracted from a similar cell concentration; D, C + 0.2 ul trypsin; E, human granulocyte extract B similar procedure to A; F, E + 0.2 ul trypsin; G, non-concanavalin A binding proteins from E; H, concanavalin A bound proteins released with Alpha-d-methyl glucoside; I, H + 0.2 ul trypsin. Separation on a pH 3.5 -10 ampholine gradient on 250u thick gel with 37S V/cm maximum and stained with silver diamine. non-concanavalin A binding proteins had proteinase binding capacity.

Discussion Affinity Immunodeletion in conjunction with ultrathin layer PAGIF separated by high voltage gradients provides a method

194 in which to identify and study the marker proteins of inflammation with a greater degree of resolving power in a single dimension. It is doubtful that the full resolution capacity of PAGIF has as yet been reached since theoretically longer gels at such voltage gradients as used in this study would yield even higher resolution. Thus, the ability to increase theoretical plates by both high voltage gradients and affinity immunodeletion has as yet not even approached its potential. However, the usefulness of this approach is apparent based on these initial results with a limited number of affinity gels and antisera applied to the specific problem of the proteins of inflammation. An unexpected finding in this study was the apparent differential binding of the M4 and M6 proteinase inhibitor (Pi)allele products. The significance of this observation requires further study and additional lectins to determine the responsible mechanism. The use of the silver diamine stain technique gives a further advantage in being able to study proteins present in sub-nanogram quantities. Thus, not only may small amounts of proteins be titered by varying the amounts of antisera, but the reverse is also possible. Thus, monospecific antisera or monoclonal antisera deletion by specific antigen may be quantified by this technique. This may prove of particular value with small proteins or those with high isoelectric points such as relaxin where it may be possible to quantify it by deletion « titration of specific antibody followed by ultrathin layer PAGIF and silver staining. This would appear, on the surface, as an attractive alternative in some cases to radioimmunoassay. Silver staining must be regarded with some caution since there are a variety of methods presently in use.

These range from

that described herein which has been made non-specific for proteins and stains all zones black,to those described by Adams and Sammons (3). Gelcode provides colors ranging from

195 yellow, orange, red to blue for the various proteins present in body tissues and fluids.

A simplified photo sensitive

stain has been described by Merrill et al

(9, 10) which on

ultrathin gels is not as sensitive as that reported,here

al-

though a recent modification to a non-light sensitive variation of this technique with ferric chromate increase sensitivity

(10).

It should also be noted that stains employing

too much sodium hydroxide, used on ultrathin gels fixed to glass or polyester with Polyfix 1000, will cause swelling and loosening of the gel, thus causing loss of the whole separation. While the basic concept of the Affinity-Imraunodeletion

tech-

nique was to more fully extend the capability of high voltageultrathin-layer PAGIF as a clinical tool for the location and identification of proteins, it is obvious that this procedure, in its various possible permutations and combinations, should serve equally well in solving some of the present riddles resulting from the information explosion potential of two-dimensional

electrophoresis.

Acknowledgements The authors wish to acknowledge the expert technical assistance of Ms. Margaret Ann Simmons and the secretarial

assis-

tance of Ms. Brenda Altman. We also wish to acknowledge the generous donations of many of the specific reagents used in this study by Marine Colloids Division of FMC.

196 References 1. 2. 3. 4. 5.

Merril, C. R., Switzer, R. C. and Van Keuren, M. L.: Nat. Acad. Sc. USA 98, 231-237 (1979). Allen, R. C.: Electrophoresis 32-37 (1980).

Proc.

Adams, L. D. and Sammons, D. W. Electrophoresis '81, Editors, Allen, R. C. and Arnaud, P.: Walter de Gruyter, Berlin, 155-165 (1981). Anderson, N. G. and Anderson, N. L.: Anal. Biochem. 85, 331-341 (1978).

6.

Anderson, N. G. and Anderson, N. L.: Anal. Bioahem. 85, 341-354 (1978). Taylor, J., Anderson, N. L. , and Anderson, N. G.: Electrophoresis '81 - Editors, Allen, R. C. and Arnaud, P.: Walter de Gruyter, Berlin, 383 (1981).

7.

Swendson, H.:

8.

Allen, R. C. and Arnaud, P.: Electrophoresis '81 - Editors Allen, R. C. and Arnaud, P., Walter de Gruyter, Berlin, 167-179 (1981). Merril, C. R. Goldman, D., Sedman, S. A. and Ebert, N. H.: Science 2TL, 1437-1438 (1981). Merril, C. R., Personal Communication.

9. 10.

Acta Chem. Scand. L5, 325-341 (1961).

A SURVEY OF THE MEASUREMENT, DISTRIBUTION OF VALUES AND PHENOTYPES OF THE HAPTOGLOBINS

O. Hever National Institut for Rehabilitation Medicine, H-1528, Budapest, Hungary

It was some 30 years ago that Max-Fernand Jayle, discoverer of haptoblogin (Hp), published his iodometric titration method for the gunatification of Hp levels in biological fluids.

In

the ensuing period the following principles have been applied for the neasurement of Hp.

Figure 1.

Principles of Hp measurement 1. 2.

Instability of glycoproteins Complex formation with hemoglobin

M a r k e r Proteins in Inflammation © 1982 by W a l t e r d e G r u y t e r &. C o . , Berlin • N e w Y o r k

198 3.

Antigenicity

4.

Conjugate with enzymes - ELISA

5.

Marking with radioisotopes

1. The glycoproteins show an instability against some chemicals. The first precipitation method was based on the selective precipitation of seromucoids, included Hp, by the cetavlon/cetyl-trimethyl-ammonium bromide. These types of procedure are not in use now. The precipitates contain not only Hp but a large quantity of different glycoproteins, too. 2. The Hp forms a stable complex with the hemoglobin (Hb) and this binding is strictly stoichiometric. The complex has many properties which enable the elaboration of quantitative methods. Some of these are very popular being simple, quick and not expensive. Detailed data concerning these properties will be treated later. 3. The Hp has an excellent antigenicity. Good immunochemical measurement can be realized with antibodies against Hp produced in sheep, goat, etc. In the popularity of these methods an important phenomenon plays a great role: the complex formation has no influence on the Hp - anti Hp reaction. 4. The Hp can be conjugated with enzymes. This conjugate with alkaline phosphatase makes it possible to elaborate a procedure based on the ELISA principle. An advantage of this method is its high sensitivity. The appearance of industrial products in this field could make this procedure economic also in routine work. 5. Radioisotopes are also applied for the measurement of Hp. The Hp may be marked among others with C, J, Se, on the other hand, one can proceed by measuring the complex of 59 which the.Hb is marked, e.g. with Fe. These methods are used only in scientific work.

199

Hp-Hb Possibilities

COMPLEX

of Hp

MotecUar

Instability acidification

—

r'Uphk

Agar Agorair CAF

Figure 2. Methods based on the properties of the Hp-Hb complex. 1.

Chromatography

2.

Electrophorese

3.

Spectrophotometry

4.

Iodometry - Colorimetry

5.

Precipitation by chemicals

Numerous procedures are based on the different properties of the complex in Figure 2. 1.

1.

The size of the complex molecule - rel. mol. mass in the

order of 100,000 or multiples - gives a reliable and simple way to separate it from free Hb - rel. mol. mass 65,000 - by chromatography. photometry.

This method is generally coupled with

spectro-

Although it is applied mainly in scientific work,

some of its simpler modifications are used in hemotology and neonatology, e.g. is cases with low Hp level or with

intravas-

cular complex formation cauaed by hemolysis. 2.

The electrophoretic mobility of the complex is slower than

that of the free Hb.

The Hp level of a serum can be evaluated

200

by adding Hb solutions in increasing concentrations and then by separating electrophoretically the complex from the free Hb. This procedure consumes much work and time. Its only advantage is the simultaneous detection of methaemalbumin if present. '3. The Hb bound in complex with Hp is protected against the degradation caused by acid pH, while the free Hb under the same conditions will be decomposed into hemin and gives no more absorbance in the Soret band. This phenomenon can be easily used in spectrophotometric procedures. 4. The most interesting particular feature of the complex is its high peroxidase activity (p.a.). The discovery of the Hp is due exactly to this property. The first method based on this is the iodometric titration of Jayle, as alluded in the introduction. The method can be carried out in two ways: a saturation method to determine that volume of serum which binds exactly a known quantity of Hb; and the method of activation that is to measure the quantity of the iodine liberated by the p.a. of the complex present in a given volume of serum. This modification is good and simple enough for routine analyses and may also be used in series. Moreover, it is considered presently as a reference method. With the application of an electron donor method which produces a color reaction product during oxydation a possibility is given for the elaboration of colorimetric methods. The guaiacol method of Connel and Smithies had great success as proved by its numerous modifications. The colorimetric methods were practicable procedures for laboratory automation for the measurement of Hp in biological fluids. The first adaptation was developed by Moretti, and then by Burrows and Hosten on the Technicon AutoAnalyzer. Nowadays there are published numerous adaptations for different analyzers and semi-automatic photometers as well. The following remarks should be made on the subject of p.a.:

201

Under certain conditions, as in Jayle's method of saturation, the p.a. of phenotype Hp 1-1 proved to exceed with 5 per cent that of the two other phenotypes. Certainly, this difference is not negligible, but because of its small value it has no clinical significance. During the photometric procedure a progressively decreasing part of the Hb remains free up to the point of saturation. It has also its own p.a.; therefore, the curve saturation does not pass the origin in Figure 3. Recently, Marklund has observed that ABTS (2.21-azino-di/3ethyl benzthiazoline-sulfonate-6) specificaly inhibits the p.a. of the free Hb, while it does not block that of the complex. This is a new possibility to eliminate from the system the inert p.a. of Hb. It should be taken into consideration also that in the case of hemolysis the Hp bound intravascularly to Hb passes into the blank test and increases the value of the latter. Concerning the complex, the idea was raised again to apply chemical precipitation. This is supported by the publications recommending rivanol (2-etoxi-6, 9- diaminoacridine lactate) and chlorhexidine for this purpose. These procedures did not come into general use because of the heterogenity in glycoproteins of the precipitate obtained.

qo 0,1 Q2 q3 OA QS Q6 Q7 0.8 09 Ifl Serum ml

Figure 3.

Curve of saturation of Hb solution: photometry

202

Immunochemical methods 1.

Radial immunodiffusion - RID

2.

Nephelometry

An essential advantage of immunochemical methods is that they are not influenced by hemolysis, because Hp will be bound to it its antibody even if it has formed an earlier complex with Hb. The RID method is very slow and provides results only after 48 hours.

Moreover, laboratories which are using this method,

are obliged to determine also the phenotype of the serum. Namely, the results have to be corected with factors corresponding to the individual phenotypes.

In addition, there is

a third disadvantageous factor, the results of RID, as compared to those of other methods, show a proportional bias in the direction of higher valued in Figure 4.

Figure 4.

Proportional bias of the results of RID.

Nephelometry has more advantages:

measuring is quick, the

knowledge of the phenotype is only facultative and the results show a good correlation with those of other methods. nephelometry is already adapted to automatic

Also,

analyzers.

203 At this time, it is necessary to make some pertinent remarks. Engler et a^L. showed in the course of laser nephelometric studies that phenotype Hp 1-1 gives a somewhat higher value than expected. The reason for this is most likely the size of the molecule. This difference is distinct, but small, and consequently it renders uncertain the clinical estimation of results only in borderline cases. In any case, a correction factor must be used in research work. Here I would like to allude to the higher p.a. of Hp 1-1 already mentioned. The immunochemical determination of Hp has another problem area: the question of the Hp standards as documented by Engler in the course of comparative studies. Character of the distribution of Hp values The Hp system is a genetically determined group of serum, the phenotypes of which influence the blood level both in physiologic and the pathologic conditions. The phenotype influences also the character of the distribution of values, which is log- normal or between log-normal and normal. Also, the population study of Harduin et^ a^L. carried out in Cherbourg support this convincingly. In our population in Hungary, we have classified the values also as a function of the phenotype. The Hp 1-1 showed a skew to the left and the Hp 2-1 and 2-2 a skew to the right in Figure 5. The value of g^ was in most cases significant. The character of these curves proved to be leptikurtic. The value of g2 was, in a number of cases, significant. Thus, the curve obtained in population studies is composed by the superposition of three different curves.

We also have to

take in consideration that the curve given by the individual phenotypes is in itself also heterogenous.

The curve of Hp

1-1 is the result of the superposition of its 3 subtypes, the proportion being 1:2:2.

On the other hand, the curve of Hp

204

DISTRIBUTION

OF Hp VALUES

ACCORDING

TO

PHENOTYPES

g=

-0,5

1-1 Figure 5.

OA

2-1

Q7

2-2

The distribution of haptoblobin values according to phenotype.

2-1 and 2-2 is formed by the superimposition of 6 subtypes respectively. However, in the case of the two latter phenotypes, the variants amount to a small percentage, as will be documented later. They influence the curve not at all or to an insignificant degree. That means that the knowledge of the mean phenotypes is recommended, at least in the clinical borderline cases, but indispensable in population studies and in the scientific work. Thus, we arrive at the problem of the determination of the Hp phenotypes. Determination of the Hp phenotypes With the starch gel electrophoresis, we demonstrate the three main phenotypes and correspondingly two main groups of alleles. The Hp"*" alleles determine the production of monomeric and the 2

Hp alleles that of polymeric Hp molecules. Consequently, the Hp 1-1 phenotypes are homozygous monomeric, the Hp 2-2 homozygous polymeric and the Hp 2-1 heterozygous monomeric-polymeric. The procedure itself is relatively easy, quick, not

205

HAPTOGLOBIN

Starch

PHENOTYPES

/A/;

oe/|

H | Hp 1-1

Position of the different hp*

polypeptide chains

|j

INSULM: iHfllmr^MtpHm'i

f Detectable

M S alets. i

Figure 6.

tSphem

Haptoglobin types schematieally presented for starch gel and polyacrylamide gel electrophoresis.

expensive and reliable. This technique is rather simple: Hb is added in surplus to the serum, i.e. the performed complex migrates in the starch gel. After the electrophoresis, the phenotypes are detected on the basis of their p.a. According to our present knowledge, the whole Hp system consists of six alleles and correspondingly of 21 phenotypes in Figure 6.

The Hp molecules determined by the different

alleles differ only in their alpha-chains. The beta-chains is the same in every Hp molecule. The Hp 2 type alpha-chains

206

are formed by the non-homologous crossing over of the Hp"*" type alpha-chains: hp IF and hp IS. Thus, four variants are possible: hp*2FF, hpo(2FS, hpc(2SG and hpe(.2SS. Of these the hp c(2FS and hpo(2SF cannot be separated with our present methods. Therefore, the corresponding alleles are written together in this form: H p ^ F S . In practice, we can demonstrate five alleles and 15 phenotypes. The existance of subtypes was verified by Smithies et^ al_. in starch gel containing a high concentration of urea. sent, we use the method of polyacrylamide gel - PAGE.

At pre-; This

technique, however, is very complicated and time consuming. First, * we have to purify the serum Hp with ion-exchange chromatography. By a procedure of alkylation and reductive cleavage the Hp will be decomposed into its chains. This preparation is submitted to electrophoretic separation. For the determination of the exact position of the different alphachains insulin serves as a marker in Figure 6.

Figure 7. Hp 1-1 subtypes: from the botton upwards first band the insulin as marker; upper lines are the Hp alphapolypeptide chains; on the top are the beta chains. Subtypes from the left to the right: 1F-1S, 1F-1F, 1S-1S, IF—IS, 1F-1F, 1F-1S.

207

Figure 8. Subtypes from the left to the right: 2/FS/-1S, 2/FS/-2FF, 2FF-1S, 2SS-1S.

In Figure 7, the following subtypes may be seen: HplF-lF, Hp 1F-1S, Hp 1S-1S, Hp 2/FS/-1F, Hp 2/FS/-1S. Figure 8 illustrates among otherS^a rare subtype: Hp 2FF-2/FS. The second sample from the left. Table 1 Gene Frequencies of Subtypes in the Hungarian Population

Hp subtyping of 675 Hungarian non related individuals Gene freqvencies: Hp,F -0,11845 Hp ,s -0,22080 Hp2FF -0,00375 Hp 2,FS,-0,65550 Hp iSS -0,00150

208

The frequency of subtypes is a problem, which has to be considered. In Hungary, the subtypes of 675 non-related persons were determined. The data are summarized in Table 1. These data are informative on the frequency of the different variants. It may be seen that the incidence rate of the variants 2 of Hp alleles is very low. Summarizing our experiences gained in the determination of Hp subtypes,we may state that the detection of main Hp phenotypes for clinical requirements is completely sufficient and consequently the application of starch gel technique is satisfactory in this field. It is evident that from the beginning we were forced to give a survey on the fundamental questions of this subject. It was impossible to mention all the authors according to their merits or to discuss technical details. Likewise, we were unable to report on certain recent publications.

RECENT FINDINGS ON THE BIOLOGICAL ROLE OF HAPTOGLOBIN IN RATS

R. Engler + Laboratoire des protéines de la réaction inflammatoire, UER Biomédicale des Saints Pères, Département de Biochimie, 45, Rue des Saints Pères, 75720 Paris, Cedex 06, France

Review

In every inflammatory process, either due to bacterial, viral, tumoral, chemical, physical (burns, irradiation) or traumatic injury, two kinds of response can be distinguished : - a local reaction, including vasodilation, edema, platelet aggregation, leukocyte migration, fibrin deposition, release of histamine, serotonin, kinins, prostaglandins, superoxide ions and lysosomal enzymes originating from tissues and leukocytes. - a systemic reaction, which includes, in addition to fever, pain, and hyper leukocytosis, an increase in the concentration of some proteins called acute phase reactant proteins (Figure 1). In rats, the known acute-phase reactant proteins include alpha^ globulin of Darcy (tij-AP), alphaj-antitrypsin (c^jAT), alphaj-serum amyloid P component (SAP), haptoglobin (Hp), ceruloplasmin (Cp), alpha2~macroglobulin (cC,-M), hetnopexin (Hpx) and fibrinogen (Fib).

In animals, the subcutaneous injection of turpentine (5 ml/Kg) produces a model of inflammatory reaction.

It is difficult to assess which event occurs first in the inflammatory reaction. According to HOUCK (1), the cell pain, which is manifested by an increase in the permeability of the lysosomal membrane or the

This review is dedicated to the memory of Professor M.F. Jayle and to Christian Lombart, expert of haptoglobin in rats.

M a r k e r P r o t e i n s in I n f l a m m a t i o n © 1982 by W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k

210

LIVER

$

/

co o o a S s 8'

£

lysosomal / >staglandins ®JJ?V mes

Figure 1. Schematic diagram of the inflammatory reaction.

>

histamine serotonine kinins

INFLAMMATORY

REACTION

breakage of the latter, could represent the primary event of the inflammatory response. This event occurs very early (as soon as a few hours following the time of injury). The liver is informed of the occurence of this reaction through a "messenger" whose nature is still speculative. Recent data (2,3) suggest the existence of a leukocyte proteidic factor (Leukocytic Endogeneous Mediator, LEM) whose presence in the systemic circulation can trigger a n increase of the synthesis of inflammatory proteins by hepatocytes. This represents a de novo synthesis and does not correspond to release of proteins previously stored inside the hepatocyte (4). The mechanisms of synthesis have begun to be elucidated

(activation

of RNA polymerases (5), increase of protein synthesis (6), hypertrophy of the endoplasmic reticulum and Golgi apparatus and glycosylation of polypeptide chains (7). Studies in rats of four acute-phase reactant proteins (fibrinogen, orosomucoid,o(2"fflacroglobulin, haptoglobin ) demonstrate that the biosynthetic mechanisms and their plasma increase occur simultaneously (6). Of the acute phase reactant proteins in rats, Hp is

211 interesting in several respects. These include three functions, or pathophysiological mechanisms, hemoglobin binding, desialylation and inhibition of the enzymatic activity of the lysosomal cathepsins B and L.

During the occurence of hemolysis, Hp binds hemoglobin (Hb) and forms a complex (Hp-Hb). During the process of intravascular hemolysis, this complex is cleared very rapidly f r o m the circulation (8). A membrane receptor located o n the hepatocytes, w h i c h is different from b o t h the asialoglycoprotein receptor and the receptor for hemopexin-heme complex, is responsible for the clearance of the Hp-Hb complex (9). Complex formation b e t w e e n Hb and Hp is responsible for a conformational change of the latter following exposure of a recognition site for the membrane receptors. D u ring extra-vascular or intra tissue hemolysis, frequently associated w i t h inflammatory reactions, haptoglobin binds the released hemoglobin. The microsomal enzyme, hemec< methenyl oxygenase (10) w h i c h is distributed in a number of organs, is m o r e active o n the Hp-Hb complex than on free Hb. This explains w h y part of the released Hb is locally degraded, mostly by macrophages. W h e n hemolysis is more important, diffusion of Hb in the intravascular compartment occurs, and the Hp-Hb complex is cleared by the hepatocyte.

The asialohaptoglobin (AsHp) is bound to a lectin of the hepatocyte m e m brane and endocytosed by a m e c h a n i s m similar to most asialoglycoproteins. Its clearance from the vascular compartment is very rapid (11). The m e c h a nism w h i c h initiates asialoglycoproteins in plasma is still unknown. But, during inflammatory processes, the plasma values of asialoglycoproteins are increased (12). This raises the question of the role of lysosomal neuraminidases w i t h i n the inflammatory tissue site. W e have recently studied the endocytosis of rat As-Hp by isolated rat hepatocytes. W h e n As-Hp is bound to Hb as AsHp-Hb complex, the binding by isolated hepatocytes is only 50 % of that of Hp-Hb complex (13). This has to be compared w i t h the work of G A N o n asialo-C^j-AT-trypsin complex (16). This complex w h e n injected in rats, disappears from the circulation w i t h a half-life similar to that of C0.05

P0.05

S

.o

I 1 Total Protein

k h

P> 0.05

c • _ o IO

M

••e

SIgA

Ig 6

D

NORMAL PCM

Albumin

Figure 8. Nasal washing in 13 children with PCM, where SIgA, IgG, albumin, and total protein values are expressed as a percentage of total protein. (From Suskind RM: Malnutrition and the Immune Response. In Suskind RM (ed.): Textbook of Pediatric Nutrition. New York, Raven Press, 19 80.) Although concentrations of total protein, IgA and albumin measured in all nasal washings were reduced in children with PCM, only the SIgA concentration was significantly lower in the children with PCM than in the control children.

When the

data were pooled for given periods, the relative concentra-

309

tions of SIgA on days 1 to 8 and on days 9 to 70 were significantly lower than that of the controls. The SIgA concentrations in samples taken on days 71 to 84 and on follow-up, however, were higher than admission values and not significantly different from those of the controls. The mean serum IgA in the same malnourished children on admission was significantly higher than that of normal children of the same age. During hospitalization the relatively high SIgA levels decreased to that of the controls. Electroimmunodiffusion of the serum samples failed to disclose the presence of the secretory component (SC). McMurray et al (65) noted that the secretory IgA levels in saliva and tears of both the severely and moderately malnourished children were significantly depressed while the IgG levels in these secretions were significantly elevated. Other anti-infective proteins such as lysozymes and aminopeptidase were present in normal concentrations. Chandra (82) also demonstrated that malnourished children respond to immunization with live attenuated measles or polio virus with significantly lower levels of secretory IgA than controls. The mechanism by which secretory IgA production is decreased in PCM in yet unclear. It is unlikely that the decreased levels result from an impairment of local protein synthesis or local proteolysis since other proteins such as IgE, and albumin lysozymes are reported in normal concentrations. Sirisinha (81) and Chandra (39) excluded reabsorption of secretory IgA through the mucus membrane by showing that the reaction between the patient's serum and antiserum to the secretory component was negative. The most likely explanation for the decreased secretory IgA is a selective depression of IgA synthesis in the submucosa or a reduction in the synthesis of the secretory component by epithelial cells or both (39). This hypothesis is consistent with the pathological finding in

310 PCM of a loss of intestinal epithelial cells with mucosal thickening in association with atrophy of such GI associated lymphoid tissue as the appendix, Payer's patches, and the tonsils (13). The consequences of a reduced secretory IgA are not completely known, however, Hayworth et al (83) did find that malnourished children had significantly greater bacterial contamination of the jejunum that did controls suggesting defective mucosal clearance and/or secretory antibody mechanisms. One might speculate that the combination of an atrophic small intestine in association with decreased secretory IgA may result in systemic spread of microorganisms that are abnormally found in the small bowel of these malnourished children. The spread of these organisms may indeed lead to the gram negative septicemia which is so often seen in inadequately treated children. In addition, it may lead to the increased level of food antibodies seen in malnourished children. D.

Polymorphonuclear

Leukocyte

Although polymorphonuclear leukocyte function has been extensively studied in malnourished children the results have not always been clear. The results of the studies have often been contradictory because of the poorly defined patient population who were inadequately nutritionally assessed and who were not well-defined in terms of the presence or absence of superimposed infection. Several phases of phagocyte function have been studied in the malnourished child including mobilization, chemotaxis, phagocytosis or ingestion of particles, and postphagocytic function including phagocytic vacuole formation, and specific metabolic changes leading to intracellular killing of the microorganism. 1.

Mobilization

Chandra et al (84) were among the first to evaluate the mobil-

311 ization of polymorphonuclear leukocytes from marginal bone marrow and other pools. They demonstrated that with epinephrine which leads to splenic constriction and release of leukocytes from other sites that there was a normal increase in peripheral blood leukocytes in untreated children with protein-calorie malnutrition (PCM). In addition, they found that with the administration of Pseudomonas Polysaccaride that the neutrophilia which normally occurs as a result of the administration of this polysaccaride did not occur in children with PCM suggesting inadequate marrow reserves of polymorphonuclear leukocytes. 2.

Chemotaxis and the inflammatory response

Chemotaxis or the ability of the polymorphonuclear leukocyte to migrate towards an external stimulus is of fundamental importance to a normal inflammatory response. This imflammatory response should result in localization of invading pathogens, and activation of systems in order to deal with infecting organisms. One might expect that children with kwashiorkor who have superficial pyogenic infections and who are unable to develop suppurative lesions, would have a depressed polymorphonuclear leukocyte response. Freyre et al (85) who studied the inflammatory response in 33 uninfected Peruvian children with protein-calorie malnutrition found that these children had a normal polymorphonuclear leukocyte response. However, there was a significant depression in the macrophage response in these same malnourished children. This is a pattern which is similar to that seen in neonates and was found in children with kwashiorkor as well as those with marasmus. Kulapongs et al placed Rebuck skin windows on the forearms of nine Thai children with PCM on admission and after recovery (86).

Well-nourished children with infections and normal con-

trols were also studied.

A 1 cm superficial abrasion was made

312 on the volar surface on the forearm and a coverslip was removed after 30 min. Additional coverslips were placed over the abrasion at 2, 4, 6, 8, 12, and 24 hr. Each coverslip was stained with Wright's stain and evaluated for cellularity. A differential count was done on each slide. In children with PCM, there was no deficit in the cellular response, nor was there a deficit in the percentage of polymorphonuclear leukocytes (Fig. 9).

HOURS

Figure 9. Percent macrophage and polymorphonuclear leukocyte response in Rebuck Skin windows in children with kwashiorkor, and in three well-nourished control groups. Mean + SE measured from 2 to 24 hr after the initial skin abrasion. (From Kulapongs et al., ref. 86).

313 However, the children with PCM did not appear to have a normal mononuclear response in that the percentage of monocytes at 18 and 24 hr was significantly less than in the well-nourished children or in those recovered from PCM. These results confirmed those of Freyer et al (85). In earlier studies Gray (87) using the Rebuck skin window noted that the percentage of monocytes in the peritoneal exudate was reduced by 50% in the protein-calorie malnourished animals. The defects in macrophage mobilization noted by Freyer and Kulapongs were readily reversible with nutritional rehabilitation. Douglas and Schopfer (18) looked at in vitro chemotaxis of polymorphonuclear leukocytes from protein-calorie malnourished patients. They noted at 30, 60 and 120 minutes, that the polymorphonuclear leukocyte migration was delayed, i.e., there was a diminished number of cells migrating toward the chemotacic factor derived from Escherichia coli from the malnourished children as compared to well-nourished controls. However, by 180 minutes there was no difference between the cells from the malnourished and from the well-nourished controls. The authors concluded that, although overall chemotaxis was normal, there was a defect in the early phase of polymorphonuclear leukocyte chemotaxis. Rosen et al (88) made a similar observation, while Rich et al (89) found that the migration index of polymorphonuclear leukocytes from malnourished Ghanaian children was not significantly different from that of normal controls. Therefore, although in vivo studies indicate a defect in macrophage rather than polymorphonuclear leukocyte mobilization the in vitro data suggests an early defect in polymorphonuclear chemotaxis which is not sustained beyond the in vitro experiment. 3.

Phagocytosis

Most studies to date have found that phagocytosis by the poly-

314 morphonuclear leukocyte using a variety of particles is not significantly affected in malnourished children. Douglas and Schopfer (90) found that in isolated polymorphonuclear leukocytes and monocytes from children with kwashiorkor the percentage of cells phagocytosing and the number of particles phagocytosed per cell when these cells were incubated with latex particles were normal. Tejada et al also found that phagocytosis of Staph aureus by polymorphonuclear leukocytes from malnourished children was normal (91) . Schopfer et al found that the phagocytosis of Candida albicans by PMNs from children with PCM was normal (92) as was the engulfment of antibody coated erythrocytes by monocytes (93). Munson et al (94) also observed that the phagocytosis of microorganisms by circulating polymorphonuclear leukocytes was not effected in PCM, while Seth and Chandra (9 5) found normal phagocytosis of microorganisms by PMNs from malnourished Indian children. In addition, they described normal or slightly increased opsonic activity of the serum from malnourished children as compared with normal controls, findings similar to those reported in healthy males undergoing a prolonged fast (96). Macrophages are the phagocytes which provide primary defense against facultative intracellular organisms such as Salmonella typhimurium, Brucella and Mycobacterium tuberculosis. In addition, they play an important role in antigen recognition and processing, with subsequent interaction with T and B-cells to initiate cellular and humoral antibody responses. Indeed very few studies have been done in human populations looking at macrophage function because of the large quantity of cells required. Those in vivo studies by Freyer (85) and Kulapongs (86) indicated a rather delayed response on the part of the macrophage to an external stimulus. On the other hand, animal studies have demonstrated a rather normal macrophage function in PCM. Keusch et al (9 7) found normal phagocytosis of latex particles by isolated peritoneal cells from malnourished rats.

315 12 5 Saba and DiLuzio (9 8) using clearance of I-labeled triolein found a significantly longer plasma triolein half-life in starved animals than in well-nourished controls, thus indicating a depressed overall clearance by the RE system of malnourished rats. Splenic phagocytosis was normal suggesting that the delayed clearance of material from the vascular space was primarily due to impaired liver uptake. Prior opsonization of the test emulsion significantly increased the rate of clearance for all periods of starvation, suggesting that the depressed RE system was due not to an intrinsic phagocytic cell defect, but rather to a plasma opsonic defect. Likewise Ratnaker et al (99) found that protein deficient monkeys had a marked depression of phagocytic 32 activity as noted by clearance of intravenously administered p -labeled E. coli. The time required for blood cultures from deficient animals to become sterile was two and one-half times that required by controls• Deo et al (100) found that in the protein deficient monkey there was a significant decrease in the percentage of Kupffer cells phagocytosing colloidal carbon and a striking decrease in the amount of carbon phagocytosed per cell. This suggested that the overall clearance was impaired both by a decreased number of phagocytosing cells and impaired function of those phagocytic cells which remained. Price and Bell (101) showed that protein-deprived mice had a decreased capacity for detoxifying bacterial endotoxin as a result of depressed RE cell function. In addition, they found that there was a decrease in the number of peritoneal macrophages as measured by clearance of 125 I-labeled E. coli from the bloodstream. Passwell et al (10 2) also found an impaired RES phagocytic function due not only to a reduction in the number of macrophages, but to an impairment of macrophage function as well. 125 Coovadia et al (103) found the clearance of I-labeled polyvinylpyrrolidone was reduced when dietary protein was reduced to less than 4%.

With this reduction there was also lymphoid

316 atrophy so that the relative clearance corrected for cellularity was actually increased in the protein-calorie malnourished group. Thus Coovadia et al (103) felt that the depression was due to a loss of macrophages with those remaining demonstrating a compensatory increase in individual function. 4.

Postphagocytic events

Douglas and Schopfer (18) noted that the electron microscopic studies of circulating neutrophils revealed rather immature cells with prominent Golgi zones, granule pheomorphisms and the presence of prominent rough endoplasmic reticulum suggesting that the phagocyte had been activated by excessive antigenic stimulation. They subsequently found that additional EM studies of phagocytes revealed no qualitative difference between those from malnourished and control children with regard to vacuole formation after the phagocytosis of Candida albicans, E. coli or Staph aureus or after the ingestion of several microbial species in protein-deficient rats (18). a.

Intracellular killing

Although the studies evaluating chemotaxis and phagocytosis have indicated relatively normal polymorphonuclear leukocyte function with regard to these two activities, there is conflicting evidence regarding the intracellular killing function of polymorphonuclear leukocytes. A number of investigators have reported normal intracellular killing by the phagocytes of malnourished children (104). Arbeter et al noted that the intracellular killing activity was unaffected in children and adults with PCM unless iron deficiency was also found (105). Keusch et al (97) found only a slightly decreased intracellular killing of E. coli, Staph aureus and Salmonella by isolated macrophages from protein-deficient rats. Likewise Bhuyan et al (10 4) found that peritoneal macrophages from protein-

317 deficient rabbits exhibited efficient and unimpaired bactericidal activity. On the other hand, Palmblad (96) found that when normal male volunteers had fasted for ten days there was a significant decrease in bacterial killing by PMNs incubated with Staph aureus in vitro. Likewise, Seth and Chandra (95) noted an inefficient killing of phagocytosed bacteria by PMNs from patients with PCM, as did Douglas and Schopfer who found impaired bacterial killing activity of PMNs isolated from malnourished children when incubated with Staph aureus, E. coli and Candida albicans (90,106,18). PMNs from children with kwashiorkor were noted to demonstrate normal killing capacity during the first 30 minutes of incubation, but a significantly reduced killing capacity at 60 to 160 minutes (18,25,90,106). Rosen et al (88) on the other hand reported decreased PMN function only in patients with kwashiorkor who had evidence of an intercurrent infection. They felt that it was the infection, not the PCM, per se, which was the most significant factor in causing the impaired bactericidal function. Because of the controversy regarding leukocyte function, a study was undertaken in Northern Thailand to evaluate this parameter in 42 malnourished children (16 with marasmus, 14 with marasmus-kwashiorkor, and 12 with kwashiorkor). Patients were studied on admission, days 15, 28, 49, and 50. Isolated polys were suspended with E. coli in 0.1% gel Hanks solution at a bacteria-to-phagocyte ratio of 1.1. The incubation mixture was sampled at 0, 60, and 120 min. Culture plates were counted after an 18-hr incubation and the percentage killing calculated by dividing the number of viable bacteria remaining at 120 min by the initial number of bacteria added to the culture media. Controls were run with each patient's sample. The mean + 2SD PKF value for 199 controls was 9 7.7+9.9. Therefore, any value of less than 88% was considered abnormal. Only 1 out of 16 marasmic children (6%) had an abnormal PKF

318 on admission, while 2 out of 14 (14%) marasmic-kwashiorkor and 3 out of 12 (25%) kwashiorkor had abnormal PKF (Fig. 10).

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Figure 13. Serum CH50 activity in units/ml in healthy and febrile controls and in children with marasmus and marasmuskwashiorkor (M-MK), and kwashiorkor (K) on admission and with recovery (mean + SEM). (From Suskind et al., ref. 121) The percentage of children with depressed hemolytic activity decreased from 60% to less than 30% on day 8. When 33 serums from healthy and febrile controls were each mixed with a

331 standard serum of 920 to 1,280 units/ml, the CH 5 Q activity was always equal to or greater than 4 80 units/ml.

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