Marker Proteins in Inflammation: Volume 3 Proceedings of the Third Symposium, Lyon, France, June 26–28, 1985 9783110860757, 9783110106398

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Marker Proteins in Inflammation Volume 3

Marker Proteins in Inflammation Volume 3 Proceedings of the Third Symposium Lyon, France, June 26-28,1985 Editors J. Bienvenu • J. A. Grimaud • P Laurent

W DE G Walter de Gruyter • Berlin • New York 1986

Editors Jacques Bienvenu, M. D. Jean Alexis Grimaud, M. D. Philippe Laurent, M. D. Institut Pasteur de Lyon 77, Rue Pasteur F-69365 Lyon Cédex 07 France

CIP-Kurztitelaufnahme der Deutschen Bibliothek Marker proteins in inflammation : proceedings of the . . . symposium. Berlin ; New York : de Gruyter Vol. 3. Proceedings of the third symposium, Lyon, France, June 26-28,1985. 1986.-XVI, 693 p. ISBN 311010639 6

ISBN 311010639 6 Walter de Gruyter • Berlin • New York ISBN 0-89925-223-0 Walter de Gruyter, Inc., New York Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.

P R E F A C E

The Third Symposium on Markers of Inflammation, held in Lyon in the l a s t week of June 1985, was an international meeting that brought together some 300 s c i e n t i s t s and physicians. During t h i s Symposium which was sponsored by the "Groupe d'Etude et de Recherche sur

les Marqueurs de 1'Inflammation - GERMI",

under i t s president Professor J.L. Touraine, the participants exchanged a l o t of new information about progress in the study of inflammation d i s o r d e r s . Recent discoveries about the fundamental aspects and the c l i n i c a l

relevance of chemi-

cal messengers that communicate between c e l l s of the lymphoid system, such as i n t e r l e u k i n s and lymphokins, have caused great excitement. The complex effects of the b i o l o g i c a l actions of INTERLEUKINS include most of the c l i n i c a l features of inflammation, such as swelling of t i s s u e s and j o i n t s , pain, fever, modulation of immune response, and also the breakdown, repair and remodelling of injured t i s s u e s . Speakers from several

countries

described the p u r i f i c a t i o n of i n t e r l e u k i n s . They reported on the cloning of genes by the new methods of molecular b i o l o g y , and discussed the opportunities for production by biotechnology and the potential applications to the t r e a t ment of disease. Basic and c l i n i c a l

research on several inflammatory diseases was reported, for

example a disease called AMYLOIDOSIS. In t h i s disease certain blood proteins suddenly increase in concentration. Sometimes, these proteins are deposited as f i b r i l s in t i s s u e s such as l i v e r , kidney and heart, and t h i s may eventually cause organ f a i l u r e . Methods to purify and measure these markers in the blood and t h e i r c l i n i c a l usefulness were presented. The second day emphasized the molecular pathology of CONNECTIVE TISSUE MATRIX. Connective t i s s u e c e l l s '

populations and extracel1ular components are involved

from the very beginning of the inflammatory process. Research devoted to ext r a c e l l u l a r matrix has accelerated greatly over the past ten years. The growing f e e l i n g that almost every aspect of matrix research can be p r o f i t a b l y studied at the c e l l u l a r level has promoted u l t r a s t r u c t u r a l tissual

i n v e s t i g a t i o n s and

immunolabelling in d i f f e r e n t experimental model and human diseases.

During t h i s same period, the physico-chemical studies of p a r t i c u l a r matrix

VI components came to i n t e r e s t greatly cell b i o l o g i s t s and pathologists t r y i n g to v i s u a l i z e what kind of micro-domain might surround c e l l s involved in i n flammatory processes. L i v e r , lung, pancreas, colon, dentin, c a r t i l a g e ,

...

are target organs for inflammation and f i b r o s i s . Studies on granulomas ( s c h i s t o s o m i a s i s offers an e x c i t i n g model) are now leading to new developments where e x t r a c e l l u l a r matrix changes must be considered as an important additional factor implicated in the natural history of t h e i r formation. Chronic i n flammatory i n f i l t r a t e s contain factors generated during the c e l l u l a r immune response producing collagen accumulation and/or f i b r o b l a s t and myofibroblast stimulation. Recent progress on the characterization and synthesis of these mediators has opened new perspectives on a possible control of post-inflammatory f i b r o s i s and led t h i s f i e l d of i n v e s t i g a t i o n into a mostly exciting phase. The l a s t day was devoted to the design of synthetic DRUGS that may prevent or a l l e v i a t e inflammatory conditions. The application of biological agents such as interferon and monoclonal antibodies in immunopathology was also discussed. One of the aims of GERMI meetings i s to bring together in a common e f f o r t s c i e n t i s t s and c l i n i c i a n s from d i f f e r e n t countries for an exchange of experience and to focus on advances in the study and treatment of both acute and chronic inflammatory diseases. This aim has been l a r g e l y reached when we cons i d e r that more than 80 papers appear in the present proceedings. The Editors wish to express t h e i r sincere gratitude to Professor Frank W. Putnam of the department of Molecular Biology and Biochemistry, Indiana University (Bloomington, Indiana, USA) who acted as an expert president of t h i s Symposium. We would l i k e to thank the Vice Presidents of the Symposium: Doctor J.T. Whicher ( B r i s t o l , U.K.) and Professor M. Perrin Fayolle (Lyon, France) whose knowledge of the topics was greatly appreciated. The following i n s t i t u t i o n s provided f i n a n c i a l support: GERMI, I n s t i t u t Pasteur de Lyon et du Sud E s t , Fondation Merieux, Société Française d'Immunologie, Hospices C i v i l s de Lyon, Biomërieux, Hyland-Travenol, Hoechst Behring, Orimbio, B i o l y o n , Rhone Poulenc, Beckman, Sebia, Diagnostica Merck, Corning Medical.

We wish to express our profound gratitude to the organizing committee of the Symposium for t h e i r outstanding e f f o r t . This includes Helene Bernon, Abel

VII R o u l l e t , Professor M. Carraz, and Professor J . L . Touraine. The publication of these proceedings was made possible by the collaboration of authors and the s t a f f of Walter de Gruyter. Lyon, January 1986

J. Bienvenu J.A. P.rimaud P. Laurent

C O N T E N T S I N T R O D U C T O R Y

L E C T U R E

Progress in plasma protein related to inf larrmation. F.W. Putnam S E C T I O N

I

I N T E R L E U K

INS

The biological effects of purified and recaitoinant hunan interleukin 1. Ch. A. Dinarello

17

Are there defects in the production of interleukin 1 in disease ? J.T. Whicher, C. Westacott.

27

Negative acute phase protein : an interleukin 1 independent mechanism ? A. Fleck, M.A. Myers, Vasantha Nagendran.

37

Prostacyclin production frcm vascular endothelium interleukin 1. J.J.F. Belch, D. Shapiro, A. Shenkin, R.D. Sturrock.

41

is

enhanced

by

Effects of drugs altering arachidonic acid metabolism on interleukin 1 release from monocyte-1ike cells. D. Shapiro, J.J.F. Belch, R.D. Sturrock, A. Shenkin.

43

Murabutide induced production of non pyrogenic interleukin 1 by hunan monocytes and rabbit macrophages. C. Damais, G. Riveau, M. Parant, L. Chedid.

47

Does protracted IL-1 production cause a depression of cell mediated imnunity ? Vasantha Nagendran, M.A. Myers, A. Fleck, M.S. Pulley, D.C. Dunonde. Stable E rosettes : marker of activated hunan T-lymphocytes. A. Gaspar, P. Laurent, J. Marichy.

59

Production, purification and biological activities of natural hunan interleukin 2. L. Rimsky, H. Wakasugi, P. Robin, J.F. Lagabrielle, I). Fradelizi, J. Bertoglio.

63

Interleukin 2 (IL2) production by lymphocytes of hunan breast cancer patients. R. Lidereau, J. Oglobine, J. Anione, M. Renaud, M. Martinière, M.H. Chámpeme, A. Desplaces.

77

Prothymocytes in mouse fetal liver produce interleukin 2. M. Romano, J. Zenmour, J.L. Touraine.

83

X S E C T I O N AMYLOID

II P R O T E I N S

I s o l a t i o n and physico-chemical c h a r a c t e r i s t i c s of hunan serun amyloid P component from normal and acute phase s e r a . L. M i r i b e l , P. Arnaud.

89

Serum amyloid P carponent evaluation. B.A. F i e d e l , C . S . L . Ku.

99

(SAP)

and

hemostasis

:

a

continuing

Mechanism of induction of synthesis of serun amyloid P carponent (SAP) by mouse hepatocytes in c u l t u r e . R . F . Mortensen. R e a c t i v i t y of hunan serun amyloid P p r o t e i n (SAP) with concanavalin A. A. Mackiewicz, S . Mackiewicz, J . A . K i n t , J . G . Leroy, I . Vandermeulen. A new binding s p e c i f i c i t y practical applications.

for

serim

amyloid

P

component

105

117

and i t s

D. Serban, Ch. Rordorf-Adam.

125

Structure and Ffunction of Serun Amyloid A protein (SAA). Benson, . E . Dwulet, B. Kluve-Beckerman, M.Aldo-Benson. M.D.

129

Serun Amyloid A protein (SAA) - a s e n s i t i v e inflanmation marker. G. Marhaug, M. Ostensen, G. Husby, A. Husebekk, K. S l e t t e n .

139

SAA as a marker of inflammatory d i s e a s e . R . E . Chambers, J . T . Whicher.

145

Clinical usefulness of SAA and disorders : a conparative study.

CRP measuranents

in

inf lamnatory

C . P . J . Maury.

153

Iirmunonephelcmetric SAA measurement H. Bernon, J . Bienvenu, P. Laurent, M. Peyret.

157

S E C T I O N OTHER

III

HUMAN

ACUTE

PHASE

P R O T E I N S

Does hunan C-Reactive Protein c i r c u l a t e in ccmplexed form ? Equilibrium chromatography and d i a l y s i s s t u d i e s . M. Pontet, J . P . T r e s c a , M. O l l i v i e r , R. Engler.

XI

Evaluation of a q u a n t i t a t i v e l a t e x assay f o r CRP determination by l a s e r nephelometry : r e s u l t s of a c o l l a b o r a t i v e study. F. D a t i , W. Kapmeyer, A. Adam, J . Bienvenu, R.L. Hunbel, W. M u l l e r .

169

Alpha 1 - a n t i t r y p s i n and haptoglobin in normal and a l l e r g i c responses. M.A. Santos Rosa, M.F. Gareào, A.J.A. Robalo Cordeiro, C. Guimaràes, F. Campilho, J . Fleming T o r r i n h a .

175

Hie molecular v a r i a n t s of alpha 1 - a c i d g l y c o p r o t e i n : study of the g l y c o s y l a t e d dimeric, polymeric, and albimin-corrplex forms. L. M i r i b e l , P. Arnaud.

183

Fibronectin fragments act as cryoprecipi t a t ion. J . C . Renversez, C. Revol, M.J. V a l l é .

195

promoters

of

inmunoglobul in

F i b r o n e c t i n level in amniotic f l u i d . A.M. Mangé, F. B e t t r a y , F. Muí 1er.

201

HieAdam, himan J kal 1 ikrein-kininogen A. . Damas, J . Lecomte. - system.

205

Protease a c t i v i t é s in the cytosol of b r e a s t t u n o r s . J . Oglobine, C. Bouchet, F. Spyratos, K. Hacène, Desplaces.

C.

Brault,

A.

Serim p r o t e i n p r o f i l e e v o l u t i o n during moderate m a l n u t r i t i o n . M. David, A. Lobera, E. Legrand. SECTION

IV

ANIMAL

ACUTE

PHASE

PROTEINS

Biochemical c h a r a c t e r i s t i c s of acute phase p r o t e i n s in the r a t . R. Engler, F. Mege.

231

Synthesis of two r a t acute phase r e a c t a n t s : alpha 1 - a c i d g l y c o p r o t e i n and C3 by hepatocytes c o - c u l t u r e d with a l i v e r e p i t h e l i a l c e l l . J . P . Lebreton, M. Oaveau, M. Hiron, M. Fontaine, D. G i l b e r t , D. Biou, Ch. Guguen-Guillouzo.

24 3

Effects of 17-beta-oestradiol, 17-alpha-ethyniloestradiol, 5 - a l p h a - d i h y d r o t e s t o s t e r o n e and t u r p e n t i n e - o i l on the s y n t h e s i s of C3 and alpha 1 - a c i d glycoprotein in the r a t . M. Daveau, M. Hiron, M. Fontaine, J . P . Lebreton, D. Biou. 247 Production of acute phase p r o t e i n s by hepatocytes maintained in co-culture. A. Guillouzo, F. D e l e r s , G. B a f f e t , R. Engler, C. Guguen-Guillouzo

253

XII

Biosynthesis and secretion of haptoglobin by chicken embryo hepatocyte primary culture. F. Delers, R. Engler. 261 The Brown Norway deficient rat as a tool for the study involvement of the kinin system in sane physiological processes. J. Damas, A. Adam. Transferrin and alpha 2-macroglobulin-I reactants in the mouse. P.M.H. Heegaard, T.C. Bog Hansen.

are

of the 26 5

circulating acute-phase 275

A comparative study on the physico-chemical properties of himan, rabbit and rat C-reactive protein and serum amyloid P-ccmponent. M. Ollivier, M. Pontet, R. Engler. S e n m amyloid P component in the assessment of arthritis activity in autoimmune MRL/lpr/lpr mice. C. Rordorf-Adam, D. Serban, A. Pataki. 2 97 Hie effects of different drugs upon the level of serun amyloid P carponent in autoimmine MRL lpr/lpr mice. C. Rordorf-Adam, D. Serban, M. Gruninger, B.F. Rordorf. 303

S E C T I O N

V

C O N N E C T I V E M A T R I X AND I N F L A M M A T O R Y P R O C E S S E S The extracellular matrix and the inflaimiatory process. J. Labat-Robert, L. Robert, W. Hornebeck.

311

Structure andW.E. function of D.M. connective tissue proteoglycans. J.R. Hassel, Horton, Noonan, K.J. Doege, G.W. Laurie.

327

Isolation and characterization of fibronectin from mouse plasma. G. Caillot, G. Picelli, D.J. Hartmann, G. Ville.

337

Type V collagen : heterogeneity in different tissues. J. Rauterberg, D. Troyer.

343

Type VI collagen and inflammation. R. Garrone, J. Tiollier.

357

Basement membranes and tumoral invasion in the manmary gland. C. Clavel, P. Birembaut, J.J. Adnet, J.M. Foidart.

363

Fibroblasts and fibrocytes. Ch. M. Lapiere.

369

Cytoskeletal and cytocontractile features of myofibroblasts. G. Gabbiani.

375

XIII Collagen metabolism in the aorta of spontaneously hypertensive rats : variations in enzyme activities according to blood pressure and age. C. Falcy, A. Grochulski, N. Gilson, A.M. Borsos, N. Christeff, J.P. Dupeyron, J. Peyroux, M. Sternberg, J. Far jane 1, A. Rat trier.

387

Collagen metabolism in the heart of spontaneously hypertensive rats : variations in enzyme activities at different ages. C. Falcy, N. Gilson, A.M. Borsos, N. Christeff, J.P. Dupeyron, J. Peyroux, M. Sternberg, A. Rattner, J. Farjanel. Cell-cell and cell-matrix interactions in normal and cirrhotic liver. M. Rojkind.

3 9 9

Light and electron microscopic innnmolocalization of fibronectin and type I collagen in adult rat hepatocytes during primary culture. B. Clement, M . Rissel, S. Peyrol, J.A. Grimaud, A. Guillouzo.

411

Sinusoids and the Disse space in patients with liver diseases. P. Biouliac-Sage, C. Balabaud, J. Dubroca, L. Boussarie, J.A. Grimaud, Ph. Latry, H . Lamouliatte, A. Quinton.

417

Origin of collagens and fibronectin in normal and fibrotic liver. B. Clement, S. Peyrol, J.P. Campion, Y. Deugnier, J.A. Grimaud, A. Guillouzo.

433

Measurement of serun procollagen type III N-teiminal propeptide in patients with alcoolic liver disease. D.J. Hartmann, J.C. Trinchet, B. Galet, P. Callard, B. Nusgens; Ch.M. Lapiere, M. Beaugrand, G. Ville.

^43

The schistosome egg granuloma as a model of cell-mediated inflammation synthesis and degradation of connective tissue matrix. D.L. Boros.

4 4 9

Fibrous resorption in schistosomal granuloma. Z.A. Andrade, J.A. Grimaud.

461

Antibody to collagen CB-peptides alpha 2-CB(3,5) : a marker of type I collagen breakdown. H. Bnonard, S. Peyrol, S. Guerret-Stocker, M. Druguet, J.A. Grimaud.

473

The effect of malotilate in an experimental model of hepatic fibrosis induced by heterologous serun in the rat. J.M. Dunont, M.F. Maignan, D. Perrissoud. Development of fibrosis in hepatic alveolar echinococcosis : a sequential study in mice infected with Echinococcus multilocularis. D.A. Vuitton, Van-Pierre Tran, S. Guerret-Stocker, J.A. Grimaud, M. Liance, R. Houin.

XIV I n t r a a l v e o l a r f i b r o s i s : a r e v e r s i b l e fotm of pulmonary f i b r o s i s . S. Peyrol, J . F . C o r d i e r , J . A . Grimaud.' D i s t i n g u i s h i n g p a t t e r n s of a l v e o l i t i s and f i b r o s i s in disease. J . F . C o r d i e r , S. Peyrol, C. Takiya, J.A. Grimaud, J . Brune.

hunan

501 lung

P a n c r e a t i c connective m a t r i x changes in chronic p a n c r e a t i t i s . R. Kennedy, L. Uscanga, R. Choux, H. S a r l e s , D.E. Bockman, J . A . Grimaud.

SECTION

507

^21

VI

IMMUNOMODULAT

ION

Monoclonal a n t i b o d i e s t a r g e t i n g f o r cancer inmunotherapy. M.J. Ehibleton, R.W. Baldwin.

529

Inmunosuppressive p r o p e r t i e s of muramyl p e p t i d e s . C. L e d e r e , L. Chedid. The m u l t i p l e a cJt .i vWijdenes. i t i e s of i n t e r f e r o n garmia. J . Banchereau, Purine metabolism and inmunomodulation of monocyte f u n c t i o n s . M. P e t r i n i , P. Goldsclmit-Clermont, F. Ambrogi. I s o p r i n o s i n e ( i n o s i p l e x ) : an inmunological and c l i n i c a l review. J . Wybran.

543

551 571 579

Imuthiol. G. Renoux.

591

Inmunomodulation of macrophage f u n c t i o n s by a t r i - p e p t i d e CIKP) derived frcm IgG. C. A u r i a u l t , M. Joseph, El Hassan Hinmi, A. Capron, A. T a r t a r e .

599

Phospholipids and membrane phenomena involved in the r e l e a s e of mediators of anaphylaxis. Modulation by S-Adenosyl-L-Hcmocysteine and i t s s t r u c t u r e analogs. Y. Pacheco, N. B i o t , M. P e r r i n F a y o l l e , P. Fonlupt, A.F. P r i g e n t , H. Pacheco.

607

E f f e c t of the imnuncmodulator RU 41740 on the p r o l i f e r a t i o n of c u l t u r e d mouse embryo f i b r o b l a s t s . S. Durant, F. Homo-del arche, D. Duval, P. Stnets.

621

Evaluation of the thymus r o l e in the LH g e n e t i c hypertension by thymostimulin CTP-1). J.C.A. F r e i c h e , A. B a t a i l l a r d , J . L . Touraine, M. Vincent, J . Sassard.

625

XV TWo i n - v i t r o e f f e c t s of 40 639 RP in immunity mediated c e l l s . B. Dauvergne, F. Touraine, C. Quincy, J . L . Touraine, F. Floch.

631

Various imnunomodulating e f f e c t s of L-methionine i n - v i t r o . B. Dauvergne, F. Touraine, A. Galy, A. Guichard, J . L . Touraine, S. Ferrant.

635

SECTION

VII

LECTINS

AND

BIOLOGICAL

Identification of lectin like irrmunoa f f i n o e l e c t rophor es i s . R.M.F. Guinet, V. Rogemond, H. P e r r o l l e t . Lectin-induced egg j e l l y .

agglutination

of

A C T I V I T I E S bacterial

adhesins

by 641

microorganisms

in urodelan amphibian

A. Chesnel, H. Lerivray, P. Jego.

647

F i bCaron, r o n e c t i nR. asJ o au bmodel M. e r t . of m u l t i f u n c t i o n a l Modulation of phenobarbital. D. Monnet, J . Fournet.

glycosylation Feger,

D.

of

Biou,

lectin

rat

alpha

G.

Durand,

653 1-acid

glycoprotein

by

P. Cardon, Y. Leroy, B.

Study of the s p e c i f i c i t y of a r a t b r a i n e x t r a c t l e c t i n . R. J o u b e r t , M. Caron, M.A. Deugnier, J . C . Bisconte. V a r i a t i o n s in the p r o p o r t i o n s of microheterogeneous alpha 1 - a c i d g l y c o p r o t e i n in a l c o h o l i c c i r r h o s i s . N. S e t a , G. Durand, J . Agneray, J . Feger, M. Corbie.

661 667

forms of human 673

Assay of PHA m i t o g e n i c i t y on hunan lynphocytes a f t e r e l e c t r o - b l o t t i n g . B. Beal, P. Laurent, A. Gaspar.

679

AUTHOR

683

SUBJECT

INDEX INDEX

687

INTRODUCTORY

LECTURE

PROGRESS IN PLASMA PROTEINS RELATED TO INFLAMMATION

Frank W. Putnam Department of B i o l o g y , Indiana U n i v e r s i t y Bloomington, IN 47405, USA Introduction I t i s a p l e a s u r e and an honor for me to g i v e the i n t r o d u c t o r y Third Symposium. years.

l e c t u r e to the

We can r e f l e c t with pride on the advances of the past

four

I n d e e d , i n the two y e a r s s i n c e the Second Symposium on I n f l a m m a t i o n

Markers, a t r u l y major development has occurred i n knowledge of the m o l e c u l a r b i o l o g y o f a c u t e phase p r o t e i n s and o f the p o l y p e p t i d e l y m p h o k i n e s s u c h as interleukins

which

are

associated

with

i n c r e a s e i n k n o w l e d g e about i n t e r l e u k i n s

inflammation.

I n some ways

the

i s more s t a r t l i n g and s i g n i f i c a n t

than the advances made with acute phase p r o t e i n s ; for the l a t t e r act mainly as markers

of

mediators.

inflammation,

whereas the i n t e r l e u k i n s

As a protein chemist,

the many-named,

are o f t e n the

actual

i t g i v e s me great s a t i s f a c t i o n to see that

and often o v e r l a p p i n g f a c t o r s of immunology and inflammation

are b e i n g p u r i f i e d and shown to be p o l y p e p t i d e s o f d e f i n e d s t r u c t u r e , the genes for which are being cloned and sequenced. biochemical

p r o p e r t i e s , t h e i r chemical

and t h e i r r o l e s

And e q u a l l y important,

their

r e l e v a n c e to the acute phase response,

in inflammation are being c l a r i f i e d ,

as we s h a l l

hear today.

This g i v e s us hope, not o n l y for understanding the b a s i c biochemical

processes

but a l s o f o r i n t e r v e n t i o n to p r e v e n t or reduce i n f l a m m a t i o n by

rational

a p p r o a c h e s o f immunopharmacology. T h i r d Symposium.

These r e p r e s e n t the main themes o f o u r

However, r a t h e r than i n t r o d u c e the c h i e f t o p i c s o f our

Symposium and then t o t r y to a n t i c i p a t e t h e i r h i g h l i g h t s , I h a v e c h o s e n t o r e v i e w r e c e n t p r o g r e s s i n a more l i m i t e d a r e a , n a m e l y , the p l a s m a p r o t e i n s r e l a t e d t o i n f l a m m a t i o n w i t h e m p h a s i s on r e c e n t work o f my own l a b o r a t o r y . This review i s based on the r e c e n t l y p u b l i s h e d Volume IV of my t r e a t i s e , The Plasma P r o t e i n s , which s h o u l d be c o n s u l t e d for references to the work of many laboratories

that

I c i t e (1).

I n the p a s t

few y e a r s

there

has been g r e a t

progress

in e l u c i d a t i n g

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the

4 structure of plasma proteins, e s p e c i a l l y those of man.

The role of antibodies

in inflammatory processes is being made more clear both by the advances in the structural

study of immunoglobulins

antibodies.

Sequence determination

and by the widespread of the 5 classes

use of

monoclonal

of both human and mouse

immunoglobulins is now complete, but the v o l u m e of sequence data continues to rise exponentially. of m o n o c l o n a l

The same logarithmic law governs the rise in applications

antibodies

in

research,

for d i a g n o s t i c

applications,

and

p o t e n t i a l l y for therapeutic uses.

Sequence

analysis

of

plasma

proteins

entering a similar exponential of sophisticated

other than

phase (Fig. 1).

new methods of

immunoglobulins

First this came about

rapid automatic

sequencing

is

now

because

of proteins.

Now

these methods are hard pressed to keep up with the results of gene cloning and nucleotide

sequencing

techniques which began about

1980.

The consequence

is

that we now know the primary structure for at least 50 of the 100 or so human p l a s m a proteins,

and the carbohydrate

structure

for 25.

The genes for about

40 p l a s m a p r o t e i n s h a v e b e e n c l o n e d , and the c D N A s e q u e n c e and t h e g e n o m i c structure reported in many cases.

Furthermore, we are learning the chromosomal

location of the genes for many human plasma proteins. for the plasma protein, on the s a m e c h r o m o s o m e , transferrin, are a l l

the

and m a y be u n d e r a c o m m o n c o n t r o l .

transferrin

receptor,

and the

l o c a t e d on h u m a n c h r o m o s o m e 3.

the grand design

In some cases, the gene

its receptor, and a related growth factor are

governing

related

For

located example,

p97 m e l a n o m a

antigen

T h i s b e g i n s to g i v e us i n s i g h t

the system that promotes tissue growth and

on the one hand, and inflammation and necrosis on the other.

Another

is the coordinate regulation of expression of serum albumin and t h e g e n e s f o r w h i c h are l o c a t e d o n l y

into

repair example

a

fetoprotein

13.5 kB a p a r t on the s a m e

chromosome.

The lipoproteins represent another example.

Amino Acid Sequence of Human Acute Phase

Proteins

W e n o w k n o w t h e c o m p l e t e a m i n o acid s e q u e n c e of a l m o s t al 1 t h e m a j o r a c u t e phase proteins of man and a l s o the sequence of several proteins of the rat and the mouse. a m i n o acid s e q u e n c e of h u m a n

of the acute phase (AP)

As I reported at the Second Symposium,

a^-acid glycoprotein («^S),

( a j - A T ) , C - r e a c t i v e p r o t e i n (CRP), h a p t o g l o b i n

«j-antitrypsin

(Hp), and f i b r i n o g e n

had already been determined by methods of protein sequencing.

the

(F11)

At that time, I

5

Fig. 1. Increase in amino acid sequence data (excluding immunoglobulins) for all plasma proteins of all species. Updated from Fig. 4, p. 22 of Putnam (1). The dates for publication of the sequence of human plasma proteins are indicated by standard symbols for the proteins. For acute phase reactants the symbols are defined in the text. The Greek letters at the abscissa identify the dates when the complete amino acid (protein) sequences were published for the human light (K , x) and heavy (Y » P . E, O J , A 2» 9 5 % p u r e h u m a n SAP w i t h a y i e l d of 65%. contaminants

Sepharose

in the

covalent polymers. physico-

inflammatory

sera

92

669 K»440 K ^ 232 K»140 K ^

1

2

3

4

5

6

Fig. 1 - Immunoelectrophoresis of human control plasma (lanes 1 and 4), of fractions pooled and concentrated from Sepharose 4B (lane 2) and Sepharose 2B (lane 3). T, antiserum to total human serum, SAP, specific antiserum to SAP protein. Anode at the right.

1 -

2 SAP 3 4

..

^

S * ® ^ "

Fig. 2 - Polyacrylamide gradient gel electrophoresis of purified fractions of SAP. Lanes 1 and 2: low and high M.W. markers. Lanes 3 and 4: fractions pooled and concentrated from Sepharose 4B, lanes 5 and 6 from Sepharose 2B. Anode at the bottom. The gel was stained with 0.5% Coomassie Blue R250 in ethanol:watertacetic acid (9:9:2 v/v).

93

— 4 5

PL

Fig. 3A - SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of SAP p u r i f i e d fraction. Lane 1: reduced sample. Lane 2: non reduced sample. Lane 3: non reduced and non heated sample. L a n e s 4 and 5: high and low M . W . m a r k e r s . Stained with C o o m a s s i e Blue. A n o d e at the bottom. B

- T r a n s b l o t on n i t r o c e l l u l o s e m e m b r a n e following S D S - P A G E . The p r i m a r y a n t i s e r u m used was d i r e c t e d to human SAP. L a n e s 1 to 3 as in 3-A.

was explored. M.W.

No d i f f e r e n c e could be o b s e r v e d

in their

In c o n t r a s t , studies p e r f o r m e d on a n a l y t i c a l

respective

IEF g e l s

showed

a h e t e r o g e n e o u s p a t t e r n w h i c h c o n s i s t e d of about 10 to 12 b a n d s (Fig. 4A). according

In a d d i t i o n , a c l e a r l y d i f f e r e n t p a t t e r n was to the o r i g i n of the serum,

c a t h o d a l w h e n isolated from i n f l a m m a t o r y by the i m m u n o b l o t of an a n a l y t i c a l

IEF

serum. This was

(Fig. 4B).

The

p o i n t s of the p r o t e i n were found to be close to those reported normal

(2,12)

i.e., between 5.0 and 6.2 w h e n

serum and b e t w e e n pH 5.5 and 6.7 w h e n

m a t o r y serum.

evident

i.e., SAP b a n d s were

confirmed

isoelectric already

isolated

isolated

more

from

from

inflam-

This d i f f e r e n c e , w h i c h seems to affect equally

w h o l e p a t t e r n , could be related to a change

in the

the

glycosylation

94

A

Fig.

B

4A - A n a l y t i c a l IEF on p u r i f i e d SAP f r a c t i o n s . Lanes 1, 2 and 12: SAP isolated from i n f l a m m a t o r y h u m a n serum. L a n e s 3 to 9: SAP isolated from normal human serum using S e p h a r o s e 4B. Lanes 10 and 11: SAP isolated from n o r m a l h u m a n serum using S e p h a r o s e 2B. T h i c k n e s s of the g e l : 0.2 m m ; a c r y l a m i d e and bis a c r y l a m i d e : T5C2.5LKB a m p h o l i n e s 3.5-10 6% - u r e a : 8M. The gel was run for 3 h o u r s at 3W c o n - s t a n t p o w e r , then silver stained (35). A n o d e at the top. B

- T r a n s b l o t on n i t r o c e l l u l o s e m e m b r a n e after an analytical IEF: same c o n d i t i o n s as above e x c e p t for the LKB ampholines: 3.5-10 (3%), 4-6 (1.7%) and 5-8 (1.7%). L a n e s 1 and 2: SAP isolated from i n f l a m - m a t o r y human serum. Lanes 3 to 6: SAP isolated from normal human serum.

of the p r o t e i n during

the p r o c e s s of i n f l a m m a t i o n .

r e s u l t s have been reported for a l p h a ^ - a n t i t r y p s i n protein

(30-31).

already

reported

Since some m i c r o h e t e r o g e n e i t y (7,29)

in c o n t r a s t

to CRP,

for SAP has

to further e x p l o r e

analytical

IEF involves amino acids s u b s t i t u t i o n s

significance

alpha^-glycobeen

it could be of

importance

if the h e t e r o g e n e i t y

in the g l y c o s y l a t i o n of the protein.

Similar and

In a d d i t i o n ,

observed and/or

in

changes

it will be of

to d e v e l o p t e c h n i q u e s w h i c h will allow d i r e c t

study

95 of SAP in plasma samples, despite its low concentration.

The

progress of immunoblot following IEF, especially using Zeta bind membranes

(32, and Miribel and Arnaud, in preparation)

with more refined and specific staining techniques cate that this will be feasible soon.

together

(33-34)

indi-

Then, the effects of the

inflammation process on the molecule will be possible to study with respect to time and type of triggering mechanism, as already performed for classical acute-phase reactants

(30,31).

Finally,

our result, which do not exclude the existence of a polymorphism in the primary structure of SAP as well as the suggested

associ-

ation of some forms of SAP with the development of amyloidosis (29), raises the question of the significance of SAP during the process of

inflammation.

Acknowledgement Publication No. 734 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.

Supported in part by grants from the French Foreign

Office, from the Philippe Foundation, Inc., and from Labcatal Laboratories, Montrouge, France.

L. Miribel was a postdoctoral

fellow of the College of Graduate Studies and University

Research,

Medical University of South Carolina.

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2.

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3.

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1983.

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313.

1981.

SERUM AMYLOID EVALUATION

P COMPONENT

(SAP)

AND

HEMOSTASIS:

A

CONTINUING

B . A . Fiedel D e p a r t m e n t of I m m u n o l o g y / M i e r o b i o l o g y , R u s h M e d i c a l C e n t e r , C h i c a g o , I l l i n o i s , 6 0 6 1 2 , USA ( P r e s e n t A d d r e s s : D i v i s i o n of I m m u n o l o g y , J a m e s N . G a m b l e I n s t i t u t e of M e d i c a l R e s e a r c h , C i n c i n n a t i , O h i o , 1)5219, U S A )

C . S . L . Ku D e p a r t m e n t of B i o c h e m i s t r y , M o l e c u l a r a n d C e l l B i o l o g y , Northwestern University, Evanston, Illinois, 60201, USA.

Introduction

R e c e n t l y , we (1) a n d o t h e r s (2) i n v e s t i g a t e d a r o l e f o r S A P in coagulative processes. In p a r t i c u l a r , we f o u n d t h a t S A P c o u l d i n t e r f e r e w i t h t h e c o n v e r s i o n of f i b r i n o g e n to f i b r i n , a n a c t i v i t y e n h a n c e d by t h e p r e s e n c e o f h e p a r i n , a n d c o u l d a l t e r f i b r i n - f i b r i n polymerization. T h e p u r p o s e of t h i s p r e s e n t a t i o n is to e l a b o r a t e u p o n d a t a o b t a i n e d f r o m our c o n t i n u i n g e v a l u a t i o n of the e f f e c t s of S A P u p o n h e m o s t a t i c e v e n t s . T h e q u e s t i o n s we a s k e d i n c l u d e d : is t h e a n t i c o a g u l a n t a c t i v i t y e x p r e s s e d b y S A P r e l a t e d t o c h a n g e s in a n t i - t h r o m b i n I I I b e h a v i o r , d o e s S A P i n c o r p o r a t e i n t o f o r m i n g c l o t s , d o e s it a f f e c t n o r m a l c l o t r e t r a c t i o n , a n d d o e s it h a v e a n effect upon Factor XIII activity?

Materials

and

Methods

S e r u m a m y l o i d P c o m p o n e n t was i s o l a t e d as d e s c r i b e d (1,3). APTT , PT a n d t h r o m b i n c l o t t i m e s w e r e a l s o p e r f o r m e d a s d e s c r i b e d ( 1 ) , as w e r e f i b r i n o p e p t i d e A a n d f i b r i n p o l y m e r i z a t i o n a s s a y s . Antit h r o m b i n I I I a c t i v i t y w a s d e t e r m i n e d u s i n g a k i t p r o v i d e d by Abbott Laboratories and used chromogenic substrate methodology; c l o t r e t r a c t i o n w a s a s s e s s e d a c c o r d i n g to ( 1 , 1 ) .

Results

and

Discussion

W e h a v e s h o w n t h a t S A P a n d h e p a r i n o p e r a t e d s y n e r g i s t i c a 1 1 y to i n c r e a s e c l o t t i m e s in c i t r a t e d c e l l - f r e e p l a s m a ( C F P ) w h e n C F P c o a g u l a t i o n w a s i n i t i a t e d t h r o u g h t h e i n t r i n s i c , e x t r i n s i c or terminal pathways; data illustrating these points are p r e s e n t e d Tables I-III.

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

in

100 TABLE APTT

CLOT

TIMES

SAP

IN T H E

HEP

I

PRESENCE

+/-

OF

SAP

Clot

Ug/ml

AND

HEPARIN

Time

Average

(Seconds) of

_

_

42 . 0

-

+

60.6

+ + + +

62.9 60 . 2

10 25 40 60 80

110.8 138.0 167.6 >>200 (no

+ +

1 00

TABLE TISSUE

HEP

+ /-

PRESENCE

Clot Times (Seconds) Average of T h r e e

Ug/ml -

34.2 -

-

53.9

+ + +

10 20 40 60

62.3 80.7 153.2 187.4

+ +

100

>>200

TABLE THROMBIN

SAP Ug/ml

c lot)

11

T H R O M B O P L A S T I N T I M E IN T H E OF SAP AND HEPARIN

SAP

Three

TIMES

IN

HEP

THE

+/-

_ -

+

20

+

25

+

(no

clot)

III

PRESENCE

OF

SAP

Clot

AND

HEPARIN

Time

32.6; 51.2; 57.4; >>200

(Seconds)

33.1 53.4 62.1

(no

clot)

H o w S A P and h e p a r i n s y n e r g i z e d to b r i n g a b o u t i n c r e a s e d c l o t t i n g t i m e s w a s f i r s t a p p r o a c h e d by a s k i n g w h e t h e r SAP a n d / o r h e p a r i n could alter r e g u l a t o r y p a t h w a y s and/or d e - s t a b i l i z e systems c r i t i c a l to the f o r m a t i o n of a p a t e n t c l o t . As p r e s e n t e d in T a b l e I V , it b e c a m e e v i d e n t t h a t SAP and h e p a r i n r e d u c e d the g e n e r a t i o n of f i b r i n o p e p t i d e A (F PA ) from f i b r i n o g e n .

101 TABLE

IV

E F F E C T O F SAP A N D H E P A R I N ON T H R O M B I N C L O T T I M E , R E L E A S E O F FI B R I N O P E P T I D E A AND T H R O M B I N A C T I V I T Y Plasma

Clot

Control + SAP (50 y g / m l ) + Heparin (0.08 U / m l ) + SAP (50 y g / m l ) Heparin (0.08 U / m l )

Times

(s)

Released F PA ( y g / m l )

Thrombin Activity (U/ml)

23 ..9 26 ,.8

1 5 1 6

(100?) (106%)

0 .22 . 0 .27 ,

48 ,. 2

9

( 6 0?)

0. , 1 7

200 ,. 0

5

(

0.. 25

32?)

T h a t SAP a n d h e p a r i n r e d u c e d FPA l e v e l s w a s an i m p o r t a n t f i n d i n g s i n c e r e d u c e d FPA l e v e l s t r a n s l a t e into l e s s a v a i l a b l e f i b r i n for cross-linking, hence a less patent clot. S A P and h e p a r i n a l s o interfered with fibrin monomer p o l y m e r i z a t i o n ; thus, these stages of c o a g u l a t i o n a p p e a r e d to be t h o s e p r e d o m i n a t e l y a f f e c t e d . W h e t h e r S A P m o d i f i e d the r e g u l a t o r y i n f l u e n c e of a n t i - t h r o m b i n III ( A T - I I I ) on t h r o m b i n a c t i v i t y r e s u l t i n g in r e d u c e d FPA p r o d u c t i o n was tested next. As d e p i c t e d in T a b l e V, SAP at c o n c e n t r a t i o n s w h i c h i n c r e a s e d c l o t t i m e s in CFP (20-1 0 0 y g / m l ; T a b l e s I - I I I ) r e d u c e d s l i g h t l y , r a t h e r t h a n e n h a n c e d , A T - I I I a c t i v i t y as w o u l d h a v e b e e n p r e d i c t e d f r o m the FPA d a t a . T h u s , SAP a p p e a r e d to b e a n t i - h e p a r i n in the A T - I I I a s s a y .

TABLE EFFECTS SAP

(yg/ml) 0 50 100 150 250

V

O F S A P ON A N T I T H R O M B I N IN N O R M A L P L A S M A

III

? Antithrombin

ACTIVITY III

Activity

92.5 98.0 86.0 8 4.0 85.5

F i b r o n e c t i n a n d f i b r i n ( - o g e n ) are k n o w n to be i n c o r p o r a t e d into f o r m i n g c l o t s a n d c r o s s - l i n k e d t h r o u g h the a c t i o n of c o a g u l a t i o n Factor XIII. As a p p r o p r i a t e l y a g g r e g a t e d S A P h a s b e e n r e p o r t e d to b i n d w i t h f i b r o n e c t i n (5), a n d n e p h e l o m e t r i c a n a l y s e s s u g g e s t an i n t e r a c t i o n b e t w e e n SAP and f i b r i n o g e n (1), we q u e s t i o n e d w h e t h e r S A P c o u l d d e p o s i t in a f o r m i n g c l o t and i n t e r f e r e w i t h c l o t retraction. As p r e s e n t e d in T a b l e V I , S A P a l o n e i n c o r p o r a t e d i n t o f o r m i n g c l o t s , a n d t h i s w a s p o t e n t i a t e d by h e p a r i n . Furthermore, S A P and h e p a r i n s y n e r g i s t i c a 1ly r e d u c e d c l o t r e t r a c t i o n by - 1 9 ? o v e r the c o n t r o l ( T a b l e V I I ) . S A P a l o n e at s u p r a - p h y s i o 1 o g i c a l c o n c e n t r a t i o n (200 y g / m l ) was i t s e l f an i n h i b i t o r of c l o t retraction.

102 TABLE

VI

I N C O R P O R A T I O N OF 1 2 5 I - S A P C L O T S IN T H R O M B I N - T R E A T E D

SAP

(ug/ml)

%

INTO FIBRIN FIBRINOGEN*

Incorporation

Control

0

+ SAP (30 y g / m l ) Heparin

17 .9

+ SAP (50 y g / m l )

45 .1

+ SAP (50 y g / m l ) Heparin

61.5

*The concentration was 1 U/ml.

of

TABLE

heparin

used

VII

E F F E C T OF S A P , H E P A R I N AND S A P / H E P A R I N C L O T R E T R A C T I O N OF N O R M A L P L A S M A

Platelet

Rich

Plasma

Control + SAP (80 p g / m l ) + SAP ( 2 0 0 u g / m l ) + H e p a r i n (0.02 U / m l ) + SAP (80 y g / m l ) H e p a r i n (0.02 U / m l )

% Clot Retraction

ON

%

Inhibition

57 50 43 57

0 12.5 25 . 0 0

46

19.0

C r i t i c a l to the d e v e l o p m e n t of c l o t r e t r a c t i l e f o r c e is coagulation Factor XIII (FXIII). W h e n we a s s e s s e d w h e t h e r S A P a n d / o r h e p a r i n c o u l d m o d i f y F X I I I a c t i v i t y ( T a b l e V I I I ) , we o b s e r v e d t h a t e a c h SAP and h e p a r i n i n h i b i t e d p u r i f i e d F X I I I , a s t h e y did in c o m b i n a t i o n ; h o w e v e r , in c o m b i n a t i o n , S A P r e d u c e d s u b s t a n t i a l l y the m o r e n e g a t i v e i n f l u e n c e of h e p a r i n on F X I I I activity. This r e p r e s e n t e d , along with A T - I I I , a second antih e p a r i n e x p r e s s i o n of S A P .

103 TABLE EFFECTS

Factor

XIII

Vili

O F SAP A N D H E P A R I N ON T H E OF PURIFIED FACTOR X H I a I n c o r p o r a t i o n of Monodansyl Group (uMole/hr) Representative Assay

ACTIVITY

% C h a n g e in Activity

Control

1 .98

0

+

1.28

-36

+ SAP (100 p g / m l )

1 .62

-18

+

1.70

- 1 1)

Heparin (0.1 U / m l )

SAP/Heparin

T h e d a t a p r e s e n t e d h e r e i n i l l u s t r a t e t h a t the p r e v i o u s l y a p p r e c i a t e d a n t i c o a g u l a n t p r o p e r t i e s of S A P in the p r e s e n c e of h e p a r i n r e l a t e to a n i n h i b i t i o n in f i b r i n g e n e r a t i o n f r o m fibrinogen and decreased f i b r i n - f i b r i n p o l y m e r i z a t i o n . This a f f e c t c a n be e x t e n d e d f u r t h e r to c l o t r e t r a c t i o n , p e r h a p s as a c o n s e q u e n c e of SAP d e p o s i t i n g ( b i n d i n g ) in f o r m i n g c l o t s , b u t n o t to the m o d i f i c a t i o n by SAP of A T - I I I or F X I I I a c t i v i t y w h e r e its e x p r e s s e d n a t u r e is a n t i - h e p a r i n .

Ac k n o w l e d g e m e n t F u n d e d , in p a r t , by a g r a n t ( H L - 2 3 1 5 7 ) f r o m the N I H . B A F is r e c i p i e n t of R e s e a r c h C a r e e r D e v e l o p m e n t A w a r d H L - 0 0 6 1 4 f r o m the NIH. C S L K p e r f o r m e d a s p e c t s of t h i s w o r k as p a r t i a l f u l f i l l m e n t of the r e q u i r e m e n t s for the P h . D . D e g r e e in the D e p a r t m e n t of Immunology/Microbiology, Rush Medical Center, Chicago, USA.

References 1.

K u , C . S . L . and B . A. F i e d e l . 1 9 8 3 . M o d u l a t i o n of f i b r i n f o r m a t i o n by h u m a n s e r u m a m y l o i d P c o m p o n e n t (SAP) a n d h e p a r i n . J. E x p . M e d . 1 5 8 : 7 6 7 - 7 8 0 .

clot

2.

P e p y s , M . B . , G . J . B e c k e r , R . F . D y c k , A. M c C r a w , P. H i l g a r d , R . E . M e r t o n a n d D . P . T h o m a s . 1 9 8 0 . S t u d i e s of h u m a n s e r u m a m y l o i d P - c o m p o n e n t (SAP) in r e l a t i o n to c o a g u l a t i o n . C l i n i c a Chimica Acta 1 05:83-91 .

104 3.

F i e d e l , B . A . , C . S . L . K u , J . M . I z z i a n d H. G e w u r z . 1 9 8 3 . S e l e c t i v e i n h i b i t i o n of p l a t e l e t a c t i v a t i o n by the a m y l o i d coraponent of s e r u m . J. I m m u n o l . 131 : 1 11 6 - 1 1 1 9 .

i) .

Bowie, E . J . W . 1971. Mayo C l i n i c L a b o r a t o r y M a n u a l Hemostasis. Saunders, Philadelphia. pp29~31•

5.

de B e e r , F . C . , M . L . B a l t z , S. H o l f o r d , A . F e i n s t e i n a n d P e p y s . 1 9 8 1 . F i b r o n e c t i n a n d C M - b i n d i n g p r o t e i n are s e l e c t i v e l y b o u n d by a g g r e g a t e d a m y l o i d P c o m p o n e n t . J . Med. 154:1131-1119.

P-

of M.B. Exp.

MECHANISM OF INDUCTION OF SYNTHESIS OF SERUM AMYLOID P-COMPONENT (SAP) BY MOUSE HEPATOCYTES IN CULTURE

Richard F . Mortensen Department of M i c r o b i o l o g y , Ohio State U n i v e r s i t y Columbus, Ohio 43210, USA

Introduction A systemic inflammatory response i s characterized by the s y n t h e s i s of a group of blood p r o t e i n s c o l l e c t i v e l y termed acute phase reactants (APRs).

Serum

amyloid P-component (SAP) i s the major APR of mice and i s a member of the pentraxin family of p r o t e i n s , which includes C - r e a c t i v e protein (CRP), the prototypical

APR of man ( 1 ) .

Mouse SAP i s a 230 Kd a l p h a - g l y c o p r o t e i n composed

of two i n t e r a c t i n g p e n t r a x i n s , each containing f i v e i d e n t i c a l 28 Kd subunits (2, 3).

SAP has been p u r i f i e d on the b a s i s of i t s Ca + + -dependent binding

s p e c i f i c i t y for the galactan in agarobiose that i s s u b s t i t u t e d with pyruvate at the 4 , 6-enol p o s i t i o n ( 4 ) .

The induction of new APR s y n t h e s i s by the l i v e r

has long been appreciated as an event r e q u i r i n g a blood born mediator(s)

(5).

The i d e n t i f i c a t i o n of one of the mediators as i n t e r l e u k i n 1 ( I L 1) was f i r s t made using the induction of serum amyloid A (SAA) protein s y n t h e s i s by mouse hepatocytes treated with monokines (6) and l a t e r with p a r t i a l l y p u r i f i e d IL 1 (7).

E a r l i e r work by Kampschmidt ( r e v . i n 8) a l s o c l e a r l y implicated leukocyte

endogenous mediator, which l a t e r was found to be i d e n t i c a l or s i m i l a r to IL 1, i n APR s y n t h e s i s in the r a b b i t . We have recently e s t a b l i s h e d in v i t r o c u l t u r e c o n d i t i o n s for the induction and s y n t h e s i s of mouse SAP by hepatocytes ( 9 ) .

C o c u l t u r i n g the hepatocytes with

e i t h e r inflammatory ( e l i c i t e d ) or f u l l y activated macrophages (M4>s) g r e a t l y enhanced SAP production; furthermore, the a d d i t i o n of p u r i f i e d mouse IL 1 a l s o r e s u l t e d in a s u b s t a n t i a l

increase in the s y n t h e s i s of SAP ( 1 0 ) .

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

The mechanism

106 whereby IL 1 and/or ftys increase SAP synthesis may depend on an increase in the rate of SAP synthesis per c e l l , an increase in the number of SAP-secreting hepatocytes (recruitment), or p o s s i b i l y both.

The experiments described herein

were designed to determine the mechanism of the SAP response. Methods and Materials The methodology for c u l t u r i n g isolated mouse hepatocytes under conditions that result in SAP induction and synthesis i s described elsewhere ( 9 ) .

A s i n g l e cell

assay for detecting individual SAP-secreting hepatocytes was developed by using a sandwich ELISA procedure.

The assay i s based on the "capture" of the

SAP-secreted by a s i n g l e hepatocyte by s o l i d phase anti-SAP and then l o c a l i z i n g the enzyme (horseradish peroxidase, HRP) substrate reaction in an agarose s o l u t i o n ( F i g . 1). The reagents used are:

monospecific rabbit anti-mouse SAP (IgG) ( 3 ) , a

b i o t i n y l a t e d IgG antibody of the same s p e c i f i c i t y , avidin-HRP and o-phenylenediamine as the chromogen.

The dark spots observed represent the

" f o o t p r i n t " of a s i n g l e SAP-secreting hepatocyte.

Hepatocytes incubated for 30

hrs at 2.5 x 10 3 cells/cm 2 or 2 x 104 cells/35 mm t i s s u e culture plate gave c l e a r l y separated spots that permitted accurate counting.

Mouse SAP in the

culture f l u i d was quantitated by competitive ELISA as described previously (10). P u r i f i e d mouse IL 1 was obtained by the procedure described by Mizel (11) using supernatants from the superinduced P388Di (adherent) macrophage l i n e (12). s i n g l e preparation of 320 units/ml was used.

A

A macrophage supernatant was also

obtained from a 30 hr culture of t h i o g l y c o l 1 ate e l i c i t e d (3 days) peritoneal exudate adherent c e l l s (85-92% cytoplasmic esterase p o s i t i v e ) from C57BL/10ScN (LPS-unresponsive) mice.

IL 1 and the M(> culture supernatant were

chromatographed on a B i o - S i l TSK-250 (G 3000 SW) HPLC column (Bio-Rad, Richmond, CA).

107

T TT T T T

Ariti SAP A n t i b o d y

^

Hepatocytes SAP

VYVT

T TT

Biotinylated A n t i b o d y

4 * T T Figure 1.

r

Avidin-HPR

1

4

Procedure for detecting a single SAP-producing hepatocyte by a Reverse Enzyme-Linked Immunosorbent Spot (RELISPOT) assay.

RESULTS Macrophage mediated recruitment of hepatocytes.

Previous studies by us showed

that elicited Ms added to isolated hepatocytes resulted in increased SAP production (10); however, we did not know whether the increase in SAP levels reflected an increase in the fraction of SAP-secreting hepatocytes.

Using a

single-cell assay to detect the number of such cells, a 10-fold increase in the number of SAP-secreting hepatocytes was observed in the presence of elicited M$s at a ratio of 8/1 of hepatocytes/M)>s (Fig. 2). not alter the number of SAP-secreting cells.

Resident peritoneal Ms did

Inflammatory peritoneal M$s that

accumulate in response to a stimulus are not fully activated and secrete small amounts of IL 1 (13); however, the addition of these cells to hepatocytes at ratios as low as 1/32 (M$/hepatocyte) generated a significant increase in the number of SAP-secreting hepatocytes (Fig. 2).

The addition of syngeneic

108

x

50

CM

CO Ui I>o

40

0

H < Q. LU 1 O

30

Hi

0

HI CO 1

20

CL
s from C57BL/10SN mice cultured with syngeneic C57BL/10Sc hepatoyctes at 2xl0 4 hepatocytes/2.5xl0 3 M+ s in 35 mm plates.

Table 2.

Recruitment of Hepatocytes into SAP Synthesis by a Mf Culture Supernatant SAP Secreting Hepatocytes

Mi> Culture Supernatant (Dilution)

IL 1 (units/ml)

undi1.

6.0

7

(±D

1/5

3.0

10

(±3)

1/10

1.5

57

(±6)

1/20

0.75

41

(±4)

1/40

0.37

29

(±2)

1/80

0.18

23

(±6)

per 2 x l 0 4 C e l l s (SO)

measured by the standard thymocyte p r o l i f e r a t i o n assay for IL 1 (13). Both the

culture supernatant and IL 1 preparation were separated on the

b a s i s of size by HPLC and the f r a c t i o n s tested for t h e i r a b i l i t y to not only r e c r u i t , but also induce SAP s y n t h e s i s .

Monokines with a Mr of 40 Kd and 79 Kd

were most active in the r e c r u i t i n g assay ( F i g . 3 ) .

The IL 1 preparation

110

displayed much less r e c r u i t i n g a c t i v i t y .

The most active f r a c t i o n in the LAF

assay contained proteins of 16 Kd M r ; however, only the 32 Kd M r proteins displayed s i g n i f i c a n t r e c r u i t i n g a c t i v i t y ( F i g . 3 ) . Table 3.

When SAP l e v e l s in culture

Recruitment of Hepatocytes into SAP Synthesis By P u r i f i e d Mouse IL 1 IL 1

SAP-Secreting Hepatocytes

units/ml

per 2xl0 4 C e l l s (SO)

0

6 (±3)

13.0

18 (±7)

6.5

25 (±6)

3.2

15 (±5)

1.6

8 (±2)

0.8

7 (±2)

Figure 3.

Hepatocyte r e c r u i t i n g a c t i v i t y of a Mt> culture supernatant and

p u r i f i e d IL 1 separated by HPLC gel

filtration.

111

supernatants were measured in response to the same f r a c t i o n s containing monokines, the most s i g n i f i c a n t SAP production was induced by the 16 Kd protein within the IL 1 preparation ( F i g . 4 ) .

The enhanced SAP production by the 35 Kd

and 71 Kd monokines in the W)> culture supernatant most l i k e l y i s a r e f l e c t i o n of increased number of hepatocytes recruited into SAP s y n t h e s i s .

Thus,

p u r i f i e d IL 1 displayed minimal r e c r u i t i n g a c t i v i t y , but could induce an increase in the amount of SAP synthesized per c e l l . 16 Kd

K' Figure 4.

SAP production by hepatocytes in response to proteins present in

a Mtf> culture supernatant or an IL 1 preparation separated by size on HPLC. Effect of Anti-Mouse IL 1.

Since non IL 1 monokines appear to be

responsible for the recruitment of hepatocytes, a well-characterized goat anti-mouse IL 1 was used to test i t s effect on both hepatocyte recruitment

112 and SAP production (14).

In preliminary experiments a d i l u t i o n of 1/5000 was

found to neutralize the LAF a c t i v i t y of the p u r i f i e d IL 1 preparation. Treatment of IL 1 (4 units/ml) and the M)> culture supernatant with a n t i - I L 1 (1/500) resulted in i n h i b i t i o n of SAP production, but did not a l t e r hepatocyte recruitment a c t i v i t y (Table 4 ) .

Table 4.

Additional properties of the r e c r u i t i n g

Effect of Goat Anti-Mouse IL 1 on SAP Production and Hepatocyte Recruitment SAP-Secreting

Addition to Hepatocytes

AntiMo IL 1

SAP ng/ml

IL 1 (4 units/ml)

None(a)

323 (±15)

25 (±3)

1/500

132 (±33)

31 (±7)

235 (±15)

100 (±14)

246 (±24)

101 (±12)

-Culture Supernatant

a

None 1/500

Cells/2xl04

Controls contained a 1/500 d i l u t i o n of preimmune serum.

monokines are s e n s i t i v i t y to heat (70° C, 15 min), i n a c t i v a t i o n by proteinase-K and reduction in a c t i v i t y by exposure to pH 2 and pH 10.

The monokine-driven

r e c r u i t i n g a c t i v i t y was not s e n s i t i v e to either phenylglyoxal treatment which abolishes IL 1-driven LAF a c t i v i t y (13) or anti-a/g interferon antibodies.

The

r e s u l t s suggest that IL 1 alone does not drive APR s y n t h e s i s , but requires the additional monokines that i n i t i a l l y recruit hepatocytes

into subsequent IL

1-dependent induction of new SAP s y n t h e s i s .

Discussion

The increase in the blood concentration of APRs t r a d i t i o n a l l y was thought to reflect increased synthesis and secretion by each l i v e r parenchymal cell.

Since hepatocytes have been found to be heterogenous with respect to

113 serum p r o t e i n synthesis (15), i t

seemed l o g i c a l

that hepatocytes might also be

heterogenous in t h e i r response to the mediator(s) Evidence f o r sequential born mediators was f i r s t (16, 17).

inducing APR s y n t h e s i s .

recruitment of hepatocyte synthesis of an APR by blood shown f o r rabbit CRP by Kushner and his

colleagues

An increase in the number of hepatocytes s e c r e t i n g f i b r i n o g e n

and haptoglobulin

(19) were shown some years ago using

(18)

immunofluorescent

s t a i n i n g methods. The f i n d i n g s reported here show that recruitment can be examined in v i t r o with an a p p r o p r i a t e l y

s e n s i t i v e method for enumerating

single

hepatocytes s e c r e t i n g an APR. A scheme d e p i c t i n g the r o l e of IL 1 and the other monokines in the induction of ARP synthesis i s shown in Figure 5.

Multiple biological

inflammation have been described (reviewed in 20). M4>s need to be a c t i v a t e d to secrete s u b s t a n t i a l

functions

E l i c i t e d or

f o r IL 1 i n

inflammatory

amounts of IL 1 (21);

we have r e c e n t l y reported that p u r i f i e d mouse SAP s e l e c t i v e l y

however,

induces IL 1

INTRACELLULAR PATHOGENS or INFLAMMATORY STIMULUS

l

SAA FIBRINOGEN

Figure 5.

The c e l l u l a r and molecular components that p a r t i c i p a t e

induction of synthesis of an APR.

in the

114 production by inflammatory MJ>s under conditions that excluded endotoxin contamination (22).

Thus SAP at least in the mouse may function as an

amplifier of the acute phase response and i n d i r e c t l y contribute to heightened host resistance.

Further studies of the molecular events leading to the

induction of APR synthesis may provide a basis for modifying the inflammatory response.

Acknowledgement

This work was supported by USPHS grant CA 30015.

I thank Dr. Steven Mizel

for

h i s generous g i f t of goat anti-mouse IL 1.

References

(1)

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(2)

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(4)

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(5)

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(6)

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(7)

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(15)

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1982.

J . Immunol. 131:1834.

Ann. N.Y. Acad. S e i .

J . Am. Med. Assoc.

J. Clin. Invest.

187, 128.

45:314.

Rev. I n f e c t . D i s .

1985.

C e l l . Immunol.

93, 398.

REACTIVITY OF HUMAN SERUM AMYLOID P PROTEIN (SAP) WITH CONCANNAVALIN A

A. Mackiewicz, S. Mackiewicz Department of Immunology and Rheumatology, Medical Academy, Poznan, Poland J.A. Kint, J.G. Leroy Department of Pediatrics, Medical School, Gent State University, Belgium I. Vandermeulen I.W.O.N.L. - Belgium

Introduction Serum amyloid P component (SAP), a human serum protein with a molecular weight of 230.000 daltons, consists of 10 identical subunits. The latter polypeptide chains are arranged in two parallel rings, resulting in a regular pentagon (1). Calcium dependent binding to agarose was among the first described proporties of SAP (2). This binding has recently been shown to be due to the presence of pyruvated galactose in the agarose preparation (3). SAP is also identical to Clt, one of the lesser known factors in the complement chain (4). The exact amino acid sequence of human SAP is known (5). Although it has been surmised that human SAP is a glycoprotein (6), no experimental data about its carbohydrate content have been published. The present study is a first attempt towards obtaining some information on the identity and structural features of human SAP. Affinity electrophoresis with lectins as ligands is a reliable method for the detection of carbohydrate residues in proteins and for discovering microheterogeneity in a class of glycoproteins (7) (8).

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

118

Material and Methods Reagents : Electrophoretic buffers : Veronal Calcium (pH = 8 , 6 0,025 ionic strength). Tris-EDTA-borate (TEB) (pH = 8,6 - 0,025 ionic strenght), Agarose A (Mr = - 0.13, Pharmacia, Sweden), Agarose Janssen Chimica (Mr = - 0.10, Jansen Chimica, Belgium). Antibodies : goat antihviman SAP, IgG fraction (Atlantic Antibodies, U.S.A.) rabbit antihuman SAP gamma globulin fraction (Dako, Denmark). Lectins : soy bean agglutinin (SBA) was from Pharmacia, Sweden. Concanavalin A (Con A) was purified from jack bean by affinity chromatography on Sephadex G 75 as described (9). Lentil lectin (L.C.A.) from Lens culinaris seeds and Pisum sativum agglutinin (PSA) from garden peas were purified according to the same procedure. Solanum tuberosum agglutinin (STA) was kindly provided by Dr. E. Van Driessche (University of Brussels, Belgium). Alpha-methyl-D-mannoside (alpha MM) was purchased from Jansen Chimica, Belgium and N-acetyl-D-glucosamine from Sigma, U.S.A. Hydrolyzed agarose : 20 % agarose A was incubated in 2N HCL under nitrogen atmosphere for 4 hours at 60°C. The pH was then adjusted to 8,6 with solid Na2HP04 and concentrated NaOH. Excess of salts was removed by Amberlite Monobed MB-3 resin (B.D.H., Poole, England). Hydrolysed agarose when mixed with agarose A or agarose Jansen did not influence the electrophoretic mobility of serum proteins, as checked by zone electrophoresis. Purification of SAP : SAP was purified from pleura fluid of two adult patients suffering from lung cancer by means of calcium dependent affinity chromatography (2). Crossed Immunoelectrophoresis (CIEP) : was performed according to Axelsen et al. (10). All experiments were run in parallel in 1 % agarose Jansen and 1 % agarose A in veronal calcium and TEB buffers. Increasing amounts (0,1 % - 0,2 % - 0,4 % - 0,8 % 1,6 %) of hydrolyzed agarose were added to the first dimension gel in order to estimate the influence on SAP binding. The first dimension was run at 10 Volt-cm~l for 40 minutes, the second dimension in the antibody containing gel at 2 Volt-cm-^ for 16 hours. This second dimension gel contained 5 microliter of anti human SAP antiserum (Daka or Atlantic Antibody) per milliliter of gel. After electrophoresis the plates were pressed, dried and

119

stained with Coomassie Brillant Blue R 250 (Sigma). Affinity-electrophoresis (Aff-EP) : was performed as described by Bog-Hansen (11). All experiments were performed in 1 % agarose A and veronal-calcium buffer. The first dimension gel contained one of five lectins (Con A, L.C.A., S.B.A., S.T.A., P.S.A.) in a concentration of 1 mg per ml and - except controls - 0,4 % of the hydrolyzed agarose. The second dimension gel contained anti SAP antibodies (5 jil per ml of gel) and 10 % of alpha MM or 10 % of N-acetyl-D-glucosamine, except for the control experiment when sugars werd omitted. Electrophoresis and processing of the gel was performed as described for CIEP.

Results Crossed immuno-electrophoresis Non purified pleura fluid SAP is resolved into a retarded and a non-retarded fraction when a Veronal-calcium buffer is used. The resolution into two fractions of crude pleura liquid SAP was seen in agarose A, but not in agarose Jansen (Figure 1).

1A

1B

Figure 1. - Crossed immuno electrophoresis of SAP from pleura fluid in Veronal-calciumbuffer. First dimension:l % agarose without any addition. Second dimension:1 % agarose with anti SAP antiserum (see material and methods). A = agarose A; First dimension : Ca+2-Veronal buffer. B = agarose Jansen; First dimension : Ca+2-Veronal buffer.

120

Purified SAP does not show any precipitate in the second dimension of agarose Jansen. In agarose A however, an elongated precipitate appears (Figure 2 A). Upon addition of increasing amounts of hydrolyzed agarose in the first dimension gel in veronal calcium buffer (agarose A), the migration of purified SAP increases (figure 2). In CIEP with agarose Jansen, hydrolyzed agarose causes the appearance of a SAP-anti SAP precipitate as well as changes in the SAP mobility similar to those observed with agarose A.

2A

29.

Figure 2. - Crossed Immunoelectrophoresis of purified SAP in agarose A. First dimension : increasing amounts of hydrolyzed agarose. A = control, without hydrolyzed agarose. B = 0,1 % hydrolyzed agarose. C = 0,2 % hydrolyzed agarose. D = 0,U % hydrolyzed agarose.

In control experiments with either purified or non-purified SAP in TEB buffer, always a single peak of SAP with alpha-1-mobility is obtained in agarose A or in agarose Jansen. Affinity electrophoresis Free Con A in the first dimension gel (Figure 3). Non-purified SAP yields a low peak at the position of the too of the affinity precipitate when alpha MM is omitted from the second dimension gel. In the experiment with purified SAP no affinity precipitate in the first dimension and no SAP anti-SAP precipitate

121

in the second dimension gels are seen. Addition of 10 % alpha MM to the second dimension gel causes the appearance of two SAP peaks : variant 1 is retarded while variant 2 precipitates at the application well. The retardation of variant 1 and the relative amount of variant 2 increases while the relative amount of variant 1 decreases with an increase of the Con A concentration. Other lectins Application of LCA, SBA, PCA and STA in affinity electrophoresis does not result in any ratardation of the SAP migration or in its precipitation during the first dimension run. However, when non-purified SAP was examined, affinity precipitates with other glycoproteins appear with LCA and PSA.

3A

Figure 3. -

3B

Con The A = B = C = The and

3C

A affinity electrophoresis of purified SAP. first dimension gel contains con A : 0,125.10-5M 0,250.10-5M 0,500.10-5M second dimension gel contains anti SAP antiserum (0,5 10 % alpha-methyl-manno side.

Discussion 1. Calcium dependent interaction between agarose and SAP. The property that SAP binds strongly to agarose has been used for its affinity purification (2). Our results confirm the

122

conclusion of others (1) that the quality of the agarose itself is an important factor for the binding of SAP : the higher the pyruvate content of the agarose, the higher its binding capacity for SAP (3). In the agarose from one manufacturer (Pharmacia) about 50 % of the SAP gives an interaction with the agarose, while in the agarose from the other manufacturer almost 100 % of the SAP is retarded (Fig. 1A-1B). However this interaction is mucht stronger for purified SAP than for SAP in crude pleura fluid. 2. Interaction between SAP and lectins. Human, mouse and rat SAP are all glycoproteins, but their exact carbohydrate composition has not yet been determined (6). The carbohydrate moieties of plaice SAP have been identified : 3 mannose, 2 galactose, 3 N-acetylglucosamine and 2 sialic acid residues per glycosylated polypeptide subunit. Our findings indicate that among 5 lectins with different sugar specificity only Con A showed an interaction with SAP. Suprisingly, two other lectins with similar specificity (LCA and PSA) did not show any SAP binding, although both lectins produced a clearly visible affinity precipitate with the other glycoproteins present in the pleura fluid. As has been shown by Debray et al. (12) lectins with D-mannose specificity can recognize fine differences in more complex carbohydrate structures. Different microheterogeneity pattern have been found when analyzing alpha fetoprotein in affinity electrophoresis with Con A and L.C.A. (7). Studies of alpha-l-acid glycoprotein revealed 3 variants of this glycoprotein in affinity-electrophoresis with Con A where as the same technique with L.C.A. gave no interaction at all between the lectin and the glycoprotein (Kint, unpublished results).

123

References 1. Painter, R.H., I. De Escallon, A. Massey, L. Pinteric, S.B. Stern. 1982. In : "C-reactive Protein and the plasma protein response to tissue in jury". (I. Kushner, J.E. Volanakis and H. Gewürz, eds.). Annals of the New York Academy of Sciences 389, 199-215. 2. Assimeh, S.N., D.H. Bing, R.H. Painter. 113, 225-234.

1974.

J. Immunol.

3. Hind, C.R.K., P.M. Collins, D. Renn, R.D. Cook et al. J. Exp. Med. 15j), 1058-1069. 4. Painter, C.H.

1977.

1984.

J. Immunol. 119, 2203-2205.

5. Anderson, J.K., J.E.M. Mole. 1982. Ann. N.Y. Acad. Sei. 289, 216-223. J.A. Taylor, 1983. Ph.D. Thesis, University of London. 6. Baltz, M., F.C. de Beer, A. Feinstein, E.A. Munn, C.P. Milstein, T.C. Fletcher, J.F. March, J. Taulor, C. Bruton, J.R. Clomp, A.J.S. Davies, M.B. Pepijs. 1982. Ann. N.Y. Acad. Sei. 389, 49-74. 7. Mackiewicz, A., J. Breborowicz. 1981. In : "Lectins-Biology, Biochemistry, Clinical Biochemistry". (T.C. Bog-Hansen, ed.), (W. de Gruyter, publisher), 1, 315-326. 8. Bog-Hansen, T.C., K. Takeo. 67-71.

1980.

J. Electrophoresis 1,

9. Entlicjer, G., J.V. Kostir, J. Kocourek. Biophys. Acta 221, 272-281.

1970.

Biochim.

10. Axelsen, N.H., E. Bock.

1972.

11. Bog-Hansen, T.C.

Anal. Bioch. 56, 480-488.

1973.

J. Immunol. Methods 1, 109.

12. Debray, H., D. Decout, G. Stecher, G. Spik, J. Montreuil. 1981. Eur. J. Biochem. 117, 41-55.

A NEW BINDING SPECIFICITY FOR SERUM AMYLOID P COMPONENT ITS P R A C T I C A L A P P L I C A T I O N S

D . S e r b a n and

AND

Ch.Rordorf-Adam

P h a r m a D i v i s i o n , R - 1 0 5 6 . 1 . 7 8 , C i b a - G e i g y L t d . , 4002 Switzerland

Basel,

Int roduct i on Serum amyloid P-component protein

(SAP) is the c i r c u l a t o r y

S A P is a normal

serum p r o t e i n in h u m a n s ,

s l i g h t l y r i s i n g in i n f l a m m a t i o n an a c u t e - p h a s e

reactant

its

(1), w h e r e a s

in the m o u s e

(3, 4). R e c e n t l y ,

it b e h a v e s

like

(2).

in a

C^-binding

calcium-dependent

the c a r b o h y d r a t e m o i e t y of

r e c o g n i z e d b y S A P has b e e n i d e n t i f i e d W e have r e p o r t e d that

the

(AP).

concentration

It has b e e n shown that S A P b i n d s to f i b r o n e c t i n , p r o t e i n , e l a s t i n fibers and a g a r o s e manner

form of

found in a m y l o i d d e p o s i t s , a m y l o i d P - c o m p o n e n t

agarose

(5).

(TNP)-conjugated proteins 2+ are r e c o g n i z e d b y b o t h h u m a n and m o u s e S A P in a C a -depending

reaction

trinitrophenyl

(6). B a s e d on this i n t e r a c t i o n we d e v e l o p e d a s e n s i -

tive E L I S A

for the d e t e c t i o n of SAP.

Result s P u r i f i e d SAP (human or m o u s e ) depending manner,to

bound,

in a

concentration-

i n s o l u b i 1 i z e d T N P - K L H . T h e S A P b o u n d to

the T N P - K L H w a s d e t e c t e d by indirect E L I S A , w e l l s sequentially

i n c u b a t e d w i t h rabbit

horseradish peroxidase

being

anti-SAP antiserum

linked a n t i - r a b b i t

s e n s i t i v i t y of the assay w a s 0.6 ng/ml

and

IgG c o n j u g a t e .

SAP.

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

The

126 T h e b i n d i n g of SAP to T N P - K L H w a s chelator

inhibited by a calcium

(EDTA), by p e c t i n , a g a l a c t o s e - r i c h

polysaccharide,

b y K L H and T N P - K L H a n d b y two soluble h a p t e n s ,

phosphorylcho-

line and p - a m i n o p h e n y l a r s o n i c acid. S u r p r i s i n g l y , p i c r i c was a weak

inhibitor

(Table

acid

1).

Table 1 I n h i b i t i o n of P u r i f i e d M o u s e SAP B i n d i n g to

Insolubi1ized

TNP-KLH Inhibitor

C o n c e n t r a t i o n at inhibition

picric acid phosphory1cho1ine p-aminophenylarsonic pect i n KLH

22.0

x 10

4.6

x 10

_3 _3 _3

0.13 x 10

a

1 . 0 x 1 0 ®

3

2.5

TNP-KLH a

acid

half-maximum

(M)

a

x

10"8

0.86 x 1 0 ~ 8

B a s e d on a m o l e c u l a r w e i g h t of 25,000 d (pectin), (KLM) and 43 T N P m o l e c u l e s

In o r d e r to test the a s s a y

100,000

for the d e t e c t i o n of

unpurified

SAP, two g r o u p s of five C 5 7 B L / 6 m i c e w e r e

i n j e c t e d i.p. w i t h

e i t h e r saline or 10 ug LPS. A f t e r 48 hrs,

the m i c e w e r e

b l e d a n d the 10 samples, d i l u t e d 1:2500, w e r e a s s a y e d S A P on insolubi1i zed T N P - K L H . T h e results

(Table 2)

that our E L I S A s y s t e m can be u s e d for d e t e c t i o n of mouse

SAP.

d

/ 100,000 d K L M

for

indicates unpurified

127 Table 2 P r e s e n c e of T N P - B i n d i n g S A P in C 5 7 B L / 6 SAP3

Treatment none LPS a

Serum

25 ±

3 |ig/ml (0,.39 ± 0. 13)

100 ± 10 ug/ml

(1.. 65 ± 0.36)

The v a l u e s g i v e n are the a v e r a g e s of 5 d i f f e r e n t C57BL/6

samples of

s e r u m tested ± s t a n d a r d d e v i a t i o n . T h e v a l u e s

in b r a c k e t s represent

given

OD^g2>

We

found that u n p u r i f i e d h u m a n SAP w a s also e a s i l y 4 d e t e c t e d even at a 1:10 d i l u t i o n of serum (to be r e p o r t e d e1sewhere).

T h e T N P - s p e c i f i c i t y of SAP w a s

further d e m o n s t r a t e d b y

affini-

ty c h r o m a t o g r a p h y on T N P - S e p h a r o s e . The p a s s a g e of b o t h h u m a n 2+ and m o u s e serum, in the p r e s e n c e of C a , through TNP-columns, removed more

than 90 % of the a p p l i e d SAP

(6).

1. P e p y s , M . B . et al., 1978. N a t u r e 273,

168.

2. P e p y s , M . B . a n d M . L . B a l t z ,

Immunol. 34,

Re ferences

1983. A d v .

3. de B e e r , F . C . , M . L . B a l t z , S . H o l f o r d , A . F e i n s t e i n M . B . P e p y s , 1981. J. Exp. M e d . 154, 1134. 4. P e p y s , M . B . et al., 1977. Lancet

1,

5. H i n d , C . R . K . et al. 1984. J . E x p . M e d . 6. S e r b a n , D . and C h . R o r d o r f - A d a m , Bi o c h e m .

141.

and

1029. 159,

1058.

1985, s u b m i t t e d to A n a l .

STRUCTURE AND FUNCTION OF SAA

M.D. Benson, F.E. Dwulet, B. Kluve-Beckerman, M. Aldo-Benson Department of Medicine, Indiana University School of Medicine Indianapolis, Indiana 46223

Introduction Serum amyloid A (SAA) is an acute phase protein which is synthesized by the liver and secreted into the plasma where it circulates as part of the high density lipoprotein (HDL) complex (1,2,3). Normally it is present in plasma in concentrations around 1 to 4 ug/ml, but during times of inflammation, either acute or chronic, the plasma SAA level may rise to 300 to 500 yg/ml. This acute phase response is associated with synthesis of specific mRNA for SAA by hepatocytes (4) and is under the control of interleukin 1 (IL 1) or a similar monokine that is produced by monocyte/macrophages. This mechanism of protein synthesis induction appears to be the same for C—reactive protein (CRP) and probably alpha 1 acid glycoprotein (6). These proteins stand apart from other acute phase reactants by the magnitude of their response to inflammation. It is this unique response that makes these proteins of great interest to the student of inflammatory processes and at the same time raises questions about their importance in the biological mechanisms of response to injury. To solve some of these problems we have studied SAA at the primary structure level and searched for presumptive biologic functions in the immune system.

Structure of SAA Protein AA is the major constituent of secondary (reactive) amyloid, and this has allowed isolation of the protein in sufficient quantities to do structural analyses. The complete amino acid sequence of amyloid protein A (AA) was first reported by Levin et al. in 1972 (7) (Figure 1). Since that time a few AA

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

130

proteins have been completely sequenced but not enough data are available to analyze the heterogeneity that probably exists. In 1982 Parmelee et al. reported the entire sequence of SAA which is a single chain polypeptide with 104 amino acid residues (8). The first 76 amino acids are homologous to AA and it is presumed that AA is a degradation product of the serum protein. Two amino acids were found at positions 52 and 57 of SAA isolated from pooled serum suggesting that there are at least 2 gene products in the serum during acute phase response. Work by other authors has shown as many as 6 species of SAA when isolated by ion exchange chromatography (9). It would appear that much of this heterogeneity is explained by variations at the amino terminal end of the SAA molecule. It is now recognized that while human protein AA has an arginine amino terminus, a sizeable fraction of the serum protein has a serine amino terminus (position 2) and indeed a smaller fraction has phenylalanine at the amino terminus (position 3). It is not yet clear whether this represents different gene products or represents a post translational modification of the molecule. Studies at the gene level will probably be required to answer this question. SAA has been identified in a number of species and most of these species have been shown to develop reactive amyloidosis. Primary structure analysis of human, monkey, mink and guinea pig AA proteins has shown a striking degree of homology particularly in the portion of the molecule from residue 35 through 60 (10,11,12). To study this in more depth we have undertaken a phylogenetic analysis of the AA proteins and isolated amyloid protein AA from the CBA/J mouse, the Abyssinian cat, and spontaneous amyloid in the dog. Comparison of these structures to the data known for human, monkey, mink and duck amyloid protein AA has confirmed the conservation of homology in the region from residues 35 through 60 (13,14,15,16) (Figure 1). At the same time the amino terminal and carboxyl terminal portions of the molecule show heterogeneity among species suggesting normal phylogenetic divergence. The fact that there is a strongly conserved region of the SAA molecule suggests that there is an important function for the protein. This important function has not been obvious.

131

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«9

< 9 I < 9 < E < 3
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< > < >

< >

i/i u> v> >->->-

-I -1 -I

UI a

•

3
< « m LU Ul Ui je X K ) Z o t- Z z V 3 (J S 8 < 3 o 3 X £ Q u £ (9 £ a

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3 < i -I 0 1 3
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ra : Ol •H a 3 ra = u a) >

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O o

4-1 o rH (0 s

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485

Fig. 2: Effect of malotilate on hepatic alterations induced by repeated pig serum injections in the rat

Fig. 2a: Liver section from control rat, treated with malotilate (100 mg/kg) for 10 weeks. (HE stain x 160)

Fig. 2b: Liver section from pig serum intoxicated rats for 10 weeks showing connective tissue septa linking portal tracts and central veines. (HE stain x 160)

Fig. 2c: Liver section from pig serum and malotilate (20 mg/kg) treated rats. Neither septal fibrosis nor necrosis was observed. (HE stain x 160)

486 Discussion

20 injections of pig serum produced marked hepatic fibrosis in the rat, as shown by both biochemical and histological examinations. This appeared in the absence of large hepatocellular damage. Our results are in accordance with previous data [12]. Malotilate treatment prevented almost completely the development of fibrosis and the impairment of hepatic excretion function. The two dosages (20 and 100 mg/kg) were almost equally effective.

The hepatic fibrosis described in animals chronically immunized with pig serum may be an example of immune complex mediated collagen deposition [13]. A type III immune reaction would occur when soluble antigens combine with antibody to form immune complexes in either the interstitial space or the intravascular space, then the inflammatory response would trigger the collagen deposition in the liver.

The inhibitory effect of malotilate on fibrogenesis could be mediated through an action on the immune system, the inflammatory response and/or the collagen metabolism.

No data are yet available concerning the effect of malotilate on the immune system. Regarding its effect on the inflammatory stage, malotilate inhibited specifically the in vitro proliferation of liver connective tissue cells, isolated from fibrotic livers of patients with severe schistosomiasis [14]. Preliminary experiments on human embryo fibroblast chemotaxis, using fibronectin as chemoattractant

[15], suggested that malotilate decreased the

fibroblast migration. These results are interesting since the

487 relationship between connective

tissue c e l l s and f i b r o n e c t i n , as a

c h e m o t a c t i c factor, has b e e n p o s t u l a t e d [16]. M a l o t i l a t e c o u l d t h e r e f o r e

in the pig s e r u m m o d e l

interfere o n the

r e s p o n s e and e s p e c i a l l y o n the c e l l s i n v o l v e d p r o c e s s by d e c r e a s i n g unlikely synthesis

inflammatory

in the

fibrogenic

their migration and/or proliferation.

that m a l o t i l a t e

i n t e r f e r e s d i r e c t l y w i t h the

It is

collagen

in the n o r m a l s i t u a t i o n . In v i t r o , m a l o t i l a t e had no

s p e c i f i c e f f e c t on c o l l a g e n b i o s y n t h e s i s skin fibroblasts;

nor d i d it m o d i f y ,

in c u l t u r e s of

in young g r o w i n g

normal

rats,

the

h y d r o x y p r o l i n e c o n t e n t of skin, lung or liver, n e i t h e r the d a i l y u r i n a r y e x c r e t i o n of h y d r o x y p r o l i n e

[17].

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

r e m a i n s u n c l e a r , but our

e x p e r i m e n t has b r o u g h t new insight into the e f f i c a c y of the d r u g : it is d e m o n s t r a t e d that m a l o t i l a t e p r e v e n t s the d e v e l o p m e n t of hepatic fibrosis

in two e x p e r i m e n t a l m o d e l s

m e c h a n i s m s : CCl^

(necrosis) and pig serum

induced by

(immune reaction)

does not require m e t a b o l i c a c t i v a t i o n and i n v o l v e s only n e c r o s is.

different which

few cell

488

References 1.

Kitagawa, H., Saito, H., Sugimoto, T., et al. Effects of diisopropyl-1.3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs and heavy metals. J. Toxicol. Sei. 7, 123-134 (1982).

2.

Younes, M. and Siegers, C.P. Effect of malotilate on paracetamol induced hepatotoxicity. Tox. Letters 25, 143-146 (1985).

3.

Sugimoto, T., Imaizumi, Y. and Kasai, T. Treatment of carbon tetrachloride fatty liver with diisoproply-1.3 dithiol-2-ylidene malonate (NKK-105). Japan. J. Pharmacol. 28, 126 (1978).

4.

Dumont, J.M., Maignan, M.F., Herbage, D. and Perrissoud, D. Effect of malotilate on carbon tetrachloride induced liver injury in the rat. EASL Satellite Symposium, Bern, 1984.

5.

Zimmerman, H.J. Experimental hepatotoxicity. In: Experimental production of diseases, part 5, Liver, ed. D. Eichler, Springer-Verlag, Berlin-Heidelberg-New York, 1976.

6.

Tamayo, R.P. Is cirrhosis of the liver experimentally produced by CC1. an adequate model of human cirrhosis. Hepatology, 3, 112-120 (1983).

7.

Parronetto, F. and Popper. H. Chronic liver injury induced by immunologic reactions. Am. J. Pathol. 49, 1087-1101 (1966).

8.

Folch, H., Lees, M. and Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-510 (1957).

9.

Prockop, D.J. and Udenfriend, S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem. 1, 228-239 (1960).

10

Perrissoud, D. L'electrophorese en gel de Polyacrylamide. In: New Trends in the therapy of liver diseases. Int. Symp. Tirrenia, 1974, pp. 38-54, Karger, Basel, 1975.

11. Heath, D.F. Normal or log-normal: appropriate distributions. Nature, 213, 1159-1160 (1967). 12. Ballardini, G., Esposte, F.D., Bianchi, F.B. et al. Correlation between Ito cells and fibrogenesis in an experimental model of hepatic fibrosis. A sequential stereological study. Liver 3, 58-63 (1983). 13. Thomas, H.C. Immunological aspects of hepatic fibrosis. Ital. J. Gastroenterol. 12, 33-36 (1980).

489 14. Borojevic, R., Vinhas, S.A., Domont, G.B. and Grimaud, J.A. Effect of malotilate on human connective tissue cells in vitro. EASL Satellite Symposium, Bern, 1984. 15. Mensing, H., Pontz, B.F., Muller, P.K. and Gauss-Muller, V. A study on fibroblast Chemotaxis using fibronectin and conditioned medium as chemoattractants. Eur. J. Cell. Biol. 29, 268-273 (1983). 16. Cenacchi, G., Ballardini, G., De Giorgi, L.B. et al. Relationship between connective tissue cells and fibronectin in a sequential model of experimental hepatic fibrosis. Virchows Arch. (Cell Pathol.) 43, 75-84 (1983). 17. Ryhanen, L. and Hamalainen, I. Effect of malotilate on the metabolism of collagen. EASL Satellite Symposium, Bern, 1984.

DEVELOPMENT OF FIBROSIS IN HEPATIC ALVEOLAR ECHINOCOCCOSIS A SEQUENTIAL STUDY IN MICE INFECTED WITH ECHINOCOCCUS MULTILOCULARIS

D.A. Vuitton, Van-Pierre Tran

Service d ' H é p a t o l o g i e , CHU, F-25030 Besançon cedex F r a n c e

S. G u e r r e t - S t o c k e r , J.A. Grimaud Laboratoire

de

Pathologie

Cellulaire

du

Foie,

CNRS

ERA

819,

Institut

Pasteur,

F-69365 Lyon cedex 7, F r a n c e M. Liance, R. Houin

L a b o r a t o i r e de Parasitologic, CHU, F-94010 C r e t e i l cedex F r a n c e

Introduction A dense fibrosis destroying normal s t r u c t u r e s of t h e liver is one of t h e of

human

h e p a t i c alveolar

hallmarks

echinococcosis (AE). This very s e v e r e p a r a s i t i c

disease

is due to t h e development in t h e liver of t h e larval f o r m of t h e c e s t o d e Echinococcus

multilocularis (Em). The p a r a s i t i c development

is a s s o c i a t e d

to an

important

g r a n u l o m a t o u s response of t h e host and an intense fibrogenesis which is responsible for the main complications of t h e disease, such as bile duct obstruction and p o r t a l hypertension. Immunopathological studies in human AE (1) suggest t h a t a) t h e development of fibrosis is r e l a t e d to t h e immune response of t h e host, mainly at the b o r d e r line b e t w e e n

granuloma

and

normal

liver

parenchyma,

and

b) increased

fibroge-

nesis is present in t h e e n t i r e liver, even in a r e a s d i s t a n t from t h e foci of p a r a s i t i c lesions. Various species- and strains- r e l a t e d sensitivities to Em have been

shown

(2, 3), and we have developed a murine model of AE in order to assess t h e r e l a t i o n ship b e t w e e n t h e cellular i m m u n e response of t h e host and t h e i n t r a h e p a t i c development of t h e Em larvae (3). Fibrogenesis could play an i m p o r t a n t role in modulating the

larval

development,

and

be directly

dependent

upon

functional activities

of

cells of t h e granuloma, and p a r t i c u l a r l y m a c r o p h a g e s . In order to p r e c i s e this r e l a tionship

between

fibrosis,

p a r a s i t e i t s e l f , a sequential

periparasitic

granulomatous

infiltration,

and

the

larval

immunopathological and q u a n t i t a t i v e study of collagen

M a r k e r Proteins in Inflammation, Vol. 3 © 1986 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

492 d e p o s i t s in t h e liver has b e e n p e r f o r m e d in e x p e r i m e n t a l l y i n f e c t e d m i c e of 2 inbred s t r a i n s d i f f e r i n g by t h e i r r e s p e c t i v e s e n s i t i v i t y and r e s i s t a n c e to Em.

M a t e r i a l and Methods Animals Mice of t h e inbred s t r a i n s AKR and C57BL10 w e r e used for this study ( I F F A - C R E D O L ' A r b r e s l e , F r a n c e ) . These t w o s t r a i n s h a v e been previously shown t o be " s e n s i t i v e " and " r e s i s t a n t "

to

Em g r o w t h

was p e r f o r m e d by d i r e c t previously

described

intrahepatic

injection

respectively

intrahepatic

in d e t a i l (3). C o n t r o l of

saline,

with

(3) (fig

1). I n f e c t i o n with

Em

larvae

i n j e c t i o n , under l a p a r o t o m y , as it has the

m i c e of same

the same

surgical

strains

procedure.

been

received Autopsies

5 m i c e of e a c h s t r a i n and t h e i r c o n t r o l s w e r e p e r f o r m e d a f t e r 7, 20, 30, 60, and

180 days

of

i n f e c t ion.

In t h e

"resistant"

C57BL10

strain,

additional

an of 120

studies

w e r e p e r f o r m e d on t h e 270th d a y . The liver was r e m o v e d and w e i g h t e d a f t e r m a c r o scopical

examination.

apparently

"normal"

Samples areas,

of

were

liver, frozen

taken in

from

liquid

parasitic

nitrogen

and

lesions

and

stored

at

from -80°C.

Fig 1. Histological a s p e c t s of t h e lesions observed in t h e liver, on t h e '/th m o n t h a f t e r i n f e c t i o n with Em l a r v a e ; a) in AKR m i c e . Epithelioid cells (EC) a r e lining t h e g e r m i n a l layer (arrow) of a l a r g e p a r a s i t i c v e s i c l e (PV) (Masson's t r i c h r o m e x 625). b) in C57BL10 m i c e . An i m p o r t a n t a r e a of n e c r o s i s (N) is surrounding t h e g e r m i n a l layer (arrow) of a small PV. ( H e m a t o x y l i n - E o s i n - S a f r a n ; x 625). G + F = polymorphous g r a n u l o m a and fibrosis.

493 I m m u n o f l u o r e s o e n c e s t u d i e s with a n t i - c o l l a g e n t y p e s a n t i s e r a .

Polyclonal

monospecific antibodies against

t y p e I, III and IV c o l l a g e n s ,

propeptide

III, f i b r o n e c t i n and laminin w e r e used on 5 Jim thick u n f i x e d f r o z e n liver s e c t i o n s . P r e p a r a t i o n and p u r i f i c a t i o n of t h e m o n o s p e c i f i c a n t i b o d i e s a r e d e s c r i b e d (U, 5). F l u o r e s c e i n Pasteur,

France)

isothiocy a n a t e or

rabbit

(FITC) labelled

anti-goat

IgG

(Nordic,

sheep anti-rabbit Tilburg)

elsewhere

IgG

(Institut

immunoglobulins

used, a c c o r d i n g t o t h e origin of t h e m o n o s p e c i f i c a n t i s e r u m , a t

were

1/20 dilution. All

readings w e r e p e r f o r m e d on a L e i t z - D i a l u x f l u o r e s c e n c e m i c r o s c o p e .

Quantitative

determination

of

collagen-protein

and

total-protein

c o n t e n t on

liver

sections. The

micromethod

for

determination

of

the

total-

and

collagen—protein

content

in p a r a f f i n e m b e d d e d h u m a n liver s e c t i o n s d e v e l o p e d by Lopez de Leon and Rojkind (6) was

adapted

performed

to

on two

frozen 10 p m

sections

of

mouse liver.

Briefly, determinations

thick f r o z e n liver s e c t i o n s . P a r a s i t i c a r e a s ,

were

apparently

" n o r m a l " liver of i n f e c t e d m i c e , and liver of c o n t r o l m i c e w e r e a n a l y s e d

respecti-

vely. Slides w i t h liver s e c t i o n s w e r e i n c u b a t e d for t w o hours in a solution of F a s t Green

FCF

and

temperature.

Sirius red F3 BA in p i c r i c acid with c o n t i n u o u s shaking a t

acid and F a s t g r e e n . The l a t t e r c o n t r o l slide was used t o c a l c u l a t e t h e of

absorbance

staining

room

Two c o n t r o l slides per s a m p l e w e r e i n c u b a t e d r e s p e c t i v e l y with p i c r i c of

Fast

was eluded

green

with

at

1 ml of

540 nm. NaOH

After

3 washings with

0.1 N (0.5 ml) and

A b s o r b a n c e was r e a d on a s p e c t r o p h o t o m e t e r

at

percentage

distilled

methanol

water,

(0.5

ml).

540 nm (red Sirius + Fas t g r e e n ,

25 - 30 %) and 605 nm ( F a s t green only). The r a t i o b e t w e e n and green allowed us t o c a l c u l a t e t h e a m o u n t of c o l l a g e n

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

red

p r o t e i n (pg) per mg of

t o t a l p r o t e i n s in t h e liver s e c t i o n s . Four assays w e r e m a d e per liver s a m p l e , and t h e mean p r o t e i n c o n t e n t s w e r e o b t a i n e d f r o m 3 m i c e of t h e s a m e group.

Results

Immunofluorescence studies Seven in t h e

days a f t e r

infection,

2 s t r a i n s of

f i b r o g e n e s i s was conspicuous

mice. Concentric collagen

around

parasitic

d e p o s i t s w e r e heavily

vesicles

labelled

with

a n t i - t y p e III p r o c o l l a g e n and t y p e III collagen a n t i s e r a . At this s t a g e , t h e p r e d o m i nant f e a t u r e was the development

of t h e f i b r o n e c t i n f r a m e w o r k , having t h e s h a p e

494

Fig 2. F i b r o n e c t i n n e t w o r k on t h e 7th day a f t e r i n t r a h e p a t i c l a r v a e a) in AKR m i c e ; b) in C57BL10 m i c e . ( x 625).

infection

with

Em

Fig 3. D e t a i l of collagen d e p o s i t s a r o u n d a large p a r a s i t i c vesicle in AKR m i c e , a t one month a f t e r i n t r a h e p a t i c i n f e c t i o n with Em l a r v a e , a) labelled with a n t i t y p e III c o l l a g e n , b) labelled with a n t i - t y p e 111 p r o - c o l l a g e n . Two main lines of deposition a r e n o t i c e a b l e : along t h e p a r a s i t i c v e s i c l e (PV), and a t t h e b o r d e r l i n e with liver c e l l s ( x 625).

495

F i g . k. Aspects of the p e r i p a r a s i t i c h e p a t i c fibrosis in C57BL10 m i c e , on the 4 t h m o n t h a f t e r i n f e c t i o n w i t h Em l a r v a e , a) L a b e l l i n g w i t h a n t i - t y p e 1 collagen a n t i serum (x 625) ; b) Vascular neogenesis in the p e r i p a r a s i t i c g r a n u l o m a : vessels are labelled w i t h a n t i - l a m i n i n a n t i s e r u m (PV = p a r a s i t i c vesicle ; x 250).

F i g . 5. C o m p a r i s o n b e t w e e n thickness of the p e r i p a r a s i t i c i n f i l t r a t e a) in A K R m i c e , on the 6 t h m o n t h a f t e r i n f e c t i o n w i t h Em l a r v a e ( l a b e l l i n g w i t h a n t i - t y p e I collagen a n t i s e r u m ) ; b) in C 5 7 B L 1 0 m i c e , on the 9th m o n t h a f t e r i n f e c t i o n (labelling w i t h a n t i - t y p e III collagen a n t i s e r u m ; PV = p a r a s i t i c vesicle ; x 625).

496 of a thin n e t m a d e of c o n c e n t r i c f i b e r s in AKR m i c e , and of c o a r s e - f i b r e d bundles in C57BL10 m i c e (fig 2 a and b). One m o n t h a f t e r i n f e c t i o n , t w o main

locations

of c o l l a g e n d e p o s i t s around p a r a s i t i c lesions w e r e o b s e r v e d in AKR m i c e : a) along t h e i n t e r n a l and e x t e r n a l sides of t h e f i r s t layer of h i s t i o c y t i c c e l l s ( t h e so-called "epithelioid cells"), lining t h e p a r a s i t i c vesicles, and b) a t t h e borderline

between

c e l l s of t h e g r a n u l o m a and p a r e n c h y m a l liver c e l l s (fig 3 a and b); t h e s e

collagen

d e p o s i t s w e r e heavily labelled with a n t i - t y p e III c o l l a g e n and p r o c o l l a g e n (no l a b e l ling with a n t i - t y p e I c o l l a g e n was o b s e r v e d in this s t r a i n ) . B e t w e e n t h e s e t w o main lines

of

collagen

deposition,

clusters

of

collagen

deposits

were

concentrically

s c a t t e r e d along t h e c e l l s of t h e g r a n u l o m a . At this s t a g e , collagen was a l r e a d y o r g a n i zed

in bundles

with anti-type

around

the

parasitic

lesions

of

I collagen (fig 4a). This l a t t e r

C57BL10

mice,

and

labelled

labelling w a s p a r t i c u l a r l y

also

important

on t h e 4th m o n t h a f t e r i n f e c t i o n in this s t r a i n , while in AKR m i c e , this was seen only on t h e 6th m o n t h . The 4th m o n t h - l e s i o n s w e r e c h a r a c t e r i z e d by a very " a c t i v e " aspect and

of

fibrogenesis

anti-laminin

in t h e

showed

an

2 strains, obvious

and

labelling

development

of

with

anti-type

vascular

IV c o l l a g e n

n e o g e n e s i s (fig

4b).

A p r o g r e s s i v e and d e f i n i t i v e e x t e n s i o n of f i b r o s i s and of p a r a s i t i c lesions was o b s e r ved in A KR s t r a i n up t o 6 m o n t h s , t h e a v e r a g e delay leading to " s p o n t a n e o u s " d e a t h f r o m t h e d i s e a s e in t h e s e m i c e . On t h e o p p o s i t e , as e a r l y as t h e 6th m o n t h , r e g r e s sion

of

fibrosis,

and

disappearance

of

type

1-collagen

was

observed

in

C57BL10

i n f e c t e d m i c e (fig 5 a and b).

D e t e r m i n a t i o n of t o t a l - and c o l l a g e n - p r o t e i n c o n t e n t . R e s u l t s of t h e q u a n t i t a t i v e analysis of fibrosis in t h e liver of i n f e c t e d and c o n t r o l m i c e of t h e 2 s t r a i n s under study a r e p r e s e n t e d in fig 6. Sham o p e r a t i o n with i n j e c tion of saline in t h e liver of m i c e w a s followed by a t r a n s i e n t i n c r e a s e of collagen p r o t e i n s which r e a c h e d its m a x i m a l value a t 2 m o n t h s in AKR m i c e and a t 1 m o n t h in

C57BL10

AKR

mice.

areas

was

mice.

This

value

A progressive observed

was

significantly

increase

in t h e

of

higher

in C57BL10

collagen-protein

2 strains a f t e r

content

intrahepatic

of

i n j e c t i o n of

mice the

than

in

parasitic

Em

larvae

:

it r e a c h e d its m a x i m a l v a l u e a t 6 m o n t h s a i t e r i n f e c t i o n in t h e 2 s t r a i n s . At this s t a ge,

collagen-protein

content

of

the

pathological

areas

was

significantly

than that observed

in t h e " n o r m a l " a r e a s of t h e liver in t h e s a m e m i c e

liver"), and

liver

the

mean

in t h e

collagen-protein

of

control content

mice of

("control

liver").

the pathological

("normal

B e f o r e t h e 6th

a r e a s w a s lower

higher month,

than

that

d e t e r m i n e d in " n o r m a l liver", or " c o n t r o l liver", but t h e d i f f e r e n c e was not significant

except

for

collagen-protein

the

1st and

2nd

months

in C57BL10

mice.

The

a m o u n t in t h e pa r a s i t i c a r e a was lower in C57BL10

mean

peak

m i c e t h a n in

of

497 AKR m i c e , but t h e d i f f e r e n c e was not s i g n i f i c a n t . The survival of C57BL10

mice

allowed us t o q u a n t i f y liver p r o t e i n - c o n t e n t a t t h e 9th m o n t h : t h e r e s u l t s showed a significant decrease assessment

obtained

collagen-protein

of c o l l a g e n - p r o t e i n c o n t e n t which c o n f i r m e d t h e q u a l i t a t i v e with

content

immunofluorescence of

"normal

liver"

study

in

(fig

infected

5b and

mice

6b). Analysis of

showed

an

increased

f i b r o g e n e s i s in t h e liver d i s t a n t f r o m t h e p a r a s i t i c lesions. In AKR m i c e , it c o n s i s t e d of

a plateau,

A spontaneous

significantly regression

higher of

than

in c o n t r o l

fibrosis, beginning

at

mice the

up t o 6 m o n t h s (fig 4th

month,

was

6a).

observed

in t h e "normal liver" of i n f e c t e d C57BL10 m i c e (fig 6b).

Fig. 6. C o u r s e of h e p a t i c fibrosis in AKR and C57BL10 m i c e ( r e s p e c t i v e l y s i t i v e " and " r e s i s t a n t " to Em) and their s h a m - o p e r a t e d c o n t r o l s .

"sen-

498 Discussion

This

sequential

that

a

study

strong

and d e v e l o p m e n t dary

immune

granuloma

present

in

studies to

The the

as

a

along main

the

the

first

hypothetical

sequence

deposition

granulomas of

("loose

fibrosis

with

connective

experimental

showed

hours of

of

of

and

events

type-Ill

bundles

on

days

of

the

matrix

type-I

It

their

suggests

internal

the

hypothesis

(7).

However,

the

amorphous

side, of

lack

nature

labelling

of

a

non-specific

been

obtained

the

mouse

this

area

of

of of

this

with

foci

an

to confirm

increased of

sections appeared of

fibrosis

in c o n t r o l matory "normal"

of

with has

in t h e

be

antiserum

aspects

was

shown

anti-laminin

in

layer

location, A

( f i g ¿ib) ;

binding

of

Em

AE

(8)

liver

in

in

the

antiadsor-

IgG. the

(1).

development

in

the

The

infection

liver.

infected

have

;

could have been

of

fibrosis

in h u m a n

entire

liver,

micromethod

was

particularly

AE which suggested in

for

portal

spaces

protein

interesting, the

distant

measurement

in

mice.

An

increased

fibrogenesis

presence from on

However,

mice

was

collagen-protein

higher,

and

its

peak

content was

of

the

delayed

the

liver-

evolution

was

observed

m i c e ; t h i s s u g g e s t s t h a t s h a m - i n j e c t i o n w a s f o l l o w e d by a d i s c r e t e

reaction

how-

host-IgG

to be r e l i a b l e and quite s u i t a b l e to study sequentially the

after

and

frequent

antibodies

experimental

antiserum,

at

laminated

observed

The

layer",

favor

explained.

these

murine fibrosis.

could

particular

because

:

the

("dense

in of

"laminated

be

(1) of

area

fibrosis

the

to

the

remodelling

central

the

and

AE

periphery

subsequent

of

Earlier

performed

human

the

liver

already

confirmed

These

mouse-laminin.

previous observations

lesions

a

this

remain

been

in

at

origin in

excluded

the

the

components

week

analysis of

of

host

layer

be

1st

and

Em.

fibrosis

anti-laminin cannot

should

periparasitic

of

the

of

of

cell-

of

infection.

collagen

studies

formation

about

vesicles

present

fibrogenesis

parasitic

al.

cells

in t h e e a r l y d e v e l o p m e n t

layer

laminated

the

parasitic

Quantitative assessment in o r d e r

et

immunization

antibodies,

the

remodelling

b e d on t h e s e p a r a s i t e - b o u n d

of

to

germinal

labelling

after

surface IgG

the

Mehlhorn

the

ever,

to

contribution

lining

after

the

the

the

the role of "epithelioid cells",

also

after

cells, secon-

due to a

of

borderline

day

sequential

AE demonstrated

their

the

procollagen

in

Moreover,

fibrosis

inoculation

organization"),

hypothesis

type-I collagen, were

different

from

and

collagen

liver

at

the

murine intrahepatic

involvement

7th

Em

the

suggested

organization").

and

made

collagen

of

except

after of

observations

connective

matrix

vesicles,

appearance

The

model

particular

of collagen,

granulomas

confirmed

immunocompetent

t h e value of

the

parasitic

AE

peri-parasitic

experimental

It

the

sequences

fibronectin.

early

in

between

components

particularly

an

useful

peri-parasitic

during

assess

exists

reaction.

located

parenchyma.

fibrogenesis

o f f i b r o s i s . It s u b s t a n t i a t e d

echinococcosis

mediated

of

relationship

inflam-

apparently in

C57BL10

499 mice,

as c o m p a r e d

to that

A significantly increased

observed

in c o n t r o l

m i c e of t h e s a m e s t r a i n (fig 6b).

f i b r o g e n e s i s in t h e " n o r m a l " liver

was o b s e r v e d on

the

6th month in t h e AKR m i c e only ; a t this s t a g e , it r e t u r n e d t o n o r m a l values in C57BL10

mice.

This d i f f e r e n c e suggests t h a t

a persistent

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

"normal"

p o r t a l s p a c e s was only observed when a s i g n i f i c a n t p a r a s i t i c mass - and a s i g n i f i c a n t g r a n u l o m a t o u s i n f i l t r a t e - w e r e still p r e s e n t in t h e liver. In f a c t , t h e main i n t e r e s t of our r e s u l t s c o n s i s t e d of t h e d i f f e r e n t c o u r s e s of fibrosis in t w o s t r a i n s of m i c e d i f f e r i n g by t h e i r r e c e p t i v i t y t o t h e Em l a r v a e . C o m p a r i s o n of quantitative of

lower

month,

data

showed

intensity

were

in C57BL10

also

f i b r o g e n e s i s of

that

lower

the

initial

collagen

mice.

The

in this s t r a i n

deposition

maximal

than

in AKR

C57BL10 s t r a i n a p p e a r e d

in

parasitic

values,

observed

mice.

However,

areas on

was

the

6th

capacity

t o be higher than t h a t of t h e

of

AKR

s t r a i n . This was d e m o n s t r a t e d by t h e c o l l a g e n - p r o t e i n c o n t e n t of t h e " n o r m a l liver" of

these

mice

after

i n f e c t i o n or s h a m - o p e r a t i o n .

Moreover,

they w e r e

noticeable

q u a l i t a t i v e d i f f e r e n c e s when t h e 2 s t r a i n s w e r e c o m p a r e d : c o l l a g e n - b u n d l e - f o r m a tion

and r e m o d e l l i n g

mice,

while

as e a r l y and

as

the

definitive

exhibited

of

thickness 4th

type-I

to

collagen

the

month.

deposition

a regressive

of

fibrosis with

of

collagen.

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

AKR

of

and

could

be

of

mice exhibited

type-I

collagen.

observations deposition, different

collagen

and

f i b r o s i s with e a r l y

These

synthesis

type-I

granuloma-

On

suggest

in t h e

deposits-

an e x t e n s i v e the

formation

and/or

occurred earlier

collagen

that

other immune

2 strains.

reduced late

C57BL10

of

earlier

mice

disappearance

mechanisms

activity

The

C57BL10

f i b r o s i s with

hand,

but s u b s e q u e n t

functional

in was

cells

leading involved

development

of

a w e l l - o r g a n i z e d fibrosis could be p a r t i a l l y responsible for t h e l i m i t a t i o n of p a r a s i t i c growth to

be

in

the resistant

stronger

foot-pad

C57BL10 s t r a i n .

in this

viewed and

as

schistosomiasis

of

compared

shown

(10).

The

to that respective

observed capacity

of

different

in a g r a n u l o m a t o s t i m u l a t e f i b r o g e n e s i s has r e c e n t l y been

(11). Analysis of

isolation

and

has been

mice

such

antigens

immunity

delayed-type-hypersensitivity para-

models,

Em

by t h e

in AKR

cell-types present

to

Cell-mediated

when assessed

(9). The role of lymphokines in f i b r o g e n e s i s has been e m p h a s i z e d in t h e o t h e r sitic

response

strain,

factors

t h e r e l a t i o n s h i p b e t w e e n c e l l - t y p e s and stimulating

bring m o r e p r e c i s i o n s a b o u t r e c e p t i v i t y

fibrogenesis

in

granulomatous

of t h e host and its c a p a c i t y

e f f i c a c i o u s f i b r o u s b a r r i e r a g a i n s t t h e d e v e l o p m e n t of Em.

re-

collagen-deposits, lesions

could

t o mount an

500 Acknowledgements

We wish t o

thank

Mrs Belin for m a n u s c r i p t p r e p a r a t i o n

and Mr T h i e b a u t

for

the

photomicrographs.

References

1. Vuitton, D.A., S. G u e r r e t - S t o c k e r , 3.P. C a r b i l l e t , G. Mantion, J . P . Miguet, G r i m a u d : Z. P a r a s i t e n k d . (in press).

J.A.

2. Webster, G.A., T.W.M. C a m e r o n . 1961. C a n . 3. Zool. 39, 877. 3.

Liance, M., D.A. Vuitton, S. G u e r r e t - S t o c k e r , R. Houin. 1984. E x p e r i e n t i a 40, 1436.

3.P.

Carbillet,

J.A.

Grimaud,

4. G r i m a u d , 3.A., M. D r u g u e t , S. P e y r o l , O. C h e v a l i e r , D. H e r b a g e , N. El B a d r a w y . 1980. 3. H i s t o c h e m . C y t o c h e m . 28, 1145. 5.

Linck,

G., S. S t o c k e r ,

3.A. G r i m a u d ,

A. P o r t e .

1983. H i s t o c h e m i s t r y

77,

323.

6. L 6 p e z - d e - L e 6 n , A., M. Rojkind : 3. H i s t o c h e m . C y t o c h e m . (in press). 7. Mehlhorn, H., 3. E c k e r t , R . C . A . Thompson. 1983. Z. P a r a s i t e n k d . 69, 749. 8. Ali-Khan, Z., R. Siboo. 1981. Exp. P a r a s i t o l .

159.

9. L i a n c e , M., D.A. Vuitton, D. R i v o l l e t , 3. Breuil, R. Houin : P r o c . of t h e Xlllth Int. Cong. Hydatidology (in press). 10.Wyler, D.3., S.M. Wahl, L.M. Wahl. 1978. S c i e n c e 202, 438. 11.Claman, H.N. 1985. Immunol. Today 6, 192.

INTRAALVEOLAR FIBROSIS : A REVERSIBLE FORM OF PUMONARY FIBROSIS

S. Peyrol

1

, J.F. Cordier

2

, J.A. Grimaud

1

1. Laboratoire de Pathologie Cellulaire du Foie, CNRS ERA 819, Institut Pasteur, 77 rue Pasteur, 69365 Lyon cedex 7 (France) 2. Hôpital Cardiovasculaire et Pneumoloqique Louis Pradel, 28 avenue du Doyen Lépine, Lyon (France)

Intraalveolar fibrosis has been described as a "second form of scarring" (1) in various lung disorders (idiopathic pulitDnary fibrosis, hypersensitivity pneumonia (2), histiocytosis X) and also following accidental or experimental paraquat intoxication (3). On histological sections of the lung, intraalveolar fibrosis consisted of widespread connective buds present in most, but not all, alveolar lumens, appearing as spherical nodules of variable size (50-100 n) which widened the intraalveolar spaces (fig. 1a). Recently, intraalveolar fibrosis was described as a characteristic feature of two forms or organizing pneumonia of unknown etiology, cryptogenic organizing pneumonia (COP) and bronchiolitis obliterans pneumonia ; the latter differed only by the extent of the lesions in the bronchiolar compartment. The clinical features (dyspnea, coughing, malaise, bilateral opacities on the chest X-ray's) were resolved under corticotherapy suggesting that the interstitial fibrotic lesion probably disappeared ; a relapse might be observed after stopping treatment. We studied an open lung biopsy obtained from a patient with COP during a period of relapse. Attention was focused on the connective framevrork of the intraalveolar fibrotic buds, in order to characterize the distinctive features possibly involved in the process of reversibility.

The biopsy was investigated with specific histological methods which allowed us to determine (i) the nature of the connective matrix components of the intraalveolar fibrotic buds and (ii) the pattern and organization of their deposits in relation to the cell populations involved. 1. Trichrome and Gordon-Sweets staining of paraffin embedded sections emphasized the polymorphic aspects of the intraalveolar buds ;

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

502

Fig. 1 : Intraalveolar fibrotic nodule. a. Trichrome staining ; x 675 b. Selective staining of the fibrillar connective framework (Gordon-Sweets staining) ; x 700 2. Inmunofluorescent staining on fresh frozen tissue sections allowed us to identify, in situ, immunoglobulins and the main matricial proteins (collagen types, fibronectin and laminin, proteoglycans), and also determine their relative contribution to the intraalveolar connective framework. 3. Ultrastructural observation demonstrated how the matricial conponents vrere associated into a complex connective matrix, and permitted the characterization of the cell populations involved. Intraalveolar connective buds displayed different patterns of organization from one place to the other. ALVEOLITIS was always prominent : alveolar macrophages (fig. 2a), lymphocytes and plasma cells (fig. 2b) formed large intraalveolar cell clusters where iirtnunoglobulins (mainly IgG) were present (fig. 3a) , either in the cells or localized in the extracellular space as a fluffy material (fig. 2a). At this stage, a fibrillar connective framework was appearing among immunocompetent cells, as a delicate network growing through the inflammatory focus (fig. 1b) .

503

Fig. 2 : Alveolitis. a. Alveolar macrophage ( -^h ) in alveolar lumen ; extracellular fluffy material ( 0 ), probably immunoglobulins polymers ; x 4 350 b. Plasma cells in the alveolar lumen ; x 4 200 In neighbouring intraalveolar buds, FIBROSIS probably succeeded the inflammatory stage ; conspicuous spherical fibrotic nodules consisted of a loose matricial network of fibrillar and amorphous material circumscribed and stretched by spindle-sphaped fibroblast-like cells (fig. 1a). Type III collagen (fig. 3b) and fibronectin (fig. 3c) were the main components when compared to the other matricial proteins (type I collagen, type IV collagen and laminin, proteoglycans) which had also been detected, but in smaller quantities, using monospecific inmune sera as tissular markers. In some areas of the biopsy, fibrotic nodules of neighbouring intraalveolar spaces appeared joined by fibrous connective septa leading to the formation of an extensive intraalveolar fibrotic field, and suggesting that, in a later stage, progressive confluence of the intra alveolar fibrotic buds took place by an active fibrogenetic process. In confluent fibrosis too, type III collagen (fig. 4b) and fibronectin (fig. 4a) exceeded the type I collagen.

504

Fig. 3 : Imrrtunofluorescent labeling of the intraalveolar connective bud components ; x 750 a. Immunoglobulins G-A-M b. Collagen type III c. Fibronectin

Fig. 4 : Confluent fibrotic nodules ; imrrtunofluorescent labeling of matricial components ; x 300 a. Fibronectin b. collagen type III

Fig. 5 : Intraalveolar fibrosis ; cytoplasmic process of a myofibroblast ( ) with prominent contractile filamentous apparatus ( ) ; loose extracellular connective matrix ( * ) ; x 7 500 Fig. 6 : Polymorphism of the matricial components : fibrils (1), microfibrillar clusters (2), interfibrillar meshwork (3) ; x 56 000 Fig. 7 : Myofibrocyte with prominent erqastoplasm ( ) mentous apparatus ( ) ; x 22 000

and contractile fila-

Fig. 8 : Myofibroclast ; part of the cytoplasm ; inclusions containing partially damaged collagen fibrils ( ) ; x 27 000

506 Electron microscope observation emphasized two peculiar characteristics of the CONNECTIVE MATRIX in the intraalveolar fibrotic buds (characteristics which remained unchanged in confluent fibrotic fields) : LOOSE ASPECT and POLYMORPHISM. The loose aspect, already noticed by light microscopy, appeared as an irregular arrangement of fibrillar proteins forming sinuous bundles (fig. 5). At least, three norphologically different kinds of molecular complexes were involved in this matricial arrangement (fig. 6) : collagen fibrils (types I and III), microfibrillar clusters of fibronectin and a delicate arborescent interfibrillar meshvrork, probably proteoglycans. The sinuous appearance of the extracellular connective matrix was probably related to the anchorage of the fibrillar network to the membrane and to the associated basement membrane of the unique connective cell type present in the intraalveolar fibrotic buds and septa ; this cell type was identified as MYOFIBROBLAST. These cells were recognized with their contractile filamentous apparatus associated with the cytoplasmic membrane (fig.5-7). They displayed, in various degrees, either prominent ergastoplasm (fig. 7) or multiple inclusions containing partially damaged collagen fibers (fig. 8), which probably represented the expression of the fibrocytic or fibroclastic modulation of the same cell type. Clinical evidence of complete healing with corticotherapy confirms the previous findings that such morphological features may be considered as RELEVANT SIGNS FOR THE REMODELING ABILITY AND REVERSIBILITY OF FIBROSIS (4). In conclusion, studying COP opens special investigative areas concerning : - fibrosis initiated by inflammation in response to an unknown etiologic agent, - the origin of the myofibroblastic cell line, - the significance of the matricial components' polymorphism (collagens, glycoproteins, proteoglycans...) , - the process of collagen resorption. References 1. Basset F., J. Lacronique, V. Ferrans, Y. Fukuda, R. Crystal. 1984. ftmer. Rev. respir. Dis. 129, part II, A-72. 2. Kawanami 0., F. Basset, R. Barrios, J. Lacronique, V. Ferrans, R. Crystal. 1983. Amer. J. Pathol. J_1_0, 275. 3. Fukuda Y., V. Ferrans, C. Schoenberger, R. Rennard, R. Crystal. 1985. Amer. J. Pathol. Vl8, 452. 4. Grimaud J.A., 1983. Contr. Microbiol. Immunol. 7, 190.

DISTINGUISHING PATTERNS OF ALVEOLITIS AND FIBROSIS IN HUMAN LUNG DISEASE

J.F. Cordier, S. Peyrol, C. Takiya, J.A. Grimaud, J. Brune Department of Pneuraology, Hospital Louis Pradel, 69394 LYON Cedex 3, FRANCE Department of Pathology, CNRS ERA Ö19, Institut Pasteur, 69365 LYON Cedex 7, FRANCE

Although alveolitis precedes the matricial stage of most fibrotic processes in the lung, tne cell-matrix interactions which lead from alveolitis to fibrosis vary from disease to disease. To illustrate this, we shall analyse three conditions where both the pattern of alveolitis and the site of fibrosis differ : idiopathi pulmonary fibrosis (IPF), sarcoidosis and cryptogenic organising pneumonitis (COP).

ALVEOLITIS Alveolitis consists of the accumulation within alveolar structures of immune and inflammatory cells (lymphocytes, macrophages and neutrophil polymorphs)(Fig 1). Cell-matrix interactions are mediated by the release of substances such as free oxygen radical ana proteases which can injure parenchymal cells and the connective matrix, as well as factors directing cell movement, activation or differentiation (1). Alveolitis can be stimulated by many agents including bacteria, organic or inorganic dusts, and toxic gases. Often, however, the stimulus is not known. In infectious diseases, alveolitis general

Marker Proteins in Inflammation, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

508

Fig

1 - ALVEOLITIS (Sarcoidosis). Presence (M) in alveolar septum (AS). One epithelial c e l l s ( E p ) . *Alveolar

of lymphocytes (L) and lymphocyte is seen between lumen (X 7000)

Fig

2 - STRUCTURE OF THE ALVEOLAR SEPTUM. S.A. Col : collagenj Exchange zone (arrow) lary (X 14000)

;

: supportive * Alveolar

axis lumen

macrophages alveolar

j ;

E : C. :

elastin capil-

509 ly resolves when the causative organism disappears. Alveolitis of unknown origin, as in IPF, may persist and in such cases usually leads to the fibrosis of "fibroproliferative disease".

SITE OF FIBROSIS The connective matrix of the lung is essential for maintaining its mechanical properties and providing maximal gas-exchange surfaces. Alveolar structures consist of a supportive axis, limited by the epithelial cell layers and composed of interstitial cells and polymorphic fibrillar deposits of collagen and elastin ; and an exchange zone, where alveolar capillaries and epithelial cells are closely associated for easy gas diffusion, being separated only by their basement membranes (Fig 2). The junctions between endothelial cells are leaky, allowing intravascular fluids and molecules to reach the interstitial spaces, whereas tight occluding junctions between alveolar cells prevent the leakage of most substances into the alveolar lumen. Fibrotic changes occuring in alveolar structures will decrease both lung compliance and oxygen diffusion. The degree of functional impairment depends upon both the type of fibrosis and its location within alveolar structures (Fig 3). In IPF, fibrosis is interstitial and diffuse

(Fig 4). All alveolar structures are

involved, resulting in severe clinical impairment due to the loss of gas-exchange units. In sarcoidosis, on the other hand, fibrosis is restricted to the interstitial zones with granulomas and is thus localised. Finally, in COP, intra-alveolar fibrosis is prominent whereas interstitial fibrosis is generally mild (Fig 5).

510

Fig 3 - DIFFERENT

LOCALIZATIONS

OF PULMONARY

FIBROSIS.

3a. Idiopathic pulmonary fibrosis : diffuse fibrotic of alveolar septa. (Masson's trichrome ; X 160)

thickening

Sb. Sarcoidosis : fibrosis restricted to coallescent (arrow) (Masson's trichrome ; X 160)

granulomas

3c. Cryptogenic organizing pneumonitis : Intraalveolar fibrosis (arrow). Thickened alveolar septa due to the presence of inflammatory cells. (Masson's trichrome j X 200)

Fig 4 - FIBROTIC ALVEOLAR S.A. : supportive

SEPTUM : * Alveolar axis (X ¿300)

lumen ; C. : capillary j

511

Fig 5 - Schematic representation of the different locations of pulmonary fibrosis ; A : normal alveolar structure. B : interstitial diffuse fibrosis with widened fibrotic alveolar wall and reduction of air spaces. C : interstitial localized fibrosis respecting most areas of alveolar wall. D : intraalveolar fibrosis without significant impairment of alveolar wall

IDIOPATHIC PULMONARY FIBROSIS IPF is characterized by increasing dyspnea and usually leads to death within a few years. The alveolitis of IPF involves both immune and inflammatory mechanisms (1). Interactions between macrophages, T-lymphocytes, and B-lymphocytes result in the production of immune complexes which activate alveolar macrophages. These release a neutrophil chemotactic factor which recruits circulating neutrophils which in turn release oxidants capable of injuring endothelial and alveolar cells

and components of

the connective matrix. The oxidants may also inactivate alpha-1antitrypsin. The neutrophils may also release proteases such as collagenase which break down the connective matrix (Fig 6).

512

mm

T-LYMPHOCYTES ALVEOLAR MACROPHAGE

Immune

B-LYMPHOCYTES