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The significance of fibronectin in rheumatoid arthritis
The Significance
of Fibronectin
in Rheumatoid
Arthritis
By D. L. Scott and K. W. Walton
R
HEUMATOID ARTHRITIS (RA) is characterized by its chronicity. Many typical features result from chronic inflammation, with progressive destruction of normal synovial structure and changes in the connective tissue matrix involving collagen, noncollagenous structural glycoproteins, and probably other components, including proteoglycans and glycosaminoglycans. Although immunologic changes have been at the center of research into RA in the last two decades, changes in the connective tissue matrix are a constant feature and may be of equal significance in pathogenesis. Fibronectin is one of the noncollagenous structural glycoproteins, and in recent years its roles in the organization of connective tissue and in the relationship of cells to the connective tissue matrix have been defined. These roles are especially relevant in a chronic inflammatory disorder such as RA. In this review we will briefly describe the structure and function of fibronectin, its relationship to the pathologic features of RA, and its interactions with other elements of connective tissue. Finally, we will consider how such changes in the connective tissue matrix may influence the clinical features of the disease. STRUCTURE AND FUNCTIONS OF FIBRONECTIN
The discovery of fibronectin, which was initially termed cold-insoluble globulin, dates from Cohn’s studies of plasma proteins in the 1940s and the consequent investigations of the various Cohn fractions.‘*2 However, it was only after a series of studies in the 1970s at different centers From the Department of Investigative Pathology, Rheumatism Research Wing, The Medical School, University of Birmingham, Birmingham, England. Supported by the Arthritis and Rheumatism Council and the West Midlands Regional Health Authority. D. L. Scott BSc MD MRCP: Lecturer, and K. W. Walton MD PhD DSc FRCPath: Professor, the Department of Investigative Pathology. Rheumatism Research Wing. The Medical School, University of Birmingham. Address reprint requests to D. L. Scott, M.D.. Department of Investigative Pathology, Rheumatism Research Wing, The Medical School, University of Birmingham, Birmingham, England 815 2TJ. o I984 by Grune & Stratton, Inc. 0049-0172/84/1303-0003$2.00/0 244
that fibronectin’s important role as a plasma and connective tissue protein was recognized. During this period the protein was known by a large and confusing number of synonyms. Fortunately the problem of nomenclature has been resolved and it is now known by one name-fibronectin.3 The discovery of fibronectin and its more recent history have been extensively reviewed.“8 We will therefore concentrate on those aspects of its biochemistry that are directly relevant to its involvement in the pathogenesis of RA. There are two forms of fibronectin: Plasma fibronectin is soluble, whereas tissue fibronectin is an insoluble matrix protein. There are a number of biochemical differences between these forms of fibronectin, although their general structure is similar’ and they show immunologic cross-reactivity. Fibronectin is a glycoprotein (molecular weight, 440,000) with two polypeptide chains that are linked by a disulphide bridge; the two chains may be slightly different. The chains are composed of several domains with different functional roles. This includes binding sites for cells, heparin, hyaluronic acid, and collagen.‘G’2 Fibronectin will also bind both fibrinogen and fibrin, which is influenced by factor XIII.13 In common with many proteins, fibronectin is degraded by a variety of different proteolytic enzymes;‘4*‘5 there is some evidence that its sugar residues protect it against degradation.16 The different functional roles of fibronectin (Table 1) can be readily understood in relation to its structure. It has several interactions with cells. The first to be studied in detail was its ability to promote the adhesion of fibroblasts to gelatin-coated surfaces. I7 There is now evidence that fibronectin mediates the adhesion of a variety of cells, including fibroblasts, endothelial cells, and macrophages to the surrounding matrix”-*’ and also has a role in cell-to-cell interactions. Cell surface fibronectin and fibronectin receptors modulate these roles.2’V22 The second main function of fibronectin is that as a component of the extracellular matrix. The extracellular matrix is a complex structure and contains a variety of macromolecules, including collagens, elastin, proteoglycans, and noncollageSeminars in Arthritis and Rheumatism, Vol. 13, No. 3 (February), 1984
245
FIBRONECTININ RA
Table 1. Biochemical Properties of Fibronectin Relevant in RA Examples
Property Cell interactions Macromolecular
Cell-substratum bind-
ing
adhesion
Binding to collagen, fibrin, complement (Cl,), hyaluronic acid, and sulphated glycosaminoglycans
Opsonization
Clearance of collagenous and
ditions, all of which are severe.27-29 This is thought to occur as the result of fibronectin being consumed as part of the uptake of fibrin and other particulate debris by the reticuloendothelial system in severe disease. There is evidence that monocytes and other cells have cell-surface fibronectin receptors that may modulate its role as an opsonin.2’
other particulate debris by re-
FIBRONECTIN AND RA
ticuloendothelial system and other phagocytic cells
nous structural glycoproteins. This latter group of proteins includes fibronectin and a number of other proteins;23*24the extraction procedures for these are complex and denaturing and their exact composition is debatable.25 Ail these macromolecules can be produced by fibroblasts and other connective tissue cells (Fig. 1). The binding of fibronectin to the macromolecules of the extracellular matrix is mediated by the specific binding sites in its different domains. Finally, fibronectin is a major “nonimmunologic” opsonin. This is thought to be the most important role of plasma fibronectin,26 and changes in fibronectin levels in the blood are seen in a variety of situations. In experimental animals this is seen after the intravenous administration of gelatin particles and is a feature of the clearance of a number of macromolecules from the circulation, including high molecular weightxharged polysaccharides (for example, dextran sulphate; molecular weight, 500,000). In humans a low plasma fibronectin level is seen in disseminated intravascular coagulation, fulminant hepatic failure, and certain other con-
A series of studies in recent years have established that fibronectin is a component of synovial fluid, the rheumatoid synovium, and the rheumatoid pannus.3g40 These studies have suggested it may have significant pathogenic roles. The sites at which fibronectin has been demonstrated in rheumatoid joints are summarized in Fig. 1. Synovial Fluid Fibronectin
The fibronectin concentration of synovial fluid is significantly higher in RA than in other arthropathies, with mean levels two to three times those of plasma. The various reports of plasma and synovial fluid fibronectin levels are summarized in Table 2. In individual patients there may be no relationship between synovial fluid and plasma fibronectin levels.30x3’Synovial fluid fibronectin is unrelated to the degree of joint inflammation, the acute phase response, the total synovial fluid protein level, and levels of specific proteins in synovial fluid such as albumin, IgG, and C3.30132These findings suggest that the local production of fibronectin by synoTable 2. Fibronectin Levels in Plasma and Synovial Fluid MCWl
Svnovlal
fluid
SOUMX
Normal plasma
RA plasma
Flbronectw Level (g/Lb 0.28
Reference Matsuda et al.99
0.33
Mosher and Williams*’
0.32
Scott et al.30
0.29
Gressner et al.33
0.32
Scott et aL3’ Clemmensen and Ander-
0.33
son”
RA synovial fluid
Svnowum
Pannus
0.70
Scott et aL3’
0.44
Vartio et al.34
0.32’
Gressner et al.32
0.81
Clemmensen and Ander-
0.70
Carsons et al.”
CBrtilaqe
Fig. 1. The sites et which fibronectin is present in the rheumatoid synovium.
so?
*Included other arthropathies.
246
vial lining cells and vascular endothelium cells is an important source. There is some evidence that synovial fluid fibronectin consists of several different molecular weight species, with some of lower molecular weight compared to the fibronectin of plasma.32 This may result from partial degradation within the joint or by the local synthesis of a defective molecule. Many enzymes, including plasmin and leukocyte proteases, can degrade fibronectin which may be of significance within the joint. Fibronectin in Cryoprecipitates
Fibronectin is a component of synovial fluid and serum cryoprecipitates in RA.35-38Cryoprecipitates containing fibronectin also contain immunoglobulins and a variety of other proteins, including fibrinogen and fibrin degradation products. The presence of fibronectin in cryoprecipitates may result from a variety of molecular interactions. It binds to fibrinogen and fibrin monomer in the cold3’-this was the basis of its original identification as cold insoluble globulin.
SCOlT AND WALTON
However, it can also bind to the collagenlike fragment of Clq40 and may react with certain immunoglobulins.38 Fibronectin may play a role in the formation of cryoprecipitates, especially in synovial fluid. If it is selectively removed from serum and synovial fluid prior to cooling to 4 OC, the amount of cryoprecipitate formed is often markedly reduced, although this varies from patient to patient.36 Fibronectin is not specific to rheumatoid cryoprecipitates and is found in those isolated from patients with a variety of other connective tissue diseases and arthropathies. Fibronectin and Rice Bodies
Intraarticular rice bodies were first described by Reise 4’ in association with tuberculous arthritis, but they are more commonly associated with RA.42.43Their name is derived from their resemblance to grains of polished rice, although they vary considerably in size and consistency (Fig. 2). The majority of rice bodies consist of fibrinous material. Some contain areas of collagen and
FIBRONECTIN
247
IN RA
mononuclear cells. Fibronectin codistributes with both fibrin and areas of immature collagen within rice bodies, as shown in Fig. 2. The mode of development of rice bodies is controversial. One view is that the earliest rice bodies are predominantly fibrinous, and at a later stage, collagen may be formed due to synthesis by mononuclear cells that are entrained within the rice bodies.42 Alternatively the hypoxia of RA synovial fluid and the associated focal necrosis of synovial tissue44,72may cause synovial microinfarction; this will then provide the initial source of the collagen core of rice bodies, which is subsequently enriched by superficial fibrin coating. It is likely that both these processes occur to some extent. In any event, fibronectin, with its specific binding to fibrin and collagen, will have a central role in the development of rice bodies. Rice bodies within a joint act as an irritant. One reason may be persistence of fibrin4j another, the antigenicity of collagen fragments and altered co1lagen.46However, they can be removed by lavage with a wide bore needle, which gives symptomatic improvement.42 Fibronectin in the Synovial Membrane
Fibronectin is a structural component of normal connective tissue24 and is found in reticulin fibers, as part of the perivascular connective tissue, and in relationship to certain connective tissue cells. It is present in this distribution in the normal synovium. However, in an inflammatory arthritis such as RA there are extensive and significant changes in its distribution. In RA the synovial lining cells contain fibronectin intracellularly, in relation to their cell processes, and pericellularly. Immediately beneath the proliferated synovial cell lining layer there is a meshwork of reticulin fibers that contain fibronectin, which is also present around the many small blood vessels in this region (Fig. 3). Lymphocytes and other inflammatory cells that infiltrate the synovial membrane are not directly related to fibronectin, although it is present in the reticulin fibers that subdivide areas of inflammatory cell infiltration. Fibronectin also codistributes with fibrin on the surface of the synovium and in deeper areas of the synovium.47 These findings are not specific for RA and have been seen in other forms of inflammatory synovitis.48
Fig. 3. Section of rheumatoid synovium stained by antifibronectin by using PAP technique, showing positive reaction of synovial cell lining layer and perhrascular and reticular staining deeper in synovium. (x222)
The rheumatoid pannus is an area of connective tissue proliferation and remodeling. Fibronectin is present in all areas of the pannus, including its junctions with cartilage and other structures within the joint.49 It is especially marked in the more cellular and vascular areas of the pannus, where it forms a coarse extracellular meshwork. In normal cartilage it may not be demonstrable in the matrix, although there is some perichondrocyte fibronectin that is demonstrable after hyaluronidase treatment. However, marginal areas of articular cartilage next to the pannus that show loss of proteoglycans do contain immunoreactive fibronectin within their matrix. Fibronectin is also related to the development of fibrosis within the joint. In the early stages of the deposition of fibrous tissue, fibronectin shows close codistribution with immature collagen that is demonstrated by various histologic stains, such as Martius Scarlet Blue and van Gieson fluid (Fig. 4). However, as the fibrous tissue condenses and the collagen becomes mature, this codistribution is lost and mature fibrous tissue does not contain immunoreactive fibronectin. Pari passu with its relationship to fibrosis, fibronectin is codistributed with collagen type III in reticulin fibers5’ and collagen type IV in basement membranes,” and this is a feature of the synovium in RA as at other sites.47 Studies in Experimental
Models
A variety of experimental models have given further information on the relationship of fibronectin to the pathologic changes of RA. The best
SCOTT AND WALTON
Fig.4. Synovial biopsy of rheumatoid synovium. showing “micronodules” in superficial synovium: (A) stained with Martius Scarlet Blue-micronodule stained pale blue in the original (as for “immature”collagenJ; (Bj same field in adjacent section, stained with rabbit antifibronectin and then FITC-labeled antibody to rabbit immunoglobulin, demonstrating the contribution of fibronectin with “immature” collagen by immunofluorescence. (x498)
known animal model for chronic synovitis is antigen-induced arthritis in rabbits. In its original form this used fibrin as an antigen5* but a modification is the use of ova1bumin.53 Following intraarticular challenge with ovalbumin in sensitized animals, the relationship of fibronectin to the time course of the arthritis can be evaluated by immunohistologic techniques. Initially there is a widespread deposition of fibronectin throughout the inflamed synovium; this is seen 48 hours after challenge and represents the deposition of plasma fibronectin as one component of an inflammatory exudate. As organization and remodeling of the joint occurs over the next few weeks the early, marked extracellular deposits of fibronectin disappear; many areas of the synovium contain little fibronectin, although it persists around small blood vessels, at some sites of the hyperplastic synovial lining membrane, and in fibrin deposits. When the chronic stage develops (at about three months) the synovium contains extensive deposits of fibronectin. It is seen in the synovial lining cell layer, which is markedly hyperplastic, codistributing with fibrin, and in relation to immature collagenous tissue.s4 These changes are similar to those seen in human disease in the chronic phase. The changes in the cartilage in RA, seen at the cartilage-pannus junction, can be studied experi-
mentally by using the mode1 devised by Jubb and Fel1.55 When cocultures of cartilage and synovium are grown the cartilage shows a series of changes mediated by the synovial tissue, although in this situation vascular tissue can also induce the changes.56 Immunohistologic studies show that normal cartilage contains relatively little fibronectin, although hyaluronidase treatment shows that some perichondrocyte fibronectin is present. However, in this model there is deposition of fibronectin in the cartilage matrix over 14 days which is concomittant with loss of matrix proteog1ycans.57 This suggests that various stimuli can cause chondrocytes to produce fibronectin. Studies designed to elucidate the control of synovial fluid fibronectin levels and to determine the reason for their elevation in RA, are difficult to perform. However, the induction of a subcutaneous air pouch in rats provides a structure that is similar to the synovial membrane58 which can be used to investigate allergic inflammation.” Preliminary studies in our laboratories have shown that during the induction of chronic allergic inflammation in the rat air pouch, fibronectin is present in the pouch fluid and in the walls of the pouch; this approach may elucidate the factors that lead to and control local production and secretion of fibronectin into a mesenchymal tissue space.
FIBRONECTIN
IN RA
FIBRONECTIN AND CONNECTIVE TISSUE CHANGES IN RA
Most chronic diseases are associated with marked changes in the connective tissue matrix. As an important extracellular connective tissue protein, fibronectin is involved in the pathologic changes of a variety of diseases in addition to RA. These include colitis,60 cirrhosis,6’ glomerular disorders,62 and progressive systemic sclerosis.” In many of these situations there are marked changes in the distribution of collagenthe most important structural protein. There are at least five types of collagen. Collagens type I and III are the major collagens of synovial joints,64 and there is some evidence that the amount of collagen III is increased in RA.65 The relationship between fibronectin and collagens may well be an important factor in determining whether there is continuing granulation tissue formation or whether fibrosis and scar formation predominate. The rheumatoid pannus has similarities to granulation tissue, which shows three phases: (1) a cellular proliferative phase; (2) a stage of synthesis and secretion of extracellular connective tissue components; and (3) a separation phase, often with fibrosis. This may be disordered in RA and in certain other diseases.66 The consecutive phases of granulation tissue evolution can be reproduced experimentally.67 The proliferation begins with the development of capillary buds and the accumulation of hematogenie cells, leukocytes, and macrophages which migrate to the extracellular space. In the proliferative phase there is an increase in the numbers of fibroblasts; in the active phase they predominate among cells and are active in synthesizing and secreting the fibrous extracellular components of connective tissue. Although the synthesis of collagen and other connective tissue components is qualitatively the same in granulation tissue as in normal connective tissue, the reparative fibroblasts are different. For example, they differ in their reactivity to macrophage factors.68 The role of fibronectin in experimental granulation tissue has been studied in the spongeinduced granuloma.69*70 Fibronectin is present around the invading fibroblasts in the initial
249
stages, whereas interstitial collagens type I and II appear two to seven days later. When the interstitial collagen has matured into bundles, fibronectin diminishes or disappears. These results suggest that in this experimental situation fibronectin acts as a primary matrix for the organization of collagenous tissue during the repair process. Although these experimental findings have similarities to the observations made in the rheumatoid synovium and pannus, the striking difference in RA is the persistence of both extracellular fibronectin and cellular proliferation in the chronic stage of the disease. Fibronectin has a role in the differentiation of various tissues; and this is comparable with its role in granulation tissue formation. For example, a recent immunohistologic study by Weiss and Reddi” has shown that fibronectin is involved in various stages of endochondral bone formation following experimental implantation of a demineralized bone matrix, including the stages of differentiation of chondrocytes, osteoblasts, and marrow cells. But in this situation the ubiquitous presence of fibronectin is relatively nonspecific for any given cell type. The appearance of fibronectin in the rheumatoid pannus, which we have previously described, may also be related to dedifferentiation of the tissue-a view stressed by Fassbender in his concept of “mesenchymal transformation” of the connective tissue elements of the pannus.” The appearance of fibronectin at the cartilage pannus junction, which we have also described in RA, is possibly another feature of dedifferentiation of chondrocytes which has been described in a variety of experimental situations. However, it is probable that the relationship between chondrocytes and fibronectin is complex since another related glycoprotein, termed chondronectin, has also been isolated from articular cartilage.” Although fibronectin is involved in the deposition of connective tissue, tissue destruction is the other major feature of the pannus, especially cartilage destruction. This is principally mediated by collagenase.74 Explants of synovial tissue, grown in culture, produce large quantities of neutral collagenase.75 This enzyme is similar to that found in synovial fragments that are capable of degrading cartilage collagen. It is
250
SC0-l-r AND WALTON
located at the cartilage-pannus junction in RA.76 A variety of other enzymes are produced by the cells of the rheumatoid pannus which are also able to degrade the connective tissue matrix.” Thus, there is a delicate balance in the pannus between connective tissue production and destruction. FIBRONECTIN AND THE CONTROL OF THE CHRONIC INFLAMMATORY
RESPONSE
Chronic inflammation is a complex reaction that varies depending on the site and nature of the underlying disease process. It is controlled by an intricate web of factors, including prostaglandins, various kinins, and polypeptide factors such as lymphokines. Fibronectin is yet another cell product, not of lymphocytes but of mesenchymal cells (fibroblasts, endothelial cells, and macrophages) which is involved in inflammation. Some of the interrelated polypeptides and proteins that control the chronic inflammatory response have been isolated in experimental models and have been demonstrated to affect the formation and proliferation of granulation tissue.“-” Such factors are known to be present in rheumatoid joints. For example, Kulonen and Potila” have shown that rheumatoid synovial fluid can stimulate collagen synthesis in the presence of macrophages in a similar manner to an extract from a carbon tetrachloride-damaged liver and the connective tissue activating peptide of Castor and Lewis.79 Fibroblasts, the principal cells involved in the synthesis of components of the connective tissue matrix, are influenced by both monokines and lymphokines. Indeed, the close proximity of lymphoid components and fibroblasts in inflammatory sites has been recognized for many years. In vitro studies have shown that fibroblast chemotaxis is influenced by soluble factors that are released by macrophages.83 Once fibroblasts have entered the inflammatory site they proliferate and synthesize collagen and other components of the connective tissue matrix. This is controlled by a macrophage-derived fibroblastactivating factor.84 Similar factors are also released by lymphocytes, although these vary in their molecular size and certain other characteristics.*’ In some experimental situations the initial spreading of fibroblasts in serum-free medium
depends on the secretion of fibronectin.86 On the other hand, in other situations fibronectin bound to collagen can inhibit the migration of human skin fibroblasts and stimulate the migrations of other cells.87 This ability to subserve different functions in different situations is typical of fibronectin and is also seen in a number of lymphokines. McAuslan et al.‘* have proposed that in the process of neovascularization, variant endothelial cells have a special function in laying down fibronectin on which an endothelium can become established. This is one of its major functions. Endothelial cells at the site of an inflammatory process are one source of local production of fibronectin.89V90This local production has, in some experiments, been differentiated from plasma deposition, although both contribute to fibronectin in the extracellular matrix.” In this context it is relevant that dexamethasone inhibits the development of a well-defined cellular matrix of fibronectin92 and that interferon affects the distribution of cell surface fibronectin in human fibroblast cultures, changing it from a network of fibers to an array of filaments.93 There is also evidence that the presence of proteoglycans can greatly modify the interrelationships between collagen, fibronectin, and fibroblasts; Rich et a1.94 have shown that cartilage proteoglycans greatly inhibit fibroblast from spreading on fibronectin-coated collagen substances. This shows the complexity of the molecular interactions that are involved. It also suggests one mechanism that inhibits the spreading of cells onto normal cartilage. A failure of this inhibition may be important in the spread of cells in the invasion of cartilage by the pannus. However, Clemmensen et a1.95recently suggested that the normal cartilage matrix actually contains large amounts of fibronectin in complex with hyaluronic acid and that this is unmasked by treatment with hyaluronidase-the apparent deficit in most circumstances resulting from insufficient hyaluronidase treatment. ACUTE AND CHRONIC CHANGES IN RHEUMATOID SYNOVITIS
In acute cytes logic
RA there are often recurrent episodes of inflammatory synovitis (involving lymphoand plasma cells and a variety of immunosuperimposed on chronic changes),
FIBRONECTIN IN RA
251
changes, which are especially prominent in the pannus. Neither the acute nor chronic changes are “specific” for RA;96 indeed pannus formation occurs in various arthropathies. However, the severity of joint destruction and its widespread nature are characteristic of RA. The pathologic changes may be reflected in clinical and laboratory assessments of the disease. Clinical measurements of the degree of inflammatory synovitis and estimations of laboratory variables such as erythrocyte sedimentation rate (ESR) and C-reactive protein levels are closely related and can be influenced by treatment with second-line drugs such as gold.97 Joint destruction, the end result of chronic connective tissue changes, can be assessed radiologically. But roentgenographic changes follow a different time course from the acute phase changes. The evidence that they are influenced by treatment with second-line drugs is debatable.98 The present treatment of rheumatoid disease appears to concentrate on resolving episodes of severe acute inflammation. However, we consider that persistent chronic inflammation with connective tissue remodeling and progressive joint destruction (which can be visualized radiographically) will have a major influence upon the patient’s functional abilities and the outcome of the disease. New therapeutic avenues are undoubtedly needed and we suggest that it is in the control of chronic inflammation and connective tissue changes that these should be aimed.
Fibronectin is principally involved in the chronic phase of rheumatoid synovitis, especially in the initiation of connective tissue remodeling. Therefore in attempting new approaches to the management of rheumatoid disease, agents that influence the deposition of fibronectin and its relationship with cells and other connective tissue elements may profoundly influence the course of chronic inflammation and indeed of the disease process itself. This approach should merit consideration and could lead to a new direction in the treatment of joint destruction.
CONCLUSIONS
These various studies, taken together, show that fibronectin plays an important role in a number of the humoral and tissue changes that occur in RA. Its widespread distribution in the synovium and synovial fluid are compatible with the view that it has diverse functions in the pathologic changes of RA, a view in keeping with its known complex cellular and biochemical interactions. We suggest that in the RA joint its main roles are, first, opsonization of fibrinous debris and other particulate material that are shed into the synovial fluid, and second, a controlling function in the reorganization of the connective tissue matrix that is a characteristic feature of the synovium.
REFERENCES I. Cohn EJ. Properties and functions of plasma proteins with consideration of methods for their separation and purification. Chemical Reviews 1941;28:395417. 2. Cohn EJ. Chemical, physiological and immunological properties and clinical uses of blood derivatives. Experimentia 1947;3:125-36. 3. Kuusela P, Ruoslahti E, Engvall E, et al. Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry 1976;1:639-42. 4. Yamada KM, Olden K. Fibronectins-Adhesive glycoproteins of cell surface and blood. Nature 1978;275: 179-84. 5. Yamada KM. Fibronectin: transformationSensitive cell surface protein. In: Pick E, ed. Lymphokine Reports. New York: Academic Press, I980:23 l-53. 6. Mosesson MW, Amrani DL. The structure and biologic activities of plasma fibronectin. Blood 1980;56: 145-58. 7. Ruoslahti E, Engvall E, Hayman EG. Fibronectin: Current concepts of its structure and function. Collagen Research 1981;1:95-128.
8. Mosher DF. Fibronectin. Prog Haemost Thromb 1980;5:111-51. 9. Yamada KM, Kennedy DW. Fibroblast cellular and plasma fibronectins are similar but not identical. J Cell Biol 1979;80:492-8. 10. Stathakis NE, Mosesson MW. Interactions among heparin, cold insoluble globulin, and fibrinogen in formation of the heparin-precipitable fraction of plasma. J Clin Invest 1977;60:855-65. 1 I. Yamada KM, Kennedy DW, Kimata K, et al. Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J Biol Chem 1980;255:6055-63. 12. Dessau W, Adelmann BC, Timpl R, et al. Identification of the sites in collagen-Chains that bind serum antigelatin factor (cold insoluble globulin). Biochem J 1978;169:55-9. 13. Mosher DF. Cross-linking of cold-insoluble globulin by fibrin-stablizing factor. J Biol Chem 1975;250:6614-21, 14. McDonald JA, Baum BJ, Rosenberg DM, et al.
252
Destruction of a major extracellular adhesive glycoprotein (fibronectin) of human fibroblasts by neutral proteases from polymorphonuclear leucocyte granules. Lab Invest 1979;40:35&7. 15. Blumberg PM, Robbins PW. Effect of proteases on activation of resting chick embryo fibroblasts and on cell surface proteins. Cell 1975;6: 13747. 16. Olden K, Pratt RM, Yamada KM. Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin. Proc Natl Acad Sci USA 1979;76:3343-7. 17. Klebe RJ. Isolation of a collagen-dependent cell attachment factor. Nature 1974;250:248-5 1. 18. Grinnell F, Hays DG, Minter D. Cell-adhesion and spreading factor: Partial purification and properties. Exp Cell Res 1977; I IO: 175-90. 19. Birdwell CR, Gospodarowicz D, Nicholson GL. Identification, localization and role of fibronectin in cultured bovine endothelial cells. Proc Nat1 Acad Sci USA 1978;75:3273-7. 20. Alitalo K, Hovi T, Vaheri A. fibronectin is produced by human macrophages. J Exp Med 1980;15 I :602-l 3. 21. Bevilacqua MP, Amrani D, Mosesson MW, et al. Receptors for cold-insoluble globulin (plasma fibronectin) on human monocytes. J Exp Med 1981;153:42-60. 22. Grinnell F, Feld MK. Spreading of human fibroblasts in serum-free medium: Inhibition by dithiotheitol and the effect of cold insoluble globulin (plasma fibronectin). J Cell Physiol 1980;104:321-34. 23. Anderson JC. Glycoproteins of the connective tissue matrix. Int Rev Connect Tissue Res 1976;7:251-322. 24. Stenman S, Vaheri A. Distribution of a new major connective tissue protein, fibronectin, in normal human tissues. J Exp Med 1978;147:1054-64. 25. Bach PR, Bentley JP. Structural glycoproteins, fact or artefact. Connect Tissue Res 1980;7:185-96. 26. Saba TM, Jaffe E. Plasma fibronectin (opsonic glycoprotein): Its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function. Am J Med 1980;68:577-94. 27. Mosher DF, Williams EM. Fibronectin concentration is decreased in plasma of severely ill patients with disseminated intravascular coagulation. J Lab Clin Med 1978; 91~729-35. 28. Stathakis NE, Fountas A, Tsianos E. Plasma fibronectin in normal subjects and in various disease states. J Clin Pathol 1981;34:504-8. 29. Gonzalez-Calvin J, Scully MF, Sanger Y, et al. Fibronectin in fulminant heparin failure. Br Med J 1982;285: 1231-2. 30. Scott DL, Farr M, Crockson AP, et al. Synovial fluid and plasma fibronectin levels in rheumatoid arthritis. Clin Sci 1982;62:71-6. 31. Carsons S, Mosesson MW, Diamond HS. Detection and quantitation of fibronectin in synovial fluid of patients with rheumatoid disease. Arthritis Rheum 1981;24:1261-7. 32. Clemmensen I, Andersen RB. Different molecular forms of fibronectin in rheumatoid synovial fluid. Arthritis Rheum 1982;25:25-3 1. 33. Gressner VAM, Wallraff P. Der Einsatz der Lasernephelometri zur Bestimmung und rcchnerunter-stiizten Auswertung der Fibronectinkonzentration in verschiedenem kiir
SCO-IT AND WALTON
perfliissigkeiten. 805.
J Clin Chem
Clin Biochem
1980;18:797-
34. Vartio T, Vaheri A, Von Essen R, et al. Fibronectin in synovial fluid and tissue in rheumatoid arthritis. Eur J Clin Invest 1981;11:207-12. 35. Scott DL, Almond TJ, Naqvi SNH, et al. The significance of fibronectin in cryoprecipitation in rheumatoid arthritis and other disorders. J Rheumatol 1982;9:514-8 36. Beaulieu AD, Valet JP, Storey J. The influence of fibronectin on cryoprecipitate formation in rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum 1981;24:1383-8. 37. Anderson B, Rucker M, Entwhistle R, et al. Plasma fibronectin is a component of cryoglobulins from patients with connective tissue and other diseases. Ann Rheum Dis 1981;40:5&4. 38. Wood G, Rucker M, Davies JW, et al. Interaction of plasma fibronectin with selected cryoglobulins. Clin Exp Immunol 1980;40:358-64. 39. Mosesson MW. Structure of human plasma coldinsoluble globulin and the mechanism of its precipitation in the cold with heparin or fibrin-fibrinogen complexes. Ann NY Acad Sci 1978;312:11--30. 40. Menzell EJ, Smolen JS, Liotta L, et al. Interaction of fibronectin with Cl, and its collagen-like fragment (CLF). FEBS Lett 1981;129:188-92. 41. Reise H. Die Reiskorpchen in tuberculijs erkrunken synovalsacken. Deutsch Z Chir 1895;42:1-99. 42. Popert AJ, Scott DL, Wainwright AC, et al. The frequency of occurrence, mode of development and significance of rice-bodies in rheumatoid joints. Ann Rheum Dis 1982;41:109-17. 43. Berg E, Wainwright R, Barton B, et al. On the nature of rheumatoid rice bodies. An immunologic, histochemical and electron microscope study. Arthritis Rheum 1977;20:1343-9. 44. McCarty DJ, Cheung HS. Origin and significance of rice bodies in synovial fluid. Lancet 1982;2:7 15-6. 45. Glynn LE. The chronicity of inflammation and its significance in rheumatoid arthritis. Ann Rheum Dis 1968;27:105-21. 46. Trentham DE, Dynesius RA, Rocklin RE, et al. Cellular sensitivity to collagen in rheumatoid arthritis. N Engl J Med 1978;299:327-32. 47. Scott DL, Wainwright A, Walton KW, et al. The significance of fibronectin in rheumatoid arthritis and osteoarthrosis. Ann Rheum Dis 1981;40:142-53. 48. Revel1 PA, Mayston V, Davies PG. Fibronectin in the synovium of chronic inflammatory joint disease. Ann Rheum Dis 1983;42:222. 49. Scott DL, Delamere JP, Walton KW. The distribution of fibronectin in the rheumatoid pannus. Br J Exp Pathol 1981;62:362-8. 50. Unsworth DJ, Scott DL, Almond TJ, et al. Studies on reticulin I: Serological and immunohistological investigation of the occurrence of collagen type III, fibronectin and the non-collagenous glycoprotein of Pras and Glynn in reticulin. Br J Exp Pathol 1982;63:154-66. 5 1. Bosselli JM, Macarak EJ, Clark CC, et al. Fibronectin: Its relationship to basement membranes. I. Light microscopic studies. Collagen Research 1981;5:391-404.
FIBRONECTIN IN RA
52. Dumonde DC, Glynn LE. The production of arthritis in rabbits by an immunological reaction to fibrin. Br J Exp Pathol 1962;43:373-83. 53. Consden R, Doble A, Glynn LE. et al. Production of chronic arthritis with ovalbumin. Its retention in the rabbit knee joint. Ann Rheum Dis 1971;30:307-15. 54. Scott DL, Almond TJ, Walton KW, et al. Fibronectin in antigen induced arthritis in the rabbit. J Pathol 1983 (in press) 55. Fell HB, Jubb RW. The effect of synovial tissue on the breakdown of articular cartilage in organ culture. Arthritis Rheum 1977;20:1359-71. 56. Jubb RW. Breakdown of articular cartilage by vascular tissue. J Pathol 1982;136:333-43. 57. Scott DL, Almond TJ, Walton KW, et al. Involvement of fibronectin in fibrosis and opsonisation in rheumatic diseases. Ann Rheum Dis 1983;42:221-2. 58. Edwards TCW, Sedgwick AD, Willoughby DA. The formation of a structure with the features of synovial lining by subcutaneous injection of air: An in vivo tissue culture system. J Pathol I98 I ; 134: 147-56. 59. Yoshino S, Bacon PA, Blake DR, et al. A model of persistent, antigen-induced chronic inflammation in the rat air pouch (submitted for publication). 60. Scott DL, Morris CJ, Blake AE, et al. Distribution of fibronectin in the rectal mucosa. J Clin Pathol 1981;34:74958. 61. Hahn E, Wick G, Pencev D, et al. Distribution of basement membrane proteins in normal and fibrotic human liver: Collagen type IV, laminin and fibronectin. Gut 1980;2 I :63-7 1 62. Scheinman Jl, Foidart JM, Gehron-Robey P, et al. The immunohistology of glomerular antigens. Clin lmmunol lmmunopathol 1980;15:175-89. 63. Cooper SM. Keyser AJ, Beaulieu AD, et al. Increase in tibronectin in the deep dermis of involved skin in progressive systemic sclerosis. Arthritis Rheum 1979;22:983-7. 64. Gay S, Gay RE, Miller EJ. The collagens of the joint. Arthritis Rheum 1980;23:93741. 65. Eyre DR. Muir H. Type Ill collagen: A major constituent of rheumatoid and normal human synovial membrane. Conn Tissue Res 1976;4: I l-6. 66. Jayson MIV (ed). Symposium on the fibrotic process. Ann Rheum Dis (Supp12). 1977 67. Kulonen E. Experimental granuloma as a tool in connective tissue research. In: Hall DA, ed. The methodology of connective tissue research. Oxford: Joynson--Bruvvers, 1976:29-36. 68. Aalto M, Potila M, Kulonen E. Effects of silica treated macrophages on the synthesis of collagen and other proteins in vitro. Exp Cell Res 1976;97: 193-202. 69. Kurkinen M, Vaheri A, Roberts PJ, et al. Sequential appearance of hbronectin and collagen in experimental granulation. Lab Invest 1980,43:47-57. 70. H#lund B, Clemmensen I, Junker P, et al. Fibronectin in experimental granulation tissue. Acta Pathol Microbial lmmunol Stand 1982;90A:15965. 71. Weiss RE, Reddi AH. Appearance of fibronectin during the differentiation of cartilage, bone and bone marrow. J Cell Biol 1981;88:630-6.
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72. Fassbender HG. The pathology of rheumatic Loewi G, Trans. Berlin: Springer Verlag, 1975.
diseases.
73. Hewitt AT, Kleinman HK, Pennypacker JP, et al. Identification of an adhesion factor for chondrocytes. Proc Nat] Acad Sci USA 1980;77:385-8. 74. Woolley DE, Glanville RW, Crossley MJ, et al. Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen. Eur J Biochem 1975;54:61 l2. 75. Dayer JM, Goldring SR, Robinson DR, et al. Cell-cell interactions and collagenase production. In: Wooley DE, Evanson JM, eds. Collagenase in normal and pathological connective tissues. Chichester: John Wiley & Sons, 1980:83104. 76. Woolley DE, Crossley MJ, Evanson JM. Collagenase at sites of cartilage erosion in the rheumatoid joint. Arthritis Rheum 1977;20:1231-9. 77. Vaes G, Hauser D, Huybrecht-Godin G, et al. Cartilage degradation by macrophages, fibroblasts and synovial cells in culture. An in vitro model suitable for studies on rheumatoid arthritis. In: Willoughby DA, Girond JP, Velo GP, eds. Perspectives in inflammation. Lancaster: MTP Press, 1977: 115-26. 78. Aalto M, Kulonen E. Inhibition of protein synthesis in tendon cells by extracts from experimental granulation tissue. FEBS Lett 1974;49:7&2. 79. Castor CW, Lewis RB. Connective tissue activation x current studies on the process and of its mediators. Stand J Rheumatol 1976;5(Suppl 12):41-54. 80. Aalto M, Kulonen E. Fractionation of connectivetissue-activating factors from the culture medium of silicatreated macrophages. Acta Pathol Microbial Stand 1979; 87C:241-50. 81. Aalto M, Turakainen H, Kulonen E. Effect of SiO, liberated macrophage factor on protein synthesis in connective tissue in vitro. Stand J Clin Lab Invest 1979;39:205-13. 82. Kulonen E, Potila M. Macrophages of connective tissue components. Acta Stand 1980;88(3:7-13.
and the synthesis Pathol Microbial
83. Wahl SM. Wahl LM. Modulation of fibroblast growth and function of monokines and lymphokines. In: Pick E, ed. Lymphokines, Vol 2. New York: Academic Press 1981:179-202. 84. Wahl SM, Wahl LM, McCarthy JB, et al. Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide. J lmmunol 1979;122:2226-31. 85. Wahl SM. Wahl LM, McCarthy JB. Lymphoctye mediated activation of fibroblast proliferation and collagen production. J lmmunol 1978;121:942-6. 86. Grinnell F, Feld MK. Spreading of human fibroblasts in serum free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin. J Cell Physiol 1980;104:321-34. 87. Schor SL, Schor SM, Bazill GW. The effects of fibronectin on the migration of human foreskin fibroblasts and Syrian hamster melanoma gels of native collagen fibres. J Cell Sci 1981;48:301-14. 88. McAuslan BR, Aannan GN, Reilly W, et al. Variant endothelial cells. Fibronectin as a transducer of signals for
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migration and neovascularisation. J Cell Physiol 1980;104:177-86. 89. Clark RAF, Dvorak HF, Colvin RB. Fibronectin in delayed hypersensitivity skin reactions: Association with vessel permeability and endothelial cell activation. J Immunol 1981;126:787-93. 90. Clark RAF, Quinn JH, Winn HJ, et al. Fibronectin is produced by blood vessels in response to injury. J Exp Med 1982;156:646-51. 91. Hayman EG, Ruoslahti E. Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix. J Cell Biol 1979;83:255-9. 92. Mareau N, Goyette R, Valet JP, et al. The effect of dexamethasone on formation of a fibronectin extracellular matrix by rat hepatocytes in vitro. Exp Cell Res 1980;125:497-502. 93. Pfeffer LM, Wang E, Tamm I. Interferon effects on microfilament organization, cellular fibronectin distribution, and cell motility in human fibroblasts. J Cell Biol 1980;85: 9-17.
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94. Rich AM, Pearlstein E, Weissmann G, et al. Cartilage proteoglycans inhibit fibronectin-mediated adhesion. Nature 1981;293:2246. 95. Clemmensen I, Hdlund B, Johansen N, et al. Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique. Histochemistry 1982;76:51-6. 96. Gardner DL. General pathology of the peripheral joints. In: Sokoloff L, ed. The joints and synovial fluid, vol 2. New York: Academic Press 1980:316-427. 97. McConkey B, Davis P, Crockson RA, et al. Effects of gold dapson and prednisolone on serum C-reactive protein and haptoglobin and the erythrocyte sedimentation rate in rheumatoid arthritis. Ann Rheum Dis 1979;38:141-4. 98. Scott DL, Grindulis KA, Struthers GR, et al. The progression of radiological changes in rheumatoid arthritis. Ann Rheum Dis 1983 (in press) 99. Matsuda B, Yoshida N, Aoki N, et al. Distribution of cold-insoluble globulin in plasma and tissues. Ann NY Acad Sci 1978;312:56-73.
of Fibronectin
in Rheumatoid
Arthritis
By D. L. Scott and K. W. Walton
R
HEUMATOID ARTHRITIS (RA) is characterized by its chronicity. Many typical features result from chronic inflammation, with progressive destruction of normal synovial structure and changes in the connective tissue matrix involving collagen, noncollagenous structural glycoproteins, and probably other components, including proteoglycans and glycosaminoglycans. Although immunologic changes have been at the center of research into RA in the last two decades, changes in the connective tissue matrix are a constant feature and may be of equal significance in pathogenesis. Fibronectin is one of the noncollagenous structural glycoproteins, and in recent years its roles in the organization of connective tissue and in the relationship of cells to the connective tissue matrix have been defined. These roles are especially relevant in a chronic inflammatory disorder such as RA. In this review we will briefly describe the structure and function of fibronectin, its relationship to the pathologic features of RA, and its interactions with other elements of connective tissue. Finally, we will consider how such changes in the connective tissue matrix may influence the clinical features of the disease. STRUCTURE AND FUNCTIONS OF FIBRONECTIN
The discovery of fibronectin, which was initially termed cold-insoluble globulin, dates from Cohn’s studies of plasma proteins in the 1940s and the consequent investigations of the various Cohn fractions.‘*2 However, it was only after a series of studies in the 1970s at different centers From the Department of Investigative Pathology, Rheumatism Research Wing, The Medical School, University of Birmingham, Birmingham, England. Supported by the Arthritis and Rheumatism Council and the West Midlands Regional Health Authority. D. L. Scott BSc MD MRCP: Lecturer, and K. W. Walton MD PhD DSc FRCPath: Professor, the Department of Investigative Pathology. Rheumatism Research Wing. The Medical School, University of Birmingham. Address reprint requests to D. L. Scott, M.D.. Department of Investigative Pathology, Rheumatism Research Wing, The Medical School, University of Birmingham, Birmingham, England 815 2TJ. o I984 by Grune & Stratton, Inc. 0049-0172/84/1303-0003$2.00/0 244
that fibronectin’s important role as a plasma and connective tissue protein was recognized. During this period the protein was known by a large and confusing number of synonyms. Fortunately the problem of nomenclature has been resolved and it is now known by one name-fibronectin.3 The discovery of fibronectin and its more recent history have been extensively reviewed.“8 We will therefore concentrate on those aspects of its biochemistry that are directly relevant to its involvement in the pathogenesis of RA. There are two forms of fibronectin: Plasma fibronectin is soluble, whereas tissue fibronectin is an insoluble matrix protein. There are a number of biochemical differences between these forms of fibronectin, although their general structure is similar’ and they show immunologic cross-reactivity. Fibronectin is a glycoprotein (molecular weight, 440,000) with two polypeptide chains that are linked by a disulphide bridge; the two chains may be slightly different. The chains are composed of several domains with different functional roles. This includes binding sites for cells, heparin, hyaluronic acid, and collagen.‘G’2 Fibronectin will also bind both fibrinogen and fibrin, which is influenced by factor XIII.13 In common with many proteins, fibronectin is degraded by a variety of different proteolytic enzymes;‘4*‘5 there is some evidence that its sugar residues protect it against degradation.16 The different functional roles of fibronectin (Table 1) can be readily understood in relation to its structure. It has several interactions with cells. The first to be studied in detail was its ability to promote the adhesion of fibroblasts to gelatin-coated surfaces. I7 There is now evidence that fibronectin mediates the adhesion of a variety of cells, including fibroblasts, endothelial cells, and macrophages to the surrounding matrix”-*’ and also has a role in cell-to-cell interactions. Cell surface fibronectin and fibronectin receptors modulate these roles.2’V22 The second main function of fibronectin is that as a component of the extracellular matrix. The extracellular matrix is a complex structure and contains a variety of macromolecules, including collagens, elastin, proteoglycans, and noncollageSeminars in Arthritis and Rheumatism, Vol. 13, No. 3 (February), 1984
245
FIBRONECTININ RA
Table 1. Biochemical Properties of Fibronectin Relevant in RA Examples
Property Cell interactions Macromolecular
Cell-substratum bind-
ing
adhesion
Binding to collagen, fibrin, complement (Cl,), hyaluronic acid, and sulphated glycosaminoglycans
Opsonization
Clearance of collagenous and
ditions, all of which are severe.27-29 This is thought to occur as the result of fibronectin being consumed as part of the uptake of fibrin and other particulate debris by the reticuloendothelial system in severe disease. There is evidence that monocytes and other cells have cell-surface fibronectin receptors that may modulate its role as an opsonin.2’
other particulate debris by re-
FIBRONECTIN AND RA
ticuloendothelial system and other phagocytic cells
nous structural glycoproteins. This latter group of proteins includes fibronectin and a number of other proteins;23*24the extraction procedures for these are complex and denaturing and their exact composition is debatable.25 Ail these macromolecules can be produced by fibroblasts and other connective tissue cells (Fig. 1). The binding of fibronectin to the macromolecules of the extracellular matrix is mediated by the specific binding sites in its different domains. Finally, fibronectin is a major “nonimmunologic” opsonin. This is thought to be the most important role of plasma fibronectin,26 and changes in fibronectin levels in the blood are seen in a variety of situations. In experimental animals this is seen after the intravenous administration of gelatin particles and is a feature of the clearance of a number of macromolecules from the circulation, including high molecular weightxharged polysaccharides (for example, dextran sulphate; molecular weight, 500,000). In humans a low plasma fibronectin level is seen in disseminated intravascular coagulation, fulminant hepatic failure, and certain other con-
A series of studies in recent years have established that fibronectin is a component of synovial fluid, the rheumatoid synovium, and the rheumatoid pannus.3g40 These studies have suggested it may have significant pathogenic roles. The sites at which fibronectin has been demonstrated in rheumatoid joints are summarized in Fig. 1. Synovial Fluid Fibronectin
The fibronectin concentration of synovial fluid is significantly higher in RA than in other arthropathies, with mean levels two to three times those of plasma. The various reports of plasma and synovial fluid fibronectin levels are summarized in Table 2. In individual patients there may be no relationship between synovial fluid and plasma fibronectin levels.30x3’Synovial fluid fibronectin is unrelated to the degree of joint inflammation, the acute phase response, the total synovial fluid protein level, and levels of specific proteins in synovial fluid such as albumin, IgG, and C3.30132These findings suggest that the local production of fibronectin by synoTable 2. Fibronectin Levels in Plasma and Synovial Fluid MCWl
Svnovlal
fluid
SOUMX
Normal plasma
RA plasma
Flbronectw Level (g/Lb 0.28
Reference Matsuda et al.99
0.33
Mosher and Williams*’
0.32
Scott et al.30
0.29
Gressner et al.33
0.32
Scott et aL3’ Clemmensen and Ander-
0.33
son”
RA synovial fluid
Svnowum
Pannus
0.70
Scott et aL3’
0.44
Vartio et al.34
0.32’
Gressner et al.32
0.81
Clemmensen and Ander-
0.70
Carsons et al.”
CBrtilaqe
Fig. 1. The sites et which fibronectin is present in the rheumatoid synovium.
so?
*Included other arthropathies.
246
vial lining cells and vascular endothelium cells is an important source. There is some evidence that synovial fluid fibronectin consists of several different molecular weight species, with some of lower molecular weight compared to the fibronectin of plasma.32 This may result from partial degradation within the joint or by the local synthesis of a defective molecule. Many enzymes, including plasmin and leukocyte proteases, can degrade fibronectin which may be of significance within the joint. Fibronectin in Cryoprecipitates
Fibronectin is a component of synovial fluid and serum cryoprecipitates in RA.35-38Cryoprecipitates containing fibronectin also contain immunoglobulins and a variety of other proteins, including fibrinogen and fibrin degradation products. The presence of fibronectin in cryoprecipitates may result from a variety of molecular interactions. It binds to fibrinogen and fibrin monomer in the cold3’-this was the basis of its original identification as cold insoluble globulin.
SCOlT AND WALTON
However, it can also bind to the collagenlike fragment of Clq40 and may react with certain immunoglobulins.38 Fibronectin may play a role in the formation of cryoprecipitates, especially in synovial fluid. If it is selectively removed from serum and synovial fluid prior to cooling to 4 OC, the amount of cryoprecipitate formed is often markedly reduced, although this varies from patient to patient.36 Fibronectin is not specific to rheumatoid cryoprecipitates and is found in those isolated from patients with a variety of other connective tissue diseases and arthropathies. Fibronectin and Rice Bodies
Intraarticular rice bodies were first described by Reise 4’ in association with tuberculous arthritis, but they are more commonly associated with RA.42.43Their name is derived from their resemblance to grains of polished rice, although they vary considerably in size and consistency (Fig. 2). The majority of rice bodies consist of fibrinous material. Some contain areas of collagen and
FIBRONECTIN
247
IN RA
mononuclear cells. Fibronectin codistributes with both fibrin and areas of immature collagen within rice bodies, as shown in Fig. 2. The mode of development of rice bodies is controversial. One view is that the earliest rice bodies are predominantly fibrinous, and at a later stage, collagen may be formed due to synthesis by mononuclear cells that are entrained within the rice bodies.42 Alternatively the hypoxia of RA synovial fluid and the associated focal necrosis of synovial tissue44,72may cause synovial microinfarction; this will then provide the initial source of the collagen core of rice bodies, which is subsequently enriched by superficial fibrin coating. It is likely that both these processes occur to some extent. In any event, fibronectin, with its specific binding to fibrin and collagen, will have a central role in the development of rice bodies. Rice bodies within a joint act as an irritant. One reason may be persistence of fibrin4j another, the antigenicity of collagen fragments and altered co1lagen.46However, they can be removed by lavage with a wide bore needle, which gives symptomatic improvement.42 Fibronectin in the Synovial Membrane
Fibronectin is a structural component of normal connective tissue24 and is found in reticulin fibers, as part of the perivascular connective tissue, and in relationship to certain connective tissue cells. It is present in this distribution in the normal synovium. However, in an inflammatory arthritis such as RA there are extensive and significant changes in its distribution. In RA the synovial lining cells contain fibronectin intracellularly, in relation to their cell processes, and pericellularly. Immediately beneath the proliferated synovial cell lining layer there is a meshwork of reticulin fibers that contain fibronectin, which is also present around the many small blood vessels in this region (Fig. 3). Lymphocytes and other inflammatory cells that infiltrate the synovial membrane are not directly related to fibronectin, although it is present in the reticulin fibers that subdivide areas of inflammatory cell infiltration. Fibronectin also codistributes with fibrin on the surface of the synovium and in deeper areas of the synovium.47 These findings are not specific for RA and have been seen in other forms of inflammatory synovitis.48
Fig. 3. Section of rheumatoid synovium stained by antifibronectin by using PAP technique, showing positive reaction of synovial cell lining layer and perhrascular and reticular staining deeper in synovium. (x222)
The rheumatoid pannus is an area of connective tissue proliferation and remodeling. Fibronectin is present in all areas of the pannus, including its junctions with cartilage and other structures within the joint.49 It is especially marked in the more cellular and vascular areas of the pannus, where it forms a coarse extracellular meshwork. In normal cartilage it may not be demonstrable in the matrix, although there is some perichondrocyte fibronectin that is demonstrable after hyaluronidase treatment. However, marginal areas of articular cartilage next to the pannus that show loss of proteoglycans do contain immunoreactive fibronectin within their matrix. Fibronectin is also related to the development of fibrosis within the joint. In the early stages of the deposition of fibrous tissue, fibronectin shows close codistribution with immature collagen that is demonstrated by various histologic stains, such as Martius Scarlet Blue and van Gieson fluid (Fig. 4). However, as the fibrous tissue condenses and the collagen becomes mature, this codistribution is lost and mature fibrous tissue does not contain immunoreactive fibronectin. Pari passu with its relationship to fibrosis, fibronectin is codistributed with collagen type III in reticulin fibers5’ and collagen type IV in basement membranes,” and this is a feature of the synovium in RA as at other sites.47 Studies in Experimental
Models
A variety of experimental models have given further information on the relationship of fibronectin to the pathologic changes of RA. The best
SCOTT AND WALTON
Fig.4. Synovial biopsy of rheumatoid synovium. showing “micronodules” in superficial synovium: (A) stained with Martius Scarlet Blue-micronodule stained pale blue in the original (as for “immature”collagenJ; (Bj same field in adjacent section, stained with rabbit antifibronectin and then FITC-labeled antibody to rabbit immunoglobulin, demonstrating the contribution of fibronectin with “immature” collagen by immunofluorescence. (x498)
known animal model for chronic synovitis is antigen-induced arthritis in rabbits. In its original form this used fibrin as an antigen5* but a modification is the use of ova1bumin.53 Following intraarticular challenge with ovalbumin in sensitized animals, the relationship of fibronectin to the time course of the arthritis can be evaluated by immunohistologic techniques. Initially there is a widespread deposition of fibronectin throughout the inflamed synovium; this is seen 48 hours after challenge and represents the deposition of plasma fibronectin as one component of an inflammatory exudate. As organization and remodeling of the joint occurs over the next few weeks the early, marked extracellular deposits of fibronectin disappear; many areas of the synovium contain little fibronectin, although it persists around small blood vessels, at some sites of the hyperplastic synovial lining membrane, and in fibrin deposits. When the chronic stage develops (at about three months) the synovium contains extensive deposits of fibronectin. It is seen in the synovial lining cell layer, which is markedly hyperplastic, codistributing with fibrin, and in relation to immature collagenous tissue.s4 These changes are similar to those seen in human disease in the chronic phase. The changes in the cartilage in RA, seen at the cartilage-pannus junction, can be studied experi-
mentally by using the mode1 devised by Jubb and Fel1.55 When cocultures of cartilage and synovium are grown the cartilage shows a series of changes mediated by the synovial tissue, although in this situation vascular tissue can also induce the changes.56 Immunohistologic studies show that normal cartilage contains relatively little fibronectin, although hyaluronidase treatment shows that some perichondrocyte fibronectin is present. However, in this model there is deposition of fibronectin in the cartilage matrix over 14 days which is concomittant with loss of matrix proteog1ycans.57 This suggests that various stimuli can cause chondrocytes to produce fibronectin. Studies designed to elucidate the control of synovial fluid fibronectin levels and to determine the reason for their elevation in RA, are difficult to perform. However, the induction of a subcutaneous air pouch in rats provides a structure that is similar to the synovial membrane58 which can be used to investigate allergic inflammation.” Preliminary studies in our laboratories have shown that during the induction of chronic allergic inflammation in the rat air pouch, fibronectin is present in the pouch fluid and in the walls of the pouch; this approach may elucidate the factors that lead to and control local production and secretion of fibronectin into a mesenchymal tissue space.
FIBRONECTIN
IN RA
FIBRONECTIN AND CONNECTIVE TISSUE CHANGES IN RA
Most chronic diseases are associated with marked changes in the connective tissue matrix. As an important extracellular connective tissue protein, fibronectin is involved in the pathologic changes of a variety of diseases in addition to RA. These include colitis,60 cirrhosis,6’ glomerular disorders,62 and progressive systemic sclerosis.” In many of these situations there are marked changes in the distribution of collagenthe most important structural protein. There are at least five types of collagen. Collagens type I and III are the major collagens of synovial joints,64 and there is some evidence that the amount of collagen III is increased in RA.65 The relationship between fibronectin and collagens may well be an important factor in determining whether there is continuing granulation tissue formation or whether fibrosis and scar formation predominate. The rheumatoid pannus has similarities to granulation tissue, which shows three phases: (1) a cellular proliferative phase; (2) a stage of synthesis and secretion of extracellular connective tissue components; and (3) a separation phase, often with fibrosis. This may be disordered in RA and in certain other diseases.66 The consecutive phases of granulation tissue evolution can be reproduced experimentally.67 The proliferation begins with the development of capillary buds and the accumulation of hematogenie cells, leukocytes, and macrophages which migrate to the extracellular space. In the proliferative phase there is an increase in the numbers of fibroblasts; in the active phase they predominate among cells and are active in synthesizing and secreting the fibrous extracellular components of connective tissue. Although the synthesis of collagen and other connective tissue components is qualitatively the same in granulation tissue as in normal connective tissue, the reparative fibroblasts are different. For example, they differ in their reactivity to macrophage factors.68 The role of fibronectin in experimental granulation tissue has been studied in the spongeinduced granuloma.69*70 Fibronectin is present around the invading fibroblasts in the initial
249
stages, whereas interstitial collagens type I and II appear two to seven days later. When the interstitial collagen has matured into bundles, fibronectin diminishes or disappears. These results suggest that in this experimental situation fibronectin acts as a primary matrix for the organization of collagenous tissue during the repair process. Although these experimental findings have similarities to the observations made in the rheumatoid synovium and pannus, the striking difference in RA is the persistence of both extracellular fibronectin and cellular proliferation in the chronic stage of the disease. Fibronectin has a role in the differentiation of various tissues; and this is comparable with its role in granulation tissue formation. For example, a recent immunohistologic study by Weiss and Reddi” has shown that fibronectin is involved in various stages of endochondral bone formation following experimental implantation of a demineralized bone matrix, including the stages of differentiation of chondrocytes, osteoblasts, and marrow cells. But in this situation the ubiquitous presence of fibronectin is relatively nonspecific for any given cell type. The appearance of fibronectin in the rheumatoid pannus, which we have previously described, may also be related to dedifferentiation of the tissue-a view stressed by Fassbender in his concept of “mesenchymal transformation” of the connective tissue elements of the pannus.” The appearance of fibronectin at the cartilage pannus junction, which we have also described in RA, is possibly another feature of dedifferentiation of chondrocytes which has been described in a variety of experimental situations. However, it is probable that the relationship between chondrocytes and fibronectin is complex since another related glycoprotein, termed chondronectin, has also been isolated from articular cartilage.” Although fibronectin is involved in the deposition of connective tissue, tissue destruction is the other major feature of the pannus, especially cartilage destruction. This is principally mediated by collagenase.74 Explants of synovial tissue, grown in culture, produce large quantities of neutral collagenase.75 This enzyme is similar to that found in synovial fragments that are capable of degrading cartilage collagen. It is
250
SC0-l-r AND WALTON
located at the cartilage-pannus junction in RA.76 A variety of other enzymes are produced by the cells of the rheumatoid pannus which are also able to degrade the connective tissue matrix.” Thus, there is a delicate balance in the pannus between connective tissue production and destruction. FIBRONECTIN AND THE CONTROL OF THE CHRONIC INFLAMMATORY
RESPONSE
Chronic inflammation is a complex reaction that varies depending on the site and nature of the underlying disease process. It is controlled by an intricate web of factors, including prostaglandins, various kinins, and polypeptide factors such as lymphokines. Fibronectin is yet another cell product, not of lymphocytes but of mesenchymal cells (fibroblasts, endothelial cells, and macrophages) which is involved in inflammation. Some of the interrelated polypeptides and proteins that control the chronic inflammatory response have been isolated in experimental models and have been demonstrated to affect the formation and proliferation of granulation tissue.“-” Such factors are known to be present in rheumatoid joints. For example, Kulonen and Potila” have shown that rheumatoid synovial fluid can stimulate collagen synthesis in the presence of macrophages in a similar manner to an extract from a carbon tetrachloride-damaged liver and the connective tissue activating peptide of Castor and Lewis.79 Fibroblasts, the principal cells involved in the synthesis of components of the connective tissue matrix, are influenced by both monokines and lymphokines. Indeed, the close proximity of lymphoid components and fibroblasts in inflammatory sites has been recognized for many years. In vitro studies have shown that fibroblast chemotaxis is influenced by soluble factors that are released by macrophages.83 Once fibroblasts have entered the inflammatory site they proliferate and synthesize collagen and other components of the connective tissue matrix. This is controlled by a macrophage-derived fibroblastactivating factor.84 Similar factors are also released by lymphocytes, although these vary in their molecular size and certain other characteristics.*’ In some experimental situations the initial spreading of fibroblasts in serum-free medium
depends on the secretion of fibronectin.86 On the other hand, in other situations fibronectin bound to collagen can inhibit the migration of human skin fibroblasts and stimulate the migrations of other cells.87 This ability to subserve different functions in different situations is typical of fibronectin and is also seen in a number of lymphokines. McAuslan et al.‘* have proposed that in the process of neovascularization, variant endothelial cells have a special function in laying down fibronectin on which an endothelium can become established. This is one of its major functions. Endothelial cells at the site of an inflammatory process are one source of local production of fibronectin.89V90This local production has, in some experiments, been differentiated from plasma deposition, although both contribute to fibronectin in the extracellular matrix.” In this context it is relevant that dexamethasone inhibits the development of a well-defined cellular matrix of fibronectin92 and that interferon affects the distribution of cell surface fibronectin in human fibroblast cultures, changing it from a network of fibers to an array of filaments.93 There is also evidence that the presence of proteoglycans can greatly modify the interrelationships between collagen, fibronectin, and fibroblasts; Rich et a1.94 have shown that cartilage proteoglycans greatly inhibit fibroblast from spreading on fibronectin-coated collagen substances. This shows the complexity of the molecular interactions that are involved. It also suggests one mechanism that inhibits the spreading of cells onto normal cartilage. A failure of this inhibition may be important in the spread of cells in the invasion of cartilage by the pannus. However, Clemmensen et a1.95recently suggested that the normal cartilage matrix actually contains large amounts of fibronectin in complex with hyaluronic acid and that this is unmasked by treatment with hyaluronidase-the apparent deficit in most circumstances resulting from insufficient hyaluronidase treatment. ACUTE AND CHRONIC CHANGES IN RHEUMATOID SYNOVITIS
In acute cytes logic
RA there are often recurrent episodes of inflammatory synovitis (involving lymphoand plasma cells and a variety of immunosuperimposed on chronic changes),
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changes, which are especially prominent in the pannus. Neither the acute nor chronic changes are “specific” for RA;96 indeed pannus formation occurs in various arthropathies. However, the severity of joint destruction and its widespread nature are characteristic of RA. The pathologic changes may be reflected in clinical and laboratory assessments of the disease. Clinical measurements of the degree of inflammatory synovitis and estimations of laboratory variables such as erythrocyte sedimentation rate (ESR) and C-reactive protein levels are closely related and can be influenced by treatment with second-line drugs such as gold.97 Joint destruction, the end result of chronic connective tissue changes, can be assessed radiologically. But roentgenographic changes follow a different time course from the acute phase changes. The evidence that they are influenced by treatment with second-line drugs is debatable.98 The present treatment of rheumatoid disease appears to concentrate on resolving episodes of severe acute inflammation. However, we consider that persistent chronic inflammation with connective tissue remodeling and progressive joint destruction (which can be visualized radiographically) will have a major influence upon the patient’s functional abilities and the outcome of the disease. New therapeutic avenues are undoubtedly needed and we suggest that it is in the control of chronic inflammation and connective tissue changes that these should be aimed.
Fibronectin is principally involved in the chronic phase of rheumatoid synovitis, especially in the initiation of connective tissue remodeling. Therefore in attempting new approaches to the management of rheumatoid disease, agents that influence the deposition of fibronectin and its relationship with cells and other connective tissue elements may profoundly influence the course of chronic inflammation and indeed of the disease process itself. This approach should merit consideration and could lead to a new direction in the treatment of joint destruction.
CONCLUSIONS
These various studies, taken together, show that fibronectin plays an important role in a number of the humoral and tissue changes that occur in RA. Its widespread distribution in the synovium and synovial fluid are compatible with the view that it has diverse functions in the pathologic changes of RA, a view in keeping with its known complex cellular and biochemical interactions. We suggest that in the RA joint its main roles are, first, opsonization of fibrinous debris and other particulate material that are shed into the synovial fluid, and second, a controlling function in the reorganization of the connective tissue matrix that is a characteristic feature of the synovium.
REFERENCES I. Cohn EJ. Properties and functions of plasma proteins with consideration of methods for their separation and purification. Chemical Reviews 1941;28:395417. 2. Cohn EJ. Chemical, physiological and immunological properties and clinical uses of blood derivatives. Experimentia 1947;3:125-36. 3. Kuusela P, Ruoslahti E, Engvall E, et al. Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry 1976;1:639-42. 4. Yamada KM, Olden K. Fibronectins-Adhesive glycoproteins of cell surface and blood. Nature 1978;275: 179-84. 5. Yamada KM. Fibronectin: transformationSensitive cell surface protein. In: Pick E, ed. Lymphokine Reports. New York: Academic Press, I980:23 l-53. 6. Mosesson MW, Amrani DL. The structure and biologic activities of plasma fibronectin. Blood 1980;56: 145-58. 7. Ruoslahti E, Engvall E, Hayman EG. Fibronectin: Current concepts of its structure and function. Collagen Research 1981;1:95-128.
8. Mosher DF. Fibronectin. Prog Haemost Thromb 1980;5:111-51. 9. Yamada KM, Kennedy DW. Fibroblast cellular and plasma fibronectins are similar but not identical. J Cell Biol 1979;80:492-8. 10. Stathakis NE, Mosesson MW. Interactions among heparin, cold insoluble globulin, and fibrinogen in formation of the heparin-precipitable fraction of plasma. J Clin Invest 1977;60:855-65. 1 I. Yamada KM, Kennedy DW, Kimata K, et al. Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J Biol Chem 1980;255:6055-63. 12. Dessau W, Adelmann BC, Timpl R, et al. Identification of the sites in collagen-Chains that bind serum antigelatin factor (cold insoluble globulin). Biochem J 1978;169:55-9. 13. Mosher DF. Cross-linking of cold-insoluble globulin by fibrin-stablizing factor. J Biol Chem 1975;250:6614-21, 14. McDonald JA, Baum BJ, Rosenberg DM, et al.
252
Destruction of a major extracellular adhesive glycoprotein (fibronectin) of human fibroblasts by neutral proteases from polymorphonuclear leucocyte granules. Lab Invest 1979;40:35&7. 15. Blumberg PM, Robbins PW. Effect of proteases on activation of resting chick embryo fibroblasts and on cell surface proteins. Cell 1975;6: 13747. 16. Olden K, Pratt RM, Yamada KM. Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin. Proc Natl Acad Sci USA 1979;76:3343-7. 17. Klebe RJ. Isolation of a collagen-dependent cell attachment factor. Nature 1974;250:248-5 1. 18. Grinnell F, Hays DG, Minter D. Cell-adhesion and spreading factor: Partial purification and properties. Exp Cell Res 1977; I IO: 175-90. 19. Birdwell CR, Gospodarowicz D, Nicholson GL. Identification, localization and role of fibronectin in cultured bovine endothelial cells. Proc Nat1 Acad Sci USA 1978;75:3273-7. 20. Alitalo K, Hovi T, Vaheri A. fibronectin is produced by human macrophages. J Exp Med 1980;15 I :602-l 3. 21. Bevilacqua MP, Amrani D, Mosesson MW, et al. Receptors for cold-insoluble globulin (plasma fibronectin) on human monocytes. J Exp Med 1981;153:42-60. 22. Grinnell F, Feld MK. Spreading of human fibroblasts in serum-free medium: Inhibition by dithiotheitol and the effect of cold insoluble globulin (plasma fibronectin). J Cell Physiol 1980;104:321-34. 23. Anderson JC. Glycoproteins of the connective tissue matrix. Int Rev Connect Tissue Res 1976;7:251-322. 24. Stenman S, Vaheri A. Distribution of a new major connective tissue protein, fibronectin, in normal human tissues. J Exp Med 1978;147:1054-64. 25. Bach PR, Bentley JP. Structural glycoproteins, fact or artefact. Connect Tissue Res 1980;7:185-96. 26. Saba TM, Jaffe E. Plasma fibronectin (opsonic glycoprotein): Its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function. Am J Med 1980;68:577-94. 27. Mosher DF, Williams EM. Fibronectin concentration is decreased in plasma of severely ill patients with disseminated intravascular coagulation. J Lab Clin Med 1978; 91~729-35. 28. Stathakis NE, Fountas A, Tsianos E. Plasma fibronectin in normal subjects and in various disease states. J Clin Pathol 1981;34:504-8. 29. Gonzalez-Calvin J, Scully MF, Sanger Y, et al. Fibronectin in fulminant heparin failure. Br Med J 1982;285: 1231-2. 30. Scott DL, Farr M, Crockson AP, et al. Synovial fluid and plasma fibronectin levels in rheumatoid arthritis. Clin Sci 1982;62:71-6. 31. Carsons S, Mosesson MW, Diamond HS. Detection and quantitation of fibronectin in synovial fluid of patients with rheumatoid disease. Arthritis Rheum 1981;24:1261-7. 32. Clemmensen I, Andersen RB. Different molecular forms of fibronectin in rheumatoid synovial fluid. Arthritis Rheum 1982;25:25-3 1. 33. Gressner VAM, Wallraff P. Der Einsatz der Lasernephelometri zur Bestimmung und rcchnerunter-stiizten Auswertung der Fibronectinkonzentration in verschiedenem kiir
SCO-IT AND WALTON
perfliissigkeiten. 805.
J Clin Chem
Clin Biochem
1980;18:797-
34. Vartio T, Vaheri A, Von Essen R, et al. Fibronectin in synovial fluid and tissue in rheumatoid arthritis. Eur J Clin Invest 1981;11:207-12. 35. Scott DL, Almond TJ, Naqvi SNH, et al. The significance of fibronectin in cryoprecipitation in rheumatoid arthritis and other disorders. J Rheumatol 1982;9:514-8 36. Beaulieu AD, Valet JP, Storey J. The influence of fibronectin on cryoprecipitate formation in rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum 1981;24:1383-8. 37. Anderson B, Rucker M, Entwhistle R, et al. Plasma fibronectin is a component of cryoglobulins from patients with connective tissue and other diseases. Ann Rheum Dis 1981;40:5&4. 38. Wood G, Rucker M, Davies JW, et al. Interaction of plasma fibronectin with selected cryoglobulins. Clin Exp Immunol 1980;40:358-64. 39. Mosesson MW. Structure of human plasma coldinsoluble globulin and the mechanism of its precipitation in the cold with heparin or fibrin-fibrinogen complexes. Ann NY Acad Sci 1978;312:11--30. 40. Menzell EJ, Smolen JS, Liotta L, et al. Interaction of fibronectin with Cl, and its collagen-like fragment (CLF). FEBS Lett 1981;129:188-92. 41. Reise H. Die Reiskorpchen in tuberculijs erkrunken synovalsacken. Deutsch Z Chir 1895;42:1-99. 42. Popert AJ, Scott DL, Wainwright AC, et al. The frequency of occurrence, mode of development and significance of rice-bodies in rheumatoid joints. Ann Rheum Dis 1982;41:109-17. 43. Berg E, Wainwright R, Barton B, et al. On the nature of rheumatoid rice bodies. An immunologic, histochemical and electron microscope study. Arthritis Rheum 1977;20:1343-9. 44. McCarty DJ, Cheung HS. Origin and significance of rice bodies in synovial fluid. Lancet 1982;2:7 15-6. 45. Glynn LE. The chronicity of inflammation and its significance in rheumatoid arthritis. Ann Rheum Dis 1968;27:105-21. 46. Trentham DE, Dynesius RA, Rocklin RE, et al. Cellular sensitivity to collagen in rheumatoid arthritis. N Engl J Med 1978;299:327-32. 47. Scott DL, Wainwright A, Walton KW, et al. The significance of fibronectin in rheumatoid arthritis and osteoarthrosis. Ann Rheum Dis 1981;40:142-53. 48. Revel1 PA, Mayston V, Davies PG. Fibronectin in the synovium of chronic inflammatory joint disease. Ann Rheum Dis 1983;42:222. 49. Scott DL, Delamere JP, Walton KW. The distribution of fibronectin in the rheumatoid pannus. Br J Exp Pathol 1981;62:362-8. 50. Unsworth DJ, Scott DL, Almond TJ, et al. Studies on reticulin I: Serological and immunohistological investigation of the occurrence of collagen type III, fibronectin and the non-collagenous glycoprotein of Pras and Glynn in reticulin. Br J Exp Pathol 1982;63:154-66. 5 1. Bosselli JM, Macarak EJ, Clark CC, et al. Fibronectin: Its relationship to basement membranes. I. Light microscopic studies. Collagen Research 1981;5:391-404.
FIBRONECTIN IN RA
52. Dumonde DC, Glynn LE. The production of arthritis in rabbits by an immunological reaction to fibrin. Br J Exp Pathol 1962;43:373-83. 53. Consden R, Doble A, Glynn LE. et al. Production of chronic arthritis with ovalbumin. Its retention in the rabbit knee joint. Ann Rheum Dis 1971;30:307-15. 54. Scott DL, Almond TJ, Walton KW, et al. Fibronectin in antigen induced arthritis in the rabbit. J Pathol 1983 (in press) 55. Fell HB, Jubb RW. The effect of synovial tissue on the breakdown of articular cartilage in organ culture. Arthritis Rheum 1977;20:1359-71. 56. Jubb RW. Breakdown of articular cartilage by vascular tissue. J Pathol 1982;136:333-43. 57. Scott DL, Almond TJ, Walton KW, et al. Involvement of fibronectin in fibrosis and opsonisation in rheumatic diseases. Ann Rheum Dis 1983;42:221-2. 58. Edwards TCW, Sedgwick AD, Willoughby DA. The formation of a structure with the features of synovial lining by subcutaneous injection of air: An in vivo tissue culture system. J Pathol I98 I ; 134: 147-56. 59. Yoshino S, Bacon PA, Blake DR, et al. A model of persistent, antigen-induced chronic inflammation in the rat air pouch (submitted for publication). 60. Scott DL, Morris CJ, Blake AE, et al. Distribution of fibronectin in the rectal mucosa. J Clin Pathol 1981;34:74958. 61. Hahn E, Wick G, Pencev D, et al. Distribution of basement membrane proteins in normal and fibrotic human liver: Collagen type IV, laminin and fibronectin. Gut 1980;2 I :63-7 1 62. Scheinman Jl, Foidart JM, Gehron-Robey P, et al. The immunohistology of glomerular antigens. Clin lmmunol lmmunopathol 1980;15:175-89. 63. Cooper SM. Keyser AJ, Beaulieu AD, et al. Increase in tibronectin in the deep dermis of involved skin in progressive systemic sclerosis. Arthritis Rheum 1979;22:983-7. 64. Gay S, Gay RE, Miller EJ. The collagens of the joint. Arthritis Rheum 1980;23:93741. 65. Eyre DR. Muir H. Type Ill collagen: A major constituent of rheumatoid and normal human synovial membrane. Conn Tissue Res 1976;4: I l-6. 66. Jayson MIV (ed). Symposium on the fibrotic process. Ann Rheum Dis (Supp12). 1977 67. Kulonen E. Experimental granuloma as a tool in connective tissue research. In: Hall DA, ed. The methodology of connective tissue research. Oxford: Joynson--Bruvvers, 1976:29-36. 68. Aalto M, Potila M, Kulonen E. Effects of silica treated macrophages on the synthesis of collagen and other proteins in vitro. Exp Cell Res 1976;97: 193-202. 69. Kurkinen M, Vaheri A, Roberts PJ, et al. Sequential appearance of hbronectin and collagen in experimental granulation. Lab Invest 1980,43:47-57. 70. H#lund B, Clemmensen I, Junker P, et al. Fibronectin in experimental granulation tissue. Acta Pathol Microbial lmmunol Stand 1982;90A:15965. 71. Weiss RE, Reddi AH. Appearance of fibronectin during the differentiation of cartilage, bone and bone marrow. J Cell Biol 1981;88:630-6.
253
72. Fassbender HG. The pathology of rheumatic Loewi G, Trans. Berlin: Springer Verlag, 1975.
diseases.
73. Hewitt AT, Kleinman HK, Pennypacker JP, et al. Identification of an adhesion factor for chondrocytes. Proc Nat] Acad Sci USA 1980;77:385-8. 74. Woolley DE, Glanville RW, Crossley MJ, et al. Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen. Eur J Biochem 1975;54:61 l2. 75. Dayer JM, Goldring SR, Robinson DR, et al. Cell-cell interactions and collagenase production. In: Wooley DE, Evanson JM, eds. Collagenase in normal and pathological connective tissues. Chichester: John Wiley & Sons, 1980:83104. 76. Woolley DE, Crossley MJ, Evanson JM. Collagenase at sites of cartilage erosion in the rheumatoid joint. Arthritis Rheum 1977;20:1231-9. 77. Vaes G, Hauser D, Huybrecht-Godin G, et al. Cartilage degradation by macrophages, fibroblasts and synovial cells in culture. An in vitro model suitable for studies on rheumatoid arthritis. In: Willoughby DA, Girond JP, Velo GP, eds. Perspectives in inflammation. Lancaster: MTP Press, 1977: 115-26. 78. Aalto M, Kulonen E. Inhibition of protein synthesis in tendon cells by extracts from experimental granulation tissue. FEBS Lett 1974;49:7&2. 79. Castor CW, Lewis RB. Connective tissue activation x current studies on the process and of its mediators. Stand J Rheumatol 1976;5(Suppl 12):41-54. 80. Aalto M, Kulonen E. Fractionation of connectivetissue-activating factors from the culture medium of silicatreated macrophages. Acta Pathol Microbial Stand 1979; 87C:241-50. 81. Aalto M, Turakainen H, Kulonen E. Effect of SiO, liberated macrophage factor on protein synthesis in connective tissue in vitro. Stand J Clin Lab Invest 1979;39:205-13. 82. Kulonen E, Potila M. Macrophages of connective tissue components. Acta Stand 1980;88(3:7-13.
and the synthesis Pathol Microbial
83. Wahl SM. Wahl LM. Modulation of fibroblast growth and function of monokines and lymphokines. In: Pick E, ed. Lymphokines, Vol 2. New York: Academic Press 1981:179-202. 84. Wahl SM, Wahl LM, McCarthy JB, et al. Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide. J lmmunol 1979;122:2226-31. 85. Wahl SM. Wahl LM, McCarthy JB. Lymphoctye mediated activation of fibroblast proliferation and collagen production. J lmmunol 1978;121:942-6. 86. Grinnell F, Feld MK. Spreading of human fibroblasts in serum free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin. J Cell Physiol 1980;104:321-34. 87. Schor SL, Schor SM, Bazill GW. The effects of fibronectin on the migration of human foreskin fibroblasts and Syrian hamster melanoma gels of native collagen fibres. J Cell Sci 1981;48:301-14. 88. McAuslan BR, Aannan GN, Reilly W, et al. Variant endothelial cells. Fibronectin as a transducer of signals for
254
migration and neovascularisation. J Cell Physiol 1980;104:177-86. 89. Clark RAF, Dvorak HF, Colvin RB. Fibronectin in delayed hypersensitivity skin reactions: Association with vessel permeability and endothelial cell activation. J Immunol 1981;126:787-93. 90. Clark RAF, Quinn JH, Winn HJ, et al. Fibronectin is produced by blood vessels in response to injury. J Exp Med 1982;156:646-51. 91. Hayman EG, Ruoslahti E. Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix. J Cell Biol 1979;83:255-9. 92. Mareau N, Goyette R, Valet JP, et al. The effect of dexamethasone on formation of a fibronectin extracellular matrix by rat hepatocytes in vitro. Exp Cell Res 1980;125:497-502. 93. Pfeffer LM, Wang E, Tamm I. Interferon effects on microfilament organization, cellular fibronectin distribution, and cell motility in human fibroblasts. J Cell Biol 1980;85: 9-17.
SCOTT AND WALTON
94. Rich AM, Pearlstein E, Weissmann G, et al. Cartilage proteoglycans inhibit fibronectin-mediated adhesion. Nature 1981;293:2246. 95. Clemmensen I, Hdlund B, Johansen N, et al. Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique. Histochemistry 1982;76:51-6. 96. Gardner DL. General pathology of the peripheral joints. In: Sokoloff L, ed. The joints and synovial fluid, vol 2. New York: Academic Press 1980:316-427. 97. McConkey B, Davis P, Crockson RA, et al. Effects of gold dapson and prednisolone on serum C-reactive protein and haptoglobin and the erythrocyte sedimentation rate in rheumatoid arthritis. Ann Rheum Dis 1979;38:141-4. 98. Scott DL, Grindulis KA, Struthers GR, et al. The progression of radiological changes in rheumatoid arthritis. Ann Rheum Dis 1983 (in press) 99. Matsuda B, Yoshida N, Aoki N, et al. Distribution of cold-insoluble globulin in plasma and tissues. Ann NY Acad Sci 1978;312:56-73.