996

EXTRAVASCULAR FIBRIN FORMATION AND DISSOLUTION IN SYNOVIAL TISSUE O F PATIENTS WITH OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS J. BRICE WEINBERG, ANNE M. M. PIPPEN, and CHARLES S. GREENBERG Fibrin deposition is a prominent finding in the synovium of patients with rheumatoid arthritis (RA). Macrophages are found in increased numbers in RA synovium, and these cells are known to produce a variety of procoagulant and anticoagulant molecules. Using immunohistologic techniques, the content and distribution of several important components of the coagulation system in the synovium of patients with RA, osteoarthritis (OA), or traumatic joint abnormalities requiring surgery were investigated. Samples from 3 patients from each category were examined in detail. RA synovium (compared with that of patients with OA or joint trauma) had increased numbers of macrophages and increased expressionlcontent of fibrinogen, tissue factor, factor XIII, tissue transglutaminase, crosslinked fibrin (fibrin D dimer), urokinase-type plasminogen activator, and cu,-plasmin inhibitor. Macrophage content in RA synovium was increased in both the lining cell areas and the interstitial cell areas. Fibrinogen was distributed throughout the tissue in all samples and was greater in RA synovium. In trauma and OA synovia, tissue factor was seen only in association with vessels (endothelial cells), but in RA synovium, it was markedly increased throughout the tissues. While fibrin D dimer From the Veterans Administration and Duke University Medical Centers, Durham, North Carolina. Supported in part by the Veterans AfFairs Research Service. the James Swiger Hematology Kesearch Fund, and NIH grants PO1 AI-23308,1’01 32682. HI,-38245, and P50 AR-39162. Dr. Greenberg is an Established Investigator of the American Heart Association. J. Brice Weinberg, MD; Anne M. M. Pippen, HS; Charles S. Greenberg, MD. Address reprint requests to J. Brice Weinberg, MD, Veterans Affairs and Duke University Medical Centers, 151G, Durham, NC 27705. Submitted for publication October 12, 1990: accepted in revised form February 28, 1991.

Arthritis and Rheumatism, Vol. 34, No. 8 (August 1991)

was seen in small amounts in synovial lining cell areas of trauma and OA synovia, it was present in increased amounts in the lining cell and interstitial cell areas of RA synovium. Factor XI11 and tissue transglutaminase were present in scant amounts in trauma and OA synovia, but there were increased amounts of both (especially tissue transglutaminase) in RA synovium in the vessel, lining cell, and interstitial cell areas. Urokinase and a2plasmin inhibitor were also markedly increased in RA synovium. These results suggest that in inflamed synovium, there is ongoing extravascular tissue fibrin formation and dissolution that correlates with the degree of inflammation and macrophage content. Extravascular coagulationlfibrinolysis in RA represents a potential target for therapeutic intervention in this disease.

Mononuclear phagocytes (monocytes and macrophages) are found in normal and inflamed synovial tissue (1,2). In normal synovium, many of the cells lining the joint space (the synovial lining cells or the “type A” cells [I]) are of mononuclear phagocyte origin. Rheumatoid arthritis (RA) is a systemic inflammatory disease with a synovitis characterized by synovial tissue hyperplasia, with increased numbers of synovial lymphocytes and mononuclear phagocytes, accompanied by variable degrees of synovial fibroblast proliferation (2). Cartilage and bone destruction may accompany the synovial tissue changes. Synovial macrophages, having migrated there from the blood as monocytes ( l ) , play important roles in initiating, propagating, and maintaining the inflammation (2). Various investigators have noted that fibrin is found in the synovial tissue in RA (3,4). Indeed, in most types of inflammation (especially immunologically induced inflammation such as delayed-type hypersensitivity re-

997

FIBRIN FORMATION/DISSOLUTION IN RA SYNOVIUM

Table 1. Results of immunofluorescence studies of synovial tissue from trauma patients, osteoarthritis (OA) patients, and rheumatoid arthritis (RA) Datients*

RA patients (sample no.)

OA patients (sample no.)

Trauma patients (sample no.)

33

36

86

90

I46

8 25 Elbow Knee Acet. ASNIAS 1 yr. 10 yrs. 2+ LC, 2+ LC, 2 + IC 2+ IC 3+LC, 3+LC, I + IC I + IC 4 + EC 4 + EC

14 Knee ASA 3 yrs. 1 + LC

5 Hip ASA 3 yrs. 1 + LC

I + LC

t LC

t LC

I + LC

1 + LC

0

2+ IC

3+ EC

3+ EC

I + EC

Factor XIII

I+ 1c

I + IC

2+ IC

2+ LC

2+ LC

Tissue plasminogen activator Urokinase plasminogen activator

I + EC

0

0

I + EC

1 + EC

I + LC, 1 + IC 0

14 Hand (PIP) NSAID/MTX 5 yrs. 3+ LC, 3+ 1 c 4+ LC, 4+ 1 c 4 + EC, 4 + IC, 4+ LC 3+ LC, 3+ IC 4+ EC, 4+ IC, 4+ LC 2+ LC, 2+ IC I + EC

13 Knee PredJMTX >20 yrs. 2+ LC, 2+ IC 4 + LC, 4 + IC 4+ EC, 4 + IC, 4+ LC 4+ LC

0

0

0

0

0

I + EC

1+ IC, 3+ EC

0

0

2+ IC

2

0

1 + LC, 1+ EC

15 Knee ASA/NSAID >I0 yrs. 3+ LC, 3+ IC 4 + LC, 3+ IC 4 + EC, 4 + IC, 4+ LC 2+ LC, 2+ IC 4 + EC, 4+ IC, 4+ LC 3 + LC, 3+ IC I + IC, f EC 4 + EC, 4 + IC, 4+ LC 3+ LC, 3+ IC

3+ 3+ 3+ 3+ 3+

4+ 4+ 4+ 2+

161

178

Tissue factor

1 Hand (PIP) Acet. 20 yrs. I + LC, I + 1c I + LC, I + IC 3+ EC

11 Shouldei None < 1 mo. 1 + LC, I + IC 1 + LC, 1 + IC 1 + LC

Fibrin D dimer

t LC

Tissue transglutaminase

Inflammation score Joint assessed Treatment Disease duration CD14 (Leu-M3) Fibrinogen

cu,-plasmin inhibitor

212

20

LC:

t IC

I + LC, 4 + LC, 3 + IC I + 1c 1 + EC 3+ EC, 1 + IC

EC, IC, LC LC, IC

4 + EC, 4+ IC, 4+ LC 4+ LC, 4 + IC t EC EC, IC, LC IC

* The inflammation scores, based on immunohistologic examination, were derived as described elsewhere (27,28). Other scores were based on immunofluorescence intensity, using a scale of W + ,where 0 = negative and 4+ = greatest intensity. PIP = proximal interphalangeal; Acet. = acetaminophen; ASA = acetylsalicylic acid; IAS = intraarticular steroid; NSAID = nonsteroidal antiinflammatory drug; MTX = methotrexate; Pred. = prednisone; LC = lining cells; 1C = interstitial cells; EC = endothelial cells. actions), extravascular fibrin formation is prominent ( 3 3 ) . Coagulation is apparently initiated by tissue factor (TF; thromboplastin) that is expressed on macrophages, endothelial cells, and/or fibroblasts (6,7), resulting in activation of the extrinsic pathway of coagulation with eventual formation of fibrin from fibrinogen. Fibrin can be cross-linked by transglutaminases (factor XI11 or tissue transglutaminase [TTG]) to form a relatively stable, insoluble meshwork of covalently cross-linked fibrin (8), and the fibrin can be further modified by attachment of various components of the biomatrix and/or by degradation by plasmin (9-11). Products of the coagulation reactions act to amplify the inflammatory reaction by attracting more inflammatory cells to the site through the chemotactic activities of thrombin (12) and fibrin(ogen) degradation products (13). Also, the extravascular fibrin meshwork serves as a provisional matrix onto which inflammatory and endothelial cells can adhere and migrate (5). In addition to other numerous different functions, macrophages are known to elaborate/express

several different enzymedfactors involved in the formation and dissolution of fibrin. Included are TF (7,14), prothrombinase (15), factor XI11 (16), TTG (16), and plasminogen activators (urokinase [uPA]) (17). The purpose of this study was to determine (using immunohistologic techniques) the presence and distribution of several different procoagulant and anticoagulant molecules in tissues from patients who had trauma necessitating joint surgery, and from patients with severe osteoarthritis (OA) and RA requiring joint surgery. The results demonstrate that RA synovium has increased numbers of macrophages and increased expression/content of fibrinogen, TF, factor XIII, TTG, cross-linked fibrin (fibrin D dimer), uPA, and a,-plasmin inhibitor (a2-PI).

MATERIALS AND METHODS Specimens of sterile human synovial tissue were obtained from the Duke University Department of Pathology as “discarded material.” The subjects were those requiring

WEINBERG ET AL

998 joint surgery/resection for recent severe trauma or severe RA or OA. RA and OA were diagnosed by standard criteria (18). The patients’ records were reviewed to determine disease duration, medications, and a clinical estimate of disease activity at the time of the surgery. All OA and RA patients had advanced disease. Although the “trauma” samples approximated normal samples most closely, we did not consider them actually normal because of tissue damage, hemarthrosis, etc. Synovial samples (-5 X 5 X 5 mm) were frozen rapidly in liquid nitrogen. Serial, 4 mm-thick, frozen sections were made and analyzed after staining with hematoxylin and eosin (H & E) and by indirect immunofluorescence techniques after staining as described previously (19). After H & E staining, synovial tissue samples were scored (in a blinded manner) for the degree of inflammation as described elsewhere (20). For immunofluorescence studies, the following primary antibodies were used: Leu-M3 (mouse monoclonal anti-CD14, macrophage) (21), rabbit anti-human fibrinogen (Dakopatts, Copenhagen, Denmark), mouse monoclonal Al-3 (anti-human brain TF) (22), rabbit anti-human factor XI11 a chain (Calbiochem, La Jolla, CA) (16), mouse monoclonal anti-guinea pig TTG (23), DD386/22 (mouse monoclonal anti-human fibrin D-dimer) (24), rabbit anti-human uPA and rabbit anti-human tissue PA (tPA; from Dr. H . Berger, Jr., Burroughs Wellcome, Research Triangle Park, NC) (25), and rabbit anti-human a,-PI (American Diagnostica, New York, NY) (26). Secondary antibodies were fluorescein isothiocyanate (F1TC)-conjugated goat anti-mouse immunoglobulin and FITC-conjugated goat anti-rabbit immunoglobulin (Tago, Burlingame, CA). Anti-fibrinogen reacts with both fibrinogen and fibrin. Otherwise, the primary antibodies are specific for the designated antigens, as determined by immunoblots and/or immunoprecipitations detailed elsewhere (21-26). The tissues were examined with the use of a Zeiss microscope using epifluorescence and phase-contrast optics. Controls, in which supernatant from mouse myeloma P3 cells or normal rabbit serum was used as primary “antibody” (instead of the primary antibodies designated above), showed no reactivity with the appropriate secondary antibody.

RESULTS Macrophages, as determined by CD14 expression (21), were present in low numbers scattered in the interstitium and in the lining cell areas of synovium from trauma and OA patients (Table 1). As others have noted from immunohistologic studies (27) and as we have seen from our studies quantitating macrophages in enzymatically dissociated synovial tissues (28), RA synovium had more lining cell and interstitial area macrophages than did trauma or OA synovium. Fibrinogen was distributed throughout the synovial tissue in all of the samples we examined, being most prominent in the areas lining the synovial space. There was more fibrinogen apparent in the tissues from RA

patients, with increased staining in the interstitial areas. Tissue factor, or tissue thromboplastin, was seen primarily in association with vessels (especially with or near endothelial cells) in trauma and OA synovium. In synovium from RA patients, there was a marked increase in TF in all areas of the tissue (vessels, interstitiurn, and lining cell areas) (Figure 1A). When the tissues were examined with antibody DD386/22, an antibody that recognizes fibrin D dimer (a product of fibrin that has been cross-linked by a transglutaminase [TTG or factor XIII], and then partially degraded by plasmin [24]), tissue from trauma and OA patients had very little positivity. The only positive cells were in the synovial lining cell areas (Figure 1B). However, the RA synovial tissues expressed much more fibrin D dimer in the synovial lining areas, as well as in the interstitium (Figure 1B). The enzymes capable of cross-linking fibrin are TTG and factor XI11 (8,16). Factor XI11 is produced by megakaryocytes (platelets) and mononuclear phagocytes, while TTG is produced by endothelial cells and mononuclear phagocytes (8,16). TTG was not seen or was seen only in very low amounts in synovium from trauma patients (Figure 2A). In OA patient synovium, TTG was present in very low amounts, being limited primarily to vessels (mainly endothelial cells), but in RA synovium, TTG expression was dramatically increased in the tissues (vessels, lining cell areas, and interstitial areas) (Figure 2A). Factor XIII was noted in low amounts scattered in the synovial interstitium of trauma patients (Figure 2B). In OA synovium, there was slightly more factor XIII, primarily in the synovial lining cell areas, but in RA synovium, the amount was markedly increased and present in vessels, lining cell areas, and in the interstitium (Figure 2B). Once fibrin is formed, it can be degraded by plasmin. Plasmin is formed by the action of plasminogen activator on the proenzyme plasminogen (1 1). Urokinase-type plasminogen activator is found primarily in mononuclear phagocytes (11,17), while tissuetype plasminogen activator is found primarily in endothelial cells (1 1). By immunofluorescence, tPA was not seen or was seen only in small amounts in the endothelial cell areas of tissue from patients with trauma, OA, and RA (Table 1). Urokinase-type PA was not seen in synovium from trauma patients, but it was present in synovium from OA patients, primarily in the synovial lining cell areas (macrophage areas) (Figure 3A). In synovium from RA patients, the amount of uPA was dramatically increased, being seen in vessels,

FIBRIN FORMATION/DISSOLUTION IN RA SYNOVIUM

999

A

B Figure 1. Expression of A, tissue factor and B, fibrin D-dimer in synovial tissue from trauma (“normal”) patients, osteoarthritis (OA) patients, and rheumatoid arthritis (RA) patients, by phase-contrast microscopy (top panels) and fluorescence microscopy (bottom panels). In tissue from trauma and OA patients, tissue factor is seen primarily in the vessels (endothelial cells), but in the synovium from the RA patient, tissue factor is increased and is seen in essentially all areas. Fibrin D-dimer is seen in small amounts and is limited to the lining cell areas of synovium from trauma and OA patients, but in synovium from the RA patient, there is increased content of fibrin D-dimer, and it is present in the interstitial cell area as well as the lining cell area. (Magnification x 400.)

WEINBERG ET AL

1000

A

B Figure 2. Expression of A, tissue transglutaminase and B, factor XI11 in synovial tissue from trauma (“normal”) patients, osteoarthritis (OA) patients, and rheumatoid arthritis (RA) patients, by phase-contrast microscopy (top panels) and fluorescence microscopy (bottom panels). Tissue transglutaminase is present only in scant amounts in tissue from trauma and OA patients, being limited primarily to the vessel areas, but in RA synovium, there is an increased amount, which is distributed throughout the tissue. Factor XI11 is present in increased amounts and spread diffusely throughout the RA synovium. (Magnification x 400.)

FIBRIN FORMATION/DISSOLUTION IN RA SYNOVIUM

1001

Figure 3. Expression of plasminogen activator (urokinase) in synovial tissue from trauma (“normal”) patients, osteoarthritis (OA) patients, and rheumatoid arthritis (RA) patients, by phase-contrast microscopy (top panel) and fluorescence microscopy (bottom panel). Urokinase is present in small amounts in synovia from trauma and OA patients, being seen primarily in the lining cell areas. However, in RA synovium, there is an increased amount, which is distributed throughout the tissue. (Magnification x 400.)

as well as in the lining cell and interstitial areas (Figure 3). The primary inhibitor of plasmin is a2-PI (29). There was no (or very little) a,-PI in trauma or OA tissue, but it was increased in RA synovium, being distributed generally throughout the synovium. Table 1 displays the immunofluorescence, histologic, and inflammation scores, and clinical data. Thus, in summary, synovial tissues from RA patients have increased numbers of macrophages, increased amounts of fibrinogen, TF, fibrin D dimer, TTG, factor XIII, uPA, and az-PI. This suggests that in inflamed synovium, there is ongoing extravascular tissue fibrin formation and dissolution that correlates with the degree of inflammation and the macrophage content.

DISCUSSION By interacting with B and T lymphocytes and through their secretion of numerous substances that affect the function of other cells, mononuclear phagocytes are thought to play very important roles in RA (2,28,30). Among the important macrophage-derived mediators are chemotactic factors (e.g., macrophage-

colony-stimulating factor [313 and interleukin-8 [32]), complement components, growth factors (macrophage [platelet]-derived growth factor, interleukin- 1, tumor necrosis factor [TNF], T cell growth factor p), angiogenesis factors, arachidonic acid metabolites, reactive oxygen species, and various hydrolases and proteinases (including collagenases) (30). In addition to this array of important mediators, macrophages also produce various factors that can modulate the overall clotting process (the formation and dissolution of fibrin). Monocytes and/or macrophages have been shown to produce TF (14), factor VII (33), factor V (34), a prothrombinase complex (15), factor XIII (16), TTG (16), uPA (17), and a,-macroglobulin (35). Also, other cells in the synovium (fibroblasts and endothelial cells) can produce TF, TTG, and plasminogen activators. Because of the increased vascular permeability in the inflamed tissue, there are high tissue levels of various plasma proteins in synovial tissues. Increased levels of fibrin and fibrin-like materials are present in synovial fluid and synovial tissue of RA patients (3,4). The “rice bodies” seen in RA synovial fluid are composed of fibrin (3). Anderson and Gormsen noted that the fibrin found in RA synovium

WEINBERG ET AL

was insoluble in urea, signifying that it had probably been chemically cross-linked by a transglutaminase (36). Different investigators have shown that synovial tissues from arthritis patients have fibrinolytic activities (37), and that synovial macrophages and fibroblasts display plasminogen activator/plasmin activity (38). Extravascular coagulation is a prominent feature in immunologically induced inflammation (e.g., delayed-type hypersensitivity reactions, Arthus reactions, and experimental glomerulonephritides), in wound healing, and in tumors (5,39). In these conditions, a variety of factors may be operative in causing the fibrin formation. In tumors, the neoplastic cells can supply procoagulant factors (most prominently T F [39,40]), while in immunologically induced inflammation and in healing wounds, macrophages, fibroblasts, and/or endothelial cells may supply procoagulants (5). Products of the coagulation reactions can amplify the inflammation-thrombin and fibrin(ogen) degradation products are chemotactic and may modify the function of inflammatory cells (5,12,13), transglutaminases may chemically cross-link bioactive molecules (e.g., a,-PI or fibronectin [9,11]) to biomatrix, and plasmin may modify the biomatrix and activate proteinases, including collagenase (41). Furthermore, fibrin contributes to the mass (swelling) of the inflammatory tissue, and serves as an important part of the provisional matrix onto which fibroblasts and inflammatory cells attach and migrate (5,39). Our work definitively demonstrates that inflammatory synovium from humans with RA has much fibrin accumulation. This could result from increased formation and/or decreased dissolution. Tissue factor in the trauma and OA tissues was seen in small amounts, being restricted primarily to the endothelial cell areas, but in RA tissues, it was dramatically increased, being seen in all areas. Because of the distribution of T F in the RA synovia, it appears that T F is increased in macrophages, fibroblasts, and endothelial cells. Others have noted that TF is usually not expressed in normal endothelial cells (42,43), except for small amounts in placental vessels (44). We did see TF in endothelial cells in the synovial samples from trauma patients, as well as in those from OA and RA patients. It should be noted, however, that these trauma patients could not be considered actually normal because of various degrees of tissue damage and hemarthrosis in these subjects. It has been found that RA synovial fluid and synovium contain increased amounts of T N F (45); this may be important in ex-

plaining the increased levels that we demonstrate, since T N F can increase T F expression in endothelial cells and in mononuclear phagocytes (46,47). Previous immunohistologic studies of RA synovium characterizing fibrin have not used antibodies that could clearly distinguish between fibrinogen and fibrin (4), but the antibody against fibrin D dimer demonstrates well that true fibrin is being formed in the synovial tissue, is being cross-linked, and is being acted upon (to a limited degree) by plasmin (24). The RA tissues contained high levels of fibrin D dimer, as compared with trauma and OA tissues. Remarkably, the transglutaminases TTG and factor XI11 were also increased in the RA tissues, providing the ability to cross-link the fibrin. We have noted before that tissue macrophages have more transglutaminase activity than do blood monocytes, and that blood monocytes, after in vitro “differentiation,” have more transglutaminase activity (16). The finding of increased levels of TTG and factor XI11 in the RA synovial tissues may reflect the degree of cellular “differentiation” that has taken place in the RA synovial tissue. While tPA levels were not appreciably different in the synovia of trauma, OA, and RA patients, uPA was dramatically increased in all areas of the RA synovial tissue. This finding of increased plasminogen activator activity in RA tissues correlates with that noted previously by other investigators (37,38). Cytokines (most notably, interferon- y [48]) have been shown to increase plasminogen activator levels in blood monocytes. Interferon-y (protein or messenger RNA) has been noted to be increased in RA synovial fluid and synovium (30,49). Other cytokines in the fluid (e.g., the colony-stimulating factors [SO]) may also play a role in controlling the expression of plasminogen activator. The significance of the rather selective increase in uPA compared with levels of TPA is not known. The enzymes of the coagulation and fibrinolytic pathways are regulated by several proteinase inhibitors. Alpha,-macroglobulin, a high molecular weight proteinase inhibitor of broad specificity, has been noted in RA synovial fluid and synovial tissue (51,52). Studies from our laboratory confirm increased levels in RA synovium (Hoffman M, Weinberg J: unpublished observations). Likewise, we show here that a,-PI, the most potent plasmin inhibitor (29), is increased in RA synovium. Thus, we have demonstrated that in the inflamed synovial tissue of RA patients, there is evidence of increased expression of procoag-

FIBRIN FORMATION/DISSOLUTION IN RA SYNOVIUM

ulant molecules (fibrinogen, TF, TTG, and factor XIII), increased cross-linked and plasmin-modified fibrin (fibrin D dimer), and increased anticoagulant molecules (uPA and q-PI). The remarkable coagulation/anticoagulation, fibrin formatioddegradation activity noted in RA synovium likely contributes to the local pathology and, through the actions of various generated enzymes, lymphokines, monokines, and fibrin degradatory products, perpetuates the inflammation. This extravascular coagulation/fibrinolysis in RA represents a target for therapeutic intervention. It has been noted that anticoagulant therapy can modify immunologically mediated inflammation in experimental animals and in humans. Warfarin and heparin anticoagulant treatments have been shown to diminish delayed-type hypersensitivity reactions ( 3,5) and experimental nephritis in animals (53,54). Defibrination with the snake venom enzyme ancrod has been noted to diminish the degree of experimental nephritis in animals ( 5 3 , systemic lupus erythematosus in animals (56), and glomerulonephritis in humans (57), disorders characterized by prominent extravascular fibrin deposition. Belch et a1 noted that the androgen stanolozol improved the inflammation in RA, and that this reduction of inflammation was accompanied by an increase in the plasma fibrinolytic activity (58). It is possible that agents capable of decreasing extravascular fibrin formation and dissolution might be useful in ameliorating or diminishing the synovitis in RA.

ACKNOWLEDGMENT We thank Dr. Frederick Rickles for the anti-tissue factor antibody A1-3.

REFERENCES Edwards JCW, Sedgwick AD, Willoughby DA: Membrane properties and esterase activity of synovial lining cells: further evidence for a mononuclear phagocyte subpopulation. Ann Rheum Dis 41 :282-286, 1982 Krane SM, Simon LS: Rheumatoid arthritis: clinical features and pathogenic mechanisms. Med Clin North Am 70:263-284, 1986 Jasani MK: Fibrin: metabolism, immunopathogenesis and significance in rheumatoid arthritis, Immunopathogenesis of Rheumatoid Arthritis. Edited by GS Panayi, PM Johnson. Surrey, UK, Reedbooks, Ltd., 1978 Clemmensen I, Hglund B, Andersen RB: Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid. Arthritis Rheum 26:479485, 1983

1003

5. Colvin RB: Wound healing processes in hemostasis and thrombosis, Vascular Endothelium in Hemostasis and Thrombosis. Edited by MA Gimbrone Jr. New York, Churchill Livingstone, 1986 6. Bach R: Initiation of coagulation by tissue factor. CRC Crit Rev Biochem 23:339-368, 1988 7. Edwards RL, Ewan VA, Rickles FR: Macrophage procoagulants, fibrin deposition, and the inflammatory response, The Reticuloendothelial System, Vol. 9. Edited by SM Phillips, MR Escobar. New York, Plenum Press, 1986 8. Lorand L, Conrad SM: Transglutaminases. Mol Cell Biochem 58:9-35, 1984 9. Sakata J, Aoki N: Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest 65:29&297, 1980 10. Mosher DF, Schad PE, Kleinman HK: Cross-linking of fibronectin to collagen by blood coagulation factor XIIIa. J Clin Invest 64:781-787, 1979 11. Lijnen HR, Collen D: Interaction of plasminogen activators and inhibitors with plasminogen and fibrin. Semin Thromb Hemost 8:2-10, 1982 12. Bar-Shavit R, Kahn A, Wilner GD: Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 220:728-73 1, 1983 13. Richardson DL, Pepper DS, Kay AB: Chemotaxis for human monocytes by fibrinogen-derived peptides. Br J Haematol 32507-513, 1976 14. Edwards RL, Rickles FR: Macrophage procoagulants. Prog Hemost Thromb 7: 183-209, 1984 15. Lindahl U, Pejler G, Bggwald A, Seljelid R: A prothrombinase complex of mouse peritoneal macrophages. Arch Biochem Biophys 273:18&188, 1989 16. Conkling PR, Achyuthan KE, Greenberg CS, Newcomb TF, Weinberg JB: Human mononuclear phagocyte transglutaminase activity cross-links fibrin. Thromb Res 5557-68, 1989 17. Vassalli J-D, Dayer J-M, Wohlwend A, Belin D: Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J Exp Med 159:1653-1668, 1984 18. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA: 19.58 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9:175-176, 1958 19. Hale LP, Martin ME, McCollum DE, Nunley JA, Springer TA, Singer KH, Haynes BF: Immunohistologic analysis of the distribution of cell adhesion molecules within the inflammatory synovial microenvironment. Arthritis Rheum 32:22-30, 1989 20. McCachren SS, Haynes BF, Niedel JE: Localization of collagenase in mRNA in rheumatoid arthritis synovium by in situ hybridization histochemistry. J Clin Immunol 10: 19-27, 1990 21. Dimitriu-Bona A, Burmester R, Waters SJ, Winchester

WEINBERG ET AL

1004

22.

23.

24.

25.

26.

27.

28.

29. 30.

31.

32.

33.

34.

35.

RJ: Human mononuclear phagocyte differentiation antigens. J Immunol 130:145-152, 1983 Ewan VA, Cieplinski W, Hancock WW, Boyd AW, Godschneider IG, Rickles FR: Production and characterization of a monoclonal antibody (Al-3) that binds selectively to activated monocytes and inhibits monocyte procoagulant activity. J Immunol 136:2416-2420, 1986 Birchbichler PJ, Upchurch HF, Patterson MK, Conway E: A monoclonal antibody to cellular transglutaminase. Hybridoma 4: 179-186, 1984 Rylatt DB, Blake AS, Cottis LE, Massingham DA, Fletcher WA, Masci PP, Whitaker AN, Elms M, Bunce I, Webber AJ, Wyatt D, Bundesen PG: An immunoassay for human D dimer using monoclonal antibodies. Thromb Res 31:767-768, 1983 Berger H Jr, Tuttle PR: Comparative properties of six human plasminogen activators. Prog Fibrinolysis 6:2935, 1982 Hattey E, Wojta J, Binder BR: Monoclonal antibodies against plasminogen and alpha-2-antiplasmin: binding to native and modified antigens. Thromb Res 45:485495, 1987 Burmester GR, Dimitriu-Bona A, Waters SJ, Winchester RJ: Identification of three major synovial lining cell populations by monoclonal antibodies directed to Ia antigens and antigens associated with monocytesl macrophages and fibroblasts. Scand J Immunol 17:6982, 1983 Weinberg JB, Wortham TS, Patton KL, Chitneni SR: Synovial mononuclear phagocytes in rheumatoid arthritis and osteoarthritis: quantitative and functional aspects. Submitted for publication Lijnen HR, Collen D: Alpha-2 antiplasmin. J Med 16: 225-284, 1985 Lipsky PE, Davis LS, Cush JJ, Oppenheimer-Marks N: The role of cytokines in the pathogenesis of rheumatoid arthritis. Springer Semin Immunopathol 11:123-162, 1989 Wang JM, Griffin JD, Rambaldi A, Chen ZG, Mantovani A: Induction of monocyte migration by recombinant macrophage colony-stimulating factor. J Immunol 141: 575-579, 1988 Matsushima K , Oppenheim JJ: Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL- 1 and TNF. Cytokine 1:2-13, 1989 Van Dam-Mieras MCE, Muller AD, van Deijk WA, Hemker HC: Clotting factors secreted by monocytes and macrophages: analytical considerations. Thromb Res 37:9-19, 1985 Rothberger H, McGee MP: Generation of coagulation factor V activity by cultured rabbit alveolar macrophages. J Exp Med 160:1880-1890, 1984 Hovi T, Moshe D, Vaheri A: Cultured human mono-

36.

37.

38.

39. 40.

41.

42.

43.

44.

45. 46.

47.

48.

49.

50.

cytes synthesize and secrete a2-macroglobulin. J Exp Med 145:1580-1589, 1977 Anderson B, Gormsen J: Fibrinolytic and fibrin stabilizing activity of synovial membranes. Ann Rheum Dis 29:287-293, 1970 Van de Putte LBA, Hegt VN, Overbeek TE: Activators and inhibitors of fibrinolysis in rheumatoid and nonrheumatoid synovial membranes: a histochemical study. Arthritis Rheum 20:671-677, 1977 Medcalf RL, Hamilton JA: Human synovial fibroblasts produce urokinase-type plasminogen activator. Arthritis Rheum 29:1397-1401, 1986 Dvorak HF: Tumors: wounds that do not heal. N Engl J Med 315:165&1659, 1986 Rickles FR, Edwards RL: Activation of blood coagulation in cancer: Trousseau's syndrome revisited. Blood 62:1&31, 1983 Werb Z, Mainardi CL, Vater CA, Harris ED Jr: Endogenous activation of latent collagenase by rheumatoid synovial cells: evidence for a role of plasminogen activator. N Engl J Med 296:1017-1023, 1977 Drake TA, Morrissey JH, Edgington TS: Selective expression of tissue factor in human tissues: implications for disorders of hemostasis and thrombosis. Am J Pathol 134:1087-1097, 1989 Wilcox JN, Smith KM, Schwartz SM, Gordon D: Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 86:2839-2843, 1989 Faulk WP, Labarrere CA, Carson SD: Tissue factor: identification and characterization of cell types in human placentae. Blood 76:86-96, 1990 Husby G, Williams RC Jr: Synovial localization of tumor necrosis factor in patients with rheumatoid arthritis. J Autoimmun 1:363-371, 1988 Bevilacqua MP, Pober JS, Majeua GR, Fiers W, Cotran RS, Gimbrone MA: Recombinant tumor necrosis factor induces procoagulant activity in human vascular endothelium: characterization and comparison with the actions of interleukin-1. Proc Natl Acad Sci USA 83:45334537, 1986 Conkling PR, Greenberg CS, Weinberg JB: Tumor necrosis factor induces tissue factor-like activity in human leukemia cell line U937 and peripheral blood monocytes. Blood 72: 128-133, 1988 Weinberg JB, Hobbs MM, Misukonis MA: Recombinant human gamma-interferon induces human monocyte polykaryon formation. Proc Natl Acad Sci USA 81: 4554-4557, 1984 Degre M, Mellbye 0, Clarke-Jenssen 0: Immune interferon in serum and synovial fluid in rheumatoid arthritis and related disorders. Ann Rheum Dis 42:672-676, 1983 Xu WD, Firestein GS, Taetle R, Kaushansky K, Zvaifler NJ: Cytokines in chronic inflammatory arthritis. 11. Granulocyte-macrophage colony-stimulating factor in

FIBRIN FORMATION/DISSOLUTION IN RA SYNOVIUM

51. 52.

53.

54.

55.

rheumatoid synovial effusions. J Clin Invest 832376882, 1989 Borth W: Alpha 2-macroglobulin in connective tissue matrix metabolism. Coll Relat Res 4:83-95, 1984 Borth W, Dunky A, Kleesiek K: a,-macroglobulinproteinase complexes as correlated with a,-proteinase inhibitor-elastase in synovial fluids of rheumatoid arthritis patients. Arthritis Rheum 29:319-325, 1986 Halpern B, Milliez P, Lagrue G, Fray A, Morad JC: Protective action of heparin in experimental immune nephritis. Nature 205:257-259, 1965 Vassalli P, McCluskey RT: The pathogenic role of fibrin deposition in immunologically induced glomerulonephritis. Ann N Y Acad Sci 116: 1052-1062, 1964 Naish P, Penn GB, Evans DJ, Peters DK: The effect of

1005

detibrination on nephrotoxic serum in nephritis in rabbits. Clin Sci 42:643-646, 1972 56. Cole EH, Glynn MF, Laskin CA, Sweet J , Mason N, Levy GA: Ancrod improves survival in murine systemic lupus erythematosus. Kidney Int 37:29-35, 1990 57. Kim S, Wadhwa NK, Kant KS, Pollak VE, GlasGreenwalt P, Weiss MA, Hong CG: Fibrinolysis in glomerulonephritis treated with ancrod: renal functional, immunologic and histopathologic effects. Q J Med 69: 879-905, 1988 58. Belch JJF, Madhok R, McArdls B, McLaughlin K, Kluft C, Forbes CD, Sturrock A: The effect of increasing fibrinolysis in patients with rheumatoid arthritis: a double blind study of stanozolol. Q J Med 58:19-27, 1986