CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Vol. 63, No. 2, May, pp. 155-162, 1992
Pathways of Coagulation Activation in Situ in Rheumatoid Synovial Tissue’ LEO R. ZACHARSKI,*~ FORST E. BROWN,+ VINCENT A. MEMOLI,§ WALTER KISIEL,~~ BOHDAN J. KUDRYK,~~ SANDRA M. ROUSSEAU,? JANE A. HuNT,t CHRISTOPHER DUNWIDDIE,** AND ELKA M. NUTT** Departments of *Medicine, #Surgery, and §Pathology, Dartmouth Medical School and the tVA Medical Center, White River Junction, Vermont 05009; “Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; ?/Plasma Protein-coagulation Laboratory, New York Blood Center, New York, New York 10021; and **Department of Pharmacology, Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486
Immunohistochemical techniques were applied to rheumatoid synovium in order to detect components of coagulation and fibrinolysis pathways within these tissues. These techniques revealed an intact coagulation pathway and plasminogen activator inhibitor-2 associatedwith macrophagelike cells that were present throughout these tissues, especially in subsurface areas. Cell-associated thrombin generation appeared to account for conversion of abundant fibrinogen to fibrin. Occasional macrophage-like cells also stained for urokinase but tissue-type plasminogen activator and plasminogen activator inhibitor-l were restricted to vascular endothelium. Intense synovial fibrin deposition (with the limited evidence for associatedfibrinolysis) may contribute to local inflammation and explain certain clinical features of rheumatoid arthritis. These findings suggestnovel treatment hypotheses for this disease. o 1992 Academic PESS. IIIC.
INTRODUCTION
Rheumatoid arthritis (RA) is a chronic inflammatory disorder of which the pathogenesis is not completely understood (1). Although much attention has focused on immunologic events that undoubtedly contribute to this disease (l), coagulation abnormalities have also long been known to be a feature of RA. For example, it has been postulated that deposition of fibrimogen) in synovium may contribute to the chronicity and progressive character of the tissue abnormality in RA (24). While a number of studies on peripheral blood (2, 5-7), synovial fluid (5, 7-ll), and synovial tissues (9, 11-16) have attempted to characterize the pathologic coagulation activation that is known to exist in RA, these have generally been limited to measurement of levels of fibrinogen and its derivatives. Interpretation
of studies performed on synovial tissues is difficult because of the previous lack of reagents capable of distinguishing with certainty between fibrinogen and fibrin (9, 11-16) and because of possible artefactual effects of fixation methods on results obtained (12). Little is known about the cellular basis for or pathophysiologic significance of activation of coagulation and fibrinolysis pathways in RA. Recently, specific reagents have become available that permit definition by means of immunohistochemical techniques of cellular sites of assembly of components of coagulation and fibrinolysis pathways (17-21). Such procedures also provide information on the potential reactivity of these pathways. These techniques have been applied successfully to various types of malignant tissue in order to define the contribution of the malignant cell to these reactions (17-21). Because they are also potentially capable of elucidating similar reactions in inflammatory tissues, these techniques were applied to rheumatoid synovial tissues in an attempt to clarify the possible contribution of cells within these tissues to the pathogenesis of the complex coagulopathy that exists in RA. MATERIALS
AND METHODS
Studies were performed on fresh rheumatoid synovium obtained at tenosynovectomy of the hand in patients with seropositive RA. Tissues from consecutive cases were obtained without selection on the basis of current or past treatments. Fresh frozen tissue was prepared from eight cases and AMeX (acetone-methylbenzoat+xylene)-fixed tissue (22) was prepared from five specimens from three cases. Staining procedures and controls for the avidin-biotin complex (ABC) technique (17-21) using reagents (Vectastain Kits) from Vector Laboratories, Burlingame, California, and for the indirect immunofluorescence technique (23) have been described previously. These procedures include a step in which nonspecific antibody binding to Fc recep-
1 This work was supported in part by the Department of Veterans Affairs Medical Research Service and by National Institutes of Health Grants HL35246 (W.K.) and HL21465 (B.J.K.) and by Blood Systems, Inc. (W.K.). 155
All
Copyright 0 1992 rights of reproduction
0090-1229/92 $1.50 by Academic Fksa, Inc. in any form reserved.
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tors is blocked. Antigen staining was detected by the dark brown peroxidase reaction product obtained with diaminobenzidine in the ABC procedure. This contrasted with the dark blue nuclei of cells and the pale pink appearance of unstained cells and stroma. (We have shown previously that the results obtained in these procedures are similar in AMeX-fixed and fresh frozen tissues.) Findings were compared with results obtained on tissues obtained from patients with several types of malignancies and with normal liver and placenta (17-21). The double-labeling technique was used to visualize simultaneously two different antigens in the same section. For demonstration of two antigens on the same cell within a single tissue section, an immunoenzymatic step that gave a colored reaction product was applied to unstained sections and this step was followed by the direct immunofluorescence procedure (24). Procedures employed monospecific, purified rabbit antibodies to the following: recombinant human TF (tissue factor), factor VII, factor X, the “a” subunit of factor XIII, u-PA (251, t-PA, plasminogen, and plasminogen activator inhibitor type 2 (PAI-2). The antibody to t-PA was prepared by immunizing rabbits with recombinant t-PA (Genentech, South San Francisco, CA) and isolating monospecific IgG as described previously (26). Rabbit polyclonal antibodies were prepared from protein antigens that were >99% pure. Antibodies were purified from crude antiserum either by affinity chromatography on an antigen-Sepharose column (for antibodies to factor VII and factor X) or on a protein A-Sepharose column (for antibodies to TF and u-PA). Antibody to u-PA was purified by sequential adsorption using both of the above ligands. Antibody specificity was demonstrated on the basis of activity neutralization, Western immunoblot analysis, and immunoprecipitation studies using lz51-labeled antigen or proteins labeled metabolically in uiuo using 13?Slmethionine. The antibody to recombinant TF (27) was capable of immunoprecipitating TF from cell extracts and stained a protein of about 42,000 Da on Western blots. This antibody blocked the procoagulant activity of TF (28) and binding of factor VII to TF-expressing cells (29), and colocalized TF antigen and mRNA in the same cells in human vascular tissues (28). Antibodies to t-PA and u-PA did not cross-react with the alternative plasminogen activator (25, 26). Monoclonal antibodies specific for epitopes on fibrimogen) or their degradation products included: antibody l-8C6 that requires an intact BP 14-15 Arg-Gy bond and therefore reacts with fibrinogen or fibrin I (des fibrinopeptide AA-type fibrin) but not fibrin II (des-fibrinopeptide AA des-fibrinopeptide B B-type fibrin) (30); antibody T2Gl that reacts with the amino-terminal part of the BP chain only following removal by thrombin of FPB, BP 1-14, and thus with fibrin but not fibrinogen (31); and
k i ii’
antibody C;C4 that reacts with fragment ii ,?i nbrrrii’ gen as well as D-dimer from cross-linked fibrin i)ul. nr+‘ with either fibrinogen or fibrin ‘321. The react!vr:y 11~ these antibodies in immunohistochemical procedurehas been verified independently (331. In addition ~SF’ have performed parallel studies of fresh frozen ;-inii AMeX-fixed term placenta. small cell carcinom;t of the lung (SCCLI, and renal cell carcinoma tissues char have produced comparable results. This indicated thar these fibrinogen- and fibrin-specific epitopes are pre. served upon AMeX fixation. The activated form of fat tor X (factor Xa) was detected using recombinant antistasin (rATSi as a probe together with its antibody according to techniques described previously 134) Goat antibody to PAI- was obtained from American Diagnostica, Greenwich, Connecticut. The macrophage’ specific monoclonal antibody EBM-11 was obtained from Dakopatts, Giostrup, Denmark. While the macrophage antigen recognized by this antibody has not yer. been completely identified, available data indicate that this antibody is useful for demonstration of activated macrophages in situ (35. 36). The antibody to neut.rophi1 elastase was obtained from Biodesign International, Kennebunkport, Maine; and to plasmir; antiplasmin complex neoantigen from Behring Diagnostics/Calbiochem, San Diego, California. Ant,ibodies were tested on control and tumor tissues in concentrations that provided maximum staining intensity with minimum background staining. Controls consisted of omission from the procedure of the primary antibodv and use of antibodies developed in the same species but with different or irrelevant specificities. R ESI.LTP
Numerous macrophage-like cells present in these rheumatoid synovial tissues stained for tissue factor, factor V, factor VII, factor X, factor Xa, factor XIII (a subunit), u-PA, and PAI-2. Double-labeling procedures performed using the macrophage-specific antibody EBM-11 and antibody to tissue factor showed only partial identify of cellular staining. Thus, while certain cells stained coincidently for both antigens, other cells stained for one but not the other antigen. The explanation for this finding is uncertain. These cells were scattered throughout the specimens but were the most numerous just beneath the synovial surface. The results obtained in staining procedures for cell.associated factor Xa using rATS as a probe are illustrated in Fig. 1. This figure illustrates the strict association of coagulation activation with (presumably the surfaces of) specific cells. While both u-PA and PAIwere detected, u-PA was present in a minority of cells while PAI- was present in abundance. The results for PAI- are shown in Fig. 2. Fibrinogen (i.e., antibody 1-8C6-reactive material) was present in abundance in the connective tissue
BLOOD
COAGULATION
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RHEUMATOID
SYNOVIUM
of AMeX-fixed tissue by the peroxidase technique with diaminobenzidine as a substrate FIG. 1. Specific staining for Xa on macrophage-like cells in subsurface regions in rheumatoid synovium (arrows, frame A). The appearance are shown in frame B. Hematoxylin was used to counterstain nuclei. preparatic ens from which the probe was omitted x400.
157
using rATS as a probe of unstained cells in Original magnification
158
FIG. 2. Specific staming of AMe-X-fixed tissue by the peroxidase technque w;th diammobenzldin~ a:: :A -uhstratr: using the antibody to PAIon macrophage-like cells in subsurface regions in rheumatoid synovium (arrows, frame A). The appearance of unstained control preparations from which the antibody was omitted are shown in frame B. Hematoxylin was used t.o counterstain nuclei. Original magnification X 400.
throughout the specimens. Fibrin (i.e., antibody T2Glreactive material) was present in a somewhat more restricted distribution and was particularly concentrated in subsurface areas where it appeared to virtually replace synovial villous projections (see Fig. 3).
Staining for D-dimer coincided with antibody GC4 with that of fibrin. Rare foci of staining for plasminogen and plasmin-antiplasmin complex neoantigen were observed in these areas. Staining for t-PA and PAIwas restricted to the vascular endothelium. En-
BLOOD COAGULATION
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SYNOVIUM
159
FIG. 2. Specific staining of AMe-X-fixed tissue by the peroxidase technique with diaminobenxidine as a substrate using the antibody having specificity for fibrin in the connective tissue in subsurface regions in rheumatoid synovium (straight arrows). Fibrin occupies virtually the entire area of a villous projection of synovium (curved arrows). The appearance of unstained connective tissue is illustrated in the deeper regions of the synovium (open arrows) and in Fig. 1B. Hematoxylin was used to counterstain nuclei. Original magnification x250.
dothelium also stained focally for tissue factor, factor VII, and factor X; and rarely for plasminogen and plasmin-antiplasmin complex neoantigen. Elastasepositive neutrophils were scattered throughout these specimens without apparent anatomic relationship to other features described here. The above results were consistent between all cases studied. DISCUSSION
Studies to define the occurrence and distribution of coagulation and fibrinolysis reaction pathways in tissues are capable of providing information on mechanisms by which cells trigger these pathways in rheumatoid synovium. While it has long been known that coagulation/fibrinolysis activation occurs in RA, previous studies of this reactivity have focused largely on alterations in levels of fibrin(ogen) and derivatives in the blood and synovial fluid (2, 5-16). Such changes apparently represent spillover of enzymatic activity generated by cells into these fluids and provide little direct information on primary events taking place in the inflammatory tissues themselves. Interpretation of the putative heightened fibrinolytic activity in these tissues is confounded by the apparent coexistence of increased levels of functional protease inhibitors (6-9, 13). Previous reports of “fibrin” in synovial tissue were of uncertain validity because of the previous lack of
reagents capable of distinguishing between fibrinogen and fibrin (9, 11-16). Our techniques appear to resolve these ambiguities. We have shown a dominant procoagulant pathway associated with numerous macrophage-like cells present within the synovial tissues. Evidence that this coagulation pathway is active consists of the demonstration of factor Xa (Fig. 1) on these cells and the demonstration of conversion of fibrinogen to fibrin adjacent to these cells (Fig. 3) by means of reagents that detect either factor Xa or the thrombin-specific cleavage site on the B(3 chain of fibrinogen, respectively. This indicates that enzymatically active thrombin has been generated locally. The precise characterization of these cells was not a primary goal of these studies. However, despite the fact that only partial coincidence of staining was observed in double-labeling studies performed with the macrophage antibody EBM-11; the existence of tissue factor together with factors of the prothrombinase complex (37411, factor XIII (a subunit) (421, u-PA, (43,441, and PAI- (40,411 on these cells argues strongly for their macrophage origin. Patients were entered into this study without selection on the basis of current or past treatment. It is conceivable that such treatment might have altered the findings described here but we believe this is unlikely. Unfortunately, tissues usually become available for study only relatively late in the course of the disease while medical management is usually begun
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relatively early following diagnosis. Thus, it would be unusual to have an opportunity to study tissues from previously untreated patients. It has been proposed that a protease cascade resulting in plasmin formation (45, 46) may lead to activation of collagenase that is likely responsible for the joint destruction characteristic of rheumatoid arthritis (1, 2, 47). Certain studies on cultured synovial cells have demonstrated their ability to synthesize u-PA in vitro (48-50) that might be capable of converting plasminogen to plasmin in uivo. While we have demonstrated cellular staining for u-PA in synovium, this substance was present in a minority of cells and was overshadowed by the presence of PAIthat was present in abundance (Fig. 2). It has been shown that PAI- is capable of inhibiting u-PA when both are produced by the same cell (43,44) and such inhibition may indeed occur in uivo based on our findings. A marked paucity of staining for plasminogen and for plasminantiplasmin complex neoantigen (a marker for plasmin generation) was also observed. Neutrophil proteases are also capable of converting plasminogen to plasmin (7, 51) but such a reaction is unlikely to be prominent in situ in rheumatoid synovium based on our findings. These features, together with the net accumulation of fibrinogen and fibrin, contrast distinctly with findings in other diseases (20, 21) and argue against the generation of significant free plasmin activity in rheumatoid synovial tissues. This conclusion is supported by previous studies that have shown an overall decrease in fibrinolytic activity of rheumatoid synovial tissue as assessed by the fibrin slide lysis method (13). Attention might wisely be directed toward investigation of other pathways of collagenase activation in rheumatoid synovium (52-54 ). The prominent procoagulant pathway present on macrophage-like cells in rheumatoid synovium corresponds to the increased functional coagulant activity observed on pulmonary alveolar macrophages from patients with RA (55). This procoagulant activity may contribute to the pathogenesis of RA. Thus, such fibrin formed locally may contribute directly to the synovial thickening and friability that leads to the mechanical abnormalities characteristic of rheumatoid joints (24). Fragmentation of fibrin-laden synovial villi appear to be responsible for the so-called “rice bodies” present in synovial fluid (56, 57). Because fibrincogen) and its derivatives are hydroscopic, their presence may contribute to the formation of joint effusions (58). This local fibrin appears to have been stabilized through cross-linking achieved by factor XIII available locally. The potential arthrogenic effects of fibrin in human RA (2-4) are supported by studies in animals in which fibrin implanted locally within joints induces a reaction that resembles human RA (2). Little attention has been directed to the possibility that pharmacologic limitation of coagulant activity or
I b t i,
enhancement ot hbrlnoi:~~~iti may ~me!ioi’alr ,.iir* ,,L:L~. of RA. However, the rczsults of a recent doublr i~!li’:~ clinical trial (59b have shown therapeutic heneti7.r f’rl~r: stanozolol treatment. that increases both systcrrr:i. ani: intraarticular fibrinolytic activity, in RA 16 .? $ta? i-: tically significant treatment-associated decrea:-r ;n lhs erythrocyte sedimentation rate. improvement I!.: ;ir! 1:’ ular Index, decrease lr: duration of morning ~t&les.decrease in pain. decrease in plasma fibrinogen ! rt>cei: tration. and increase in piasminogen activator :~ctiv~ I 3 were observed (59). Ir was postulated thar 1he.~1eii:k provements may be attributable to H ~rea’:menr.induced reduction in synovial fibrin (591. Findings rf:ported here provide a possible basis for these favorab!c results and suggest other novel treatment hppot.heses (59,60) for this disease These results also suggest pru cedures that may elucidate mechanisms of local mat rophage activation that may be cytokine mediat.ed i 6 l631
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57
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56. Scott, D. L., and Walton, K. W., The significance of fibronectin in rheumatoid arthritis. Senin. Arthritis Rheum. 13, 244-254, 1984. Received 1992
October
23, 1991;
accepted
with
revision
February
20,
McCarty. i) .i ,tnd I_t:cung. rice hodies in synnvial fluid.
tl
Len&
3.
Orlgit!
nilti
ii. 715-716.
.~~giilticdtl;~:
I!@.?
j8.
Colvrn. R. B.. Johnson l< i, Mihm. M. (‘.. and Dvvra~r ri r Role of the clotting system in cell-mediated hypersensi:i;,it;y Fibrin deposition in delaved skin reactions in man. ,i E.rp Alec: 13x. 686698 i 9’3 1L 59. Belch. .I. J. F , Madhok, ii. VlcArdle, H.. McLaughlin h &luli (’ Forbes, C. D.. and Slurrock. K. D.. The effect of inrreasini: iibrinolpsis in patients with rheumatoid arthritis& donhh hlind study of stanozolol Q ./. Med. 58, 19-27. 1986 60. Fritzler, M. J.. and Hart 11. ii.. Prolonged improvemen: s** Raynaud’s phenomenon and scleroderma after recombinant tisue plasminogen activator therapy. Arthritis Rheum 33, 27.S 276. 1990.
62. Chin, J., Rupp. E.. Cameron. P. M,, MacNaul, K. L., Lotkc, i’ A. Tocci. M. J., Schmidt, J. A.. and Bayne, E. K., Identification of a. high-affinity receptor for interleukin la and interleukin lfi on cultured human rheumatoid synovial cells. */. C/i/l. Irtuest. X2. 4201126,1988. 63. Res, P. C. M., Telgt, D., vanlaar, J. M., Pool, M. 0. Breedveid, F. C., and DeVries. R. P., High antigen reactivity in mononuclear cells from sites of chronic inflammation. Lancet ii, 1406~ 1408. 1990.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Vol. 63, No. 2, May, pp. 155-162, 1992
Pathways of Coagulation Activation in Situ in Rheumatoid Synovial Tissue’ LEO R. ZACHARSKI,*~ FORST E. BROWN,+ VINCENT A. MEMOLI,§ WALTER KISIEL,~~ BOHDAN J. KUDRYK,~~ SANDRA M. ROUSSEAU,? JANE A. HuNT,t CHRISTOPHER DUNWIDDIE,** AND ELKA M. NUTT** Departments of *Medicine, #Surgery, and §Pathology, Dartmouth Medical School and the tVA Medical Center, White River Junction, Vermont 05009; “Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; ?/Plasma Protein-coagulation Laboratory, New York Blood Center, New York, New York 10021; and **Department of Pharmacology, Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486
Immunohistochemical techniques were applied to rheumatoid synovium in order to detect components of coagulation and fibrinolysis pathways within these tissues. These techniques revealed an intact coagulation pathway and plasminogen activator inhibitor-2 associatedwith macrophagelike cells that were present throughout these tissues, especially in subsurface areas. Cell-associated thrombin generation appeared to account for conversion of abundant fibrinogen to fibrin. Occasional macrophage-like cells also stained for urokinase but tissue-type plasminogen activator and plasminogen activator inhibitor-l were restricted to vascular endothelium. Intense synovial fibrin deposition (with the limited evidence for associatedfibrinolysis) may contribute to local inflammation and explain certain clinical features of rheumatoid arthritis. These findings suggestnovel treatment hypotheses for this disease. o 1992 Academic PESS. IIIC.
INTRODUCTION
Rheumatoid arthritis (RA) is a chronic inflammatory disorder of which the pathogenesis is not completely understood (1). Although much attention has focused on immunologic events that undoubtedly contribute to this disease (l), coagulation abnormalities have also long been known to be a feature of RA. For example, it has been postulated that deposition of fibrimogen) in synovium may contribute to the chronicity and progressive character of the tissue abnormality in RA (24). While a number of studies on peripheral blood (2, 5-7), synovial fluid (5, 7-ll), and synovial tissues (9, 11-16) have attempted to characterize the pathologic coagulation activation that is known to exist in RA, these have generally been limited to measurement of levels of fibrinogen and its derivatives. Interpretation
of studies performed on synovial tissues is difficult because of the previous lack of reagents capable of distinguishing with certainty between fibrinogen and fibrin (9, 11-16) and because of possible artefactual effects of fixation methods on results obtained (12). Little is known about the cellular basis for or pathophysiologic significance of activation of coagulation and fibrinolysis pathways in RA. Recently, specific reagents have become available that permit definition by means of immunohistochemical techniques of cellular sites of assembly of components of coagulation and fibrinolysis pathways (17-21). Such procedures also provide information on the potential reactivity of these pathways. These techniques have been applied successfully to various types of malignant tissue in order to define the contribution of the malignant cell to these reactions (17-21). Because they are also potentially capable of elucidating similar reactions in inflammatory tissues, these techniques were applied to rheumatoid synovial tissues in an attempt to clarify the possible contribution of cells within these tissues to the pathogenesis of the complex coagulopathy that exists in RA. MATERIALS
AND METHODS
Studies were performed on fresh rheumatoid synovium obtained at tenosynovectomy of the hand in patients with seropositive RA. Tissues from consecutive cases were obtained without selection on the basis of current or past treatments. Fresh frozen tissue was prepared from eight cases and AMeX (acetone-methylbenzoat+xylene)-fixed tissue (22) was prepared from five specimens from three cases. Staining procedures and controls for the avidin-biotin complex (ABC) technique (17-21) using reagents (Vectastain Kits) from Vector Laboratories, Burlingame, California, and for the indirect immunofluorescence technique (23) have been described previously. These procedures include a step in which nonspecific antibody binding to Fc recep-
1 This work was supported in part by the Department of Veterans Affairs Medical Research Service and by National Institutes of Health Grants HL35246 (W.K.) and HL21465 (B.J.K.) and by Blood Systems, Inc. (W.K.). 155
All
Copyright 0 1992 rights of reproduction
0090-1229/92 $1.50 by Academic Fksa, Inc. in any form reserved.
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tors is blocked. Antigen staining was detected by the dark brown peroxidase reaction product obtained with diaminobenzidine in the ABC procedure. This contrasted with the dark blue nuclei of cells and the pale pink appearance of unstained cells and stroma. (We have shown previously that the results obtained in these procedures are similar in AMeX-fixed and fresh frozen tissues.) Findings were compared with results obtained on tissues obtained from patients with several types of malignancies and with normal liver and placenta (17-21). The double-labeling technique was used to visualize simultaneously two different antigens in the same section. For demonstration of two antigens on the same cell within a single tissue section, an immunoenzymatic step that gave a colored reaction product was applied to unstained sections and this step was followed by the direct immunofluorescence procedure (24). Procedures employed monospecific, purified rabbit antibodies to the following: recombinant human TF (tissue factor), factor VII, factor X, the “a” subunit of factor XIII, u-PA (251, t-PA, plasminogen, and plasminogen activator inhibitor type 2 (PAI-2). The antibody to t-PA was prepared by immunizing rabbits with recombinant t-PA (Genentech, South San Francisco, CA) and isolating monospecific IgG as described previously (26). Rabbit polyclonal antibodies were prepared from protein antigens that were >99% pure. Antibodies were purified from crude antiserum either by affinity chromatography on an antigen-Sepharose column (for antibodies to factor VII and factor X) or on a protein A-Sepharose column (for antibodies to TF and u-PA). Antibody to u-PA was purified by sequential adsorption using both of the above ligands. Antibody specificity was demonstrated on the basis of activity neutralization, Western immunoblot analysis, and immunoprecipitation studies using lz51-labeled antigen or proteins labeled metabolically in uiuo using 13?Slmethionine. The antibody to recombinant TF (27) was capable of immunoprecipitating TF from cell extracts and stained a protein of about 42,000 Da on Western blots. This antibody blocked the procoagulant activity of TF (28) and binding of factor VII to TF-expressing cells (29), and colocalized TF antigen and mRNA in the same cells in human vascular tissues (28). Antibodies to t-PA and u-PA did not cross-react with the alternative plasminogen activator (25, 26). Monoclonal antibodies specific for epitopes on fibrimogen) or their degradation products included: antibody l-8C6 that requires an intact BP 14-15 Arg-Gy bond and therefore reacts with fibrinogen or fibrin I (des fibrinopeptide AA-type fibrin) but not fibrin II (des-fibrinopeptide AA des-fibrinopeptide B B-type fibrin) (30); antibody T2Gl that reacts with the amino-terminal part of the BP chain only following removal by thrombin of FPB, BP 1-14, and thus with fibrin but not fibrinogen (31); and
k i ii’
antibody C;C4 that reacts with fragment ii ,?i nbrrrii’ gen as well as D-dimer from cross-linked fibrin i)ul. nr+‘ with either fibrinogen or fibrin ‘321. The react!vr:y 11~ these antibodies in immunohistochemical procedurehas been verified independently (331. In addition ~SF’ have performed parallel studies of fresh frozen ;-inii AMeX-fixed term placenta. small cell carcinom;t of the lung (SCCLI, and renal cell carcinoma tissues char have produced comparable results. This indicated thar these fibrinogen- and fibrin-specific epitopes are pre. served upon AMeX fixation. The activated form of fat tor X (factor Xa) was detected using recombinant antistasin (rATSi as a probe together with its antibody according to techniques described previously 134) Goat antibody to PAI- was obtained from American Diagnostica, Greenwich, Connecticut. The macrophage’ specific monoclonal antibody EBM-11 was obtained from Dakopatts, Giostrup, Denmark. While the macrophage antigen recognized by this antibody has not yer. been completely identified, available data indicate that this antibody is useful for demonstration of activated macrophages in situ (35. 36). The antibody to neut.rophi1 elastase was obtained from Biodesign International, Kennebunkport, Maine; and to plasmir; antiplasmin complex neoantigen from Behring Diagnostics/Calbiochem, San Diego, California. Ant,ibodies were tested on control and tumor tissues in concentrations that provided maximum staining intensity with minimum background staining. Controls consisted of omission from the procedure of the primary antibodv and use of antibodies developed in the same species but with different or irrelevant specificities. R ESI.LTP
Numerous macrophage-like cells present in these rheumatoid synovial tissues stained for tissue factor, factor V, factor VII, factor X, factor Xa, factor XIII (a subunit), u-PA, and PAI-2. Double-labeling procedures performed using the macrophage-specific antibody EBM-11 and antibody to tissue factor showed only partial identify of cellular staining. Thus, while certain cells stained coincidently for both antigens, other cells stained for one but not the other antigen. The explanation for this finding is uncertain. These cells were scattered throughout the specimens but were the most numerous just beneath the synovial surface. The results obtained in staining procedures for cell.associated factor Xa using rATS as a probe are illustrated in Fig. 1. This figure illustrates the strict association of coagulation activation with (presumably the surfaces of) specific cells. While both u-PA and PAIwere detected, u-PA was present in a minority of cells while PAI- was present in abundance. The results for PAI- are shown in Fig. 2. Fibrinogen (i.e., antibody 1-8C6-reactive material) was present in abundance in the connective tissue
BLOOD
COAGULATION
AND
RHEUMATOID
SYNOVIUM
of AMeX-fixed tissue by the peroxidase technique with diaminobenzidine as a substrate FIG. 1. Specific staining for Xa on macrophage-like cells in subsurface regions in rheumatoid synovium (arrows, frame A). The appearance are shown in frame B. Hematoxylin was used to counterstain nuclei. preparatic ens from which the probe was omitted x400.
157
using rATS as a probe of unstained cells in Original magnification
158
FIG. 2. Specific staming of AMe-X-fixed tissue by the peroxidase technque w;th diammobenzldin~ a:: :A -uhstratr: using the antibody to PAIon macrophage-like cells in subsurface regions in rheumatoid synovium (arrows, frame A). The appearance of unstained control preparations from which the antibody was omitted are shown in frame B. Hematoxylin was used t.o counterstain nuclei. Original magnification X 400.
throughout the specimens. Fibrin (i.e., antibody T2Glreactive material) was present in a somewhat more restricted distribution and was particularly concentrated in subsurface areas where it appeared to virtually replace synovial villous projections (see Fig. 3).
Staining for D-dimer coincided with antibody GC4 with that of fibrin. Rare foci of staining for plasminogen and plasmin-antiplasmin complex neoantigen were observed in these areas. Staining for t-PA and PAIwas restricted to the vascular endothelium. En-
BLOOD COAGULATION
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SYNOVIUM
159
FIG. 2. Specific staining of AMe-X-fixed tissue by the peroxidase technique with diaminobenxidine as a substrate using the antibody having specificity for fibrin in the connective tissue in subsurface regions in rheumatoid synovium (straight arrows). Fibrin occupies virtually the entire area of a villous projection of synovium (curved arrows). The appearance of unstained connective tissue is illustrated in the deeper regions of the synovium (open arrows) and in Fig. 1B. Hematoxylin was used to counterstain nuclei. Original magnification x250.
dothelium also stained focally for tissue factor, factor VII, and factor X; and rarely for plasminogen and plasmin-antiplasmin complex neoantigen. Elastasepositive neutrophils were scattered throughout these specimens without apparent anatomic relationship to other features described here. The above results were consistent between all cases studied. DISCUSSION
Studies to define the occurrence and distribution of coagulation and fibrinolysis reaction pathways in tissues are capable of providing information on mechanisms by which cells trigger these pathways in rheumatoid synovium. While it has long been known that coagulation/fibrinolysis activation occurs in RA, previous studies of this reactivity have focused largely on alterations in levels of fibrin(ogen) and derivatives in the blood and synovial fluid (2, 5-16). Such changes apparently represent spillover of enzymatic activity generated by cells into these fluids and provide little direct information on primary events taking place in the inflammatory tissues themselves. Interpretation of the putative heightened fibrinolytic activity in these tissues is confounded by the apparent coexistence of increased levels of functional protease inhibitors (6-9, 13). Previous reports of “fibrin” in synovial tissue were of uncertain validity because of the previous lack of
reagents capable of distinguishing between fibrinogen and fibrin (9, 11-16). Our techniques appear to resolve these ambiguities. We have shown a dominant procoagulant pathway associated with numerous macrophage-like cells present within the synovial tissues. Evidence that this coagulation pathway is active consists of the demonstration of factor Xa (Fig. 1) on these cells and the demonstration of conversion of fibrinogen to fibrin adjacent to these cells (Fig. 3) by means of reagents that detect either factor Xa or the thrombin-specific cleavage site on the B(3 chain of fibrinogen, respectively. This indicates that enzymatically active thrombin has been generated locally. The precise characterization of these cells was not a primary goal of these studies. However, despite the fact that only partial coincidence of staining was observed in double-labeling studies performed with the macrophage antibody EBM-11; the existence of tissue factor together with factors of the prothrombinase complex (37411, factor XIII (a subunit) (421, u-PA, (43,441, and PAI- (40,411 on these cells argues strongly for their macrophage origin. Patients were entered into this study without selection on the basis of current or past treatment. It is conceivable that such treatment might have altered the findings described here but we believe this is unlikely. Unfortunately, tissues usually become available for study only relatively late in the course of the disease while medical management is usually begun
160
ZACHAHSK
relatively early following diagnosis. Thus, it would be unusual to have an opportunity to study tissues from previously untreated patients. It has been proposed that a protease cascade resulting in plasmin formation (45, 46) may lead to activation of collagenase that is likely responsible for the joint destruction characteristic of rheumatoid arthritis (1, 2, 47). Certain studies on cultured synovial cells have demonstrated their ability to synthesize u-PA in vitro (48-50) that might be capable of converting plasminogen to plasmin in uivo. While we have demonstrated cellular staining for u-PA in synovium, this substance was present in a minority of cells and was overshadowed by the presence of PAIthat was present in abundance (Fig. 2). It has been shown that PAI- is capable of inhibiting u-PA when both are produced by the same cell (43,44) and such inhibition may indeed occur in uivo based on our findings. A marked paucity of staining for plasminogen and for plasminantiplasmin complex neoantigen (a marker for plasmin generation) was also observed. Neutrophil proteases are also capable of converting plasminogen to plasmin (7, 51) but such a reaction is unlikely to be prominent in situ in rheumatoid synovium based on our findings. These features, together with the net accumulation of fibrinogen and fibrin, contrast distinctly with findings in other diseases (20, 21) and argue against the generation of significant free plasmin activity in rheumatoid synovial tissues. This conclusion is supported by previous studies that have shown an overall decrease in fibrinolytic activity of rheumatoid synovial tissue as assessed by the fibrin slide lysis method (13). Attention might wisely be directed toward investigation of other pathways of collagenase activation in rheumatoid synovium (52-54 ). The prominent procoagulant pathway present on macrophage-like cells in rheumatoid synovium corresponds to the increased functional coagulant activity observed on pulmonary alveolar macrophages from patients with RA (55). This procoagulant activity may contribute to the pathogenesis of RA. Thus, such fibrin formed locally may contribute directly to the synovial thickening and friability that leads to the mechanical abnormalities characteristic of rheumatoid joints (24). Fragmentation of fibrin-laden synovial villi appear to be responsible for the so-called “rice bodies” present in synovial fluid (56, 57). Because fibrincogen) and its derivatives are hydroscopic, their presence may contribute to the formation of joint effusions (58). This local fibrin appears to have been stabilized through cross-linking achieved by factor XIII available locally. The potential arthrogenic effects of fibrin in human RA (2-4) are supported by studies in animals in which fibrin implanted locally within joints induces a reaction that resembles human RA (2). Little attention has been directed to the possibility that pharmacologic limitation of coagulant activity or
I b t i,
enhancement ot hbrlnoi:~~~iti may ~me!ioi’alr ,.iir* ,,L:L~. of RA. However, the rczsults of a recent doublr i~!li’:~ clinical trial (59b have shown therapeutic heneti7.r f’rl~r: stanozolol treatment. that increases both systcrrr:i. ani: intraarticular fibrinolytic activity, in RA 16 .? $ta? i-: tically significant treatment-associated decrea:-r ;n lhs erythrocyte sedimentation rate. improvement I!.: ;ir! 1:’ ular Index, decrease lr: duration of morning ~t&les.decrease in pain. decrease in plasma fibrinogen ! rt>cei: tration. and increase in piasminogen activator :~ctiv~ I 3 were observed (59). Ir was postulated thar 1he.~1eii:k provements may be attributable to H ~rea’:menr.induced reduction in synovial fibrin (591. Findings rf:ported here provide a possible basis for these favorab!c results and suggest other novel treatment hppot.heses (59,60) for this disease These results also suggest pru cedures that may elucidate mechanisms of local mat rophage activation that may be cytokine mediat.ed i 6 l631
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57
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October
23, 1991;
accepted
with
revision
February
20,
McCarty. i) .i ,tnd I_t:cung. rice hodies in synnvial fluid.
tl
Len&
3.
Orlgit!
nilti
ii. 715-716.
.~~giilticdtl;~:
I!@.?
j8.
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