These studies of arthritis in mice with selected alterations in fibrinogen have directly established that: (a) fibrin(ogen) is an important determinant of inflammatory joint disease, (b) fibrin(ogen) deposition within joints drives arthritic disease via mechanisms linked to the leukocyte integrin receptor αMβ2 binding motif, and (c) the impact of fibrin(ogen) on the progression of joint disease is context dependent. In the experimental setting of CIA, the genetic elimination of fibrin(ogen) significantly diminished the development of overt, clinically evident arthritis and ameliorated the entire spectrum of pathological features of disease when tissue sections were examined microscopically. However, fibrinogen deficiency did not diminish the adaptive immune responses (e.g., T cell response and anti-CII antibody production) and was compatible with leukocyte trafficking following CIA. Thus, fibrin-rich matrices appear to serve as powerful modifiers of the local inflammatory events leading to joint degeneration. While disease severity was much reduced within cohorts of Fib– mice relative to Fib+ controls, individual Fib– mice that developed disease were encountered. Thus, neither fibrinogen nor fibrin is strictly required for the development of arthritis, and inflammatory pathways must still exist that can supersede the fibrinogen-dependent process(es) in arthritis (see below). Complementary CIA studies in mice that express mutant forms of fibrinogen lacking selected integrin receptor engagement motifs demonstrated that the elimination of the motif recognized by the leukocyte integrin receptor αMβ2 was sufficient to alleviate arthritis, despite the maintenance of full clotting. In contrast, the elimination of the platelet receptor αIIbβ3 binding motif from fibrinogen had no apparent impact on the progression of inflammatory joint disease. The attenuation of collagen-induced arthritis in Fib– and Fibγ390–396A mice was also associated with a significant diminution in the expression of key cytokines known to be important in the development of disease (e.g., TNF-α, IL-1β, and IL-6). An attractive working hypothesis that has emerged is that fibrin contributes to degenerative inflammatory changes within the joint by supporting αMβ2-dependent leukocyte activation programs, including the secondary expression of proinflammatory cytokines. Consistent with this view, arthritic disease was found to be fibrin(ogen) independent in the experimental context of mice expressing exuberant human TNF-α.
A variety of mechanisms can be envisioned whereby fibrin(ogen) could influence the progression of inflammatory joint disease. First, as a key factor in the maintenance of vascular integrity and/or patency, fibrin thrombus formation within vessels serving joint tissues may further compromise already damaged arthritic joints by limiting blood flow and, ultimately, nutrient delivery and gas exchange. Second, the formation and deposition of antibodies to novel fibrin(ogen) derivatives (e.g., anti-citrullinated fibrinogen antibodies) might be a significant factor in joint disease (see below). Third, as a provisional matrix protein that supports tissue repair, the deposition and cellular organization of fibrin matrices within arthritic joints may drive inappropriate reparative processes. This could potentially lead to a vicious cycle of fibrin deposition, tissue reorganization, further loss of joint function, further mechanical and inflammatory damage, and further fibrin deposition. In this regard, fibrin and fibrin degradation products have been reported to have a remarkable array of biological activities that could contribute to inopportune reparative changes within damaged joints, including chemotactic, mitogenic, and angiogenic activities (27
). Each of these mechanisms may be an important factor in human disease. However, the findings that Fibγ390–396A
mice (a) carry levels of fibrinogen comparable to those of wild-type mice when both unchallenged and CIA challenged (present study and ref. 14
), (b) retain full clotting function (14
), and (c) exhibit a significant resistance to CIA similar to that of Fib–
mice implies that any fibrin-dependent mechanisms linked to hemostasis may be of only modest importance in the context of standard CIA.
The genetic elimination of either fibrin(ogen) or αM
has been shown to be compatible with leukocyte trafficking to multiple sites including the lung, skin, and peritoneal cavity in various experimental inflammatory contexts (14
). Here, we show that neutrophil migration into the joints of CIA-challenged mice is also not fundamentally compromised by the absence of fibrin(ogen). Rather, fibrinogen-αM
interactions appear to regulate local leukocyte function. Multiple in vitro and in vivo studies have shown that fibrin(ogen)-αM
engagement can regulate the activities of macrophages, neutrophils, and other inflammatory cells, including cell adhesion, calcium mobilization, cytokine release, chemokine release, degranulation, oxidative response, phagocytosis, activation of ERK1/2, NFκB-dependent transcription, and survival (10
). For example, fibrinogen can trigger the full activation of neutrophils primed with TNF-α through an αM
-dependent mechanism resulting in degranulation, upregulation of cell surface adhesion molecules, increased phagocytosis, and delayed apoptosis (11
). Neutrophils and macrophages expressing αM
have been strongly implicated in the pathogenesis of arthritic disease based on depletion studies (32
). Therefore, an attractive working hypothesis is that fibrin(ogen) drives inflammatory joint disease by supporting local leukocyte “target recognition” and leukocyte activation events, presumably in concert with soluble immune mediators (e.g., cytokines and chemokines). However, it should be emphasized that multiple fibrin(ogen)-dependent processes may influence arthritic disease, and the importance of these processes may vary from critical to modest based on joint location, mechanical stress, and underlying immunological derangements.
A concept that has recently gained increasing attention is that the conversion of arginine residues to citrulline by peptidylarginine deiminases on selected proteins is thought to generate novel epitopes that initiate antibody formation and subsequently result in immune complex–driven arthritis (34
). Fibrinogen has been identified as a target of this modification, and the development of anti-citrullinated fibrinogen antibodies has been documented in both human RA patients and mice subjected to CIA (35
). It was recently shown that direct administration of antibodies specific to citrullinated fibrinogen exacerbated disease initiated by an attenuated CIA protocol (38
). Whatever the importance of anti-citrullinated fibrin(ogen) antibodies as a determinant of arthritic disease, the present studies with Fib–
mice establish that, at least in mice with CIA, the formation of anti-citrulline antibodies directed specifically at fibrin(ogen) is not required for the development
of inflammatory joint disease. However, it is possible that there may be cooperation among multiple mechanisms in which fibrin(ogen) promotes the pathogenesis
of arthritis. One attractive theory that couples previous findings on anti-citrullinated fibrin(ogen) antibodies and the present findings on fibrin(ogen)-αM
interactions in arthritic disease is that local leukocyte activation events promote fibrin citrullination, secondary anti-citrullinated antibody formation, and, ultimately, immune complex–mediated arthritis, but examining this possibility will require additional analyses.
Although the development of advanced and debilitating arthritic disease is a feature common to both TNF-α–driven arthritis and CIA, these two experimental settings of joint disease have distinct etiologies, use unique mechanisms of pathogenesis, and, as shown here, have very distinct fibrin(ogen) dependencies. The contribution of proinflammatory cytokines to the pathogenesis of arthritis also is clearly distinct in CIA and Tg197 mice. For example, IL-1β has been shown to be critical for both CIA and TNF-α–driven arthritis in mice (39
). The genetic elimination of IL-6 was reported to have no impact on the development of severe arthritic disease in Tg197 mice but results in near-complete protection from arthritis in the context of CIA (6
). The distinct etiologies and cytokine-dependent pathways of disease in CIA and Tg197 mice, together with our finding that the local expression of cytokines (e.g., TNF-α, IL-1β, and IL-6) within challenged joints is sensitive to genetic alterations in fibrinogen, offer a plausible model consistent with all of our present findings and the working view that fibrin(ogen)-rich matrices can regulate inflammatory cell function. We propose that fibrinogen influences inflammatory joint disease at a control point upstream of the local expression of TNF-α and other important cytokines. Under this theory, the constitutive expression of a master immune modulator such as TNF-α would be expected to reduce or eliminate the disease dependence on fibrin(ogen), precisely what has been observed in Tg197 mice. The proposed placement of fibrin matrices upstream of the leukocyte activation events that support the secondary expression of fundamental soluble inflammatory mediators is also consistent with the profound diminution in arthritis observed in Fib–
mice in the context of CIA.
The finding that fibrin(ogen) is a powerful, but context dependent, contributor to inflammatory joint disease may provide a partial explanation for many seemingly conflicting reports on the role of fibrinolytic factors in inflammatory joint disease. Mice lacking either urokinase-type plasminogen activator or plasminogen were reported to develop greatly exacerbated AIA but markedly diminished CIA (19
). On the other hand, tissue-type plasminogen activator–deficient mice have been consistently resistant to arthritic disease challenge (19
). One potential variable that has been proposed to account for some of these seemingly incompatible findings is investigator-imposed joint trauma introduced in some settings (e.g., injection of antigen into the joint space in AIA), but not other settings (e.g., CIA) (19
). The present studies show that the importance of fibrinogen in arthritic disease can vary markedly in two distinct experimental contexts even when there is no investigator-imposed mechanical joint trauma. Similarly, plasmin-mediated proteolysis within joint spaces might be beneficial in some joint disease contexts by supporting prompt fibrin clearance, whereas excessive plasmin generation might aggravate disease in other contexts by participating in the direct or indirect proteolysis of extracellular matrix glycoproteins, collagen, or immune modulators (e.g., complement factors) (40
Fibrin deposition is carefully regulated both spatially and temporally at sites of injury, making it an ideal candidate to be a local, matrix-associated regulator of leukocyte target recognition leading to activation events in both physiological and pathological inflammatory contexts. The results of the present study directly establish that fibrin(ogen) is an important, albeit context-dependent, determinant of inflammatory joint disease. Together with our earlier finding that the elimination of the αM
binding motif on fibrinogen impedes Staphylococcus aureus
clearance in vivo (14
), the present work shows that fibrin(ogen) is central to inflammatory processes in both physiological (e.g., antimicrobial host defense) and pathological (e.g., inflammatory joint disease) contexts. Finally, the available data suggest that fibrin(ogen) may be an attractive therapeutic target in the treatment of arthritis and other inflammatory diseases. This concept is also supported by previous studies showing that ancrod-mediated depletion of fibrinogen in mice can impede AIA (40
). The findings presented here suggest that pharmacological interruption of fibrinogen-αM
interactions might be efficacious in the treatment of arthritic disease. Because the feature of fibrinogen recognized by αM
is functionally distinct from the features required for fibrin polymer formation, this could be accomplished without imposing any hemorrhagic risks.