Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin alphaMbeta2 binding motif

Abstract

Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib-) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin alphaMbeta2 binding motif (Fibgamma390-396A) or the alphaIIbbeta3 platelet integrin-binding motif (FibgammaDelta5), were challenged with collagen-induced arthritis (CIA). Fib- mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibgamma390-396A mice, which retain full clotting function. In contrast, arthritis in FibgammaDelta5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib- and Fibgamma390-396A mice with CIA displayed reduced local expression of TNF-alpha, IL-1beta, and IL-6, which suggests that alphaMbeta2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-alpha expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to alphaMbeta2-mediated inflammatory processes.

Figure 1. Fibrinogen deficiency results in diminished macroscopic CIA.

Representative experiment with cohorts of male Fib⁺ and Fib^– mice (n = 12 per group) immunized with CII. (A) Median arthritic index. (B) Median arthritic severity of paws. *P < 0.05, **P < 0.03, ***P < 0.01, Mann-Whitney U test. (C) Number of arthritic paws per mouse. Values are mean ± SEM. *P < 0.05, **P < 0.03, ***P < 0.01, Student’s t test. (D) Percentage of mice free of any observable macroscopic inflammatory joint disease in the fore and hind paws. P < 0.03, Kaplan-Meier log-rank analysis.

Figure 2. Microscopic analysis of the metacarpophalangeal joints of Fib⁺ and Fib^– mice immunized with CII.

All joints were sectioned in the plane of the fore paw. (A and B) Representative joint sections from unchallenged Fib⁺ and Fib^– mice. (C–H) Representative joint sections from CII-immunized Fib⁺ and Fib^– mice harvested at day 40 and stained with hematoxylin and eosin. CIA-challenged Fib⁺ mice generally exhibited severe joint pathology, with individuals occasionally displaying obliterated joint architecture (C) and granulation tissue invading into the underlying trabecular bone. The metacarpophalangeal joints from Fib⁺ mice often displayed significant cartilage and bone erosion (E, arrowheads) with robust inflammatory infiltrates and synovial hyperplasia (G, arrows). Asterisk in C denotes pannus tissue. Joints from CII-immunized Fib^– mice typically displayed far less overall joint space damage with only mild synovial inflammation and hyperplasia (D and F, arrows). Many metacarpophalangeal joints from CIA-challenged Fib^– mice were nearly free of microscopically apparent disease (H). (I–L) Immunohistochemical stain of fibrin(ogen) within joints of unchallenged and CII-immunized mice harvested 40 days after primary CII immunization. No fibrin deposition was observed within joints from unchallenged Fib⁺ mice (I). However, robust fibrin staining (brown, denoted by asterisk) was observed within inflamed and hyperplastic synovial tissue (J) and on articular surfaces (K, arrows) in Fib⁺ mice. As expected, no fibrin(ogen) was detectable within joint sections prepared from CII-immunized Fib^– mice (L) regardless of the degree of joint damage. Scale bar: 100 μm.

Figure 3. Diminished joint pathology within the knees of Fib^– mice immunized with CII.

(A–H) Representative hematoxylin and eosin–stained knee joint sections prepared from unchallenged (A and B) and CII-immunized (C–H) Fib⁺ and Fib^– mice. At 40 days after initial challenge, the knee joints collected from Fib⁺ mice exhibited extensive destruction of the joint, characterized by widespread synovial hyperplasia (C, arrow), and considerable erosion, if not obliteration, of cartilage on articular surfaces (C and E, arrowheads) and within the meniscus. Neutrophil-rich inflammatory exudates were frequently observed within the synovial fluid of knees collected from Fib⁺ mice with CIA (E and G). In contrast, knee joints collected in parallel from CII-immunized Fib^– mice typically displayed more normal architecture with intact and smooth articular surfaces (D, F, and H). (I–K) Immunohistochemical detection of fibrin(ogen) within knee sections prepared from Fib⁺ mice (brown stain). Note the strong fibrin(ogen) deposition on eroding articular surfaces (I, arrowheads) and as a component of neutrophil-rich rice bodies within the joint space (J and K). Scale bar: 200 μm (A–F, I, and J); 10 μm (G, H, and K).

Figure 4. Fibrinogen deficiency results in diminished microscopic evidence of disease within knee joints of CIA-challenged mice.

Knee joint sections from individual groups of Fib⁺ and Fib^– mice that were either all male (n = 22 mice, 44 knees per genotype) or all female (n = 13 mice, 26 knees per genotype) mice were processed for histological evaluation on day 40 of the CIA protocol. (A) Composite histopathology index (the sum of scores for individual disease parameters) for each Fib^– and Fib⁺ mouse immunized with CII. Symbols denote values for individual mice; bars denote median values. P < 0.002, P < 0.005 between groups in male and female mice, respectively; Mann-Whitney U test. (B) Scores for each of the knee histology evaluation parameters. Every disease parameter evaluated was significantly reduced in Fib^– relative to Fib⁺ CII-immunized mice. Data are mean ± SEM. *P < 0.05, **P < 0.0005, ***P < 0.0001, Student’s t test.

Figure 5. Diminished knee joint cartilage degradation in Fib^– mice with CIA relative to Fib⁺ mice with CIA.

(A) Representative knee joint sections prepared from Fib⁺ and Fib^– mice with CIA on day 40 of the protocol and stained with Masson’s trichrome. Note the absence of contiguous Alcian blue stain, and thus the absence of cartilage, along much of the articular surface of Fib⁺ mice, whereas the majority of the articular surface of Fib^– mice stained positive for Alcian blue. Arrows denote the position of the articular surface. (B) Percent of knee joint articular surface cartilage remaining intact and smooth on the tibias of Fib⁺ (n = 12) and Fib^– (n = 9) mice with CIA. The articular surface length staining positive for Alcian blue was measured by morphometric analysis and compared with the total length of the articular surface of the tibia. Results are shown as mean ± SEM percent of Alcian blue staining. P < 0.00001, Student’s t test.

Figure 6. Fibrinogen deficiency does not alter anti-CII antibody production, T cell reactivity, or neutrophil accumulation in joints of mice with CIA.

(A) Determination of anti-CII specific IgG antibody titers by ELISA using plasma harvested at day 40 of the CIA protocol from Fib⁺ and Fib^– mice (n = 12 per group). Titers are expressed as arbitrary units relative to titers found in wild-type DBA/1 mice at day 28 of CIA. Results are expressed as mean ± SEM. (B) Cellular response to CII. Spleen cells were harvested from Fib⁺ and Fib^– mice (n = 3 per group) at day 40 of the CIA protocol and stimulated with 30 μg/ml CII or anti-CD3 activating T cell receptor antibody. Results are mean ± SEM of [³H]TdR incorporated in cpm. (C) Determination of neutrophil-derived MPO activity in total protein extracts from hind paws of Fib⁺ and Fib^– mice that were unchallenged (n = 4 per genotype), at day 28 of CIA (n = 8 per genotype), and at day 40 of CIA (n = 16 per genotype). Results are mean ± SEM. *P < 0.03, Student’s t test.

Figure 7. Fibγ^Δ5 mice develop similar CIA to control mice.

Median arthritic index (A) and median arthritic severity (B) of paws from 2 combined experiments using cohorts of wild-type (n = 31) and Fibγ^Δ5 (n = 30) mice.

Figure 8. Diminution of CIA in Fibγ^390–396A knockin mice.

(A) Median arthritic index of paws from a representative experiment in which a cohort of wild-type DBA/1 (n = 16) and Fibγ^390–396A (n = 17) mice were immunized with CII. (B) Median arthritic severity of paws from 2 combined experiments using cohorts of wild-type DBA/1 and Fibγ^390–396A mice (n = 32 per group). *P < 0.05, **P < 0.02, ***P < 0.005, Mann-Whitney U test. (C) Representative knee joint sections from wild-type and Fibγ^390–396A mice stained either with hematoxylin and eosin or immunohistochemically for fibrin. Fibγ^390–396A mice generally had reduced knee joint pathology relative to knee joints from wild-type mice, despite the fact that fibrin deposition remained a prominent feature of knee joints from Fibγ^390–396A mice. (D) Histopathology index analysis of knee joint sections from wild-type (n = 13) and Fibγ^390–396A (n = 14) mice. P < 0.05, Mann-Whitney U test. (E) Representative knee joint sections from wild-type and Fibγ^390–396A mice at day 42 of CIA stained with Masson’s trichrome. (F) Morphometric analysis of intact tibial surface cartilage of CIA day 42 wild-type and Fibγ^390–396A mice. P < 0.00001, Student’s t test. Scale bars: 200 μm (C); 100 μm (E).

Figure 9. Paws from Fib^– and Fibγ^390–396A mice have reduced message levels of proinflammatory cytokines in CIA.

Quantitative PCR was used to determine the relative levels of TNF-α (A and D), IL-1β (B and E), and IL-6 (C and F) from total RNA isolated from hind paws of mice. Paws from unchallenged Fib⁺ (n = 8), Fib^– (n = 8), wild-type (n = 4), and Fibγ^390–396A (n = 4) mice were harvested as controls. CII-immunized Fib⁺, Fib^–, wild-type (n = 24), and CII-immunized Fibγ^390–396A (n = 23) mice were analyzed at days 28 and 42 of the CIA protocol. Cytokine levels within each sample were normalized to GAPDH detected within that sample. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, Mann-Whitney U test.

Figure 10. Fibrinogen deficiency has no effect on TNF-α–driven inflammatory joint disease.

(A and B) Scatter plot of arthritic index scores of paws from 8-week-old (A) and 10-week-old (B) Fib⁺ and Fib^– mice overexpressing human TNF-α. Bars indicate the median scores for each genotype. (C) Knee joint histology of representative sections taken from 10-week-old Fib⁺ and Fib^– mice overexpressing human TNF-α show identical joint destruction characterized by inflammatory cell infiltrate, robust synovial hyperplasia, articular cartilage erosion, and significant loss of bone. Quantitative evaluation of knee joint sections indicated a similar level of disease severity between TNF-α–overexpressing Fib⁺ and Fib^– mice. (D) Scatter plot of composite histopathology index score of knee joint sections; bars indicate median values (Fib⁺, 10.5; Fib^–, 11.5; P = 0.09, Mann-Whitney U test). (E) Mean scores for histopathology criteria of knee joint sections showed no significant difference for any individual parameter.