Differing roles for urokinase and tissue-type plasminogen activator in collagen-induced arthritis

Abstract

The plasminogen activators, urokinase PA (u-PA) and tissue-type PA (t-PA), are believed to play important roles in inflammatory cell infiltration, fibrin deposition, and joint destruction associated with rheumatoid arthritis; however, their precise roles in such processes, particularly u-PA, have yet to be defined. Using gene-deficient mice we examined the relative contribution of the PAs to the chronic systemic collagen-induced arthritis model. Based on clinical and histological assessments, u-PA-/- mice developed significantly milder disease and t-PA-/- mice more severe disease compared with the relevant wild-type mice. Fibrin deposition within joints paralleled disease severity and was particularly pronounced in t-PA-/- mice. Likewise, cytokine levels in the synovium reflected the severity of disease, with interleukin-1beta levels in particular being lower in u-PA-/- mice and increased in t-PA-/- mice. The antibody response to type II collagen was normal in both knockouts; however, T cells from u-PA-/- mice had a reduced proliferative response and produced less interferon-gamma on antigen stimulation in vitro. These results indicate that the major effect of u-PA in the collagen-induced arthritis model is deleterious, whereas that of t-PA is protective. Our data highlight the complexities of PA function, and suggest that approaches either to target u-PA or to enhance local t-PA activity in joints may be of therapeutic benefit in rheumatoid arthritis.

Figure 1.

CIA development in u-PA−/− mice and control u-PA+/+ mice. A: Severity. Results are expressed as the mean clinical score ± SEM. There is significantly milder disease in the u-PA−/− mice (n = 39) compared with the u-PA+/+ mice (n = 31) (P = 0.03, Mann-Whitney using the mean clinical score of individual mice averaged throughout days 21 to 60). B: The number (%) of individual limbs with a particular clinical score is presented. For u-PA−/− mice, there are significantly less limbs with clinical scores of 2 or 3 compared with u-PA+/+ mice (P < 0.001, multi-way chi-square). C: Paw thickness for individual limbs with a particular clinical score, expressed as the mean thickness ± SEM. For u-PA+/+ mice, there is a highly significant correlation between the paw thickness and clinical score of individual limbs (r = 0.846, P < 0.001). For u-PA−/− mice, there is a weak, but significant, correlation between the paw thickness and clinical score of individual limbs (r = 0.475, P = 0.002). *, P = 0.027, paws from u-PA−/− mice with a clinical score of 3 are significantly less swollen than paws from u-PA+/+ mice with a clinical score of 3.

Figure 2.

CIA development in t-PA−/− mice and control t-PA+/+ mice. A: Severity. Results are expressed as the mean clinical score ± SEM. There is significantly more severe disease in the t-PA−/− mice (n = 15) compared with the t-PA+/+ mice (n = 22) (P = 0.015, Mann-Whitney using the mean clinical score of individual mice averaged throughout days 21 to 60). B: The number (%) of mice that developed arthritis in a given number of limbs is presented. C: The number (%) of individual limbs with a particular clinical score is shown. For t-PA−/− mice, there are significantly more limbs with a clinical score of 3 compared with t-PA+/+ mice (P < 0.001, multi-way chi-square).

Figure 3.

Histological assessment of CIA in u-PA−/− mice. Distal interphalangeal joints from u-PA−/− mice (A, C, and E) and u-PA+/+ mice (B, D, and F). These are representative of average clinical scores in each case. A and B: H&E staining. C and D: Safranin O, fast green staining with a hematoxylin counterstain. E and F: Immunohistochemical detection of fibrin. Note that the arthritis is milder in the joints from the u-PA−/− mice and there is only very weak fibrin staining. Ca, cartilage; Sy, synovium. Original magnifications, ×125. G: Histological scores (mean ± SEM) for each histological feature (n = 26 for u-PA−/− and 18 for u-PA+/+ limbs) and for fibrin staining (n = 10 for u-PA−/− and 10 for u-PA+/+ limbs). Note that scoring for fibrin staining is 0 to 6 and for all other histological features is 0 to 3. *, P < 0.05; **, P < 0.01 u-PA−/− versus u-PA+/+ mice.

Figure 4.

Histological assessment of CIA in t-PA−/− mice. Severely affected metatarsal phalangeal joints from t-PA−/− mice (A, C, E, and G) and t-PA+/+ mice (B, D, F, and H). These represent joints with the most severe arthritis in each case. A and B: H&E staining. C and D: Safranin O, fast green staining with a hematoxylin counterstain. E and F: Immunohistochemical detection of fibrin. G and H: Control staining for fibrin. Note that the arthritis is more severe in the joint from the t-PA−/− mouse and there is more fibrin staining. Ca, cartilage; Sy, synovium. Original magnifications, ×125. I: Histological scores (mean ± SEM) for each histological feature (n = 14 for t-PA−/− and 15 for t-PA+/+ limbs) and for fibrin staining (n = 11 for t-PA−/− and 9 for t-PA+/+ limbs). Note that scoring for fibrin staining is 0 to 6 and for all other histological features is 0 to 3. *, P < 0.05 t-PA−/− versus t-PA+/+ mice.

Figure 5.

Effect of u-PA and t-PA deficiency on CII-specific immune responses in CIA. A: Cellular response to CII in u-PA−/− or u-PA+/+ mice. B: IFN-γ levels from the CII-specific T-cell culture supernatants described in A. C: Cellular response to CII in t-PA−/− or t-PA+/+ mice. D: IFN-γ levels from the CII-specific T-cell culture supernatants described in C. Inguinal lymph node cells from pools of three mice immunized for CIA were cultured in the presence of differing concentrations (0 to 100 μg/ml) of denatured CII for 72 hours. Cultures were pulsed with tritiated thymidine ([³H]TdR) for the last 16 hours (see Materials and Methods). Results are expressed as the amount of [³H]TdR incorporation (mean ± SEM) in cpm (A and C). Cells from u-PA−/− mice showed significantly less [³H]TdR incorporation than those of u-PA+/+ mice at all CII concentrations (P = 0.034). IFN-γ levels were measured by ELISA and are expressed as the mean ± SEM (pg/ml) (B and D). Cells from u-PA−/− mice produced significantly less IFN-γ than those from u-PA+/+ mice at all CII concentrations (P = 0.030). E: IgG antibody response to CII measured at the end of each experiment (day 60). Results are expressed as the mean ± SEM. F: IgG1, IgG2b, IgG2c, and IgG3 antibody response to CII in sera from u-PA+/+ and u-PA−/− mice. Results are expressed as the mean ± SEM, where the mean level of antibody for each IgG subclass was standardized such that u-PA+/+ mice had a mean level of 100 U/ml.

Figure 6.

Effect of u-PA and t-PA deficiency on proinflammatory cytokine levels in joints in CIA. TNF-α (A) and IL-1β (B) levels (ELISA) were measured at sacrifice in washouts from ankle joints of arthritic mice. Results are expressed as the mean level ± SEM for each cytokine (pg/ml). *, P < 0.05, knockout versus wild-type; **, P = 0.001, knockout versus wild-type.