Elevated matrix metalloproteinase-9 in patients with systemic sclerosis

Abstract

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = 19). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming growth factor beta. Moreover, dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1beta, tumor necrosis factor alpha, or transforming growth factor beta. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal fibroblasts in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.

Figure 1

Comparison of serum concentrations of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in patients with systemic sclerosis versus healthy controls. Data are presented as means ± standard error of the mean (Mann–Whitney rank sum test).

Figure 2

Concentrations of circulating matrix metalloproteinase-9 (MMP-9) in patients with diffuse (n = 23) or limited (n = 19) systemic sclerosis and in healthy controls (n = 32). Data are presented as means ± standard error of the mean (Mann–Whitney rank sum test).

Figure 3

Gelatinase activity of matrix metalloproteinase (MMP)-2 (72 kDa) and MMP-9 (92 kDa) in sera of patients with systemic sclerosis (SSc) and healthy controls. Sera (0.5 μl) from 20 patients with SSc and 10 healthy controls were analyzed for their MMP-2 and MMP-9 activities by gel zymography. As a positive control, supernatants from cultured HT1080 cell lines (HT) stimulated with 10 μg/ml of concanavalin A were used. Numbers in parentheses are MMP-9 concentrations (ng/ml) determined by ELISA. The figure shows representative results for serum samples from the two groups.

Figure 4

Correlation of circulating matrix metalloproteinase-9 (MMP-9) concentrations with skin fibrosis and with concentrations of transforming growth factor β (TGFβ). (a) Correlation of MMP-9 concentrations with skin scores. The extent of skin involvement of systemic sclerosis was determined by modified Rodnan scores. Broken line indicates cutoff value for patients with severe skin involvement (Rodnan score ≥20). Bars represent the mean ± standard error of the mean of MMP-9 in patients with severe versus mild-to-moderate skin involvement (Rodnan score <20). (b) Correlation of circulatory MMP-9 concentrations with TGFβ concentrations.

Figure 5

The production of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) from cultured dermal fibroblasts. Dermal fibroblasts were obtained from affected skin of two patients with systemic sclerosis (SSc) and two healthy controls. Second- or third-passage fibroblasts (5 × 10⁴ cells) were cultured for 24 hours in Dulbecco's modified Eagle's medium alone and in the presence of IL-1β (10 ng/ml), tumor necrosis factor (TNF)-α (10 ng/ml), transforming growth factor β (TGFβ) (10 ng/ml), IL-1β (10 ng/ml) plus cyclosporin A (CsA) (500 ng/ml), or TNF-α (10 ng/ml) plus CsA (500 ng/ml). The concentrations of MMP-9 in the supernatants were determined by ELISA. Data are expressed as means ± standard error of the mean (SEM) of two independent experiments performed in triplicate using different cell lines. *P < 0.05, **P < 0.01, ***P < 0.001 versus medium alone (Mann–Whitney rank sum test).