Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts S. Fuchs, MD, A. Skwara, MD,

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Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts S. Fuchs, MD, A. Skwara, MD, M. Bloch, MD, B. Dankbar, PhD Osteoarthritis and Cartilage Volume 12, Issue 5, Pages 409-418 (May 2004) DOI: 10.1016/j.joca.2004.02.005

Fig. 1 Effects of cytokines on MMP-1 secretion by synovial fibroblasts. TSC (A) and FSC (B) were incubated with or without various recombinant cytokines at concentrations of 10ng/ml. Total MMP-1 concentrations were determined in culture supernatants after 48h. Data represent median values and IQRs of stimulation experiments of 6 independent cultures each. Analysis of significance for overall group differences was performed by the Kruskal–Wallis test (P<0.001). The multiple comparisons‘ criterion was employed to identify differences between the groups (IL-1α and IL-1β vs controls, P<0.05; TNFα/IL-1β vs controls, P<0.001; TNFα/IL-1β vs IL-6, P<0.05 for both TSC and FSC). Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 2 Effects of cytokines on MMP-3 secretion by synovial fibroblasts. TSC (A) and FSC (B) were incubated with or without various recombinant cytokines at concentrations of 10ng/ml. Total MMP-3 concentrations in culture supernatants after 48h are presented as median values and IQRs of 6 independent cultures each. Analysis of significance for overall group differences was performed by the Kruskal–Wallis test (P<0.001). The multiple comparisons‘ criterion was employed to identify differences between groups (TSC: TNFα/IL-1β vs controls, P<0.01; FSC: IL-1α, IL-1β vs controls, P<0.05; TNFα/IL-1β vs controls, P<0.005; TNFα/IL-1β vs IL-6, P<0.01). Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 3 Effects of cytokines on MMP-13 secretion by synovial fibroblasts. TSC (A) and FSC (B) were incubated with or without various recombinant cytokines at concentrations of 10ng/ml. Total MMP-13 concentrations in culture supernatants after 48h are presented as median values and IQRs of six independent cultures each. Stars indicate the absence of detectable protein. Analysis of significance for overall group differences was performed by the Kruskal–Wallis test (P<0.001). The multiple comparisons‘ criterion revealed differences between groups solely in FSC (TNFα/IL-1β vs controls, TNFα, and IL-6, P=0.01). Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 4 Intracellular production of MMP-8 in synovial fibroblasts. Levels of MMP-8 were measured in cell extracts of TSC (open bars) and FSC (hatched bars) after stimulation with 10ng/ml of various cytokines for 48h. Data represent median values and IQRs of stimulation experiments using four independent cell cultures each (P<0.04 for overall group differences only in TSC, Kruskal–Wallis test). The multiple comparisons‘ criterion revealed differences between IL-1β and TNFα/IL-1β vs controls (P<0.05 and P<0.01, respectively). Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 5 Induction of MMP-8 secretion. TSC and FSC were cultured with bovine cartilage discs either directly (adherence) or separately using transwell tissue culture inserts (transwell). Enzyme concentrations were determined in coculture supernatants after stimulation with the cytokine combination TNFα/IL-1β (A) or osteoarthritic synovial fluid (1:1 dilution with medium) (B) for 72h. Data are presented as median values and IQRs of three independent cell cultures each. Concentrations given in panel B are normalized against synovial fluid concentrations of MMP-8. Stars indicate the absence of detectable protein. Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 6 Gelatin-zymography of CM derived from stimulated synovial fibroblasts. TSC and FSC were stimulated with 10ng/ml TNFα (lane 2), IL-1α (lane 3), IL-6 (lane 4), IL-1β (lane 5), and TNFα/IL-1β (lane 6) for 48h. Culture media (CM) from unstimulated control cultures (lane 1). The representative gel demonstrates increased gelatinolytic activity upon cytokine stimulation, a trend that was apparent in all other patients tested. Notably, a higher activity was evident in stimulated FSC compared to TSC. Equal loading was confirmed by similar intensities of lytic bands representing MMP-2. Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)

Fig. 7 Immunoblotting of CM derived from stimulated synovial fibroblasts. FSC were stimulated with 10ng/ml TNFα (lane 2), IL-1α (lane 3), IL-6 (lane 4), IL-1β (lane 5), and TNFα/IL-1β (lane 6) for 48h. CM from unstimulated control cultures (lane 1). Specific antibodies recognizing pro- and active forms of MMP-1 (A) and MMP-3 (B) were employed. Analysis revealed a marked increase in MMP-1 as well as MMP-3 secretion upon stimulation with all cytokines, except IL-6. In addition to their proforms, antibodies detected active forms of MMP-3 (∗) and further processed active forms of MMP-1 (#). Osteoarthritis and Cartilage 2004 12, 409-418DOI: (10.1016/j.joca.2004.02.005)