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. Author manuscript; available in PMC: 2019 Jun 21.
Published in final edited form as: Arthritis Rheum. 2013 Feb 1;65(2):481–491. doi: 10.1002/art.37738

Figure 4.

Figure 4

Type I collagen expression in fibroblasts stimulated with T lymphocyte conditioned medium. A, Healthy fibroblasts were incubated with conditioned medium originating from peripheral blood CD3+ T cells from systemic sclerosis (SSc) patients and healthy controls (n = 8 per group). T cells were cultured with or without CD3/CD28 beads and subsequently incubated in the presence or absence of tumor necrosis factor (TNF)–selective variants and soluble TNF (sTNF), each at a concentration of 100 ng/ml. Type I collagen expression was determined by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Induction of type I collagen by supernatants (SN) from activated SSc lymphocytes was significantly greater than that observed with supernatants from activated control lymphocytes. Values are the mean ± SEM. * = P < 0.05. B, SSc T cells were activated with CD3/CD28 beads and treated with TNF receptor type II (TNFRII)–selective ligands or with medium alone, and supernatants were collected. Healthy dermal fibroblasts were then cultured in the presence of the conditioned medium. Neutralizing antibodies against interleukin-6 (IL-6), IL-13, or the combination of IL-6 and IL-13 were added to the conditioned medium prior to incubation with dermal fibroblasts. A significant reduction in collagen production after dual inhibition of IL-6 and IL-13 was observed. Symbols represent individual patients; horizontal bars show the mean. C, Healthy dermal fibroblasts were cultured overnight in serum-free medium and then stimulated with IL-6 (20 ng/ml), soluble IL-6 receptor (sIL-6R) (25 ng/ml), and IL-13 (100 ng/ml) in various combinations, with (solid bars) or without (open bars) incubation with the STAT-3 inhibitor S31-201 (50 μM). After 24 hours of stimulation, cells were harvested and type I collagen expression was analyzed by qRT-PCR, with normalization to 18S and control serum-free medium as the calibrator. Selective inhibition of STAT-3 attenuated IL-6 trans-signaling but did not significantly reduce IL-13–stimulated induction of type I collagen. Values are the mean ± SEM.