Cardiac fibrosis is a key component of diabetes and involves the proliferation and differentiation of matrix-producing fibroblasts. We determined the influence of high glucose (HG) conditions on cardiac fibroblasts (CF) functions and the effects of recombinant human (rh) relaxin (RLX) in these responses. We cultured neonatal rat CF in either normal glucose (NG) or HG media. The mRNA of procollagen types I and III, and RLX-1 were assessed by real time PCR and procollagen type I C-terminal peptide (PICP) and procollagen type III amino terminal peptide (PIIINP), matrix metalloproteinases 2 (MMP2), MMP9 were assessed by enzyme linked immunosorbent assay. The results are as follows: a) CF proliferation was significantly increased by HG; rhRLX significantly inhibited HG fibroblast proliferation, while it had no marked effect on CF proliferation in NG. b) CF treated with HG significantly increased the production of PICP and PIIINP. rhRLX had no marked effect on production of PICP and PIIINP in NG. rhRLX blocked the HG-induced increases in collagen synthesis. c) The production of MMP2 and MMP9 is significantly increased by HG. rhRLX decreased overproduction of MMP2 and MMP9 in the presence of HG. d) The RLX- 1 mRNA expression of HG group was higher than in the NG group. We concluded that rhRLX could inhibit both the proliferation of CF and the synthesis of collagen under the HG condition. HG concentration could stimulate the expression of endogenous RLX.

PurposeRepeated exposure to bacterial superantigens, such as staphylococcal enterotoxin A or B (SEA or SEB), is known to induce Vβ-specific anergic T cells which suppress the proliferative and cytokine responses of naïve T cells to SEA or SEB. We sought to determine if repeated exposure to another bacterial superantigen, Toxic Shock Syndrome Toxin-1 (TSST-1), could also generate regulatory T cells.Method6-8 week-old female C57B6 mice were injected subcutaneously with 4μg TSST-1 or saline for three doses at 4-day intervals. After the 3rd injection, mice were sacrificed, splenocytes were obtained, and CD4+ T cells were purified by magnetic separation. Splenocytes from TSST-1 or saline-treated mice were stimulated with either TSST-1 (22 ng/ml) or SEB (1 μg/ml) for 48h. The suppressive effect of CD4+ T cells from TSST-1 or saline-treated mice were evaluated by co-culture with naïve splenocytes and stimulation with TSST-1 or SEB in vitro. The proliferative response was measured by carboxy-fluorescein diacetate (CFSE)-staining and flow cytometry, and various cytokines in the culture supernatant were assayed by ELISA.ResultsSplenocytes from TSST-1 treated mice produced lower levels of IL-2 and higher levels of IL-10 and IFN-γ, compared to saline-treated mice (p≤0.05), when re-stimulated with TSST-1 in vitro, consistent with a Tr1 phenotype. Stimulation with SEB in vitro induced a similar cytokine production profile as with TSST-1. Moreover, TSST-1 primed CD4+ T cells proliferated at a higher rate than saline-primed CD4+ T cells when re-stimulated with TSST-1 in vitro (74% vs. 16%; p≤0.01, paired t test), indicating that in vivo administration of TSST-1 did not result in anergy of TSST-1 responsive CD4+ T cells. Co-culture of TSST-1 primed CD4+ T cells with naïve splenocytes in the presence of TSST-1 led to decreased production of both IL-2 and IL-12, and increased production of IL-10 (p≤0.05). Furthermore, naïve CD4+ T cells proliferated in response to TSST-1 at a higher rate when co-cultured with TSST-1 primed CD4+ T cells, than with saline-primed CD4+ T cells (55% vs. 27%; p≤0.05). Co-culture of naïve splenocytes with TSST-1 primed CD4+ T cells in the presence of SEB showed a similar pattern of cytokine production as with TSST-1 stimulation.ConclusionRepeated subcutaneous injection of TSST-1 induced CD4+ regulatory T cells that were non-anergic, and mediated both antigen-specific (TSST-1) and by-stander (SEB) inhibition of IL-2 and IL-12 production by naïve splenocytes. The therapeutic application of these regulatory T cells is currently under investigation.