Aim
To investigate the effects of activated rat hepatic stellate cells (HSCs) on rat Th1/Th2 profile in vitro.
Conclusion
Activated rat HSCs lower the Th1/Th2 profile, inhibiting the Th1 response and enhancing the Th2 response, and this may be a novel pathway for liver fibrogenesis.
Methods
Growth and survival of activated HSCs and CD4(+) T lymphocytes cultured alone or together was assessed after 24 or 48 h. CD4(+) T lymphocytes were then cultured with or without activated HSCs for 24 or 48 h and the proportion of Th1 [interferon (IFN)-γ(+)] and Th2 [interleukin (IL)-4(+)] cells was assessed by flow cytometry. Th1 and Th2 cell apoptosis was assessed after 24 h of co-culture using a caspase-3 staining procedure. Differentiation rates of Th1 and Th2 cells from CD4(+) T lymphocytes that were positive for CD25 but did not express IFN-γ or IL-4 were also assessed after 48 h of co-culture with activated HSCs. Galectin-9 expression in HSCs was determined by immunofluorescence and Western blotting. ELISA was performed to assess galectin-9 secretion from activated HSCs.
Results
Co-culture of CD4(+) T lymphocytes with activated rat HSCs for 48 h significantly reduced the proportion of Th1 cells compared to culture-alone conditions (-1.73% ± 0.71%; P < 0.05), whereas the proportion of Th2 cells was not altered; the Th1/Th2 ratio was significantly decreased (-0.44 ± 0.13; P < 0.05). In addition, the level of IFN-γ in Th1 cells was decreased (-65.71 ± 9.67; P < 0.01), whereas the level of IL-4 in Th2 cells was increased (82.79 ± 25.12; P < 0.05) by co-culturing, as measured by mean fluorescence intensity by flow cytometry. Apoptosis rates in Th1 (12.27% ± 0.99%; P < 0.01) and Th2 (1.71% ± 0.185%; P < 0.01) cells were increased 24 h after co-culturing with activated HSCs; the Th1 cell apoptosis rate was significantly higher than in Th2 cells (P < 0.01). Galectin-9 protein expression was significantly decreased in HSCs only 24 h after co-culturing (P < 0.05) but not after 48 h. Co-culture for 48 h significantly increased the differentiation of Th1 and Th2 cells; however, the increase in the proportion of Th2 cells was significantly higher than that of Th1 cells (1.85% ± 0.48%; P < 0.05).