FIGURE 5.

Infliximab-induced apoptosis was mediated by activation of Rho kinase. (A) TmTNF-expressing Jurkat cells were incubated with 0.1 μM MTX for 20 h, 0.01 μM IFX for 6 h, or combination of MTX and IFX for 6 h after MTX for 14 h, in the presence (white bar) or absence (black bar) of 0.01 μM of Rho kinase inhibitor (Y-27632). Apoptotic cells were analyzed by flow cytometry using Annexin V staining. The proportion of Annexin V-positive cells was indicated. (B) TmTNF- expressing Jurkat cells were stimulated with 0.1 μM MTX for 24 h, 0.01 μM ETN for 12 h, or combination of MTX and ETN for 12 h after MTX for 12 h with or without Y-27632. (C) TmTNF-expressing Jurkat cells were stimulated with 0.1 μM MTX for 24 h, 0.01 μM CZP for 12 h, or combination of MTX and CZP for 12 h after MTX for 12 h with or without Y-27632. Values are mean ± SEM; n = 4/group in (A) and n = 5/group in (B,C). *p < 0.05, **p < 0.01, Mann Whitney U tests. (D,E) TmTNF-expressing Jurkat cells were stimulated with 0.01 μM IFX for 2 h in the presence (Rho-i + IFX) or absence (IFX) of 0.01 μM of Y-27632. Stimulated cells or unstimulated cells (Ctrl) were lysed in lysis buffer and the levels of phosphorylated JNK (p-JNK) was analyzed in western blotting (D). The ratio of p-JNK: total JNK is indicated in (E).