Figure 4

Involvement of MAPkinases in the modulation of LPS-induced IL-12 release. A. Monocytes were stimulated with PMT^wt (1 μg/ml) and/or LPS (30 ng/ml) for the indicated time points or left unstimulated. The cells were lysed and extracted with RIPA buffer as described under Experimental procedures. Total amounts of p42/44, JNK and p38 and the phosphorylation levels of the MAP kinases was determined by subsequent immunoblotting with specific antibodies (p42/44: Thr202/Tyr204 JNK: Thr183/Tyr185, p38: Thr180/Tyr182). Shown is one representative blot from three. B. Cells were pre-treated for two hours with UO 126 or JNK inhibitor II (concentrations indicated) and then stimulated with PMT^wt (1 μg/ml, 4 h) and LPS (30 ng/ml, 1 h). Unstimulated cells were used as a control. Lysates of the cells were immunoblotted with antibodies against p-p42/44, p42/44, p-c-Jun, c-Jun or actin. C. Cells were pre-treated with Ptx (200 ng/ml, overnight), UO 126 (10 μM, 2 h) or JNK inhibitor II (20 μM, 2 h), followed by stimulation with PMT^wt (1 μg/ml, 3 h pre-treatment) and LPS (30 ng/ml) overnight. The release of IL-12p40 was measured by ELISA (mean ± SD; n = 2). D. Cells that were treated like described in C and used for MLR (1x10⁴ CD14⁺ hBDMs, 1x10⁵ CD3⁺ alloreactive T cells). After three days of co-culture proliferation of T cells was analysed by [³H]-thymidine incorporation and presented as counts per minute (cpm). Results are given as mean ± SD; n = 2. Statistical significance was calculated with a student’s t test with *P < 0.05 and **P < 0.01.