Figure 6
IL-8, MCP-1 and IP-10 mRNA in fibroblasts activated by T cell contact and effect of inhibitors. Fibroblasts were plated to confluence resulting in about 1 × 106 cells per Petri dish. They were serum starved overnight, and T cell membranes equivalent to 8 × 106 cells from Th1 and Th2 clones were then added for 4 hours in 1% FCS medium. Intracellular signalling inhibitors were added one hour before adding T cell membranes. Total fibroblast RNA was isolated and mRNA levels were assessed by RNase protection assay. (a) A representative analysis from five performed. n = nil; u = U-0126 (40 μmol/l); p = PSI (40 μmol/l); j = JNK inhibitor (10, 20 and 50 μmol/l). (b) mRNA signal intensity was determined densitometrically and normalized values, computed by dividing chemokine by housekeeping probe signals, were used to evaluate the effect of T cell contact and intracellular signal inhibitors. The bars represent the percentage of the chemokine signal intensity measured in the presence of T cell membranes with intracellular signal inhibitors divided by the signal obtained in the presence of T cell membranes without inhibitors. Shown is the mean ± standard deviation of five distinct experiments. Statistically significant differences versus medium: *P < 0.05, ‡P < 0.001. ERK, extracellular signal-regulated kinase; FCS, foetal calf serum; IFN, interferon; IL, interleukin; IP, interferon-γ inducible protein; JNK, c-Jun N-terminal kinase; MCP, monocyte chemoattractant protein; PSI, proteasome inhibitor I; Th, T-helper; TNF, tumour necrosis factor.