Fig. 3. mRNA levels of inflammatory cytokines in macrophages are attenuated by MAP kinase and PI 3-kinase inhibitors, which additionally block the inhibition of fibroblast cytokine mRNA expression by DHT. Macrophage- and fibroblast-derived mRNA samples were analysed by qPCR and supernatants by ELISA. (A) The increase in macrophage IL-6 mRNA levels in response to DHT was blocked by actinomycin D, PD 98059 and wortmannin. By contrast, TNF- mRNA expression was unaffected by treatment with DHT or testosterone but was attenuated by actinomycin D, PD 98059 and wortmannin. Treatment with wortmannin tended to reduce TGF-ß1 mRNA levels that were otherwise unaffected by androgen treatment. (B) In fibroblasts, the decrease in IL-6 mRNA levels in response to DHT was blocked by wortmannin but not PD 98059. Expression of TNF- mRNA was similarly attenuated by DHT, a response that was blocked by treatment with PD 98059 or wortmannin. Co-treatment with PD 98059 or wortmannin reversed the inhibitory effect of DHT on fibroblast TGF-ß1 mRNA expression. (C) Fibroblast secretion of IL-6 and total TGF-ß1 protein levels (active and inactive) was reduced after treatment with DHT. (D-F) Fibroblast mRNA levels of IL-6 and TNF- were dramatically increased following treatment with cycloheximide. Fibroblast TGF-ß1 mRNA levels were unaffected. Data represent mean ± s.d. ^*P<0.05; ^**P<0.01. Con, control; T, testosterone; D, DHT; A, actinomycin D; P, PD 98059; W, wortmannin; CHX, cycloheximide.