EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-I 1 or phorbol 12-myristdte-13-dcetate (PMA), or PMA + IL-I to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-rnethylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20to 30-fold) the production of 1L-2 or IL4, when EL 4-6.1 cells were induced by IL-la (or IL-ID) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA + IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-I, PMA, or PMA + IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated ccIIs were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretransldtional effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-I-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-l R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-I and PMA stimulatory pathways. In addition, they suggest that the IL-I stimulatory pathway involves PKC(s) [or other undcfined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA. o 1992 WilPy-Liss, Inc.