Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a calcium ionophore A23187 and a phorbol ester

Induction of interleukin 2 (IL2) mRNA in human tonsillar lymphocytes under various conditions was examined by cytoplasmic dot hybridization using a 32P‐labeled IL 2 cDNA probe to study the signal transduction mechanisms which lead to IL2 gene expression. A tumor‐promoting phorbol ester, 12‐O‐tetradecanoyl‐phorbol 13‐ace‐tate (TPA), acted synergistically with a Ca2+ ionophore A23187 or phytohemaggluti‐nin (PHA) to induce a high level of IL2 mRNA in lymphocytes, whereas each of them by itself could not induce the mRNA production. In two‐step culture experiments the lymphocytes pulse‐incubated with TPA for 1 h (the first culture) could efficiently initiate IL2 mRNA production by subsequent culture with A23187 or PHA (the second culture). Results obtained by removal of extracellular Ca2+ from either the first or second culture revealed that Ca2+ was not necessarily required during the first culture with TPA, but it is essential in the second culture with A23187 or PHA, regardless of the presence or absence of Ca2+ in the first culture. A reagent known to be a calmodulin antagonist, N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfonamide (W‐7), almost completely inhibited the IL2 mRNA induction in A23187‐TPA‐stimu‐lated lymphocytes at a concentration of 25 μM, whereas N‐(6‐aminohexyl)‐l‐naph‐thalenesulfonamide that has much lower affinity for calmodulin than W‐7 did not inhibit at this concentration. The IL2 mRNA induction was also blocked by the addition of 50 UM of 8‐(N, N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydro‐ chloride which is known to block the release of Ca2+ from intracellular storage sites. These results show that mobilization of Ca2+ and the calmodulin‐dependent regulatory system appear to work synergistically with TPA which probably activates protein kinase C in the pathway to IL2 gene expression.