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

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Abstract
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.