CELL REGULATION, VOI. 1, 425-434, April 1990 Regulation of IL-4 lymphokine gene expression and cellular proliferation in murine T helper type 11 cells Eduardo Mufloz, Ana M. Zubiaga, Juan Mufloz and Brigitte T. Huber Department of Pathology Tufts University School of Medicine Boston, Massachusetts 02111 Activation of T cells results in the production of lymphokines and cellular proliferation. Protein ki- nase C (PKC) plays a key role in this process. It has been shown that this enzyme is essential to elicit a response to Con A or specific antigen in CD4+ T helper type 1 (Th 1) cells that secrete IL-2. We have now explored the signal transduction pathway that leads to transcription of the IL-4 gene and prolif- eration in murine CD4+ T helper type 2 (Th 2) cells. Surprisingly, we have found in two independently derived Th 2 clones that neither cellular prolifera- tion nor IL-4 lymphokine production is affected by blocking or depletion of PKC. This differential mechanism of signal transmission leading to cel- lular activation implies a new distinction between murine Th 1 and Th 2 cells. Introduction The activation of T cells by specific antigen, mitogen, or monoclonal antibodies directed against certain cell surface proteins induces the hydrolysis of phosphatidylinositol 4'5', biphos- phate (PIP2), mediated by phospholipase C (Wil- son etal., 1984; Bennett and Crooke, 1987). This hydrolysis leads to the production of second messengers such as diacylglycerol (DAG) and inositol triphosphate (InsP3) (Berridge, 1987). InsP3 increases the concentration of intracel- lular calcium ([Ca2+],) (Carafoli, 1987), whereas DAG is an essential cofactor for the activation of the enzyme protein kinase C (PKC) (Kikkawa and Nishizuka, 1986). It has been postulated that the combination of both effects leads to production of lymphokines, expression of lym- phokine receptors, and cellular proliferation (Mills et al., 1985; Isakov et al., 1987). In the murine system two types of CD4+ T helper (Th) cells have been defined, namely Th 1 and Th 2 cells, that differ in their lymphokine production and immune function (reviewed by Mosmann and Coffman, 1987). Whereas Th 1 cells produce IL-2, y-IFN, and lymphotoxin on activation, Th 2 cells secrete IL-4 and IL-5 (Cherwinsky et al., 1987). Recently, it has been reported that in Th 1 cells PKC is essential for IL-2 production and proliferation in response to T cell receptor (Tcr)-mediated activation but not for proliferation in response to exogenous IL-2 (Mills et al., 1988; Valge et aL, 1988). The mechanism of signal transduction in Th 2 cells after Tcr-mediated stimulation has not been elucidated yet. Using two different sys- tems to block the function of PKC, we have found that this enzyme plays a different regu- latory role in Th 2 than in Th 1 cells; namely, neither IL-4 lymphokine production nor cellular proliferation is dependent on the presence of PKC in Th 2 cells. These results support the idea that the differentiation of CD4+ T cells into Th 1 or Th 2 cells is an event that occurs at the level of second messengers generated by the activation of differential signaling pathways. Results Calcium ionophores induce IL-4 mRNA in D10 cells We were interested in determining what signals lead to IL-4 mRNA expression in the prototype Th 2 cell, D1O.G4.1 (D10). There is general agreement that activation of T cells through the Tcr leads to an increase in [Ca2+i as well as activation of PKC. This physiological activation signal can be mimicked by the use of ionophores in conjunction with phorbol esters (Truneh et al., 1985). We cultured resting Dl 0 cells for 6 h with these agents and measured IL-4 mRNA expression in a Northern blot. As shown in Fig- ure 1, ionomycin alone (but not PMA alone) is able to induce IL-4 message. Synergy is seen with the combination of the two agents, result- ing in IL-4 mRNA levels similar to those induced by Con A stimulation. Another Ca2l ionophore, A23187, also induced IL-4 steady-state mes- sage in D10 cells in a dose-dependent manner, supporting the idea that the IL-4 gene is posi- tively regulated by a Ca2"-dependent pathway. © 1990 by The American Society for Cell Biology 425