Ca2+-calmodulin regulates the induction and expression of macrophage cytotoxicity

Ca2+-activated calmodulin (CAM) is known to regulate cellular responses of diverse cell types to external stimuli. To examine the effects of Ca2+ influx and CAM on macrophage (MP) cytotoxicity, we used 51Cr-labeled cells as targets and in vivo or in vitro activated MPs from C57 BL/6 or BALB/c mice as effectors in a 16-h cytotoxicity assay. MPs activated in vivo by administration of vaccinia virus or in vitro with lymphokine (LK) were cytotoxic for a variety of tumor cell lines, including SV3T3, but not for normal 3T3 cells. Addition of verapamil (0.1 mM), a Ca2+ channel blocker, to MPs activated in vivo by vaccinia virus markedly reduced their cytotoxicity for SV3T3 cells. This correlated with an inhibition of Ca2+ uptake by MPs, as measured by 45Ca influx. Chlorpromazine (20 microM), trifluoperazine (20 microM), and W13 (75 microM), inhibitors of CAM activity, also suppressed MP cytotoxicity for SV3T3 cells when added to the assay, suggesting that Ca2+-activated CAM is an integral component in expression of MP cytotoxicity. To further explore the mechanism of MP cytotoxicity, supernatants from activated MPs treated with various pharmacological agents were examined for cytotoxicity. Vaccinia-activated MPs released a soluble factor(s) that was cytotoxic for SV3T3 cells. Resident MPs cultured under the same condition produced no significant cytotoxic activity. As observed with direct MP cytotoxicity, addition of a Ca2+ channel blocker or CAM inhibitors to cultured activated MPs markedly reduced the cytotoxic activity of the supernatants, suggesting an active secretory process. The requirement for Ca2+ and CAM in the expression of MP cytotoxicity was confirmed in an in vitro MP activation system. Resident cultured MPs activated by an LK preparation were reduced in their tumoricidal capability when verapamil or CAM inhibitors were added to the cytotoxicity assay. This correlated with a reduction in lytic activity of the MP culture supernatants. Further, addition of verapamil or CAM inhibitors to resident MP cultures markedly reduced the induction of tumoricidal MPs by LK, suggesting that Ca2+ and CAM are necessary for both the induction and the expression of MP cytotoxicity. Suppression of cytotoxicity of in vitro activated MP cell line B6MP102 with verapamil and CAM inhibitors confirmed that the MP was the cytotoxic cell being modulated by Ca2+-activated CAM.(ABSTRACT TRUNCATED AT 400 WORDS)