CALMODULIN REGULATION OF MACROPHAGE FUNCTIONS

Abstract

We have explored the role of calmodulin (CaM) in the regulation of a number of immunologically important cellular functions expressed by macrophages, such as phagocytosis, enzyme secretion, and migration. We chose the continuous macrophage-like cell line J774, because cloned lines derived from J774 maintain these differentiated functions in culture. The phenothiazine, trifluoperazine (Tfp), a specific inhibitor of CaM in vitro, inhibits attachment, growth, phagocytosis of opsonized and unopsonized particles, and migration of J774 without affecting secretion of lysozyme or plasminogen activator. Phosphorylation of exogenous myosin light chains of endogenous substrates in extracts of J774 is regulated by Ca('2+) and CaM and sensitive to Tfp.;We have selected variants resistant to the growth inhibitory properties of Tfp from cells mutagenized with ethylmethane sulfonate. The growth and cellular function of the variants are identical to that of the parent, J774, with the exception of growth resistance to Tfp. The CaM in the variants is unaltered in content, activity and sensitivity to inhibition by Tfp. We have identified a complex formed by {lcub}('125)I{rcub} CaM and a calmodulin binding protein (CaMBP) in activated peritoneal macrophages by a non-denaturing electrophoretic technique, that is 80-90% reduced in resident peritoneal macrophages and in the variants resistant to Tfp. This CaMBP appears to be unique to macrophages and is the major CaMBP in J774. It has been purified 46-fold from extracts of J774, and can be resolved from endogenous phosphodiesterase and protein kinase activities. The protein inhibits CaM activation of phosphodiesterase and binds CaM with a dissociation constant of 20 nM determined by sedimentation in glycerol gradients. The apparent molecular weight of the {lcub}('125)I{rcub} CaM-CaMBP complex in glycerol gradients is dependent on the relative concentrations of CaM and the CaMBP.;The results suggest that inhibition of the growth of J774 by Tfp is due to a specific interaction between the drug and CaM. In addition, the data are consistent with a model of J774 in which the major CaMBP has an inhibitory function in the regulation of growth that is relieved by its interaction with Ca('2+)/CaM, and in which CaM has an effector function in the regulation of phagocytosis and migration.