Pharmacological Modulation of Macrophage Cytotoxicity: Role of Protein Kinases

134 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.The molecular mechanism of macrophage (MP) activation to cytotoxicity has been investigated with an emphasis on the possible involvement of various classes of protein kinases.The effect of Ca$\sp{2+}$ influx and a major Ca$\sp{2+}$ binding protein, calmodulin (CAM), on MP cytotoxicity was shown by using calcium ionophore A23187 (A2) to induce MP cytotoxicity for SV3T3 cells. Addition of the Ca$\sp{2+}$ channel blocker, verapamil, to A2-activated MP markedly reduced their cytotoxic activity for SV3T3 cells. This correlated with an inhibition of Ca$\sp{2+}$ uptake by MPs, as measured by $\sp{45}$Ca influx. Inhibitors of CAM activity, chlorpromazine, trifluoperazine, and W13 also suppressed cytotoxicity of in vivo and in vitro activated MPs for SV3T3 cells.In addition, these inhibitors also suppressed the induction of tumoricidal MPs by lymphokines, suggesting that Ca$\sp{2+}$-activated CAM regulates both the induction and expression of MP cytotoxicity, most likely through a Ca$\sp{2+}$-CAM dependent kinases.To study to role of protein kinase C (PrKC) in MP cytotoxicity, MPs were treated with two pharmacological agents that mimic the action of some second messengers. At low concentration, phorbol ester TPA and A2 synergistically induce MP cytotoxicity for SV3T3. Only active phorbol esters, which are known to activated PrKC, are able to cooperate with A2 to induce killing. The combination of A2 and 4$\alpha$-phorbol didecanoate does not induce MP cytotoxicity. The data suggest that activation of PrKC and Ca$\sp{2+}$ mobilization are necessary for MP cytotoxicity.Since MP treated with sodium vanadate, a tyrosine kinase promotor, possess a 60 Kd phosphorprotein which has antigenic similarities to an avian pp60$\sp{\rm v-src}$ and was also seen expressed in response to in vivo MP activation signals, vanadate was employed to activate MPs. The addition of vanadate to MPs markedly enhanced their tumoricidal activity for SV3T3 cells. Addition of the tyrosine kinase inhibitors, N-$\alpha$-p-tosyl-L-lysine chloromethyl ketone (TLCK) and quercetin, to vanadate-induced MP in the cytotoxicity assay suppressed the cytotoxicity. TLCK and quercetin also reduced cytotoxicity of in vitro lymphokine activated MP. These results suggest that tyrosine phosphorylation may be part of the cascade of events leading to expression of MP cytotoxicity.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD