Studies on the Mechanism of Lymphocyte-Mediated Cytolysis

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Abstract
The role of divalent cations in the thymus-derived (T) lymphocyte-mediated cytolysis of DBA/2 mastocytoma cells by immune allogeneic (C57BL) splenic lymphocytes has been studied. In the presence of 0.001 M EDTA no specific cytolysis (51Cr release) was demonstrable. Cytolysis was restored upon addition of either an excess of Ca2+ or a much larger excess of Mg2+. When EDTA was added to an ongoing cytolytic event there was a considerable lag period of the order of 1 hr before inhibition was demonstrable, indicating the existence of cation dependent and independent phases of cytolysis. Although cytolysis did not occur when lymphocytes and target cells were incubated together in the initial presence of EDTA, upon readdition of cations the rate of cytolysis was increased significantly from that observed when the interacting cells were not pre-incubated with inhibitor. These findings suggest that initial steps along the cytolytic pathway proceed in the absence of cations, but fall short of cytolysis. When specific target cell lysis was inhibited by cytochalasin B or by prostaglandin E2 (PGE2), the substitution of EDTA for these inhibitors further suppressed cytolysis. On the other hand, cytolysis proceeded in an unimpeded manner when EDTA-inhibited reaction mixtures were washed and confronted with the same concentrations of cytochalasin B or PGE2 which completely inhibited de novo cytolysis. These findings demonstrate that the cytolytic event proceeds in discrete stages. The events which are inhibited by cytochalasin B or the prostaglandins are clearly distinct from, and occur before, those stages which require divalent cations and are inhibited by EDTA.

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