Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca2+](i)) and pr otein kinase C (PKC) activity are two major early mitogenic signals to init iate proliferation of human peripheral T cells. Bacterial lipopolysaccharid e (LPS) is nonmitogenic in human T cells. However, in the presence of monoc ytes, LPS becomes mitogenic to proliferate T cells. The aim of th is study was to define the incompetency of LPS on two mitogenic signals in human per ipheral T cells. T eel Is were isolated from human peripheral blood. [Ca2+] (i) and pH(i) were determined by loading the cells with the fluorescent dye s, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2',7'-bis(2-carboxyethyl)-5-( and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was d etermined by protein kinase assay and cell proliferation was estimated from the incorporation of [H-3]-thymidine. The results indicated that (1) LS (1 0 mu g/ml) stimulated PKC activity significantly within 5 min, reached a pl ateau at 30 min, and maintained that level for at least 2 h; and (2) LPS st imulated cytoplasmic alkalinization but did not affect the levels of [Ca2+] (i) and [H-3]-thymidine incorporation into T cells. Moreover, the combinati on of calcium ionophore A23187 with LPS significantly stimulated [H-3]-thym idine incorporation into T cells. Thus, the results demonstrate that LPS fa iled to proliferate T cells, probably because of a lack of the machinery ne cessary to stimulate the mitogenic signal on [Ca2+](i) elevation. (C) 2000 Wiley-Liss, Inc.