Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells.
The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca(2+)](i) and pH(i) were determined by loading the cells with the fluorescent dyes, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2',7'-bis(2-carboxyethyl)-5-(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [(3)H]-thymidine. The results indicated that (1) LPS (10 microg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca(2+)](i) and [(3)H]-thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [(3)H]-thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca(2+)](i) elevation.
Chien
E J
EJ
Department of Physiology, School of Life Science, National Yang-Ming University, Taipei, 11221, Taiwan, Republic of China. eileen@ym.edu.tw
Chien
C H
CH
Chen
J J
JJ
Wang
S W
SW
Hsieh
D J
DJ
D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D008934:Mitogens / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*
D008070:Lipopolysaccharides; D008934:Mitogens; D011493:Protein Kinase C; D002118:Calcium