Free intracellular Ca2+ regulates bacterial lipopolysaccharide induction of iNOS in human macrophages

Inducible nitric oxide synthase (iNOS) reaction is of enormous significance in innate immune protective response of host to microbial challenges. We speculate that microbial challenges, by virtue of their lipopolysaccharide (LPS) content, exert an effect on intracellular free Ca(2+) levels ([Ca(2+)]i), which evokes cellular signaling in a manner that depends on LPS type. In this study, we investigated the effect of LPS from heterogeneous sources (Chlamydia pneumoniae [cLPS], Pseudomonas aeruginosa [pLPS], and Salmonella typhimurium [sLPS]) on nitric oxide (NO) release and iNOS expression in THP-1- and U937-derived macrophages, and determined the role of free [Ca(2+)]i on LPS-mediated generation of NO and iNOS expression in these cell types. The role of free [Ca(2+)]i oscillation and the probable input of extracellular Ca(2+) influx on Ca(2+)-signals was monitored in relation to iNOS expression in LPS-stimulated macrophages. There was a significant difference in the time course release of NO in both macrophage types when stimulated with cLPS, or pLPS, or sLPS. In all instances, LPS induced a significant release of NO compared with interferon-gamma. There was a difference in the pattern of basal iNOS reaction leading to NO release compared to iNOS expression observed at 24h post-stimulation. While a biphasic pattern was observed in the manner in which free [Ca(2+)]i affected iNOS expression, inhibition of extracellular Ca(2+) influx resulted in a steady increase in iNOS expression. The implications of these findings are: moderate free [Ca(2+)]i affects macrophage release of NO and iNOS expression during LPS stimulation; also, LPS of heterogeneous sources differentially evokes macrophage iNOS reactions irrespective of macrophage types.