Differential expression of inducible nitric oxide synthase in septic shock

During cardiopulmonary bypass (CPB), the septic patient has markedly decreased peripheral vascular resistance as a consequence of endotoxin release from microorganisms. This decrease in peripheral vascular resistance is the result of endotoxin-induced nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). iNOS and eNOS are responsible for the synthesis of NO because of various stimuli, including the bacterial endotoxin, lipopolysaccharide (LPS). We tested the hypothesis that a differential expression of iNOS among human endothelial cells and murine macrophage is dependent upon exposure to endotoxin and various pro-inflammatory cytokines. Using a human endothelial cell line, ECV-304 and murine macrophage cell line, RAW 264.7, we quantified the expression of iNOS with specific FITC-conjugated antibodies using fluorescence activated cell sorter (FACS) and NO production with a Bioxytech nitric oxide spectrophotometric assay. This in vitro septic model utilized LPS supported with species-specific interferon-gamma, interleukin-1 beta, and tumor necrosis factor-alpha. The cell type were stimulated for 8 hours with combinations of the cytokines mentioned. The FACS data demonstrated a significant stimulus-dependent increase in iNOS expression among the macrophage groups; however, the stimulated endothelial cells showed no significant change in iNOS expression. The nitric oxide production data demonstrated significant increases in NO production among macrophage stimulated groups; whereas, endothelial stimulated groups exhibit no significant change. We conclude that NO secreted during septic shock is the result of activated macrophage, not the endothelium. The clinical relevance is that the more severe the infectious process, the lower the PVR may be during CPB because of increased NO production from activated macrophage.