Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes

Abstract

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG-monomethyl-L-arginine (L-NMMA)-inhibitable conversion of 14C-labeled L-arginine to L-[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L-NMMA-inhibitable nitrite (NO2-) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 x 10(6) cells/well) were unable to generate significant amounts of NO2- when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4- and 6-h elicited rat PMNs increased 21- and 42-fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4- and 6-h elicited rat PMNs, respectively. Approximately 20-30% of the total iNOS activity was Ca(2+)-dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 microM/10(6) cells for the 4- and 6-h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation-induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time-dependent fashion and that 20-30% of the total inducible NOS is Ca(2+)-dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Functional and molecular characterization of NOS isoforms in rat neutrophil precursor cells

“…2B). NOS enzymatic activity measurement with 3 H-L-Arginine (14,15,29) also exhibited same trend. Calcium independent NOS (iNOS) activity (15,29) was increased from Band 3 to neutrophils, whereas calcium dependent NOS activity remained comparable at all the stages.…”

“…The presence of nNOS and iNOS in rat peripheral PMNs has been well documented (15,27,29,30,42,43). As neutrophils precursors constitute the major fraction of bone marrow niche, NO produced from these cells might act as a paracrine effector to regulate hematopoiesis in the bone marrow.…”

“…Ability of neutrophils to synthesize NO was first discovered by its ability to relax aortic rings (11) and by the inhibition of platelet aggregation (12,13), which was abolished by NOS inhibitors. Nitrite (NO 2 2 ) content (14,15) correlate with NO production in rat and human PMNs. Neutrophils generate NO at a rate of 10-100 nmoles/5 min/10 6 cells, comparable to the endothelial cells, thus contributing much to the amount of NO in circulation (16).…”

“…Rodents are commonly exploited for the various NO-mediated investigations. However, Interspecies differences in terms of NOS expression and enzymatic NO production have been reported (15,27,28). Specifically, rat neutrophils constitutively express nNOS and generate higher levels of NO; whereas human neutrophils express low level of enzyme at mRNA and protein level.…”

Neutrophils, Nitric Oxide Synthase, and Mutations in the Mutatect Murine Tumor Model

“…57 In our limited study, we noted a correlation between the number of neutrophils and necrosis in adenocarcinoma of the lung. When these same specimens were stained for IL-8, most showed reactivity in tumor cells but there was no obvious correlation of IL-8 with neutrophil content.…”