Nitrite is produced by elicited but not by circulating neutrophils

A G Stewart,G J Dusting,R G Giarracca,T Harris,Y Lim,C G Sobey

doi:10.1155/s0962935193000481

A G Stewart, G J Dusting + Show 4 more

Open Access

https://doi.org/10.1155/s0962935193000481

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Abstract

The generation of nitrite (NO2-) was used as an index of the production of nitric oxide by human and rat polymorphonuclear leukocytes (PMN) and rat peritoneal macrophages. Human peripheral blood PMN did not produce significant levels of NO2-. Attempts to induce NO2- generation in human PMN by incubation with GM–CSF (1 nM), TNFα (0.3 nM), endotoxin (1 μg/ml) or formyl-Met-Leu-Phe (100 nM) for up to 16 h were not successful. Addition of human PMN primed by GM–CSF (1 nM) to rabbit aortic ring preparations precontracted with phenylephrine had no effect on tone. In contrast to these observations, PMN, isolated from the peritoneum of oyster glycogen treated rats, generated NO2- via a pathway sensitive to inhibition by the nitric oxide synthase inhibitor, NG-monomethyl L-arginine. However, peripheral blood rat PMN obtained from the same animals did not produce NO2-, even during prolonged incubation for periods of up to 16 h. It is suggested that detectable NO production by PMN requires NO synthase activity to be induced either by the process of PMN migration or by exposure to certain cytokines produced locally at the site of inflammation.

Highlights

Rat peritoneal macrophage Nitric oxide (NO) generation: Macrophages isolated from rats previously treated with 2% oyster glycogen generated levels of NO- which were detectable for 2 h of incubation and continued to increase up to 24h (Table 1)
The present results indicate that human peripheral blood polymorphonuclear leukocytes (PMN) do not generate detectable levels of NO- during prolonged incubation
Untreated PMN and those primed by granulocyte macrophage colony stimulating factor (GM-CSF) did not show any NO-like bioactivity when co-incubated with pre-contracted rabbit aortic strips

Summary

Introduction

Nitric oxide (NO) is a relatively long-lived free radical (half-life of 3-5 s) which is identical to, or is the active component of, endothelium derived relaxing factor. 1’2 It is claimed that NO has significant roles in haemostasis, regulation of systemic blood pressure, neurotransmission and immune responses.3’4 Many of the physiological actions of NO appear to be mediated by activation of soluble guanylate cyclase,[4] whereas the cytotoxic activities in host defence responses appear to involve toxic interactions with Fe-S proteins. 5’6 NO biosynthesis has been reported to occur in a variety of cell types including endothelial cells, vascular smooth muscle cells,[8] adrenal gland cells,[9] Kupffer cells,l cerebellar neurones,[11] macrophages[12] and polymorphonuclear leukocytes (PMN). 1>16 It has recently been suggested that the NO synthase in PMN may represent a third type of isozyme which is intermediate between that of the inducible form and the calcium/calmodulin dependent constitutive form. 17’18 It is not clear whether the neutrophil enzyme is inducible or constitutive, but its activity appears to be independent of calcium/calmodulin. 14’19 The inhibitory activity was prevented by the preincubation of PMNs with the NO synthase inhibitor, L-NMMA or oxyhaemoglobin, which binds and inactivates NO, suggesting that the inhibitory factor was NO These results are in contrast to those of Schattner and coworkers[2] who found that the inhibition of platelet aggregation by co-incubation of human PMN with platelets was not prevented by oxyhaemoglobin, suggesting that the inhibition of platelet aggregation was not due to neutrophil derived NO.[2] it has recently been suggested that platelets themselves may generate NO, and this endogenous NO may modulate platelet activity.[21] PMN derived NO has been measured by chemiluminescence,ls’[1] a technique which detects inorganic NO- as well as NO.[22] In addition, human PMN NO generation has been measured indirectly by a spectrophotometric assay based on the reaction of the Griess reagent with NO-, one of the stable NO decay products, which provides an index of NO generation.

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