Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.
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.
The close proximity of pleural mesothelial cells (PMC) and mononuclear cells during pleural inflammation suggests that leukocyte-derived products (e.g., cytokines) may play an important role in modulating PMC function. The purpose of this study was to determine whether certain cytokines and bacterial products induce PMC to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to tumor necrosis factor (TNF), interleukin-1 beta (IL-1), gamma-interferon (IFN), or lipopolysaccharide (LPS) individually and in various double and triple combinations for 6-72 h. Concentrations of nitrite and nitrate were quantified and used as indirect indices of NO production. Nitrite/nitrate accumulation was maximal at 72 h, with most of the increase occurring from 48 to 72 h. Maximal nitrite/nitrate production was observed with triple combinations with the combination of LPS, IL-1, and TNF giving the highest concentration (137.4 +/- 2.8 microM). Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce large amounts of NO in response to specific combinations of proinflammatory cytokines and LPS.
The regulation of matrix metalloproteinase activity is crucial for maintaining the proper balance of tissue remodeling vs. injury. Metalloproteinase proenzymes are activated when the active site zinc is exposed via a cysteine switch mechanism. Peroxynitrite, the product generated from the interaction between nitric oxide and superoxide, has been shown to release zinc from zinc-thiolate groups, suggesting that it might alter metalloproteinase activity. This study examined the effects of nitric oxide and superoxide generators on gelatinase A activity. Results showed that nitric oxide alone had no effect on gelatinase A activity relative to control, whereas superoxide-derived metabolites increased activity. The simultaneous generation of both nitric oxide and superoxide caused an inhibition of gelatinase A activity. This inhibition was reversed by the addition of hemoglobin, superoxide dismutase, or sodium urate, suggesting that peroxynitrite and/or peroxynitrous acid caused the inhibition. Authentic peroxynitrite also inhibited gelatinase A activity. We postulate that the relative fluxes of nitric oxide and superoxide at sites of inflammation may modulate metalloproteinase activity and thus affect matrix protein metabolism.
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