NITRIC-OXIDE SYNTHASE IN CIRCULATING VS EXTRAVASATED POLYMORPHONUCLEAR LEUKOCYTES

It is becoming increasingly apparent that certain forms of acute and c hronic inflammation are associated with enhanced production of nitric oxide (NO), Although substantial information has been obtained describ ing the regulation of NO synthase (NOS) in macrophages, little informa tion 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 b iochemical properties of the inducible NO synthase (iNOS) in circulati ng vs, extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicit ed in rats by intraperitoneal injection of 1% oyster glycogen or in hu mans by peritoneal dialysis of patients with peritonitis, Inducible NO S mRNA from circulating and elicited PMNs was quantified using slot bl ot hybridization analysis with a cDNA probe specific for iNOS, iNOS pr otein was identified using Western immunoblot analysis, and NOS activi ty was quantified by measuring the N-G-monomethyl-L-arginine (L-NMMA)- inhibitable conversion of C-14-labeled L-arginine to L-[C-14]citrullin e. In a separate series of experiments, circulating or extravasated PM Ns were cultured for 4 h and the accumulation of L-NMMA-inhibitable ni trite (NO2 .) in the supernatant was determined and used as a measure of NO production in vitro, We found that circulating PMNs (rat or huma n) 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, NOS mRNA levels in 4- and 6-h elicited rat PMNs increase d 21- acid 42-fold, respectively, when compared with circulating cells , Western blot analysis revealed the presence of iNOS protein in the e licited rat PMNs and iNOS enzymatic activity increased from normally u ndetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg f or the 4- and 6-h elicited rat PMNs, respectively, Approximately 20-30 % of the total. iNOS activity was Ca2+-dependent, Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased f rom normally undetectable amounts for circulating PMNs to approximatel y 8 and 11 mu M/10(6) cells for the 4- and 6-h elicited PMNs, respecti vely, Highly enriched preparations of extravasated human PMNs containe d neither message, protein nor iNOS enzymatic activity, Taken together our data demonstrate that inflammation-induced extravasation of rat P MNs upregulates the transcription and translation of iNOS in a time-de pendent fashion and that 20-30% of the total inducible NOS is Ca2+-dep endent, In contrast, neither circulating nor extravasated human PMNs c ontained iNOS message, protein, or enzymatic activity, These data sugg est that the human PMN iNOS gene is under very different regulation th an is the rat gene.