Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils.
The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.
Cedergren
Jan
J
Division of Rheumatology, Department of Molecular and Clinical Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. jan.cedergren@lio.se
Follin
Per
P
Forslund
Tony
T
Lindmark
Maria
M
Sundqvist
Tommy
T
Skogh
Thomas
T
D015153:Blotting, Western; D002469:Cell Separation; D005122:Exudates and Transudates / Q000166:cytology / Q000201:enzymology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000097:blood* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D012867:Skin / Q000166:cytology / Q000201:enzymology
D005456:Fluorescent Dyes; D006454:Hemoglobins; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D016650:Fluorescein-5-isothiocyanate