Expression of nitric oxide synthase in human peripheral blood mononuclear cells and neutrophils.
It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.
Amin
A R
AR
Department of Rheumatology, Hospital for Joint Diseases Orthopaedic Institute, New York, New York, USA.
Attur
M
M
Vyas
P
P
Leszczynska-Piziak
J
J
Levartovsky
D
D
Rediske
J
J
Clancy
R M
RM
Vora
K A
KA
Abramson
S B
SB
D000246:Adenosine Diphosphate Ribose / Q000097:blood; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000097:blood / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation / Q000201:enzymology; D007963:Leukocytes, Mononuclear / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000097:blood* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D000246:Adenosine Diphosphate Ribose; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases