title: Endogenously produced nitric oxide increases tumor necrosis factor-alpha production in transfected human U937 cells.
abstract: Various functions of human phagocytes are modulated by nitric oxide (NO). We transfected the human U937 monoblastoid cell line with an expression vector containing human endothelial NO synthase (eNOS) or murine inducible NOS (iNOS) cDNA to study the regulatory role of NO without the nonspecific effects associated with exogenous NO sources. Western blot confirmed expression of eNOS or iNOS in respectively transfected cells, but not in naive or empty-vector transfected cells. Transfectants expressing iNOS, a calcium-independent enzyme, but not eNOS, a calcium-dependent enzyme, spontaneously produced NO (P < .001). The NO release from iNOS-transfected cells, as measured by nitrite and nitrate accumulation and by cyclic guanosine monophosphate (cGMP) increases in rat reporter cells, was inhibitable (P < .01 for both) with N(omega)-methyl-L-arginine (L-NMA), a NOS inhibitor. The eNOS transfectants were shown to contain functional enzyme by the conversion of L-arginine to L-citrulline in fractionated cells (P = .0001) and by exposing intact cells to calcium ionophore using the cGMP reporter cell assay (P = .0001). After differentiation with phorbol-12-myristate-13-acetate (PMA), iNOS transfectants produced more tumor necrosis factor-alpha (TNF-alpha) (124.9 +/- 25.4 pg/5 x 10(5) cells per 24 hours) than did empty-vector transfected cells (21.9 +/- 1.9 pg/5 x 10(5) cells per 24 hours; P = .02). This effect was inhibited by 500 micromol/L L-NMA (54.4 +/- 3.1 pg/5 x 10(5) cells per 24 hours; P = .05). However, in the presence of high concentrations of lipopolysaccharide (1 microg/mL), which further increased NO production in iNOS transfected cells (P = .044), TNF-alpha production was similar comparing PMA-differentiated iNOS and empty-vector transfectants (12.2 +/- 0.8 and 13.1 +/- 1.7 ng/5 x 10(5) cells per 24 hours, respectively; P = .5). The results show that under certain conditions endogenously produced NO can upregulate TNF-alpha production in human phagocytes.
authors.affiliation: Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: L
authors.initials: L
authors.lastname: Yan
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.forename: S P
authors.initials: SP
authors.lastname: Rafferty
authors.forename: R A
authors.initials: RA
authors.lastname: Wesley
authors.forename: R L
authors.initials: RL
authors.lastname: Danner
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002956:Citrulline / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000473:pathology*; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate