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Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages

JB Weinberg, MA Misukonis, PJ Shami, SN Mason, DL Sauls, WA Dittman, ER Wood, GK Smith, B McDonald and KE Bachus

Department of Medicine, Veterans Affairs, Medical Center, Durham, NC.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage- mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase- polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell- permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Volume 86, Issue 3, pp. 1184-1195, 08/01/1995
Copyright © 1995 by The American Society of Hematology


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Infect. Immun.Home page
J. Turco, H. Liu, S. F. Gottlieb, and H. H. Winkler
Nitric Oxide-Mediated Inhibition of the Ability of Rickettsia prowazekii To Infect Mouse Fibroblasts and Mouse Macrophagelike Cells
Infect. Immun., February 1, 1998; 66(2): 558 - 566.
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J. Cell Sci.Home page
S. Kim, O. Bang, Y. Lee, and S. Kang
Production of inducible nitric oxide is required for monocytic differentiation of U937 cells induced by vitamin E-succinate
J. Cell Sci., January 2, 1998; 111(4): 435 - 441.
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JEMHome page
A. I. Sharara, D. J. Perkins, M. A. Misukonis, S. U. Chan, J. A. Dominitz, and J. B. Weinberg
Interferon (IFN)-{alpha} Activation of Human Blood Mononuclear Cells In Vitro and In Vivo for Nitric Oxide Synthase (NOS) Type 2 mRNA and Protein Expression: Possible Relationship of Induced NOS2 to the Anti-Hepatitis C Effects of IFN-{alpha} In Vivo
J. Exp. Med., November 3, 1997; 186(9): 1495 - 1502.
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JGPHome page
C.-W. Chow, A. Kapus, R. Romanek, and S. Grinstein
NO3--induced pH Changes in Mammalian Cells: Evidence for an NO3--H+ Cotransporter
J. Gen. Physiol., August 1, 1997; 110(2): 185 - 200.
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BloodHome page
L. Yan, S. Wang, S. P. Rafferty, R. A. Wesley, and R. L. Danner
Endogenously Produced Nitric Oxide Increases Tumor Necrosis Factor-alpha Production in Transfected Human U937 Cells
Blood, August 1, 1997; 90(3): 1160 - 1167.
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Am. J. Respir. Crit. Care Med.Home page
Y. FUJII, P. GOLDBERG, and S. N. A. HUSSAIN
Contribution of Macrophages to Pulmonary Nitric Oxide Production in Septic Shock
Am. J. Respir. Crit. Care Med., May 1, 1997; 157(5): 1645 - 1651.
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J. Biol. Chem.Home page
M. Ding, B. A.St. Pierre, J. F. Parkinson, P. Medberry, J. L. Wong, N. E. Rogers, L. J. Ignarro, and J. E. Merrill
Inducible Nitric-oxide Synthase and Nitric Oxide Production in Human Fetal Astrocytes and Microglia. A KINETIC ANALYSIS
J. Biol. Chem., April 25, 1997; 272(17): 11327 - 11335.
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