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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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S. J. LINNANE, V. M. KEATINGS, C. M. COSTELLO, J. B. MOYNIHAN, C. M. O'CONNOR, M. X. FITZGERALD, and P. MCLOUGHLIN
Total Sputum Nitrate plus Nitrite Is Raised during Acute Pulmonary Infection in Cystic Fibrosis
Am. J. Respir. Crit. Care Med.,
July 1, 1998;
158(1):
207 - 212.
[Abstract]
[Full Text]
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T. Musso, L. Calosso, M. Zucca, M. Millesimo, M. Puliti, S. Bulfone-Paus, C. Merlino, D. Savoia, R. Cavallo, A. N. Ponzi, et al.
Interleukin-15 Activates Proinflammatory and Antimicrobial Functions in Polymorphonuclear Cells
Infect. Immun.,
June 1, 1998;
66(6):
2640 - 2647.
[Abstract]
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T. Shimizu, K.-i. Kinugawa, Y. Sugishita, K. Sugishita, K. Harada, H. Matsui, O. Kohmoto, T. Serizawa, and T. Takahashi
Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes
Cardiovasc Res,
May 1, 1998;
38(2):
405 - 413.
[Abstract]
[Full Text]
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A. Gross, S. Spiesser, A. Terraza, B. Rouot, E. Caron, and J. Dornand
Expression and Bactericidal Activity of Nitric Oxide Synthase in Brucella suis-Infected Murine Macrophages
Infect. Immun.,
April 1, 1998;
66(4):
1309 - 1316.
[Abstract]
[Full Text]
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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.
[Abstract]
[Full Text]
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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.
[Abstract]
[PDF]
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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.
[Abstract]
[Full Text]
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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.
[Abstract]
[Full Text]
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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.
[Abstract]
[Full Text]
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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.
[Abstract]
[Full Text]
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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.
[Abstract]
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