HUMAN MONONUCLEAR PHAGOCYTE INDUCIBLE NITRIC-OXIDE SYNTHASE (INOS) - ANALYSIS OF INOS MESSENGER-RNA, INOS PROTEIN, BIOPTERIN, AND NITRIC-OXIDE PRODUCTION BY BLOOD MONOCYTES AND PERITONEAL-MACROPHAGES

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including sm ooth muscle relaxation, neurotransmission, and murine macrophage-media ted cytotoxicity for microbes and tumor cells. Although murine macroph ages produce NO readily after activation, human monocytes and tissue m acrophages have been reported to produce only low levels of NO in vitr o. The purpose of this study was to determine if stimulated human mono nuclear 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 i mmunoblot analyses, we show monocytes and peritoneal macrophages conta in 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 t he ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Pe ritoneal macrophages have significantly enhanced nitrite/nitrate produ ction and NOS activity after treatment with LPS and/or IFN-gamma, wher eas monocyte nitrite/nitrate production and NOS activity are not alter ed by the treatments. Monocytes cultured with various live or heat-kil led bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not pr oduce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expressi on and NO production in mouse macrophages, do not enhance NO productio n in human monocytes or macrophages. Biopterin, an obligate cofactor o f iNOS enzymatic activity, is undetectable in freshly isolated or cult ured human monocytes and peritoneal macrophages. However, replenishmen t of intracellular levels of tetrahydrobiopterin by culture with the c ell-permeable, nontoxic precursor sepiapterin does not enhance the abi lities of the human mononuclear phagocytes to produce NO in vitro. Mix ing experiments show no evidence of a functional NOS inhibitor in huma n 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. This is a US government work. There are no restrictions on its use.