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

Abstract

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.

Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll‐like receptor agonists

“…Important differences were noted between human and murine mononuclear phagocytes. For example, resting murine Mφ or Mφ‐like cell lines produce more NO than maximally activated human counterparts 37,38 . In addition, varying results on murine DC exist 39,40 and bovine Mφ and DC were previously analysed by PCR only 41 .…”

Immunology of the peritoneal cavity: Relevance for host-tumor relation

“…The antitumor activity of monocytes/macrophages in experimental rodent systems has been linked to high production of nitric oxide (NO) by activated macrophages (36) . On the other hand, human monocytes/macrophages (peripheral blood monocytes or peritoneal macrophages) are known to produce NO in quantities often around the limit of detection (45) .…”

“…IFN‐γ is a potent monocyte activator, capable of inducing both cytotoxic and cytostatic activity of monocytes/macrophages and increasing the antigen‐presenting function of these cells (35,37,38) . The antitumor efficacy of macrophages is associated with inducible nitric oxide synthase (iNOS) in rodent macrophages, but the role of iNOS and nitric oxide (NO) in the antitumor activity of human monocytes/macrophages is still under discussion (45) . In humans, the induction by IFN‐γ of indoleamine 2,3‐dioxygenase (IDO), an enzyme catalyzing the synthesis of kynurenine from tryptophan may be more important.…”

Nitric oxide produces HLA‐G nitration and induces metalloprotease‐dependent shedding creating a tolerogenic milieu

“…Both NOS 23 and HLA‐G are expressed in the placenta. In several diseases, such as coeliac disease, multiple sclerosis and HIV infection, HLA‐G 24–27 and iNOS 28–30 expression gives rise both to an increase in circulating HLA‐G and to protein nitration. It has been shown that NO can influence the function of some tolerogenic molecules such as the enzyme indoleamine 2,3‐dioxygenase 31 .…”