Macrophage Endothelial Nitric-oxide Synthase Autoregulates Cellular Activation and Pro-inflammatory Protein Expression

Abstract

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrowderived macrophages (BMDMØs) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDMØs from eNOS KO animals exhibited reduced nuclear factor-B activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDMØs at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDMØ activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.Nitric oxide (NO) 1 production by the inducible isoform of NO synthase (iNOS) plays a pivotal role in numerous and diverse pathophysiological processes, particularly as a principal mediator of the microbicidal and tumoricidal actions of macrophages (1-3). Inducible NOS is expressed in many cell types in response to a wide range of inflammatory cytokines including interleukin-1␤, interleukin-2, interferon-␥, tumor necrosis factor-␣, and bacterial metabolites such as lipopolysaccharide (LPS) (4, 5). The inherent activity exhibited by iNOS results in the production of "high-output" NO, which is cytotoxic and cytostatic to a number of pathogens and tumor cells; this is mediated via inhibition of various enzymes within target cells including complexes I and IV of the mitochondrial respiratory chain (6 -9), ribonucleotide reductase (10), aconitase (3), and glyceraldehyde-6-phosphate dehydrogenase (11) and through DNA modification (12, 13).It is now becoming clear that NO plays a pivotal role in the regulation of gene expression (14 -18). One key facet of this regulatory activity may be the control of iNOS induction (19 -22). Such a mechanism would constitute a self-regulating pathway by which NO production from this NOS isoform could be fine tuned, which is essential because iNOS is regulated transcriptionally rather than biochemically. We have reported recently that NO has a patent biphasic effect on nuclear factor-B (NF-B) activity in murine macrophages and hence possesses the ability both to up-and down-regulate the expression of a number of pro-inflammatory proteins, including enzymes such as iNOS, cyclooxygenase-2, and interleukin-6 (17). The dual effect of NO on NF-B h...

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

“…The protective effect of upregulation of the low NO output NOS isoform, eNOS, by the NO donor during the hyperglycemic insult is supported by several studies in eNOS knockout mice, which revealed that this isoform serves cardioprotective and vasoprotective functions (Connelly et al, 2003;Gewaltig and Kojda, 2002;Jablonka-Shariff and Olson, 1998). These functions include modulation of vasoactivity and blood pressure, and inhibition of platelet aggregation, leukocyte adhesion and smooth muscle cell proliferation (Limbourg et al, 2002;Jugdutt, 2002;Papapetropoulos et al, 1999).…”

Tetrahydro-4-Aminobiopterin Attenuates Dendritic Cell-Induced T Cell Priming Independently from Inducible Nitric Oxide Synthase

“…1A). Expression of constitutive NOSs present in addition to iNOS and generating NO in the absence of inflammatory stimuli has been demonstrated in various cell types including rodent macrophages (22)(23)(24). However, in dendritic cells from both wild-type and iNOS Ϫ/Ϫ mice, endothelial nitric oxide synthase as well as neuronal nitric oxide synthase mRNA expression were close to the detection limit of ϳ5 ϫ 10 Ϫ9 mRNA copies per 18 S rRNA (data not shown).…”

Manipulating the NF-κB pathway in macrophages using mannosylated, siRNA-delivering nanoparticles can induce immunostimulatory and tumor cytotoxic functions

“…[17][18][19] Though TAMs have adopted a protumor phenotype, we and others have shown that they retain the potential to produce cytotoxic levels of inflammation, lyse surrounding cells, and coordinate an immune response from cells of the innate and adaptive immune system. [21][22][23][24] The ability to recapitulate these cytotoxic and immunostimulatory functions in TAMs, thus creating an antitumor phenotype, would be a powerful therapeutic tool for treating tumors and metastases with a significant macrophage population. An antitumor macrophage phenotype could potentially be produced by strategically manipulating the nuclear factor-kappaB (NF-κB) signaling pathway in TAMs.…”