Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production

Abstract

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS).

Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity

“…Promastigotes of both species were used to infect human monocytic cell lines when in the stationary phase of growth. Human monocytic U937 cells (ATCC CRL-1593.2) [14], DFGiNOS U937 transfectant (clone 2), and pBABE U937 vector control transfectant were maintained in culture as previously described [11]. For infection with Leishmania, these cells were differentiated for 3 days at 5 ϫ 10 5 cells/mL in Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS) containing 10 -7 M 1,25-dihydroxy-vitamin D 3 (VD3; Calbiochem) and 10 -7 M retinoic acid (RA; Sigma), in the absence of geneticin for all cell lines; lipopolysaccharide (LPS; from Escherichia coli 0:55-B.5, Difco Laboratories; 1 µg/mL) was added for the last 12-15 h of incubation.…”

“…Cell culture supernatants were assayed for NO 2 Ϫ by incubating them with Griess reagents as described elsewhere [11]. Absorbance was measured at 550 nm using a 690-nm reference filter.…”

“…We reported recently that U937 cells transfected with human hepatic iNOS cDNA (DFGiNOS U937) were capable of high- output NO synthesis when supplemented with BH 4 [11]. This unique property allows NO production to be switched on as desired in order to mimic the natural induction process.…”

“…We thus addressed the question of the sensitivity of intracellular Leishmania parasites to NO produced by human U937 monocytic cells previously transfected with human hepatic iNOS cDNA (DFGiNOS U937) [11]. In these cells, the iNOS transgene is constitutively expressed, and the production of NO can be switched on by simply supplementing the cells with tetrahydrobiopterin (BH 4 ).…”

Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions

“…10 These data strongly suggest that the factors that regulate iNOS expression differ between mice and humans and this has been supported by studies in which it has been shown that activation of iNOS in human monocytes/macrophages is relatively refractory to lipopolysaccharide and cytokine activation, and comparatively little is known about the regulation of this enzyme in these cells. 11,12 However, iNOS can be readily induced in human cell types of varying lineages by a combination of cytokines, and in human fetal astrocytes activation by IL-1 in combination with IFN-␥ forms a potent inducing stimulus. 13 Because IL-1 and IFN-␥ are known to be up-regulated in active MS lesions, these data would suggest that astrocytes should be activated to express iNOS at these sites.…”