Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis

Abstract

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 g/ml) and interferon-␥ (100 units/ml), respond with endogenous NO ⅐ formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO ⅐ -derived apoptosis was blocked by the NO ⅐ -synthase inhibitor N Gmonomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-␥, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO ⅐ , released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO ⅐ -mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.Nitric oxide (NO) 1 is recognized for its participation in diverse biological processes in nearly all aspects of life (1-3). The formation of NO ⅐ occurs under both physiological and pathophysiological settings. The molecule is synthesized by a family of enzymes termed NO ⅐ synthases (NOS), which utilize arginine as their substrate in the generation of NO ⅐ and stoichiometric amounts of citrulline (4). For convenience, two types of NOS are recognized; constitutive isoforms, which are active for a relatively short time in response to intracellular Ca 2ϩ fluctuations, and a cytokine-inducible isoform. For the latter to be active, mRNA translation and protein synthesis are required. The inducible NOS generates large amounts of NO ⅐ for an extended period. However, once NO ⅐ is produced by the action of NOS, it is extremely susceptible to both oxidation and reduction. This results in the concomitant formation of species with NO ϩ -like activity (nitrosonium ion) or NO Ϫ (nitroxyl anion), respectively (5). In addition to reacting with oxygen, superoxide, and transition metals, NO ⅐ causes biological signaling via interactions with sulfhydryl groups. Because multiple NO surrogates are formed, transduction pathways are classified as either cyclic GMP-dependent or -independent. Cyclic GMP formation is initiated by NO ⅐ binding to the heme group of soluble guanylyl cyclase, thus causing enzyme activati...

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis

“…It has been demonstrated that, whereas murine-and bovine-stimulated macrophages can produce copious amounts of NO, human, caprine, lapine and porcine cells may not be able to do so. 19,20 Thus, although it is clear that stimulation of murine macrophages with LPS and IFN-g provokes NO production from iNOS, which is sufficient to induce apoptosis, [21][22][23][24] it is by no means certain that this is also the case in human cells. Nevertheless, despite the relative inability of human macrophages to produce endogenous NO upon external stimulation, they appear to respond to exogenous sources of NO in much the same way as those from other species.…”

“…25 Therefore, observations using NO donors in murine cells could still be extrapolated to human cells, and may provide useful guidance on the potential therapeutic benefits of using NO in inflammatory conditions. Several NO donors have been demonstrated to elicit apoptosis in RAW 264.7 cells, including sodium nitroprusside (SNP), 26 Snitroso-N-acetylpenicillamine (SNAP), [26][27][28] and the NONOates SPER/NO, 21,22,26,27,29 DEA/NO and DETA/NO. 26 In addition, Sandoval et al 30 demonstrated increased apoptosis in RAW 264.7 cells upon exposure to peroxynitrite (ONOO À ; 100-300 mM over 4 h and 10-100 mM over 14 h).…”

“…This discrepancy may well be the result of the different concentrations of ONOO À used in the above studies. In addition, Brune et al 22 reported that RAW 264.7 cells that have higher levels of O 2 À are resistant to iNOS-mediated apoptosis, suggesting that ONOO À plays no role in mediating NO-induced apoptosis in murine macrophages, and indeed may have a protective role. Although Brockhaus and Brune 31 showed that overexpression of superoxide dismutase (SOD) in RAW 264.7 macrophages inhibited NO-mediated apoptosis, which implies a role for ONOO À , on further investigation they found this not to be the case.…”

“…Contrasting studies in macrophage cell lines 22,29,32 suggest that the redox status of the cell may partially determine the effects of NO. An inhibitory effect of endogenous NO on J774 cell apoptosis can be unmasked when O 2 À is scavenged, 32 and over-expression of SOD also protects RAW 264.7 cells against apoptosis induced by endogenous or exogenously supplied NO.…”

“…31 These studies suggest a role for ONOO À in mediating NO-induced apoptosis. Conflicting evidence suggests that RAW 264.7 cells that overproduce O 2 À are resistant to NO-mediated apoptosis, 22 and von Knethen et al 98 observed that O 2 À activates NF-kB, thus mediating survival in these cells. Furthermore, Brockhaus and Brune 31 found that ONOO À had no role in NO-evoked apoptosis, despite the protective effect of SOD in RAW 264.7 cells.…”