Activation of murine microglial cell lines by lipopolysaccharide and interferon‐γ causes NO‐mediated decreases in mitochondrial and cellular function

Abstract

Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO-mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N-guanidino monomethyl L-arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex-specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

“…For instance, LPS/IFN-c-induced activation of iNOS in both enriched primary microglial cultures and the N11 microglial cell line elicits a 40% decrease in GSH that can be blocked by the inhibition of iNOS (44,200,214). Similar observations have been recorded using N9 microglia, although it was noted that mitochondrial GSH was unaffected by the drop in total GSH after LPS/IFN-c treatment (266).…”

Neuroactive steroids, their metabolites, and neuroinflammation

“…When stimulated, microglia acquire an ameboid morphology and present an upregulated catalog of surface molecules (Oehmichen & Gencic 1975, Cho et al 2006, such as MHC, chemokine receptors, and CD14 (Rock et al 2004). This activated state also coincides with the production of soluble mediators both detrimental, such as pro-inflammatory cytokines, reactive oxygen species, and reactive nitrogen species (Sawada et al 1989, Moss & Bates 2001, Viviani et al 2001, Liu et al 2002, Tansey et al 2007, and beneficial (i.e. anti-inflammatory cytokines and tropic factors) (Morgan et al 2004, Liao et al 2005, Muller et al 2006Fig.…”

Role of Microglia in Central Nervous System Infections

“…Animal species differences, however, appear to prevail in the generation of the RNI nitric oxide (NO) by microglia. While microglia isolated from rat and mouse (167,256) brains yield substantial amounts of NO when activated, the picture is less clear for human microglial cells. On stimulation with cytokines and LPS, human microglia release little or no appreciable NO (47,99,168,282), a finding which is consistent with the hyporesponsiveness of the human inducible NO synthase (iNOS) gene reported for other populations of human mononuclear phagocytes.…”