TGFβ1 and TNFα potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

Abstract

Nitric oxide (NO) synthase-2 (NOS-2), a key source of NO at sites of neuroinflammation, is induced in astrocyte cultures treated with lipopolysaccharide (LPS) plus interferon-γ (IFNγ). A recent study examining the regulation of astrocytic NOS-2 expression demonstrated that transforming growth factor-β1 (TGFβ1) potentiated LPS plus IFNγ-induced NOS-2 expression via expansion of the pool of astrocytes that express NOS-2. Results in the current report indicate that this population-based mechanism of increasing NOS-2 expression is not restricted to TGFβ1, since it also accounts for the potentiation of NO production in astrocyte cultures by tumor necrosis factor-α (TNFα). In contrast to TGFβ1, which required 24 hr preincubation for optimal potentiation of NO production, TNFα was maximally effective when added concurrently with LPS plus IFNγ. Nevertheless, under conditions that optimally potentiated NO production, both cytokines recruited similar numbers of astrocytes to express NOS-2 (% NOS-2-positive cells after LPS plus IFNγ alone or with TNFα or TGFβ1 was 9.5 ± 1.2, 25.3 ± 2.9, and 32.4 ± 3.0, respectively). Interestingly, stimulation of astrocytes in the presence of both TGFβ1 and TNFα additively increased the number of astrocytes that expressed NOS-2 protein (% NOS-2-positive cells was 61.0 ± 4.2) relative to each cytokine alone. Potentiation of NO production by either TNFα or TGFβ1 was not ablated by neutralizing antibodies to TGFβ1 or TNFα, respectively. Thus, the two cytokines act independently to recruit separate pools of astrocytes to express NOS-2. These results are consistent with the notion that astrocytes possess an innate heterogeneity with respect to responsiveness to these cytokines. KeywordsLPS; cytokines; heterogeneity; NO; iNOS; nitric oxide synthase Nitric oxide (NO) is an endogenously derived free radical gas that affects a spectrum of homeostatic and physiologic processes, including blood flow and coagulation, normal brain and heart function, and innate immunity (Belge et al. 2005;Ignarro et al. 1999;MacMicking et al. 1997;Yun et al. 1996). However, it can be harmful under certain pathophysiological conditions. Within the CNS, aberrant NO production has been linked to cerebrovascular dysfunction ( Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NO is derived from L-arginine by the catalytic action of three different NOS isoforms (Stuehr 1999). NOS-1 and -3 are constitutively expressed in neurons and endothelial cells, respectively, and thus were initially termed nNOS and eNOS. In contrast, NOS-2 is not constitutively expressed but can be induced ...

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