TGF beta 1 and TNF alpha potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

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-gamma (IFN gamma). A recent study examining the regulation of astrocytic NOS-2 expression demonstrated that transforming growth factor-beta1 (TGF beta 1) potentiated LPS plus IFN gamma-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 TGF beta 1, since it also accounts for the potentiation of NO production in astrocyte cultures by tumor necrosis factor-alpha (TNFalpha). In contrast to TGF beta 1, which required 24h preincubation for optimal potentiation of NO production, TNF alpha was maximally effective when added concurrently with LPS plus IFN gamma. 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 IFN gamma alone or with TNFalpha or TGF beta 1 was 9.5+/-1.2, 25.3+/-2.9, and 32.4+/-3.0, respectively). Interestingly, stimulation of astrocytes in the presence of both TGF beta 1 and TNFalpha 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 TNF alpha or TGF beta 1 was not ablated by neutralizing antibodies to TGF beta 1 or TNFalpha, 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.