TGFβ1 and TNFα 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-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 h 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.