CD40 and tumour necrosis factor-α co-operate to up-regulate inducuble nitric oxide synthase expression in macrophages

Regulation of inducible nitric oxide synthase (NOS2) expression is important given the role of this enzyme in inflammation, control of infections and immune regulation. In contrast to tumour necrosis factor-α (TNF-α) alone or CD40 stimulation alone, simultaneous stimulation of mouse macrophages through CD40 ligation and TNF-α led to up-regulation of NOS2 and nitric oxide production. This response was of functional relevance because CD40/TNF-α-stimulated macrophages acquired nitric oxide-dependent anti-Leishmania major activity. CD40 plus TNF-α up-regulated NOS2 independently of interferon-γ, interferon-α/β and interleukin-1. TNF receptor-associated factor 6 (TRAF6), an adapter protein downstream of CD40, appears to be required for NOS2 up-regulation because a CD40-TRAF6 blocking peptide inhibited up-regulation of NOS2 in CD40/TNF-α-stimulated macrophages. CCAAT/enhancer-binding protein-β (C/EBPβ), a transcription factor activated by TNF-α but not CD40, was required for NOS2 up-regulation because this enzyme was not up-regulated when C/EBPβ(-/-) macrophages received CD40 plus TNF-α stimulation. These results indicate that CD40 and TNF-α co-operate to up-regulate NOS2, probably via the effect of TRAF6 and C/EBPβ.