2004;304(5675):1328C1331

2004;304(5675):1328C1331. 24 hr preincubation for optimal potentiation of NO production, TNF was LTX-401 maximally effective when added concurrently with LPS plus IFN. Nevertheless, under conditions that optimally potentiated NO production, both cytokines recruited comparable numbers of astrocytes to express NOS-2 (% NOS-2-positive cells after LPS plus IFN alone or with TNF or TGF1 was 9.5 1.2, 25.3 2.9, and 32.4 3.0, respectively). Interestingly, activation of astrocytes in the presence of both TGF1 and TNF 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 or TGF1 was not ablated by neutralizing antibodies to TGF1 or TNF, respectively. Thus, the two cytokines take action independently to recruit individual pools Mouse monoclonal to ALCAM 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. Keywords: LPS, 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 (Thiel and Audus 2001), pathogenesis of autoimmune-mediated demyelination (Smith and Lassmann 2002), and neurodegeneration associated with cerebral ischemia as well as Parkinson and Alzheimer diseases (Iadecola et al. 1997; Liberatore et al. 1999; Nathan et al. 2005). The cytotoxic actions of NO involve oxidative mechanisms that are precipitated by reactive metabolites, such as s-nitrosothiol and/or peroxynitrite (Moncada and Bolanos 2006), which can cause functional alterations in the cytoplasm (Chung et al. 2005; Chung et al. 2004), endoplasmic reticulum (Uehara et al. 2006), mitochondria (Barsoum et al. 2006; Moncada and Bolanos 2006), and nucleus (Dawson and Dawson 2004). Whether NO is beneficial or detrimental depends on the context in which it is produced and on the isoform of nitric oxide synthase (NOS; EC 1.14.13.39) that catalyze its formation (Iadecola 1997). 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 in the beginning termed nNOS and eNOS. In contrast, NOS-2 is not constitutively expressed but can be induced in many cell types by inflammatory mediators and, as such, was initially termed inducible NOS or iNOS. NO production by NOS-1 and -3 is usually coupled to elevated intracellular calcium by transient binding of calmodulin. However, calmodulin is usually tightly associated with NOS-2 at basal intracellular calcium LTX-401 levels, leading to sustained catalytic activity. The copious amount of NO derived from this isozyme, while an important component of the armamentarium against invading pathogens (MacMicking et al. 1997), accounts for much of the pathophysiology attributed to NO. This notion is supported by results from numerous studies demonstrating that selective inhibition of NOS-2 or NOS-2 gene deletion attenuates the neuropathological sequelae seen in several models of neurological diseases/disorders (Cross et al. 1994; Iadecola et al. 1997; Iadecola et al. 1995; Liberatore et al. 1999; MacMicking et al. 1995; Nathan et al. 2005; Thiemermann et al. 1993). Astrocytes are among the resident cells of the CNS that express NOS-2 under inflammatory conditions. Inflammatory mediators induce NOS-2 expression in astrocyte cultures (Galea et al. 1992; Hewett et al. 1993; Simmons and Murphy 1992) and NOS-2 has been detected in astrocytes in several CNS inflammatory conditions 0127:B8, Difco Laboratories, Inc.) plus IFN (3 ng/mL; recombinant mouse; R&D Systems) was used to induce expression of NOS-2 in mouse LTX-401 main astrocyte cultures since either LPS or IFN alone are poor stimuli (Hamby et al. 2006; Hewett et al. 1993; Vidwans et al. 2001). These concentrations were empirically determined to provide a saturating response with respect to astrocytic NO production (Hamby et al. 2006). Activation medium consisted LTX-401 of DMEM supplemented with 5%.