Nitric oxide generated by the inducible form of nitric oxide synthase (iNOS) may contribute to the pathogenesis of multiple sclerosis (MS). In this report, we studied postmortem tissues of MS patients for the expression of iNOS by in situ hybridization and immunocytochemistry. Immunocytochemistry for nitrotyrosine, a putative footprint for peroxynitrite formation was also performed. In acute MS lesions, intense reactivity for iNOS mRNA and protein was detected in reactive astrocytes throughout the lesion and in adjacent normal appearing white matter. Staining of macrophages, inflammatory cell infiltrates, and endothelial cells was variable from case to case, but generally detected only in acute lesions. In chronic MS lesions reactive astrocytes at the lesion edge were positive for iNOS whereas the lesion center was nonreactive. Normal appearing white matter demonstrated little reactivity, as did tissues from noninflamed control brains. Staining for nitrotyrosine was also detected in acute but not chronic MS lesions, and displayed a diffuse parenchymal, membranous, and perivascular pattern of immunoreactivity. These results support the conclusion that iNOS is induced in multiple cell types in MS lesions and that astrocyte-derived nitric oxide could be important in orchestrating inflammatory responses in MS , particularly at the blood-brain barrier. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that is thought to be mediated by an autoimmune attack directed against components of the myelin sheath. MS lesions are characterized by loss of myelin, oligodendrocytes, and axons associated with a mononuclear inflammatory infiltrate and a reactive gliosis. Although the mechanisms that lead to loss of function associated with these events remain poorly understood, the activation of T cells and macrophages that secrete freely diffusable factors has been widely implicated. Included in these factors are the proinflammatory cytokines interleukin (IL)-1, tumor necrosis factor-␣, IL-12, and interferon (IFN)-␥, and reactive oxygen and reactive nitrogen species. All of these factors have been shown to be elevated in active MS lesions, and animal models support a role for them in disease pathogenesis. 1,2 The anti-proliferative and/or cytotoxic effects of nitric oxide (NO) have been associated with the persistent production of high levels of NO that occurs after the activation of the inducible form of nitric oxide synthase (iNOS). 3 The expression of this enzyme in various cell types is known to be transcriptionally regulated and to be activated by a combination of pro-inflammatory signals such as ligands that activate toll-like receptors and/or cytokines such as IL-1, tumor necrosis factor-␣, and interferon-␥ (IFN-␥). 3 NO by itself demonstrates only weak toxic activity, but congeners formed by auto-oxidation such as NO 2⅐ , N 2 O 3 , and S-nitrosothiols enhance its cytotoxic potential. The toxicity of NO is also greatly enhanced when it combines with O 2Ϫ to generate peroxynitrite (ONOOϪ),...

In mammalian astrocytes, calcium waves are transmitted between cells via both a gap junction-mediated pathway and an extracellular, P2 receptor-mediated pathway, which link the cells into a syncytium. Calcium waves in astrocytes have also been shown to evoke calcium transients in neurons, and activity in neurons can elicit calcium waves in astrocytes. In this study, we show that in primary human fetal astrocytes, the P2 receptor-mediated and gap junctionmediated pathways are differentially regulated by the cytokine IL-1␤. Confocal microscopy of astrocytes loaded with Indo-1 demonstrated that intercellular calcium wave transmission in IL-1␤-treated cultures was potentiated compared with controls. However, transmission of calcium waves via the gap junction-mediated pathway was strikingly reduced. The major component of functional gap junctions in human fetal astrocytes was demonstrated to be connexin43 (Cx43), and there was a marked reduction of junctional conductance, loss of dye coupling, loss of Cx43 protein, and down-regulation of Cx43 mRNA expression after IL-1␤ treatment of cultures. Conversely, transmission of calcium waves via the P2 receptormediated pathway was potentiated in IL-1␤-treated cultures compared with controls. This potentiation was associated with an increase in the number of cells responsive to UTP, and with a transient increase in expression of the P2Y 2 purinoceptor mRNA. Because in inf lammatory conditions of the human central nervous system IL-1␤ is produced both by resident glia and by invading cells of the immune system, our results suggest that inf lammatory events may have a significant impact on coordination of astrocytic function and on information processing in the central nervous system. Intercellular calcium signaling has been shown to occur in a variety of cells in culture and may represent a general mechanism for cell-cell communication (1). Two models have been proposed by which intercellular calcium waves may be transmitted in populations of nonexcitable cells. In one model, the increase in intracellular calcium concentration is transmitted by diffusion of inositol triphosphate (IP 3 ) and͞or calcium between adjacent cells via gap junctions composed of connexin subunits (2, 3). In the second, the calcium wave is mediated by autocrine or paracrine activity of secreted tri-and dinucleotide phosphates on P2 receptors, which may further be divided into the metabotropic P2Y receptor family and the ionotropic P2X receptor family (4). In the central nervous system (CNS), intercellular calcium waves have been observed in neurons (5), astrocytes (6, 7), and oligodendrocytes (8), and although these observations were originally made in populations of cells in culture, more recently intercellular calcium waves have also been observed in more integrated systems, for example in astrocytes in brain slice cultures (9), in neurons in cerebral cortical slices (5), and in glial cells in an intact preparation of retina (10). Intercellular calcium signaling in astrocytes has attracted par...

In human astrocytes, interleukin-1beta (IL-1beta) is a potent inducer of genes associated with inflammation. In this study, we tested the hypothesis that in primary cultures of human fetal astrocytes signaling by the P2 purinergic nucleotide receptor pathway contributes to, or modulates, cytokine-mediated signal transduction. Calcium imaging studies indicated that most cells in culture responded to ATP, whereas only a subpopulation responded to UTP. Pretreatment of astrocytes with P2 receptor antagonists, including suramin and periodate oxidized ATP (oATP), resulted in a significant downregulation of IL-1beta-stimulated expression of nitric oxide, tumor necrosis factor (TNFalpha), and IL-6 at both the protein and mRNA levels, without affecting cell viability. In cells transiently transfected with reporter constructs, IL-1beta demonstrated more potent activation of the transcription factors nuclear factor -kappaB (NF-kappaB) and activator protein-1 (AP-1) than TNFalpha. However, pretreatment with oATP downregulated activation of NF-kappaB and AP-1 by IL-1beta or TNFalpha. Electromobility shift assays using oligonucleotides containing specific NF-kappaB binding sequences confirmed that pretreatment with oATP or apyrase attenuated cytokine-mediated induction of this transcription factor. From these data, we conclude that P2 receptor-mediated signaling intersects with that of IL-1beta and TNFalpha to regulate responses to cytokines in the CNS. Because inflammation, trauma, and stress all lead to the release of high levels of extracellular nucleotides, such as ATP and UTP, signaling via P2 receptors may provide a mechanism whereby cells can sense and respond to events occurring in the extracellular environment and can fine tune the transcription of genes involved in the inflammatory response.

Nitric oxide generated from the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of multiple sclerosis. Because significant species- and cell-specific differences exist in the expression of iNOS, we used primary human glial cell cultures to screen for an inhibitor of iNOS expression. Remarkably, among numerous soluble factors tested, interferon-beta (IFN-beta) alone showed a selective and potent inhibition of interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma)-induced iNOS expression in astrocytes. Inhibition of iNOS may provide a mechanism by which IFN-beta can ameliorate inflammation and cytotoxicity in the central nervous system of patients with multiple sclerosis.