Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-gamma receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.     

Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis

Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, prevented the clinical development of experimental autoimmune encephalomyelitis (EAE) with a reduction in inflammation and demyelination. Administration of AG reduced the expression of nitrosotyrosine in inflammatory lesions in the central nervous system. Cytokine expression, determined by semiquantitative PCR, revealed increased expression of IFN-gamma, IL-10, and TGF-beta, which was associated with protection from EAE, and reduced TNF-alpha, associated with the development of EAE. Furthermore, AG blocked the secretion of nitric oxide, TNF-alpha, and PGE2 in astrocyte cultures. AG did not influence the proliferation response of T cells to a pathogenic epitope of myelin basic protein. Down-regulation of nitric oxide by AG has widespread consequences for cytokine production in central nervous system inflammation and prevents EAE.     

Selective inhibition of human glial inducible nitric oxide synthase by interferon-beta: implications for multiple sclerosis

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.     

Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis

Previous studies examining the effect of nitric oxide synthase (NOS) inhibition on the course of experimental allergic encephalomyelitis (EAE) have yielded conflicting results. This may relate to the use of nonspecific inhibitors and to differences between active and adoptive EAE. We examined the effect of treatment with L-N-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of the cytokine-inducible isoform of NOS, on the clinical course of active and adoptive EAE in Lewis rats. We find that while L-NIL treatment of recipients is protective in adoptive EAE, treatment of active EAE with L-NIL leads to a marked accentuation of disease expression. In L-NIL-treated animals treated with myelin basic protein/complete Freund's adjuvant (MBP/CFA), disease onset is accelerated and clinical symptoms are more severe. Accentuation of integrated disease scores is seen even if L-NIL treatment is started 5 days following immunization. The histological findings in involved spinal cords from L-NIL-treated animals with active EAE are similar to those from untreated animals with similar clinical scores. L-NIL treatment of MBP/CFA-immunized animals does not prevent recovery from clinical symptoms, nor does it allow for reinduction of disease in animals previously immunized with MBP/CFA. Treatment of F344 rats, a strain which is relatively nonsusceptible for EAE, with L-NIL results in consistent evidence of EAE following immunization with MBP/CFA. These findings, together with our previous work on interstitial nephritis, support a role for endogenously generated NO in immunoregulation of T cell responses following immunization with antigen in CFA, and suggest that inducibility of NOS expression may be an important susceptibility factor for autoimmunity.     

The interplay of nitric oxide and peroxynitrite with signal transduction pathways: implications for disease

Since the discovery that at least one form of endothelium derived relaxing factor is nitric oxide (NO), numerous studies have uncovered diverse roles for this free radical in a variety of physiological and pathophysiological processes. NO production, a process mediated by a family of enzymes termed NO synthases, has been detected in most cell types. Many of the effects of NO are thought to be mediated through its direct interaction with specific and defined cell signaling pathways. The nature of such interactions are highly dependent on the concentration of NO and cell type. Furthermore, specific NO derived reaction products, such as peroxynitrite, also have the potential to effect cell signal transduction events. As with NO, this can occur through diverse mechanisms and depends on concentration and cell type. It is perhaps not surprising that the reported effects of NO in different disease states are often conflicting. In this brief overview, a framework for placing these apparently disparate properties of NO will be described and will focus on the effects of NO and peroxynitrite on signaling pathways.     

1,25-Dihydroxyvitamin D3 inhibits the expression of inducible nitric oxide synthase in rat central nervous system during experimental allergic encephalomyelitis

The inducible form of nitric oxide synthase (iNOS) generates nitric oxide of which the excessive production is associated with central nervous system (CNS) inflammatory diseases. The investigation of iNOS expression during experimental allergic encephalomyelitis (EAE) of the Lewis rat demonstrated iNOS immunoreactivity and mRNA both during inflammatory bursts (days 12 and 23 post-immunization) and during the remission phase (day 18). iNOS expression was region-specific and expanded with time along a caudo-rostral axis, thus, correlating with the development of inflammatory infiltrates. Whereas cells of the monocyte/macrophage lineage continuously contributed to iNOS expression, astrocytes only expressed iNOS immunoreactivity or mRNA during the relapse (day 23). In order to investigate possible regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-D3) on iNOS expression, rats were treated with the hormone after the beginning of clinical signs (days 11, 13, 19, 21 and 23 post-immunization), and areas of the CNS were examined at day 23. 1,25-D3 exerted a drastic inhibitory effect on iNOS expression, both at the protein and the mRNA levels. However, this effect was region-specific, and was most pronounced in the cerebellum and brainstem, but non-existent in cerebral cortex. iNOS down-regulation occurred in macrophages, activated microglia and astrocytes. The inhibition of iNOS expression in some CNS structures could account for the improvement of clinical signs observed in EAE-rats treated with 1,25-D3. Since 1,25-D3 can be synthesized by activated macrophages or microglia, our results support the hypothesis that this hormone might be implicated in the control of the CNS-specific immune responses. 1,25-D3 or its analogues could, thus, be of therapeutic value in the management of iNOS-associated diseases of the CNS.     

Prevention of acute autoimmune encephalomyelitis and abrogation of relapses in murine models of multiple sclerosis by the protease inhibitor D-penicillamine

The in vitro activity of gelatinase B, an enzyme whose appearance in the cerebrospinal fluid is associated with inflammatory diseases of the central nervous system, was dose-dependently inhibited by the antirheumatic D-penicillamine. Inhibition of gelatinase B in electrophoretically pure preparations and in cell culture supernatants and human body fluids was obtained at dosages reached in the circulation of patients treated with a peroral dosis of 750 mg D-penicillamine per day. In mice, developing acute demyelination, D-penicillamine significantly reduced the mortality and morbidity rates of experimental allergic encephalomyelitis (EAE). In chronic relapsing EAE in Biozzi AB/H mice, an animal model for relapses in multiple sclerosis (MS), it attenuated the exacerbations, even when the treatment was started after the primary full-blown disease had developed. We infer protease inhibition as the mechanism of action of D-penicillamine and suggest that its use may be effective as peroral treatment for MS.     

Reciprocal stimulation between TNF-alpha and nitric oxide may exacerbate CNS inflammation in experimental autoimmune encephalomyelitis

Nitric oxide (NO) and TNF-alpha are both highly active pleotypic modulators of cell function that are abundantly generated during inflammation. Experiments in animal systems have linked the generation of NO and TNF-alpha to autoimmune pathogenesis, and blockade of either NO or TNF-alpha has been shown to impede disease development. In this study, we show that NO and TNF-alpha can act mutually to stimulate each other's production. While IFN-gamma alone induces NO release from microglia, astrocytes are provoked into significant NO production only by a combination of IFN-gamma and TNF-alpha. Since both TNF-alpha and NO are abundantly generated during T-glial cell interaction, we asked whether and how NO affects TNF-alpha production. Using an in vitro system in which TNF-alpha secretion is induced in MBP-reactive T cells by co-culture with syngeneic astrocytes, we observed that the efficiency of TNF-alpha secretion was markedly increased, in a dose-dependent fashion, by addition of micromolar concentrations of a chemical generator of NO donor, sodium nitroprusside (SNP). Similarly, low concentrations of SNP significantly enhanced the IL-2 dependent growth of MBP-reactive T cells. These results suggest that autoimmune pathogenesis initiated by inflammatory responses within the CNS may result in part from a vicious cycle in which TNF-alpha and NO mutually provoke each other's production.     

MRI studies of multiple sclerosis: implications for the natural history of the disease and for monitoring effectiveness of experimental therapies

The use of magnetic resonance imaging (MRI) in multiple sclerosis (MS) has increased in our understanding of the natural history of the disease course and has provided and important tool for the analysis of new experimental therapies. Studies using MRI as well as pathological studies of MS indicate that the first event in the development of a new MS lesion as seen on T2 weighted images is disruption of the blood brain barrier (BBBD) which can be demonstrated by areas of increased signal on T1 weighted images done after the administration of gadolinium DTPA. When GdDTPA enhanced MRIs are used to monitor disease activity in patients with mild relapsing remitting MS, a considerable degree of disease activity is observed in clinically stable patients. These findings indicate that MS is an active and progressive disease in most patients even during the earliest phases of the disease and before significant clinical disability has occurred. MRI is also an important tool in evaluating new therapies. Using simple baseline vs treatment designs evidence for an effect of a new treatment on MRI parameters such as Gd-DTPA enhanced measure of BBBD can be achieved using a small study cohort and over a short duration. Together these advances should lead to more rapid progress in the understanding of MS and in identifying new treatments.     

Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study

Experimental allergic encephalomyelitis (EAE) is a demyelinating autoimmune disorder that can be induced in susceptible mice by T lymphocytes sensitized to central nervous system (CNS) myelin components and is a prime animal model for the human CNS demyelinating disorder, multiple sclerosis (MS). Although CNS inflammation in which T lymphocytes and activated macrophages are the predominant cell types is observed in mice with EAE and in humans with MS, the exact mechanisms underlying the CNS damage and demyelination are not understood. Nitric oxide (NO), a gaseous free radical, has recently been shown to be a cytolytic product of activated macrophages. Using electron paramagnetic resonance spectroscopy, the nitric oxide free radical complexed with iron-sulfur proteins has been identified in affected spinal cords of mice with EAE, concurrent with the diminution of iron-sulfur proteins. These results indicate NO may play a role in the disease process of EAE, and perhaps MS.     

Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide

Electrolytic lesions of the dorsal hippocampus (DH) produce deficits in both the acquisition and expression of conditional fear to contextual stimuli in rats. To assess whether damage to DH neurons is responsible for these deficits, we performed three experiments to examine the effects of neurotoxic N-methyl-D-aspartate (NMDA) lesions of the DH on the acquisition and expression of fear conditioning. Fear conditioning consisted of the delivery of signaled or unsignaled footshocks in a novel conditioning chamber and freezing served as the measure of conditional fear. In Experiment 1, posttraining DH lesions produced severe retrograde deficits in context fear when made either 1 or 28, but not 100, days following training. Pretraining DH lesions made 1 week before training did not affect contextual fear conditioning. Tone fear was impaired by DH lesions at all training-to-lesion intervals. In Experiment 2, posttraining (1 day), but not pretraining (1 week), DH lesions produced substantial deficits in context fear using an unsignaled shock procedure. In Experiment 3, pretraining electrolytic DH lesions produced modest deficits in context fear using the same signaled and unsignaled shock procedures used in Experiments 1 and 2, respectively. Electrolytic, but not neurotoxic, lesions also increased pre-shock locomotor activity. Collectively, this pattern of results reveals that neurons in the DH are not required for the acquisition of context fear, but have a critical and time-limited role in the expression of context fear. The normal acquisition and expression of context fear in rats with neurotoxic DH lesions made before training may be mediated by conditioning to unimodal cues in the context, a process that may rely less on the hippocampal memory system.     

Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.     

The effect of nitric oxide and peroxynitrite on apoptosis in human polymorphonuclear leukocytes

In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled nitric oxide is now a widely used therapy in patients with acute lung injury, and its effects on apoptosis may be important. We investigated the effect of nitric oxide and peroxynitrite on apoptosis in lipopolysaccharide stimulated polymorphonuclear leukocytes as a model of nitric oxide-treated lung injury. Cells were incubated for up to 16 h with and without 1.7 microg/ml lipopolysaccharide and the nitric oxide donor GEA-3162 or the peroxynitrite donor SIN-1. Apoptosis was assessed using flow cytometry following annexin-V staining, after 4, 6, 8, and 16 h. Data were assessed using Kruskal-Wallis analysis of variance or Mann-Whitney U-test as appropriate. Annexin-V staining increased spontaneously over 16 h in untreated cells (p = .0002) and incubation with either 1000 microM SIN-1 or 10 microM GEA-3162 increased annexin staining at early time points in nonactivated cells. Apoptosis was attenuated when cells were exposed to lipopolysaccharide and both nitric oxide and peroxynitrite dose dependently inhibited this suppression at all time points and was most apparent at 16 h (p = .004 and .001, respectively). Exposure of activated neutrophils to exogenous nitric oxide or peroxynitrite has marked influences on apoptosis. This work has implications for the modulation of neutrophil function within the lung in patients with lung injury who receive inhaled nitric oxide therapy.     

Hyperbaric oxygen treatment in acute experimental allergic encephalomyelitis. Contribution of magnetic resonance imaging study

Magnetic resonance imaging (MRI) has been performed to determine the efficacy of hyperbaric oxygen treatment (HBO) on experimental allergic encephalomyelitis (EAE) in Lewis rats. The animals were exposed for 2 x 3 h in a 24-h period at 2 ATA pure O2. Three HBO treatment protocols were used: a 10-day preventive treatment (beginning on the 1st day postimmunization), a 3-day preventive treatment (beginning on the 1st day postimmunization), and a 10-day symptomatic treatment (beginning on the 11th day postimmunization). Based on clinical and MRI observations, this study demonstrates: (i) that HBO treatment does not reduce the blood-brain barrier (BBB) disturbance and cerebral edema in EAE, (ii) that in an opposite way, it provokes reversible BBB breakdown, and (iii) that preventive HBO treatments results in modification of the course of EAE, possibly by immunosuppression effect during the initial sensitization step.     

Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO-). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO- generated from Angeli's salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO- and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.     

Differential sensitivity of the tyrosyl radical of mouse ribonucleotide reductase to nitric oxide and peroxynitrite

Ribonucleotide reductase is essential for DNA synthesis in cycling cells. It has been previously shown that the catalytically competent tyrosyl free radical of its small R2 subunit (R2-Y.) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate a loss of R2-Y. induced either by .NO or peroxynitrite in vitro. The .NO effect is reversible and followed by an increase in ferric iron release from mouse protein R2. A similar increased iron lability in radical-free, diferric metR2 protein suggests reciprocal stabilizing interactions between R2-Y. and the diiron center in the mouse protein. Scavenging of R2-Y. by peroxynitrite is irreversible and paralleled to an irreversible loss of R2 activity. Formation of nitrotyrosine and dihydroxyphenylalanine was also detected in peroxynitrite-modified protein R2. In R2-overexpressing tumor cells co-cultured with activated murine macrophages, scavenging of R2-Y. following NO synthase II induction was fully reversible, even when endogenous production of peroxynitrite was induced by triggering NADPH oxidase activity with a phorbol ester. Our results did not support the involvement of peroxynitrite in R2-Y. scavenging by macrophage .NO synthase II activity. They confirmed the preponderant physiological role of .NO in the process.