Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADPribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS ؊/؊ ) and corresponding wild-type animals (PARS ؉/؉ ), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH 2 BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Nitric oxide (NO), superoxide, and their cytotoxic reaction product peroxynitrite (ONOO Ϫ ) are terminal mediators of cellular injury in various forms of inflammation. In vitro studies employing conventional inhibitors of the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30) suggested that the oxidative injury in response to oxy radicals and peroxynitrite is related to DNA single-strand breakage and consequent activation of PARS (1, 2). Massive ADPribosylation of nuclear proteins by PARS then results in cellular energy depletion and injury, reminiscent of necrosis (1-3). However, objections can be raised against the conclusions of these studies, because the commonly used relatively high concentrations of PARS inhibitors (e.g., nicotinamide and benzamide analogs), have additional effects as free radical scavengers, and have short cellular residence time (4-6).More recently a potent pharmacologically active inhibitor of PARS, the lipophilic 6-iodo-5-amino-1,2-benzopyrone (INH 2 BP), was developed (7, 8). Moreover, a genetically engineered mouse line that lacks PARS is now available: a fibroblast cell line from these animals can be used for in vitro investigations (9). These tools allow a direct testing of the role of PARS. The present work was designed to elucidate (i) whether inhibition of PARS by INH 2 BP protects against cellular oxidant injury triggered by peroxynitrite, a cytotoxic oxidant produced by the reaction of superoxide and NO (10-14); (ii) whether the PARS The results of the current study support the role of PARS activation in the peroxynitrite-mediated cellular oxidant injury and inflammation, and they demonstrate that either deletion of PARS or its selective inhibition by INH 2 BP protects against inflammatory cell injury.

SUMMARYThe mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 m) of peroxynitrite led to apoptosis, whereas higher concentrations (50 m) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS ) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H 2 O 2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energyconserving step, enabling the cell to complete the process of apoptosis.

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO−), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO− or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO−-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO−, or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.

1 Ligands of the various adenosine receptor subtypes modulate the production of pro-and antiin¯ammatory cytokines. Here we evaluated the e ect of adenosine and various ligands of the adenosine receptor subtypes (A 1 , A 2 , A 3 ) on the chemokine macrophage in¯ammatory protein (MIP) 1a production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A 3 adenosine receptor agonist inhibits MIP-1a production and a ects the course of in¯ammation in collagen-induced arthritis. 2 In the cultured macrophages, the A 3 receptor agonist N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and, less potently, the A 2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS; 1 ± 200 mM) dose-dependently suppressed the production of MIP-1a. The selective A 1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1 ± 200 mM) was ine ective, and adenosine was a weak inhibitor. The inhibition of MIP-1a production by the A 3 and A 2 agonist was associated with suppression of its steady-state mRNA levels. 3 Based on the in vitro data, we concluded that activation of A 3 , and to a lesser extent A 2 adenosine receptors suppresses MIP-1a expression. Since IB-MECA was the most potent inhibitor of MIP-1a expression, we next investigated whether it a ects the production of other pro-in¯ammatory mediators. We observed that IB-MECA (1 ± 300 mM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4 Since MIP-a is a chemokine which enhances neutrophil recruitment into in¯ammatory sites, we investigated whether the A 3 agonist IB-MECA a ects the course of in¯ammation, MIP-a production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint in¯ammation. IB-MECA inhibited the formation of MIP-1a, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil in®ltration. 5 We conclude that adenosine receptor agonists, most notably the A 3 agonist IB-MECA suppress the production of MIP-a, and exert anti-in¯ammatory e ects. Therefore, stimulation of adenosine receptor subtypes A 3 and A 2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic in¯ammatory disorders.