Peroxynitrite formation within the central nervous system in active multiple sclerosis

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Abstract

Peroxynitrite, generated by the reaction of nitric oxide (NO) with superoxide at sites of inflammation, is a strong oxidant capable of damaging tissues and cells. Detection of nitrotyrosine (NT) at inflammatory sites serves as a biochemical marker for peroxynitrite-mediated damage. In this study, NT was detected immunohistochemically within autopsied CNS tissues from six of nine multiple sclerosis (MS) patients, and in most of the MS sections displaying inflammation. Nitrite and nitrate, the stable oxidation products of NO and peroxynitrite, respectively, were measured in cerebrospinal fluid samples obtained from MS patients and controls. Levels of nitrate were elevated significantly during clinical relapses of MS. These data suggest that peroxynitrite formation is a major consequence of NO produced in MS-affected CNS and implicate a role for this powerful oxidant in the pathogenesis of MS.

Introduction

The free radical nitric oxide (NO), generated by the isoform of nitric oxide synthase whose expression is inducible by inflammatory cytokines (iNOS or NOS II), is thought to be produced in lesions of multiple sclerosis (MS) (Bo et al., 1994; Brosnan et al., 1994; Bagasra et al., 1995; De Groot et al., 1997). In addition, iNOS and increased levels of NO have been found in the central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a commonly used animal model for MS (Lin et al., 1993; Hooper et al., 1995). The exact role of NO in these disorders is not fully understood as the inhibition of iNOS has been shown to either protect against or exacerbate EAE, depending upon the choice of inhibitor, the dosing regimen and the animal species used (Cross et al., 1994; Zielasek et al., 1995; Zhao et al., 1996; Brenner et al., 1997; Hooper et al., 1997). The pharmacologic and biochemical evidence in vivo for a pathologic role of NO is supported by the in vitro demonstration of NO cytotoxicity towards oligodendroglia (Merrill et al., 1993).

The toxicity of NO is greatly enhanced when it combines with superoxide (SO) to generate peroxynitrite anion (ONOO–), in a rapid reaction that occurs at diffusion-controlled rates (Radi et al., 1991; Huie and Padmaja, 1993; Pryor and Squadrito, 1995). Protonation transforms peroxynitrite into a powerful oxidant (HOONO) capable of damaging protein, lipid, intact membranes, and DNA. In fact, many of the toxic effects formerly attributed to excess NO have now been shown to be mediated by peroxynitrite (Castro et al., 1994; Hausladen and Fridovich, 1994). Activated macrophages, abundant in lesions of MS, are a potential source of SO via oxidative metabolism, as are activated endothelial cells, B lymphocytes, and astrocytes (Maly et al., 1989; Heales et al., 1994; Kooy and Royall, 1994; Kobayashi et al., 1995). Inducible NOS is expressed by astrocytes and microglia/macrophages in MS tissues (Bo et al., 1994; Brosnan et al., 1994; Bagasra et al., 1995). In addition, human glial cell cultures containing both astrocytes and microglia have been shown to generate peroxynitrite following exposure to the combination of pro-inflammatory cytokines, IL-1β and interferon gamma (Ding et al., 1997). We hypothesized that peroxynitrite might be generated during the inflammatory response of developing MS lesions.

To test this hypothesis, two complementary methods were used to detect and semi-quantitate the generation of peroxynitrite within the CNS of MS patients. The first involved the detection of nitrotyrosine (NT), a relatively specific and stable biochemical marker for peroxynitrite (Beckman et al., 1994; Haddad et al., 1994b; Crow and Ischiropoulos, 1996). Frozen CNS sections obtained at autopsy from nine patients with MS, and five control patients were examined immunohistochemically for the presence of NT. NT immunoreactivity was observed in sections from six of the nine definite MS CNS tissues examined, and in 10 of 14 CNS sections displaying inflammation or active demyelination.

Since the protonated form of peroxynitrite (HOONO) decomposes predominantly to form nitrate, the second method for assessing peroxynitrite production in the CNS was to measure nitrate levels in cerebrospinal fluid (CSF) samples from 30 MS patients and 48 control subjects (Ignarro et al., 1993; Stern et al., 1996). CSF levels of nitrate were significantly elevated in association with clinical relapses of MS. Thus, our studies provide strong evidence for the production of peroxynitrite in the CNS of MS patients with active disease, implicating it in the pathogenesis of MS.

Section snippets

Human autopsy specimens

Fresh CNS tissue (brain, optic nerves, brainstem and spinal cord) was examined and flash-frozen and embedded in Optimal Cooling Temperature (O.C.T.) compound (Miles, Elkhart, IN) at the time of autopsy. When MS lesions were identified by gross inspection, these were sampled. All MS patient autopsies derived from patients classified clinically as secondary chronic progressive, with the exception of case #76 which was primary progressive (Lublin and Reingold, 1996). Control subject CNS was

Immunohistochemical detection of nitrotyrosine in CNS tissues

Twenty-one regions of the CNS from nine definite MS patients, and 16 regions of the CNS from five non-MS control subjects (one of whom was diagnosed in life with MS, but had an arteriopathy with leukoencephalopathy at autopsy) were examined in blinded fashion for evidence of NT immunoreactivity. The post-mortem times prior to tissue acquisition ranged from 3.5 h to 10 h for the MS patients and from 3 h to 7 h for the control subjects. Of the 21 areas of CNS from the nine MS patients studied, 11

Discussion

The present studies provide strong evidence for the generation of peroxynitrite in the CNS of MS patients during active disease. Using either of two complementary methods (nitrotyrosine detection in MS lesions, and nitrate assays of CSF), there was an association of disease activity with the production of peroxynitrite. This information confirms and expands the previous report of NT immunostaining in brain samples of two MS patients (Bagasra et al., 1995). Several groups of investigators have

Conclusion

The present studies provide evidence for the generation of peroxynitrite in the CNS of MS patients. Importantly, generation of peroxynitrite was associated with activity of disease, being observed predominantly in the setting of inflammation and active demyelination. Although it remains to be demonstrated what roles peroxynitrite might play in MS disease pathogenesis, based upon its known actions it is likely that peroxynitrite contributes to tissue destruction and cellular injury. The present

Acknowledgements

The authors thank Manuel San and Denise Dorsey for excellent technical assistance and Drs. John L. Trotter, Jeri-Anne Lyons, Michael K. Racke and Amy Lovett-Racke for critical discussions. The Nikon photographic microscope used was purchased with a grant from the Multiple Sclerosis Foundation. This work was supported by grants from the National Multiple Sclerosis Society (RG-2621 and RG 2934 to A.H.C.) and Monsanto.

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