Extensive peroxynitrite activity during progressive stages of central nervous system inflammation
Introduction
Free-radical production is thought to be involved in inflammatory processes (Winrow et al., 1993), both exacerbating inflammation and effecting tissue damage. Central nervous system (CNS) tissue is particularly vulnerable to oxidative damage (Konat and Wiggins, 1985; Fisher et al., 1993), suggesting that this could be an important factor in the pathogenesis of CNS inflammation in multiple sclerosis (MS) and its model experimental autoimmune encephalomyelitis (EAE) (Weiner and Hafler, 1988). Several reports indirectly implicate free-radical involvement in autoimmune CNS inflammation. For instance, antioxidant treatment ameliorates EAE (Ruuls et al., 1995; Hansen et al., 1995).
The free-radical species that has been most convincingly linked to MS or EAE activity is nitric oxide (NO), through studies on inducible NO synthase (iNOS) or its inhibitors (Merrill et al., 1993; Ludowyk et al., 1993; Bo et al., 1994, Cross et al., 1994; Zhao et al., 1996). Direct measurement of NO levels in CNS revealed that during inflammation, the NO concentration was increased substantially (Hooper et al., 1995). Furthermore, encephalitogenic T-cells stimulate NO production by macrophages (Misko et al., 1995). Although NO has been implicated in glial cell damage (Mitrovic et al., 1994), it is improbable for NO itself to be the major effector during CNS inflammation and tissue damage, since it displays low toxicity (Beckman, 1994). NO may even protect against lipid peroxidation (Hogg et al., 1993), or be involved in immunoregulation (Matthys et al., 1995). On the other hand, NO reacts strongly with the free radical superoxide (O2−) to form the powerful oxidant peroxynitrite (ONOO−), at a rate constant which is three times faster than the rate at which superoxide dismutase (SOD) scavenges superoxide (Beckman et al., 1994; Beckman, 1994; Miller et al., 1995; Szabo et al., 1995). Elevated production of both superoxide and NO by inflammatory cells during EAE has been reported (MacMicking et al., 1992), thus setting the stage for peroxynitrite formation.
Peroxynitrite may decay homolytically into the most reactive oxygen free radical species known, the hydroxyl radical. However, the oxidizing reactions of peroxynitrite itself appear more damaging to tissues, because these reactions occur much faster than the hydroxyl formation (Beckman, 1994). Peroxynitrite induces lipid peroxidation and may thus be involved in demyelination. It also efficiently oxidizes DNA and proteins, thereby damaging cells, and is far more toxic than NO (Zingarelli et al., 1996). NO-induced neuronal damage is also mediated by peroxynitrite (Ohkuma et al., 1995). Nitrotyrosine (NT) formation is used as a footprint of peroxynitrite activity. Here we demonstrate that NT formation occurs extensively in the CNS during progressive stages of inflammatory disease, which suggests a role for peroxynitrite during progressive disease activity, as discussed.
Section snippets
Disease induction
`Hyperacute' EAE was induced by a combined transfer and immunization procedure using SJL/J mice (Jackson Laboratories, Bar Harbor, Maine) at 8–12 weeks of age. Spleen cells from mice immunized 7–10 days earlier with 1 mg of mouse spinal cord homogenate emulsified in complete Freund's adjuvant, were cultured in the presence of the synthetic peptide corresponding to the amino-acid sequence 139–151 of myelin proteolipid protein at 1 μM. After 10 days, 5×107 resting mononuclear cells were injected
Results
The Ab specific for NT recognizes proteins with peroxynitrite-induced nitrated tyrosine residues. We used this Ab to examine whether peroxynitrite activity can be identified during acute CNS inflammation.
Discussion
The studies described here demonstrate a widespread distribution of NT formation in the CNS during active stages of EAE. Since NT formation occurs mainly through peroxynitrite, not NO or NO2 (Beckman et al., 1992), these studies indicate that peroxynitrite formation through the reaction of NO with superoxide is very prominent during the progressive stages of disease. On the other hand, less peroxynitirite activity is observed during disease regression, even when infiltration and clinical
Acknowledgements
The authors are grateful for the skillful assistance of L. Pen, A. Gilmore, W. Wei and S.Q. Garcia, and for the helpful discussions with Dr. Jean E. Merrill. Supported in part by NMSS grant PP0455.
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