Bilirubin as a potent antioxidant suppresses experimental autoimmune encephalomyelitis: implications for the role of oxidative stress in the development of multiple sclerosis

Abstract

Increasing evidence shows that oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In recent years, bilirubin has been demonstrated to be a potent antioxidant in vitro. In this study, we administered bilirubin to rats with acute and chronic EAE. Bilirubin prevented both acute and chronic EAE effectively. More significantly, bilirubin suppressed ongoing clinical EAE and halted EAE progression when given after disease onset. Subsequent histological examination showed that if administered to rats before the onset of EAE, bilirubin interfered with the invasion of inflammatory cells into the central nervous system (CNS) because it protected the blood–brain barrier (BBB) from free radical-induced permeability changes. However, in some cases, inflammation still occurred even when no clinical illness was observed. In rats with treatment initiated after the onset of EAE, despite the clinical improvements, treatment with bilirubin did not reduce the degree of CNS inflammation, or change cytokine expression in CNS lesions, indicating a lack of immunosuppressive effect of this treatment. By contrast, bilirubin treatment significantly alleviated oxidative damage in the spinal cord, and the clinical signs of EAE correlated well with the degree of oxidative injury in the lesions. Our results suggest that free radicals play an important role in the final effector stages of EAE, and that antioxidant therapies may have potential for the treatment of MS.

Introduction

Multiple sclerosis (MS) is believed to be a T cell-mediated autoimmune inflammatory disease of the central nervous system (CNS) (Raine, 1994). Despite the progress in recent years, a therapy that halts the disease is not yet available. Experimental autoimmune encephalomyelitis (EAE) shares similar neuropathology and immunologic dysfunctions with MS, and serves as an animal model of this disease. The pathology of EAE is characterized by the loss of blood–brain barrier (BBB) integrity, CD4⁺ T cell and macrophage infiltration of CNS, and demyelination with various degrees of axonal damage Martin and McFarland, 1995, Ferguson et al., 1997. The pathogenic T cells initiate a cascade of proinflammatory cytokines, which activate microglia, recruit additional inflammatory cells including macrophages, and lead to the disease Raine, 1994, Martin and McFarland, 1995. Although it is becoming increasingly clear that CD4⁺ T cells play a central role in the induction of EAE, the exact sequence of events as well as the molecular mediators of myelin destruction is not completely understood.

Recent studies suggest that oxidative stress is important in the etiology of EAE, and that it contributes directly to CNS damage Benveniste, 1997, Smith et al., 1999. It has been shown that CNS cells, notably oligodendrocytes and neurons, are highly vulnerable to oxidative damage Smith et al., 1999, Hollensworth et al., 2000. Free radicals are produced massively both in MS and EAE (Smith et al., 1999). Their consequence, the oxidative damage to membrane lipids, proteins, and DNA of cells, has been demonstrated in MS and EAE lesions Toshniwal and Zarling, 1992, Vladimirova et al., 1998, Lu et al., 2000. It has also been indicated that free radicals are required for the phagocytosis of myelin by macrophages (van der Goes et al., 1998), which represents the final pathway for myelin removal and degradation. Recently, we demonstrated that heme oxygenase-1, a potent endogenous antioxidant enzyme, was induced and played an important protective role in EAE (Liu et al., 2001). Although the targeted induction of heme oxygenase-1 overexpression by hemin was only partially effective in the treatment of this disease, the inhibition of this enzyme by tin mesoporphyrin was fatal to all the treated rats. This result suggested that oxidative stress plays an important role in the development of EAE, and that antioxidant treatment might prove to be an effective therapy for EAE.

In recent years, bilirubin has been demonstrated to be a powerful antioxidant substance in vitro. Bilirubin and its serum albumin complex are superoxide scavengers and peroxyl radical-trapping antioxidants (Marilena, 1997). Bilirubin suppresses oxidation more strongly than many other antioxidants, including α-tocopherol (Vitamin E), SOD, and catalase, especially under pathological conditions Stocker et al., 1987, Wu et al., 1991. However, it has never been used for treatment in vivo. In this study, we demonstrate that bilirubin significantly reduces the clinical signs of EAE when administered both before and after the onset of the disease. Furthermore, we show that free radicals play an important role in the etiology of EAE, and may represent the final effectors of demyelination.