Inflammatory cell expression of cyclooxygenase-2 in the multiple sclerosis lesion

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Abstract

Multiple sclerosis (MS) is a progressive immune-mediated disease characterized by the loss of the oligodendrocytes that constitute the myelin sheath. Recent reports show that glutamate-mediated excitotoxic death of oligodendrocytes contributes to pathogenesis in demyelinating disease. A link between the immune-mediated inflammatory response and glutamate-mediated excitotoxicity of oligodendrocytes could involve the interaction of inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2). Both enzymes are tightly coupled to neuronal excitotoxic death. We examined tissue from two controls and seven MS patients with chronic active lesions to determine the extent of COX-2 and iNOS expression. In contrast to the lack of expression in controls, there was a marked induction of COX-2 in all these MS lesions. COX-2 was frequently expressed in association with iNOS. COX-2 was found in areas that contained catabolites of myelin basic protein, indicating recent demyelination. COX-2 expression was found near damaged oligodendrocytes in cells that expressed the macrophage/microglia marker CD64, indicating that a substantial portion of the COX-2 in the lesions was expressed in immune-derived cells. We discuss these findings in the context of how COX-2 could be coupled with iNOS to contribute to excitotoxic death and damage of oligodendrocytes.

Introduction

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that often presents in young adults. This disease is characterized by the damage and loss of the oligodendrocytes (oligos) that myelinate axons and facilitate neurotransmission.

Much of the focus of earlier studies regarding MS and the animal model of this disease, experimental allergic encephalomyelitis (EAE), centered on the role of inflammatory and immune system interactions. The steps leading from the immune-mediated inflammatory response to death and damage of oligos are not known. One component thought to contribute to oligodendrocyte damage and death is nitric oxide (NO) (Mitrovic et al., 1995), a diffusible product synthesized by the inducible form of the enzyme nitric oxide synthase (iNOS).

Expression of iNOS has been observed in MS lesions in regions where NO-derived reactants such as nitrotyrosine can also be detected Bo et al., 1994, Cross et al., 1998, Hill et al., 2004, Liu et al., 2001, Oleszak et al., 1998. iNOS expression can also be seen with reactive astrocytes and immune cell infiltrates. Other reactants, myelin degradation products, myelin basic protein (MBP) fragments, and lipids are also associated with iNOS expression (Hill et al., 2004). These findings suggest that iNOS could contribute to the pathogenesis of MS. Inhibitors of iNOS can also attenuate the pathogenesis in the mouse model of Theiler's virus induced demyelinating disease (TMEV-IDD) (Rose et al., 1998) and EAE (Cross et al., 1994).

Another protein often associated with iNOS is the inducible isoform of the enzyme cyclooxygenase (COX-2). Expression of COX-2 and iNOS is tightly coupled and thought to contribute to pathogenesis in colon cancer (Rao et al., 2002), diabetes (Joussen et al., 2002), heart disease (Shinmura et al., 2002) and stroke Nogawa et al., 1997, Nogawa et al., 1998. Experimentally, nitric oxide is known to stimulate the enzymatic activity of COX in vitro (Salvemini et al., 1995) and in vivo (Marnett et al., 2000).

Three isoforms of COX have been identified; a constitutive form (COX-1), an inducible form (COX-2) (Smith et al., 1996) and a splice variant of COX-1 designated as COX-3 (Chandrasekharan et al., 2002). In the CNS, COX-2 expression is increased in neurons following seizures (Yamagata et al., 1993) and ischemia (Nogawa et al., 1997). COX-2 has also been observed in the CNS of mice in the EAE model of MS (Aloisi et al., 2000). COX-2 catalyzes the initial reactions in the synthesis of prostanoids (PGs), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2α (PGF), prostacyclin (PGI2), and thromboxane (TxA2) from arachidonic acid (Smith et al., 1996). Reactive oxygen species (ROS) are also generated by COX as a side-product in the synthesis of prostanoids (Smith et al., 1996). Expression and activity of COX-2 is associated with loss of neurons in animal models of stroke (Nogawa et al., 1997) and is tightly linked to iNOS activity (Nagayama et al., 1999). Inhibitors of COX-2 that are neuroprotective against ischemic insult in wild-type mice, have no effect in iNOS knockout mice (Nagayama et al., 1999). Therefore, COX-2 and iNOS can act in synergy to facilitate excitotoxicity.

In this study, we examined the expression of COX-2 in MS lesions and determined the extent to which this enzyme is found associated with iNOS. Our findings are discussed in the context of how the expression of iNOS and COX-2 could contribute to loss and damage of oligos, which has recently been shown to occur through glutamate-mediated excitotoxicityPitt et al., 2000, Smith et al., 2000, Werner et al., 2000.

Section snippets

CNS tissue

MS brain tissues were obtained from the Rocky Mountain MS Center (Englewood, CO; courtesy of Dr. Ron Murray) and normal brain tissues were obtained from the National Neurological Research Specimen Bank at UCLA (courtesy of Dr. Wallace Tourtellotte). The tissues used in this research are exempt from IRB review as defined by DHHS federal regulation 45CFR46. The MS specimens that were requested from the tissue banks were chosen on the basis of having significant inflammation around plaques as

Expression of COX-2 and iNOS in MS lesions

We have demonstrated that iNOS is present in the active edges of MS demyelinating lesions and is prominent in regions of active inflammation (Fig. 1) (Hill et al., 2004). In addition, iNOS is predominantly produced in cells co-labeled with markers including CD64, the high affinity Fcγ receptor associated with phagocytotic macrophages (Dougherty et al., 1987). A subset of these cells showed reactivity for MBP fragment, consistent with recent phagocytosis of myelin (Groome et al., 1988). These

Discussion

In this study, we observed that COX-2 was extensively expressed in MS lesions. COX-2 was frequently expressed in association with iNOS, which is thought to contribute to the progression of MS. Co-association of COX-2 and iNOS was found near regions showing active demyelination suggesting that these inducible enzymes involved in inflammation could contribute to the pathology of MS.

We observed that active phagocytic macrophages/microglia (CD64+) expressed COX-2 in the MS lesion. CD64, which is

Acknowledgments

We would like to thank Ikuo Tsunoda, James Burns and Robert Fujinami for their helpful comments in reading the manuscript. This work is supported by a VA Merit grant to JWR and a grant from the National Multiple Sclerosis Society to JWR and NGC.

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