Regional and temporal expression patterns of interleukin-10, interleukin-10 receptor and adhesion molecules in the rat spinal cord during chronic relapsing EAE
Introduction
Multiple sclerosis (MS) is a chronic inflammatory disease characterized by pronounced focal inflammation mainly in the white matter of the brain and spinal cord, destruction of myelin, and axonal and neuronal degeneration. Clinical symptoms often follow a relapsing-remitting time course Steinman, 2001, Wingerchuk et al., 2001. Therefore, rat models of chronic relapsing experimental autoimmune encephalomyelitis (cr-EAE) have been developed. cr-EAE can be induced by immunization of Dark Agouti (DA) rats with myelin oligodendrocyte glycoprotein (MOG), and is characterized by multiple phases of neurological deficits and recovery, the presence of perivascular infiltrates in the CNS mainly in the spinal cord, and demyelination. The neuropathology as seen in this model closely resembles that seen in MS patients (Storch et al., 1998).
Early events in the pathogenesis of MS and EAE are breakdown of the blood–brain barrier (BBB), and multifocal infiltration of activated immune cells, mainly T cells and monocytes, into the brain and spinal cord. Adhesion of circulating leukocytes to the cerebral endothelium and their subsequent migration across the BBB is regulated by various adhesion molecules that are expressed on endothelial cells, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which interact with their respective ligands on leukocytes, leukocyte function-associated antigen-1 (LFA-1), and very late antigen-4 (VLA-4) (Archelos et al., 1999).
ICAM-1 has been found in active MS lesions Sobel et al., 1990, Cannella and Raine, 1995 and is upregulated on cerebral endothelial cells during acute and chronic EAE, in particular during active phases of disease Wilcox et al., 1990, Cannella et al., 1991, O'Neill et al., 1991. In acute EAE, it appears to play an important role as administration of antibodies against ICAM-1 effectively reduces CNS leukocyte infiltration and clinical severity Archelos et al., 1993, Willenborg et al., 1993. Also, VCAM-1 is expressed on cerebral endothelial cells in MS and EAE lesions Dopp et al., 1994, Steffen et al., 1994, Cannella and Raine, 1995. The involvement of the VCAM/VLA-4 pathway in acute EAE is further supported by the suppression of neurological symptoms and neuropathology by antibodies against VLA-4 Yednock et al., 1992, Baron et al., 1993, Kent et al., 1995, van der Laan et al., 2002.
The infiltrated lymphocytes and macrophages, but also resident microglial and astroglial cells, produce cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, chemokines and other inflammatory mediators (e.g. nitric oxide, reactive oxygen species) which may contribute to CNS tissue damage (Merrill and Benveniste, 1996). Inflammatory cytokines may further contribute to the pathogenesis of MS and EAE by inducing the expression of adhesion molecules on endothelial cells and glial cells Wong and Dorovini-Zis, 1992, De Vries et al., 1994, Shrikant et al., 1994, Shrikant et al., 1995.
The mechanisms regulating disease progression in MS and EAE are poorly understood. The immunoregulatory cytokine interleukin-10 (IL-10) is considered to play an inhibitory role in the pathogenesis of MS and EAE. This is based on the finding that IL-10 can suppress the expression of inflammatory mediators by activated immune cells and glial cells Ledeboer et al., 2000, Moore et al., 2001, and the spontaneous recovery as seen in acute monophasic EAE models is associated with elevated expression of IL-10 mRNA Kennedy et al., 1992, Issazadeh et al., 1995. However, administration of recombinant IL-10 in various EAE models has generated conflicting results regarding the outcome of disease Rott et al., 1994, Cannella et al., 1996, Genain et al., 1996, Xiao et al., 1998, as did targeted delivery of the IL-10 gene or protein to the CNS Mathisen et al., 1997, Shaw et al., 1997, Croxford et al., 1998, Croxford et al., 2001, Cua et al., 2001. Nonetheless, IL-10-deficient mice are more susceptible to EAE, develop a more severe disease, and show no spontaneous remission as compared to wild-type controls Bettelli et al., 1998, Samoilova et al., 1998. Conversely, IL-10 transgenic mice are resistant to the development of EAE Bettelli et al., 1998, Cua et al., 1999. Taken together, these data are consistent with the view that endogenous IL-10 signaling is involved in suppressing the onset and/or progression of the disease.
A putative mechanism of action whereby IL-10 influences the ongoing inflammatory cascade in the CNS is suppression of cytokine-induced expression of adhesion molecules on cerebral endothelial cells and glial cells, as shown in vitro Krakauer, 1995, Shrikant et al., 1995. In support of this, IL-10-deficient mice show an upregulation of brain endothelial VCAM-1 expression (Sans et al., 2001). In the present study, we investigated whether endogenous expression of IL-10 and/or IL-10 signaling in the CNS is associated with decreased local expression of adhesion molecules in EAE in vivo. Thereto, we assessed the endogenous expression of IL-10 and its receptor (IL-10R1, the ligand-binding chain), ICAM-1 and VCAM-1 in the spinal cord of rats suffering from cr-EAE. Because cytokine expression and histopathology in the spinal cord show regional differences in this model (Wierinckx et al., personal communication), expression of these genes and their gene products was studied in several spinal cord regions of rats in different phases of disease. Furthermore, we correlated the individual expression levels of these genes with each other and with the severity of neurological symptoms to elucidate possible gene interactions determining the chronic relapsing disease course as seen in this model.
Section snippets
Animal model of cr-EAE
Adult male DA rats (240–290 g; Harlan, Horst, The Netherlands) were used for the induction of cr-EAE. Recombinant protein corresponding to the N-terminal sequence of rat MOG (amino acids 1–125) was expressed in Escherichia coli and purified to homogeneity by Ni-chelate chromatography using a 6-His tag (Amor et al., 1994). The purified protein, dissolved in 6 M of urea, was dialyzed against 10 mM of sodium acetate buffer (NaAc; pH 3.0) to obtain a soluble preparation that was stored at −80 °C.
Neurological symptoms
The clinical course of cr-EAE is shown in Fig. 1. All rats immunized with rat recombinant MOG developed neurological symptoms of EAE (100% incidence), consisting of progressive weight loss, limp tail and moderate to severe paraparesis or paraplegia. First neurological symptoms were observed 9.4±0.2 days p.i. (mean±S.E., n=35). All rats gradually recovered from the first disease phase (lowest score reached at day 14.1±0.3 p.i., n=23), followed by a relapse with mean day of onset 17.2±0.5 p.i. (n
Discussion
In the present study, we demonstrate that cr-EAE in rats is associated with a marked upregulation of mRNA expression levels for IL-10 receptor and adhesion molecules ICAM-1 and VCAM-1 in the spinal cord, and a more limited upregulation of IL-10 mRNA levels. Unexpectedly, expression levels of IL-10 and IL-10 receptor mRNA correlated positively with adhesion molecule expression at the different phases of cr-EAE studied. Furthermore, marked spatiotemporal differences in expression levels were
Acknowledgements
The authors wish to thank Drs. C. Linington and A. Stefferl (Max Planck Institute for Neurobiology, Department of Neuroimmunology, Martinsried, Germany) for the kind gift of the rrMOG1–125 plasmid and valuable help with MOG preparation. The authors also thank Dr. A.B. Smit (Research Institute Neurosciences, Department of Molecular Neurobiology, Faculty of Earth and Life Sciences, Free University Amsterdam) for allowing the use of the ABI PRISM 7700 Sequence Detection System. This work was
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