Regional and temporal expression patterns of interleukin-10, interleukin-10 receptor and adhesion molecules in the rat spinal cord during chronic relapsing EAE

https://doi.org/10.1016/S0165-5728(03)00031-6Get rights and content

Abstract

Adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mediate leukocyte infiltration into the CNS, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Because exogenous interleukin-10 (IL-10) inhibits ICAM-1 and VCAM-1 expression and clinical EAE, we hypothesize that endogenous IL-10 signaling may suppress expression of adhesion molecules. In a rat model of chronic relapsing EAE, expression levels of IL-10 and its receptor (IL-10R1), ICAM-1 and VCAM-1 mRNA in the spinal cord are markedly increased, whereas levels of IL-10 mRNA remain relatively low. The temporal pattern of mRNA and protein expression showed marked differences between spinal cord levels. During relapse, IL-10, IL-10R1, ICAM-1, VCAM-1 mRNA levels and neurological scores show positive correlations. We conclude that endogenous IL-10 is not a crucial factor inhibiting adhesion molecule expression in this model.

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

References (52)

  • J.E Merrill et al.

    Cytokines in inflammatory brain lesions: helpful and harmful

    Trends Neurosci.

    (1996)
  • E.B Samoilova et al.

    Acceleration of experimental autoimmune encephalomyelitis in interleukin-10-deficient mice: roles of interleukin-10 in disease progression and recovery

    Cell. Immunol.

    (1998)
  • P Shrikant et al.

    Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma in astrocytes

    J. Neuroimmunol.

    (1994)
  • N Tanuma et al.

    Competitive PCR quantification of pro- and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis

    J. Neuroimmunol.

    (1997)
  • N Tanuma et al.

    Characterization of acute versus chronic relapsing autoimmune encephalomyelitis in DA rats

    J. Neuroimmunol.

    (2000)
  • C.E Wilcox et al.

    Endothelial cell expression of the intercellular adhesion molecule-1 (ICAM-1) in the central nervous system of guinea pigs during acute and chronic relapsing experimental allergic encephalomyelitis

    J. Neuroimmunol.

    (1990)
  • D.O Willenborg et al.

    ICAM-1-dependent pathway is not critically involved in the inflammatory process of autoimmune encephalomyelitis or in cytokine-induced inflammation of the central nervous system

    J. Neuroimmunol.

    (1993)
  • D.M Wingerchuk et al.

    Multiple sclerosis: current pathophysiological concepts

    Lab. Invest.

    (2001)
  • D Wong et al.

    Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide

    J. Neuroimmunol.

    (1992)
  • B.G Xiao et al.

    Suppression of acute and protracted-relapsing experimental allergic encephalomyelitis by nasal administration of low-dose IL-10 in rats

    J. Neuroimmunol.

    (1998)
  • S Amor et al.

    Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice

    J. Immunol.

    (1994)
  • J.J Archelos et al.

    Inhibition of experimental autoimmune encephalomyelitis by an antibody to the intercellular adhesion molecule ICAM-1

    Ann. Neurol.

    (1993)
  • S.E Baranzini et al.

    Transcriptional analysis of multiple sclerosis brain lesions reveals a complex pattern of cytokine expression

    J. Immunol.

    (2000)
  • J.L Baron et al.

    Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma

    J. Exp. Med.

    (1993)
  • E Bettelli et al.

    IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice

    J. Immunol.

    (1998)
  • P.A Calabresi et al.

    Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosis treated with interferon beta-1b

    Ann. Neurol.

    (1997)
  • Cited by (47)

    • Toll-like receptor 2 and 4 antagonism for the treatment of experimental autoimmune encephalomyelitis (EAE)-related pain

      2021, Brain, Behavior, and Immunity
      Citation Excerpt :

      Upon arrival, the rats were randomly assigned to either the myelin oligodendrocyte glycoprotein (MOG) or saline group. For Experiments 1–8, the MOG assigned rats received a 4 μg injection of recombinant rat MOG1-125 (VU University Medical Center, Netherlands, gifted by Dr. Anne-Marie Van Dam) in a vehicle consisting of sodium acetate (pH = 3) and incomplete Freund’s adjuvant (Sigma; St. Louis, MO; (Ledeboer et al., 2003). This dose of MOG is a lower dose than that which we have found induces typical EAE motor symptoms, including full hindlimb paralysis/partial upper limb paralysis (i.e. 16 µg, the dose employed for Experiment 9).

    • Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone

      2021, Behavioural Brain Research
      Citation Excerpt :

      All animals were between 10–12 weeks of age at the time of injection. Motor behavior was scored on weekdays prior to (+)-NTX dosing and daily after the initiation of (+)-NTX dosing in all rats to assess the severity of their EAE symptoms as previously described [43–46]. The motor score quantified physical paralysis and scoring was based on the following designations: 0 = no signs of paralysis, 1 = partial tail paralysis, 2 = full tail paralysis, 3 = hind limb weakness, 4 = partial hind limb paralysis, 5 = full hind limb paralysis, 6 = partial upper limb paralysis.

    • Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy

      2017, Brain, Behavior, and Immunity
      Citation Excerpt :

      A single intrathecal treatment of XT-101-R or vehicle control was administered on the first day that motor deficits were observed. The rats receiving vehicle control demonstrated the relapsing remitting motor impairment (Fig. 1A) as described previously (Ledeboer et al., 2003; Loram et al., 2015; Sloane et al., 2009). However, treatment with XT-101-R significantly attenuated the clinical score (Fig. 1A and B; P < 0.05).

    • Alterations in microglial phenotype and hippocampal neuronal function in transgenic mice with astrocyte-targeted production of interleukin-10

      2015, Brain, Behavior, and Immunity
      Citation Excerpt :

      Interleukin 10 (IL-10) is one of the most crucial immunoregulatory cytokines in the periphery where in general it has anti-inflammatory functions (Couper et al., 2008; Moore et al., 2001). In the central nervous system (CNS), IL-10 expression has been reported to be upregulated under a variety of neuroinflammatory and pathological situations including traumatic brain injury (Kamm et al., 2006), excitotoxicity (Gonzalez et al., 2009), middle cerebral artery occlusion (MCAO) (Zhai et al., 1997), Alzheimer’s disease (Apelt and Schliebs, 2001), multiple sclerosis (Hulshof et al., 2002) and experimental autoimmune encephalomyelitis (EAE) (Ledeboer et al., 2003). Noteworthy, and consistent with the anti-inflammatory role attributed to IL-10 in the periphery (Couper et al., 2008; Moore et al., 2001), upregulation of IL-10 expression after EAE occurs mostly during the recovery phase (Issazadeh et al., 1995; Ledeboer et al., 2003) and in the MCAO model coincides with a decrease of pro-inflammatory mediators (Zhai et al., 1997).

    View all citing articles on Scopus
    View full text