Methadone diminishes neuroinflammation and disease severity in EAE through modulating T cell function

https://doi.org/10.1016/j.jneuroim.2012.10.015Get rights and content

Abstract

Methadone is known to exert modulatory effects on the immune system. We investigated the potential effects of methadone on infiltration of inflammatory cells into the spinal cord, as well as the proliferative and cytokine responses of T cells in MOG35–55-induced experimental autoimmune encephalomyelitis in mice. Methadone significantly suppressed clinical signs of the disease and level of inflammatory cytokines (p < 0.05) produced by T cells. Moreover, invasion of inflammatory cells into the spinal cord was significantly decreased by methadone (p < 0.05). Our data point to therapeutic effects of methadone and highlight the beneficial role of opioid receptor signaling in the context of autoimmune neuroinflammation.

Introduction

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS). Neuropathologically, MS is characterized by leukocyte infiltration of the CNS white matter, leading to local demyelination and axonal loss. The inflammatory infiltrate in MS is mainly composed of T cells and monocyte-derived macrophages, together with locally activated microglia. While modifying disease symptoms, currently available therapies have failed to cure MS and efforts continue to develop therapies targeting the chief underlying pathogenic processes. Numerous studies have shown that T cells play a crucial role in MS pathogenesis and hence, a large body of work has focused on strategies to module T cell function during disease process (Hemmer et al., 2002, McFarland and Martin, 2007, Zozulya and Wiendl, 2008). Experimental autoimmune encephalomyelitis (EAE), a well known animal model of MS, recapitulates some of the pathogenic and clinical features of MS, and it has been widely used in developing new treatments for the disease (Steinman and Zamvil, 2005, Mix et al., 2008).

Different opioids have been reported to exert immunosuppressive and modulatory effects on a variety of immune cells including T cells, macrophages and NK cells (Govitrapong et al., 1998, Gomez-Flores and Weber, 2000, McCarthy et al., 2001, Budd, 2006, Sacerdote, 2006). Moreover, they have been shown to diminish the levels of inflammatory cytokines including IL-2, IFN-γ and TNF-α while enhancing the levels of anti-inflammatory cytokines like IL-4 during immune/inflammatory processes. The overall effect of opioids on the immune system is to shift immune responses from Th1 to Th2 (Roy et al., 2001, Roy et al., 2004). Similar to other opioids, methadone, a potent μ-opioid receptor agonist, has been demonstrated to have immunosuppressive/modulatory effects (Li et al., 2002, Amirshahrokhi et al., 2008, Toskulkao et al., 2010). Despite these findings, there is limited information with regard to the role of methadone in MS disease process. Herein, we investigated the effects of methadone on MS-related neuroinflammation, using MOG-induced EAE animal model.

Section snippets

Materials

Mice were purchased from Pasture Institute, Iran. RPMI cell culture medium, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Germany). Hooke kit for EAE induction [myelin oligodendrocyte glycoprotein (MOG)35–55/Complete Freund's Adjuvant emulsion (CFA) and pertussis toxin (PTX):5 ×] and MOG35–55 for in vitro stimulation of cells were purchased from Hooke laboratories (Hooke laboratories, EK-0115, Lawrence, MA, USA). Reagents required for

Methadone administration ameliorates clinical signs of EAE

Effects of methadone administration on clinical signs and severity of EAE were evaluated from 7th to 35th day after immunization by scoring animals in each group and mean daily clinical score of the disease and CDI were calculated. Daily clinical score of the disease was inhibited significantly by methadone at effector phase of the disease (p < 0.05, days 22–35 after immunization, Fig. 1a). Methadone-treated animals showed significantly lower EAE disease severity as measured by CDI, 28.77 ± 3.5

Discussion

The results of this study demonstrate that methadone has regulatory effects on EAE disease process. Methadone diminished the clinical severity of EAE, an effect that was associated with the modulation of the immune system activity. The inhibitory effects of methadone started at the effector phase of the disease and continued to the chronic phase. It is notable that methadone suppressed EAE at the chronic phase but not at the peak of the disease. This finding might indicate that the potential

Acknowledgments

This work was supported by the grant from Tehran University of Medical Sciences and facilities at the Shefa Neuroscience Research Center. The authors would like to thank Professor Ali Gorji. This work has never been done without his support. We are also grateful of Dr Farshid Noorbakhsh's scientific support and help throughout the whole experiments.

References (26)

  • T. Toskulkao et al.

    Alteration of lymphocyte opioid receptors in methadone maintenance subjects

    Neurochem. Int.

    (2010)
  • A.L. Zozulya et al.

    The role of CD8 suppressors versus destructors in autoimmune central nervous system inflammation

    Hum. Immunol.

    (2008)
  • P. Govitrapong et al.

    Alterations of immune functions in heroin addicts and heroin withdrawal subjects

    J. Pharmacol. Exp. Ther.

    (1998)
  • Cited by (24)

    • Diosgenin ameliorates cellular and molecular changes in multiple sclerosis in C57BL/6 mice

      2021, Multiple Sclerosis and Related Disorders
      Citation Excerpt :

      For this purpose, the level of inflammation was scored according to the following criteria: 0, no inflammation; 1, cellular infiltrates only around blood vessels and meninges; 2, mild cellular infiltrates in the parenchyma (1–10/section); 3, moderate cellular infiltrates in the parenchyma (11–100/section); and 4, severe cellular infiltrates in the parenchyma (> 100/section) (Zhang et al., 2014). Furthermore, white matter demyelination was scored in LFB-stained sections as 1 for normal myelination, 2 for mild or minor demyelination (> 50% myelin staining preserved), or 3 for moderate to severe demyelination (< 50% myelin staining preserved), as reported before (Kafami et al., 2013). In addition, the axonal loss was assessed using the following scale: 0, no loss; 1, a few foci of superficial loss involving less than 25% of tissue; 2, foci of deep axonal loss, encompassing over 25% of tissue; and 3, diffuse and widespread axonal loss (Yin et al., 2010).

    • Paricalcitol improves experimental autoimmune encephalomyelitis (EAE) by suppressing inflammation via NF-κB signaling

      2020, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      LPS and CD3/CD28 T cell activator were obtained from Sigma Aldrich (USA) and STEMCELL Technologies (Canada), respectively, to stimulate the cells. Clinical calculation was analyzed according to the clinical signs of neurological deficits showing as followings: grade 0, no abnormality; grade 1, complete tail paralysis; grade 2, complete tail paralysis and hind limb weakness; grade 3, complete hind limb paralysis; grade 4, tetraplegia; grade 5, moribund state or death [22]. Protein samples were collected from tissues or cells using Whole Cell Lysis Assay or Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN BioTECH, China), followed by centrifugation (13,000 g) for 15 min at 4 °C.

    • Immunotoxicology of Drugs of Abuse

      2018, Comprehensive Toxicology: Third Edition
    • S-allyl cysteine improves clinical and neuropathological features of experimental autoimmune encephalomyelitis in C57BL/6 mice

      2018, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Mice were daily assessed to record behavioral and neurological signs until day 18 after the first immunization. The signs of EAE were scored as reported before [26,27] as follows: 0, no sign; 0.5, half paralyzed tail; 1, fully paralyzed tail; 1.5, fully paralyzed tail and weak or altered gait; 2, fully paralyzed tail and hind limb weakness; 2.5, unilateral hind limb paralysis; 3, complete hind limb paralysis; 3.5, complete hind limb paralysis and forelimb weakness; 4, complete paralysis; 5, moribund state or death. On the 18th day, after last behavioral assessment, spinal cords were isolated and collected for further biochemical and pathological studies.

    • Piperlongumine attenuates experimental autoimmune encephalomyelitis through inhibition of NF-kappaB activity

      2017, Free Radical Biology and Medicine
      Citation Excerpt :

      A masked investigator examined and scored mice daily for clinical signs of neurological deficit according to the following scale: grade 0, no abnormality; grade 0.5, partial tail paralysis; grade 1, complete tail paralysis; grade 1.5, complete tail paralysis and clumsy gait; grade 2, complete tail paralysis and hind limb weakness; grade 2.5, unilateral hind limb paralysis; grade 3, complete hind limb paralysis; grade 3.5, complete hind limb paralysis and fore-limb weakness; grade 4, tetraplegia; grade 5, moribund state or death. Data were plotted as daily mean clinical score for all animals in a particular treatment group, including the scores of asymptomatic mice (score=0) [23–25]. All other analyses were carried out on the 29th day.

    • Interaction between the protective effects of cannabidiol and palmitoylethanolamide in experimental model of multiple sclerosis in C57BL/6 mice

      2015, Neuroscience
      Citation Excerpt :

      Mice were daily assessed to record behavioral and neurological signs until 28 days after the first immunization. The signs of EAE were scored as follows: 0, no signs; 0.5, half paralyzed tail; 1, fully paralyzed tail; 1.5, fully paralyzed tail and weak or altered gait; 2, fully paralyzed tail and hind limb weakness; 2.5, unilateral hind limb paralysis; 3, complete hind limb paralysis; 3.5, complete hind limb paralysis and fore-limb weakness; 4, complete paralysis; 5, moribund state or death (Kafami et al., 2013). On twenty-eighth day, spinal cords were collected for further pathological and gene expression studies.

    View all citing articles on Scopus
    1

    Both Laya Kafami and Ifa Etesami contributed equally to the work.

    View full text