Evidence that opioids may have toll-like receptor 4 and MD-2 effects

https://doi.org/10.1016/j.bbi.2009.08.004Get rights and content

Abstract

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Introduction

Recent evidence indicates that glia can exhibit proinflammatory responses to opioids, contributing to a reduced opioid analgesia and development of tolerance and dependence, albeit by a previously uncharacterized mechanism (Hutchinson et al., 2007, Watkins et al., 2005). In vivo opioid-induced proinflammatory glial activation has been inferred from: (a) morphine-induced upregulation of microglial (Cui et al., 2008, Hutchinson et al., 2009) and astrocytic (Hutchinson et al., 2009, Song and Zhao, 2001) activation markers, (b) morphine-induced upregulation and/or release of proinflammatory cytokines (Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c, Hutchinson et al., 2009, Johnston et al., 2004, Raghavendra et al., 2002, Raghavendra et al., 2004), (c) enhanced morphine analgesia by coadministering the microglial attenuators minocycline (Cui et al., 2008, Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c) or AV411 (Hutchinson et al., 2009), and the astrocyte inhibitor fluorocitrate (Song and Zhao, 2001), (d) enhanced morphine analgesia by blocking proinflammatory cytokine actions (Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c, Shavit et al., 2005), and (e) opioid-induced selective activation of microglial p38 MAPK and associated enhanced morphine analgesia (Cui et al., 2006). As such, opioid-induced proinflammatory glial activation is characterized by a cellular phenotype of enhanced reactivity and propensity to proinflammation in response to exposure of glia to opioids.

In vitro studies support that opioids can alter the function of microglia and astrocytes (Dobrenis et al., 1995, El-Hage et al., 2005, Horvath and DeLeo, 2009, Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c, Lipovsky et al., 1998, Narita et al., 2006, Peterson et al., 1998, Stefano, 1998, Takayama and Ueda, 2005). Also, morphine can sensitize (“prime”) microglia in vitro to over-respond to subsequent stimuli, thereby generating exaggerated release of neuroexcitatory substances (Chao et al., 1994).

As microglia and astrocytes can express mRNA for mu, delta, and kappa opioid receptors (Ruzicka and Akil, 1997), opioids have been thought to exclusively influence glia via these receptors. However, opioid receptor knockout mouse studies of opioid-induced peripheral immune function modulations reveal both opioid receptor dependent (Gaveriaux-Ruff et al., 1998) and independent actions (Gaveriaux-Ruff et al., 2001).

Opioids may potentially activate glia through mechanisms distinct from classical opioid receptors. While classical opioid receptors are stereoselective, as they bind (−)-opioid isomers but not (+), several studies report (+)-isomer glial effects for both opioid agonists and antagonists. For example, (+)-opioid agonists suppress (−)-opioid analgesia (Wu et al., 2007), an effect attributed to glial activation based on propentofylline blockade (Wu et al., 2005) and independent of classical μ-opioid receptors in knockout mice studies (Wu et al., 2006a, Wu et al., 2006b). It has also been reported that morphine administered to triple opioid receptor knockouts can induce hyperalgesia (Juni et al., 2007), supporting the studies reviewed above that suggest that a non-classical opioid receptor may exist that opposes analgesia.

Intriguingly, it has recently been reported that (+)-opioid antagonists attenuate the reduction in opioid analgesia that occurs in response to glial activation by lipopolysaccharide (LPS) (Wu et al., 2006a, Wu et al., 2006b). This is exciting because it suggests the novel possibility that opioid agonists may actually signal, not only via classical opioid receptors, but also through the LPS receptor, toll-like receptor 4 (TLR4). TLR4 is an innate immune receptor, also capable of recognizing endogenous danger signals, whose signaling via the Toll/Interleukin-1 receptor (TIR) domain results in a profound proinflammatory signal. Thus, opioid effects via TLR4 could potentially provide an explanation for opioid-induced proinflammatory glial activation (Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c, Hutchinson et al., 2009, Johnston et al., 2004, Raghavendra et al., 2002, Song and Zhao, 2001). The present series of in vivo, in vitro, and in silico studies were designed to provide an initial exploration of this issue.

Section snippets

Subjects

Pathogen-free adult male Sprague–Dawley rats (n = 6 rats/group for each experiment; 300–375 g; Harlan Labs, Madison, WI, USA) were used. Pathogen-free male Balb/c wildtype and TLR4 knockout mice, back crossed onto Balb/c 10 times were used for the TLR4 knockout studies (n = 6 mice/group for each experiment; 24–32 g; kindly gifted by Dr. Simon Phipps and sourced from Prof. Akira). This TLR4 knockout strain has an established track record in the TLR4 literature (Hoshino et al., 1999). Mice and rats

Experiment 1. In vitro studies of TLR4 signaling in RAW264.7 macrophages

As noted above, we have recently reported that naloxone can non-stereoselectively inhibit TLR4-mediated LPS signaling in vitro, using a human TLR4 transfected human embryonic kidney (HEK293-hTLR4) cell line that generates secreted alkaline phosphatase (SEAP) as a reporter protein in response to human TLR4 activation (Hutchinson et al., 2008a, Hutchinson et al., 2008b, Hutchinson et al., 2008c). (+)- and (−)-naloxone appears to be capable of acting as TLR4 antagonists based on their

Discussion

The present series of studies utilized a combination of in vitro, in vivo, and in silico techniques to explore whether opioids may potentially influence TLR4 signaling. Evidence was found suggestive that TLR4 signaling can occur in response to clinically-employed opioid agonists, their non-opioid (+)-isomers, and the opioid-inactive metabolite morphine-3-glucuronide, but not other classes of typical/atypical analgesics or glial attenuators. Also, the opioid-inactive (+)-naloxone and

Acknowledgments

This work was supported by an International Association for the Study of Pain International Collaborative grant, American Australian Association Merck Company Foundation Fellowship, National Health and Medical Research Council CJ Martin Fellowship (ID 465423; M.R.H.) and NIH Grants DA015642, DA017670, DA024044, DE017782, T32 GM-065103, and DE017782. This work was partially supported by the by the NIH Intramural Research Programs of the National Institute on Drug Abuse and the National Institute

References (54)

  • C.K. Hwang et al.

    Transcriptional regulation of mouse mu opioid receptor gene by PU.1

    J. Biol. Chem.

    (2004)
  • A. Juni et al.

    Nociception increases during opioid infusion in opioid receptor triple knock-out mice

    Neuroscience

    (2007)
  • Y. Li et al.

    Morphine promotes apoptosis via TLR2, and this is negatively regulated by beta-arrestin 2

    Biochem. Biophys. Res. Commun.

    (2009)
  • D.Y. Liang et al.

    Chronic pain and genetic background interact and influence opioid analgesia, tolerance, and physical dependence

    Pain

    (2006)
  • M.M. Lipovsky et al.

    Morphine enhances complement receptor-mediated phagocytosis of Cryptococcus neoformans by human microglia

    Clin. Immunol. Immunopathol.

    (1998)
  • S.L. Liu et al.

    A novel inhibitory effect of naloxone on macrophage activation and atherosclerosis formation in mice

    J. Am. Coll. Cardiol.

    (2006)
  • M. Narita et al.

    Increased level of neuronal phosphoinositide 3-kinase gamma by the activation of mu-opioid receptor in the mouse periaqueductal gray matter: further evidence for the implication in morphine-induced antinociception

    Neuroscience

    (2004)
  • P.K. Peterson et al.

    The opioid–cytokine connection

    J. Neuroimmunol.

    (1998)
  • R.D. Polakiewicz et al.

    mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control

    J. Biol. Chem.

    (1998)
  • B.B. Ruzicka et al.

    The interleukin-1beta-mediated regulation of proenkephalin and opioid receptor messenger RNA in primary astrocyte-enriched cultures

    Neuroscience

    (1997)
  • Y. Shavit et al.

    Interleukin-1 antagonizes morphine analgesia and underlies morphine tolerance

    Pain

    (2005)
  • P. Song et al.

    The involvement of glial cells in the development of morphine tolerance

    Neurosci. Res.

    (2001)
  • G.B. Stefano

    Autoimmunovascular regulation: morphine and anandamide and ancodamide stimulated nitric oxide release

    J. Neuroimmunol.

    (1998)
  • L.R. Watkins et al.

    Glia: novel counter-regulators of opioid analgesia

    Trends Neurosci.

    (2005)
  • H.E. Wu et al.

    Stereoselective action of (+)-morphine over (−)-morphine in attenuating the (−)-morphine-produced antinociception via the naloxone-sensitive sigma receptor in the mouse

    Eur. J. Pharmacol.

    (2007)
  • H.E. Wu et al.

    Dextro- and levo-morphine atten0uate opioid delta and kappa receptor agonist produced analgesia in mu-opioid receptor knockout mice

    Eur. J. Pharmacol.

    (2006)
  • C.C. Chao et al.

    Priming effect of morphine on the production of tumor necrosis factor-alpha by microglia: implications in respiratory burst activity and human immunodeficiency virus-1 expression

    J. Pharmacol. Exp. Ther.

    (1994)
  • Cited by (423)

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text