Evidence that opioids may have toll-like receptor 4 and MD-2 effects
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
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These authors contributed equally to this work.