Immune cell-derived opioids protect against neuropathic pain in mice

Abstract

The analgesic effects of leukocyte-derived opioids have been exclusively demonstrated for somatic inflammatory pain, for example, the pain associated with surgery and arthritis. Neuropathic pain results from injury to nerves, is often resistant to current treatments, and can seriously impair a patient's quality of life. Although it has been recognized that neuronal damage can involve inflammation, it is generally assumed that immune cells act predominately as generators of neuropathic pain. However, in this study we have demonstrated that leukocytes containing opioids are essential regulators of pain in a mouse model of neuropathy. About 30%-40% of immune cells that accumulated at injured nerves expressed opioid peptides such as beta-endorphin, Met-enkephalin, and dynorphin A. Selective stimulation of these cells by local application of corticotropin-releasing factor led to opioid peptide-mediated activation of opioid receptors in damaged nerves. This ultimately abolished tactile allodynia, a highly debilitating heightened response to normally innocuous mechanical stimuli, which is symptomatic of neuropathy. Our findings suggest that selective targeting of opioid-containing immune cells promotes endogenous pain control and offers novel opportunities for management of painful neuropathies.

Figure 1. Expression of opioid peptides in immune cells accumulating at the nerve injury site.

(A) Flow cytometric quantification of leukocytes (stained with CD45 Ab) and of CD45⁺ cells expressing opioids (stained with 3E7 Ab) at nerves and in hind paws. ipsi and contra, nerves ipsilateral and contralateral to surgeries, respectively; Nonoperated, nerves or paws from nonoperated animals. *P < 0.001, ^†P = 0.002; ^‡P < 0.001, ^§P = 0.002, ^¶P = 0.039 versus nonoperated; ^#P = 0.021, **P < 0.001, ^††P = 0.012, 2 days versus 14 days (t test). (B) Representative flow cytometric analysis of CD45⁺ cells (with CD45-phytoerythrin–cyanine dye 5 [Cy5] Ab) and of CD45⁺3E7⁺ cells (with 3E7-phytoerythrin Ab). Middle panels at 2 days and 14 days show control staining specificity of 3E7 Ab. (C) Representative double-immunofluorescence images showing coexpression of β-endorphin (END), Met-enkephalin (ENK), or dynorphin A (DYN) with CRF receptors (CRFR) in leukocytes (arrows) at injured nerves at 2 days and 14 days following CCI. Scale bars: 50 μm.

Figure 2. Analgesic effects produced by CRF injected at the site of nerve injury.

(A) Dose-response relationships of CRF-induced analgesia measured 30 minutes after CRF application, in paws ipsilateral to CCI at 2 days (5–20 ng) and at 14 days (20–100 ng) following CCI (P < 0.001 and P = 0.003, respectively; ANOVA, linear regression). No significant changes were observed in paws contralateral to CCI (P > 0.05, ANOVA). (B) The time course of CRF-induced analgesia in paws ipsilateral to CCI at 2 days (20 ng) and at 14 days (100 ng) following CCI (*P < 0.05 compared with 0 time point; repeated-measures ANOVA, Dunnett’s test). There were no significant changes in control groups (P > 0.05 compared with 0 time point; repeated-measures ANOVA). (C) Dose-dependent reversibility of CRF-induced (20 ng at 2 days or 100 ng at 14 days) analgesia by coinjection of CRF receptor antagonist α-helical CRF (0.125–2 ng), in paws ipsilateral to CCI at 2 days and 14 days (P < 0.001; ANOVA, linear regression). (D) Dose-dependent reversibility of CRF-induced (20 ng at 2 days or 100 ng at 14 days) analgesia by coinjection of Abs against β-endorphin (Anti-END; 0.015–0.25 μg), Met-enkephalin (Anti-ENK; 0.0625–0.5 μg), or dynorphin A (Anti-DYN; 0.5–4 μg), in paws ipsilateral to CCI at 2 days and 14 days after nerve injury (P < 0.001; ANOVA, linear regression).

Figure 3. Lack of analgesic effects of CRF injected at sites distant to nerve injury.

(A) Effects of CRF (20 ng at 2 days or 100 ng at 14 days) injected s.c. at the neck on mechanical allodynia in paws ipsilateral to CCI as a function of time, at 2 days and 14 days after nerve injury. (B) Effects of CRF (20 ng at 2 days or 100 ng at 14 days) injected into paws innervated by injured nerves on mechanical allodynia in these paws, as a function of time, at 2 days and 14 days after nerve injury. There were no significant changes after any treatments (P > 0.05 compared with 0 time points; repeated-measures ANOVA).

Figure 4. Contribution of opioid-containing immune cells to analgesia produced by CRF injected at the site of nerve injury.

(A) Representative immunostaining images showing expression of ICAM-1 in blood vessels (in green) of injured nerves and in morphologically defined immune cells (arrows) at the injured nerves at 2 days and 14 days after CCI. Scale bars: 50 μm. (B) Decrease in the numbers of all leukocytes (CD45⁺) and of opioid-containing leukocytes (CD45⁺3E7⁺) at the injured nerves by i.p. treatment with mAb against ICAM-1 (anti–ICAM-1; 150 μg per mouse). *P < 0.001, ^†P = 0.036, ^‡P = 0.018, control versus anti–ICAM-1 (t test). (C) Lack of significant changes in mechanical allodynia in paws ipsilateral to CCI at 2 and 14 days following nerve injury by anti–ICAM-1 (12.5–150 μg per mouse, i.p.; P > 0.05, repeated-measures ANOVA). (D) Dose-dependent reversibility of CRF-induced (20 ng at 2 days or 100 ng at 14 days) analgesia by anti–ICAM-1 (12.5–150 μg per mouse, i.p.) in paws ipsilateral to CCI (P < 0.001; ANOVA, linear regression).

Figure 5. Contribution of opioid receptors expressed in injured nerves to analgesia produced by CRF injected at the site of injury.

(A) Representative double-immunofluorescence images showing μ-, δ-, and κ-opioid receptor expression in sensory fibers (as determined by their coexpression with CGRP [arrows]) in the injured nerves at 2 and 14 days following CCI. Images are taken proximal to the most proximal ligature. Scale bars: 50 μm. (B) Left panels at 2 days and 14 days following CCI: Dose-dependent reversibility of CRF-induced (20 ng at 2 days or 100 ng at 14 days) analgesia by coinjection of antagonists selective to μ- (CTOP; 0.015–0.25 μg), δ- (ICI 174,864; 0.0625–2 μg), or κ-opioid (norBNI; 0.5–10 μg) receptors, as well as by an opioid receptor antagonist with limited access to the CNS, naloxone methiodide (NLXM; 0.625–5 μg) (P = 0.001 for CTOP at 2 days and P = 0.002 for CTOP at 14 days; P = 0.016 for ICI 174,864 at 2 days and P = 0.004 for ICI 174,864 at 14 days; P < 0.001 for norBNI and NLXM; ANOVA, linear regression). Right panels at 2 days and 14 days following CCI: Lack of reversibility of near-nerve CRF-induced (20 ng at 2 days or 100 ng at 14 days) analgesia s.c. at the neck injected with the most effective near-nerve doses of CTOP (0.25 μg), ICI 174,864 (2 μg), norBNI (10 μg), and NLXM (5 μg) (P > 0.05; ANOVA). The effects were measured in paws ipsilateral to CCI.