Adoptive transfer of peripheral immune cells potentiates allodynia in a graded chronic constriction injury model of neuropathic pain

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Abstract

Recent evidence demonstrates that peripheral immune cells contribute to the nociceptive hypersensitivity associated with neuropathic pain by infiltrating the central nervous system (CNS). We have recently developed a rat model of graded chronic constriction injury (CCI) by varying the exposure of the sciatic nerve and control non-nerve tissue to surgical placement of chromic gut. We demonstrate that splenocytes can contribute significantly to CCI-induced allodynia, as adoptive transfer of these cells from high pain donors to low pain recipients potentiates allodynia (P < 0.001). The phenomenon was replicated with peripheral blood mononuclear cells (P < 0.001). Adoptive transfer of allodynia was not achieved in sham recipients, indicating that peripheral immune cells are only capable of potentiating existing allodynia, rather than establishing allodynia. As adoptively transferred cells were found by flow cytometry to migrate to the spleen (P < 0.05) and potentiation of allodynia was prevented in splenectomised low pain recipients, adoptive transfer of high pain splenocytes may induce the migration of host-derived immune cells from the spleen to the CNS as observed by flow cytometry (P < 0.05). Importantly, intrathecal transfer of CD45+ cells prepared from spinal cords of high pain donors into low pain recipients led to potentiated allodynia (P < 0.001), confirming that infiltrating immune cells are not passive bystanders, but actively contribute to nociceptive hypersensitivity in the lumbar spinal cord.

Research highlights

► Intraperitoneal adoptive transfer of peripheral immune cells potentiates chronic constriction injury-induced allodynia. ► Peripheral immune cells only capable of potentiating existing allodynia, rather than establishing allodynia. ► Intraperitoneal adoptive transfer of high pain splenocytes may induce the migration of host-derived immune cells from the spleen to the CNS. ► Intrathecal transfer of “pain-activated” CD45+ cells results in potentiated allodynia.

Introduction

Neuropathic pain is initiated by nerve damage or disease and is associated with spontaneous pain and hypersensitivity to noxious (hyperalgesia) and non-noxious (allodynia) stimuli. This altered sensory processing may be persistent, even in the absence of ongoing injury. Pharmacotherapy is unsatisfactory for many patients and the dearth of effective novel therapies reflects an incomplete understanding of the mechanisms underlying neuropathic pain. A growing body of evidence supports a neuro-immune model of neuropathic pain, whereby glia are viewed as integral members of the tripartite synapse and are posited to make a causal contribution to exaggerated pain states (Araque et al., 1999, Milligan and Watkins, 2009).

The central nervous system (CNS) is no longer considered to be an entirely immunologically privileged organ, especially with respect to entry of activated T lymphocytes (Mason et al., 1986, Hickey et al., 1991, Furtado et al., 2008, Wilson et al., 2010) and research over the last decade has implicated immune cells in many animal models of pain hypersensitivity that resemble human neuropathic pain (Liu et al., 2000, Perkins and Tracey, 2000, Hu and McLachlan, 2002, Hu et al., 2007, Li et al., 2007, Morin et al., 2007, Shaw et al., 2008). The presence of infiltrating T lymphocytes in the lumbar spinal cord of nerve-injured rodents suggested that there might be an adaptive immune component in the pathogenesis of neuropathic pain (Hu and McLachlan, 2002, Sweitzer et al., 2002a, Hu et al., 2007, Cao and DeLeo, 2008, Cao et al., 2009). These data were supported by the observation that nociceptive hypersensitivity was attenuated in nerve-injured T lymphocyte deficient rodents (Moalem et al., 2004, Kleinschnitz et al., 2006, Cao and DeLeo, 2008, Costigan et al., 2009). Using DNA microarray, Costigan et al. (2009) have recently advanced the understanding of peripheral immune cells in neuropathic pain by comparing the profiles of RNA transcripts from pooled ipsilateral lumbar dorsal horns of adult rats that develop allodynia following nerve injury and from neonatal rats that are not disposed to development of allodynia. Pathway analysis of the 148 genes found to be differentially regulated revealed pronounced differences in the signalling pathways of microglia and T lymphocytes. A critical role for T lymphocyte signalling was supported by the observation that allodynia was attenuated in nerve-injured Rag1 and interferon-γ receptor 1 (IFN-γR1) gene knockout (GKO) mice compared to wild-type controls (Costigan et al., 2009).

The comparison of nerve-injured neonatal and adult rats by Costigan et al. (2009) introduced variables related to neural and immune system immaturity. In particular, neonatal rodents are effectively T lymphocyte-deficient, so that differences in expression of T lymphocyte-related genes in the spinal cords between neonatal and adult rats may be explained on this basis. We aimed to advance these findings, using a model in which test and control animals differ only with respect to specific experimental interventions. Our novel model of graded neuropathy (Grace et al., 2010) allows comparison of varying degrees of allodynia in intact adult animals, facilitating investigation of subtle neuro-immune mechanisms responsible for neuropathic-like pain. Here we demonstrate that CNS infiltration by immune cells from the spleen may be causally involved in the nociceptive hypersensitivity associated with graded chronic constriction injury (CCI). Supportive evidence was obtained by adoptive transfer of spleen cells or peripheral blood mononuclear cells from ‘high pain’ donors to syngeneic ‘low pain’ recipients, which was found to potentiate allodynia. We conclude that immune cells are critical contributors to allodynia in our model of graded neuropathy, a behavioural correlate of neuropathic pain.

Section snippets

Animals

Inbred male Dark Agouti (DA CD45.1; 10–12 week old) and outbred male Sprague–Dawley (SD; 12 week old) rats were purchased from Animal Resource Centre (Perth, WA, Australia). Male DA CD45.2 rats (10–12 week old) were bred at the University of Adelaide as described (Spargo et al., 2006). These congenic DA lines are histocompatible (Spargo et al., 2006), differing only in expression of functionally normal CD45 alleles that can be distinguished by a monoclonal antibody and thus can be used to

Experiment 1: allodynia is potentiated by transfer of splenocytes from high pain donors to low pain recipients

A role for immune cells in the mechanisms responsible for nociceptive hypersensitivity was investigated in our graded CCI model. As this experimental design was the first of its kind, it was necessary to use a wide, non-specific population of immune cells, such as that in the spleen. Therefore, splenocytes (2 × 108) from high pain DA strain (N4S0) donors were transferred i.p. to low pain (N1S3) syngeneic recipients (representative pre adoptive transfer allodynia shown for N1S3 and N4S0 rats [Fig.

Discussion

Our graded CCI model (Grace et al., 2010) has allowed us to select conditions that induce either mild or severe allodynia. Rats with mild allodynia are ideal experimental subjects in which to examine whether cells and/or soluble factors from syngeneic high pain donors can transfer allodynia, in that they represent an intact biological system that is not affected by differences between neonatal and adult animals or by GKO. Involvement of the immune system in the pathogenesis of CCI-induced

Conflict of interest

All authors declare that there are no financial or commercial conflicts of interest.

Acknowledgments

Mr. Peter Grace is the recipient of a Faculty of Health Sciences Divisional Ph.D. Scholarship. Dr. Mark Hutchinson is a NHMRC C.J. Martin Fellow (ID 465423). This study was funded by the Pain and Anaesthesia Research Clinic, University of Adelaide. Special thanks to Ms. Yuen Hei Kwok for assistance during surgeries.

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