IFN-γ Mediates T Cells Contribution To The Chronicity Of Inflammatory Pain

ObjectivesPathological pain(or chronic pain) has become the global burden of disease, in which the inflammatory pain is the main type. Previous studies have shown that microglia and astrocytes in the spinal cord both could produce and release a variety of pro-inflammatory cytokines, chemoattractant factors and neurotrophic factors after tissue injury or inflammation. Microglia and astrocytes interact with each other, constitute the inflammatory micro- environment, enhance excitatory synaptic transmission ultimately(or central sensitization), and the organism manifests hyperalgesia and allodynia. However, the key mechanism involved in maintenance of central sensitization or chronicity of pain is as yet obscure.The neurodegenerative disorder, which pathogenesis involves auto-antigen specific T cell, often accompanies with pain. Th1 cells and Th17 cells both help to the evolution of multiple sclerosis. Growing evidence has shown T cells participate in the neuropathic pain and mainly help to the chronicity of neuropathic pain. Th1 cells are the most likely subtype in the pathology of pain, and the role of Th17 cells is yet to be explored. The research on the contribution of T cells to inflammatory pain and the involved mechanism is still little.Peripheral T cells regulating pain in spinal cord must pass through the blood-CNS barrier and infiltrate into the spinal cord. Activated microglia and astrocytes could increase the permeability of blood-CNS barrier and chemotaxis to peripheral immune cells by releasing inflammatory mediators(TNF-α, IL-1β, CCL2, etc.). At the same time, astrocyte end-feet processes cover over the Blood-CNS barrier. Furthermore, astrocytes and microglia could upregulate the expression of MHCⅡafter inflammatory insult,and may be the antigen presenting cells of central nervous system. The above points out that the activation of glial cells, especially astrocytes, affords the prerequisite for T cells infiltrating into spinal cord with immune effect and the contribution to maintenance of pathological pain. MethodsThis study selected monoarthritis rats as inflammatory pain model in vivo and primary astrocytes in vitro. The experimental methods included immunofluorescence histochemistry, flow cytometry, western blot. The aim of the study was to explore the contribution to inflammatory pain, the molecular mechanism and the impact of glial cells on T cells. The details were as follows1. The number of T cells and the activation of glial cells in the spinal cord of monoarthritis ratsAfter establishment of MA model, the number, distribution and activation of T cells were inspected in the spinal cord separated from MA group and sham group on day 7 and 10. At the same time, the expression of Iba1 in microglia and GFAP in astrocytes was determined. The kinetics of the number of T cells and activation of glial cells was noted.2. The effect of activated glial cells on the number and activation of T cellsAfter inhibition on the activation of microglia and astrocytes by pharmacological methods, PWT, the number of T cells and the expression of IFN-γ in the spinal cord were examined. The effect of the activation of glial cells on T cells was studied.3. The correlation between the expression of IFN-γ and activation of astrocytes in spinal cordAfter alteration on the level of IFN-γ by pharmacological methods in spinal cord of MA and naive rats, PWT and GFAP were inspected. The phosphorylation of NF-κBp65 in primary cultured astrocytes after incubation with IFN-γ was investigated. The aim was to testify the effect of IFN-γ on astrocytes. Results1. The number of T cells and the activation of glial cells in the spinal cord of monoarthritis ratsT cells infiltration into spinal cord and the activation of glial cells after inflammation: the number of T cells in the spinal cord of MA rats increased on day 7, 10, especially on day 10. T cells were mainly distributed and concentrated in the superficial spinal dorsal horn. IFN-γ was exclusively co-localized with CD3-positive T cells and IL-17, was mainly co-localized with GFAP-positive cells. The expression of Iba1 and GFAP increased on post-operation day 7, 10, especially on day 7 and 10 respectively.2. The effect of activated glial cells on the number and activation of T cellsAstrocytes regulated the expression of IFN-γ in spinal cord: multiple intrathecal administrations of microglial inhibitor minocycline with the first injection on day 3 had no obvious effect on the allodynia, the number of T cells and the level of IFN-γ. And inversely, astrocytic inhibitor fluorocitrate used in the same way reduced IFN-γ in the presence of reduced PWT.3. The correlation between the expression of IFN-γ and activation of astrocytes inspinal cordIFN-γ involved in the modulation of pain and activation of astrocytes: compared with PBS group, PWT upregulated and the expression of GFAP decreased after IFN-γ antibody treatment. Compared with NS group, exogenous IFN-rdirectly induced increase of PWT and GFAP expression, enhanced the level of NF-κB phosphorylation in vivo and in vitro respectively.ConclusionsThe number of T cells and activation of astrocytes showed similar kinetics after inflammation. Th1 cell was most likely involved in modulation of pain. The alteration on the level of IFN-γ in spinal cord elicited the change of mechanical hyperalgesia, and IFN-γ directly activated astrocytes in vivo and vitro. Astrocytes contributed to the maintenance of inflammatory pain, and regulated the expression of IFN-γ in spinal cord.Taken together, the study demonstrated that Th1 cells contributed to the evolution of inflammatory pain by producing IFN-γ and activating astrocytes. And inversely, activated astrocytes regulated Th1 cells producing IFN-γ. Th1 cells and astrocytes collaborated to promote the chronicity of inflammatory pain.