| The P2X7 receptor (P2X7R) belongs to the family of ionotropic P2X receptors, which are ATP-gated ion channels. P2X7R has been proposed to serve as a regulator of inflammation. To determine if the P2X7R regulates autoimmune mediated inflammation, we tested whether the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, was modified in P2X7R -/- mice. The results showed that the clinical and pathological signs of EAE were more severe in P2X7R-/- mice than in WT mice. More numerous perivascular lesions and neuronal damage were found in the brain in the P2X7R-/- mice, reflecting a more severe inflammatory process. Bone marrow (BM) radiation chimeras experiment indicated that the genotype of the BM cells regulated disease susceptibility.; In vitro, proliferation assays and TUNEL staining showed that spleen and lymph node cells from P2X7R-/- mice had a higher proliferative activity in response to myelin oligodendrocyte glycoprotein (MOG) peptide, and that lymphocytes in these cultures showed less evidence of apoptosis. FACS analysis of cells isolated from the CNS showed significantly fewer Annexin V/PI+ lymphocytes in the CNS of P2X7R -/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. Cytokine assays of culture supernatants showed significantly decreased expression of IFNgamma, IL-6, TNFalpha and IL-13 in MOG-activated spleen cells from P2X7R-/- mice. QPCR and protein assays for in vivo cytokine expression of the spinal cord showed that protein for IL-1beta, but not mRNA, was significantly reduced in the CNS of P2X7R-/- mice with EAE. However, both mRNA and protein levels for IFNgamma and IL-6 were significantly reduced in the same samples. Thus, the data for IFNgamma are consistent with the data obtained from the in vitro cell proliferation assays. Coculture of P2X7R-/- macrophages (adherent cells) with WT lymphocytes (suspension cells) and vice-versa, showed that the enhanced proliferative activity resided within the P2X7R -/- lymphocyte population, and correlated with reduced levels of IFNgamma, nitric oxide and apoptosis of lymphocytes. We conclude that the P2X7 R regulates apoptosis in activated lymphocytes and that its loss leads to unregulated expansion of antigen-specific T cells and exacerbation of autoimmune disease. |
