Japanese Journal of Ophthalmology

, Volume 62, Issue 3, pp 398–406 | Cite as

Crucial role of P2X7 receptor for effector T cell activation in experimental autoimmune uveitis

  • Atsunobu TakedaEmail author
  • Hisakata Yamada
  • Eiichi Hasegawa
  • Mitsuru Arima
  • Shoji Notomi
  • Sayaka Myojin
  • Takeru Yoshimura
  • Toshio Hisatomi
  • Hiroshi Enaida
  • Ryoji Yanai
  • Kazuhiro Kimura
  • Tatsuro Ishibashi
  • Koh-Hei Sonoda
Laboratory Investigation



To investigate the roles of P2X7 receptors (P2RX7) in the pathogenesis of experimental autoimmune uveoretinitis (EAU).

Study design



Either wild-type (P2rx7 +/+ ) or P2rx7-deficient (P2rx7 −∕− ) mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20. Severity of EAU was evaluated clinically and histopathologically. The induction of IRBP-specific proliferation and cytokines in draining lymph nodes was assessed by enzyme-linked immunosorbent assays (ELISA). The frequency of activation markers was examined by flow cytometry. Furthermore, inhibitory roles of systemic administration of Brilliant Blue G (BBG), an antagonist for P2RX7, in EAU were also assessed in the wild-type mice.


The severity of EAU in P2rx7 −∕− mice was reduced as compared with that in P2rx7 +/+ mice, both clinically and histopathologically. IRBP-specific proliferation in P2rx7 −∕− on day 16 was slightly decreased compared to that in P2rx7 +/+ mice. The induction of IRBP-specific interferon (IFN)-γ and interleukin (IL)-17 in P2rx7 −∕− mice on day 16 was lower than that in P2rx7 +/+ mice. The up-regulation of surface expression of activation markers such as CD25, CD44, and CD69 in response to TCR stimulation in P2rx7 −∕− mice was decreased as compared with that in P2rx7 +/+ mice. Furthermore, neutralization of P2RX7 in vivo by BBG suppressed EAU clinically and histopathologically. IRBP-specific IFN-γ and IL-17 induction in BBG-treated mice was significantly lower than that in vehicle-treated mice.


The results suggest that P2RX7 is a novel preventative therapeutic target for uveitis as it suppresses the effector functions of both Th1 and Th17 cell responses.


P2RX7 CD4+ T cells Uveitis EAU BBG 



We thank to Ms.Michiyo Takahara, Ms.Yuka Matsutani, and Ms.Yukari Mizuno for their technical support throughout the experiments. This work was supported by JSPS KAKENHI grant number 15K10896 (grant C to A.T.) and Grant Number 10294943 (grants B to K-H.S.) from the Ministry of Education, Science, Sports and Culture, Japan and grants from the Japan Foundation for Applied Enzymology (AT; Osaka, Japan) and Charitable Trust Fund for Ophthalmic Research in Commemoration of Santen Pharmaceutical’s Founder (AT; Tokyo, Japan). A.T. designed and performed experiments, analyzed data, and wrote the manuscript. H.Y., E.H., S.M., and T.Y. performed experiments and analyzed data. A.M., S.N, T.H., R.Y., K.K. and H.E. analyzed data. T.I. designed experiments and analyzed data. K-H. S. was the principal investigator, designed experiments, analyzed data, and wrote the manuscript. All authors had the opportunity to discuss the results and comment on the manuscript.

Conflicts of interest

A. Takeda, None; H. Yamada, None; E. Hasegawa, None; M. Arima, None; S. Notomi, None; S. Myojin, None; T. Yoshimura, None; T. Hisatomi, None; H. Enaida, None; R. Yanai, None; K. Kimura, None; T. Ishibashi, None; K. Sonoda, None.


  1. 1.
    Kerr EC, Copland DA, Dick AD, Nicholson LB. The dynamics of leukocyte infiltration in experimental autoimmune uveoretinitis. Prog Retin Eye Res. 2008;27:527–35.CrossRefPubMedGoogle Scholar
  2. 2.
    Forrester JV. Uveitis: pathogenesis. Lancet. 1991;338:1498–501.CrossRefPubMedGoogle Scholar
  3. 3.
    Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, et al. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med. 2008;205:799–810.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sun B, Sun SH, Chan CC, Wiggert B, Caspi RR. Autoimmunity to a pathogenic retinal antigen begins as a balanced cytokine response that polarizes towards type 1 in a disease-susceptible and towards type 2 in a disease-resistant genotype. Int Immunol. 1999;11:1307–12.CrossRefPubMedGoogle Scholar
  5. 5.
    Takeda A, Hasegawa E, Fukuhara T, Hirakawa S, Yamada H, Yang Y, et al. EBI3 is pivotal for the initiation of experimental autoimmune uveitis. Exp Eye Res. 2014;125:107–13.CrossRefPubMedGoogle Scholar
  6. 6.
    Abbracchio MP, Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther. 1994;64:445–75.CrossRefPubMedGoogle Scholar
  7. 7.
    North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002;82:1013–67.CrossRefPubMedGoogle Scholar
  8. 8.
    Surprenant A, North RA. Signaling at purinergic P2X receptors. Annu Rev Physiol. 2009;71:333–59.CrossRefPubMedGoogle Scholar
  9. 9.
    Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, et al. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood. 2001;97:587–600.CrossRefPubMedGoogle Scholar
  10. 10.
    Surprenant A, Rassendren F, Kawashima E, North RA, Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science. 1996;272:735–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Yip L, Cheung CW, Corriden R, Chen Y, Insel PA, Junger WG. Hypertonic stress regulates T-cell function by the opposing actions of extracellular adenosine triphosphate and adenosine. Shock. 2007;27:242–50.CrossRefPubMedGoogle Scholar
  12. 12.
    Loomis WH, Namiki S, Ostrom RS, Insel PA, Junger WG. Hypertonic stress increases T cell interleukin-2 expression through a mechanism that involves ATP release, P2 receptor, and p38 MAPK activation. J Biol Chem. 2003;278:4590–6.CrossRefPubMedGoogle Scholar
  13. 13.
    Corriden R, Insel PA, Junger WG. A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells. Am J Physiol Cell Physiol. 2007;293:C1420–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Tsukimoto M, Tokunaga A, Harada H, Kojima S. Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors. Biochem Biophys Res Commun. 2009;384:512–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Yip L, Woehrle T, Corriden R, Hirsh M, Chen Y, Inoue Y, et al. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors. FASEB J. 2009;23:1685–93.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lang PA, Merkler D, Funkner P, Shaabani N, Meryk A, Krings C, et al. Oxidized ATP inhibits T-cell-mediated autoimmunity. Eur J Immunol. 2010;40:2401–8.CrossRefPubMedGoogle Scholar
  17. 17.
    Schenk U, Frascoli M, Proietti M, Geffers R, Traggiai E, Buer J, et al. ATP inhibits the generation and function of regulatory T cells through the activation of purinergic P2X receptors. Sci Signal. 2011;4:ra12.CrossRefPubMedGoogle Scholar
  18. 18.
    Foster CS, Kothari S, Anesi SD, Vitale AT, Chu D, Metzinger JL, et al. The Ocular Immunology and Uveitis Foundation preferred practice patterns of uveitis management. Surv Ophthalmol. 2016;61:1–17.CrossRefPubMedGoogle Scholar
  19. 19.
    Mesquida M, Molins B, Llorenc V, Hernandez MV, Espinosa G, Dick AD, et al. Current and future treatments for Behcet’s uveitis: road to remission. Int Ophthalmol. 2014;34:365–81.CrossRefPubMedGoogle Scholar
  20. 20.
    Arida A, Sfikakis PP. Anti-cytokine biologic treatment beyond anti-TNF in Behcet’s disease. Clin Exp Rheumatol. 2014;32:S149–55.PubMedGoogle Scholar
  21. 21.
    Okada AA, Goto H, Ohno S, Mochizuki M. Multicenter study of infliximab for refractory uveoretinitis in Behcet disease. Arch Ophthalmol. 2012;130:592–8.CrossRefPubMedGoogle Scholar
  22. 22.
    Takeuchi M, Kezuka T, Sugita S, Keino H, Namba K, Kaburaki T, et al. Evaluation of the long-term efficacy and safety of infliximab treatment for uveitis in Behcet’s disease: a multicenter study. Ophthalmology. 2014;121:1877–84.CrossRefPubMedGoogle Scholar
  23. 23.
    Kuroyanagi K, Sakai T, Kohno H, Okano K, Akiyama G, Aoyagi R, et al. Association between the major histocompatibility complex and clinical response to infliximab therapy in patients with Behcet uveitis. Jpn J Ophthalmol. 2015;59:401–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Ramos-Casals M, Brito-Zeron P, Munoz S, Soria N, Galiana D, Bertolaccini L, et al. Autoimmune diseases induced by TNF-targeted therapies: analysis of 233 cases. Medicine (Baltimore). 2007;86:242–51.CrossRefPubMedGoogle Scholar
  25. 25.
    Rutgeerts P, Feagan BG, Lichtenstein GR, Mayer LF, Schreiber S, Colombel JF, et al. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn’s disease. Gastroenterology. 2004;126:402–13.CrossRefPubMedGoogle Scholar
  26. 26.
    van der Laken CJ, Voskuyl AE, Roos JC, Stigter van Walsum M, de Groot ER, Wolbink G, et al. Imaging and serum analysis of immune complex formation of radiolabelled infliximab and anti-infliximab in responders and non-responders to therapy for rheumatoid arthritis. Ann Rheum Dis. 2007;66:253–6.CrossRefPubMedGoogle Scholar
  27. 27.
    Taylor AW, Yee DG, Nishida T, Namba K. Neuropeptide regulation of immunity. The immunosuppressive activity of alpha-melanocyte-stimulating hormone (alpha-MSH). Ann N Y Acad Sci. 2000;917:239–47.CrossRefPubMedGoogle Scholar
  28. 28.
    Kitaichi N, Namba K, Taylor AW. Inducible immune regulation following autoimmune disease in the immune-privileged eye. J Leukoc Biol. 2005;77:496–502.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Thurau SR, Chan CC, Nussenblatt RB, Caspi RR. Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals. Clin Exp Immunol. 1997;109:370–6.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Enaida H, Hisatomi T, Hata Y, Ueno A, Goto Y, Yamada T, et al. Brilliant blue G selectively stains the internal limiting membrane/brilliant blue G-assisted membrane peeling. Retina. 2006;26:631–6.PubMedGoogle Scholar
  31. 31.
    Mochizuki N, Yamamoto T, Enaida H, Ishibashi T, Yamashita H. Long-term outcomes of 3 surgical adjuvants used for internal limiting membrane peeling in idiopathic macular hole surgery. Jpn J Ophthalmol. 2014;58:455–61.CrossRefPubMedGoogle Scholar
  32. 32.
    Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, et al. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. Proc Natl Acad Sci USA. 2009;106:12489–93.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kimbler DE, Shields J, Yanasak N, Vender JR, Dhandapani KM. Activation of P2X7 promotes cerebral edema and neurological injury after traumatic brain injury in mice. PLoS One. 2012;7:e41229.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Giuliani AL, Sarti AC, Falzoni S, Di Virgilio F. The P2X7 receptor-interleukin-1 liaison. Front Pharmacol. 2017;8:123.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Gulen MF, Bulek K, Xiao H, Yu M, Gao J, Sun L, et al. Inactivation of the enzyme GSK3alpha by the kinase IKKi promotes AKT-mTOR signaling pathway that mediates interleukin-1-induced Th17 cell maintenance. Immunity. 2012;37:800–12.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Chang J, Burkett PR, Borges CM, Kuchroo VK, Turka LA, Chang CH. MyD88 is essential to sustain mTOR activation necessary to promote T helper 17 cell proliferation by linking IL-1 and IL-23 signaling. Proc Natl Acad Sci USA. 2013;110:2270–5.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Castrichini M, Lazzerini PE, Gamberucci A, Capecchi PL, Franceschini R, Natale M, et al. The purinergic P2x7 receptor is expressed on monocytes in Behcet’s disease and is modulated by TNF-alpha. Eur J Immunol. 2014;44:227–38. Scholar
  38. 38.
    Martin BN, Wang C, Zhang CJ, Kang Z, Gulen MF, Zepp JA, et al. T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis. Nat Immunol. 2016;17:583–92.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Aswad F, Dennert G. P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes. Cell Immunol. 2006;243:58–65.CrossRefPubMedGoogle Scholar
  40. 40.
    Adriouch S, Dox C, Welge V, Seman M, Koch-Nolte F, Haag F. Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. J Immunol. 2002;169:4108–12.CrossRefPubMedGoogle Scholar
  41. 41.
    Rao NA. Role of oxygen free radicals in retinal damage associated with experimental uveitis. Trans Am Ophthalmol Soc. 1990;88:797–850.PubMedPubMedCentralGoogle Scholar
  42. 42.
    Sharp AJ, Polak PE, Simonini V, Lin SX, Richardson JC, Bongarzone ER, et al. P2x7 deficiency suppresses development of experimental autoimmune encephalomyelitis. J Neuroinflamm. 2008;5:33.CrossRefGoogle Scholar
  43. 43.
    Matute C, Torre I, Perez-Cerda F, Perez-Samartin A, Alberdi E, Etxebarria E, et al. P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis. J Neurosci. 2007;27:9525–33.CrossRefPubMedGoogle Scholar
  44. 44.
    Notomi S, Hisatomi T, Kanemaru T, Takeda A, Ikeda Y, Enaida H, et al. Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death. Am J Pathol. 2011;179:2798–809.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Lee J. Use of antioxidants to prevent cyclosporine a toxicity. Toxicol Res. 2010;26:163–70.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Japanese Ophthalmological Society 2018

Authors and Affiliations

  • Atsunobu Takeda
    • 1
    • 5
    Email author
  • Hisakata Yamada
    • 2
    • 5
  • Eiichi Hasegawa
    • 1
  • Mitsuru Arima
    • 1
  • Shoji Notomi
    • 1
  • Sayaka Myojin
    • 1
  • Takeru Yoshimura
    • 1
  • Toshio Hisatomi
    • 1
  • Hiroshi Enaida
    • 4
  • Ryoji Yanai
    • 3
  • Kazuhiro Kimura
    • 3
  • Tatsuro Ishibashi
    • 1
  • Koh-Hei Sonoda
    • 1
  1. 1.Department of Ophthalmology, Graduate School of Medical ScienceKyushu UniversityFukuokaJapan
  2. 2.Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of BioregulationKyushu UniversityFukuokaJapan
  3. 3.Department of Ophthalmology, Graduate School of MedicineYamaguchi UniversityUbeJapan
  4. 4.Department of Ophthalmology, Faculty of MedicineSaga UniversitySagaJapan
  5. 5.Clinical Research CenterNational Hospital Organization, Kyushu Medical CenterFukuokaJapan

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