Of Antigen Receptor-Driven T Cells Hypoxia-Inducible Factor Regulates Survival
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Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells Yuichi Makino, Hiroshi Nakamura, Eiji Ikeda, Kei Ohnuma, Kenji Yamauchi, Yutaka Yabe, Lorenz Poellinger, Yasunori This information is current as Okada, Chikao Morimoto and Hirotoshi Tanaka of September 28, 2021. J Immunol 2003; 171:6534-6540; ; doi: 10.4049/jimmunol.171.12.6534 http://www.jimmunol.org/content/171/12/6534 Downloaded from References This article cites 46 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/171/12/6534.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells1 ‡,Kenji Yamauchi ء,Eiji Ikeda,† Kei Ohnuma ء,Hiroshi Nakamura ء,Yuichi Makino and ء,Yutaka Yabe,‡ Lorenz Poellinger,§ Yasunori Okada,† Chikao Morimoto ءHirotoshi Tanaka2 Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We dem- onstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1␣ (HIF-1␣), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1␣ in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated Downloaded from hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1␣-AM cascade, defining a previously unknown mode of regulation of peripheral immunity. The Journal of Immunology, 2003, 171: 6534–6540. successful immune response is achieved by rapid mo- different compartments of the body, T cells are likely to encounter bilization of circulating peripheral T cells and antigen- significant differences in oxygen tension, i.e. ϳ100 mm of Hg http://www.jimmunol.org/ driven expansion of a certain fraction of the cells in situ. (14% O ) in arterial blood and 40 mm of Hg (5–6% O ) or less in A 2 2 Subsequently, accumulated T cells may need to be cleared away to the tissue interstitium (9). Moreover, activated T cells accumulate prevent a harmful over-response or for preservation of homeostasis and function in an area of inflammation or tumor growth, both of within the T cell compartment of peripheral immunity. An apo- which are known to be hypoxic (10). Responses of T cells to hyp- 3 ptotic process termed activation-induced cell death (AICD) trig- oxia, therefore, may be essential not only for adaptation but also gered by repeated Ag challenge via the TCR/CD3 complex, is for their functional performance. believed to be a mechanism for efficient elimination of activated T Cellular adaptation to hypoxic conditions involves a transcrip- cells (1, 2). Dysregulation of AICD in T cells has been shown to tional response pathway mediated by the hypoxia-inducible factor by guest on September 28, 2021 result in autoimmune diathesis (3), failure in transplantation tol- (HIF)-1, a heterodimeric complex of the basic helix-loop-helix erance (4), or immunosuppression by an environmental toxin such (bHLH) PAS (Per, Arnt, Sim) domain proteins HIF-1␣ and as dioxin (5). AICD in T cells has been suggested to be mediated HIF-1 (Arnt) (11–13). Two distinct mechanisms are important mainly by Fas (CD95)/Fas-ligand (Fas-L) interaction (6), and also for regulation of HIF-1 activity by oxygen. Under normoxic con- has been modulated by environmental constituents (7, 8). The ␣ mechanism of regulation of AICD in situ, however, remains ditions, HIF-1 is targeted by the von Hippel-Lindau protein largely unknown. It should be noted that during traffic through (pVHL) for ubiquitylation and rapid proteasomal degradation (14, 15). pVHL binding is mediated through hydroxylation of specific prolyl residues within the N-terminal transactivation domain ␣ ء Division of Clinical Immunology, Advanced Clinical Research Center, Institute of (TAD) of HIF-1 by a set of dioxygenases that have an absolute Medical Science, University of Tokyo, †Department of Pathology, Keio University requirement of dioxygen, iron, and 2-oxoglutarate as cosubstrates School of Medicine, ‡Department of Orthopedics, Kyosai Tachikawa Hospital, To- kyo, Japan; and §Department of Cell and Molecular Biology, Medical Nobel Institute, (16–18). Upon a decrease in available oxygen levels, there is a Karolinska Institutet, Stockholm, Sweden corresponding decrease in prolyl hydroxylation of HIF-1␣, result- Received for publication July 9, 2003. Accepted for publication October 13, 2003. ing in release of pVHL and dramatic stabilization of HIF-1␣ pro- The costs of publication of this article were defrayed in part by the payment of page tein. In addition, hypoxia induces interaction between the C-ter- charges. This article must therefore be hereby marked advertisement in accordance minal TAD (C-TAD) of HIF-1␣ and transcriptional coactivators with 18 U.S.C. Section 1734 solely to indicate this fact. (19–21). Under normoxic conditions, this interaction is blocked by 1 This work was supported by Japan Society for the Promotion of Science, Kanagawa Academy of Science and Technology, The Vehicle Racing Commemorative Foun- hydroxylation of an asparagine residue within the C-TAD of dation, the Cell Science Research Foundation, and the Ministry of Education, Culture, HIF-1␣ (22). HIF-1␣-mediated signaling serves as a master regu- Sports, Science, and Technology of Japan. lator in oxygen homeostatic processes, from sensing to responding 2 Address correspondence and reprint requests to Dr. Hirotoshi Tanaka, Division of to changes in environmental oxygen tension. Expression of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Sci- ence, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 Japan. HIF-1␣ has been documented in a wide variety of mammalian E-mail address: [email protected] cells including immune cells (23). A recent study has indicated that 3 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas genetic disruption of HIF-1␣ expression resulted in abnormal B ligand; HIF, hypoxia-inducible factor; bHLH, basic helix-loop-helix; PAS, Per-Arnt- Sim; pVHL, von Hippel-Lindau; TAD, transactivation domain; C-TAD, C-terminal cell development and autoimmunity (24), and that selective dele- TAD; AM, adrenomedullin; CGRP, calcitonin gene-related peptide; PI, propidium tion of HIF-1␣ in granulocytes and macrophages/monocytes leads iodide; VEGF, vascular endothelial growth factor; CRLR, calcitonin receptor-like receptor; RAMP, receptor activity-modifying protein; HRE, hypoxia response ele- to impairment of inflammatory responses such as motility and in- ment; GLUT, glucose transporter. vasiveness of, and bacterial killing by, those cells (25). The role of Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 6535 HIF-1␣ in regulation of function of peripheral T cells, however, is RT-PCR analysis largely unknown. Total RNA was isolated from T cells by guanidine isothiocyanate lysis/ In this study, we described that AICD of human peripheral T phenol chloroform extraction, followed by removal of contaminating cells by TCR/CD3 engagement was suppressed under hypoxic DNA. First-strand cDNA was synthesized using 2 g of DNase-treated conditions and HIF-1 target gene product adrenomedullin (AM) total RNA as a template in 20 l of reaction mixture containing 50 mM Tris-HCl (pH 8.3), 3 mM MgCl , 10 mM DTT, 75 mM KCl, 1 mM dNTPs, increased survival of T cells in an autocrine fashion. Therefore, 2 ␣ 0.1 mM oligo dT (25) primer, and 50 U Superscript II (Invitrogen, Carls- HIF-1 -AM cascade-mediated control of T cell survival may con- bad, CA) at 42°C for 50 min. PCR was carried out in a total volume of 30 stitute a novel milieu for regulation of T cell-mediated immune l in a mixture composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 response in situ. mM MgCl2, 200 M dNTPs, 0.25 M each of the sense and antisense primers, 1 U ExTaq DNA polymerase (TaKaRa, Ohtsu, Japan). The amount of cDNA, as judged by the intensity of the amplified -actin signal, Materials and Methods was comparable among the preparations. Amplification by 27 cycles of Abs and reagents 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min was performed after 3 min of denaturing of the samples at 94°C, and shown to be within linear range mAb against human CD3 (UCHT1) was purchased from BD PharMingen or nonsaturated conditions for -actin amplification. Identities of the PCR (San Diego, CA). Anti-human Fas Ab (CH11) was from Medical & Bio- products were confirmed by sequencing. PCR primer pairs for amplifica- logical Laboratories (Nagoya, Japan).