Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1α (HIF-1α), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1α 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 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-1α-AM cascade, defining a previously unknown mode of regulation of peripheral immunity.
A successful immune response is achieved by rapid mobilization of circulating peripheral T cells and antigen-driven expansion of a certain fraction of the cells in situ. Subsequently, accumulated T cells may need to be cleared away to prevent a harmful over-response or for preservation of homeostasis within the T cell compartment of peripheral immunity. An apoptotic process termed activation-induced cell death (AICD)3 triggered by repeated Ag challenge via the TCR/CD3 complex, is believed to be a mechanism for efficient elimination of activated T cells (1, 2). Dysregulation of AICD in T cells has been shown to result in autoimmune diathesis (3), failure in transplantation tolerance (4), or immunosuppression by an environmental toxin such as dioxin (5). AICD in T cells has been suggested to be mediated mainly by Fas (CD95)/Fas-ligand (Fas-L) interaction (6), and also has been modulated by environmental constituents (7, 8). The mechanism of regulation of AICD in situ, however, remains largely unknown. It should be noted that during traffic through different compartments of the body, T cells are likely to encounter significant differences in oxygen tension, i.e. ∼100 mm of Hg (14% O2) in arterial blood and 40 mm of Hg (5–6% O2) or less in the tissue interstitium (9). Moreover, activated T cells accumulate and function in an area of inflammation or tumor growth, both of which are known to be hypoxic (10). Responses of T cells to hypoxia, therefore, may be essential not only for adaptation but also for their functional performance.
Cellular adaptation to hypoxic conditions involves a transcriptional response pathway mediated by the hypoxia-inducible factor (HIF)-1, a heterodimeric complex of the basic helix-loop-helix (bHLH) PAS (Per, Arnt, Sim) domain proteins HIF-1α and HIF-1β (Arnt) (11, 12, 13). Two distinct mechanisms are important for regulation of HIF-1 activity by oxygen. Under normoxic conditions, HIF-1α is targeted by the von Hippel-Lindau protein (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 (TAD) of HIF-1α by a set of dioxygenases that have an absolute requirement of dioxygen, iron, and 2-oxoglutarate as cosubstrates (16, 17, 18). Upon a decrease in available oxygen levels, there is a corresponding decrease in prolyl hydroxylation of HIF-1α, resulting in release of pVHL and dramatic stabilization of HIF-1α protein. In addition, hypoxia induces interaction between the C-terminal TAD (C-TAD) of HIF-1α and transcriptional coactivators (19, 20, 21). Under normoxic conditions, this interaction is blocked by hydroxylation of an asparagine residue within the C-TAD of HIF-1α (22). HIF-1α-mediated signaling serves as a master regulator in oxygen homeostatic processes, from sensing to responding to changes in environmental oxygen tension. Expression of HIF-1α has been documented in a wide variety of mammalian cells including immune cells (23). A recent study has indicated that genetic disruption of HIF-1α expression resulted in abnormal B cell development and autoimmunity (24), and that selective deletion of HIF-1α in granulocytes and macrophages/monocytes leads to impairment of inflammatory responses such as motility and invasiveness of, and bacterial killing by, those cells (25). The role of HIF-1α in regulation of function of peripheral T cells, however, is largely unknown.
In this study, we described that AICD of human peripheral T cells by TCR/CD3 engagement was suppressed under hypoxic conditions and HIF-1 target gene product adrenomedullin (AM) increased survival of T cells in an autocrine fashion. Therefore, HIF-1α-AM cascade-mediated control of T cell survival may constitute a novel milieu for regulation of T cell-mediated immune response in situ.
Materials and Methods
Abs and reagents
mAb against human CD3 (UCHT1) was purchased from BD PharMingen (San Diego, CA). Anti-human Fas Ab (CH11) was from Medical & Biological Laboratories (Nagoya, Japan). Anti-human HIF-1α Ab (Ab463) was from Abcam (Cambridge, U.K.). Recombinant human IL-2 was obtained from PeproTech (London, U.K.) and PHA-M was from Sigma-Aldrich (St. Louis, MO). Synthetic peptides of human AM and human calcitonin gene-related peptide (CGRP) 8–37 were provided by Peptide Institute (Minoh, Japan). Other chemicals were purchased from Sigma-Aldrich unless specified.
Cell culture and activation
Fresh PBMC were prepared from heparinized blood of healthy volunteers by Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation and suspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS and antibiotics. Adherent cells were removed by incubation on plastic dishes for 1 h at 37°C, and the rest of the cells were separated on nylon wool columns to obtain T cell-rich fraction. For activation, T cell blasts were generated by stimulation of the T cell fraction (1 × 106 cells/ml) with 5 μg/ml PHA-M for 48 h. The mitogen was then washed out, and the cells were maintained in the medium with 10 ng/ml recombinant human IL-2. TCR/CD3 engagement of the cells (1 × 106 cells/ml) was performed in the presence of 10 ng/ml IL-2 on 96-well plates coated with CD3 Ab (5 μg/ml). Jurkat T cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in fully supplemented RPMI 1640 medium. A Jurkat T cell derivative stably integrated with cDNA encoding HIF-1α/1-396/VP16 AD chimeric protein, Jurkat-HIF-1α, was established by means of G418 selection. Exposure of the cells to various oxygen concentrations was carried out as described previously (14).
Determination of cell death and viability
The cells were harvested after culture under the indicated conditions and incubated with Annexin VFITC (0.25 μg/ml) and propidium iodide (PI) (50 μg/ml) in binding buffer (10 mM HEPES, 140 mM sodium chloride, 2.5 mM calcium chloride) 30 min before flow cytometric analysis for detection of apoptotic/necrotic cells. Numbers and the ratio of the viable cells were determined by means of trypan blue dye exclusion and counting on a hemocytometer.
Immunodetection of HIF-1α protein was performed as described previously (16). Briefly, whole cell extracts of T cells were prepared in lysis buffer (25 mM HEPES, 100 mM NaCl, 5 mM EDTA, 100 μM orthovanadate, and 0.5% Triton X-100, pH 7.9), followed by centrifugation for 30 min at 14,000 rpm. One hundred micrograms of whole cell extract were separated by SDS-PAGE and blotted onto polyvinylidine difluoride filters. The filters were incubated with anti HIF-1α Ab (Abcam) in TBS containing 1% nonfat milk at 4°C overnight, followed by anti-mouse Ig-HRP conjugate (Amersham Biosciences) in the same buffer. Immunocomplexes were visualized by ECL (Amersham Biosciences).
Samples of the synovial tissue were fixed overnight in 4% parafolmaldehyde at 4°C, dehydrated in alcohol, and embedded in paraffin. Four-micrometer sections were then deparaffinized and incubated with monoclonal anti-HIF-1α Ab and alkaline phosphatase-conjugated anti mouse Ig Ab, or polyclonal anti-human CD3 Ab (DAKO, Glostrup, Denmark) and histofine simple stain MAX PO (Nichirei, Tokyo, Japan). Nonimmunized mouse Ig was used as a negative control for the primary Ab.
Total RNA was isolated from T cells by guanidine isothiocyanate lysis/phenol chloroform extraction, followed by removal of contaminating DNA. First-strand cDNA was synthesized using 2 μg of DNase-treated total RNA as a template in 20 μl of reaction mixture containing 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 10 mM DTT, 75 mM KCl, 1 mM dNTPs, 0.1 mM oligo dT (252, 200 μM dNTPs, 0.25 μM 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 β-actin signal, was comparable among the preparations. Amplification by 27 cycles of 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 or nonsaturated conditions for β-actin amplification. Identities of the PCR products were confirmed by sequencing. PCR primer pairs for amplification of each gene are as following; Fas, sense: 5′-CCAAGTGACTGACATCAACTC-3′, antisense: 5′-ACTCTTTGCACTTGGTGTTGC-3′, Fas-L, sense: 5′-GGAATGGGAAGACACCTATG-3′, antisense: 5′-GCACTGGTAAGATTGAACAC-3′; bcl-2, sense: 5′-TGAACTGGGGGAGGATTGTG-3′, antisense: 5′-GCCAGGAGAAATCAAACAGA-3′; bcl-xL sense: 5′-CCCAGAAAGGATACAGCTGG-3′, antisense; 5′-GCGATCCGACTCACCAATAC-3′; vascular endothelial growth factor (VEGF), sense: 5′-TGCCTTGCTGCTCTACCTCC-3′, antisense: 5′-TCACCGCCTCGGCTTGTCAC-3′; glucose transporter (GLUT)-1, sense: 5′-CTTTCTCCAGCCAGCAATGA-3′, antisense: 5′-TGGATCCTGAGTCGAAGTCT-3′, GLUT-3, sense: 5′-GATGCTGGAGAGGTTAAGGT-3′, antisense: 5′-ACTTCCACCCAGAGCAAAGT-3′; AM, sense: 5′-AAGAAGTGGAATAAGTGGGCT-3′, antisense: 5′-TGGCTTAGAAGACACCAGAGT-3′; calcitonin receptor-like receptor (CRLR), sense: 5′-GATGCTCTGTGAAGGCATTT-3′, antisense: 5′-CAGAATTGCTTGAACCTCTC-3′, receptor-activity-modifying protein (RAMP)1, sense: 5′-GAGACGCTGTGGTGTGACTG-3′, antisense: 5′-TCGGCTACTCTGGACTCCTG-3′; RAMP2, sense: 5′-GGACGGTGAAGAACTATGAG-3′, antisense: 5′-ATCATGGCCAGGAGTACATC-3′; β-actin, sense: 5′-CCTCGCCTTTGCCGATCC-3′, antisense: 5′-GGATCTTCATGAGGTAGTCAGTC-3′.
DNA microarray analysis
Expression of HIF-1α in T cells accumulating in the interstitium of inflammatory tissue
We first examined expression of HIF-1α in T cells accumulating in a hypoxic inflammatory tissue. Immunohistochemical analysis demonstrated expression of HIF-1α in infiltrating CD3-positive T cells in the synovial tissue of a patient with rheumatoid arthritis (Fig. 1⇓). The largest portion of HIF-1α staining was found in the cytoplasmic compartment of T cells, and a nuclear signal was only occasionally detected (Fig. 1⇓B). The affected skin in a patient with dermatomyositis also revealed accumulation of CD3-positive T cells expressing HIF-1α (data not shown). Given these results, we hypothesized that HIF-1α might have a role in regulation of T cell-mediated immune reactions in the inflammatory tissues.
Hypoxia represses AICD of human peripheral T cells with concomitant expression of HIF-1α
To explore the potential role of HIF-1α in regulation of T cell function in the microenvironment of the tissue, we investigated the effect of hypoxia on AICD of peripheral T cells. PHA-activated human peripheral blood T cells were maintained with IL-2 supplementation, followed by incubation with an immobilized mAb against CD3 either under normoxic (21% O2) or hypoxic (1% O2) conditions. Staining with annexin V and PI revealed massive apoptotic cell death of T cells induced by TCR/CD3-mediated activation under normoxic conditions, indicating the induction of AICD. To our surprise, this TCR/CD3-triggered AICD of T cells was dramatically attenuated by exposure of the cells to hypoxia (Fig. 2⇓A). When the cells were incubated at various oxygen concentrations (ranging from 21 to 1% O2), the survival rates of T cells after TCR/CD3 ligation were gradually increased as oxygen tension decreased, whereas survival of T cells without TCR/CD3 ligation showed proportional reduction (Fig. 2⇓B). This influence of the oxygen concentration on T cell viability became evident when the oxygen concentration was decreased to 7 or 5%, corresponding to that often encountered in the tissue interstitium. These results indicate that not only activation through the Ag receptor but also the oxygen concentration play critical roles in determination of peripheral T cell life.
HIF-1α mRNA was constitutively expressed in human peripheral T cells (Fig. 2⇑C). In contrast, it was not possible to detect HIF-1α protein by immunoblot in the extracts from unstimulated T cells even if the cells had been exposed to hypoxia (1% O2 (Fig. 2⇑C). However, Ab-mediated TCR/CD3 engagement resulted in dramatic accumulation of HIF-1α protein upon exposure of the cells to hypoxic conditions corresponding to tissue oxygen levels (below 7% O2), indicating that expression of HIF-1α protein in peripheral T cells requires not only hypoxia but also additional signals following TCR/CD3-mediated stimulation (Fig. 2⇑C and data not shown). Because Ab-cross-link of Fas molecules did not induce HIF-1α protein accumulation either under normoxic or hypoxic conditions (Fig. 2⇑C), it may not be the cell death executing components but activation pathways after TCR/CD3 ligation that are involved in HIF-1α induction. Activation of T cells by a combination of PMA and a calcium ionophore ionomycin mimicked TCR/CD3 ligation with regard to cooperation with hypoxia in HIF-1α protein accumulation (Fig. 2⇑D). PMA/ionomycin-mediated AICD was gradually decreased along with the reduction of oxygen levels and expression of HIF-1α (Fig. 2⇑E). Taken together, we may conclude that HIF-1α plays an essential role in regulation of AICD of T cells in the hypoxic tissue microenvironment.
AM suppresses AICD of peripheral T cells via autocrine regulatory mechanism
To examine the mechanism of HIF-1α-mediated attenuation of AICD, we performed gene expression profiling experiments using DNA microarray analysis and RT-PCR assays of peripheral T cells cultured in the absence or presence of TCR/CD3 ligation and exposure to hypoxia. As shown in Table I⇓, a variety of changes in expression profile of T cell-related genes were induced by those treatments. Among them, expression of Fas mRNA was detected irrespective of cellular treatment. Fas-L mRNA expression was induced by TCR/CD3 ligation, whereas hypoxic culture had no effect on this induction response, suggesting that hypoxia does not likely interfere with TCR/CD3-mediated activation of T cells leading to Fas-L expression. mRNA levels of Bcl-2 remained constant, whereas Bcl-xL mRNA expression was induced by TCR/CD3 activation but not by hypoxia. These classical apoptosis-regulating factors, thus, do not show any correlation with HIF-1α protein levels. Expression of mRNA for GLUT-1, -3, and VEGF were induced by hypoxia but not altered after TCR/CD3 stimulation. In contrast, hypoxia had only a modest effect on AM mRNA induction, and the combination of hypoxia and TCR/CD3 stimulation resulted in a robust enhancement of AM gene expression in T cells (Fig. 3⇓A and Table I⇓). These results may indicate a close correlation between AM expression and HIF-1α protein accumulation in T cells. It is therefore likely that AM may represent a HIF-1α target gene involved in determination of cell survival of activated T cells. In fact, RT-PCR analysis demonstrated mRNA expression of the components of the receptor complex for AM, CRLR and RAMP 1 and 2 (26) in T cells (Fig. 3⇓B). Blocking of the AM receptor by a fragment of the calcitonin gene-related peptide CGRP 8-37 abolished the effect of hypoxia on T cell survival (Fig. 3⇓C), strongly suggesting that AM receptor-mediated signal transduction is essential for hypoxia-dependent rescue of T cells from AICD. In excellent agreement with this model, incubation of T cells with synthetic AM peptides bypassed requirement of hypoxia for survival, and inhibited AICD under normoxic conditions (Fig. 3⇓D). Taken together, these results suggest that HIF-1α target gene product AM plays an important role in T cell survival counteracting AICD under hypoxic conditions possibly via an autocrine loop mechanism.
Constitutively active HIF-1α induces AM expression under normoxic conditions to protect T cells from AICD
To directly monitor the involvement of HIF-1α mediated induction of AM expression in the regulation of AICD in T cells, we generated Jurkat T cells which demonstrate functional activity of HIF-1α even under normoxic conditions. To this end, we constructed a chimeric protein, HIF-1α/1-396/VP16AD, consisting of the N-terminal bHLH/PAS domain of HIF-1α and the constitutively active TAD of the viral transcription factor VP16 (Fig. 4⇓A). Transient expression of HIF-1α/1-396/VP16AD in Jurkat cells resulted in constitutive activation of HIF-1α regulated reporter gene expression (Fig. 4⇓B). Jurkat cells stably expressing HIF-1α/1-396/VP16AD, designated Jurkat-HIF-1α cells, also showed enhanced expression of HIF-1-regulated reporter gene activity at normoxia (Fig. 4⇓C). In a similar fashion, Jurkat-HIF-1α cells expressed not only AM but also CRLR and RAMP2 mRNA even under normoxic conditions (Fig. 4⇓D). In clear contrast to the parental Jurkat cells, Jurkat-HIF-1α cells showed resistance to TCR/CD3-triggered cell death at normoxia. Moreover, blocking of the AM receptor by CGRP 8–37 impaired the survival of Jurkat-HIF-1α cells (Fig. 4⇓E). In conclusion, sequential expression of HIF-1α and AM under conditions of hypoxia critically controls the viability of Ag receptor-activated T cells, participating in maintenance of T cell-mediated immune reactions in the hypoxic microenvironment.
In this study, we presented that the hypoxic condition corresponding to the oxygen levels in the tissue microenvironment protects peripheral T cells from Ag receptor stimulation-triggered AICD, indicating a possible role of local oxygen concentrations in regulation of immune reactions.
AICD of peripheral T cells is likely to be coregulated by a variety of factors including environmental constituents. These regulatory mechanisms involve costimulatory molecules (7), humoral factors, or chemicals (5), all of which have been shown to involve the Fas/Fas-L system or Bcl-family proteins. Similarly, but mechanistically distinct from AICD, Ag receptor stimulation in naive T cells has been shown to result in apoptosis, in which NF-κB and p73 play a critical role (27). In the present study, we have demonstrated that hypoxia attenuates AICD in peripheral T cells through HIF-1α-mediated expression of AM possibly via an autocrine regulatory loop mechanism. Strikingly, among the hypoxia-inducible and cell death/survival-regulating genes tested, only AM gene expression was correlated with protein levels of HIF-1α in T cells, similar to the observation that AM gene expression is strictly dependent not on the HIF-1β subunit but on HIF-1α subunit expression in ES cells (28). Although the mechanism of this deviation in target gene induction by HIF-1α in peripheral T cells is elusive, the potential role of the HIF-1-AM regulatory pathway in control of T cell survival was further illustrated by experiments in which a constitutively active form of HIF-1α induces resistance to AICD. In the endometrial cancer cells, induction of Bcl-2 by AM has been shown to play an important role in inhibition of severe hypoxia-mediated apoptosis (29). In contrast, we did not find up-regulation of Bcl-2 in the present study on T cell AICD, indicating that the AM-Bcl-2 cascade may not be critically involved in regulation of peripheral T cell death triggered by Ag receptor stimulation. Indeed, roles of AM and subsequent cellular signaling in controlling cell survival have been demonstrated to be varied, either proapoptotic or antiapoptotic, between cell types and cellular stimuli inducing cell death (29, 30, 31, 32). In any case, further studies are clearly needed for identification of a downstream signal of AM for T cell survival. Recently, Conforti et al. (33) have demonstrated that hypoxia down-regulates Kv1.3 channels in T cells and modulates T cell proliferation possibly via membrane-delimited mechanisms such as membrane depolarization or activating the calcium release-activated channel, which was seen only when T cells are activated by membrane-associated stimulation including TCR ligation (33). Because stimuli that bypass the membrane such as PMA/ionomycin (33) similarly induced HIF-1α expression and consequent AICD inhibition in our study, a mechanism independent of Kv1.3 channel-modulation might be involved in HIF-1-AM-mediated T cell survival control under the hypoxic condition. Of interest, in the myeloid lineage of the immune cells, HIF-1α has been shown to be essentially involved in the metabolic switch for glycolytic energy production, a pivotal process for myeloid cell functions, and thus is critically implicated in regulation of innate immune responses (25). In contrast, as demonstrated in the present study, hypoxia-dependent regulation of apoptosis in Ag receptor-activated T cells via the HIF-1α-AM pathway may be a mechanism for control of acquired immune systems. Distinct use of HIF-1α and its downstream signals for a different function/component of the immune system, thus, may constitute a critical mechanism for fine tuning of local inflammatory responses.
In mice splenic T cells, hypoxia-independent up-regulation of HIF-1α mRNA by TCR/CD3 stimulation has been demonstrated, which is achieved by up-regulation of the alternatively spliced shorter isoform of HIF-1α mRNA (23). This shorter isoform seen in the mice has not been found in humans, and human HIF-1α mRNA corresponds to a “constitutive” longer isoform in mice (34), indicating the presence of a distinct mode of HIF-1α induction in human T cells from that in mice. In fact, we observed a constitutive expression of HIF-1α mRNA in human peripheral T cells irrespective of TCR/CD3 stimulation, and instead, demonstrated significant up-regulation of HIF-1α in protein levels upon TCR/CD3 activation under hypoxic conditions. To our surprise, in human peripheral T cells, hypoxia is not sufficient and an additional TCR/CD3 stimulation was required for HIF-1α protein accumulation, in clear contrast to cancer-derived cell lines in which HIF-1α can be induced by solely hypoxic treatment (15). Similarly, histological analyses have revealed that the HIF-1α protein was seldom recovered in normal human tissues although physiological tissue oxygen concentration is known to be low enough to induce HIF-1α (9, 35). However, in vivo studies have demonstrated unique aspects of HIF-1α expression in inflammation (36), ischemia (37), and during development (38), all of which are indicated to involve additional oxygen-independent pathways of HIF-1α regulation. Therefore, for normal tissues including T cells, not only hypoxia-dependent but also hypoxia-independent mechanisms might be prerequisite for induction of HIF-1α, allowing rationale regulation of HIF-1α expression and subsequent adaptation to the hypoxic microenvironment. As seen in the present study, disunity in HIF-1α appearance both in the cytoplasm and the nucleus of T cells in rheumatoid arthritis synovium may indicate a possible heterogeneity of regulatory pathways leading to HIF-1α accumulation even in the same tissue.
There has been compelling evidence that physicochemical components of the tissue microenvironment such as osmolarity and temperature are crucial determinants of immune reactions (39, 40, 41, 42). Considering that immune cells often infiltrate into a hypoxic area, hypoxia-mediated regulation of the immune cells (43, 44, 45, 46) may be an efficient strategy for microenvironmental control of immune reactions in situ. In this line, survival control of T cells by hypoxia might represent such a rational mechanism for control of peripheral immunity. Under hypoxic conditions and in the absence of TCR/CD3 stimulation, peripheral T cells are indicated to be prone to die, whereas hypoxic T cells in the presence of TCR/CD3 stimuli might be protected from death. This mechanism may provide the basis of the scenario that Ag-specific T cells are preferentially allowed to survive and nonspecifically migrated T cells would be purged in the tissue microenvironment. In conclusion, oxygen concentration is suggested to be a critical regional regulator of T cells, and strict regulation of HIF-1α function and subsequent AM expression in a hypoxia- and T cell activation-dependent manner may define the mechanism determining the selection of specific T cell populations in situ. Obviously, detailed analyses on wider aspects of T cell functions using, e.g., fractionated and/or cloned T cells would be extremely important and further elucidation of distinct roles of the HIF-1-AM cascade in T cell-mediated pathophysiology would provide novel possibilities for the development of immunomodulatory therapeutic strategies.
We thank Dr. Hitoshi Abe (Department of Pathology, Keio University School of Medicine) for the excellent technical assistance and Dr. Yoshiko Kanemoto (Maruho, Co. Ltd) for cooperation in the initial course of study.
↵1 This work was supported by Japan Society for the Promotion of Science, Kanagawa Academy of Science and Technology, The Vehicle Racing Commemorative Foundation, the Cell Science Research Foundation, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
↵2 Address correspondence and reprint requests to Dr. Hirotoshi Tanaka, Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 Japan. E-mail address:
↵3 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas 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 TAD; AM, adrenomedullin; CGRP, calcitonin gene-related peptide; PI, propidium iodide; VEGF, vascular endothelial growth factor; CRLR, calcitonin receptor-like receptor; RAMP, receptor activity-modifying protein; HRE, hypoxia response element; GLUT, glucose transporter.
- Received July 9, 2003.
- Accepted October 13, 2003.
- Copyright © 2003 by The American Association of Immunologists