Macrophage migration inhibitory factor counterregulates dexamethasone-mediated suppression of hypoxia-inducible factor-1 alpha function and differentially influences human CD4+ T cell proliferation under hypoxia.
ABSTRACT Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4(+) T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4(+) T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4(+) T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4(+) T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.
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ABSTRACT: Bone cells respond to the integrated effects of local and systemic regulation. Here we show that hypoxia and the stress hormones PGE2 and glucocorticoid interact in complex ways in osteoblasts, converging on insulin like growth factor I (IGF-I) expression. Whereas hypoxia alone rapidly increased transcription factor HIF activity, it suppressed DNA synthesis, had no significant effects on protein synthesis or alkaline phosphatase activity, and drove discrete changes in a panel of osteoblast mRNAs. Notably, hypoxia increased expression of the acute phase response transcription factors C/EBPδ which can induce IGF-I in response to PGE2, but conversely prevented the stimulatory effect of PGE2 on IGF-I mRNA. However, unlike its effect on C/EBPδ, hypoxia suppressed expression of the obligate osteoblast transcription factor Runx2, which can activate an upstream response element in the IGF-I gene promoter. Hypoxic inhibition of IGF-I and Runx2 were enforced by glucocorticoid, and continued with prolonged exposure. Our studies thus reveal that IGF-I expression is stratified by two critical transcriptional elements in osteoblasts, which are resolved by the individual and combined effects of hypoxic stress and stress hormones. In so doing, hypoxia suppresses Runx2, limits the enhancing influence of PGE2, and interacts with glucocorticoid to reduce IGF-I expression by osteoblasts.Gene 01/2014; · 2.20 Impact Factor
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ABSTRACT: Hypoxia and lymphangiogenesis are closely related processes that play a pivotal role in tumor invasion and metastasis. Intratumoral hypoxia is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells, insufficient blood supply and poor lymph drainage. Hypoxia induces functional responses in lymphatic endothelial cells (LECs), including cell proliferation and migration. Multiple factors (e.g., ET-1, AP-1, C/EBP-δ, EGR-1, NF-κB, and MIF) are involved in the events of hypoxia-induced lymphangiogenesis. Among them, HIF-1α is known to be the master regulator of cellular oxygen homeostasis, mediating transcriptional activation of lymphangiogenesis via regulation of signaling cascades like VEGF-A/-C/-D, TGF-β and Prox-1 in experimental and human tumors. Although the underlying molecular mechanisms remain incompletely elucidated, the investigation of lymphangiogenesis in hypoxic conditions may provide insight into potential therapeutic targets for lymphatic metastasis.Cancer letters 12/2013; · 4.86 Impact Factor
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ABSTRACT: Background Glucocorticoid functions are markedly impaired in patients with chronic obstructive pulmonary disease (COPD). The phosphodiesterase 4 inhibitor roflumilast N-oxide (RNO) is the active metabolite of roflumilast approved as a treatment to reduce the risk of exacerbations in patients with severe COPD. Objective We sought to characterize the differential effects of RNO versus corticosteroids and their potential additive/synergistic effect in neutrophils from patients with COPD, thus providing scientific rationale for the combination of roflumilast with corticosteroids in the clinic. Methods Peripheral blood neutrophils were isolated from patients with COPD (n = 32), smokers (n = 7), and healthy nonsmokers (n = 25). Levels of IL-8, matrix metallopeptidase 9 (MMP-9), and biomarkers of glucocorticoid resistance were determined by using ELISA and RT-PCR. Neutrophils were incubated with dexamethasone (0.1 nmol/L to 1 μmol/L), RNO (0.1 nmol/L to 1 μmol/L), or the combination of 1 nmol/L RNO plus 10 nmol/L DEX and stimulated with LPS (1 μg/mL) or cigarette smoke extract 5%; levels of IL-8, MMP-9, and other biomarkers were measured at the end of the incubation period. Results Peripheral neutrophils from patients with COPD showed a primed phenotype with an increased basal release of IL-8 and MMP-9 and expressed a corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase δ, macrophage migration inhibitory factor, and glucocorticoid receptor β expression and a decrease in HDAC activity and mitogen-activated protein kinase phosphatase 1 expression. RNO demonstrated robust anti-inflammatory effects on neutrophils from patients with COPD, reversing their resistance to corticosteroids. The combination of RNO and dexamethasone showed additive/synergistic effects, which were consistent with the reversal of corticosteroid-resistant molecular markers by RNO. Conclusion RNO reverses corticosteroid resistance and shows strong anti-inflammatory effects alone or in combination with corticosteroids on neutrophils from patients with COPD.The Journal of allergy and clinical immunology 01/2014; · 12.05 Impact Factor
The Journal of Immunology
Macrophage Migration Inhibitory Factor Counterregulates
Dexamethasone-Mediated Suppression of Hypoxia-Inducible
Factor-1a Function and Differentially Influences Human
CD4+T Cell Proliferation under Hypoxia
Timo Gaber,*,†,‡Saskia Schellmann,*,†Kerem B. Erekul,* Monique Fangradt,*,†
Karolina Tykwinska,*,†Martin Hahne,*,†,xPatrick Maschmeyer,*,†,‡Markus Wagegg,*,†,‡
Cindy Stahn,*,†Paula Kolar,*,†Rene ´ Dziurla,* Max Lo ¨hning,*,†Gerd-Ru ¨diger Burmester,*
and Frank Buttgereit*,†,‡
Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration in-
hibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcrip-
tion factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate
MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary
human CD4+T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and
the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4+T cells. In this article, we show
that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4+T cells, as well as in
Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1a expression and HIF-1 target gene
induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation.
Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-
mediated suppression of MIF and HIF-1a expression. Based on these data, we suggest that hypoxia significantly affects the
expression of HIF-1a in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4+T cells, thus
influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF
could be useful targets to optimize GC therapy when treating inflammation.
a result of an injuredvascular network, tumor cell progression, and/
The Journal of Immunology, 2011, 186: 764–774.
common feature of injured tissues, tumors, and inflam-
mation is the change in oxygen tension toward hypoxia:
oxygen supply is unable to meet the cellular demand as
or distinct influx of metabolically active inflammatory cells (1). In
response to hypoxia, major transcriptional changes occur leading
to cellular adaptation processes that are controlled by the trans-
cription factor hypoxia-inducible factor (HIF)-1. HIF-1 is a het-
erodimeric protein that is composed of an oxygen-sensitive a
subunit and a constitutively expressed b subunit. In nonhypoxic
cells, HIF-1a is tagged by O2-dependent hydroxylation via prolyl
hydroxylases (PHDs)1–3, prior to binding of the von Hippel–
Lindau tumor-suppressor protein (pVHL), which targets HIF-
1a for proteasomal degradation. Another mechanism of inhibiting
HIF-1a function is mediated by factor inhibiting HIF (FIH),
which prevents the transcriptional activation of HIF-1a by block-
ing interaction with the coactivators p300 and CREB binding
protein (2). However, under hypoxia, HIF-1 ensures cellular ad-
aptation and functional integrity by inducing/enhancing the syn-
thesis of proteins, such as erythropoietin, glucose transporter
type 1, glycolytic enzymes (PGK1 and LDHA), vascular endothe-
lial growth factor (VEGF), and matrix metalloproteinases, which
are implicated in angiogenesis, energy metabolism, apoptosis, me-
tastasis, and invasion (2). Thus, HIF-1 links cancer and inflam-
Macrophage migration inhibitory factor (MIF) is a multifunc-
tional protein involved in a broad range of inflammatory activities.
MIF counteracts the anti-inflammatory effects of glucocorticoids
(GCs), participates in the regulation of cell proliferation and dif-
ferentiation, and plays a role in the progress of septic shock, chro-
nicinflammation, tissuedamage, andautoimmunediseases (3).Be-
cause inflammation is a critical component of tumor progression,
*Department of Rheumatology and Clinical Immunology, Charite ´ University Hospi-
tal, 10117 Berlin, Germany;†German Rheumatism Research Center, 10117 Berlin,
Germany;‡Berlin-Brandenburg Center of Regenerative Therapies, 13353 Berlin, Ger-
many; andxBerlin-Brandenburg School of Regenerative Therapies, 13353 Berlin,
1T.G. and S.S. contributed equally to this article.
Received for publication October 20, 2009. Accepted for publication November 12,
This work was supported by Research Grant 01GS0110/01GS0160/01GS0413
through the National Genome Research Network from the German Federal Ministry
of Education and Research (to T.G.) and by the Berlin-Brandenburg Center of Re-
generative Therapies (to T.G., M.H., P.M., and M.W.).
Address correspondence and reprint requests to Dr. Timo Gaber, Department of
Rheumatology and Clinical Immunology, Charite ´ University Hospital, Charite ´platz
1, 10117 Berlin, Germany. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: ChIP, chromatin immunoprecipitation; DEX, dexa-
methasone; DRFZ, German Rheumatism Research Center; FIH, factor inhibiting
hypoxia-inducible factor; GC, glucocorticoid; GCR, glucocorticoid receptor; GIF,
glycosylation-inhibiting factor; HIF, hypoxia-inducible factor; HRE, hypoxia re-
sponse element; MIF, macrophage migration inhibitory factor; mTOR, mammalian
target of rapamycin; PBA, PBS/BSA/azide; PHA-L, phytohemagglutinin-L; PHD,
prolyl hydroxylase; pVHL, von Hippel–Lindau tumor-suppressor protein; qPCR,
quantitative real-time PCR; rh, recombinant human; shRNA, short hairpin RNA;
VEGF, vascular endothelial growth factor.
MIF represents an important link between inflammation and tumor
biology, similar to HIF.
The functional relationship between HIF-1 and MIF has been
investigated in human cancer cell lines and in murine cancer mod-
els, indicating an HIF-dependent, as well as an HIF-independent,
induction of MIF (4, 5) and an indirect protein interaction of HIF
and MIF (6, 7). In our study, we investigated the HIF/MIF inter-
action, focusing on nontransformed primary human cells of the
adaptive immune response; additionally, we addressed the effect
of GCs under hypoxic conditions on this relationship.
GCs represent avery important and frequently used class of anti-
inflammatory and immunosuppressive drugs. Our understanding of
the action of GCs has advanced in the last few years with regard to
recent insights into signaling, regulation of transcription processes,
and gene expression (genomic GC effects); dosage–plasma level–
effect relationships; nongenomic GC effects; and new GC receptor
(GCR) ligands (8, 9). However, there is only scattered information
available on their effects under conditions of restricted oxygen
availability. This is surprising because this topic is of clinical im-
portance for the reasons given above. Controversial results obtained
from tumor cell lines with known defects in cell-signaling trans-
duction and transcriptional regulation point to a cell type-specific
GC action on HIF-1–mediated function under hypoxia (10–12).
To our knowledge, this is the first report of a functional re-
lationship between MIF and HIF-1 in primary human Th cells,
with focus on the interplay with the synthetic GC dexamethasone
cell adaptation toward hypoxic areas (e.g., tumors, inflamed and
injured tissues), which may be important with respect to impaired
tissue regeneration, such as wound healing after GC therapy (13).
Based on our findings, we suggest that hypoxia affects CD4+
Th cell function and regulation of HIF-1a expression in a MIF-
dependent manner, thus influencing the IL-2–mediated lympho-
proliferative response and DEX action via the GCR.
Materials and Methods
Abs and reagents
DEX, the GCR antagonist RU486, and phytohemagglutinin-L (PHA-L)
were purchased from Sigma-Aldrich. For immunoblotting, mouse anti–
HIF-1a mAb and anti–b-actin were purchased from BD Transduction
Laboratories and Sigma-Aldrich. Anti-MIF and anti–HIF-2a were pur-
chased from R&D Systems. Mouse anti-Jun B and anti–NF-kB(p65)
mAbs and polyclonal goat anti-lamin B Ab were purchased from Santa
Cruz Biotechnologies. Secondary HRP-labeled Abs, goat anti-mouse IgG,
and donkey anti-goat IgG were obtained from Promega. For chromatin
immunoprecipitation (ChIP), rabbit polyclonal anti-HIF-1a Ab was pur-
chased from Abcam. For in vitro assays, recombinant human (rh)MIF was
purchased from R&D Systems, and anti-CD74 blocking Ab was obtained
from Santa Cruz Biotechnologies. For surface staining of CD3, CD4,
CD25, and CD74, anti-human CD4-FITC and anti-human CD3-PE were
obtained from the German Rheumatism Research Center (DRFZ), and
anti-human CD25-allophycocyanin and anti-human CD74-Cy5 were pur-
chased from ImmunoTools and Santa Cruz Biotechnologies; the latter was
labeled at DRFZ. For intracellular staining of IL-2, FITC-conjugated anti-
human IL-2 was obtained from BD Pharmingen.
Cell isolation and cell culture
Human CD4+T cells (.99% purity and .95% viability) were prepared as
described previously (14). Cells were resuspended in RPMI 1640 sup-
plemented with 10% (v/v) heat-inactivated FCS (Sigma-Aldrich), 100 U/
ml penicillin G, 100 mg/ml streptomycin (both from PAA Laboratories),
and 50 mM 2-ME (Sigma-Aldrich).
Induction of hypoxia and stimulation
T cells were incubated in a hypoxic chamber (Binder) at 5% CO2and ,1%
O2, balanced with N2. Normoxic controls were incubated at 5% CO2in
a humidified atmosphere with 18% O2. Mitogen stimulation was done
using 5 mg/ml PHA-L (Sigma-Aldrich).
For the analysis of rapid GC effects, CD4+T cells were incubated in
a water-jacket chamber sealed with a Clark-type oxygen electrode (Strath-
kelvin Instruments, North Lanarkshire, U.K.), which continuously moni-
tored cellular oxygen consumption as a decrease of the oxygen concen-
tration. The Clark-type oxygen electrode has a small slot for introduction of
a 22-gauge needle, which enabled us to apply drugs and obtain samples
without influencing the oxygen concentration of the analyzed cell suspen-
sion. Detailed information is given in Supplemental Fig. 1.
RNA isolation and quantitative real-time PCR
by reverse transcription using TaqMan Reverse Transcription Reagents
(Applied Biosystems). Quantitative real-time PCR (qPCR) was carried out
using the LightCycler Fast-Start DNA Master SYBR Green I Kit (Roche).
Data were normalized to the expression of b-actin (ACTB). As a second
“so-called” housekeeping gene, HPRT was tested to verify the accuracy of
normalization. All primers used were obtained from TIB Molbiol (Table I).
Immunoblot of HIF-1a, MIF, lamin B, and b-actin
Cell lysis. For whole-cell extracts of T cells, 106cells were lysed in 20 ml
Laemmli buffer. For the preparation of nuclei, the Nuclear Extract Kit from
Active Motif was used on T cells, according to the manufacturer’s in-
Immunodetection of proteins.Twenty microlitersof whole-cellextract or 10
mg nuclear/cytoplasmic fraction was separated by SDS-PAGE and blotted
onto polyvinylidene difluoride membranes (Millipore). Blotted proteins
were analyzed, as indicated, and visualized by enzymatic chemilumines-
cence (Amersham Biosciences).
Quantification of cytokine production
Intracellular cytokine staining. Intracellular cytokine staining was per-
formed based on the paraformaldehyde-saponin procedure (15). After
PHA stimulation of CD4+T cells and 66 h of hypoxic incubation, the cells
were restimulated for an additional 6 h with 10 ng/ml PMA and 1 mg/ml
ionomycin. Finally, protein transport was blocked by adding brefeldin A at
10 mg/ml for the final 3 h to obtain intracellular cytokine accumulation.
Cells were washed with PBS and fixed for 20 min at room temperature
using 2% (v/v) formaldehyde (Merck KGaA). Cells were stained at room
temperature for 15 min in PBS/BSA/azide (PBA) with 0.5% (w/v) saponin
(Sigma-Aldrich). After blocking nonspecific binding with 5 mg/ml poly-
clonal human IgG (Grifols), we stained the cells for 15 min using 2.5 mg/
ml FITC-conjugated anti-human IL-2 (clone MQ1-17H12, isotype rIgG2a;
BD Pharmingen). The cells were washed with PBA/saponin and then
stored in PBA until FACS analysis.
Secreted MIF. Culture supernatants of CD4+T cells (106cells/ml), incubated
under normoxic or hypoxic conditions, were immediately frozen and stored
at 270˚C. Secreted MIF was quantified by multiplex suspension array (Bio-
Quantification of proliferation
CFSE (Molecular Probes Europe, Leiden, The Netherlands) was used to
measure the quantitative extent of proliferation in the PHA-L–activated
CD4+T cells. The cells were labeled before activation in a 1.5-mM so-
lution for 3.5 min at room temperature. Cell activation was performed
using PHA-L (5 mg/ml).
Lentiviral-based short hairpin RNA-mediated knockdown of
Based on pLentiLox 3.7 (Addgene plasmid 11795), short hairpin RNA (shRNA)
II), as previously described (16). Lentiviral stocks were obtained by calcium
phosphate cotransfection of HEK293 cells with the lentiviral packaging
plasmids pVSVG and pPAX2. The medium was replaced after 4 h, and viral
supernatants were collected 48–72 h later. Jurkat T cells were infected by
polybrene (Sigma-Aldrich), followed by replacement of the viral supernatant
with fresh, fully supplemented culture medium after 4 h. shRNA construct-
containing cells were enriched via coexpressed GFP using flow cytometry
(.95% GFP+). Enriched cells were used for 20-h hypoxic/normoxic in-
cubation using the hypoxic incubator (Binder).
HIF-1 promoter activity
shRNA construct-containing Jurkat T cells were transfected with 1 mg/
transfection Cignal HIF reporter plasmid mixture (Cignal HIF Reporter
The Journal of Immunology 765
[luc] Kit: CCS-007L; SA Biosciences). The HIF reporter plasmid mix-
ture contains an HIF-responsive luciferase construct premixed in a 40:1
ratio with a constitutively expressing Renilla luciferase construct. The
HIF-responsive luciferase construct encodes the firefly luciferase reporter
gene under the control of a minimal CMV promoter and an optimized
number of tandem repeats of the hypoxia response element (HRE). The
Renilla luciferase construct serves as an internal control for normalizing
transfection efficiencies and monitoring cell viability.
Transfection was performed using nucleofection (Amaxa), according to
the manufacturer’s instructions for Jurkat T cells (transfection reagent C;
program C-016; Nucleoporator II). After 48 h of transfection, cells were
incubated under hypoxia (1% O2) for 20 h. Dual Luciferase assay (Pro-
mega) was performed, according to the manufacturer’s instructions. Pro-
moter activity values are expressed as arbitrary units using a Renilla re-
porter for internal normalization.
CD4+T cells were incubated under hypoxic conditions for 20 h. ChIP
assay was performed, according to the manufacturer’s instructions (Upstate).
The HIF-1–DNA fragments were precipitated by polyclonal HIF-1a rabbit
Ab (Abcam). After removal of cross-linking, PCRs were performed with
50 ng DNA by initial denaturation of DNA at 95˚C for 3 min, followed by
40amplification cycles(95˚C for 20 s, 63.7˚Cfor 15 s, and 72˚C for 30 s) and
final elongation at 72˚C for 10 min. The primer sets used were MIF forward:
59-GCGGTGACTTCCCCACTC-39 and MIF reverse: 59-ATGGCAGAAG-
GACCAGGAG-39 (chr22: 22,566,483–22,566,660).
Data are reported as the mean 6 SD of at least three experiments. Differ-
ences between normally distributed groups were compared using the Stu-
dent t test, and thosein nonnormally distributedgroupswere comparedwith
the Mann–Whitney U test for independent groups and with the Wilcoxon t
test for dependent samples. Multiple comparisons were analyzed by one- or
two-way ANOVA, as indicated, with the Bonferroni multiple-comparison
post hoc test. The p values ,0.05 were considered statistically significant.
HIF-1a and HIF-1 target genes are induced under hypoxia in
primary human CD4+T cells and Jurkat T cells
To clarify the relationship between HIF and MIF in T lympho-
cytes, we first analyzed whether Th cells are capable of express-
ing HIF-1a, the master regulator of the hypoxic response, under
hypoxia alone or only upon hypoxia plus mitogenic stimulation.
Therefore, we cultured isolated peripheral blood CD3+CD4+
T cells, untreated or treated with PHA, under normoxic or hypoxic
conditions. At the transcriptional level, we found no significant
differences in the amounts of HIF-1a mRNA (HIF1A) after 6 h or
even after 48 h under hypoxia compared with normoxia (Fig. 1B,
Supplemental Fig. 2). At the protein level, we observed an in-
duction of HIF-1a in quiescent cells and a translocation of HIF-1a
to the nucleus, as shown by immunoblot (Fig. 1A). Upon PHA
the nucleus and in the cytoplasmic fraction (Fig. 1A). We also ob-
served that this increase in HIF-1a expression in the nucleus under
hypoxia resulted in a significant induction of HIF-1 target gene
transcription, such as shown for GLUT1, LDHA, PGK1, and VEGF
(Fig. 1B). In contrast, HIF-2a protein was not expressed in unsti-
mulated or PHA-stimulated primary human CD4+T cells under
normoxic or hypoxic incubation conditions (Supplemental Fig. 3).
in A, mRNAwas obtained and analyzed for HPRTand HIF1A, as well as HIF-1 targets GLUT1, LDHA, PGK1, and VEGF by qPCR normalized to ACTB and
normoxia (mean 6 SD; normoxia = 1 as indicated by the dashed line; n = 4). C, Jurkat T cells were incubated for 20 h under normoxic or hypoxic conditions.
Nuclear extracts (NE) and cytosolic extracts (CE) obtained were blotted and analyzed by immunodetection for HIF-1a and b-actin (one of two independently
normalized to ACTB and normoxia (mean 6 SD; normoxia = 1 as indicated by the dashed line; n = 4). *p , 0.05, **p , 0.01, ***p , 0.001; one-sample t test.
HIF-1a and HIF-1 target genes are induced under hypoxia in primary human CD4+T cells and Jurkat T cells. A, Primary human CD4+T cells
766RELATIONSHIP AMONG HIF-1a, MIF, AND GCR IN Th CELLS
1a protein (Fig. 1C) and HIF-1 target genes GLUT1, LDHA,
PGK1, and VEGF after 20 h under hypoxia (Fig. 1D). The latter
finding enabled us to use Jurkat T cells as a model for in-depth
analysis of the HIF-1 response in some of the experiments. Fur-
thermore, VEGF was chosen as the representative HIF-1 target
gene (primer sets used are given in Table I).
MIF induction under hypoxia in primary human CD4+T cells
and Jurkat T cells is mediated via HIF-1a
Next, we analyzed whether this induction of HIF under hypoxia
was accompanied by an induction of MIF, as already shown in tumor
cells (4, 6, 17). Under hypoxia, Th cells significantly (up to 2-fold)
upregulated MIF mRNA levels after 6 h when left unstimulated
and up to 4-fold with TCR engagement compared with normoxic
mediated upregulation of MIF mRNA increased up to 4-fold with-
out stimulation and up to 10-fold with PHA stimulation, in a time-
dependent manner, after 48 h (Supplemental Fig. 4). In addition,
hypoxia upregulated MIF mRNA levels after 20 h in Jurkat T cells
compared with normoxia (p = 0.0053; Fig. 2B). With regard to
the protein level, we observed a pronounced increase in secreted
MIF after 24 h (Fig. 2C). Focusing on the HIF-1–MIF relationship,
we analyzed whether HIF-1 induced MIF in human T cells. Using
shRNA-mediated RNA interference (Table II) in a T cell model
(Jurkat T cells), we efficiently knocked down HIF-1a on the
mRNA (p , 0.0001) and protein levels (Fig. 2D, 2F). The loss of
HIF-1a efficiently blocked HRE-mediated reporter gene activity
(Fig. 2G). Furthermore, loss of HIF-1a resulted in a significantly
reduced HIF target gene expression of GLUT1, LDHA, PGK1
(data not shown), and VEGF (p , 0.0001; Fig. 2H), as well as
transcript expression of MIF, after 20 h (p , 0.0001; Fig. 2E).
To verify that HIF-1 is responsible for the induction of MIF in pri-
binds to the proximal promoter region of MIF (Fig. 2I). In addition,
HIF-1a knockdown led to a reduction in MIF at the protein level
MIF is a key regulator of HIF-1a and HIF-1 target gene
expression in primary human CD4+T cells and Jurkat T cells
Because MIF is a cellular factor released from cells, we next in-
vestigated the impact of rhMIF on primary human CD4+T cells.
Addition of rhMIF (100 ng/ml) led to an increase in HIF-1a expres-
sion after20 h ofincubation underhypoxia (Fig. 3A, 3B). Incontrast,
inhibition of MIF action, by the addition of the small-molecule
inhibitor of MIF (ISO-1; 10 mM), or blocking MIF by adding anti–
MIF-IgG (2 mg/ml) resulted in reduced HIF-1a protein expression,
but it did not influence HIF-1a transcript abundance (Supplemental
Fig. 2). In addition, we knocked down MIF expression by shRNA-
mediated RNA interference (Table II) in Jurkat T cells at the mRNA
(Fig. 3C) and protein levels (Fig. 3G). MIF knockdown did not re-
sult in a downregulation of HIF-1a mRNA (Fig. 3D), but it signifi-
cantly reduced HIF-1a protein expression (Fig. 3G), HIF-1 reporter
gene assay activity (Fig. 3E), and HIF-1 target gene expression of
down MIF expression re-established HIF-1a protein expression
MIF stimulates HIF-1a expression via CD74 under hypoxia
involving ERK/mammalian target of rapamycin activity
complemented by PI3K upon mitogen stimulation
MIF was demonstrated in cell lines (THP-1, Raji) and murine and
human monocytes/macrophages to act via binding to the MHC
class II invariant chain CD74 (18). Therefore, we first confirmed
the expression of CD74 on primary human CD4+T cells (Fig.
3H). Second, by blocking CD74 using an anti–CD74-IgG during
hypoxia (20 h) before hypoxic treatment, we reduced the amount
of HIF-1a protein expression in CD4+T cells in vitro (Fig. 3I).
The reduction in HIF-1a protein expression was not due to a re-
duced mRNA level (data not shown). Furthermore, addition of
rhMIF (100 ng/ml) did not abolish this effect.
It was shown that HIF-1a protein synthesis is upregulated mainly
via the PI3K/mammalian target of rapamycin (mTOR) pathway
(19) and that MIF induces angiogenesis and HIF-1a in a MAPK-
dependent manner in the MCF-7 cell line (6, 20). Therefore, we an-
alyzed these pathways by using specific inhibitors (Fig. 3J). We first
examined the effect of the specific mTOR inhibitor rapamycin on
HIF-1a accumulation in peripheral blood CD4+T cells. Rapamycin
completely inhibited the induction of HIF-1a protein expression in
quiescent and mitogen-stimulated T cells under hypoxic conditions.
The same effect was observed using the MEK inhibitor U0126. In
contrast, the PI3K inhibitor Ly294002 inhibited the hypoxic induc-
tion of HIF-1a protein expression in mitogen-stimulated T cells only.
The observed effects on HIF-1a protein expression were not due to
Table I.Primer sets for qPCR
Gene SymbolGene NameForward PrimerReverse Primer
Lactate dehydrogenase A
Macrophage migration inhibitory
Phosphoglycerate kinase 1
Vascular endothelial growth
Factor inhibiting hypoxia-
von Hippel–Lindau tumor-
GC receptor a
HIF prolyl hydroxylase 1
HIF prolyl hydroxylase 2
HIF prolyl hydroxylase 3
The Journal of Immunology767
increased cell death or apoptosis after application of the respective
pathway inhibitors (Supplemental Fig. 5), and they likely did not
result from modifying the expression of short-lived proteins, which
was controlled for by NFkBp65 or Jun B (Supplemental Fig. 6).
Therefore, we assumed that ERK/mTOR is the major pathway
regulating HIF-1a protein synthesis in T cells, independent of
activation state, whereas the PI3K pathway complements ERK/
mTOR signaling in PHA-engaged T cells.
incubated for 6 h under normoxicor hypoxic conditions and stimulatedwith PHA-L (5 mg/ml)or left untreated(A), and Jurkat T cells were incubatedfor 20 h
undernormoxicorhypoxicconditions(B).mRNAlevels ofMIFand HPRT,asa secondhousekeepinggene,wereanalyzedby qPCRandnormalized toACTB
andnormoxia(mean 6SD;normoxia=1 asindicated bythedashed line).**p ,0.01,***p ,0.001;one-sample t test.C,MIF secretedfrom primaryhuman
CD4+T cells incubated for 24 h under normoxic or hypoxic conditions and stimulated as in A (n = 4). *p , 0.05, **p , 0.01; two-way ANOVA with the
Bonferroni multiple-comparison post hoc test. mRNA obtained from anti–HIF-1a–shRNA-vector(pLL)–transfected Jurkat T cells was analyzed by qPCR for
HIF-1a(D)andMIFtranscriptabundancenormalizedto mRNAobtainedfromcontrol(scr)-shRNA-vector-transfectedJurkatTcells (E)(mean6SD;n=4).
***p , 0.001; one-way ANOVAwith Bonferroni multiple-comparison post hoc test. F, Whole-cell extracts obtained from anti–HIF-1a– and control (scr)-
shRNA–vector-transfected Jurkat T cells were analyzed by immunodetection for HIF-1a, b-actin, and MIF after 20 h of incubation under hypoxia (one of
three independently performed experiments). G, Analysis of HRE-driven reporter gene activity (normalized to constitutively expressed Renilla luciferase) in
anti–HIF-1a/control-shRNA–vector-transfected Jurkat T cells (mean 6 SD). ***p , 0.001; two-way ANOVAwith the Bonferroni multiple-comparison post
hoc test. H, qPCR analysis of mRNA obtained as in D and E for HIF-1 target gene expression of VEGF (mean 6 SD). ***p , 0.001; one-way ANOVAwith
Bonferroni multiple-comparison post hoc test. I, ChIP of HIF-1a of cell extracts from primary human Th cells was analyzed by PCR for the proximal
promoter region of MIF on chromosome 22 region 22,566,483–22,566,660. H2O, no template control; PCR control, human genomic DNA; ChIP control,
unspecific rabbit IgG (all after 20 h of hypoxic incubation without stimulation; one of three independently performed experiments).
MIF induction under hypoxia is mediated via HIF-1a in primary human CD4+T cells and Jurkat T cells. Primary human CD4+T cells were
Table II. Sequences of cloned short hairpin oligo nucleotides (backbone: pLL)
Construct Name Short Hairpin Oligonucleotides
59-T gCTATCgAgAAgATCAgCC TTCAAgAgA ggCTgATCTTCTTTAgC TTTTTTC-39
59-T CCgCTggAgACACAATCATAT TTCAAgAgA ATATgATTgTgTCTCCAgCgg TTTTTTC-39
59-T CCAgTTATgATTgTgAAgTTA TTCAAgAgA TAACTTCACAATCATAACTgg TTTTTTC-39
59-T CCGATGTTCATCGTAAACA TTCAAGAGA TGTTTACGATGAACATCGG TTTTTTC-39
59-T AACAACTCCACCTTCGCCTAAGA TTCAAGAGA TCTTAGGCGAAGGTGGAGTTGTT TTTTTTC-39
Target (nontarget in case of pLL-scr) sequences are underlined.
768 RELATIONSHIP AMONG HIF-1a, MIF, AND GCR IN Th CELLS
MIF impact on CD4+T cell proliferation depends on oxygen
We then asked what functional effects extracellular MIF has on
human CD4+T cells under hypoxic conditions. Therefore, we
analyzed activation-induced proliferation and CD25 expression
under normoxic and hypoxic conditions. Under normoxic con-
ditions, CD4+T cell proliferation was significantly reduced when
MIF signaling was blocked via CD74 (p , 0.001), whereas the
addition of rhMIF did not influence T cell proliferation (Fig. 4A,
4B). In contrast, under hypoxia, PHA-stimulated CD4+T cell
proliferation was significantly reduced compared with normoxia
(p , 0.05), and it remained almost unaffected when MIF signaling
was blocked via CD74 (compared with PHA-stimulated hypoxic
CD4+T cells). Furthermore, the addition of rhMIF significantly
reduced T cell proliferation compared with PHA-stimulated CD4+
T cells under hypoxia (Fig. 4B). These findings correlated with an
altered frequency of CD25-expressing CD4+T cells and CD25
surface expression (Fig. 4C, 4D). Under normoxia, the proportion
of CD25-expressing CD4+T cells upon PHA stimulation was
significantly enhanced when CD74 was blocked, whereas the ad-
dition of rhMIF had no effect. In contrast, under hypoxia, PHA
stimulation resulted in a significant increase in CD25-expressing
CD4+T cells. Blocking CD74 and treatment with rhMIF did not
significantly influence CD25 expression under hypoxia.
Interestingly, when blocking CD74 under normoxia, surface ex-
pression of CD25 by PHA-activated CD4+T cells increased signif-
icantly (Fig. 4D). Hypoxia alone also increased the surface ex-
pression of CD25 by PHA-stimulated CD4+T cells, which was
decreased when CD74 was blocked and increased when rhMIF was
added. With respect to IL-2 production by activated Th cells, we
observed a significant reduction in IL-2–producing PHA-activated
Hypoxia alone significantly decreased the frequency of IL-2–pro-
ducing PHA-stimulated CD4+T cells, which was further reduced
when rhMIF was added. In contrast to normoxia, the frequency of
obtained from primary human Th cells that were treated with the MIF inhibitor ISO-1 (10 mM), anti–MIF-IgG, or rhMIF (100 ng/ml) and incubated for 20 h
under hypoxic conditions were analyzed by immunodetection for HIF-1a and b-actin (one of at least three independently performed experiments). mRNA
obtained from anti–MIF-shRNA–vector(pLL)-transfected Jurkat T cells was analyzed by qPCR for MIF (C) and HIF-1a (D) transcript abundance nor-
malized to mRNA obtained from control (scr)-shRNA–vector-transfected Jurkat T cells (mean 6 SD; n = 4). ***p , 0.001; one-way ANOVA with
Bonferroni multiple-comparison post hoc test. E, Analysis of HRE-driven reporter gene activity (normalized to constitutive expressed Renilla luciferase) in
anti-MIF/control-shRNA–vector-transfected Jurkat T cells (mean 6 SD). ***p , 0.001; two-way ANOVA with Bonferroni multiple-comparison post hoc
test. F, qPCR analysis of mRNA obtained as in C and D for HIF-1 target gene expression of VEGF. **p , 0.01, ***p , 0.001; one-way ANOVA with
Bonferroni multiple-comparison post hoc test. G, Whole-cell extracts obtained from anti–MIF-shRNA–vector(pLL)-transfected Jurkat T cells were ana-
lyzed by immunodetection for HIF-1a, b-actin, and MIF after a 20-h incubation under hypoxia treated previously with rhMIF (100 ng/ml) as indicated (one
of at least three independently performed experiments). H, Analysis of CD74 expression on primary human CD4+T cells after a 20-h hypoxic or normoxic
incubation without stimulation by flow cytometry (unstained and block control from normoxic CD4+T cells; one of at least three independently performed
experiments). I, Whole-cell extracts obtained from primary human Th cells pretreated with anti–CD74-IgG (2 mg/ml) or rhMIF (100 ng/ml) after a 20-h
incubation under hypoxia were analyzed by immunodetection for HIF-1a and b-actin (one of at least three independently performed experiments). J,
Whole-cell extracts obtained from primary human Th cells pretreated with rapamycin (25 mM), U0126 (25 mM), or Ly294002 (10 mM) after a 20-h
incubation under hypoxia were analyzed as described in I.
MIF acts via CD74 and is a key regulator of HIF-1a stabilization and HIF-1 target gene expression. A and B, Whole-cell extracts that were
The Journal of Immunology769
IL-2–producing PHA-stimulated CD4+T cells did not decrease
when CD74was blocked compared withthe corresponding control.
These data suggested that MIF may play a dual role when
influencing Th cell proliferation via CD74 by maintaining/en-
hancing T cell proliferation via IL-2 under normoxia but not un-
der hypoxia. In addition, we assumed that the increase in CD25-
expressing Th cells and CD25 expression per cell may be due
to a feedback loop to overcome inhibition of IL-2–producing
T cells and T cell proliferation.
DEX dose dependently abrogates hypoxia-induced HIF-1a
Keeping in mind thatMIFacts inconcert with GCs tocontrol T cell
activation [e.g., MIF antagonizes GC-mediated inhibition of T cell
proliferation, as shownby Bacher et al. (21)], we studied the effects
of the GC DEX on the expression of HIF-1a. In untreated and
PHA-stimulated Th cells, DEX dose dependently abrogated HIF-
1a expression under hypoxic conditions at the protein level but
not at the mRNA level (Fig. 5A, 5B). This inhibition of the HIF-1a
protein expression was blocked by the GCR antagonist RU486.
Thus, the inhibitory effect of DEX on HIF-1a protein expression
is mediated through the GCR. Furthermore, antagonizing DEX in
PHA-activated Th cells abolished the inhibition of HIF-1a protein
expression (Fig. 5A). Suppression of HIF-1a by DEX resulted in
a significant downregulation of HIF-1 target gene induction (e.g.,
VEGF) under hypoxia in a GCR-dependent manner (Fig. 5C).
Abrogation of HIF-1a by DEX, as well as its induction by the
combination of DEX and RU486, in PHA-treated Th cells did not
result from the induction or repression of inhibitors of HIF-1a,
such as pVHL, FIH, and PHD1-3. Transcript expression of the
molecules increased slightly as a result of hypoxia but remained
independent of GC treatment, with the exception of mitogen-
stimulated hypoxia-induced PHD1 expression (Fig. 5E–G). How-
ever, stimulated hypoxia-induced PHD1 expression decreased
after DEX treatment, which was blocked by the combination of
DEX and RU486 (Fig. 5G). Therefore, the dose-dependent regu-
lation of PHD1 by DEX did not contribute to the abrogation of
HIF-1a by DEX or its induction by the combination of DEX and
RU486. Moreover, PHD1 is regulated in a HIF-1 target gene
fashion, such as observed for VEGF (Fig. 5C).
Hypoxia also increased the expression of GCR mRNA, whereas
GC treatment, as well as antagonizing GCR binding by DEX, in-
creased GCR mRNA in quiescent cells but not in PHA-sti-
mulated Th cells (Fig. 5D). Therefore, the reduction in HIF-1a
expression might be a rapid event.
To analyze the rapid effects of DEX on established HIF-1a
expression in primary CD4+T cells under a stable hypoxic envi-
ronment, we used a Clark-type electrode within a sealed chamber;
this enabled us to induce hypoxia in the T cell suspension, con-
tinuously monitor the oxygen concentration of the T cell suspen-
sion (including a hypoxia phase), and add drugs and remove
samples via a fine slot for the introduction of a 22-gauge needle.
Under these controlled, stable, hypoxic conditions that were
achieved after cellular respiration had removed dissolved ox-
ygen, we found a very rapid reduction in HIF-1a expression 10
and 20 min postapplication of DEX, which was not observed
analyzed by flow cytometry after a 96-h normoxic or hypoxic incubation with and without PHA stimulation and initial treatment with anti–CD74-IgG (2 mg/
ml) or rhMIF (100 ng), as indicated. CD4+T cell proliferation is demonstrated as representative CFSE staining of a single donor after exclusion of dead cells
via propidium iodide staining (A) and the percentage of divided cells (B) (mean 6 SD; n = 6). Analysis of the frequency of CD25+CD4+T cell population (C)
and the amount of CD25 surface expression by flow cytometry (D) after a 96-h normoxic or hypoxic incubation with and without PHA stimulation and initial
treatment with anti–CD74-IgG (2 mg/ml) or rhMIF (100 ng), as indicated (mean 6 SD; n = 3). E and F, Analysis of the frequency of IL-2+CD4+T cell
populationbyflowcytometryaftera 72-hnormoxicorhypoxic incubationwithandwithoutPHAstimulationandinitialtreatmentwithanti–CD74-IgG(2 mg/
ml) or rhMIF (100 ng), as indicated. IL-2+CD4+T cells are shown as a representative graph of IL-2 staining of a single donor (E) and as a bar graph of four 4
experiments (F) (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001; two-way ANOVA with Bonferroni multiple-comparison post hoc test.
rMIFacts via CD74 anddifferentially influences T cell proliferationunder normoxiaand hypoxia. A and B, CFSEstaining ofCD4+T cells was
770RELATIONSHIP AMONG HIF-1a, MIF, AND GCR IN Th CELLS
with the vehicle control (DMSO) or the mixture of DEX and
RU486 (Fig. 5H). These results suggested that MIF may over-
come the DEX-mediated reduction in HIF-1a expression.
MIFovercomes DEX-mediated reduction in HIF-1a expression
We analyzed the effects of DEX at very high, but clinically rel-
evant, concentrations on MIF expression itself. DEX significantly
downregulated the expression of MIF at mRNA and protein levels
underhypoxia.Thiseffectwas efficiently blockedbythe GCantag-
onist RU486, suggesting a GCR-mediated signaling (Fig. 6A, 6B).
When adding increasing amounts of MIF to primary human CD4+
T cells prior to the addition of DEX at 1025M, we observed a
MIF-mediated counterregulation of the inhibitory effect of DEX
on HIF-1a expression (Fig. 6C) and on HIF-1 target gene indu-
ction, such as shown for VEGF (Fig. 6D). Blocking CD74 resulted
in a similar inhibitory effect on hypoxia-induced VEGF expres-
sion, which was not reversible by the addition of rhMIF (Fig. 6D).
Summarizing the data obtained, we propose a model of MIF-
mediated induction and DEX-mediated suppression of HIF-1a
expression as shown in Fig. 7.
Inflammation, autoimmune disorders, and tumorigenesis share
common features, such as hypoxia and the pathogenetic involve-
ment of HIF-1 and MIF. In this study, we comprehensively in-
vestigated the MIF/HIF relationship and the effect of GCs in
human primary nontumor CD4+Th cells and Jurkat T cells. The
following major findings emerged: Th cells are capable of in-
ducing HIF-1a and HIF-1 target genes under hypoxia with and
without PHA stimulation; the induction of MIF under hypoxia is
primary human Th cells treated with 1025, 1028, or 10211M DEX alone, in combination with the GCR antagonist RU486 (10 mM), or those left untreated
and incubated for 6 h under normoxic or hypoxic conditions were analyzed by immunodetection for HIF-1a and b-actin (one of at least three independently
performed experiments). B–G, Quiescent and PHA (5 mg/ml)-stimulated primary human CD4+T cells were incubated for 20 h under normoxic or hypoxic
conditions and treated with 1025M DEX alone or DEX in combination with RU486 (10 mM) or were left untreated. mRNA expression of HIF1A (B), HIF-
1 target gene VEGF (C), GCR (D), or suppressors of HIF-1a stabilization, including pVHL (E), FIH (F), and PHD1–3 (G) were analyzed by qPCR (mean 6
SD; n = 4). *p , 0.05, **p , 0.01, ***p , 0.001; two-way ANOVAwith the Bonferroni multiple-comparison post hoc test. H, Quiescent primary human
Th cells incubated for 6 h under hypoxic conditions were treated with 1025M DEX alone or DEX in combination with the GCR antagonist RU486 (10 mM)
or were left untreated. Whole-cell extracts were obtained after 10 and 20 min of drug treatment and analyzed by immunodetection for HIF-1a and b-actin
(one of two independently performed experiments).
DEX dose dependently abrogates hypoxia-induced HIF-1a. A, Whole-cell extracts obtained from quiescent and PHA (5 mg/ml)-stimulated
The Journal of Immunology771
part of the HIF-1 activity; MIF, in turn, is a key regulator of the
hypoxia-induced HIF-1a protein expression involving the MIF
receptor CD74, thus forming an autocrine positive-feedback loop
(Fig. 7); hypoxia-induced HIF-1a expression in resting T cells
involves ERK/mTOR activity, complemented by the PI3K path-
way upon mitogen stimulation; MIF influences IL-2 production/
signaling and Th cell proliferation via CD74: it maintains/
enhances T cell proliferation under normoxia but not under hyp-
oxia, where it instead seems to inhibit proliferation; and DEX
is able to abolish MIF and HIF expression rapidly in a dose-
dependent and GCR-dependent manner, which can be reversed by
extracellularly administered MIF.
In contrast to previous studies by Makino et al. (22) and
Nakamura et al. (19), we demonstrated that HIF-1a expression
and nuclear translocation are inducible under hypoxia, even with-
out mitogen stimulation or TCR engagement. This resulted in
a pronounced impact on HIF target gene expression in primary
human CD4+Th cells. In addition, a further increase in HIF-1a at
the protein level under PHA stimulation in primary Th cells and
Jurkat T cells confirmed our previous studies (23) (Fig. 1). We also
demonstrated that hypoxia alone is able to induce MIF expression
and secretion in primary nontransformed human Th cells, which is
further upregulated by mitogen stimulation (Fig. 2). This is of
clinical importance in several autoimmune diseases and in cancer
biology where MIF is highly expressed and was shown to trigger
the innate immune response and the adaptive immune response
(reviewed in Ref. 3). Furthermore, we provided evidence that HIF-
1 is responsible for the induction of MIF under hypoxia in human
T cells, as shown by HIF-1a knockdown in Jurkat T cells (Fig. 2).
This finding is supported by the study of Baugh et al. (4), who
showed that hypoxia-induced MIF expression is driven by HIF-1
in three tumor cell lines. However, hypoxia-induced HIF-1–
dependent MIF expression seems to be a cell type-specific feature
and remains controversial (5, 6).
To test the functional impact of MIF under hypoxia on HIF-1a
expression in primary Th cells, we blocked MIF signaling using
the MIF inhibitor ISO-1, as well as MIF-neutralizing Abs (Fig.
3A, 3B). We used shRNA-mediated RNA interference in Jurkat
T cells. Indeed, loss of MIF resulted in a clear inhibition of HIF-
1a protein expression, whereas rhMIF increased HIF-1a protein
expression in primary Th cells. Inhibition of MIF in Jurkat T cells
using RNA interference also efficiently reduced HIF-1a protein
production (Fig. 3C–G). Interestingly, we did not find any re-
duction in HIF-1a mRNA expression, but rather found a slight but
by qPCR (A), and secreted MIF was determined by multiplex suspension array (B) (mean 6 SD; n = 4). *p , 0.05, **p , 0.01; one-way ANOVA with
or DEX in combination with the indicated amounts of rhMIF or left untreated, were analyzed by immunodetection for HIF-1a and b-actin (one of at least
three independently performed experiments). D, Primary human CD4+T cells were incubated for 20 h under normoxic or hypoxic conditions. Th cells were
left untreated or were treated with 200 ng/ml rhMIF, 2 mg/ml anti-CD74-IgG, or 1025M DEX alone or in combination, as indicated. HIF-1 target gene VEGF
was analyzed by qPCR (mean 6 SD; normoxia = 1 as indicated by the dashed line; n = 3). *p , 0.05, **p , 0.01; one-sample t test.
DEX abrogates the hypoxia-inducedexpressionof theHIF-1 target MIF, which,inversely, cancounteractDEX-mediatedinhibitionof HIF-1a.
772 RELATIONSHIP AMONG HIF-1a, MIF, AND GCR IN Th CELLS
nonsignificant increase, indicating a MIF-dependent posttrans-
lational regulation of HIF-1a protein expression (Fig. 3). How-
ever, the reduction in HIF-1a protein expression could be reversed
by extracellularly applied MIF. The underlying mechanism seems
to be similar to that reported for the breast cancer cell line MCF-7
by Oda et al. (6). Nevertheless, to our knowledge, the results re-
present the first evidence of MIF-mediated HIF-1a regulation in
nontransformed primary Th cells.
In line with the notion that primary T cells behave like oncogenic
transformed cells in terms of MIF-dependent HIF-1a regulation, we
demonstrated that MIFacts through CD74 underhypoxia. Hypoxia-
mediated HIF-1a protein expression involves ERK/mTOR activity
in resting T cells, and it is complemented by PI3K activity upon
mitogen stimulation (Fig. 3H, 3J). These findings confirm the
observations of Nakamura et al. (19), who reported a TCR-driven
stimulation of HIF-1a protein synthesis via PI3K/mTOR. In addi-
tion,our resultsextendprevious studies byshowingthe involvement
of the hypoxia-induced protein synthesis of HIF-1a via the MAPK/
mTOR pathway in primary human Th cells.
With regard to the influence of extracellular MIF on specific
functions of human CD4+T cells under hypoxic conditions, we
observed a dual role for MIF, which strongly depends on oxygen
availability. In this study, we showed that MIF is necessary for op-
timal T cell proliferation under normoxia but not under hypoxia,
where it instead inhibits proliferation (Fig. 4A, 4B). In addition,
inhibition of T cell proliferation by blocking the MIF receptor
CD74 under normoxia was accompanied by enhanced expression
of the IL-2R CD25 (Fig. 4C, 4D), suggesting an adaptive process
to overcome the reduction in the frequency of IL-2–producing
Th cells and Th cell proliferation (Fig. 4A, 4B, 4E, 4F). CD25 ex-
pression was enhanced under hypoxia, as reported previously (24).
However, in this study, CD25 upregulation was almost independent
of the addition of MIF or blocking CD74, presumably reflecting
adaptations to the reduced proportion of IL-2–producing Th cells
and the decreased proliferation under limiting oxygen availability.
Several reports described a suppressive activity for a cysteiny-
lated MIF-related protein called glycosylation-inhibiting factor
(GIF) (25–28). MIF and GIF share an identical gene, but the re-
spective proteins vary in structure and function as the result of
different posttranslational modifications. It was demonstrated that
the cysteinylated factor GIF (Cys-60), but not the noncystein-
ylated MIF, exhibited immunosuppressive effects, such as suppres-
sion of Th2 responses by inhibiting the initiation of IL-4 pro-
duction (26, 27). Protein cysteinylation is a type of oxidation that
results from the toxicity of reactive oxygen species (29). Hypo-
xia was found to increase intracellular reactive oxygen species
in stimulated human T cells (24). Therefore, we suggest that the
hypoxic microenvironment found in tumors and severely inflamed
tissue may increase the amount of secreted suppressive cysteiny-
lated GIF (C60MIF/GIF), which decreases the T cell proliferation
rate, whereas normoxia increases the ratio of MIF/(C60MIF/GIF),
which supports T cell proliferation, as demonstrated by Bacher
et al. (21). This notion is supported by the finding that tumor-
derived MIF inhibits T lymphocyte activation (30).
These considerations led to the anti-inflammatory hypoxia hy-
potheses by Sitkovskyet al., focusing on the anti-inflammatoryrole
of HIF-1a in T cells, such as shown for murine HIF-1a(2/2)
T cells (31). Supporting the latter idea, our data indicated that MIF
reduces T cell proliferation under hypoxia.
Our findings are supported by MIF(2/2) tumors, which showed
pronounced infiltration of CD8+and CD4+T cells (32). The HIF-
1a–mediated inhibition may be additive or synergistic with im-
munosuppression caused by hypoxia-induced extracellular aden-
osine, which is protecting tumors by inhibiting the incoming
antitumor T cells via their A2Aadenosine receptors (33).
was shown to induce MIF, which is capable of antagonizing GC
(21). Therefore, we studied the effects of the GC DEX on the ex-
pression of HIF-1a. DEX treatment led to a clear dose-dependent
and rapid inhibition of HIF-1a expression; this inhibition was GCR
dependent and impacted HIF-1a–mediated target gene expression.
We did not find an effect of DEX treatment on the gene ex-
pression of HIF-1a or suppressors of HIF-1a, such as PHD1–3,
FIH, and pVHL, or on GCR expression itself, which could explain
the reduction in HIF-1a protein expression (Fig. 5). Therefore,
we suggested a rapid DEX-mediated induction of activity of HIF-
1a suppressors or a rapid DEX-mediated inhibition of hypoxia-
induced signaling, because it has been reported for the immuno-
suppressive effects of GC, which are mediated through rapid in-
hibition of TCR downstream kinases Lck and Fyn (34).
The first evidence for an interaction between HIF and GCR was
provided by Kodama et al. (11), who observed an induction of HIF
expression and target gene induction in HeLa cells. In contrast,
Wagner et al. (12) demonstrated a DEX-mediated inhibition of
induced HIF-1 target gene expression under hypoxia in HEPG2
cells. Furthermore, they described a retention of HIF-1a in the
cytoplasm, suggesting a block of nuclear import. However, we
found a clear inhibition of HIF-1a protein expression, which
resulted in reduced HIF-1 target gene expression, such as for
VEGF (Fig. 5). Interestingly, we also found that PHD1 was reg-
ulated in a similar manner as VEGF. del Peso et al. (35) showed
that PHD1 was upregulated by hypoxia in HeLa cells. Fig. 5G also
shows that PHD2 and PHD3 are not regulated in primary human
CD4+T cells. This is somewhat in contrast to the data published
by del Peso et al. (35), but these investigators showed a respective
regulation in transformed cells. In summary, the results obtained
in this study are important with regard to impaired tissue rege-
neration, such as wound healing, after GC therapy (13).
In addition, we found a clear inhibition of the HIF-1 target MIF
dependent manner (Fig.6). Wedid notobservean inductionof MIF
by low-dose GC exposure. In contrast, Leng et al. (36) recently
reported that low concentrations of DEX induced MIF secretion
from GC-sensitive CEM-C7 T cells but not from GC-insensitive
CEM-C1 T cells by a bell-shaped dose response, thereby confirm-
ing the observations of Calandra et al. (37) with regard to the
pression of HIF-1a expression.
Model of MIF-mediated induction and DEX-mediated sup-
The Journal of Immunology773
release of MIF by GC-exposed monocytes/macrophages. The
impact of the hypoxic environment applied in our experimental
setting might explain the divergence of the results. In contrast to
(17) demonstrated a repression of MIF-promoter activity in
GC-exposed CEM-C7A T cells, which is in line with our results.
Moreover, we provide evidence for MIF signaling to overcome
GC-suppressed HIF-1a expression (Fig. 6). The MIF-mediated in-
duction and DEX-mediated suppression of HIF-1a expression may
be deduced from the fine-tuning of phosphorylation events during
CD74 signaling, as described in the model shown in Fig. 7. Under
hypoxia, MIF is released from intracellular stores and induces sig-
stabilization of HIF-1a. HIF-1a is imported into the nucleus and
as MIF itself. This autoamplifying feedback loop is interrupted
by high doses of GCs via the GCR or the inhibition of HIF-1a
expression/stabilization under normoxia (Fig. 7).
These findings are of clinical importance because they reflect
the function of therapeutically administered high doses of GCs
during treatment of inflammatory processes in which hypoxia is
a critical part of pathogenesis. Furthermore, our findings suggest
that targeting HIF or MIF (C60MIF/GIF) may be useful to promote
antitumor immune responses, optimize GC therapy, and control
for brilliant technical assistance. We also acknowledge Luk Van Parijs for
the use of the plasmid pLL3.7.
The authors have no financial conflicts of interest.
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774RELATIONSHIP AMONG HIF-1a, MIF, AND GCR IN Th CELLS