NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A2A receptors

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Cellular Signalling xxx (2014) xxx–xxx

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Contents lists available at ScienceDirect

Cellular Signalling

j ourna l homepage: www.e lsev ie r .com/ locate /ce l l s ig

NF-κB enhances hypoxia-driven T-cell immunosuppression viaupregulation of adenosine A2A receptors

OFLaurie Bruzzese, Julien Fromonot, Youlet By, Josée-Martine Durand-Gorde, Jocelyne Condo, Nathalie Kipson,

Régis Guieu, Emmanuel Fenouillet, Jean Ruf ⁎Aix-Marseille Université (AMU) and Institut de Recherche Biomédicale des Armées (IRBA), UMR MD2, Faculté de Médecine Nord, Marseille, France

Abbreviations: A2AR, adenosine A2A receptor; ADA,hypoxia-inducible factor 1α; IKK, IκB kinase compthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NF-κB, nal blood lymphocytes; PDE4, phosphodiesterase 4; PHA,tein kinase A; PMA, phorbol myristate acetate.⁎ Corresponding author at: Faculté de Médecine No

F-13916 Marseille Cedex 20, France. Tel.: +33 491 32E-mail address: [email protected] (J. Ruf).

0898-6568/$ – see front matter © 2014 Published by Elsehttp://dx.doi.org/10.1016/j.cellsig.2014.01.024

Please cite this article as: L. Bruzzese, et alreceptors, Cellular Signalling (2014), http://d

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Article history:Received 22 October 2013Received in revised form 20 January 2014Accepted 24 January 2014Available online xxxx

Keywords:AdenosineAdenosine A2A receptorCell viabilityChemical hypoxiaHydrogen sulfide

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RHypoxia affects inflammation bymodulating T-cell activation via the adenosinergic system.We supposed that, inturn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditionsinvolves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α(HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factorsand four adenosinergic signaling parameters – namely adenosine, adenosine deaminase (ADA), adenosine A2A

receptor (A2AR) and cAMP – in T-cells stimulated using phorbol myristate acetate and phytohemagglutininand submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viabilitywas more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB acti-vation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulatedhypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB acti-vation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activat-ing NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosinelevel remained unchanged due to ADA regulation; and iv) the presence ofH2S, an endogenous signalingmoleculein inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcrip-tion factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the oppo-site effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMPanalogwhichpreferentially activates cAMP-dependent protein kinaseA (PKA), increased T-cell immunosuppres-sion whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together,these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. Thisprocess upregulates A2AR expression and enhances cAMP production and PKA activation, resulting inadenosinergic T-cell immunosuppression that can be modulated via H2S.

© 2014 Published by Elsevier Inc.

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UNCO1. Introduction

T-cells are exposed to gradients of low oxygen tension in inflamedtissues. Although hypoxia greatly decreases T-cell activation and viabil-ity, little is known about the effect of T-cell stimulation on the cellularresponse to hypoxia. Yet, hypoxia and inflammation are closely relatedat the molecular, cellular, and pathophysiological levels. On the onehand, hypoxia can induce inflammation. On the other hand, inflamedtissues attract infiltrating T-cells which, upon stimulation, secrete in-flammatory cytokines that cause vasculopathy, including blood vessel

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adenosine deaminase; HIF-1α,lex; MTT, 3-(4,5-dimethyl-2-uclear factor κB; PBL, peripher-phytohemagglutinin; PKA, pro-

rd, Boulevard Pierre Dramard,43 82; fax: +33 959 80 18 96.

vier Inc.

., NF-κB enhances hypoxia-dx.doi.org/10.1016/j.cellsig.20

stenosis and microthrombosis, and hence a decrease in oxygen supply[see [1] for a recent review].

The molecular mechanismsmediating hypoxia-related inflammato-ry responses are not delineated but they likely involve activation of twotranscription factors: nuclear factor κB (NF-κB) and hypoxia-induciblefactor 1α (HIF-1α) [2]. However, previous reports, which examinedthe relationship between the NF-κB and HIF-1α pathways in mediatingthe hypoxia-related inflammation process, remained inconclusive. Forexample, using in cellulo systems, it was reported that HIF-1α activatesNF-κB [3], that NF-κB controls HIF-1α transcription [4] and that activa-tion of HIF-1α may be concurrent to inhibition of NF-κB [5].

In resting cells, NF-κB is sequestered in the cytoplasmby an inhibito-ry IκB protein. Following infection and inflammation, the IκB kinasecomplex (IKK) becomes activated, which leads to phosphorylation-induced degradation of the IκB inhibitor and subsequent translocationof NF-κB to the nucleus [6,7]. HIF-1 is an heterodimer consisting ofoxygen-regulated HIF-1α and constitutively expressed HIF-1β subunits[8]. HIF-1α is rapidly degraded under normoxic conditions. In contrast,

riven T-cell immunosuppression via upregulation of adenosine A2A

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the subunit is stabilized and dimerizeswithHIF-1βwhenO2-dependentprolyl hydroxylases that target its O2-dependent degradation domainare inhibited under hypoxic conditions [9–11]. Thus, the activityof NF-κB and HIF-1α, which control numerous genes, is post-translationally regulated.

Adenosine is a purine nucleoside acting as an intercellular mediatorin hypoxia and inflammation. Adenosine is secreted by mononuclearcells via transporters and is also produced in the extracellular environ-ment fromATP released by ectoATPases/apyrases and ectonucleotidases.During hypoxia, adenosine accumulates following HIF-1α-mediated re-pression of the transcriptional activation of both adenosine kinase [12]and equilibrative nucleoside cell transporter [13]. Adenosine excess isregulated by degradation into inosine by adenosine deaminase (ADA)which is localized into the cell or associated to surface CD26 [14]. Aden-osine binding to its A2A receptor subtype (A2AR) triggers a rise in intra-cellular cAMP level via stimulation of adenylyl cyclase and GS-proteins[15]. Then, cAMP activates protein kinase A (PKA), which regulates aplethora of biological processes [16].

Exposure of mice to hypoxic atmosphere increases blood adenosinelevels and potently inhibits inflammatory tissue injury in an A2AR-dependent manner [17]. Tumors, which may have hypoxic microenvi-ronment resulting from tumor-vessel leakiness and vascular distortion,were found to contain high levels of adenosine [18]. Such high concen-trations of adenosine may contribute to protect tumors from immuneattack because blockade of the adenosine-A2AR pathway dramaticallyimproves tumor regression by immune cells [19]. Thus, the adenosine-A2AR pathway seems to be involved in hypoxia-mediated immunosup-pression although the molecular mechanisms underlying this processremain to be investigated.

We previously reported that under hypoxia, adenosine is secretedand binds to A2AR on T-cells in an autocrine manner to drive the cAMPimmunosuppressive signal [20]. In the current study, we investigatedthe impact of NF-κB and HIF-1α activity as part of the hypoxia-drivenadenosinergic signaling in T-cells treated with cobalt chloride (CoCl2),a compound that mimics hypoxia by preventing HIF-1α fromproteosomal degradation [21,22]. We found that T-cells display in-creased sensitivity to hypoxia following stimulation by upregulatingNF-κB and HIF-1α factors, which results in increased adenosinergic im-munosuppression via A2AR upregulation, cAMP production and PKA ac-tivation. We showed that this situation can be reversed using H2S, asignal molecule involved in inflammation and which also exertscytoprotectant activity [23].

2. Materials and methods

2.1. Peripheral blood lymphocytes (PBL) preparation

Blood sampleswere collected frombrachial vein of 3 healthy donors.Peripheral bloodmononuclear cells were isolated using cell preparationtubes containing sodium citrate, a polyester gel and a density gradientliquid and according to the manufacturer's instructions (BD VacutainerCPT, Beckton Dickinson, Franklin lakes, NJ). Cells were then resuspend-ed (3 × 106 cells/ml) in RPMI 1640 medium supplemented with2 mM L-glutamine, 10% fetal calf serum and penicillin/streptomycin(100 U/ml, 100 μg/ml) prior to incubation for 1 h at 37 °C under 5%CO2 in 25 cm2

flask. Adherent monocytes were discarded and PBLpresent in culture supernatant were examined using the cell viabilityassay (see below). The experiments were undertaken with the under-standing and written consent of each donor; the study methodologiesconformed to the standards set by the Declaration of Helsinki andwere approved by the local ethics committee.

2.2. Cell culture conditions

CEM cells, a human CD4+ T-cell line expressing A2AR [24], were cul-tured in RPMI 1640mediumas described above. Cells (1 × 106 cells/ml)

Please cite this article as: L. Bruzzese, et al., NF-κB enhances hypoxia-dreceptors, Cellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.20

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were seeded in 25 cm2flasks (15 ml/flask) and stimulated, or not, using

phorbol myristate acetate (PMA; 50 ng/ml) and phytohemagglutinin(PHA; 5 μg/ml) (Sigma-Aldrich, St. Louis, MO), a combination recom-mended by the manufacturer, as reported in [25]. Hypoxic injury wasachieved by adding either 100 or 200 μM CoCl2 to the culture mediumfor 6 h. To test the effect of H2S, cells were treated using culturemediumsupplemented with PMA and PHA, and either 25 or 50 μM NaHS for30 min prior to starting the hypoxic treatment by adding 200 μMCoCl2. CoCl2 andNaHS effective doseswere determined using the cell vi-ability assay (see below) in preliminary experiments. After a 6 h incuba-tion in various conditions (with or without PMA and PHA, CoCl2 orNaHS), living cells were counted using the Trypan Blue dye exclusionmethod and cell pellets (3 × 106) were frozen at−80 °C until use.

2.3. Cell viability assay

Monitoring of cell viability was performed in 24-well plates usingthe 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-mide (MTT) assay. MTT produces a yellowish solution that is convertedto dark blue, water-insoluble, MTT formazan by oxidoreductase en-zymes of living cells [26]. MTT (Sigma-Aldrich; 0.5 mg in 100 μl ofPBS, pH7.3) was added to each well containing 1 × 106 cells/ml 3 hprior to the end of the 6 h incubation period in culturemedium contain-ing 50–800 μMCoCl2 in the presence, or not, of PMA and PHA. Using thesame procedure, we tested the cytoprotective effect of H2S by incubat-ing cells in medium containing, or not, PMA and PHA and 25–200 μMNaHS for 30 min prior to addition of 200 μM CoCl2, the half-effectiveconcentration deduced from the previous experiment. In subsequenttests, and prior to addition of 200 μM CoCl2, cells were treated for30 min in culture medium containing PMA and PHA, and gradedamounts of various agents: i) CGS-21680, a specific A2AR agonist;and ii) ZM-241385, a specific A2AR antagonist, both purchased fromSigma-Aldrich; iii) Rolipram, a selective, cell permeable, inhibitor ofcAMP-specific phosphodiesterase 4 (PDE4); iv) 8-Bromoadenosine3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP), a cell-permeablecAMP analog which preferentially activates cAMP-dependent PKA; andv) H-89 dihydrochloride hydrate (H-89), a cell-permeable potent andselective inhibitor of cAMP-dependent PKA, these latter being pur-chased from Enzo Life Sciences, Farmingdale, NY. These agents were ti-trated in preliminary experiments in order to determine a range ofactive concentrations that were not toxic for CEM cells. After treatment,cells were pelleted (10,000 g; 15min) and supernatantswere discarded.Formazan crystals associated to the cell pellets were dissolved using1 ml dimethylsulfoxide prior to optical density reading at 550 nm.

2.4. Western blot analysis

HIF-1α, NF-κB and A2AR expression was assessed using a semi-quantitativeWestern blot procedure as previously described [24]. Brief-ly, cell pellets were lysed using a 4% SDS aqueous solution supplement-ed with a protease inhibitor cocktail (Sigma-Aldrich) and a 15 minsonication treatment at 47 kHz. For HIF-1α and A2AR expression, lysatesof cells (2 × 106 and 0.5 × 106, respectively) were diluted in loadingbuffer (65.2 mM Tris–HCl buffer, pH8.3, containing 10% glycerol, 0.01%bromophenol blue and 5% 2-mercaptoethanol). For NF-κB expression,nuclear extracts were used: lysates of cells (3 × 106) were first centri-fuged for 15min at 15,000 g to discard the cytosolic content and the pel-lets were resuspended in loading buffer. Samples were then analyzedusing 12% SDS-polyacrylamide gel electrophoresis and electroblottedonto polyvinylidene difluoride membrane. Blots were incubated for20 min with the mouse monoclonal antibodies of interest: anti-HIF-1α (clone 241809, R&D Systems, Minneapolis, MN; 1/500 dilution),anti-NF-κB p65 (clone NF-12, Sigma-Aldrich; 1/1000 dilution) andanti-A2AR (Adonis [27]; 1 μg/ml). Anti-β actin (clone AC-15, Sigma-Aldrich; 0.5 μg/ml) and anti-histoneH (cloneH11-4,Millipore, Billerica,MA; 1/500 dilution) were used as total and nuclear protein loading


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controls, respectively. Blots were then processed using horse-radishperoxidase labeled anti-mouse antibodies and chemiluminescent sub-strate (SuperSignal West Femto, Pierce Biotechnology, Rockford, IL).Densitometry analysis was performed using a Kodak Image Station440CF (Eastman Kodak Company, Rochester, NY) and the ImageJ soft-ware (NIH).

2.5. Adenosine assay

Intracellular adenosine concentration was measured as previouslydescribed [20] using anHPLC systemequippedwith a diode array detec-tor (Chromsystems, Munich, Germany). To prevent adenosine degrada-tion, pellets of frozen cells (3 × 106)weremixedwith500 μl of cold stopsolution (0.2 mM dipyridamole, 4.2 mM Na2EDTA, 5 mM erythro-9-(2-hydroxy-3-nonyl) adenine, and 79mMα–βmethylene adenosine 5′di-phosphates and 1 IU/ml heparin sulfate in NaCl 0.9%). Proteins wereprecipitated using 1 N perchloric acid and supernatant was injectedinto a LiChrospher C18 column (Merck, Darmstad, Germany). Adeno-sine was identified by its elution time and spectrum. Measurementwas made by comparison of peak areas versus those obtained usingadenosine standard solutions. The intra-assay and inter-assay coeffi-cients of variation ranged from 1% to 3%.

2.6. ADA activity

ADA activity measurement was carried out as previously described[20]. Briefly, cells (1.5 × 106) were lysed using 250 μl of ultrapurewater, mixedwith 750 μl of 28mMadenosine in 0.9% NaCl and incubat-ed for 36 min at 37 °C. The reaction was stopped by immersion of thesamples in ice water and ammonium production resulting from adeno-sine degradation by ADA was determined using a Synchron LX 20analyser (Beckman Coulter Inc, Villepinte, France). The intra-assay andinter-assay coefficients of variation ranged from 3% to 5%.

2.7. cAMP assay

The level of cAMP present in cell pellet was measured using theAmersham Biotrak kit (GE Healthcare Life Sciences, Buckinghamshire,UK) that combines the use of a peroxidase-labeled cAMP conjugateand a specific antiserum immobilized on microplates. Briefly, cells(1 × 106) were lysed using 100 μl of 0.25% dodecyl trimethyl ammoni-um bromide in 50 mM sodium acetate buffer, pH6.0 prior to transfer tomicroplatewells in order to carry out the competitive enzyme immuno-assay according to the manufacturer's instructions.

2.8. Statistics

Experiments were done in triplicate. Data (mean ± SD) were com-pared using student t-test. Differences with P b 0.05 were consideredstatistically significant.

3. Results

3.1. Cell stimulation increases sensitivity to hypoxia

Tomimic the hypoxic cellular stress conditionswhich arise in vivo atinflammatory sites, we designed an in cellulo model where mitogen-stimulation reflected inflammatory conditions and CoCl2 treatment in-duced hypoxia. Both freshly isolated human lymphocytes (PBL) and ahuman lymphoma T-cell line (CEM) were used. PBL were used to ad-dress the native cell behavior, CEM cells being used for cell materialavailability and experimental reproducibility. Wemimicked the inflam-matory contextwheremultiple signalingpathways are activated using alectin, PHA, and anorganic compound, PMA, a combination that iswide-ly used to potently stimulate PBL [28] and CEM cells [25]. WemimickedHIF-mediated hypoxia using CoCl2 treatment, which results in prolyl


hydroxylases inhibition, cobalt probably taking the place of iron withinthe enzymatic site, and hence preventing proteosomal degradation ofHIF-1α [29]. In pilot experiments, cells were submitted to gradeddoses of CoCl2 during 3, 6, 12, and 24 h. The 6 h-treatment durationwas chosen thereafter because it enabled maximal NF-κB and HIF-1αactivity together with significant effect of CoCl2 on cell viability as mea-sured using the MTT assay (data not shown). As shown in Fig. 1, CoCl2treatment altered cell viability in a dose-dependent manner and stimu-lation further increased the effect of CoCl2. Similar resultswere obtainedusing PBL (Fig. 1A) and CEM cells (Fig. 1B). Based on these data,we usedin the subsequent experiments two suboptimal concentrations (100and 200 μM) of CoCl2.

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OF3.2. Cell stimulation induces nuclear translocation of NF-κB and enhances

CoCl2-induced HIF-1α expression

In the canonical pathway, NF-κB is a dimer composed of p50 and p65subunits that is sequestered in the cytoplasm of resting cells as aninactive form complexed with the inhibitor protein IκB [30,31]. Uponactivation, IKK phosphorylates IκB, triggering its ubiquitination andproteasomal degradation, and NF-κB p50/p65 dissociates from IκBprior to migration to the nucleus where it directly binds to its cognateDNA sequences [32]. Accordingly, we interpreted here as a sign of acti-vation the increase in NF-κB p65 in the nuclear extract of stimulatedCEM cells only (Fig. 2A). CoCl2 treatment further increased in a dose-dependent manner NF-κB nuclear translocation.


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Fig. 2. Transcription factor activation and adenosinergic signaling in hypoxic T-cells. Stimulated (black bars) and unstimulated (open bars) CEM T-cells submitted to CoCl2 were tested forNF-κB p65 nuclear expression (A), HIF-1α expression (B), adenosine level (C), ADA activity (D), A2AR expression (E) and cAMP production (F). NF-κB p65, HIF-1α and A2AR levels weremeasured following densitometry analysis using the NIH image software (pixels of the specific peak versus the sum of peaks). Representative blots of triplicates for NF-κB (A), HIF-1α(B) and A2AR (E) analysis are shown. Adenosine, ADA and cAMP levels are expressed in μM/3 × 106 cells, international units (IU)/1.5 × 106 cells and fmol/1.5 × 106 cells, respectively.Results are mean ± SD, n = 3. P b 0.05, compared with stimulated (#) and unstimulated (*) cells in control conditions (0 μM CoCl2).


UNCORegarding HIF-1α, the subunit is stabilized by cobalt treatment

like under hypoxia and it dimerizes with the constitutively expressedHIF-1β. CoCl2 treatment weakly increased in a dose-dependent mannerHIF-1α expression in resting CEM cells (Fig. 2B). T-cell stimulation in-creased HIF-1α expression and strengthened the effect of CoCl2.

3.3. Cell stimulation modulates the HIF-1α-mediated adenosinergic signal

We next examined whether T-cell stimulation modulates the hyp-oxic effect of HIF-1α on the lymphocyte adenosinergic system. In im-mune cells, adenosine exerts suppressive effects that are mainlymediated through A2AR signaling with concomitant cAMP production[33,34]. CoCl2 treatment of resting CEM cells did not significantly affectadenosine production (Fig. 2C) and ADA activity (Fig. 2D). In contrast,ADA activity, but not adenosine level, increased in stimulated cells andfurther increased in a dose-dependent manner following CoCl2 treat-ment (Fig. 2C, D). CoCl2 treatment resulted in both stimulatedand resting cells in a dose-dependent expression of A2AR whichwas greater following stimulation (Fig. 2E). Production of cAMPdisplayed a pattern similar to that of A2AR expression (Fig. 2F). To-gether, these results show that only adenosine level remained


unchanged in stimulated cells under hypoxic conditions as a resultof ADA regulation, suggesting that cAMP level was essentially con-trolled via A2AR expression.

3.4. H2S represses the NF-κB and HIF-1α pathways

We next investigated the role of NF-κB activation on HIF-1α-mediated adenosinergic immunosuppression using NaHS, a donor ofH2S that protects cells against chemical hypoxia-induced injuries by af-fecting NF-κB nuclear translocation and the activity of cyclooxygenasesCOX-2 [35]. In vivo, H2S mainly results from cysteine metabolism in ac-tivated T-cells [36] and it acts on NF-κB pathway and activity of numer-ous kinases as a signal molecule involved in inflammation [37,38]. H2Scan also be produced from NaHS in cellulo and we used this character-istic to test its capacity to protect cells from hypoxia. As shown inFig. 3, the decrease in cell viability resulting from a 200 μM CoCl2 treat-ment for 6 h of stimulated cells was reversed in a dose-dependentman-ner when pretreatment using NaHS was performed. Such efficient cellprotection was observed using PBL (Fig. 3A) and CEM cells (Fig. 3B)and this led us to use 25 and 50 μM H2S thereafter.


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Fig. 3. Effect of H2S on viability of hypoxic stimulated T-cells. Stimulated PBL (A) andCEM T-cells (B) were pretreated for 30 min using increasing concentrations of NaHSprior to addition of 200 μM CoCl2 for 6 h. Data of the MTT test are given as in Fig. 1(mean ± SD, n = 3). P b 0.05, compared with CoCl2-treated (#) and untreated (*) PBLor CEM cells in control conditions (0 μM NaHS).


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Based on the same rationale, we tested the effect of H2S on NF-κBand HIF-1α activity. Pretreatment of stimulated CEM cells using NaHSfor 30 min prior to exposure to 200 μM CoCl2 markedly inhibited ex-pression of both intranuclear NF-κB p65 subunit (Fig. 4A) and cellularHIF-1α (Fig. 4B) in a dose-dependent manner. These results suggestthat H2S exerted its anti-inflammatory effect in CoCl2-treated cells viarepression of both NF-κB and HIF-1α pathways.

3.5. H2S abrogates the adenosinergic signal

Based on these data, we used NaHS to address the effects onadenosinergic parameters of the downregulation of NF-κB and HIF-1α.NaHS pretreatment enhanced adenosine production in CEM cells(Fig. 4C) whereas it decreased ADA activity (Fig. 4D), A2AR expression(Fig. 4E) and cAMP production (Fig. 4F). That adenosine increase(Fig. 4C) did not result in cAMP production (Fig. 4E) suggests thatcAMP signal may be controlled rather via A2AR expression than viaadenosine level, which further supports the data presented inSection 3.3.

3.6. A2AR induces T-cell immunosuppression

Adenosine produced following hypoxia stimulates A2AR on T-cellsand its action can be either increased or decreased using specific A2ARligands. Accordingly, we used specific A2AR agonist (CGS-21680) and


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antagonist (ZM-24138) compounds [39] to further validate our obser-vation that A2AR were involved in inhibition of T-cell proliferation fol-lowing CoCl2 treatment. In spite of the presence of adenosine instimulated cell medium after a 6 h incubation time with 200 μM CoCl2,addition of CGS-21680 decreased in a dose-dependent manner the via-bility of CEM cells. ZM-241385 exerted, as expected, the opposite effect(Fig. 5A).

3.7. The cAMP/PKA pathway modulates A2AR-induced immunosuppression

Signaling via the A2AR is expected to activate adenylate cyclase inT-cells and to increase production of cytosolic cAMP that is regardedas a negative regulator of T-cell activation [40]. Production of cAMP iscontrolled by various phosphodiesterases which degrade intracellularcAMP to 5′-AMP, in particular the cAMP-specific PDE4 that is preferen-tially expressed in immune and inflammatory cells [41,42]. The abilityof cAMP to activate PKA is well documented [43]. In order to evaluatethe intracellular signaling pathway involved inA2AR-mediated immuno-suppression,we tested the effect of CoCl2 treatment on CEM cell viabilityin the presence of two types of cAMP-enhancing agents and a specificPKA inhibitor. We found that: i) 8-Br-cAMP, a cell-permeable cAMP an-alog which preferentially activates cAMP-dependent PKA, further de-creased cell viability following CoCl2 treatment (Fig. 5B); ii) Rolipram,a selective, cell permeable inhibitor of cAMP-specific PDE4 [44], had asimilar effect; and iii) H-89, a cell-permeable, potent, selective inhibitorof the cAMP-dependent PKA that acts by competitive inhibition at theATP-binding sites in the catalytic subunit of the enzyme [45], reversedthe effect of CoCl2 treatment (Fig. 5B). A dose-dependent relationshipwas observed using these compounds. Together, these data indicatethat the cAMP/PKA pathway played a major role in T-cell immunosup-pression mechanism generated by hypoxia.

4. Discussion

We used an established method of chemically-induced hypoxia toaddress the effect of stimulation on adenosinergic signaling in T-cells.First, we found that viability of stimulated PBL was more affectedupon CoCl2 treatment than viability of their resting counterparts. Wethen used a T-cell line to delineate the process and showed thatthe increased sensitivity to hypoxia was dependent on NF-κB ac-tivation which, in turn, enhanced both HIF-1α production and T-celladenosinergic signaling. Second, we further documented this observa-tion by showing that H2S, a molecule known to protect cells againsthypoxia notably via the NF-κB pathway [38,46], reversed the hypoxiceffect in stimulated T-cells via downregulation of the NF-κB pathway.Fig. 6 summarizes these results: stimulation of T-cells activates theNF-κB pathway which, in hypoxic conditions, enhances the HIF-1α-driven adenosinergic signaling. Adenosine production, maintained at abasal level by ADA, stimulates overexpressed A2AR and, consequently,enhances cAMP production. Thus, here, this is the NF-κB-induced A2ARoverexpression, and not a rise in adenosine level, that leads to increasedcAMP immunosuppressive signal that blocks NF-kB activation via PKA[47].

NF-κB and HIF-1α pathways are intimately associated and a signifi-cant cross-talk exists at various levels. A number of stimuli have beenshown to upregulate HIF-1α mRNA expression via NF-κB-dependentmechanisms [48,49]. Basal NF-κB level is required for constitutive HIF-1α expression [50] and a recent publication showed that hypoxia-induced HIF-1α expression depends on an intact canonical NF-κBsignaling [51]. Our data are consistent with these findings and theydemonstrate in addition that cell stimulation increases NF-κB activity,which results in enhanced expression of hypoxia-mediated HIF-1α.Conversely, in our conditions, NF-κB was not found to be subjected toregulation by HIF-1α as reported in other systems [3] as we observedthat CoCl2 treatment specifically induced HIF-1α and did not increaseNF-κB activation. This result confirms that while hypoxia is a strong


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Fig. 4. Effect of H2S on transcription factor activation and adenosinergic signaling in hypoxic stimulated T-cells. Stimulated CEM T-cells pretreated (black bars) or not (open bars)for 30 min using 25 and 50 μM NaHS prior to addition of 200 μM CoCl2 for 6 h were tested for NF-κB activation (A), HIF-1α production (B), adenosine level (C), ADA activity(D), A2AR expression (E) and cAMP production (F). Results are expressed as in Fig. 2. P b 0.05, compared with CoCl2-treated (#) and untreated (*) cells in control conditions(0 μM NaHS).


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activator of HIF-1α-dependent pathways, it does not efficiently activateNF-κB signaling [50], which was achieved by the stimulating agents weused.Most importantly, these data indicate that theNF-κB-mediated in-flammatory pathway has a significant impact upon hypoxia-mediatedadenosinergic signaling in T-cells and, ultimately, suggest that this path-way affects cell regulation and activity.

Adenosine exhibits anti-inflammatory and immunosuppressive ef-fects on various immune cell types and lymphopoiesis [52–54] andcAMP exerts negative effects on lymphocyte survival, proliferation andeffector function (e.g. cytokine production) [55]. Here, we found thatadenosine level in stimulated cells submitted to hypoxia remained un-changed probably because of its degradation via ADA, which counter-acts the classic HIF-1α-induced adenosine production [14]. However,it is worth considering that intracellular cAMP production increasedhere concomitantly with A2AR expression in a context where adenosinelevel was shown to remain unchanged, supporting our previous asser-tion that maximal level of A2AR response in T-cell is more dependenton A2AR cell-surface expression than on a rise of adenosine level [56].The importance of A2AR plays in our observations was also illustrated


by the finding that CGS-21680 and ZM-241385 significantly affectedcell viability.

The positive regulation of A2AR expression by pro-inflammatorystimuli is inhibited by the IKK inhibitor, suggesting a role for NF-κB inthe upregulationmechanism of A2AR gene transcription [57]. Moreover,a previous study on A2AR gene expression and its regulation in inflam-matory conditions confers a major role to NF-κB binding to its proximalpromoter [58]. Our results further support the conclusion that NF-κB isinvolved in upregulation of the A2AR gene expression during hypoxia. Inaddition, we demonstrate that ADA activity increases following NF-κBactivation. Albeit counteracting excessive adenosine production, ADAwas also recently found to behave as an allosteric modulator whichcan enhance adenosine affinity and receptor activity [59]. This mecha-nism can explain the increase of cAMP levels observed here despitethe absence of a concomitant increase in adenosine production. By ele-vating cAMP levels and activating PKA, adenosinewas reported to inter-fere with activation of the IKK complex or with IKK-IκB interaction,selectively suppressing the NF-κB pathway [47]. The finding that NF-κB, a major pro-inflammatory protein, is inhibited by cAMP/PKA via


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ECA2AR stimulation further illustrates the current opinion that the

adenosinergic system is pivotal for lymphocyte regulation. A2AR expres-sion and cAMP production level in stimulated T-cells submitted to

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hypoxia also support the general view of a suppressive response as evi-denced here by monitoring T-cell viability. Finally, the beneficial effectof NaHS on survival of stimulated hypoxic PBL and CEM cells via the neg-ative action of H2S on NF-κB activation confirms and expands the dataabout a cAMP-mediated anti-apoptotic NF-kB downregulation feedbackmechanism which induces T-cell anergy [60,61].

That the intracellular cAMP pathway plays a key role in suppressionof CEM cells was shown here using selective cAMP elevating agents,namely 8-Br-cAMP, a cAMP analog, and Rolipram, a PDE4 inhibitor.These data are consistent with the reports that cAMP elevating agentsinhibit T-cell proliferation [62,63] and repress NF-kB-dependent tran-scription by various mechanisms [64]. For example, PDE4 blockade in-hibits cytokine secretion and proliferation in T-cells notably via NF-kBinhibition [65]. We also evidenced here that the transient rise in cAMPactivated PKA [16] using the PKA inhibitor H-89 that reversed thechanges in cell viability induced by hypoxia. Inhibition of T-cell prolifer-ation and cytokine production via cAMP-activated PKA is controversialhowever because of the failure of H89 to block these effects in somereports [66,67]. Yet, our data are in agreement with the findingsthat cAMP-mediated repression of T-cell activation may represent aphysiologic negative feedback control mechanism [68] that is requiredfor T-cell activation [69]. They also support the observation that themain cAMP receptor in lymphoid cells is PKA, a key regulator of T-cellimmune function at various levels [41].

The functions of NF-κB in apoptosis and tumor development arecontroversial. NF-κB is generally considered as an anti-apoptoticagent [6,70] but it was also inferred that it promotes apoptosis ina cell-type and stimulus-dependent manner [71]. Other reportsdescribed a pro-apoptotic role of NF-κB in DNA damage-triggeredapoptosis [72] and in death receptor-induced apoptosis [73]. Here,NF-κB appeared to be rather a pro-apoptotic/immunosuppressivefactor as we found that inhibition of NF-κB suppressed HIF-1α-mediated adenosinergic signaling, a process which leads to deleteri-ous cAMP elevation. Hypoxic immunosuppression that is detrimen-tal to T-cell cancer destruction [18,19] is therefore related toactivation of the NF-κB pathway that is sensible to H2S, as shownhere. Inhibition of this pathway via modulation of endogenous H2Sproduction or local injection of H2S-diffusing drugs may thereforehelp to treat hypoxic tumors.

HIF-1α Adenosine

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tudy, stimulation of T-cells in inflamed areas ismimicked using PMA+PHA treatment andnt NF-κB activation and adenosinergic immunosuppression of T-cells. A negative feedbackP production and PKA activation. + on solid arrows: activation, − on hatched arrows:


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Acknowledgments

We thank Michelle Blas for her expertise in the adenosine assay.

References

[1] H.K. Eltzschig, P. Carmeliet, N. Engl. J. Med. 364 (2011) 656–665.[2] C.T. Taylor, J. Physiol. 586 (2008) 4055–4059.[3] S.R. Walmsley, C. Print, N. Farahi, C. Peyssonnaux, R.S. Johnson, T. Cramer, A.

Sobolewski, A.M. Condliffe, A.S. Cowburn, N. Johnson, E.R. Chilvers, J. Exp. Med.201 (2005) 105–115.

[4] R.S. Belaiba, S. Bonello, C. Zahringer, S. Schmidt, J. Hess, T. Kietzmann, A. Gorlach,Mol. Biol. Cell 18 (2007) 4691–4697.

[5] A. Carbia-Nagashima, J. Gerez, C. Perez-Castro, M. Paez-Pereda, S. Silberstein, G.K.Stalla, F. Holsboer, E. Artz, Cell 131 (2007) 309–323.

[6] M. Karin, Y. Cao, F.R. Greten, Z.W. Li, Nat. Rev. Cancer 2 (2002) 301–310.[7] H. Hacker, M. Karin, Sci. STKE 357 (2006) re13.[8] G.L. Wang, G.L. Semenza, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4304–4308.[9] P.H. Maxwell, M.S. Wiesener, G.W. Chang, S.C. Clifford, E.C. Vaux, M.E. Cockman, C.C.

Wykoff, C.W. Pugh, E.R. Maher, P.J. Ratcliffe, Nature 399 (1999) 271–275.[10] G. Semenza, Cell 107 (2001) 1–3.[11] C.J. Schofield, P.J. Ratcliffe, Nat. Rev. Mol. Cell Biol. 5 (2004) 343–354.[12] J.C. Morote-Garcia, P. Rosenberger, J. Kuhlicke, H.K. Eltzschig, Blood 111 (2008)

5571–5580.[13] J.C. Morote-Garcia, P. Rosenberger, N.M. Nivillac, I.R. Coe, H.K. Eltzschig, Gastroenter-

ology 136 (2009) 607–618.[14] H.K. Eltzschig, M. Faigle, S. Knapp, J. Karhausen, J. Ibla, P. Rosenberger, K.C. Odegard,

P.C. Laussen, L.F. Thompson, S.P. Colgan, Blood 108 (2006) 1602–1610.[15] M. Klinger, M. Freissmuth, C. Nanoff, Cell. Signal. 14 (2002) 99–108.[16] R. Kopperud, C. Krakstad, F. Selheim, S.O. Doskeland, FEBS Lett. 546 (2003) 121–126.[17] A. Choukèr, M. Thiel, D. Lukashev, J.M.Ward, I. Kaufmann, S. Apasov, M.V. Sitkovsky,

A. Ohta, Mol. Med. 14 (2008) 116–123.[18] J. Blay, T.D. White, D.W. Hoskin, Cancer Res. 57 (1997) 2602–2605.[19] A. Ohta, E. Gorelik, S.J. Prasad, F. Ronchese, D. Lukashev, M.K. Wong, X. Huang, S.

Caldwell, K. Liu, P. Smith, J.F. Chen, E.K. Jackson, S. Apasov, S. Abrams, M.Sitkovsky, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 13132–13137.

[20] Y. By, L. Jacquin, F. Franceschi, J.M. Durand-Gorde, J. Condo, P. Michelet, R. Guieu, J.Ruf, Purinergic Signal 8 (2012) 661–667.

[21] K. Kanaya, T. Kamitani, Biochem. Biophys. Res. Commun. 306 (2003) 750–755.[22] Y. Yuan, G. Hilliard, T. Ferguson, D.E. Millhorn, J. Biol. Chem. 278 (2003)

15911–15916.[23] H. Kimura, Amino Acids 41 (2011) 113–121.[24] Y. By, J.M. Durand-Gorde, J. Condo, P.J. Lejeune, E. Fenouillet, R. Guieu, J. Ruf, Hum.

Immunol. 71 (2010) 1073–1076.[25] P.C. Cogswell, M.W. Mayo, A.S. Baldwin Jr., J. Exp. Med. 185 (1997) 491–497.[26] M.V. Berridge, P.M. Herst, A.S. Tan, Biotechnol. Annu. Rev. 11 (2005) 127–152.[27] Y. By, J.M. Durand-Gorde, J. Condo, P.J. Lejeune, B. Mallet, P. Carayon, R. Guieu, J. Ruf,

Mol. Immunol. 46 (2009) 400–405.[28] K.E. Sullivan, J. Cutilli, L.M. Piliero, D. Ghavimi-Alagha, S.E. Starr, D.E. Campbell, S.D.

Douglas, Clin. Diagn. Lab. Immunol. 7 (2000) 920–924.[29] A.C. Epstein, J.M. Gleadle, L.A. McNeill, K.S. Hewitson, J. O'Rourke, D.R. Mole, M.

Mukherji, E. Metzen, M.I. Wilson, A. Dhanda, Y.M. Tian, N. Masson, D.L. Hamilton,P. Jaakkola, R. Barstead, J. Hodgkin, P.H. Maxwell, C.W. Pugh, C.J. Schofield, P.J.Ratcliffe, Cell 107 (2001) 43–54.

[30] M. Buelna-Chontal, C. Zazueta, Cell. Signal. 25 (2013) 2548–2557.[31] M. Tiwari, S.Mikuni, H. Muto,M. Kinjo, Biochem. Biophys. Res. Commun. 436 (2013)

430–435.[32] G. Ghosh, V.Y. Wang, D.B. Huang, A. Fusco, Immunol. Rev. 246 (2012) 36–58.[33] A. Ohta, M. Sitkovsky, Nature 414 (2001) 916–920.[34] T. Raskovalova, X. Huang, M. Sitkovsky, L.C. Zacharia, E.K. Jackson, E. Gorelik,

J. Immunol. 175 (2005) 4383–4391.

UNC


ED P

RO

OF

[35] C. Yang, Z. Yang, M. Zhang, Q. Dong, X. Wang, A. Lan, F. Zeng, P. Chen, C. Wang, J.Feng, PLoS ONE 6 (2011) e21971.

[36] T.W. Miller, E.A. Wang, S. Gould, E.V. Stein, S. Kaur, L. Lim, S. Amarnath, D.H. Fowler,D.D. Roberts, J. Biol. Chem. 287 (2012) 4211–4221.

[37] H.O. Pae, Y.C. Lee, E.K. Jo, H.T. Chung, Arch. Pharm. Res. 32 (2009) 1155–1162.[38] L. Li, P. Rose, P.K. Moore, Annu. Rev. Pharmacol. Toxicol. 51 (2011) 169–187.[39] B.B. Fredholm, A.P. IJzerman, K.A. Jacobson, J. Linden, C.E. Müller, Pharmacol. Rev. 63

(2011) 1–34.[40] E. Bjørgo, K. Moltu, K. Taskén, Handb. Exp. Pharmacol. 204 (2011) 345–363.[41] E. Bjørgo, S.A. Solheim, H. Abrahamsen, G.S. Baillie, K.M. Brown, T. Berge, K.

Okkenhaug, M.D. Houslay, K. Taskén, Mol. Cell. Biol. 30 (2010) 1660–1672.[42] M.D. Houslay, Trends Biochem. Sci. 35 (2010) 91–100.[43] R. Mosenden, K. Taskén, Cell. Signal. 23 (2011) 1009–1016.[44] C. Lugnier, Pharmacol. Ther. 109 (2006) 366–398.[45] R.A. Engh, A. Girod, V. Kinzel, R. Huber, D. Bossemeyer, J. Biol. Chem. 271 (1996)

26157–26164.[46] C. Yang, H. Ling, M. Zhang, Z. Yang, X. Wang, F. Zeng, C. Wang, J. Feng, Mol. Cell 31

(2011) 531–538.[47] S. Minguet, M. Huber, L. Rosenkranz, W.W.A. Schamel, M. Reth, T. Brummer,

Eur. J. Immunol. 35 (2005) 31–41.[48] S. Frede, C. Stockmann, P. Freitag, J. Fandrey, Biochem. J. 396 (2006) 517–527.[49] P. van Uden, N.S. Kenneth, S. Rocha, Biochem. J. 412 (2008) 477–484.[50] J. Rius, M. Guma, C. Schachtrup, K. Akassoglou, A.S. Zinkernagel, V. Nizet, R.S.

Johnson, G.G. Haddad, M. Karin, Nature 453 (2008) 807–811.[51] S.F. Fitzpatrick, M.M. Tambuwala, U. Bruning, B. Schaible, C.C. Scholz, A. Byrne, A.

O'Connor, W.M. Gallagher, C.R. Lenihan, J.F. Garvey, K. Howell, P.G. Fallon, E.P.Cummins, C.T. Taylor, J. Immunol. 186 (2011) 1091–1096.

[52] S.G. Apasov, M.V. Sitkovsky, Int. Immunol. 11 (1999) 179–189.[53] S. Apasov, J.F. Chen, P. Smith, M. Sitkovsky, Blood 95 (2000) 3859–3867.[54] S. Majumdar, B.B. Aggarwal, Oncogene 22 (2003) 1206–1218.[55] M. Neumann, T. Grieshammer, S. Chuvpilo, B. Kneitz, M. Lohoff, S.A. chimpl, B.R.

Franza Jr., E. Serfling, EMBO J. 14 (1995) 1991–2004.[56] L. Jacquin, F. Franceschi, Y. By, J.M. Durand-Gorde, J. Condo, J.C. Deharo, P. Michelet,

E. Fenouillet, R. Guieu, J. Ruf, FEBS Open Biol. 3 (2013) 1–5.[57] L.J. Murphree, G.W. Sullivan, M.A. Marshall, J. Linden, Biochem. J. 391 (2005)

575–580.[58] S. Morello, K. Ito, S. Yamamura, K.Y. Lee, E. Jazrawi, P. Desouza, P. Barnes, C. Cicala,

I.M. Adcock, J. Immunol. 177 (2006) 7173–7183.[59] E. Gracia, K. Pérez-Capote, E. Moreno, J. Barkešová, J. Mallol, C. Lluís, R. Franco, A.

Cortés, V. Casadó, E.I. Canela, Biochem. J. 435 (2011) 701–709.[60] P.E. Zarek, C.T. Huang, E.R. Lutz, J. Kowalski, M.R. Horton, J. Linden, C.G. Drake, J.D.

Powell, Blood 111 (2008) 251–259.[61] L. Himer, B. Csoka, Z. Selmeczy, B. Koscso, T. Pocza, P. Pacher, Z.H. Nemeth, E.A.

Deitch, E.S. Vizi, B.N. Cronstein, G. Hasko, FASEB J. 24 (2010) 2631–2640.[62] M.M. Bartik, G.P. Bauman, W.H. Brooks, T.L. Roszman, Cell. Immunol. 158 (1994)

116–130.[63] M.A. Giembycz, C.J. Corrigan, J. Seybold, R. Newton, P.J. Barnes, Br. J. Pharmacol. 118

(1996) 1945–1958.[64] R.D. Ye, Am. J. Physiol. Lung Cell. Mol. Physiol. 279 (2000) L615–L617.[65] J.L. Jimenez, C. Punzon, J. Navarro, M.A. Munoz-Fernandez, M. Fresno, J. Pharmacol.

Exp. Ther. 299 (2001) 753–759.[66] P.J. Bryce, M.J. Dascombe, I.V. Hutchinson, Immunopharmacology 41 (1999)

139–146.[67] K.J. Staples, M. Bergmann, K. Tomita, M.D. Houslay, I. McPhee, P.J. Barnes, M.A.

Giembycz, R. Newton, J. Immunol. 167 (2001) 2074–2080.[68] L. Li, C. Yee, J.A. Beavo, Science 283 (1999) 848–851.[69] K. Tasken, E.M. Aandahl, Physiol. Rev. 84 (2004) 137–167.[70] A. Plantivaux, E. Szegezdi, A. Samali, L. Egan, Ann. N. Y. Acad. Sci. 1171 (2009) 38–49.[71] S.K. Radhakrishnan, S. Kamalakaran, Biochim. Biophys. Acta 1766 (2006) 53–62.[72] S. Karl, Y. Pritschow, M. Volcic, S. Häcker, B. Baumann, L. Wiesmüller, K.M. Debatin,

S. Fulda, J. Cell. Mol. Med. 13 (2009) 4239–4256.[73] C. Jennewein, S. Karl, B. Baumann, O. Micheau, K.M. Debatin, S. Fulda, Oncogene 31

(2012) 1468–1474.


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NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A2A receptors

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