human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their...

Human dendritic cells differentiated in hypoxiadown-modulate antigen uptake and change theirchemokine expression profile

Angela Rita Elia,*,†,1 Paola Cappello,*,†,1 Maura Puppo,‡ Tiziana Fraone,*,† Cristina Vanni,‡

Alessandra Eva,‡ Tiziana Musso,§ Francesco Novelli,*,† Luigi Varesio,‡ and Mirella Giovarelli*,†,2

*Center for Experimental Research and Medical Studies, San Giovanni Battista Hospital, Torino, Italy; †Departmentof Medicine and Experimental Oncology, University of Torino, Torino, Italy; ‡Laboratory of Molecular Biology,G. Gaslini Institute, Genova, Italy; and §Department of Public Health and Microbiology, University of Torino,Torino Italy

Abstract: Dendritic cells (DCs) are the most po-tent antigen-presenting cells and fine-tune the im-mune response. We have investigated hypoxia’seffects on the differentiation and maturation ofDCs from human monocytes in vitro, and haveshown that it affects DC functions. Hypoxic imma-ture DCs (H-iDCs) significantly fail to capture an-tigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression ofCD206. Moreover, H-iDCs released higher levelsof CXCL1, VEGF, CCL20, CXCL8, and CXCL10but decreased levels of CCL2 and CCL18, whichpredict a different ability to recruit neutrophilsrather than monocytes and create a proinflamma-tory and proangiogenic environment. By contrast,hypoxia has no effect on DC maturation. Hypoxicmature DCs display a mature phenotype and acti-vate both allogeneic and specific T cells like nor-moxic mDCs. This study provides the first demon-stration that hypoxia inhibits antigen uptake byDCs and profoundly changes the DC chemokineexpression profile and may have a critical role inDC differentiation, adaptation, and activation ininflamed tissues. J. Leukoc. Biol. 84: 000–000;2008.

Key Words: antigen-presenting cells � phagocytosis/endocytosis� cytokines � T cell activation � chemokine receptors

INTRODUCTION

Dendritic cells (DCs) are the most important link betweenthe innate and the acquired immune response, and they areinvolved in the initiation of both types of immunity. CD14�

and CD34� precursors from the bone marrow reach theirtarget tissues via the bloodstream and take up residence atsites of potential pathogen entry in a physiological stagespecialized for Ag capture [1]. Tissue injury and inflamma-tion cause dramatic changes throughout the microenviron-ment, including the release of inflammatory mediators, such

as cytokines and chemokines that shape protective immuneresponses and culminate in pathogen elimination. A com-mon denominator of many inflammatory processes, includ-ing cardiovascular, hematological and pulmonary disorders,dermal wounds, rheumatoid arthritis, and microbial infec-tions, and an important regulator of gene expression, is lowpartial oxygen pressure (pO2, 0-20 mm Hg) [2]. Hypoxia alsooccurs in solid tumors, where it has been associated withmalignant progression, metastasis and resistance to radioand chemotherapy [3–7]. For this reason, understanding ofmechanisms that allow adaptation to hypoxia on the part ofimmune cells migrating from lymphoid organs to tissues insearch of pathogens is important for developing new strat-egies for inflammatory pathogenesis and hypoxic canceroustissues. The molecular signaling pathways mediating geneinduction by hypoxia have been elucidated in detail andextensively reviewed [8]. Hypoxia-inducible transcriptionalfactor 1 (HIF-1) has been well described as a key factor inthe cellular adaptation to hypoxic conditions, including cellsurvival, angiogenesis and switch to glycolysis [9]. Studiesof myeloid- and lymphoid-specific HIF-1� knockout micedemonstrated that HIF-1� has different functions in varioustypes of immune cells [10]. Although HIF-1� is essential formyeloid cell-mediated inflammation [11, 12], it plays aninhibitory role in T cell functioning [13, 14]. Hypoxia mod-ulates the expression of proangiogenic factors, inflammatorymediators, and cytokines/chemokines in endothelial cellsand monocytes/macrophages, as well as in neoplastic cells[for a review, see Sitkovsky [10]]. The adaptive response tohypoxia favors both neoangiogenesis and the recruitment ofscavengers. However, while much is known about the effectsof hypoxia on monocytes and macrophages, its effects on thedifferentiation of monocytes into immature DCs (iDCs), their

1 These authors contributed equally to this work.2 Correspondence: Department of Medicine and Experimental Oncology,

University of Torino, Corso Raffaello 30, Torino 10126, Italy. E-mail:[email protected]

Received February 4, 2008; revised July 16, 2008; accepted August 5,2008.

doi: 10.1189/jlb.0208082

0741-5400/08/0084-0001 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 84, December 2008 1

Uncorrected Version. Published on August 25, 2008 as DOI:10.1189/jlb.0208082

Copyright 2008 by The Society for Leukocyte Biology.

functional properties, and maturation have not been fullyinvestigated.

In this paper, we show that the prolonged exposition tohypoxia affects DC phenotype and functions. We provide thefirst demonstration that hypoxia inhibits Ag uptake, a typi-cal function of iDCs, and profoundly changes their chemo-kine expression profile. Human monocytes induced to dif-ferentiate in vitro into iDCs under hypoxic conditions (H-iDCs) display a typical DC morphology and express CD1a, amarker of DCs. In addition, H-iDCs display higher levels ofHLA class II, costimulatory molecules and chemokine re-ceptors CCR5 and CXCR4 in comparison to normoxic iDCs(N-iDCs) [15]. H-iDCs are as efficient as N-iDCs in inducingactivation of T cells in response to both specific Ag oralloantigens. However, H-iDCs significantly fail to capturedextran, BSA, LPS, zymosan, through down-modulation ofthe RhoA/Ezrin-Radixin-Moiesin (ERM) pathway and ex-pression of the lectin receptor CD206. Moreover, theychange their chemokine pattern secretion. By comparisonwith N-iDCs, they produce higher amounts of CCL20,CXCL1, CXCL8, and CXCL10, but lower levels of CCL2and CCL18, which may predict a different ability to recruitneutrophils and monocytes into hypoxic areas. Furthermore,hypoxia negatively regulates the release of the anti-inflam-matory cytokine IL-10 (never previously reported). By con-trast, hypoxia does not affect the DC maturation induced bya cocktail of proinflammatory stimuli, namely TNF-�, IL-1�, IL-6, and prostaglandin (PGE)2. Hypoxic mature DCs(H-mDCs) efficiently activate both allogeneic and specific Tcells like normoxic mature DC (N-mDC).

These data suggest that hypoxia has a critical role in DCdifferentiation, adaptation, and activation in inflamed tissues:whether it serves to limit self-Ag uptake and recruit neutro-phils, i.e., scavengers known to better survive in hypoxicconditions, hypoxia may even contribute to the failure-of-immune response against tumors by down-modulating DC abil-ity to capture tumor Ags in the microenvironment.

MATERIALS AND METHODS

Generation of DCs

Human PBMC were isolated from venous blood of voluntary healthy donors byHistopaque density gradient centrifugation (Sigma, Milan, Italy). Monocyteswere purified with a monocyte isolation kit II (Miltenyi Biotech, Calderara diReno, Bologna, Italy) by depletion of nonmonocytes (negative selection). Theresulting preparations were consistently �90% CD14� as determined byFACS analysis (FACSCalibur, BD Bioscences, Milan, Italy). To generate DC,we used a protocol for “fast DCs” [modified from [16]]. Monocytes wereincubated in six-well culture plates (1.5�106 cells/ml) in RPMI 1640medium-10% FBS-certified heat inactivated (GIBCO, Invitrogen, Milan, Italy)and 50 �g/ml gentamycin (Schering-Plough, Milan, Italy), supplemented with100 ng/ml of (GM-CSF) and 100 ng/ml of IL-4 (both PeproTech Inc., byTebu-bio, Milan, Italy) for 2 days. For a further 48 h they were incubated incomplete RPMI 1640 medium supplemented with 100 ng/ml of GM-CSF and100 ng/ml of IL-4, to generate iDCs or with proinflammatory mediators: TNF-�(50 ng/ml), IL-1� (50 ng/ml), IL-6 (10 ng/ml) (all Peprotech Inc.), and PGE2

(1 �M) (Sigma) to generate mDCs [17]. When indicated, we compared these4-day iDCs with those generated with a “conventional” protocol (6-day iDCs).Monocytes were incubated in six-well culture plates (1.5�106 cells/ml) in

RPMI 1640 medium-10% FBS certified heat-inactivated (GIBCO, Invitrogen)and 50 �g/ml gentamycin (Schering-Plough), supplemented with 100 ng/ml ofGM-CSF and 100 ng/ml of IL-4 (both PeproTech Inc., by Tebu-bio, Milan,Italy) for 3 days. On day 3, two-thirds of the medium were replaced by freshmedium containing GM-CSF and IL-4. On day 6, cells were harvested andused for the experiments.

To generate macrophages, CD14� cells were incubated in six-well cultureplates (7.5�105 cells/ml) in complete RPMI 1640 medium-10% FBS certifiedheat inactivated, supplemented with 100 ng/ml of M-CFS (Peprotech Inc.) for4 days. Fresh M-CFS was added after 2 days. Cells were then observed with aninvert microscope (Olympus CX41, Germany) at �200, and images wererecorded as .jpg files.

Culture conditions

For the normoxic condition, cultures were maintained at 37°C in a humidifiedincubator containing 20% O2, 5% CO2, and 75% N2. For the hypoxic condi-tion, cells were cultured and handled at 37°C in a humidified, anaerobic workstation incubator (Bug Box; ALC International, Cologno Monzese, Milan, Italy)flushed for 20 min at a dynamic pressure of 35 psi and a flow rate of 25 l/minwith a gas mixture of 1% O2, 5% CO2, and 94% N2. All reagents, medium, andcytokines used for the treatments of hypoxic cells were allowed to equilibratein the anaerobic work station incubator for 2 h before use.

Flow cytometry

Human iDCs and mDCs generated in normoxic or hypoxic conditions werewashed and subsequently treated with 1% paraformaldehyde (PFA) for 15 minand resuspended in PBS (Sigma) supplemented with 0.2% BSA and 0.01%sodium azide, and incubated with fluorochrome-conjugated mAb and isotype-matched negative controls (DakoCytomation, Milan, Italy) after blocking non-specific sites with rabbit IgG (Sigma) for 30 min at 4°C. The following FITC orPE-conjugated monoclonal antibodies (mAbs) were used: anti-CD14, anti-CD83, anti-CD86, (BD Biosciences); anti-CD1a (Serotec, by SPACE Import-Export, Milan, Italy); anti-CD40 (Immunotech, Beckman Coulter, Milan, Italy),anti-CD80, anti-HLA-DP, DQ, DR (Ancell, by VinciBiochem, Florence, Italy);anti-CCR5, anti-CCR6, anti-CCR7, and anti-CXCR4 (R&D Systems, bySPACE Import-Export, Milan, Italy); anti-CD206 purified (Serotec) followed byFITC conjugated anti-mouse IgG (DakoCytomation). FACS analysis was per-formed with a FACSCalibur and CELLQuest software (BD Biosciences). Cellswere gated according to their light-scatter properties to exclude cell debris andcontaminating lymphocytes.

Migration assay

N-iDC and H-iDC migration was measured in duplicate with a transwell system(24-well plates; 8.0-�m pore size; Corning Costar by CELBIO, Milan, Italy)under normoxic and hypoxic condition, respectively. Six hundred microlitersRPMI medium with or without 5, 50, and 250 ng/ml recombinant human CCL4or CXCL12 (both Peprotech, Inc.) were added to the lower chamber. Wells withmedium only were used as a control for spontaneous migration. A total of 2.5 �105 cells in 100 �l were added to the upper chamber and incubated at 37°Cfor 2 h. Cells that migrated into the lower chamber were harvested, concen-trated to a volume of 200 �l, and counted by flow cytometry. Events wereacquired for a fixed time of 60 s. The counts fell within a linear range of thecontrol titration curves obtained by testing increasing cell concentrations. Themean number of spontaneously migrated cells was subtracted from the totalnumber of cells that migrated in response to the chemokine. Values are givenas the mean number of migrated cells � SE.

MLR assay

T cells were purified through a nylon wool column (SciGene Corporation,Sunnyvale, CA, USA) and placed in 96-well plates at 1 � 105 cells/well withallogeneic pretreated with 1% PFA N-iDCs, H-iDCs, N-mDCs, and H-mDCs inincreasing concentrations (1,250-5,000). After 4 days, 1 �Ci (0.037 MBq) oftritiated [3H] TdR (Amersham Biosciences, Milan, Italy) was added to eachwell, and incubation was prolonged for a further 16 h. Cells were directlycollected with a CELLharvester (Packard Instrument, Milan, Italy) on UNIfilterplates (Packard) and 3HTdR uptake was quantitated (TopCount microplatesscintillation counter; Packard). All tests were performed in triplicate.

2 Journal of Leukocyte Biology Volume 84, December 2008 http://www.jleukbio.org

CD8� T cell clone culture

CD8� T clones HLA-A2� specific for influenza matrix Flu-MA58-66 peptide(GILGFVFTL; Primm, Milan, Italy) provided by Dr. Claudia Giachino (Uni-versity of Turin, Italy) were expanded in RPMI 1640 supplemented with 5%AB human serum (BioWhittaker Europe, Les Verviers, Belgium) and rIL-2(200 U/ml; PeproTech Inc.). Every four weeks, they were expanded as de-scribed [18]. They were used for the functional test 2 wk after stimulation.Tyrosinase1-9 (MLLAVLYCL) (Primm) was used as unrelated control peptide.

ELISPOT assay

Elispot plates (MAIPS; Millipore, Milan, Italy) were coated with primaryanti-human IFN-� mAb (5 �g/�l) (Endogen, by Temaricerca, Bologna, Italy) at4°C overnight. N-iDCs, N-mDC, H-iDCs and H-mDC previously loaded with 3�g/ml of Flu-MA58-66 or tyrosinase1-9, as unrelated control peptide, for 4 h at37°C in their respective culture conditions, and subsequently treated with 1%PFA, were added to CD8� clones in quadruplicate. The plates were thenincubated in normoxic conditions for 24 h. A biotinylated secondary anti-IFN-� mAb (1 �g/ml) (Endogen) was added and plates were incubated at 37°Cfor 2 h. The IFN-� spots were developed using the AEC substrate (Sigma). Thespots were counted by computer assisted image analysis (Transtec 1300ELISpot Reader, AMI Bioline, Buttigliera Alta, Torino, Italy).

Primary immune response

One � 106 N-iDCs and H-iDCs previously loaded with 3 �g/ml of Flu-MA58-66

for 4 h at 37°C in their respective culture conditions and subsequently treatedwith 1% PFA were washed and cocultured with 10�106 autologous naı̈ve Tcells from an HLA-A2� healthy donor in a 6-well plate in complete medium.CD45RA� T cells were previously isolated from thawed PBMCs from the samedonor by positive immunoselection with magnetic beads (Miltenyi Biotec). Theresulting preparation was 100% CD45RA�, as determined by FACS analysis.After 7 days, T cell activation was determined by Elispot assay by usingHLA-A2�-matched T2 cell line as antigen-presenting cells loaded with Flu-MA58-66, and tyrosinase1-9 as unrelated control peptide.

Ag uptake assay

Two � 105 N-iDCs or H-iDCs were incubated with FITC-labeled Ags: dextran,BSA, LPS from Escherichia coli 0111:B4, (Sigma), and Alexa 488 zymosan(Molecular Probes, by Invitrogen, Milan, Italy), at 37°C or 4°C for differenttimes in normoxic or hypoxic conditions, respectively. After several washeswith cold phosphate buffered solution, fluorescence was measured by FACS-Calibur to reveal Ag uptake. Endocytosis comparison between 4-day iDCs and6-day iDCs was done with FITC-labeled dextran and BSA (Sigma), at 37°C or4°C for 30 min in normoxic or hypoxic conditions, respectively, and Ag uptakewas evaluated as above as described.

In some experiments, 2 � 105 N-iDCs were pretreated for 5 h with C3transferase (C3T) (Cytoskeleton, by Tebu-bio, Milan, Italy), an inhibitor of RhoGTPase (0.1-2 �g/ml) at 37°C. After treatment, cells were incubated withFITC-labeled Ags (dextran, BSA, LPS) for 30 min at 37°C still in the presenceof the inhibitor. Cells were washed several times with cold PBS and fluores-cence was measured using a FACScalibur to reveal Ag uptake.

Detection and measurement of proteins

To evaluate cytokines and chemokines produced by resting or stimulatedN-iDCs and H-iDCs, on day 3, medium was replaced with fresh medium plusGM-CSF and IL-4 or plus proinflammatory mediators, namely TNF-�, IL-1�,IL-6, and PGE2, as described to generate fast mDCs, both in normoxia andhypoxia, respectively, for further 24 h. For the hypoxic condition, mediumcontaining cytokines or proinflammatory mediators was kept in the work stationincubator for 2 h before the use. On day 4, cytokines and chemokines werequantified in resting and stimulated N-iDC and H-iDC supernatants by ELISA(R&D Systems) in accordance with the manufacturer’s instructions. The kitmeasured vascular endothelial growth factor (VEGF)-A.

On day 4, N-iDCs and H-iDCs were lysed in a buffer containing 20 mM Tris(tris(hydroxymethyl)aminomethane)-HCl, 150 mM NaCl, 1 mM EGTA (ethyl-eneglycoltetraacetic acid), 1 mM EDTA (ethylenediaminetetraacetic acid), 1%Triton-X, 1 mM glycerolphosphate, 1 mM Na3VO4, 2.5 mM sodium pyrophos-

phate, 0.5 mM NaF, 1 mM AEBSF, 10 �g/ml each of aprotinin and leupeptin.The proteins were detected by Western blot (WB) analysis with: polyclonal Abanti-phospho-Moesin to detect p-ERM, polyclonal Ab anti-ezrin, polyclonalanti-hypoxia inducing factor (HIF)-1� and anti-RhoA mAb followed by sec-ondary horseradish peroxidase-conjugated Abs anti-rabbit and anti-mouse IgG(all from Santa Cruz, by DBA, Milan, Italy). WB images were acquired with an“ImageScanner” (GE Healthcare Bio-Sciences, Milan, Italy), recorded in TIFFformat with “ImageMaster” Labscan ver. 3.000 software (GE Healthcare Bio-Sciences) and analyzed using Image Master 2D Elite ver. 3.1 software (GEHealthcare Bio-Sciences).

Activated RhoA in H-iDC, N-iDC, and N-iDCs pretreated at 37°C for 5 hwith 0.1, 0.5, and 2 �g/ml C3 transferase (Cytoskeleton) lysates was detectedwith the G-Lisa RhoA Activation assay (Cytoskeleton), as indicated by themanufacturer.

Statistical analysis

The significance of differences in cpm from the 3HTdR uptake assay, in thenumber of spots by CD8� clones and T cells in Elispot, was evaluated with anunpaired Student’s t test (GraphPad Prism 4, GraphPad Software Inc., SanDiego, CA, USA) with P 0.05 as the significance cutoff. The significance ofdifferences in the membrane marker expression and cytokine and chemokinerelease by DCs was evaluated with a nonparametric Mann Whitney U test (95%of confidence of interval) with P 0.05 as the significance cutoff.

RESULTS

Hypoxia induces human monocytes todifferentiate into iDCs able to respond tochemotactic stimuli

IDCs differentiated from human monocytes after 4 days’ cul-ture with GM-CSF plus IL-4 under hypoxic conditions showedhigh CD1a and CD40 surface expression, even though theymaintained CD14, a monocyte marker (Fig. 1A). However, themean of fluorescence intensity of CD14 was strongly reducedon H-iDCs after 4-day culture in comparison to fresh CD14�

monocytes: it was 19 � 6 on H-iDCs vs. 67 � 5 on freshCD14� cells. In addition, H-iDCs displayed higher levels ofCD86 costimulatory molecules (Fig. 1A) and of HLA class IIand of CD80 [15] compared with that of N-iDCs.

Hypoxia up-modulated the lymphoid chemokine receptorCXCR4 and the inflamed chemokine receptor CCR5 [15],whereas CCR7 was not induced (Fig. 1A). H-iDCs display anactivated, though not mature phenotype. This effect of hypoxiawas constantly observed in samples obtained from six healthydonors, as shown in Table 1.

Because a known effect of hypoxia is stabilization of theHIF-1� transcription factor, we assessed its expression in bothN-iDCs and H-iDCs. High levels were found in H-iDCs,whereas it was not detectable in N-iDCs (Fig. 1B).

Both H-iDCs and N-iDCs are loosely adherent and morpho-logically similar, including the formation of a ruffled andlobular cytoplasm with spikes and semicircular extrusions(Fig. 1C) but are strikingly different from macrophages.These results obtained from three independent donors indi-cate that hypoxia does not prevent the differentiation ofmonocytes into DCs.

We also evaluated the functional efficiency of chemokinereceptors expressed by H-iDCs, as shown in Fig. 2. H-iDCsmigrated in response to different concentrations (5-250 ng/ml)of CCL4, a specific agonist of CCR5 and of CXCL12, a specific

Elia et al. PLEASE SUPPLY SHORT TITLE RUN FOOT 3

agonist of CXCR4, whereas N-iDCs only migrated in responseto CCL4, in agreement with their membrane expression ofCCR5 and CXCR4, respectively (Table 1).

H-iDCs are able to stimulate T cells

H-iDCs and N-iDCs equally induce cell proliferation by allo-geneic T cells, as shown by the MLR assay (Fig. 3A). More-over, T cells from healthy donors alloactivated with H-iDCs or

N-iDCs showed a similar ability to produce IFN-�, whereasthere was no secretion of IL-4 after 72 h culture (Fig. 3B).

We also determined whether hypoxia modulates the abilityof iDCs to present specific peptides to T cells. HLA-A2-restricted CD8� clones specific for Flu-MA58-66 peptide (GIL-GFVFTL) were stimulated with HLA-A2 matched H-iDCs orN-iDCs previously loaded with Flu-MA58-66 and tyrosinase1-9

peptide in hypoxia or normoxia, at different T:DC ratios. Asshown in Fig. 3C, these clones are equally triggered to releaseIFN-� by Flu-MA58-66-loaded H-iDCs or N-iDCs, but not bytyrosinase1-9-loaded H-iDCs or N-iDCs.

To evaluate the ability of H-iDCs to induce a primaryimmune response, we cocultured N-iDCs and H-iDCs, previ-ously loaded with Flu-MA58-66 in normoxia or hypoxia, withautologous naı̈ve T cells from an HLA-A2� healthy donor. Asshown in Fig. 3D, T cells recovered from both cocultures areequally activated to produce IFN-� in response to HLA-A2-matched T2 cells pulsed with Flu-MA58-66 peptide, but not toT2 pulsed with tyrosinase1-9, an irrelevant peptide. Thus, H-iDCs and N-iDCs are equally able to trigger T cell reactivity.

Hypoxia inhibits Ag uptake by iDCs

We used flow cytometry to evaluate macropinocytosis andendocytosis by iDCs in terms of their ability to take up dextran,LPS, BSA, and zymosan after normoxic or hypoxic incubation(Fig. 4, A and B). At 4°C, cell metabolism is inhibited andDCs are unable to capture Ags. However, at 37°C, the uptake

Fig. 1. Phenotype of monocyte-derived iDCs generated un-der hypoxic conditions. (A) CD14� cells were cultured for 4days with GM-CSF and IL-4 in normoxic and hypoxic con-ditions, and their membrane marker expression was deter-mined by flow cytometry. The thin line shows the negativecontrol, the thick line N-iDCs and the gray peak H-iDCs.One of six independent experiments with different donors isshown. Cells were electronically gated according to theirlight-scatter properties to exclude cell debris and contami-nating lymphocytes. (B) Immunoblot for HIF-1�. Proteinswere extracted from both whole H-iDCs and N-iDCs andsubjected to immunoblot analysis using a polyclonal anti-HIF-1� Ab. �-actin loading control is shown. (C) Morphol-ogy of N-iDCs (left panel), H-iDCs (middle panel) andmacrophages (right panel). Inverted microscopy of cells inculture at �200 magnification. The results of one represen-tative experiment out of three performed are shown.

TABLE 1. Membrane Marker Expression by N-iDCs and H-iDCs

Marker

Culture conditions

Normoxia Hypoxia

CD14 6 � 2a 58 � 7*CD1a 87 � 4 83 � 4CD40 93 � 2 91 � 1CD80 22 � 6 51 � 9*CD86 28 � 9 55 � 17CD83 4 � 1 9 � 2*CCR5 42 � 5 84 � 4*CXCR4 6 � 5 64 � 8*CCR7 3 � 1 3 � 1

a % of positive cells � SE. The surface expression of all markers wasdetermined by flow cytometry after 4 days’ culture under hypoxic and normoxicconditions, as specified in Materials and Methods. Data indicate the mean ofpositive cells � SE from 6 donors. *, P 0.05, values significantly differentfrom N-iDCs.


of all 4 Ags by the N-iDCs was significant after 5 min andprogressively increased until 60 min, whereas the H-iDCs werenever able to take up dextran and less able to take up LPS(65% less) and BSA (80% less) and phagocyte zymosan (80%less) (Fig. 4B). Because dextran can also be captured by C-typelectin receptors such as CD206 [19], we checked if its inhib-ited uptake could be ascribed to down-modulation of CD206 byhypoxia. Indeed, hypoxia significantly decreased the expres-sion of CD206 on iDCs (Fig. 4C).

To prove that hypoxia Ag uptake inhibition is not dependenton the DC procedure, we investigated whether the endocyticability of 4-day iDCs and 6-day “conventional” iDCs wasequally decreased. We cultured human monocytes with GM-CSF and IL-4 for 4 and 6 days in hypoxic and normoxic

conditions, and compared their ability to take up dextran andBSA in hypoxia and normoxia, respectively, after 30 min.Four-day N-iDCs behaved as “conventional” 6-day N-iDCsin endocytosing these Ags, whereas both 4-day H-iDCs and6-day H-iDCs are equally impaired in take up dextran andBSA (Fig. 4D).

Hypoxia down-regulates activity of Rho GTPaseand ERM proteins

Because the small GTP binding protein Rho (RhoGTPase) hasbeen reported to be involved in regulating cytoskeletal dynam-ics essential to endocytosis, we evaluated the effect of hypoxiaon its activation. As shown in Fig. 5, total RhoA levels inlysates from N-iDCs and H-iDCs are similar (A, upper panel),but hypoxia reduced the levels of activated RhoA by 60%(�3.4%; three experiments), as evaluated by colorimetry(Fig. 5A). To date, in vitro, as well as in vivo analyses, havesuggested an intimate relationship between activation of theRho pathway and activation of ERM proteins [20]. We evalu-ated the activation of ERM proteins and observed that hypoxiareduces ERM phosphorylation (25%) (Fig. 5B). This couldaffect activation of RhoA GTPase involved in Ag uptake.

To determine whether there is a direct relation betweendown-modulation of RhoA and the inhibition of Ag uptake, wetreated N-iDCs with C3T, a specific Rho inhibitor, at differentconcentrations. As shown in Fig. 5C, 0.5 �g/ml of C3T issufficient to abrogate dextran uptake and reduce the LPSuptake by 65%, as in hypoxia. Even the BSA capture byC3T-treated N-iDCs was reduced but not fully abrogated. Thus theAg-uptake down-modulation by C3T parallels that observed inH-iDCs. Indeed, 0.5 �g/ml of C3T decreases the active form ofRhoA of 50% in N-iDCs (0.48�0.05 vs. 0.24�0.02).

Hypoxia significantly changes the expressionprofile of cytokines and chemokines by iDCs

Cytokines and chemokines were quantified by ELISA in su-pernatants from both resting and stimulated H-iDCs and N-iDCs collected after 24 h of culture in the presence of freshmedium containing GM-CSF plus IL-4 (“resting iDC”) or theproinflammatory mediators used to obtained the fast mDC(“stimulated iDC”). Resting H-iDCs released higher levels ofVEGF, CCL20, CXCL1, CXCL8, and CXCL10, but decreasedlevels of CCL2, CCL18, TNF-�, and IL-10 (Fig. 6A). After24 h of culture in the presence of proinflammatory mediators,stimulated H-iDCs and N-iDC released higher levels of VEGF,CCL20, CXCL1, CXCL8, and CXCL10 than resting H-iDC andN-iDC (Fig. 6B). The only statistically significant differencebetween stimulated H-iDC and N-iDC was the VEGF, CXCL1,and CXCL8 release.

Hypoxia does not affect the maturation of DCsand their ability to trigger T-cell responses

To check whether hypoxia modulates DC maturation, we cul-tured monocytes in the presence of GM-CSF and IL-4 for 2days and then added proinflammatory stimuli for a further 2days to generate mature DCs in both hypoxic and normoxicconditions. H-mDCs showed a phenotype quite similar to that

Fig. 2. Chemotactic response of N-iDCs and H-iDCs. Activity of CCRspresent on both N-iDCs (open bars) and H-iDCs (black bars) was evaluated intriplicate toward a chemokine gradient (5, 50, and 250 ng/ml) with thetranswell system. H-iDC generation and chemotaxis were performed underhypoxic conditions with no exposure to normoxia. The stimuli used weremedium CCL4 (A) and CXCL12 (B). The results are expressed as the averagenumber of cells �103 � SE that migrated to the lower chamber of thetranswell. The results of one representative experiment out of three performedare shown. *, P value 0.05 for chemotactic response significantly differentfrom the medium control.


of N-mDCs. They were both CD83� and expressed higherlevels of HLA class II and costimulatory molecules (Fig. 7A)compared with H-iDCs and N-iDCs, respectively. Both N-mDCs and H-mDCs display a decrease of CCR5 and an in-crease of CXCR4 and CCR7 expression (Fig. 7A), in keepingwith their maturation stage.

As expected, both H-mDCs and N-mDCs are equally effi-cient in inducing allogeneic T cell proliferation as shown byMLR assay (Fig. 7B) and CD8� clone activation (Fig. 7C).Flu-MA58-66-loaded H-mDCs and N-mDCs, in fact, inducedIFN-� secretion by CD8� clones in response to specific pep-tide-loaded DCs but not to tyrosinase1-9-loaded DCs (Fig. 7C).

DISCUSSION

This study defines the effects of chronic hypoxia during differ-entiation of human monocytes into iDCs and provides the firstevidence that it down-modulates Ag uptake by iDCs and sig-nificantly changes their cytokine and chemokine expressionprofile. We recently reported that H-iDCs display an activatedphenotype with an increased surface expression of HLA classII and CD80 costimulatory molecules compared with N-iDCs[15]. Here, we show that H-iDCs also express CD40 at thesame levels of N-iDCs, suggesting a similar ability to interactwith CD40L upon encounter with T cells, but enhanced levelsof CD86. Hypoxia also modifies the chemokine receptor ex-

pression pattern by up-regulating the lymphoid chemokinereceptor CXCR4 and the inflamed chemokine receptor CCR5[15] that are able to drive chemotaxis to specific agonists.CXCR4 is usually up-regulated after the DC maturation [21,22]. It is reported that hypoxia blocks DC ability to migratethrough the Matrigel due to down-modulation of matrix metal-loprotease-9 [23] and up-modulation of tissue inhibitor metal-loprotease-1 [24]. On the other hand in a tumor or inflamedmicroenvironment, several cells produce metalloproteases thatdegrade matrix [25–27] and enable DCs to migrate as theirreceptors are functionally active. CXCR4 increase by hypoxiain monocytes, macrophages, endothelial cells, and cancer cells[28] is evidence of the ability of hypoxia to regulate cellmigration and influence organization of the host response ininflammatory and neoplastic diseases.

We demonstrate that hypoxia does not change the ability ofiDCs to activate T cells, nor their Ag-presenting function.Despite a more activated phenotype, H-iDCs are equally ableto induce proliferation of allogeneic T cells and their IFN-�production compared with N-iDCs and are similar to N-iDCs instimulating both HLA-A2 matched CD8� clones and naı̈ve Tcells to produce IFN-� in the Elispot assay in response to aspecific peptide. This apparent discrepancy could be due to thefact that H-iDCs do not express CD83, a molecule up-regulatedon functionally mature DCs. The central role of CD83 indelivering costimulatory signals for the activation of naı̈ve andmemory T cells in humans has been demonstrated through

Fig. 3. H-iDCs induce proliferation and activation of T cells. (A) One �105 nylon wool purified allogeneic Tcells were cultured with decreasing concentrations of PFA-fixed N-iDCs (open bars) and H-iDCs (solid bars) for5 days, and for a further 16 h in the presence of 3HTdR. The results with six donors independently tested arerepresented as mean of cpm � SE. All tests were performed in triplicate. (B) IFN-� and IL-4 production byallogeneic T cells cultured with PFA-fixed N-iDCs (open squares) and H-iDCs (solid squares). Results arereplicates of six donors tested independently. The horizontal bar marks the mean. (C) One � 103 CD8� T cellclones specific for influenza matrix Flu-ma58-66 peptide were cultured with PFA-fixed N-iDCs and H-iDCs fromHLA-A2 matched healthy donors, previously loaded with specific peptide Flu- MA 58-66 (open bars and solidbars, respectively) or irrelevant peptide tyrosinase1-9 (dotted bars and hatched bars) in normoxia and hypoxia, inElispot plates in quadruplicate for 24 h. The graph represents the number � SE of IFN-�-secreting CD8� T cells,namely spots, in the presence of loaded iDCs, less the spots in the presence of unloaded iDCs. One of twoexperiments with different donors is shown. (D) One � 105 CD45RA� T cells were cultured with autologousPFA-fixed N-iDCs (open bars) and H-iDCs (solid bars) previously loaded with Flu- MA 58-66 peptide in normoxiaand hypoxia for 7 days. Recovered T cells were stimulated with HLA-A2-matched T2 cells previously loaded withspecific peptide Flu-MA58-66 or irrelevant peptide tyrosinase1-9 in Elispot plates for 24 h. The graph representsthe number � SE of IFN-� secreting T cells, namely spots, in the presence of loaded T2 cells, less the spots inthe presence of unloaded T2 cells. One of two experiments with different donors is shown.


RNA interference inhibition in mDCs and overexpression byelectroporated iDCs [29, 30].

Hypoxia does not affect DC maturation, as monocytes in-duced to differentiate in iDCs in prolonged hypoxic conditionswere able to mature when proinflammatory stimuli were addedto GM-CSF and IL-4. H-mDCs, in fact, increased HLA class IIand costimulatory molecule expression compared with H-iDCsand expressed CXCR4 and CCR7 like N-mDCs. H-mDCs andN-mDCs were equally able to activate allogeneic T cells toproliferate and CD8� T cell clones to secrete IFN-� in re-sponse to specific peptide.

The most interesting feature of H-iDCs is the loss of theirtypical Ag uptake ability. IDCs take up Ags via several path-ways [31], such as macropinocytosis and endocytosis via thelectin C-type receptors, but lose this ability on maturation [32].We demonstrate that LPS, BSA, and zymosan uptake arestrongly decreased by 65% to 80%, while that of dextran iscompletely inhibited. The impairment of endocytic ability is aspecific effect of hypoxia. We compared, in fact, endocyticability of 4-day iDCs and “conventional” 6-day iDCs generatedboth in normoxia and hypoxia. “Conventional” H-iDCs werecompletely unable to take up dextran and deficient in taking upBSA like fast H-iDCs. Such an experiment proves that hypoxiaAg uptake inhibition is not dependent on the DC procedure.Annulment of dextran uptake by H-iDCs may be due to hy-poxia-induced down-modulation of CD206, which is equally

involved in dextran internalization [19]. We are currently in-vestigating other endocytosis receptors that may be involved,including the macrophage mannose receptors [32] andDEC205 [33], as well as other cytoskeletal-dependent mecha-nisms. We always observed a marked decrease of CD206 as apercentage of positive cells and/or mean of fluorescence onboth N-mDCs and H-mDCs (69%�27 with 47�31 MFI forN-mDCs and 69%�22 with 32�20 MFI for H-mDCs), whichdid not capture more Ags, as expected. Ag uptake inhibitionmay also be due to down-modulation of Rho GTPase and ERMactivity. Rho-family GTPases, in fact, regulate cytoskeletalrearrangements [34] and are involved in Ag uptake mecha-nisms. We demonstrate that H-iDCs display a significant de-crease of the activated RhoA. Moreover, N-iDCs treated withC3T, a specific inhibitor of Rho activation, were no longer ableto take up Ag, suggesting a direct relation between the down-modulation of activated RhoA and the Ag uptake inhibition.An intimate relationship has been proposed between the Rhopathway and activation of the ERM proteins [20]. When RhoAis inactive, in fact, it resides in the cytoplasm bound to itsinhibitor, called Rho guanine nucleotide dissociation inhibitor(RhoGDI). ERM proteins activated by threonine phosphoryla-tion interact with RhoGDI [20] and detach GDI from Rho,which can thus migrate to the membrane, bind GTP, andbecome activated. We observed that hypoxia reduces ERM

Fig. 4. Hypoxia inhibits Ag uptake by iDCs. Two � 105 4-days N-iDCs (e) or H-iDCs (f) were incubated with FITC-dextran (1 mg/ml), -BSA (1 mg/ml), -LPS(100 �g/ml) and Alexa 488-zymosan (20 particles/cell) at 4°C and 37°C for 5, 15, 30, and 60 min in normoxic and hypoxic conditions and then analyzed by flowcytometry. (A) Geometric mean of fluorescence intensity (MFI) of cells that take up Ags at different time points, less that of cells incubated at 4°C (�GeoMean).One of three independent experiments with different donors is shown. (B) Means � SE of percentage of inhibition of Ag uptake by H-iDCs compared with N-iDCsfrom three independent experiments after 30 min of incubation with Ags at 37°C. Inhibition was calculated by evaluating �GeoMean. (C) CD206 expression onH-iDCs (shaded peak) and N-iDCs (thick line) determined by flow cytometry. The thin solid line shows the isotype control. One of three independently tested donorsis shown. (D) Two � 105 4-day and 6-day N-iDCs (e) or H-iDCs (f) were incubated with FITC-dextran (1 mg/ml) and -BSA (1 mg/ml), at 4°C and 37°C for 30min in normoxic and hypoxic conditions and then analyzed by flow cytometry. Geometric mean of fluorescence intensity (MFI) of cells that take up Ags, less thatof cells incubated at 4°C (�GeoMean). Mean � SE from three independent experiments with different donors is shown.


phosphorylation. This could affect activation of the RhoA GT-Pase involved in Ag uptake.

Local tissue hypoxia may be one of the main signals ofexcessive tissue damage, and this would initiate biochemicalprocesses that down-regulate activated immune cells, preventtheir continuing cytotoxic action, and thereby protect tissuesthat are still healthy [35]. These mechanisms could havedifferent effects on cells of the innate immune system. While itis reported that hypoxia causes an increase in phagocytosis by

macrophages [12], we demonstrate that it down-modulates theability to capture Ag by iDCs. The increase in phagocytosisand the decrease in Ag-uptake are two opposed features typicalfor activated macrophages and DCs, respectively [7, 36–38].

Lastly, we found that hypoxia increases CXCL1, VEGF,CCL20, CXCL8, and CXCL10 secretion by iDCs, and loweredthat of TNF-�, IL-10, CCL2, and CCL18. These data suggestthat hypoxia affects the DC ability to regulate initial recruit-ment tissue infiltration in hypoxic areas, as well as in neoplas-tic and non-neoplastic inflammatory sites in a multistep pro-cess [39]. Our analysis of hypoxia-regulated gene expression inH-iDCs has revealed an array of genes for chemokines amongthe many other genes that appear to be altered by hypoxia [15].By decreasing CCL18 production, hypoxia reduces the migra-tion of naive T cells and iDCs [40]: this may avoid theirinteraction, which may induce tolerance or generation of Tregulatory cells in inflamed and tumor sites. The decrease ofCCL18 parallels that of IL-10. The effect of hypoxia on the

Fig. 6. Hypoxia changes cytokine and chemokine production by iDCs.Supernatants from resting N-iDCs (open bars) and H-iDCs (solid bars) fromsix donors (A), collected after 24 h in the presence of fresh mediumcontaining GM-CSF and IL-4, and from stimulated N-iDCs (open bars) andH-iDCs (solid bars) with proinflammatory mediators (B) were individuallyanalyzed for VEGF, IL-10, TNF-�, CCL2, CCL18, CCL20, CXCL1,CXCL8, and CXCL10 production by ELISA. Results are represented as themean � SE. *, P 0.05 Values significantly different from those forN-iDCs. N.D., not done because TNF-� is present in the culture mediumas proinflammatory stimulus.

Fig. 5. Hypoxia down-modulates RhoA and ERM activation. (A) The graphreports the absorbance related to the activated RhoA from N-iDC and H-iDClysates evaluated by a colorimetric assay. Mean of O.D. at 490 nm � SE from3 donors tested independently. Cell lysates from N-iDCs (lane 1) and H-iDCs(lane 2) were analyzed by WB with anti-RhoA mAb (inset). One of threeindependently tested donors is shown. (B) Phosphorylation of ERM was de-tected by WB with anti-phospho-moesin Ab (one experiment representative of3 reproducible ones) in N-iDC and H-iDC lysates. Numbers indicate therelative values from the densitometric analysis (normalized to Ezrin) relative tonormoxia, which was assigned a value of 1. (C) Rho inhibition leads to the lossof Ag capture. Two � 105 N-iDCs were pretreated with different concentrationsof C3T, a Rho inhibitor, for 5 h, then incubated with FITC-dextran (e) (1mg/ml), -BSA (�) (1 mg/ml), -LPS (◊) (100 �g/ml) for 30 min and analyzed byflow cytometry. Inhibition was calculated by evaluating �GeoMean. One ofthree independently tested donors is shown.


regulation of IL-10 has not been previously reported. IL-10 isa potent anti-inflammatory cytokine that down-modulates othercytokines and cell surface receptors [41]. Several transcriptionfactors have been implicated in the induction of IL-10 [42, 43],but little is known about its down-regulation. Down-modulationof IL-10 may thus favor a prevalently proinflammatory envi-ronment. Both IL-10 and CCL18 are associated with an alter-native polarization (type II) of APCs and are down-modulatedfollowing DC maturation by proinflammatory stimuli [40],whereas CCL20 is usually increased. In our system, H-iDCsproduced higher levels of CCL20, as reported for monocytes[44], and differed from alternative activated or tolerogenic DCsin this respect. This production may enable them to recruit Tmemory and mature DCs [45]. H-iDCs seem more efficient inattracting neutrophils than monocytes by releasing CXCL1 and

CXCL8 [46], since CCL2 production is decreased, as reportedin macrophages [47], and Th1 cells through CXCL10 [48].Neutrophils are efficient scavengers and escape apoptosis inhypoxic conditions [49]. They are thus intrinsically welladapted to operate in oxygen-challenged environments. More-over, H-iDCs, like other cell types [50], release a number ofmolecules with angiogenic impact, such as VEGF, CXCL1, andCXCL8 [4, 45], and thus contribute to the creation of a proin-flammatory and proangiogenic environment. Stimulation ofboth N-iDCs and H-iDCs with proinflammatory stimuli equallydecreased CCL18 production and increased CCL20, CXCL8,and CXCL10. VEGF and CXCL1 secretion is strongly in-creased by both N-iDCs and H-iDCs, even if much more by thelatter ones. This tight and complex level of control exerted bylow O2 tension is clearly of pathophysiological significance as

Fig. 7. Phenotype of H-mDCs vs. N-mDCs and their ability to activate T cells. (A) To obtain mDCs, CD14� cells were cultured for 2 days withGM-CSF and IL-4 followed by incubation with proinflammatory mediators: TNF-�, IL-1�, IL-6, and PGE2 (1 �M) for another 48 h. The surface expressionof all markers was analyzed by flow cytometry after 4 days’ culture. The thin line shows the negative control, the thick line shows N-mDCs, and the graypeak shows H-mDCs. One of six independent experiments with different donors is shown. Cells were electronically gated according to their light-scatterproperties to exclude cell debris and contaminating lymphocytes. (B) One � 105 nylon wool purified allogeneic T cells were cultured with decreasingconcentrations of PFA-fixed N-mDCs (open bars) and H-mDCs (solid bars) for 5 days and for a further 16 h in the presence of 3HTdR. The results fromfour donors independently tested are represented as mean of cpm � SE. All tests were performed in triplicate. (C) One � 103 CD8� T cell clones specificfor influenza matrix Flu- MA58-66 were cultured with PFA-fixed N-mDCs and H-mDCs from HLA-A2-matched healthy donors, previously loaded withspecific peptide Flu- MA58-66 (open bars and solid bars respectively) or irrelevant peptide tyrosinase1-9 (dotted bars and hatched bars) in normoxia andhypoxia, in Elispot plates in quadruplicate for 24 h. The graph represents the number � SE of IFN-� secreting CD8� T cells, namely spots, in the presenceof loaded iDCs, less the spots in the presence of unloaded iDCs. One of two experiments with different donors is shown.


an important mechanism of regulation of leukocyte traffickingand function at the sites of inflammation.

These data suggest that hypoxia’s critical role in DC differ-entiation, adaptation, and activation in inflamed tissues servesto limit self-Ag uptake and recruit neutrophils, i.e., scavengersknown to survive better in hypoxic conditions.

ACKNOWLEDGMENTS

We thank Dr. John Iliffe for critically reading the manuscriptand Dr. Claudia Giachino for providing the CD8� T clonesHLA-A2� specific for influenza matrix Flu-MA58-66 peptide.This work was supported by grants from Italian Association forCancer Research, the Italian Ministry for Education, the Uni-versities and Research, Regione Piemonte, Progetti di RicercaFinalizzata e Applicata, Fondi Incentivazione della Ricerca diBase and Progetti di Rilevante Interesse Nazionale, CompagniaSan Paolo, special project “Oncology,” and Fondazione Itali-ana per la Lotta al Neuroblastoma, Genoa Italy. P. C. wassupported by fellowship from FIRC. T. F. was supported byFondazione Bossolasco.

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