Published online before print August 25, 2008
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,1,1
,2
* Center for Experimental Research and Medical Studies, San Giovanni Battista Hospital, Torino, Italy;
Department of 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
2 Correspondence: Department of Medicine and Experimental Oncology, University of Torino, Corso Raffaello 30, Torino 10126, Italy. E-mail: mirella.giovarelli{at}unito.it
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: antigen-presenting cells • phagocytosis/endocytosis • cytokines • T cell activation • chemokine receptors
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knockout mice demonstrated that HIF-1 has different functions in various types of immune cells [10
]. Although HIF-1 is essential for myeloid cell-mediated inflammation [11
, 12
], it plays an inhibitory role in T cell functioning [13
, 14
]. Hypoxia modulates the expression of proangiogenic factors, inflammatory mediators, and cytokines/chemokines in endothelial cells and monocytes/macrophages, as well as in neoplastic cells [for a review, see Sitkovsky [10
]. The adaptive response to hypoxia favors both neoangiogenesis and the recruitment of scavengers. However, while much is known about the effects of hypoxia on monocytes and macrophages, its effects on the differentiation of monocytes into immature DCs (iDCs), their functional properties, and maturation have not been fully investigated.
In this paper, we show that the prolonged exposition to hypoxia affects DC phenotype and functions. We provide the first demonstration that hypoxia inhibits Ag uptake, a typical function of iDCs, and profoundly changes their chemokine expression profile. Human monocytes induced to differentiate in vitro into iDCs under hypoxic conditions (H-iDCs) display a typical DC morphology and express CD1a, a marker of DCs. In addition, H-iDCs display higher levels of HLA class II, costimulatory molecules and chemokine receptors CCR5 and CXCR4 in comparison to normoxic iDCs (N-iDCs) [15
]. H-iDCs are as efficient as N-iDCs in inducing activation of T cells in response to both specific Ag or alloantigens. However, H-iDCs significantly fail to capture dextran, BSA, LPS, zymosan, through down-modulation of the RhoA/Ezrin-Radixin-Moiesin (ERM) pathway and expression of the lectin receptor CD206. Moreover, they change their chemokine pattern secretion. By comparison with N-iDCs, they produce higher amounts of CCL20, CXCL1, CXCL8, and CXCL10, but lower levels of CCL2 and CCL18, which may predict a different ability to recruit neutrophils and monocytes into hypoxic areas. Furthermore, hypoxia negatively regulates the release of the anti-inflammatory cytokine IL-10 (never previously reported). By contrast, hypoxia does not affect the DC maturation induced by a cocktail of proinflammatory stimuli, namely TNF-, IL-1β, IL-6, and prostaglandin (PGE)2. Hypoxic mature DCs (H-mDCs) efficiently activate both allogeneic and specific T cells like normoxic mature DC (N-mDC).
These data suggest that hypoxia has a critical role in DC differentiation, adaptation, and activation in inflamed tissues: whether it serves to limit self-Ag uptake and recruit neutrophils, i.e., scavengers known to better survive in hypoxic conditions, hypoxia may even contribute to the failure of immune response against tumors by down-modulating DC ability to capture tumor Ags in the microenvironment.
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(50 ng/ml), IL-1β (50 ng/ml), IL-6 (10 ng/ml) (all Peprotech Inc.), and PGE2 (1 µM) (Sigma) to generate mDCs [17
]. When indicated, we compared these 4-day iDCs with those generated with a "conventional" protocol (6-day iDCs). Monocytes were incubated in six-well culture plates (1.5x106 cells/ml) in RPMI 1640 medium-10% FBS certified heat-inactivated (GIBCO, Invitrogen) and 50 µg/ml gentamycin (Schering-Plough), supplemented with 100 ng/ml of GM-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 fresh medium containing GM-CSF and IL-4. On day 6, cells were harvested and used for the experiments. To generate macrophages, CD14+ cells were incubated in six-well culture plates (7.5x105 cells/ml) in complete RPMI 1640 medium-10% FBS certified heat inactivated, supplemented with 100 ng/ml of M-CFS (Peprotech Inc.) for 4 days. Fresh M-CFS was added after 2 days. Cells were then observed with an invert microscope (Olympus CX41, Germany) at x200, and images were recorded as .jpg files.
Culture conditions
For the normoxic condition, cultures were maintained at 37°C in a humidified incubator containing 20% O2, 5% CO2, and 75% N2. For the hypoxic condition, cells were cultured and handled at 37°C in a humidified, anaerobic work station 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/min with a gas mixture of 1% O2, 5% CO2, and 94% N2. All reagents, medium, and cytokines used for the treatments of hypoxic cells were allowed to equilibrate in the anaerobic work station incubator for 2 h before use.
Flow cytometry
Human iDCs and mDCs generated in normoxic or hypoxic conditions were washed and subsequently treated with 1% paraformaldehyde (PFA) for 15 min and 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 nonspecific sites with rabbit IgG (Sigma) for 30 min at 4°C. The following FITC or PE-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, by SPACE Import-Export, Milan, Italy); anti-CD206 purified (Serotec) followed by FITC conjugated anti-mouse IgG (DakoCytomation). FACS analysis was performed with a FACSCalibur and CELLQuest software (BD Biosciences). Cells were gated according to their light-scatter properties to exclude cell debris and contaminating lymphocytes.
Migration assay
N-iDC and H-iDC migration was measured in duplicate with a transwell system (24-well plates; 8.0-µm pore size; Corning Costar by CELBIO, Milan, Italy) under normoxic and hypoxic condition, respectively. Six hundred microliters RPMI medium with or without 5, 50, and 250 ng/ml recombinant human CCL4 or CXCL12 (both Peprotech, Inc.) were added to the lower chamber. Wells with medium only were used as a control for spontaneous migration. A total of 2.5 x 105 cells in 100 µl were added to the upper chamber and incubated at 37°C for 2 h. Cells that migrated into the lower chamber were harvested, concentrated to a volume of 200 µl, and counted by flow cytometry. Events were acquired for a fixed time of 60 s. The counts fell within a linear range of the control titration curves obtained by testing increasing cell concentrations. The mean number of spontaneously migrated cells was subtracted from the total number of cells that migrated in response to the chemokine. Values are given as 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 x 105 cells/well with allogeneic pretreated with 1% PFA N-iDCs, H-iDCs, N-mDCs, and H-mDCs in increasing concentrations (1,250-5,000). After 4 days, 1 µCi (0.037 MBq) of tritiated [3H] TdR (Amersham Biosciences, Milan, Italy) was added to each well, and incubation was prolonged for a further 16 h. Cells were directly collected with a CELLharvester (Packard Instrument, Milan, Italy) on UNIfilter plates (Packard) and 3HTdR uptake was quantitated (TopCount microplates scintillation counter; Packard). All tests were performed in triplicate.
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 (University 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 described [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 primary anti-human IFN-
mAb (5 µg/ml) (Endogen, by Temaricerca, Bologna, Italy) at 4°C overnight. N-iDCs, N-mDC, H-iDCs and H-mDC previously loaded with 3 µg/ml of Flu-MA58-66 or tyrosinase1-9, as unrelated control peptide, for 4 h at 37°C in their respective culture conditions, and subsequently treated with 1% PFA, were added to CD8+ clones in quadruplicate. The plates were then incubated in normoxic conditions for 24 h. A biotinylated secondary anti-IFN- mAb (1 µg/ml) (Endogen) was added and plates were incubated at 37°C for 2 h. The IFN- spots were developed using the AEC substrate (Sigma). The spots were counted by computer assisted image analysis (Transtec 1300 ELISpot Reader, AMI Bioline, Buttigliera Alta, Torino, Italy).
Primary immune response
One x 106 N-iDCs and H-iDCs previously loaded with 3 µg/ml of Flu-MA58-66 for 4 h at 37°C in their respective culture conditions and subsequently treated with 1% PFA were washed and cocultured with 10x106 autologous naïve T cells 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 same donor by positive immunoselection with magnetic beads (Miltenyi Biotec). The resulting preparation was 100% CD45RA+, as determined by FACS analysis. After 7 days, T cell activation was determined by Elispot assay by using HLA-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 x 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 different times in normoxic or hypoxic conditions, respectively. After several washes with cold phosphate buffered solution, fluorescence was measured by FACSCalibur to reveal Ag uptake. Endocytosis comparison between 4-day iDCs and 6-day iDCs was done with FITC-labeled dextran and BSA (Sigma), at 37°C or 4°C for 30 min in normoxic or hypoxic conditions, respectively, and Ag uptake was evaluated as above as described.
In some experiments, 2 x 105 N-iDCs were pretreated for 5 h with C3 transferase (C3T) (Cytoskeleton, by Tebu-bio, Milan, Italy), an inhibitor of Rho GTPase (0.1-2 µg/ml) at 37°C. After treatment, cells were incubated with FITC-labeled Ags (dextran, BSA, LPS) for 30 min at 37°C still in the presence of the inhibitor. Cells were washed several times with cold PBS and fluorescence was measured using a FACScalibur to reveal Ag uptake.
Detection and measurement of proteins
To evaluate cytokines and chemokines produced by resting or stimulated N-iDCs and H-iDCs, on day 3, medium was replaced with fresh medium plus GM-CSF and IL-4 or plus proinflammatory mediators, namely TNF-, IL-1β, IL-6, and PGE2, as described to generate fast mDCs, both in normoxia and hypoxia, respectively, for further 24 h. For the hypoxic condition, medium containing cytokines or proinflammatory mediators was kept in the work station incubator for 2 h before the use. On day 4, cytokines and chemokines were quantified in resting and stimulated N-iDC and H-iDC supernatants by ELISA (R&D Systems) in accordance with the manufacturer’s instructions. The kit measured 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 (ethyleneglycoltetraacetic acid), 1 mM EDTA (ethylenediaminetetraacetic acid), 1% Triton-X, 1 mM glycerolphosphate, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate, 0.5 mM NaF, 1 mM AEBSF, 10 µg/ml each of aprotinin and leupeptin. The proteins were detected by Western blot (WB) analysis with: polyclonal Ab anti-phospho-Moesin to detect p-ERM, polyclonal Ab anti-ezrin, polyclonal anti-hypoxia inducing factor (HIF)-1 and anti-RhoA mAb followed by secondary 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 TIFF format with "ImageMaster" Labscan ver. 3.000 software (GE Healthcare Bio-Sciences) and analyzed using Image Master 2D Elite ver. 3.1 software (GE Healthcare Bio-Sciences).
Activated RhoA in H-iDC, N-iDC, and N-iDCs pretreated at 37°C for 5 h with 0.1, 0.5, and 2 µg/ml C3 transferase (Cytoskeleton) lysates was detected with the G-Lisa RhoA Activation assay (Cytoskeleton), as indicated by the manufacturer.
Statistical analysis
The significance of differences in cpm from the 3HTdR uptake assay, in the number of spots by CD8+ clones and T cells in Elispot, was evaluated with an unpaired Student’s t test (GraphPad Prism 4, GraphPad Software Inc., San Diego, CA, USA) with P < 0.05 as the significance cutoff. The significance of differences in the membrane marker expression and cytokine and chemokine release by DCs was evaluated with a nonparametric Mann Whitney U test (95% of confidence of interval) with P < 0.05 as the significance cutoff.
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Figure 1. Phenotype of monocyte-derived iDCs generated under hypoxic conditions. (A) CD14+ cells were cultured for 4 days with GM-CSF and IL-4 in normoxic and hypoxic conditions, and their membrane marker expression was determined by flow cytometry. The thin line shows the negative control, the thick line N-iDCs and the gray peak H-iDCs. One of six independent experiments with different donors is shown. Cells were electronically gated according to their light-scatter properties to exclude cell debris and contaminating lymphocytes. (B) Immunoblot for HIF-1. Proteins were extracted from both whole H-iDCs and N-iDCs and subjected to immunoblot analysis using a polyclonal anti-HIF-1 Ab. β-actin loading control is shown. (C) Morphology of N-iDCs (left panel), H-iDCs (middle panel) and macrophages (right panel). Inverted microscopy of cells in culture at x200 magnification. The results of one representative experiment out of three performed are shown.
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Table 1. Membrane Marker Expression by N-iDCs and H-iDCs
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transcription factor, we assessed its expression in both N-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 morphologically similar, including the formation of a ruffled and lobular cytoplasm with spikes and semicircular extrusions (Fig. 1C) but are strikingly different from macrophages. These results obtained from three independent donors indicate that hypoxia does not prevent the differentiation of monocytes into DCs.
We also evaluated the functional efficiency of chemokine receptors expressed by H-iDCs, as shown in Fig. 2 . H-iDCs migrated in response to different concentrations (5-250 ng/ml) of CCL4, a specific agonist of CCR5 and of CXCL12, a specific agonist of CXCR4, whereas N-iDCs only migrated in response to CCL4, in agreement with their membrane expression of CCR5 and CXCR4, respectively (Table 1) .
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Figure 2. Chemotactic response of N-iDCs and H-iDCs. Activity of CCRs present on both N-iDCs (open bars) and H-iDCs (black bars) was evaluated in triplicate toward a chemokine gradient (5, 50, and 250 ng/ml) with the transwell system. H-iDC generation and chemotaxis were performed under hypoxic conditions with no exposure to normoxia. The stimuli used were medium CCL4 (A) and CXCL12 (B). The results are expressed as the average number of cells x10–3 ± SE that migrated to the lower chamber of the transwell. The results of one representative experiment out of three performed are shown. *, P value < 0.05 for chemotactic response significantly different from the medium control.
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, whereas there was no secretion of IL-4 after 72 h culture (Fig. 3B)
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Figure 3. H-iDCs induce proliferation and activation of T cells. (A) One x105 nylon wool purified allogeneic T cells were cultured with decreasing concentrations of PFA-fixed N-iDCs (open bars) and H-iDCs (solid bars) for 5 days, and for a further 16 h in the presence of 3HTdR. The results with six donors independently tested are represented as mean of cpm ± SE. All tests were performed in triplicate. (B) IFN- and IL-4 production by allogeneic T cells cultured with PFA-fixed N-iDCs (open squares) and H-iDCs (solid squares). Results are replicates of six donors tested independently. The horizontal bar marks the mean. (C) One x 103 CD8+ T cell clones specific for influenza matrix Flu-ma58-66 peptide were cultured with PFA-fixed N-iDCs and H-iDCs from HLA-A2 matched healthy donors, previously loaded with specific peptide Flu- MA 58-66 (open bars and solid bars, respectively) or irrelevant peptide tyrosinase1-9 (dotted bars and hatched bars) in normoxia and hypoxia, in Elispot 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 two experiments with different donors is shown. (D) One x 105 CD45RA+ T cells were cultured with autologous PFA-fixed N-iDCs (open bars) and H-iDCs (solid bars) previously loaded with Flu- MA 58-66 peptide in normoxia and hypoxia for 7 days. Recovered T cells were stimulated with HLA-A2-matched T2 cells previously loaded with specific peptide Flu-MA58-66 or irrelevant peptide tyrosinase1-9 in Elispot plates for 24 h. The graph represents the number ± SE of IFN- secreting T cells, namely spots, in the presence of loaded T2 cells, less the spots in the presence of unloaded T2 cells. One of two experiments with different donors is shown.
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by Flu-MA58-66-loaded H-iDCs or N-iDCs, but not by tyrosinase1-9-loaded H-iDCs or N-iDCs.
To evaluate the ability of H-iDCs to induce a primary immune response, we cocultured N-iDCs and H-iDCs, previously loaded with Flu-MA58-66 in normoxia or hypoxia, with autologous naïve T cells from an HLA-A2+ healthy donor. As shown in Fig. 3D
, T cells recovered from both cocultures are equally activated to produce IFN- in response to HLA-A2-matched T2 cells pulsed with Flu-MA58-66 peptide, but not to T2 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 and endocytosis 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 and DCs are unable to capture Ags. However, at 37°C, the uptake of all 4 Ags by the N-iDCs was significant after 5 min and progressively increased until 60 min, whereas the H-iDCs were never 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-type lectin receptors such as CD206 [19
], we checked if its inhibited uptake could be ascribed to down-modulation of CD206 by hypoxia. Indeed, hypoxia significantly decreased the expression of CD206 on iDCs (Fig. 4C)
.
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Figure 4. Hypoxia inhibits Ag uptake by iDCs. Two x 105 4-days N-iDCs ( ) or H-iDCs ( ) were incubated with FITC-dextran (1 mg/ml), -BSA (1 mg/ml), -LPS (100 µg/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 flow cytometry. (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-iDCs from three independent experiments after 30 min of incubation with Ags at 37°C. Inhibition was calculated by evaluating GeoMean. (C) CD206 expression on H-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 donors is shown. (D) Two x 105 4-day and 6-day N-iDCs () or H-iDCs () were incubated with FITC-dextran (1 mg/ml) and -BSA (1 mg/ml), at 4°C and 37°C for 30 min in normoxic and hypoxic conditions and then analyzed by flow cytometry. Geometric mean of fluorescence intensity (MFI) of cells that take up Ags, less that of cells incubated at 4°C (GeoMean). Mean ± SE from three independent experiments with different donors is shown.
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Hypoxia down-regulates activity of Rho GTPase and ERM proteins
Because the small GTP binding protein Rho (RhoGTPase) has been reported to be involved in regulating cytoskeletal dynamics essential to endocytosis, we evaluated the effect of hypoxia on its activation. As shown in Fig. 5
, total RhoA levels in lysates 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, have suggested an intimate relationship between activation of the Rho pathway and activation of ERM proteins [20
]. We evaluated the activation of ERM proteins and observed that hypoxia reduces ERM phosphorylation (25%) (Fig. 5B)
. This could affect activation of RhoA GTPase involved in Ag uptake.
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Figure 5. Hypoxia down-modulates RhoA and ERM activation. (A) The graph reports the absorbance related to the activated RhoA from N-iDC and H-iDC lysates evaluated by a colorimetric assay. Mean of O.D. at 490 nm ± SE from 3 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 three independently tested donors is shown. (B) Phosphorylation of ERM was detected by WB with anti-phospho-moesin Ab (one experiment representative of 3 reproducible ones) in N-iDC and H-iDC lysates. Numbers indicate the relative values from the densitometric analysis (normalized to Ezrin) relative to normoxia, which was assigned a value of 1. (C) Rho inhibition leads to the loss of Ag capture. Two x 105 N-iDCs were pretreated with different concentrations of C3T, a Rho inhibitor, for 5 h, then incubated with FITC-dextran () (1 mg/ml), -BSA ( ) (1 mg/ml), -LPS ( ) (100 µg/ml) for 30 min and analyzed by flow cytometry. Inhibition was calculated by evaluating GeoMean. One of three independently tested donors is shown.
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Hypoxia significantly changes the expression profile of cytokines and chemokines by iDCs
Cytokines and chemokines were quantified by ELISA in supernatants from both resting and stimulated H-iDCs and N-iDCs collected after 24 h of culture in the presence of fresh medium containing GM-CSF plus IL-4 ("resting iDC") or the proinflammatory mediators used to obtain the fast mDC ("stimulated iDC"). Resting H-iDCs released higher levels of VEGF, CCL20, CXCL1, CXCL8, and CXCL10, but decreased levels of CCL2, CCL18, TNF-, and IL-10 (Fig. 6A
). After 24 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 and N-iDC (Fig. 6B)
. The only statistically significant difference between stimulated H-iDC and N-iDC was the VEGF, CXCL1, and CXCL8 release.
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Figure 6. Hypoxia changes cytokine and chemokine production by iDCs. Supernatants from resting N-iDCs (open bars) and H-iDCs (solid bars) from six donors (A), collected after 24 h in the presence of fresh medium containing GM-CSF and IL-4, and from stimulated N-iDCs (open bars) and H-iDCs (solid bars) with proinflammatory mediators (B) were individually analyzed for VEGF, IL-10, TNF-, CCL2, CCL18, CCL20, CXCL1, CXCL8, and CXCL10 production by ELISA. Results are represented as the mean ± SE. *, P < 0.05 Values significantly different from those for N-iDCs. N.D., not done because TNF- is present in the culture medium as proinflammatory stimulus.
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Figure 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 with GM-CSF and IL-4 followed by incubation with proinflammatory mediators: TNF-, IL-1β, IL-6, and PGE2 (1 µM) for another 48 h. The surface expression of 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 gray peak shows H-mDCs. One of six independent experiments with different donors is shown. Cells were electronically gated according to their light-scatter properties to exclude cell debris and contaminating lymphocytes. (B) One x 105 nylon wool purified allogeneic T cells were cultured with decreasing concentrations 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 from four donors independently tested are represented as mean of cpm ± SE. All tests were performed in triplicate. (C) One x 103 CD8+ T cell clones specific for influenza matrix Flu- MA58-66 were cultured with PFA-fixed N-mDCs and H-mDCs from HLA-A2-matched healthy donors, previously loaded with specific peptide Flu- MA58-66 (open bars and solid bars respectively) or irrelevant peptide tyrosinase1-9 (dotted bars and hatched bars) in normoxia and hypoxia, in Elispot 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 two experiments with different donors is shown.
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secretion by CD8+ clones in response to specific peptide-loaded DCs but not to tyrosinase1-9-loaded DCs (Fig. 7C)
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We demonstrate that hypoxia does not change the ability of iDCs to activate T cells, nor their Ag-presenting function. Despite a more activated phenotype, H-iDCs are equally able to induce proliferation of allogeneic T cells and their IFN- production compared with N-iDCs and are similar to N-iDCs in stimulating both HLA-A2 matched CD8+ clones and naïve T cells to produce IFN- in the Elispot assay in response to a specific peptide. This apparent discrepancy could be due to the fact that H-iDCs do not express CD83, a molecule up-regulated on functionally mature DCs. The central role of CD83 in delivering costimulatory signals for the activation of naïve and memory T cells in humans has been demonstrated through RNA interference inhibition in mDCs and overexpression by electroporated iDCs [29
, 30
].
Hypoxia does not affect DC maturation, as monocytes induced to differentiate in iDCs in prolonged hypoxic conditions were able to mature when proinflammatory stimuli were added to GM-CSF and IL-4. H-mDCs, in fact, increased HLA class II and costimulatory molecule expression compared with H-iDCs and expressed CXCR4 and CCR7 like N-mDCs. H-mDCs and N-mDCs were equally able to activate allogeneic T cells to proliferate and CD8+ T cell clones to secrete IFN- in response to specific peptide.
The most interesting feature of H-iDCs is the loss of their typical Ag uptake ability. IDCs take up Ags via several pathways [31 ], such as macropinocytosis and endocytosis via the lectin C-type receptors, but lose this ability on maturation [32 ]. We demonstrate that LPS, BSA, and zymosan uptake are strongly decreased by 65% to 80%, while that of dextran is completely inhibited. The impairment of endocytic ability is a specific effect of hypoxia. We compared, in fact, endocytic ability of 4-day iDCs and "conventional" 6-day iDCs generated both in normoxia and hypoxia. "Conventional" H-iDCs were completely unable to take up dextran and deficient in taking up BSA like fast H-iDCs. Such an experiment proves that hypoxia Ag uptake inhibition is not dependent on the DC procedure. Annulment of dextran uptake by H-iDCs may be due to hypoxia-induced down-modulation of CD206, which is equally involved in dextran internalization [19 ]. We are currently investigating other endocytosis receptors that may be involved, including the macrophage mannose receptors [32 ] and DEC205 [33 ], as well as other cytoskeletal-dependent mechanisms. We always observed a marked decrease of CD206 as a percentage of positive cells and/or mean of fluorescence on both N-mDCs and H-mDCs (69%±27 with 47±31 MFI for N-mDCs and 69%±22 with 32±20 MFI for H-mDCs), which did not capture more Ags, as expected. Ag uptake inhibition may also be due to down-modulation of Rho GTPase and ERM activity. Rho-family GTPases, in fact, regulate cytoskeletal rearrangements [34 ] and are involved in Ag uptake mechanisms. We demonstrate that H-iDCs display a significant decrease of the activated RhoA. Moreover, N-iDCs treated with C3T, a specific inhibitor of Rho activation, were no longer able to 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 Rho pathway and activation of the ERM proteins [20 ]. When RhoA is inactive, in fact, it resides in the cytoplasm bound to its inhibitor, called Rho guanine nucleotide dissociation inhibitor (RhoGDI). ERM proteins activated by threonine phosphorylation interact with RhoGDI [20 ] and detach GDI from Rho, which can thus migrate to the membrane, bind GTP, and become activated. We observed that hypoxia reduces ERM phosphorylation. This could affect activation of the RhoA GTPase involved in Ag uptake.
Local tissue hypoxia may be one of the main signals of excessive tissue damage, and this would initiate biochemical processes that down-regulate activated immune cells, prevent their continuing cytotoxic action, and thereby protect tissues that are still healthy [35 ]. These mechanisms could have different effects on cells of the innate immune system. While it is reported that hypoxia causes an increase in phagocytosis by macrophages [12 ], we demonstrate that it down-modulates the ability to capture Ag by iDCs. The increase in phagocytosis and the decrease in Ag-uptake are two opposed features typical for activated macrophages and DCs, respectively [7 , 36 37 38 ].
Lastly, we found that hypoxia increases CXCL1, VEGF, CCL20, CXCL8, and CXCL10 secretion by iDCs, and lowered that of TNF-, IL-10, CCL2, and CCL18. These data suggest that hypoxia affects the DC ability to regulate initial recruitment tissue infiltration in hypoxic areas, as well as in neoplastic and non-neoplastic inflammatory sites in a multistep process [39
]. Our analysis of hypoxia-regulated gene expression in H-iDCs has revealed an array of genes for chemokines among the many other genes that appear to be altered by hypoxia [15
]. By decreasing CCL18 production, hypoxia reduces the migration of naive T cells and iDCs [40
]: this may avoid their interaction, which may induce tolerance or generation of T regulatory cells in inflamed and tumor sites. The decrease of CCL18 parallels that of IL-10. The effect of hypoxia on the regulation of IL-10 has not been previously reported. IL-10 is a potent anti-inflammatory cytokine that down-modulates other cytokines and cell surface receptors [41
]. Several transcription factors have been implicated in the induction of IL-10 [42
, 43
], but little is known about its down-regulation. Down-modulation of IL-10 may thus favor a prevalently proinflammatory environment. Both IL-10 and CCL18 are associated with an alternative polarization (type II) of APCs and are down-modulated following DC maturation by proinflammatory stimuli [40
], whereas CCL20 is usually increased. In our system, H-iDCs produced higher levels of CCL20, as reported for monocytes [44
], and differed from alternative activated or tolerogenic DCs in this respect. This production may enable them to recruit T memory and mature DCs [45
]. H-iDCs seem more efficient in attracting neutrophils than monocytes by releasing CXCL1 and CXCL8 [46
], since CCL2 production is decreased, as reported in macrophages [47
], and Th1 cells through CXCL10 [48
]. Neutrophils are efficient scavengers and escape apoptosis in hypoxic conditions [49
]. They are thus intrinsically well adapted to operate in oxygen-challenged environments. Moreover, H-iDCs, like other cell types [50
], release a number of molecules with angiogenic impact, such as VEGF, CXCL1, and CXCL8 [4
, 45
], and thus contribute to the creation of a proinflammatory and proangiogenic environment. Stimulation of both N-iDCs and H-iDCs with proinflammatory stimuli equally decreased CCL18 production and increased CCL20, CXCL8, and CXCL10. VEGF and CXCL1 secretion is strongly increased by both N-iDCs and H-iDCs, even if much more by the latter ones. This tight and complex level of control exerted by low O2 tension is clearly of pathophysiological significance as an important mechanism of regulation of leukocyte trafficking and function at the sites of inflammation.
These data suggest that hypoxia’s critical role in DC differentiation, adaptation, and activation in inflamed tissues serves to limit self-Ag uptake and recruit neutrophils, i.e., scavengers known to survive better in hypoxic conditions.
Received February 4, 2008; revised July 16, 2008; accepted August 5, 2008.
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-dependent manner J. Leukoc. Biol. 82,1257-1265 regulates T cell receptor signal transduction Proc. Natl. Acad. Sci. USA 102,17071-17076 and pro-inflammatory cytokine production by antigen-presenting cells is dictated by intracellular thiol redox status regulated by oxygen tension Eur. J. Immunol. 32,2866-2873[CrossRef][Medline]-dependent NF-
B activity J. Exp. Med. 201,105-115This article has been cited by other articles:
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  H.-Y. Fang, R. Hughes, C. Murdoch, S. B. Coffelt, S. K. Biswas, A. L. Harris, R. S. Johnson, H. Z. Imityaz, M. C. Simon, E. Fredlund, et al. Hypoxia-inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia Blood, July 23, 2009; 114(4): 844 - 859. [Abstract] [Full Text] [PDF]
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