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Identification Of CC-Chemokine Ligand 20 Novel Immune-Related Genes andPrimary Human Monocytes: Modulation of Hypoxia Modifies the Transcriptome of
Luigi VaresioandAnfosso, Ulrich Pfeffer, Paolo Fardin, Florinda Battaglia
Maria Carla Bosco, Maura Puppo, Clara Santangelo, Luca
http://www.jimmunol.org/content/177/3/1941doi: 10.4049/jimmunol.177.3.1941
2006; 177:1941-1955; ;J Immunol
Referenceshttp://www.jimmunol.org/content/177/3/1941.full#ref-list-1
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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,
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Hypoxia Modifies the Transcriptome of Primary HumanMonocytes: Modulation of Novel Immune-Related Genes andIdentification Of CC-Chemokine Ligand 20 as a NewHypoxia-Inducible Gene1
Maria Carla Bosco,2* Maura Puppo,* Clara Santangelo,* Luca Anfosso,‡ Ulrich Pfeffer,†
Paolo Fardin,* Florinda Battaglia,* and Luigi Varesio*
Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize themolecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profileinduced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the geneexpression pattern were detected following 16 h exposure to 1% O2, with 536 and 677 sequences showing at least a 1.5-fold increaseand decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differencesof selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whoseresponsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation ofa significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1,MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5,LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore,we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memoryT lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. Thisstudy represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into mono-cyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mecha-nism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis ofchronic inflammatory diseases. The Journal of Immunology, 2006, 177: 1941–1955.
P eripheral blood monocytes (Mn)3 represent the earlymononuclear phagocyte component of the leukocyte infil-trate at sites of inflammation, infection, and tumor growth,
where they differentiate into inflammatory and tumor-associatedmacrophages (Mf) (1). Mn/Mf are potent regulators of immuneand inflammatory reactions. They orchestrate the coordinated re-cruitment and activation of specific leukocyte subsets at patholog-ical sites through the local secretion of low m.w. structurally re-lated proteins, termed chemokines (1). Chemokines are classifiedinto CXC, CC, C, and CX3C families, which bind to and activatemembers of a superfamily of 7-transmembrane domain, G protein-
coupled receptors differentially expressed and regulated in leuko-cytes (2). CCL20 (also known as MIP-3�, liver and activationregulated chemokine, and Exodus) is a recently described Mn-derived CC-chemokine which selectively attracts immature den-dritic cells (iDC), effector/memory T lymphocytes, and naive Bcells through its specific receptor, CCR6, expressed on these cells(for a review, see Ref. 3).
Mononuclear phagocyte reactivity in pathological tissues isfinely tuned by a complex interplay between stimulatory and in-hibitory signals of various nature that include immune-derivedstimuli (4, 5), viral/bacterial products (5, 6), cell metabolites (4, 7),and tissue-specific signals (8). A common denominator of manypathological processes and an important regulator of gene expres-sion is represented by low partial oxygen pressure (pO2) (reviewedin Ref. 9). Hypoxia occurs in cardiovascular, hematological, andpulmonary disorders, ischemic wounds, arthritic joints, atheroscle-rotic plaques, and microbial infections, and experimental and clin-ical studies point toward its fundamental role in the pathogenesisof these diseases (8–11). Areas of low pO2 are also present in solidtumors, where they have been associated with malignant progres-sion, metastasis formation, resistance to therapy, and poor clinicaloutcome (8, 12–14). Transcriptional response to hypoxia is medi-ated primarily by the hypoxia-inducible factor-1 (HIF-1), a het-erodimeric basic helix-loop-helix (bHLH) transcription factorcomposed of HIF-1� (also known as the aryl hydrocarbon receptornuclear translocator (ARNT)), the constitutive subunit, and HIF-1�, 2�, or 3�, the oxygen-sensitive subunits (9, 15). The � sub-units are posttranslationally stabilized under hypoxia and translo-cate to the nucleus where they dimerize with HIF-1�,
*Laboratory of Molecular Biology, G. Gaslini Institute, and †Functional Genomics,National Cancer Research Institute, Genova, Italy; and ‡University of Insubria, Va-rese, Italy
Received for publication November 4, 2005. Accepted for publication May 18, 2006.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Italian Association for Cancer Re-search, Fondazione Italiana per la Lotta al Neuroblastoma, San Paolo Company, Ital-ian Health Ministry, and Ministero Istruzione Universita’ e Ricerca.2 Address correspondence and reprint requests to Dr. Maria Carla Bosco, Laboratoriodi Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go GerolamoGaslini 5, 16147 Genova Quarto, Italy. E-mail address: mcbosco1@virgilio.it3 Abbreviations used in this paper: Mn, monocyte; Mf, macrophage; iDC, immaturedendritic cell; pO2, partial oxygen pressure; HIF-1, hypoxia-inducible factor-1; GO,Gene Ontology; EASE, Expression Analysis Systematic Explorer; qRT-PCR, real-time quantitative PCR; VEGF, vascular endothelial growth factor; HMG, hypoxia-modulated gene; IRS, immunoregulatory signaling; ARNT, aryl hydrocarbon receptornuclear translocator; ECM, extracellular matrix; hMDM, human monocyte-derivedmacrophage; MMP, matrix metalloproteinase; bHLH, basic helix-loop-helix.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
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transactivating the hypoxia responsive element present in the pro-moter of many O2-sensitive genes (9, 15). Regulation of HIF-1expression and activity by hypoxia is a tightly regulated processwhich results from the activity of several oxygen-dependent en-zymes and requires interaction and cooperation with various tran-scriptional cofactors and other transcription factors (15).
Mononuclear phagocytes accumulate preferentially in hypoxic/ischemic areas of diseased tissues (1), and hypoxic conditions havebeen shown to profoundly affect their proinflammatory and immu-noregulatory responses by modulating the expression of genescoding for angiogenic factors, inflammatory cytokines, and extra-cellular matrix (ECM) components/regulators (reviewed in Ref. 1).Recent evidence indicates that hypoxia can also strictly control thechemokine network in cells of the monocytic lineage not only byregulating the production of specific chemokines but also control-ling their action through the modulation of their receptors. Up-regulation of CCL3 (16), CXCL1 (1), CXCL8 (1), and CXCR4(17), and inhibition of CCL2 (16) and CCR5 (18) under hypoxiawere reported.
In the last few years, microarray technology has become animportant tool for the characterization of the molecular bases un-derlying cell response to stimulation (19). Recent investigationshave defined the transcriptional profile induced by hypoxia in invitro-derived human Mf (hMDM) (20, 21). Given their critical rolein the regulation of the initial phases of inflammation (1), it wasimportant to study primary Mn as a model of the early response tothe hypoxic environment. In this study, we report the first transcrip-tome analysis of primary human Mn following hypoxia exposure. Ourresults reveal the regulation by hypoxia of a cluster of novel genesrelevant to inflammation and immunity coding for surface molecules/markers, inflammatory cytokines/chemokines, and their receptors,and identify CCL20 as a new hypoxia-inducible gene.
Materials and MethodsCells and culture conditions
PBMC were isolated from platelet apheresis of healthy donors, obtained bythe Blood Transfusion Center of the Gaslini Institute (Genova, Italy), bydensity gradient centrifugation over a Ficoll cushion (Ficoll-Paque PLUS;Amersham Biosciences). Mn were then purified by countercurrent centrif-ugal elutriation using a Beckman JE-6 elutriation chamber and AvantiJ-20XP rotor system (Beckman Coulter), as described (22), followed byMACS magnetic bead separation (Human Monocyte Isolation kit-II; Milte-nyi Biotec). The purity of Mn preparations was �95%, as assessed bymorphology on Giemsa-stained cytocentrifuge slides and flow cytometrywith anti-CD14 mAb. Viability, determined by flow cytometry after DNAstaining with propidium iodide (5 �g/ml in PBS), was �98%.
Mn were plated in Costar plates (Celbio) in RPMI 1640 (Euroclone;Celbio) supplemented with 10% heat-inactivated FCS (HyClone; Celbio),2 mM L-glutamine, 100 U/ml penicillin, and 100 �g/ml streptomycin (Cel-bio) and maintained at 37°C in a humidified incubator containing 21% O2,5% CO2, and 75% N2, referred to as normoxic conditions. Hypoxic con-ditions (i.e., 1% O2) were achieved by incubating and handling the cells at37°C in a humidified anaerobic work station incubator (Bug Box; ALCInternational) flushed with a mixture of 94% N2, 5% CO2, and 1% O2.Culture medium was allowed to equilibrate for 3 h in a loosely capped flaskin the hypoxic incubator before cell exposure, and pO2 was monitoredusing a portable trace oxygen analyzer (Oxi 315i/set; WTW). The pO2 innormoxic medium ranged between 149 and 150 mm Hg, values that cor-respond to a 21% O2 concentration in an aqueous solution at 37°C and ata barometric pressure of 760 mm Hg, whereas the pO2 attained in themedium under hypoxic conditions was �7.1 mm Hg, which is equivalentto an O2 concentration of 1% and is in the range of the hypoxic levels foundin inflammatory tissues (8–12). The endotoxin content, determined by theLimulus amebocyte lysate test (QCL-1000; Bio-Whittaker), was �0.125endotoxin units/ml in all reagents.
RNA isolation and cRNA synthesis
Total RNA was purified from different donor-derived Mn using the RNeasyMini kit from Qiagen. The physical quality control of RNA integrity was
conducted by electrophoresis with an Agilent Bioanalyzer 2100 (AgilentTechnologies Europe). For each experimental condition, equal amounts ofMn RNA from 15 different donors were randomly pooled into three sub-sets, and the RNA pools were used for probe preparation. Briefly, 20 �g ofRNA were reverse transcribed into double-stranded cDNA on a GeneAmpPCR System 2700 thermal cycler (Applied Biosystems), using the Super-Script Double-Stranded cDNA Synthesis kit (Invitrogen Life Technolo-gies) according to the manufacturer’s instructions, except that a T7-(dT)24
primer (high purity salt-free purified) was used in place of the oligo pro-vided with the kit. cDNA was purified and used for in vitro transcriptionwith the BioArray High Efficiency RNA Transcript Labeling kit (Enzo LifeSciences) in the presence of biotin-11-CTP and biotin-16-UTP. LabeledcRNA was cleaned up using the Qiagen RNeasy Mini kit, checked forquality, and fragmented by incubation in mild alkaline buffer.
GeneChip hybridization and data analysis
Fragmented cRNA probes were used for hybridization to Affymetrix Hu-man Genome-U133A 2.0 GeneChips (Genopolis) containing 22,283 probesets corresponding to 18,400 transcripts. Each RNA pool was hybridized toan individual chip, and hybridization was performed at 45°C in the pres-ence of herring sperm DNA (0.1 mg/ml; Sigma-Aldrich). Chips were thenwashed with 6� standard saline citrate phosphate/EDTA (1� is 0.15 MNaCl, 0.01 M sodium phosphate (pH 7.4), and 1 mM EDTA), stained withstreptavidin-PE, and scanned using a confocal microscope scanner (HPGeneArray Scanner 2500) according to Affymetrix’s guidelines. Data cap-turing was conducted with standard Affymetrix analysis software algo-rithms (Microarray Suite 5.0), which selects the spots representative of atranscript and subtracts the background from the significant signals (23).Comparative analysis of hypoxic relative to normoxic expression profileswas conducted on GeneSpring 7.2 software (Silicon Genetics). Gene ex-pression data for each replicate experiment were normalized using the “perchip normalization” and “per gene normalization” algorithms implementedin the GeneSpring program. First, each signal was normalized based uponthe median signal in that chip (“per chip normalization”). Each correctedvalue was then normalized based upon the median of the measurements forthat gene in all samples (“per gene normalization”). This normalizationmethod, which removes the differing intensity scales and binding ratesfrom multiple experimental readings, allows the comparison of multipleGeneChip hybridizations (24). Finally, gene expression levels of replicateexperiments were averaged, and only genes that were modulated by at least1.5-fold in hypoxic relative to normoxic samples (means of three experi-ments) were considered differentially expressed. The significance of geneexpression differences between the two experimental conditions was cal-culated using a one-way ANOVA. Only genes that passed a Student’stwo-sample t test at a confidence level of 95% ( p value �0.05) wereconsidered significant. The complete data set for each microarray ex-periment was lodged in the ArrayExpress website �www.ebi.ac.uk/arrayexpress�. Gene Ontology (GO) data mining (25) for biological processat level 3 and Expression Analysis Systematic Explorer (EASE) biologicaltheme analysis (26) were conducted online at �http://david.niaid.nih.gov�using the Database for Annotation, Visualization, and Integrated Discovery(DAVID) 2.0 program (27).
Real-time RT-PCR
Real time quantitative PCR (qRT-PCR) of reverse-transcribed cDNA wasperformed on an I-Cycler (Bio-Rad), using iQ Supermix supplementedwith 10 nM fluorescein (Bio-Rad), 0.1� Sybr-Green I (Sigma-Aldrich),and 300 nM sense and antisense oligonucleotide primers (TIBMolbiol). Allprimer pairs (listed in Table I) were designed using Primer-3 software (28)from sequences in GenBank with a Tm optimum of 60°C and a productlength of 80–150 nt and tested before use to confirm appropriate productsize and optimal concentrations. qRT-PCR was conducted in triplicate foreach target transcript under the following cycling conditions: initial dena-turation of 3 min during which the well factor was measured, 50 cycles of15 s at 95°C followed by 30 s at 60°C. Fluorescence was measured duringthe annealing step in each cycle. After amplification, melting curves with80 steps of 15 s and 0.5°C increase were performed to monitor ampliconidentity. Expression data were normalized on the values obtained in par-allel for three reference genes (actin-related protein 2/3 complex 1B(ARPC1B); lysosomal-associated multispanning membrane protein-5(LAPTM5); and thrombospondin 1 (THBS1)) selected among those notaffected by hypoxia in the Affymetrix analysis, using the Bestkeeper soft-ware (29). Relative expression values were calculated using Q-gene soft-ware (30).
For semiquantitative PCR, reverse-transcribed cDNA was amplified intriplicate with recombinant TaqDNA polymerase (Invitrogen Life Tech-nologies) using the following cycling conditions: denaturation at 94°C,
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annealing at 61°C, and extension at 72°C for 60 s for 30 cycles. Productswere separated by electrophoresis on a 1.2% agarose gel and visualized byethidium bromide staining.
Immunocytochemistry
A total of 1 � 105 Mn were applied to polysine glass slides by cytocen-trifuging at 900 rpm for 5 min. Cytospin preparations were fixed in 4%paraformaldehyde in PBS for 30 min at room temperature, and permeabil-
ized with 0.1% Triton X-100 in PBS for 5 min. Endogenous peroxidaseswere blocked with 0.3% hydrogen peroxide for 15 min. After rinsing inPBS, the slides were preincubated for 30 min in blocking buffer (PBSsupplemented with 2% human AB serum) and then incubated for 1 h withanti-human CCL20 mAb or an isotype-matched mAb (IgG1; R&D System)in blocking buffer. mAbs were detected with DakoCytomation Envision�
System Labeled Polymer-HRP anti-mouse. Peroxidase staining was re-vealed by 3-amino-9-ethylcarbazole (DakoCytomation), as a substrate.
Table I. Primer pairs used for real-time quantitative RT-PCRa
Geneb Primers Product (bp)
Actin-related protein 2/3 complex, subunit 1B (ARPC1B*) For 5�-aacgagaacaagtttgctgtg-3� 106Rev 5�-gatgggcttcttgatgtgc-3�
Activating transcription factor 5 (ATF5) For 5�-cccaccttctttcttcagc-3� 119Rev 5�-acgaggctctggaggatg-3�
Adrenomedullin (ADM) For 5�-cctgatgtacctgggttc-3� 118Rev 5�-ttccctcttcccacgact-3�
Arginase (ARG1) For 5�-attgagaaaggctggtctgc-3� 95Rev 5�-cattagggatgtcagcaaagg-3�
B cell activation gene (RGS1) For 5�-tgctgaagtaatgcaatggtct-3� 127Rev 5�-caagccagccagaactcaat-3�
BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) For 5�-ttccatctctgctgctctc-3� 80Rev 5�-tggtggaggttgtcagac-3�
Carbonic anhydrase XII (CA12) For 5�-cttggcatctgtattgtggtg-3� 121Rev 5�-tgggcctcagtctccatc-3�
CD163 Ag (CD163) For 5�-ttcgtcgcattattcttcttgac-3� 181Rev 5�-ggcaatagaccctccaag-3�
CD69 Ag (CD69) For 5�-gaacatggtgctactcttgc-3� 81Rev 5�-ttcctctctacctgcgtatc-3�
Chemokine (CC motif) ligand 20 (CCL20) For 5�-aatttattgtgggcttcacacg-3� 115Rev 5�-acccaagtctgttttggatttg-3�
Chemokine (CC motif) ligand 23 (CCL23) For 5�-ctttgaaacgaacagcgagtg-3� 179Rev 5�-cttgtgtcccttcaccttg-3�
Chemokine (CXC motif) receptor 4 (CXCR4) For 5�-gcatctggagaaccagcg-3� 111Rev 5�-gaaacagggttccttcatgg-3�
Fc fragment of IgG, high-affinity Ia, receptor for (FCGR1A) For 5�-atcgctacacatcagcagg-3� 137Rev 5�-ctgcaagagcaactttgtttc-3�
Fc fragment of IgG, low-affinity IIc, receptor for (FCGR2A) For 5�-acttctccatcccacaagc-3� 112Rev 5�-gagcttggacagtgatgg-3�
Fc fragment of IgG, low-affinity IIb, receptor for (FCGR2B) For 5�-ttacctgtccttgccactg-3� 124Rev 5�-agtttcagcacagcctttgg-3�
Hypoxia-inducible factor 1, � subunit (HIF1A) For 5�-aaatctcatccaagaagcccta-3� 118Rev 5�-cgctttctctgagcattctg-3�
IL-1R antagonist (IL1RN) For 5�-tcatgctctgttcttgggaat-3� 132Rev 5�-gcttgtcctgctttctgttc-3�
IL-6 signal transducer (IL-6ST) For 5�-ggcctaatgttccagatcc-3� 145Rev 5�-tcatttgcttctatttccacaaca-3�
Leukocyte-associated Ig-like receptor 1 (LAIR1) For 5�-cagattccgcattgactcag-3� 122Rev 5�-gaggtttctttcaccagcag-3�
Leukocyte Ig-like receptor, subfamily B, member 7 (LIR9) For 5�-gtatggtcagaacccagtg-3� 121Rev 5�-tgcgtaatcctgaaggtgtg-3�
Leukocyte membrane Ag (CMRF-35H) For 5�-gcactacgcaaatctggag-3� 163Rev 5�-tctgagcagctatcctgttg-3�
Lysosomal-associated multispanning membrane protein-5 (LAPTM5*) For 5�-ggtcacacctctgagtatg-3� 131Rev 5�-gtggaggagaagagaaactc-3�
Macrophage migration inhibitory factor (MIF) For 5�-gtccttctgccatcatgc-3� 166Rev 5�-gaaggccatgagctggt-3�
MAX-interacting protein 1 (MXI1) For 5�-agatggaacgaatacgaatgg-3� 110Rev 5�-gggagaactctgtgctttc-3�
N-myc downstream-regulated gene 1 (NDRG1) For 5�-ccttatcaacgtgaacccttg-3� 132Rev 5�-gttactctgcatttcttccttc-3�
Secreted phosphoprotein 1 (SPP1) For 5�-tgacccatctcagaagcag-3� 111Rev 5�-atggctttcgttggacttac-3�
Stabilin 1 (STAB1) For 5�-actcttcgtccctgtcaatg-3� 157Rev 5�-tcactgatgatgaggctgag-3�
Thrombospondin 1 (THBS1*) For 5�-cagcattctccatcaggaac-3� 125Rev 5�-gaggaatggactgttgatagc-3�
Vascular endothelial growth factor (VEGF) For 5�-gcagcttgagttaaacgaacg-3� 150Rev 5�-gcagcgtggtttctgtatc-3�
Vav 3 oncogene (VAV3) For 5�-tgttgtgagacgtttggaatg-3� 112Rev 5�-tgttcgagaaagtcgtgataatg-3�
a The expected PCR product size for each gene is shown. For, forward; Rev, reverse.b The * indicates the reference genes used for data normalization.
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Slides were counterstained with hematoxylin, coverslipped with 80%glycerol in PBS, and examined with a phase contrast microscope (OlympusItalia). Photomicrographs were taken with a Zeiss camera.
ELISA
Secreted CCL20 was measured in cell-free supernatants using the Quan-tikine human CCL20 immunoassay kit from R&D Systems (Space ImportExport, according to the manufacturer’s instructions. The OD of the plateswere determined using a Spectrafluor Plus plate reader from Tecan at 450nm. All assays were done in duplicate and repeated three times.
ResultsGene expression profile of hypoxic Mn
Mn purified from 15 independent donors were cultured for 16 h in1% O2, a condition previously shown to effectively modulate geneexpression in these cells (17, 31), and the mRNA for the angio-genic factor, vascular endothelial growth factor (VEGF), was as-sessed by RT-PCR as an index of the response to hypoxia. Fig. 1shows VEGF mRNA levels in a representative subset of samples.Mn exposed to normoxic conditions expressed basal levels ofVEGF mRNA, though showing some degree of donor-to-donorvariation. Incubation under hypoxia caused a strong and consistentVEGF up-regulation in all the samples, in agreement with previousobservations (31).
The transcriptional profile of hypoxic Mn was then investigatedby microarray analysis. Equal amounts of RNA from the differentMn preparations were randomly combined into three pools thatwere independently hybridized to human Affymetrix HG-U133AGeneChips, obtaining three biological replicates for each experi-mental condition. Statistical analysis was performed as describedin Materials and Methods. Pairwise comparison among the datasets from normoxic and hypoxic samples (average of three exper-iments) revealed the differential modulation by hypoxia of a largenumber of transcripts (data not shown). After restricting the profileto those sequences exhibiting �1.5-fold expression differences inhypoxic relative to normoxic samples, we identified 536 up-regu-lated and 677 down-regulated genes. The majority of differentiallyexpressed genes were identified as unique and named in GenBank,whereas the remaining transcripts were either identified as un-named expressed sequence tags or were hypothetical. A selec-tion of hypoxia-modulated genes (HMGs) is presented in TablesII and III.
Functional classification of HMGs
Genes displaying at least 1.5-fold differential expression levelswere classified into various categories based on the biologicalfunction(s) of the encoded protein to determine the global directionof the molecular response to hypoxia. According to GO data min-ing for biological processes (Fig. 2), the transcriptional profile in-duced by hypoxia in Mn was mainly related to cell growth and/ormaintenance, signal transduction, nucleic acid, and protein metab-olism, these functional categories being the most enriched in both
up- and down-regulated genes. Moreover, hypoxic Mn showed aprominent expression of genes involved in organogenesis, re-sponse to stress, biosynthesis, and phosphorus metabolism. Inter-estingly, a significant number of HMGs coded for proteins impli-cated in cell response to external stimuli, immune response, celladhesion and motility, and cell-cell signaling, revealing a trendtoward inflammation and immunity (Fig. 2).
Comparison of microarray data with known hypoxia-inducedchanges in gene expression
In an initial verification of microarray data, we have cross-refer-enced our results with those of other studies investigating HMGs.As summarized in Table II, a large cluster of known HMGs wasaffected by hypoxia. In addition to the reference gene VEGF, wedemonstrated up-regulation of 20 genes related to angiogenesis,cell adhesion, transcription, and inflammatory responses, whichhave been previously identified as hypoxia inducible in cells of theMn lineage (Table II). The majority of them, including ad-renomedullin (ADM), arginase-1 (ARG1), coagulation factor III(F3), fibronectin-1 (FN1), IL-1� (IL-1A), IL-6, TNF-�, macro-phage migration inhibitory factor (MIF), matrix metalloprotein-ase-1 (MMP-1), osteopontin (SPP1), and VEGF receptor-1(FLT1), were reported to be increased by hypoxia in hMDM and/ormouse Mf (20, 32), but not in primary Mn. The expression ofanother 46 known HMGs characterized in cells types other thanmononuclear phagocytes was also triggered in hypoxic Mn (TableII), including classical genes involved in glycolytic metabolismand glucose transport (e.g., glucose transporter 1 and 3 (GLUT1, 3)(9, 13, 15, 33), or associated with nonglycolytic metabolism andion transport (e.g., carbonic anhydrase XII (CA12) (13, 33). Genescoding for two novel HMGs, hypoxia-inducible protein 2 (HIG2)(34) and HIF-1-responsive RTP801 (DDIT4) (15), and other HIF-1target genes implicated in HIF-1� hydroxylation, such as EGL-nine homolog-1 (EGLN1) (35) and proline 4-hydroxylases-AI,II(P4HA1, A2) (33, 36), were increased in hypoxic Mn (Table II).Finally, several genes related to apoptosis, cell cycle, transcription,and immune responses, whose responsiveness to hypoxia was pre-viously demonstrated in normal and malignant cells, were alsoup-regulated in hypoxic Mn. BACH1 transcription repressor (37),bHLH domain-containing B2 transcription factor (BHLHB2/DEC1) (15), BCL2/adenovirus E1B-interacting protein 3 (BNIP-3)(15), hepatocyte growth factor receptor (MET) (15), IL-4 (38), IL-1receptor antagonist (IL-1RN) (39), MAX-interacting protein-1(MXI1) (33), and N-myc downstream-regulated gene-1 (NDRG1)(33) represent a few examples (Table II). Interestingly, we alsodemonstrated inhibition by hypoxia of a cluster of inflammatorygenes previously found down-regulated in mMf and hMDM, suchas CCL2 (16), CCR5 (18), cathepsin C (CTSC) (21), 2,5-oligoad-enylate synthetase (OASE) (40), and the ras family member, RAB7(21). Overall, these data demonstrate that primary Mn share withMf and cells of other lineages a cluster of hypoxia-responsivegenes.
Identification of novel hypoxia-modulated genes in primary Mn
To identify genes not previously characterized in terms of respon-siveness to hypoxia, the hypoxic transcriptome of primary Mn wasfurther investigated after excluding known HMGs listed in TableII. A list of selected novel hypoxia-modulated genes is presentedin Table III. Of interest, several were enzymes or other moleculesimplicated in lipid metabolism with a role in the regulation of fattyacid and/or cholesterol biosynthesis or transport (Table III). Themost relevant are: apolipoprotein B48 receptor (APOB48R, up-regulated), apolipoprotein E (APOE, down-regulated), fatty acid
FIGURE 1. RT-PCR analysis of VEGF mRNA expression in humanMn. Human peripheral blood Mn purified from different donors were cul-tured for 16 h under normoxic () or hypoxic conditions (�), and totalRNA was reverse transcribed and tested for VEGF mRNA expression bysemiquantitative RT-PCR, as detailed in Materials and Methods. �-actinmRNA was assayed in parallel as an internal control for input RNA. Arepresentative experiment of two performed is shown
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Table II. Relative expression of genes previously reported to be modulated by hypoxiaa
Function Gene Symbolb Gene Description Fold Changec Known in Mf/Mnd References
Angiogenesis ADM* Adrenomedullin 2.8 � (hMDM, peritoneal Mf) 9, 13, 15, 20, 33CALCRL Calcitonin receptor-like 1.5 79F3* Coagulation factor III (tissue factor) 4.5 � (hMDM) 20FGF1 Acid fibroblast growth factor 1 1.9 � (hMDM) 43FLT1 Vascular endothelial growth factor receptor 1 14.8 � (hMDM) 9, 13, 15, 20MIF* Macrophage migration inhibitory factor 10.1 � (hMDM) 13, 20SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 �/(hMDM) 13, 20VEGF Vascular endothelial growth factor 7.5 � (hMDM, hMn) 9, 13, 15, 20, 33WNT5A Wingless-type 5A 1.7 � (hMDM) 20
Apoptosis BCAT1* Branched chain aminotransferase 1 3.0 33BNIP3 BCL2/adenovirus E1B 19-kDa-interacting protein 3 16.5 15BNIP3L BCL2/adenovirus E1B 19-kDa-interacting protein 3-like 4.0 33MCL1 Myeloid cell leukemia sequence 1 (BCL2-related) 1.8 15TNF* Tumor necrosis factor 1.6 � (hMDM, peritoneal mMf) 43
Cell adhesion/matrix/ F3* Coagulation factor III (tissue factor) 4.5 � (hMDM) 20MMP1 Matrix metalloproteinase 1 (interstitial collagenase) 15.6 � (hMDM) 20FN1 Fibronectin 1 2.0 � (hMDM) 20SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 �/(hMDM) 13, 20TIMP-1 Tissue inhibitor of metalloproteinase 1 1.5 � (hMDDC) 44
Cell cycle/differentiation
ADM* Adrenomedullin 2.8 � (hMDM, peritoneal Mf) 9, 13, 15, 20, 33
BCAT1* Branched chain aminotransferase 1 3.0 33CCNG2 Cyclin G2 1.8 13, 33INHBA Inhibin, � A (activin A) 4.0 � (hMDM) 20MET met protooncogene (hepatocyte growth factor receptor) 1.8 15NDRG1 N-myc downstream regulated gene 1 4.1 33NPM1* Nucleophosmin 1.5 15
Glucose transport SLC2A1 (GLUT1) Solute carrier family 2 (glucose transporter), member 1 3.3 9, 13, 15SLC2A3 (GLUT3) Solute carrier family 2 (glucose transporter), member 3 5.8 9, 13, 33
Glycolytic metabolism ALDOA Aldolase A 1.6 9, 13, 15ALDOC Aldolase C 5.3 9ENO1* Enolase 1 3.0 9, 13, 15, 33ENO2 Enolase 2 38.9 33GAPDH Glyceraldehyde-3-phosphate dehydrogenase 1.6 9, 13, 15GPI Glucose phosphate isomerase 7.2 15HK2 Hexokinase 2 1.8 9LDHA Lactate dehydrogenase A 2.1 9, 13, 15PDK1 Pyruvate dehydrogenase kinase, isoenzyme 1 24.5 33PFKFB4 6-phosphofructo-2-kinasefructose-2,6-biphosphatase 4 2.0 15PFKP Phosphofructokinase, platelet 7.0 9, 13, 15, 33PGAM1 Phosphoglycerate mutase 1 2.4 15PGK1 Phosphoglycerate kinase 1 2.6 9, 13, 15, 33TPI1 Triose-phosphate isomerase 1 2.3 9
Immune/ responses ADORA2B Adenosine A2b receptor 2.5 80ARG1* Arginase I 2.8 � (peritoneal mMf) 32CCL2 Chemokine (CC motif) ligand 2 (MCP-1) 0.6 � (mMf) 16CCR5 Chemokine (CC motif) receptor 5 0.6 � (mMf) 18CD55 (DAF) Decay accelerating factor for complement 1.8 81CTSC Cathepsin C 0.3 � (hMDM) 21CTSD Cathepsin D 0.5 82CXCR4 Chemokine (CXC motif) receptor 4 1.9 � (hMn, hMf) 15, 17, 33IL1A Interleukin 1-� 3.2 � (peritoneal, alveolar Mf) 43IL4 Interleukin 4 1.8 38IL1RN Interleukin 1 receptor antagonist 3.1 39IL6 Interleukin 6 3.8 � (peritoneal mMf) 13, 43MIF* Macrophage migration inhibitory factor 10.1 � (hMDM) 13, 20OASE1 2,5-Oligoadenylate synthetase 1 0.5 � (mMf) 40OASE2 2,5-Oligoadenylate synthetase 2 0.5 � (mMf) 40SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 �/ (hMDM) 13, 20TNF* Tumor necrosis factor 1.6 � (hMDM, peritoneal mMf) 43
Metabolism (nonglycolytic) ADM* Adrenomedullin 2.8 � (hMDM, peritoneal Mf) 13, 15, 20, 33AK3 Adenylate kinase 3 2.5 9ARGI* Arginase I 2.8 � (peritoneal mMf) 32BCAT1* Branched chain aminotransferase 1 3.0 33CA12 Carbonic anhydrase XII 3.9 13, 33EGLN1 Egl nine homolog 1 4.6 35ERO1L ERO1-like 3.3 83GBE1 Glucan (1,4-�), branching enzyme 1 3.0 33P4HA1 Procollagen-proline, 2-oxoglutarate 4-dioxygenasa I 4.5 33, 36P4HA2 Procollagen-proline, 2-oxoglutarate 4-dioxygenasea II 2.2 33, 36PAM Peptidylglycine �-amidating monooxygenase 4.1 33PTGS2 Prostaglandin synthase and cyclooxygenase 2 3.6 � (hMDM) 20VLDLR Very low density lipoprotein receptor 5.3 � (hMn, hMDM) 41
Stress response DD1T4 HIF-1 responsive RTP801 3.1 15HIG2 Hypoxia-inducible protein 2 9.7 34WSB1 SOCS box-containing WD protein SWiP-1 2.4 33
Transcription/signaling BACH1 Bach1 transcription repressor 1.8 37BHLHB2 Basic helix-loop-helix domain containing, class B, 2 2.4 15EGR1 Early growth response 1 2.2 � (hMn) 43ENO1* Enolase 1 3.0 9, 13, 15, 33ID2 Inhibitor of DNA binding 2 1.6 � (hMDM) 15, 21, 33JMJD1A Zinc finger protein 3.2 33JUN Jun oncogene 2.2 13MXI1 MAX-interacting protein 1 4.5 33RAB7 Member RAS oncogene family 0.6 � (hMDM) 21NPM1* Nucleophosmin 1.5 15SNAPC1 Small nuclear RNA activating complex, polypeptide 1 3.6 33
a Gene expression profiling was carried out independently on three RNA pools, each composed by RNA from five different donor-derived Mn cultured under normoxic and hypoxicconditions, and comparative analysis of gene expression differences between the two experimental conditions was conducted as described in Materials and Methods. A function, a commongene symbol, a brief gene description, the fold change value, and published references are specified for each gene.
b The* indicates genes with more than one function appearing in multiple functional categories.c The indicated values represent the ratio of hypoxic/normoxic signals (mean of three experiments). A mean ratio �1.5 refers to an increase in hypoxic relative to normoxic expression
and a mean ratio �0.67 refers to a decrease in relative expression.d The � sign indicates genes previously shown to be induced by hypoxia in cells of the monocytic lineage; �/ indicates genes shown to require HIF-2� overexpression. hMn, human
monocytes; hMDM, human monocyte-derived macrophages; hMDDC, human monocyte-derived dendritic cells; hMf, human macrophages; mMf, mouse macrophages.
1945The Journal of Immunology
by guest on March 13, 2018
http://ww
w.jim
munol.org/
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Tab
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expr
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onof
sele
cted
nove
lge
nes
defin
ing
the
tran
scri
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hem
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NM
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326
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Che
mok
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like
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321
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and
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NM
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211
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1.5
NM
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or(g
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bosp
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0.45
NM
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932
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ooth
elin
6.4
NM
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428
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vav
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3A
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887
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V3
vav
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coge
ne0.
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3225
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fact
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tor
11.
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M_0
1400
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culin
0.59
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624
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PI2
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sue
fact
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thw
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hibi
tor
25.
5N
M_0
0346
1Z
YX
Zyx
in0.
55N
M_0
2264
8T
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sin
116
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able
cont
inue
s)
1946 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES
by guest on March 13, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
Tab
leII
I.C
onti
nued
Up-
regu
late
dD
own-
regu
late
d
Gen
Ban
kSy
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lG
ene
Des
crip
tion
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Gen
Ban
kSy
mbo
lG
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Des
crip
tion
Fold
Dow
nb
Lip
idm
etab
olis
mN
M_0
1836
1A
GP
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5A
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acyl
tran
sfer
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n2.
0N
M_0
0199
5A
CSL
1Fa
ttyac
idac
yl-C
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ntha
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fam
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0.56
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_018
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fam
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NM
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646
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8N
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lipop
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9B
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306
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prot
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1.7
NM
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444
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BP
5Fa
ttyac
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0.62
NM
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102.
1A
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LR
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dens
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0.65
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577
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taly
tic,
�3.
0N
M_0
0463
1L
RP
8L
owde
nsity
lipop
rote
inre
cept
or-r
elat
edpr
otei
n8
0.61
M61
906
PIK
3R1
Phos
phoi
nosi
tide-
3-ki
nase
poly
pept
ide
11.
5W
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3O
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L1A
Oxy
ster
ol-b
indi
ngpr
otei
n-lik
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0.41
AK
0235
46P
LC
L2
Phos
phol
ipas
eC
-lik
e2
2.1
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7710
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phol
ipas
eC
,�
10.
58A
F297
052
PT
GIS
Pros
tacy
clin
synt
hase
(CY
P8A
1)2.
0N
M_0
0622
6P
LC
L1
Phos
phol
ipas
eC
-lik
e1
0.48
AL
1175
15P
LC
L2
Phos
phol
ipas
eC
-lik
e2
0.54
Surf
ace
mol
ecul
es/r
ecep
tors
W72
082
C1Q
R1
Com
plem
ent
com
pone
nt1q
,re
cept
or1
4.2
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695
CD
11C
Inte
grin
,�
X,
leuk
ocyt
ead
hesi
onp1
500.
61N
M_0
0613
9C
D28
CD
28A
g(T
p44)
1.7
NM
_004
334
CD
157/
BST
1B
one
mar
row
stro
mal
cell
Ag
10.
66N
M_0
0061
1C
D59
CD
59A
gp1
8-20
(pro
tect
in)
1.6
Z22
970
CD
163
CD
163
Ag
0.16
L07
555
CD
69E
arly
activ
atio
nA
gC
D69
1.8
M98
399
CD
36C
D36
Ag
(thr
ombo
spon
din
rece
ptor
)0.
41N
M_0
0178
3C
D79
AC
D79
aA
g1.
6B
G23
0614
CD
47C
D47
Ag
(int
egri
n-as
soci
ated
prot
ein)
0.55
NM
_006
889
CD
86C
D86
Ag
(B7-
2A
g)1.
9N
M_0
0387
4C
D84
CD
84A
g(l
euko
cyte
Ag)
0.31
NM
_006
678
CM
RF
35L
euko
cyte
Ig-l
ike
rece
ptor
2.1
AF2
0073
8C
LE
CSF
6C
-typ
ele
ctin
supe
rfam
ilym
embe
r6
(DC
IR)
0.56
AJ0
1010
2C
MR
F-3
5HL
euko
cyte
mem
bran
eA
g26
.0X
1435
5F
CG
R1A
Fcfr
agm
ent
ofIg
G,
Ia,
rece
ptor
for
(CD
64)
0.33
U73
304
CN
R1
CB
1ca
nnab
inoi
dre
cept
or3.
4N
M_0
2391
4G
PR
86G
prot
ein-
coup
led
rece
ptor
860.
11N
M_0
3078
1C
OL
EC
12Sc
aven
ger
rece
ptor
with
G-t
ype
lect
in.1
1.7
M90
686
HL
A-G
HL
A-G
hist
ocom
patib
ility
Ag
clas
sIG
0.61
U56
237
FC
AR
Fcfr
agm
ent
ofIg
A,
rece
ptor
for
(CD
89)
4.2
NM
_002
162
ICA
M3
Inte
rcel
lula
rad
hesi
onm
olec
ule
30.
62N
M_0
2164
2F
CG
R2A
Fcfr
agm
ent
ofIg
G,
IIa,
rece
ptor
for
(CD
32)
1.9
AI3
3520
8IT
GB
5In
tegr
in,
�5
0.17
M31
933
FC
GR
2BFc
frag
men
tof
IgG
,II
b,re
cept
orfo
r(C
D32
)4.
3N
M_0
0088
9IT
GB
7In
tegr
in,
�7
0.61
NM
_000
570
FC
GR
3AFc
frag
men
tof
IgG
,II
Ib,
rece
ptor
for
(CD
16)
1.9
AF1
0968
3L
AIR
1L
euko
cyte
-ass
ocia
ted
Ig-l
ike
rece
ptor
10.
49N
M_0
2162
4H
RH
4H
ista
min
eH
4re
cept
or1.
7A
F009
007
LIL
RB
1L
euko
cyte
Ig-l
ike
rece
ptor
B1
(CD
85j)
0.65
NM
_003
259
ICA
M5
Inte
rcel
lula
rad
hesi
onm
olec
ule
52.
0A
F004
231
LIL
RB
2L
euko
cyte
Ig-l
ike
rece
ptor
B2
(CD
85d)
0.52
L76
666
KIR
3DL
2K
iller
cell
imm
unog
lobu
lin-l
ike
rece
ptor
1.8
AF0
0963
5L
ILR
B3
Leu
kocy
teIg
-lik
ere
cept
orB
30.
56N
M_0
1652
3K
LR
F1
Kill
erce
llle
ctin
-lik
ere
cept
orF1
1.9
U82
979
LIL
RB
4L
euko
cyte
Ig-l
ike
rece
ptor
B4
0.57
NM
_006
770
MA
RC
OM
acro
phag
ere
cept
orw
ithco
llage
nous
stru
ctur
e2.
0A
F212
842
LIR
9Ig
-lik
etr
ansc
ript
11pr
otei
n(I
LT
11)
0.62
NM
_002
445
MSR
1M
acro
phag
esc
aven
ger
rece
ptor
12.
1N
M_0
0234
6L
Y6E
Lym
phoc
yte
Ag
6co
mpl
ex,
locu
sE
0.59
NM
_003
693
SCA
RF
1A
cety
lL
DL
rece
ptor
,sc
aven
ger
rece
ptor
F11.
8N
M_0
0634
3M
ER
TK
c-m
erpr
oto-
onco
gene
tyro
sine
kina
se0.
15A
I949
392
SEM
A4C
Sem
apho
rin
4C1.
7N
M_0
2306
8SN
Sial
oadh
esin
(CD
169)
0.09
NM
_006
378
SEM
A4D
Sem
apho
rin
4D(C
D10
0)1.
8N
M_0
1513
STA
B1
Stab
ilin
10.
25N
M_0
0426
3SE
MA
4FSe
map
hori
n4F
1.6
AF0
5115
1T
LR
5T
oll-
like
rece
ptor
50.
56N
M_0
1864
3T
RE
M1
Tri
gger
ing
rece
ptor
expr
esse
don
mye
loid
cells
12.
4N
M_0
1656
2T
LR
7T
oll-
like
rece
ptor
70.
24(T
able
cont
inue
s)
1947The Journal of Immunology
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Tab
leII
I.C
onti
nued
Up-
regu
late
dD
own-
regu
late
d
Gen
Ban
kSy
mbo
lG
ene
Des
crip
tion
Fold
Upb
Gen
Ban
kSy
mbo
lG
ene
Des
crip
tion
Fold
Dow
nb
Tra
nscr
iptio
nfa
ctor
sN
M_0
0188
0A
TF
2A
ctiv
atin
gtr
ansc
ript
ion
fact
or2
1.5
NM
_001
621
AH
RA
ryl
hydr
ocar
bon
rece
ptor
0.57
NM
_012
068
AT
F5
Act
ivat
ing
tran
scri
ptio
nfa
ctor
51.
8N
M_0
1486
2A
RN
T2
Ary
lhy
droc
arbo
nre
cept
ornu
clea
rtr
ansl
ocat
or2
0.37
U79
751
BL
ZF
1B
asic
leuc
ine
zipp
ernu
clea
rfa
ctor
1(J
EM
-1)
1.6
U85
962
CB
PC
RE
B-b
indi
ngpr
otei
n0.
49N
M_0
0443
0E
GR
3E
arly
grow
thre
spon
se3
2.4
NM
_004
364
CE
BP
AC
CA
AT
enha
ncer
bind
ing
prot
ein
(CE
BP)
,�
0.51
NM
_006
732
FO
SBFo
sho
mol
ogB
2.0
AV
6556
40C
EB
PD
CC
AA
T/e
nhan
cer
bind
ing
prot
ein
(C/E
BP)
,�
0.55
NM
_024
530
FO
SL2
Fos-
like
Ag
21.
7N
M_0
0490
4C
RE
B5
cAM
Pre
spon
seel
emen
t-bi
ndin
gpr
otei
n5
0.46
BC
0053
29H
SF2
Hea
tsh
ock
tran
scri
ptio
nfa
ctor
22.
7N
M_0
0039
9E
GR
2E
arly
grow
thre
spon
se2
0.55
D13
889
ID1
Inhi
bito
rof
DN
Abi
ndin
g1
2.2
NM
_005
890
GA
S7G
row
thar
rest
-spe
cific
70.
20N
M_0
0246
0IR
F4
IFN
regu
lato
ryfa
ctor
41.
5N
M_0
0153
0H
IF1A
Hyp
oxia
-ind
ucib
lefa
ctor
1,�
subu
nit
0.50
NM
_005
655
KL
F10
TG
FBin
duci
ble
earl
ygr
owth
resp
onse
4.5
NM
_002
383
MA
ZM
YC
-ass
ocia
ted
zinc
finge
rpr
otei
n0.
41N
M_0
0250
5N
FY
AN
ucle
artr
ansc
ript
ion
fact
orY
,�
1.8
NM
_000
248
MIT
FM
icro
phth
alm
ia-a
ssoc
iate
dtr
ansc
ript
ion
fact
or0.
55M
2198
5N
R2C
1T
R2
nucl
ear
rece
ptor
2C1
3.3
NM
_005
933
ML
LM
yelo
id/ly
mph
oid
orm
ixed
-lin
eage
leuk
emia
0.28
NM
_006
186
NR
4A2
Nuc
lear
rece
ptor
subf
amily
4A2
2.2
NM
_002
432
MN
DA
Mye
loid
cell
nucl
ear
diff
eren
tiatio
nA
g0.
20L
3459
8R
UN
X1
Run
t-re
late
dtr
ansc
ript
ion
fact
or1
1.9
NM
_002
467
MY
Cv-
myc
avia
nm
yelo
cyto
mat
osis
vira
lon
coge
ne0.
47L
4099
2R
UN
X2
Run
t-re
late
dtr
ansc
ript
ion
fact
or2
1.7
U19
179
NC
OA
1N
ucle
arre
cept
orco
activ
ator
10.
49N
M_0
0311
2SP
4Sp
4tr
ansc
ript
ion
fact
or1.
7N
M_0
1625
0N
DR
G2
N-m
ycdo
wns
trea
m-r
egul
ated
gene
20.
53A
F077
053
TA
F9L
Neu
rona
lce
llde
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rela
ted
prot
ein
2.9
BF2
0950
7P
C4
Act
ivat
edR
NA
poly
mer
ase
IItr
ansc
ript
ion
cofa
ctor
40.
56A
I703
074
TC
F7L
2T
rans
crip
tion
fact
or7-
like
22.
8A
V72
7449
PC
AF
p300
/CB
P-as
soci
ated
fact
or0.
65B
F056
105
TC
FL
4T
rans
crip
tion
fact
or-l
ike
41.
6B
C00
0080
PM
LPr
omye
locy
ticle
ukem
ia0.
34N
M_0
1590
5T
IF1
Tra
nscr
iptio
nal
inte
rmed
iary
fact
or1
1.7
NM
_003
120
SPI1
Vir
uspr
ovir
alin
tegr
atio
non
coge
nesp
il0.
37K
0319
9T
P53
p53
cellu
lar
tum
orA
g0.
40N
M_0
0699
1Z
NF
197
Zin
cfin
ger
prot
ein
197
0.62
aG
ene
expr
essi
onpr
ofilin
gw
asca
rrie
dou
tin
depe
nden
tlyon
thre
eR
NA
pool
s,ea
chco
mpo
sed
byR
NA
from
five
diff
eren
tdo
nor-
deri
ved
Mn
cultu
red
unde
rno
rmox
ican
dhy
poxi
cco
nditi
ons,
and
com
para
tive
anal
ysis
ofge
neex
pres
sion
diff
eren
ces
betw
een
the
two
expe
rim
enta
lco
nditi
ons
was
cond
ucte
das
desc
ribe
din
Mat
eria
lsan
dM
etho
ds.
Eac
hge
neis
give
na
repr
esen
tativ
eG
enB
ank
acce
ssio
nnu
mbe
r,a
com
mon
gene
sym
bol,
abr
ief
gene
desc
ript
ion,
and
the
fold
chan
geva
lue.
Und
erlin
edge
nes
wer
eva
lidat
edby
qRT
-PC
R.
bT
hein
dica
ted
valu
esre
pres
ent
the
ratio
ofhy
poxi
c/no
rmox
icsi
gnal
s(m
eans
ofth
ree
expe
rim
ents
).A
mea
nra
tio�
1.5
refe
rsto
anin
crea
sein
hypo
xic
rela
tive
tono
rmox
icex
pres
sion
(fol
dup
)an
da
mea
nra
tio�
0.67
refe
rsto
ade
crea
sein
rela
tive
expr
essi
on(f
old
dow
n).
1948 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES
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acyl-CoA synthase long-chain family 1 and 3 (ACSL1,3, down-regulated), fatty acid binding protein 4 (FABP4, up-regulated), lowdensity lipoprotein receptor (LDLR, down-regulated), oxysterolbinding protein-like 10 (OSBPL10, up-regulated), and steroid 27-hydroxylase (CYP27A1, down-regulated). Modulation of thesegenes under low pO2 may have implications for the pathogenesisof atherosclerosis and Alzheimer’s disease, where a role for hyp-oxia has been suggested (9, 10, 41).
Other genes differentially expressed in hypoxic Mn coded forcytoskeleton and ECM components/regulators (Table III). Induc-ible genes include adhesive proteins of the desmosome type ofcell-cell junction, actin-interacting/regulatory transmembrane mol-ecules, and secreted proteins. Of note are galectin 8 (LGALS8),MMP-16 (MT3-MMP), MMP-19, regulator of G-protein signaling1 (RGS1), tensin 1 (TNS1), and tissue factor pathway inhibitors 1and 2 (TFPI1,2). Among the most significantly down-regulatedgenes, we identified advillin (AVIL), autotaxin (ENPP2), galectin2 (LGALS2), myosin heavy polypeptide 11 (MYH11), and VAV3.Modulation of these genes is likely to regulate Mn adhesion, mo-tility, and tissue remodeling.
The hypoxic profile also revealed hypoxia inducibility of a set ofgenes with transcription regulatory activity, including activatingtranscription factor (ATF)-2 and ATF-5, Fos homolog B (FOSB),Fos-like Ag 2 (FRA2), and runt-related transcription factors 1 and2 (RUNX1,2) (Table III). Moreover, several transcription factor-and cofactor-encoding genes were inhibited by hypoxia. Of rele-vance are the aryl-hydrocarbon receptor (AHR), ARNT2, CCAAT
enhancer-binding protein � (C/EBP�), CREB-binding protein(CBP), HIF-1�, microphthalmia-associated transcription factor(MITF), MYC oncogene, nuclear receptor coactivator 1 (NCOA1/SRC-1), p53 tumor Ag (TP53), and zinc finger protein 197(ZNF197/VHLaK) (Table III). These data are indicative of major,coordinated changes in transcription and suggest the existence ofboth positive and negative O2-driven feedback regulatory mecha-nisms of hypoxia transcriptional response
Differential modulation by hypoxia of immune-related genesin Mn
As summarized in Table III, a prominent set of novel HMGs haveimmunological relevance. These include genes encoding surfaceimmunoregulatory signaling (IRS) receptors, such as early activa-tion Ag (CD69), leukocyte membrane Ag CMRF-35H, low-affinityIgG receptors Fc�RIIA,B (CD32), IgA receptor Fc�R (CD89), andtriggering receptor expressed on myeloid cells 1 (TREM1), thatwere up-regulated, and high-affinity IgG receptor Fc�RIA (CD64),histocompatibility Ag class IG (HLA-G), leukocyte-associated Ig-like receptor 1 (LAIR1), leukocyte Ig-like receptor 9 (LIR9), andB1,B2,B3,B4 (LILRB1–4, CD85), that were down-regulated. Sev-eral scavenging and pattern recognition receptors were also selec-tively induced (complement component 1q receptor 1 (C1qR1);macrophage receptor with collagenous structure (MARCO); mac-rophage scavenger receptor 1 (MSR1); scavenger receptor-FI(SCARF1); scavenger receptor with C-type lectin 1 (COLEC12/SRCL)) or repressed (CD163 Ag; stabilin, STAB-1; TLR-5 and -7)
FIGURE 2. GO data mining. The gene expression profile of hypoxic vs normoxic primary human Mn was analyzed using high-density oligonucleotidearrays, as described in Materials and Methods. Genes showing at least a 1.5-fold change in expression levels between hypoxic and normoxic cells (meanof three experiments) were selected and characterized according to their biological process classification (at level 3) in the GO database. Based on thisclassification scheme, genes were placed in more than one category if more than one function of the encoded protein had been established. Transcriptswithout a GO classification were categorized as unclassified. Bars on the right of the y-axis represent up-regulated genes; bars on the left of the y-axisrepresent down-regulated genes.
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under hypoxia (Table III). Other differentially expressed genescoded for costimulatory and adhesion molecules involved in cell-cell and cell-matrix interaction, such as bone marrow stromal cellAg (CD157), CD36, CD84, and CD86 Ags, integrin � X (CD11C),integrin �5 and �7 (ITGB5,7), ICAM3,5, semaphorin 4D (CD100),and sialoadhesin (CD169) (Table III).
The hypoxic transcriptome was also characterized by the mod-ulation of a cluster of genes coding for cytokines/chemokinesand/or their receptors. Within the chemokine system, we identifiedfor the first time CCL20, CXCL2, CXCL3, CXCL5, the fractalkinereceptor CX3CR1, and the G protein-coupled chemokine orphanreceptor (RDC1) as hypoxia-inducible genes, whereas CCL15,CCL18, CCL23, CCL8, CXCL6, CCR1, and CCR2 were the mosthighly hypoxia-repressed genes (Table III). Finally, Mn hypoxicprofile included various components of the IL-1 system (IL-1 fam-ily member 9 (IL1F9); IL-1 receptor accessory protein (IL1RAP);IL-1R associated kinase 3 (IRAK-3); IL-18R1) and members of theTNF receptor and ligand superfamilies, as well as IL-23A, IL-6signal transducer (IL-6ST), IL-13RA1, IL-21R, CSF1,3, andCSF1,3Rs (Table III).
Confirmation of microarray data by qRT-PCR analysis ofselected hypoxia-modulated genes
To validate the microarray results using a different technique,mRNA levels for a subset of known and novel hypoxia-modulatedgenes were quantified by qRT-PCR on a new RNA pool (Fig. 3),using the primer pairs listed in Table I. For this analysis, we ran-domly selected 27 genes involved in immune regulation, inflam-matory responses, and transcription. Three reference genes (TableI) were used for data normalization. We found a 100% concor-dance between qRT-PCR and Affymetrix data with respect to thedirection of the expression changes. For the majority of the genes,fold-differences were also of comparable magnitude (Fig. 3), al-though they were higher according to qRT-PCR for six genes
(CCL23, FCGR1A, LIR9, BNIP-3, Mxl1, and VEGF), in agreementwith previous findings showing that microarray can often under-estimate the extent of gene regulation compared with qRT-PCR(Varesio et al., unpublished observations). For other genes, such asCMRF-35H, FCGR2B, STAB-1, and MIF, however, higher expres-sion differences were detected by microarray. These results con-firm hypoxia responsiveness of novel genes identified bymicroarray.
Hypoxia induces CCL20 expression and secretion by human Mn
We then selected CCL20, one of the novel immune-related genesmost strongly up-regulated in hypoxic Mn, for further analysis.According to microarray and qRT-PCR data, CCL20 mRNA levelswere increased by an average of 7.4- and 5.7-fold, respectively(Fig. 3). To address the issue of donor-to-donor variability, weanalyzed CCL20 mRNA expression by RT-PCR in a subset ofsamples comprising the pools used for microarrays (Fig. 4A).CCL20 basal expression was detected in all Mn preparations cul-tured under normal pO2, although with some variations amongindividual donors. Consistent CCL20 transcript up-regulation wastriggered by hypoxia in every donor, independently of the baselinelevels (Fig. 4A), indicating the general inducibility of the gene inprimary Mn. qRT-PCR was also performed to quantify the mag-nitude of hypoxia-induced changes. As shown in Fig. 4B, the ex-tent of CCL20 mRNA increase ranged from 3.1- to 11.4-foldamong the samples examined. mRNA up-regulation was paralleledby increased protein expression, as determined by immunocyto-chemistry (Fig. 5A). Mn isolated from the three donors analyzedby qRT-PCR were cytocentrifuged and immunostained with amAb directed to CCL20. Low levels of CCL20 immunoreactivitywere detectable in the cytoplasm of normoxic Mn. Hypoxia expo-sure for 16 h resulted in a marked increase in intracellular proteincontent in all the samples. No staining was detected when an iso-type-matched control Ab was used (Fig. 5A).
FIGURE 3. qRT-PCR analysis of genes selected from the microarray profile. Equal amounts of total RNA from four donor-derived normoxic or hypoxicMn were pooled, and RNA pools were subjected to qRT-PCR analysis, as detailed in Materials and Methods. Expression changes of 27 selected genes wereevaluated in relation to the values obtained in parallel for three reference genes. Results from one representative experiment of three performed areexpressed as log2 ratio (1% O2 relative to 21% O2) and are the mean of triplicate determinations for each target transcript (f). Microarray data are shownfor comparison (u). The number associated with each bar indicates the linear fold-change of mRNA expression in hypoxic relative to normoxic cells(arbitrarily defined as equal to 1). Positive values indicate that the mRNA level of a particular gene was up-regulated, whereas negative values indicate thatthe transcript was down-regulated. Known hypoxia-inducible genes identified for the first time in cells of the Mn lineage are marked with an asterisk (�).
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Conditioned medium from normoxic and hypoxic Mn cultureswas then analyzed for CCL20 content by ELISA to determinewhether a parallel rise in chemokine secretion was triggered byhypoxia (Fig. 5B). Normoxic Mn constitutively secreted variableamounts of CCL20, which accumulated in the culture mediumranging from 10 to 410 pg/106 cells/ml in six different donors.Consistent with the mRNA data, chemokine secretion increased by2.4- to 3.5-fold upon incubation to 1% O2 for 16 h (Fig. 5B) andwas further augmented at longer time points, with almost 7-foldhigher amounts detectable in the conditioned medium at 72 h ofculture (Fig. 5C, donor 12). CCL20 is a LPS-inducible chemokine(3) and the response of Mn to LPS was evaluated in parallel ex-periments as a positive control. Interestingly, the extent of CCL20secretion elicited by hypoxia exceeded that triggered by stimula-tion with LPS at all the time points analyzed (Fig. 5C), suggestingthat hypoxia is a more potent stimulus than LPS in inducingCCL20 production by Mn.
Taken together, these results indicate that primary human Mnisolated from different donors produce variable baseline levels ofCCL20 but respond to low pO2 with consistent CCL20 up-regu-lation, confirming CCL20 as a hypoxia-inducible gene.
DiscussionMn responses that ensue following recruitment at pathologicalsites begin in the setting of reduced pO2 (1). Hypoxia regulatoryeffects on Mn gene expression have not been characterized in de-tail, and only a limited number of hypoxia-inducible genes havebeen identified in these cells (17, 31, 41). In this study, we providethe first transcriptome analysis of hypoxic primary peripheralblood human Mn.
Microarray analysis was conducted on RNA pooled from dif-ferent donor-derived Mn, whose response to hypoxia was demon-strated by VEGF up-regulation. Three pools, each composed byRNA from five different donors, were investigated per each ex-perimental condition. RNA pooling is a powerful, cost-effective,
and statistically valid mean of identifying common changes in agene expression profile. Previous studies have established the fea-sibility of using this type of approach to overcome interindividualvariability and provide reliable results that reflect gene expressionin individual donors (42). The suitability of our experimental pro-tocol for identifying bona fide hypoxia-responsive genes in pri-mary cells was inferred by the demonstration that hypoxia modu-lated 74 genes known from the literature to be responsive tohypoxia. The majority of these genes, involved in glycolysis, ap-optosis, cell cycle, and transcription, may constitute a gene clustercommonly induced by hypoxia in cells of different lineages. Thegeneral representativeness of the gene expression changes detectedby microarray was further established by qRT-PCR, which con-firmed the differential expression of 27 genes randomly selectedfrom the microarray profile in a fourth set of pooled RNAs.
Two recent reports investigated the hypoxia transcriptome ofhMDM and demonstrated induction of genes encoding angiogen-esis and ECM regulators/components (20, 21), some of whichwere also present in our profile. Expression of ADM, F3, FLT1,FN1, MIF, INHBA, MMP-1, PTGS2, SPP1, and VEGF, amongothers, was detectable at significant levels in both primary humanMn (this report) and MDM (20), and exposure to hypoxia resultedin their strong up-regulation in both cell populations. Moreover,we demonstrated up-regulation of other genes previously shown tobe inducible by hypoxia in mononuclear phagocytes and codingfor immunoregulatory/proinflammatory proteins and transcriptionfactors, such as ARG1, CXCR4, EGR1, FGF1, ID2, TIMP-1, andVLDR (17, 21, 32, 41, 43, 44). Interestingly, IL-1�, IL-6, andTNF-�, induction in Mn was observed in response to hypoxiaalone, whereas up-regulation in Mf and MDM required LPS co-stimulation or a reoxygenation period (43). Furthermore, we con-firmed in primary Mn hypoxic inhibition of a cluster of inflam-matory genes found down-regulated in mMf and hMDM, such asCCL2 (16), CCR5 (18), CTSC (21), OASE (40), and RAB7 (21).These findings indicate that hypoxia is active on different types ofmononuclear phagocytes and across species in regulating the ex-pression of selected target genes, which may be critical for theiradaptation to the hypoxic environment and ability to function in itand could be representative of the hypoxic transcriptome of cellsbelonging to the monocytic lineage. In contrast, the observationthat other genes up-regulated in hMDM (20, 21) were not inducedor were even down-regulated in primary Mn, e.g., the angiogenesisinducers endothelial cell growth factor-1 (ECGF1) and IL-18 bind-ing protein (IL-18BP), suggests that hypoxia may also activate inmononuclear phagocytes a specific transcriptional response de-pending on their differentiation stage.
Several HMGs identified in our analysis were endowed withtranscription regulatory activity and/or encoded important compo-nents of the HIF-1 transcription pathway. Hypoxia increased theexpression of P4HA1, P4HA2, and EGLN1, three members of theprolyl-hydroxylase family which mediate HIF-� hydroxylation inwell-oxygenated cells targeting the protein for proteosomal deg-radation by the von Hippel Lindau tumor suppressor protein(pVHL) (15), and of BHLHB2, a bHLH family member implicatedin the regulation of pVHL/HIF pathways (15). These data are inagreement with previous findings in other cell types (15, 35, 36)and suggest an O2-driven HIF-1-dependent autoregulatory mech-anism, required to ensure fast HIF-1� degradation upon reoxygen-ation, also in Mn. Of note is the novel evidence that hypoxia pro-motes the expression of ATF2 and ATF5, two members of the ATFfamily of transcription factors (45) required for efficient activationof gene transcription by hypoxia (15). Moreover, we demonstratedup-regulation of FosB and FRA-2, two components of the AP-1complex (45), which is activated by hypoxia and was recently
FIGURE 4. Up-regulation of CCL20 mRNA expression by hypoxia inhuman Mn. Total RNA was isolated from human peripheral blood Mncultured under normoxic () or hypoxic (�) conditions for 16 h. A, RNAfrom six different donor-derived Mn was reverse transcribed and tested forCCL20 and �-actin mRNA expression by semiquantitative RT-PCR. B,The extent of CCL20 mRNA up-regulation by hypoxia was determined byqRT-PCR analysis on the RNA isolated from the indicated donors, andrelative transcript expression was calculated in relation to the values ob-tained in parallel for three reference genes, as detailed in Materials andMethods. Results from one representative experiment are expressed as foldincrease of mRNA levels in hypoxic relative to normoxic cells (arbitrarilydefined as 1) and are the mean of triplicate determinations. Fold-changevalues are indicated by a number associated with each bar.
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shown to cooperate with HIF-1� in the transactivation of hypoxiainducible genes (46), and of JEM-1, a novel transcriptional cofac-tor which enhances AP-1 activity (47). Up-regulation of thesegenes together with down-regulation of ZnF197, a novel VHL-interacting protein that functions as a repressor of HIF-1� trans-activation (48), may represent a positive regulatory mechanism ofhypoxia transcriptional response in Mn. Down-regulation of sev-eral HIF-1 transcriptional cofactors, including CBP, C/EBP�,MITF, and NCOA1/SRC-1, was also observed in hypoxic Mn. Allthese proteins physically interact with HIF-1� enhancing its trans-activation function under hypoxia (9, 15, 49), and their down-regulation suggests the existence of a negative feedback mecha-nism to control Mn hypoxic response. Consistent with thishypothesis, we observed a parallel decrease of the mRNAs encod-ing HIF-1� and one of its dimerization partners, ARNT-2 (9)
GO data mining characterized a significant cluster of genes asbeing associated with immune regulation and inflammatory re-sponses, chemotaxis, cell adhesion, and ECM remodeling, the ma-jority of which has not been previously identified as responsive tohypoxia. Profound changes were observed in the expression ofscavenger receptors. Of relevance is the up-regulation of MSR1and SCARF1, because these molecules functions as endocytic re-
ceptors for acetylated LDL and may thus be implicated in lipid-loaded foam cell formation contributing to atherosclerotic plaquesdevelopment (50). Consistent with the view that hypoxia may exerta pathogenetic role in atherosclerosis and Alzheimer’s disease (9,10, 41) is also the down-regulation of CD163 and STAB1 scaven-ger receptors, which are endowed with atheroprotective activity(51, 52), and of various genes involved in the regulation of fattyacid and/or cholesterol biosynthesis/transport and acting as anti-atherogenic factors, e.g., LDLR, ApoE, and CYP27A1 (53, 54). Mnhypoxic profile also showed up-regulation of a number of otherpattern recognition receptors critical to host defense, the most rel-evant of which are the C1qR1, COLEC12, and MARCO. Thesemolecules bind specific Ags on bacteria facilitating their recogni-tion and phagocytosis (55–57). Interestingly, the TLR familymembers TLR5 and TLR7, whose function is also to recognizepathogen or their products and hence initiate innate immune re-sponses (58), were down-regulated by hypoxia. Differential mod-ulation of receptors with similar functions is intriguing and islikely to contribute to the fine tuning of Mn antimicrobial activitiesat sites of infection.
Myeloid cell immune functions are regulated by a balance ofinhibitory and activating signals transduced by multiple families of
FIGURE 5. Hypoxia induces CCL20 production by primary Mn. Human peripheral blood Mn purified from the indicated donors were cultured undernormoxic () or hypoxic conditions (�) for 16 h (A and B) or for different time points (C), in the presence or absence of 100 ng/ml LPS. A, CCL20 proteinexpression was evaluated on cytospin preparations by immunocytochemical analysis using a specific mAb. Staining was conducted with the Envision�
System as described in the Materials and Methods. Hematoxylin-counterstained slides were examined under a phase-contrast microscope and photomi-crographed (magnification, �40). CCL20 immunoreactivity is detectable in the cell cytoplasm (brown staining). Staining with an isotype-matched controlmAb (IgG1) is shown for donor 7. B and C, Supernatants were harvested and assayed for secreted CCL20 by ELISA. Results from one experiment,representative of three performed, in which each sample was tested in triplicate, are expressed as picogram per 1 � 106 cells/ml. The number associatedwith each bar indicates the fold induction of CCL20 secretion in hypoxic relative to normoxic cells (arbitrarily defined as equal to 1).
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cell surface IRS receptors belonging to the Ig superfamily of in-hibitory/stimulatory pairs of molecules (59). These receptors areeither characterized by ITIM/ITAM regulatory components intheir cytoplasmic domain or pair with ITAM-containing trans-membrane adapter proteins (59). Mn exposure to hypoxia resultedin the selective modulation of various members of these receptorfamilies. Of particular interest is the up-regulation of Fc�RIIA,Band down-regulation of Fc�RIA. Fc�RIIA contains a functionalITAM motif, thus triggering cell activation, whereas Fc�RIIB en-codes an ITIM component, leading to repression of cellular re-sponses (60). Fc�RIA, which associates for signaling with anITAM-containing �-chain, also belongs to the activatory Fc�Rsubclass (60). Fc�Rs involvement in various autoimmune inflam-matory states was reported (60), and our findings suggest the po-tential role of hypoxia in influencing the pathogenesis of thesediseases through the modulation of distinct Fc�R genes.
Hypoxic modulation of other ITIM/ITAM Ig family members,specifically up-regulation of FCAR, CMRF-35H, and TREM-1 anddown-regulation of LIR9 and LAIR1, was also observed. Thesemolecules mediate a variety of effector functions vital to the ad-aptative immune response, and their ligation can have both proin-flammatory and immunosuppressive consequences by differen-tially modulating the secretion of pro- or anti-inflammatorymediators (59–63). The differential expression of inhibitory andactivating isoforms of a given receptor family with similar speci-ficities on the same cell is another example of the tight regulatoryrole of hypoxia on Mn inflammatory responses. In particular, theconcomitant down-regulation of the Ig-like inhibitory receptorsLILRB1–4 and of their ligand HLA-G, a nonclassical inhibitoryHLA class I Ag, is noteworthy given the role of these molecules inimmune tolerance and immune escape (64). It is conceivable thatinhibition of these molecules may decrease the activation thresholdof Mn retained at hypoxic sites. Mn activation under conditions ofreduced O2 availability may also be controlled by the selectivedown- and up-regulation of two C-type lectin receptors, the ITIM-containing inhibitory molecule CLECSF6 (65) and CD69, a mem-ber of the NK receptor family and a potent inducer of Mn inflam-matory mediator production and cytotoxic activity (66). Althougha ligand for CD69 has not been identified, previous studies sug-gested a pathogenetic role for CD69 in certain inflammatory statescharacterized by hypoxia, such as rheumatoid arthritis, chronic in-flammatory liver diseases, and asthma (66).
Mononuclear phagocyte migratory activity is a highly regulatedprocess which depends on a defined repertoire of chemokines/re-ceptors and adhesion molecules, and dysregulated expression ofthese proteins may alter their recruitment and activation (67). Var-ious studies have investigated the mechanisms whereby mononu-clear phagocytes are retained/concentrated at hypoxic pathologicalsites (1). One possibility is that hypoxia inhibits their migration inresponse to chemokines by decreasing the expression of specificchemokine receptors, as demonstrated for CCR5 in Mf (18). Inagreement with this hypothesis and with previous findings show-ing impaired Mn migratory ability to CCL2 under conditions oflow pO2 (1), we observed CCR5, CCR1, and CCR2 down-regula-tion. This study also suggests other potential mechanisms for Mnentrapment in hypoxic areas, such as the up-regulation of RGS1, amember of a new class of G protein-signaling deactivators whichinactivates several chemotactic receptors inhibiting chemoattrac-tant-induced Mn migration (68). Furthermore, repression of thesecreted cell motility-promoting factor, ENPP2 (69), together withinduction of the antichemotactic cytokine, MIF (13), and the GROfamily chemokines, CXCL2 and CXCL3, which are specializedMn-arrest chemokines (70), may also provide a “stop” signal toMn within hypoxic tissues.
The complexity of the regulation of Mn migratory behavior byhypoxia is further emphasized by the demonstration of dysregu-lated expression of several other migration-related genes. Mn hy-poxic profile was associated with down-regulation of the adhesionmolecules CD11C, CD57, and ITGB5,7, which mediate Mn adhe-sion to the endothelium and/or to the ECM (67), and with up-regulation of both the fractalkine receptor, which binds to theCX3C chemokine fractalkine expressed on endothelial cells func-tioning as a potent adhesion molecule (71), and the RDC1 receptor,which share with CXCR4 the chemokine CXCL12/SDF-1 as anatural ligand (72). A critical role in the control of mononuclearphagocyte motility is also exerted by MMPs, a group of secretedenzymes that trigger ECM degradation facilitating leukocytemovement in tissues (73). Recent reports have shown up-regula-tion by hypoxia of MMP-1, MMP-7, and MMP12 in hMDM (20,21). Interestingly, only MMP1 was induced also in primary Mnthat showed specific up-regulation of MMP16 and MMP19 anddown-regulation of MMP25. Moreover, the MMP inhibitors,TIMP1 and TFPI2, which reduce ECM degradation inhibiting cellmigratory activity (73, 74), were also modulated in hypoxic Mn.Collectively, these studies indicate that regulation of MMPs andtheir inhibitors is a common denominator of mononuclear phago-cyte response to hypoxia, but that different components of thesefamilies are controlled depending on the cell differentiation stage.Altered expression of MMPs has been associated with a variety ofacute and chronic inflammatory states (73), and hypoxia can prob-ably play a role in the pathogenesis of these diseases by modulat-ing MMP production by infiltrating Mn
Various cytokines/chemokine and/or receptors are modulated byhypoxia in mononuclear phagocytes (1, 43). The Mn hypoxic tran-scriptome confirmed and extended those findings showing differ-ential expression of other components of the cytokine/chemokinesystem. Various members of the IL-1 and the TNFR/ligand super-families were selectively up- or down-regulated in hypoxic Mn,including molecules associated with and mediating signal trans-duction from their receptors (e.g., IL1RAP, IRAK3, and the TNFR-associated factor, TRAF). Because of their pleiotropic effects onalmost every types of cells, coordinated regulation of the TNF andIL-1 systems is likely to represent an important mechanism tocontrol the amplitude and the duration of inflammatory responses.The demonstration that hypoxia induces CSF1 and CSF3, whileinhibiting their receptors, is noteworthy given the role of thesefactors in the regulation of myeloid cell production, differentiation,and function (75) and is consistent with previous findings suggest-ing a reciprocal and divergent action of hypoxia on receptor vsligand expression (16, 18). Accordingly, IL-4 up-regulation andconcomitant inhibition of its receptor IL-13RA1 was observed.This interplay is likely to serve as a negative feedback mechanismto control the autocrine activation of producing Mn. However, notall the data presented in this study are consistent with this scenario.Concomitant down-regulation of the Mn chemoattractants CCL2and CCL8 and of their receptors CCR2 and CCR5 (2) was in factobserved in response to hypoxia. Similarly, we found down-reg-ulation of CCL15, a chemoattractant for neutrophils, monocytes,and lymphocytes (76), and CCL23, a chemokine mediating restingT cell and Mn chemotaxis (77), and of their common receptorCCR1. Collectively, these data indicate that a dynamic change inchemokine/receptor expression profile occurs in Mn within hy-poxic tissues. This tight and complex level of control exerted bylow O2 tension is clearly of pathophysiologic relevance, represent-ing an important mechanism of regulation of leukocyte traffickingand function at sites of inflammation. Because some of the mod-ulated chemokines have angiogenic activity (1, 2, 76), their altered
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expression under low pO2 may also influence neoangiogenesis inpathological tissues.
One of the most important findings of this study was the dem-onstration that hypoxia strongly induced the expression of theCCL20-encoding gene in primary Mn. Marked differences in thebasal mRNA expression were detected among individual donors.However, mRNA up-regulation under hypoxia was consistentlydemonstrated in all Mn preparation analyzed, and gene expressionresults were associated to a parallel augmentation of protein ex-pression and secretion. Previous reports have shown that theCCL20 gene is markedly up-regulated in PBMC by LPS and in-ducible in other cell types in response to various mediators ofinflammation, including cytokines, growth factors, bacterial, viraland plant products, whereas it is poorly expressed in the absenceof inflammatory stimuli (3). This study is the first to identify hyp-oxia as a new CCL20 inducer. Interestingly, the extent of CCL20up-regulation by hypoxia exceeded that triggered by LPS, suggest-ing that hypoxia is a more potent stimulus than LPS for CCL20production by Mn. Given its role in the recruitment of iDC, effec-tor/memory T lymphocytes, and naive B cells, CCL20 has beenproposed as an important mediator for both the initiation and ef-fector phases of the inflammatory reactions, linking innate andacquired immunity (3). Hence, by producing CCL20, Mn may con-trol the kinetics and composition of the cellular infiltrate undervarious inflammatory conditions and at tumor sites. The identifi-cation of CCL20 as a hypoxia-inducible gene may explain, in part,the high levels of this chemokine present in areas of inflammationand in various chronic inflammatory conditions, such as rheuma-toid arthritis, inflammatory skin disorders, and tumors (for a re-view, see Refs. 3 and 78), as these sites are known to be hypoxicand infiltrated by Mn, and is indicative of a pathogenetic role forthis molecule in these diseases. Further studies are ongoing in thelaboratory to elucidate the molecular mechanisms underlyingCCL20 induction by hypoxia.
In summary, we have described the hypoxia transcriptome ofprimary human Mn and identified a large number of genes notpreviously known to change as a result of reduced O2 concentra-tions. Our findings contribute to the definition of the gene clustercommonly induced by hypoxia in cells of different lineage. Thisstudy provides novel insights into the molecular responses to thehypoxic stress and the mechanisms linking low pO2 to the regu-lation of immune and inflammatory responses, leading to new per-spectives of the role of hypoxia in programming Mn functionswithin pathological conditions and identifying potential moleculartargets for the development of rational therapeutic approaches.
AcknowledgmentsWe thank S. Delfino for technical assistance and C. Dabizzi for secretarialwork.
DisclosuresThe authors have no financial conflict of interest.
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