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The Journal of Immunology, 2006, 177: 1941-1955.
Copyright © 2006 by The American Association of Immunologists

Hypoxia Modifies the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identification Of CC-Chemokine Ligand 20 as a New Hypoxia-Inducible Gene1

Maria Carla Bosco2,*, Maura Puppo*, Clara Santangelo*, Luca Anfosso{ddagger}, Ulrich Pfeffer{dagger}, Paolo Fardin*, Florinda Battaglia* and Luigi Varesio*

* Laboratory of Molecular Biology, G. Gaslini Institute, and {dagger} Functional Genomics, National Cancer Research Institute, Genova, Italy; and {ddagger} University of Insubria, Varese, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O2, with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a 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/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral blood monocytes (Mn)3 represent the early mononuclear phagocyte component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth, where they differentiate into inflammatory and tumor-associated macrophages (Mf) (1). Mn/Mf are potent regulators of immune and inflammatory reactions. They orchestrate the coordinated recruitment and activation of specific leukocyte subsets at pathological sites through the local secretion of low m.w. structurally related proteins, termed chemokines (1). Chemokines are classified into CXC, CC, C, and CX3C families, which bind to and activate members of a superfamily of 7-transmembrane domain, G protein-coupled receptors differentially expressed and regulated in leukocytes (2). CCL20 (also known as MIP-3{alpha}, liver and activation regulated chemokine, and Exodus) is a recently described Mn-derived CC-chemokine which selectively attracts immature dendritic cells (iDC), effector/memory T lymphocytes, and naive B cells through its specific receptor, CCR6, expressed on these cells (for a review, see Ref. 3).

Mononuclear phagocyte reactivity in pathological tissues is finely tuned by a complex interplay between stimulatory and inhibitory signals of various nature that include immune-derived stimuli (4, 5), viral/bacterial products (5, 6), cell metabolites (4, 7), and tissue-specific signals (8). A common denominator of many pathological processes and an important regulator of gene expression is represented by low partial oxygen pressure (pO2) (reviewed in Ref. 9). Hypoxia occurs in cardiovascular, hematological, and pulmonary disorders, ischemic wounds, arthritic joints, atherosclerotic plaques, and microbial infections, and experimental and clinical studies point toward its fundamental role in the pathogenesis of these diseases (8, 9, 10, 11). Areas of low pO2 are also present in solid tumors, where they have been associated with malignant progression, metastasis formation, resistance to therapy, and poor clinical outcome (8, 12, 13, 14). Transcriptional response to hypoxia is mediated primarily by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix (bHLH) transcription factor composed of HIF-1beta (also known as the aryl hydrocarbon receptor nuclear translocator (ARNT)), the constitutive subunit, and HIF-1{alpha}, 2{alpha}, or 3{alpha}, the oxygen-sensitive subunits (9, 15). The {alpha} subunits are posttranslationally stabilized under hypoxia and translocate to the nucleus where they dimerize with HIF-1beta, transactivating the hypoxia responsive element present in the promoter of many O2-sensitive genes (9, 15). Regulation of HIF-1 expression and activity by hypoxia is a tightly regulated process which results from the activity of several oxygen-dependent enzymes and requires interaction and cooperation with various transcriptional cofactors and other transcription factors (15).

Mononuclear phagocytes accumulate preferentially in hypoxic/ischemic areas of diseased tissues (1), and hypoxic conditions have been shown to profoundly affect their proinflammatory and immunoregulatory responses by modulating the expression of genes coding for angiogenic factors, inflammatory cytokines, and extracellular matrix (ECM) components/regulators (reviewed in Ref. 1). Recent evidence indicates that hypoxia can also strictly control the chemokine network in cells of the monocytic lineage not only by regulating the production of specific chemokines but also controlling 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 hypoxia were reported.

In the last few years, microarray technology has become an important tool for the characterization of the molecular bases underlying cell response to stimulation (19). Recent investigations have defined the transcriptional profile induced by hypoxia in in vitro-derived human Mf (hMDM) (20, 21). Given their critical role in the regulation of the initial phases of inflammation (1), it was important to study primary Mn as a model of the early response to the hypoxic environment. In this study, we report the first transcriptome analysis of primary human Mn following hypoxia exposure. Our results reveal the regulation by hypoxia of a cluster of novel genes relevant 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 Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and culture conditions

PBMC were isolated from platelet apheresis of healthy donors, obtained by the Blood Transfusion Center of the Gaslini Institute (Genova, Italy), by density gradient centrifugation over a Ficoll cushion (Ficoll-Paque PLUS; Amersham Biosciences). Mn were then purified by countercurrent centrifugal elutriation using a Beckman JE-6 elutriation chamber and Avanti J-20XP rotor system (Beckman Coulter), as described (22), followed by MACS magnetic bead separation (Human Monocyte Isolation kit-II; Miltenyi Biotec). The purity of Mn preparations was ≥95%, as assessed by morphology on Giemsa-stained cytocentrifuge slides and flow cytometry with anti-CD14 mAb. Viability, determined by flow cytometry after DNA staining with propidium iodide (5 µg/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 µg/ml streptomycin (Celbio) and maintained at 37°C in a humidified incubator containing 21% O2, 5% CO2, and 75% N2, referred to as normoxic conditions. Hypoxic conditions (i.e., 1% O2) were achieved by incubating and handling the cells at 37°C in a humidified anaerobic work station incubator (Bug Box; ALC International) 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 flask in the hypoxic incubator before cell exposure, and pO2 was monitored using a portable trace oxygen analyzer (Oxi 315i/set; WTW). The pO2 in normoxic medium ranged between 149 and 150 mm Hg, values that correspond to a 21% O2 concentration in an aqueous solution at 37°C and at a barometric pressure of 760 mm Hg, whereas the pO2 attained in the medium under hypoxic conditions was ~7.1 mm Hg, which is equivalent to an O2 concentration of 1% and is in the range of the hypoxic levels found in inflammatory tissues (8, 9, 10, 11, 12). The endotoxin content, determined by the Limulus amebocyte lysate test (QCL-1000; Bio-Whittaker), was <0.125 endotoxin units/ml in all reagents.

RNA isolation and cRNA synthesis

Total RNA was purified from different donor-derived Mn using the RNeasy Mini kit from Qiagen. The physical quality control of RNA integrity was conducted by electrophoresis with an Agilent Bioanalyzer 2100 (Agilent Technologies Europe). For each experimental condition, equal amounts of Mn RNA from 15 different donors were randomly pooled into three subsets, and the RNA pools were used for probe preparation. Briefly, 20 µg of RNA were reverse transcribed into double-stranded cDNA on a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems), using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen Life Technologies) 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 provided with the kit. cDNA was purified and used for in vitro transcription with the BioArray High Efficiency RNA Transcript Labeling kit (Enzo Life Sciences) in the presence of biotin-11-CTP and biotin-16-UTP. Labeled cRNA was cleaned up using the Qiagen RNeasy Mini kit, checked for quality, and fragmented by incubation in mild alkaline buffer.

GeneChip hybridization and data analysis

Fragmented cRNA probes were used for hybridization to Affymetrix Human Genome-U133A 2.0 GeneChips (Genopolis) containing 22,283 probe sets corresponding to 18,400 transcripts. Each RNA pool was hybridized to an individual chip, and hybridization was performed at 45°C in the presence of herring sperm DNA (0.1 mg/ml; Sigma-Aldrich). Chips were then washed with 6x standard saline citrate phosphate/EDTA (1x is 0.15 M NaCl, 0.01 M sodium phosphate (pH 7.4), and 1 mM EDTA), stained with streptavidin-PE, and scanned using a confocal microscope scanner (HP GeneArray Scanner 2500) according to Affymetrix’s guidelines. Data capturing was conducted with standard Affymetrix analysis software algorithms (Microarray Suite 5.0), which selects the spots representative of a transcript and subtracts the background from the significant signals (23). Comparative analysis of hypoxic relative to normoxic expression profiles was conducted on GeneSpring 7.2 software (Silicon Genetics). Gene expression data for each replicate experiment were normalized using the "per chip normalization" and "per gene normalization" algorithms implemented in the GeneSpring program. First, each signal was normalized based upon the median signal in that chip ("per chip normalization"). Each corrected value was then normalized based upon the median of the measurements for that gene in all samples ("per gene normalization"). This normalization method, which removes the differing intensity scales and binding rates from multiple experimental readings, allows the comparison of multiple GeneChip hybridizations (24). Finally, gene expression levels of replicate experiments were averaged, and only genes that were modulated by at least 1.5-fold in hypoxic relative to normoxic samples (means of three experiments) were considered differentially expressed. The significance of gene expression differences between the two experimental conditions was calculated using a one-way ANOVA. Only genes that passed a Student’s two-sample t test at a confidence level of 95% (p value <0.05) were considered significant. The complete data set for each microarray experiment was lodged in the ArrayExpress website <www.ebi.ac.uk/arrayexpress>. Gene Ontology (GO) data mining (25) for biological process at level 3 and Expression Analysis Systematic Explorer (EASE) biological theme 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 was performed on an I-Cycler (Bio-Rad), using iQ Supermix supplemented with 10 nM fluorescein (Bio-Rad), 0.1x Sybr-Green I (Sigma-Aldrich), and 300 nM sense and antisense oligonucleotide primers (TIBMolbiol). All primer 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 product length of 80–150 nt and tested before use to confirm appropriate product size and optimal concentrations. qRT-PCR was conducted in triplicate for each target transcript under the following cycling conditions: initial denaturation of 3 min during which the well factor was measured, 50 cycles of 15 s at 95°C followed by 30 s at 60°C. Fluorescence was measured during the annealing step in each cycle. After amplification, melting curves with 80 steps of 15 s and 0.5°C increase were performed to monitor amplicon identity. Expression data were normalized on the values obtained in parallel 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 not affected by hypoxia in the Affymetrix analysis, using the Bestkeeper software (29). Relative expression values were calculated using Q-gene software (30).


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  		Table I. Primer pairs used for real-time quantitative RT-PCRa

 
For semiquantitative PCR, reverse-transcribed cDNA was amplified in triplicate with recombinant TaqDNA polymerase (Invitrogen Life Technologies) using the following cycling conditions: denaturation at 94°C, annealing at 61°C, and extension at 72°C for 60 s for 30 cycles. Products were separated by electrophoresis on a 1.2% agarose gel and visualized by ethidium bromide staining.

Immunocytochemistry

A total of 1 x 105 Mn were applied to polysine glass slides by cytocentrifuging at 900 rpm for 5 min. Cytospin preparations were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide for 15 min. After rinsing in PBS, the slides were preincubated for 30 min in blocking buffer (PBS supplemented with 2% human AB serum) and then incubated for 1 h with anti-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 revealed by 3-amino-9-ethylcarbazole (DakoCytomation), as a substrate. Slides were counterstained with hematoxylin, coverslipped with 80% glycerol in PBS, and examined with a phase contrast microscope (Olympus Italia). Photomicrographs were taken with a Zeiss camera.

ELISA

Secreted CCL20 was measured in cell-free supernatants using the Quantikine human CCL20 immunoassay kit from R&D Systems (Space Import Export, according to the manufacturer’s instructions. The OD of the plates were determined using a Spectrafluor Plus plate reader from Tecan at 450 nm. All assays were done in duplicate and repeated three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Gene expression profile of hypoxic Mn

Mn purified from 15 independent donors were cultured for 16 h in 1% O2, a condition previously shown to effectively modulate gene expression in these cells (17, 31), and the mRNA for the angiogenic factor, vascular endothelial growth factor (VEGF), was assessed by RT-PCR as an index of the response to hypoxia. Fig. 1 shows VEGF mRNA levels in a representative subset of samples. Mn exposed to normoxic conditions expressed basal levels of VEGF mRNA, though showing some degree of donor-to-donor variation. Incubation under hypoxia caused a strong and consistent VEGF up-regulation in all the samples, in agreement with previous observations (31).


Figure 1
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  		FIGURE 1. RT-PCR analysis of VEGF mRNA expression in human Mn. Human peripheral blood Mn purified from different donors were cultured for 16 h under normoxic (–) or hypoxic conditions (+), and total RNA was reverse transcribed and tested for VEGF mRNA expression by semiquantitative RT-PCR, as detailed in Materials and Methods. beta-actin mRNA was assayed in parallel as an internal control for input RNA. A representative experiment of two performed is shown

 
The transcriptional profile of hypoxic Mn was then investigated by microarray analysis. Equal amounts of RNA from the different Mn preparations were randomly combined into three pools that were independently hybridized to human Affymetrix HG-U133A GeneChips, obtaining three biological replicates for each experimental condition. Statistical analysis was performed as described in Materials and Methods. Pairwise comparison among the data sets from normoxic and hypoxic samples (average of three experiments) revealed the differential modulation by hypoxia of a large number of transcripts (data not shown). After restricting the profile to those sequences exhibiting ≥1.5-fold expression differences in hypoxic relative to normoxic samples, we identified 536 up-regulated and 677 down-regulated genes. The majority of differentially expressed genes were identified as unique and named in GenBank, whereas the remaining transcripts were either identified as unnamed expressed sequence tags or were hypothetical. A selection of hypoxia-modulated genes (HMGs) is presented in Tables II and III.


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  		Table II. Relative expression of genes previously reported to be modulated by hypoxiaa

 

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  		Table III. Relative expression of selected novel genes defining the transcriptome of hypoxic primary monocytesa

 

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  		Table IIIA. Continued

 

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  		Table IIIB. Continued

 
Functional classification of HMGs

Genes displaying at least 1.5-fold differential expression levels were classified into various categories based on the biological function(s) of the encoded protein to determine the global direction of the molecular response to hypoxia. According to GO data mining for biological processes (Fig. 2), the transcriptional profile induced by hypoxia in Mn was mainly related to cell growth and/or maintenance, signal transduction, nucleic acid, and protein metabolism, these functional categories being the most enriched in both up- and down-regulated genes. Moreover, hypoxic Mn showed a prominent expression of genes involved in organogenesis, response to stress, biosynthesis, and phosphorus metabolism. Interestingly, a significant number of HMGs coded for proteins implicated in cell response to external stimuli, immune response, cell adhesion and motility, and cell-cell signaling, revealing a trend toward inflammation and immunity (Fig. 2).


Figure 2
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  		FIGURE 2. GO data mining. The gene expression profile of hypoxic vs normoxic primary human Mn was analyzed using high-density oligonucleotide arrays, as described in Materials and Methods. Genes showing at least a 1.5-fold change in expression levels between hypoxic and normoxic cells (mean of three experiments) were selected and characterized according to their biological process classification (at level 3) in the GO database. Based on this classification scheme, genes were placed in more than one category if more than one function of the encoded protein had been established. Transcripts without 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-axis represent down-regulated genes.

 
Comparison of microarray data with known hypoxia-induced changes in gene expression

In an initial verification of microarray data, we have cross-referenced our results with those of other studies investigating HMGs. As summarized in Table II, a large cluster of known HMGs was affected by hypoxia. In addition to the reference gene VEGF, we demonstrated up-regulation of 20 genes related to angiogenesis, cell adhesion, transcription, and inflammatory responses, which have been previously identified as hypoxia inducible in cells of the Mn lineage (Table II). The majority of them, including adrenomedullin (ADM), arginase-1 (ARG1), coagulation factor III (F3), fibronectin-1 (FN1), IL-1{alpha} (IL-1A), IL-6, TNF-{alpha}, macrophage migration inhibitory factor (MIF), matrix metalloproteinase-1 (MMP-1), osteopontin (SPP1), and VEGF receptor-1 (FLT1), were reported to be increased by hypoxia in hMDM and/or mouse Mf (20, 32), but not in primary Mn. The expression of another 46 known HMGs characterized in cells types other than mononuclear phagocytes was also triggered in hypoxic Mn (Table II), including classical genes involved in glycolytic metabolism and glucose transport (e.g., glucose transporter 1 and 3 (GLUT1, 3) (9, 13, 15, 33), or associated with nonglycolytic metabolism and ion transport (e.g., carbonic anhydrase XII (CA12) (13, 33). Genes coding for two novel HMGs, hypoxia-inducible protein 2 (HIG2) (34) and HIF-1-responsive RTP801 (DDIT4) (15), and other HIF-1 target genes implicated in HIF-1{alpha} 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 previously demonstrated in normal and malignant cells, were also up-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-1 receptor 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 also demonstrated inhibition by hypoxia of a cluster of inflammatory genes previously found down-regulated in mMf and hMDM, such as CCL2 (16), CCR5 (18), cathepsin C (CTSC) (21), 2,5-oligoadenylate synthetase (OASE) (40), and the ras family member, RAB7 (21). Overall, these data demonstrate that primary Mn share with Mf and cells of other lineages a cluster of hypoxia-responsive genes.

Identification of novel hypoxia-modulated genes in primary Mn

To identify genes not previously characterized in terms of responsiveness to hypoxia, the hypoxic transcriptome of primary Mn was further investigated after excluding known HMGs listed in Table II. A list of selected novel hypoxia-modulated genes is presented in Table III. Of interest, several were enzymes or other molecules implicated in lipid metabolism with a role in the regulation of fatty acid and/or cholesterol biosynthesis or transport (Table III). The most relevant are: apolipoprotein B48 receptor (APOB48R, up-regulated), apolipoprotein E (APOE, down-regulated), fatty acid acyl-CoA synthase long-chain family 1 and 3 (ACSL1,3, down-regulated), fatty acid binding protein 4 (FABP4, up-regulated), low density lipoprotein receptor (LDLR, down-regulated), oxysterol binding protein-like 10 (OSBPL10, up-regulated), and steroid 27-hydroxylase (CYP27A1, down-regulated). Modulation of these genes under low pO2 may have implications for the pathogenesis of atherosclerosis and Alzheimer’s disease, where a role for hypoxia has been suggested (9, 10, 41).

Other genes differentially expressed in hypoxic Mn coded for cytoskeleton and ECM components/regulators (Table III). Inducible genes include adhesive proteins of the desmosome type of cell-cell junction, actin-interacting/regulatory transmembrane molecules, and secreted proteins. Of note are galectin 8 (LGALS8), MMP-16 (MT3-MMP), MMP-19, regulator of G-protein signaling 1 (RGS1), tensin 1 (TNS1), and tissue factor pathway inhibitors 1 and 2 (TFPI1,2). Among the most significantly down-regulated genes, we identified advillin (AVIL), autotaxin (ENPP2), galectin 2 (LGALS2), myosin heavy polypeptide 11 (MYH11), and VAV3. Modulation of these genes is likely to regulate Mn adhesion, motility, and tissue remodeling.

The hypoxic profile also revealed hypoxia inducibility of a set of genes with transcription regulatory activity, including activating transcription factor (ATF)-2 and ATF-5, Fos homolog B (FOSB), Fos-like Ag 2 (FRA2), and runt-related transcription factors 1 and 2 (RUNX1,2) (Table III). Moreover, several transcription factor- and cofactor-encoding genes were inhibited by hypoxia. Of relevance are the aryl-hydrocarbon receptor (AHR), ARNT2, CCAAT enhancer-binding protein {alpha} (C/EBP{alpha}), CREB-binding protein (CBP), HIF-1{alpha}, 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 of both positive and negative O2-driven feedback regulatory mechanisms of hypoxia transcriptional response

Differential modulation by hypoxia of immune-related genes in Mn

As summarized in Table III, a prominent set of novel HMGs have immunological relevance. These include genes encoding surface immunoregulatory signaling (IRS) receptors, such as early activation Ag (CD69), leukocyte membrane Ag CMRF-35H, low-affinity IgG receptors Fc{gamma}RIIA,B (CD32), IgA receptor Fc{alpha}R (CD89), and triggering receptor expressed on myeloid cells 1 (TREM1), that were up-regulated, and high-affinity IgG receptor Fc{gamma}RIA (CD64), histocompatibility Ag class IG (HLA-G), leukocyte-associated Ig-like receptor 1 (LAIR1), leukocyte Ig-like receptor 9 (LIR9), and B1,B2,B3,B4 (LILRB1–4, CD85), that were down-regulated. Several scavenging and pattern recognition receptors were also selectively induced (complement component 1q receptor 1 (C1qR1); macrophage receptor with collagenous structure (MARCO); macrophage 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) under hypoxia (Table III). Other differentially expressed genes coded for costimulatory and adhesion molecules involved in cell-cell and cell-matrix interaction, such as bone marrow stromal cell Ag (CD157), CD36, CD84, and CD86 Ags, integrin {alpha} X (CD11C), integrin beta5 and beta7 (ITGB5,7), ICAM3,5, semaphorin 4D (CD100), and sialoadhesin (CD169) (Table III).

The hypoxic transcriptome was also characterized by the modulation of a cluster of genes coding for cytokines/chemokines and/or their receptors. Within the chemokine system, we identified for the first time CCL20, CXCL2, CXCL3, CXCL5, the fractalkine receptor CX3CR1, and the G protein-coupled chemokine orphan receptor (RDC1) as hypoxia-inducible genes, whereas CCL15, CCL18, CCL23, CCL8, CXCL6, CCR1, and CCR2 were the most highly hypoxia-repressed genes (Table III). Finally, Mn hypoxic profile included various components of the IL-1 system (IL-1 family member 9 (IL1F9); IL-1 receptor accessory protein (IL1RAP); IL-1R associated kinase 3 (IRAK-3); IL-18R1) and members of the TNF receptor and ligand superfamilies, as well as IL-23A, IL-6 signal transducer (IL-6ST), IL-13RA1, IL-21R, CSF1,3, and CSF1,3Rs (Table III).

Confirmation of microarray data by qRT-PCR analysis of selected hypoxia-modulated genes

To validate the microarray results using a different technique, mRNA levels for a subset of known and novel hypoxia-modulated genes were quantified by qRT-PCR on a new RNA pool (Fig. 3), using the primer pairs listed in Table I. For this analysis, we randomly selected 27 genes involved in immune regulation, inflammatory responses, and transcription. Three reference genes (Table I) were used for data normalization. We found a 100% concordance between qRT-PCR and Affymetrix data with respect to the direction of the expression changes. For the majority of the genes, fold-differences were also of comparable magnitude (Fig. 3), although they were higher according to qRT-PCR for six genes (CCL23, FCGR1A, LIR9, BNIP-3, Mxl1, and VEGF), in agreement with previous findings showing that microarray can often underestimate the extent of gene regulation compared with qRT-PCR (Varesio et al., unpublished observations). For other genes, such as CMRF-35H, FCGR2B, STAB-1, and MIF, however, higher expression differences were detected by microarray. These results confirm hypoxia responsiveness of novel genes identified by microarray.


Figure 3
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  		FIGURE 3. qRT-PCR analysis of genes selected from the microarray profile. Equal amounts of total RNA from four donor-derived normoxic or hypoxic Mn were pooled, and RNA pools were subjected to qRT-PCR analysis, as detailed in Materials and Methods. Expression changes of 27 selected genes were evaluated in relation to the values obtained in parallel for three reference genes. Results from one representative experiment of three performed are expressed as log2 ratio (1% O2 relative to 21% O2) and are the mean of triplicate determinations for each target transcript ({blacksquare}). Microarray data are shown for comparison ({cjs2108}). 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 that the transcript was down-regulated. Known hypoxia-inducible genes identified for the first time in cells of the Mn lineage are marked with an asterisk (*).

 
Hypoxia induces CCL20 expression and secretion by human Mn

We then selected CCL20, one of the novel immune-related genes most strongly up-regulated in hypoxic Mn, for further analysis. According to microarray and qRT-PCR data, CCL20 mRNA levels were increased by an average of 7.4- and 5.7-fold, respectively (Fig. 3). To address the issue of donor-to-donor variability, we analyzed CCL20 mRNA expression by RT-PCR in a subset of samples comprising the pools used for microarrays (Fig. 4A). CCL20 basal expression was detected in all Mn preparations cultured under normal pO2, although with some variations among individual donors. Consistent CCL20 transcript up-regulation was triggered by hypoxia in every donor, independently of the baseline levels (Fig. 4A), indicating the general inducibility of the gene in primary Mn. qRT-PCR was also performed to quantify the magnitude of hypoxia-induced changes. As shown in Fig. 4B, the extent of CCL20 mRNA increase ranged from 3.1- to 11.4-fold among the samples examined. mRNA up-regulation was paralleled by increased protein expression, as determined by immunocytochemistry (Fig. 5A). Mn isolated from the three donors analyzed by qRT-PCR were cytocentrifuged and immunostained with a mAb directed to CCL20. Low levels of CCL20 immunoreactivity were detectable in the cytoplasm of normoxic Mn. Hypoxia exposure for 16 h resulted in a marked increase in intracellular protein content in all the samples. No staining was detected when an isotype-matched control Ab was used (Fig. 5A).


Figure 4
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  		FIGURE 4. Up-regulation of CCL20 mRNA expression by hypoxia in human Mn. Total RNA was isolated from human peripheral blood Mn cultured under normoxic (–) or hypoxic (+) conditions for 16 h. A, RNA from six different donor-derived Mn was reverse transcribed and tested for CCL20 and beta-actin mRNA expression by semiquantitative RT-PCR. B, The extent of CCL20 mRNA up-regulation by hypoxia was determined by qRT-PCR analysis on the RNA isolated from the indicated donors, and relative transcript expression was calculated in relation to the values obtained in parallel for three reference genes, as detailed in Materials and Methods. Results from one representative experiment are expressed as fold increase of mRNA levels in hypoxic relative to normoxic cells (arbitrarily defined as 1) and are the mean of triplicate determinations. Fold-change values are indicated by a number associated with each bar.

 

Figure 5
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  		FIGURE 5. Hypoxia induces CCL20 production by primary Mn. Human peripheral blood Mn purified from the indicated donors were cultured under normoxic (–) 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 protein expression 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 photomicrographed (magnification, x40). CCL20 immunoreactivity is detectable in the cell cytoplasm (brown staining). Staining with an isotype-matched control mAb (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 x 106 cells/ml. The number associated with each bar indicates the fold induction of CCL20 secretion in hypoxic relative to normoxic cells (arbitrarily defined as equal to 1).

 
Conditioned medium from normoxic and hypoxic Mn cultures was then analyzed for CCL20 content by ELISA to determine whether a parallel rise in chemokine secretion was triggered by hypoxia (Fig. 5B). Normoxic Mn constitutively secreted variable amounts of CCL20, which accumulated in the culture medium ranging from 10 to 410 pg/106 cells/ml in six different donors. Consistent with the mRNA data, chemokine secretion increased by 2.4- to 3.5-fold upon incubation to 1% O2 for 16 h (Fig. 5B) and was further augmented at longer time points, with almost 7-fold higher amounts detectable in the conditioned medium at 72 h of culture (Fig. 5C, donor 12). CCL20 is a LPS-inducible chemokine (3) and the response of Mn to LPS was evaluated in parallel experiments as a positive control. Interestingly, the extent of CCL20 secretion elicited by hypoxia exceeded that triggered by stimulation with LPS at all the time points analyzed (Fig. 5C), suggesting that hypoxia is a more potent stimulus than LPS in inducing CCL20 production by Mn.

Taken together, these results indicate that primary human Mn isolated from different donors produce variable baseline levels of CCL20 but respond to low pO2 with consistent CCL20 up-regulation, confirming CCL20 as a hypoxia-inducible gene.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Mn responses that ensue following recruitment at pathological sites begin in the setting of reduced pO2 (1). Hypoxia regulatory effects on Mn gene expression have not been characterized in detail, and only a limited number of hypoxia-inducible genes have been identified in these cells (17, 31, 41). In this study, we provide the first transcriptome analysis of hypoxic primary peripheral blood human Mn.

Microarray analysis was conducted on RNA pooled from different donor-derived Mn, whose response to hypoxia was demonstrated by VEGF up-regulation. Three pools, each composed by RNA from five different donors, were investigated per each experimental condition. RNA pooling is a powerful, cost-effective, and statistically valid mean of identifying common changes in a gene expression profile. Previous studies have established the feasibility of using this type of approach to overcome interindividual variability and provide reliable results that reflect gene expression in individual donors (42). The suitability of our experimental protocol for identifying bona fide hypoxia-responsive genes in primary cells was inferred by the demonstration that hypoxia modulated 74 genes known from the literature to be responsive to hypoxia. The majority of these genes, involved in glycolysis, apoptosis, cell cycle, and transcription, may constitute a gene cluster commonly induced by hypoxia in cells of different lineages. The general representativeness of the gene expression changes detected by microarray was further established by qRT-PCR, which confirmed the differential expression of 27 genes randomly selected from the microarray profile in a fourth set of pooled RNAs.

Two recent reports investigated the hypoxia transcriptome of hMDM and demonstrated induction of genes encoding angiogenesis and ECM regulators/components (20, 21), some of which were also present in our profile. Expression of ADM, F3, FLT1, FN1, MIF, INHBA, MMP-1, PTGS2, SPP1, and VEGF, among others, was detectable at significant levels in both primary human Mn (this report) and MDM (20), and exposure to hypoxia resulted in their strong up-regulation in both cell populations. Moreover, we demonstrated up-regulation of other genes previously shown to be inducible by hypoxia in mononuclear phagocytes and coding for immunoregulatory/proinflammatory proteins and transcription factors, such as ARG1, CXCR4, EGR1, FGF1, ID2, TIMP-1, and VLDR (17, 21, 32, 41, 43, 44). Interestingly, IL-1{alpha}, IL-6, and TNF-{alpha}, induction in Mn was observed in response to hypoxia alone, whereas up-regulation in Mf and MDM required LPS costimulation or a reoxygenation period (43). Furthermore, we confirmed in primary Mn hypoxic inhibition of a cluster of inflammatory genes found down-regulated in mMf and hMDM, such as CCL2 (16), CCR5 (18), CTSC (21), OASE (40), and RAB7 (21). These findings indicate that hypoxia is active on different types of mononuclear phagocytes and across species in regulating the expression of selected target genes, which may be critical for their adaptation to the hypoxic environment and ability to function in it and could be representative of the hypoxic transcriptome of cells belonging to the monocytic lineage. In contrast, the observation that other genes up-regulated in hMDM (20, 21) were not induced or were even down-regulated in primary Mn, e.g., the angiogenesis inducers endothelial cell growth factor-1 (ECGF1) and IL-18 binding protein (IL-18BP), suggests that hypoxia may also activate in mononuclear phagocytes a specific transcriptional response depending on their differentiation stage.

Several HMGs identified in our analysis were endowed with transcription regulatory activity and/or encoded important components of the HIF-1 transcription pathway. Hypoxia increased the expression of P4HA1, P4HA2, and EGLN1, three members of the prolyl-hydroxylase family which mediate HIF-{alpha} hydroxylation in well-oxygenated cells targeting the protein for proteosomal degradation by the von Hippel Lindau tumor suppressor protein (pVHL) (15), and of BHLHB2, a bHLH family member implicated in the regulation of pVHL/HIF pathways (15). These data are in agreement with previous findings in other cell types (15, 35, 36) and suggest an O2-driven HIF-1-dependent autoregulatory mechanism, required to ensure fast HIF-1{alpha} degradation upon reoxygenation, also in Mn. Of note is the novel evidence that hypoxia promotes the expression of ATF2 and ATF5, two members of the ATF family of transcription factors (45) required for efficient activation of gene transcription by hypoxia (15). Moreover, we demonstrated up-regulation of FosB and FRA-2, two components of the AP-1 complex (45), which is activated by hypoxia and was recently shown to cooperate with HIF-1{alpha} in the transactivation of hypoxia inducible genes (46), and of JEM-1, a novel transcriptional cofactor which enhances AP-1 activity (47). Up-regulation of these genes together with down-regulation of ZnF197, a novel VHL-interacting protein that functions as a repressor of HIF-1{alpha} transactivation (48), may represent a positive regulatory mechanism of hypoxia transcriptional response in Mn. Down-regulation of several HIF-1 transcriptional cofactors, including CBP, C/EBP{alpha}, MITF, and NCOA1/SRC-1, was also observed in hypoxic Mn. All these proteins physically interact with HIF-1{alpha} enhancing its transactivation function under hypoxia (9, 15, 49), and their down-regulation suggests the existence of a negative feedback mechanism to control Mn hypoxic response. Consistent with this hypothesis, we observed a parallel decrease of the mRNAs encoding HIF-1{alpha} and one of its dimerization partners, ARNT-2 (9)

GO data mining characterized a significant cluster of genes as being associated with immune regulation and inflammatory responses, chemotaxis, cell adhesion, and ECM remodeling, the majority of which has not been previously identified as responsive to hypoxia. Profound changes were observed in the expression of scavenger receptors. Of relevance is the up-regulation of MSR1 and SCARF1, because these molecules functions as endocytic receptors for acetylated LDL and may thus be implicated in lipid-loaded foam cell formation contributing to atherosclerotic plaques development (50). Consistent with the view that hypoxia may exert a pathogenetic role in atherosclerosis and Alzheimer’s disease (9, 10, 41) is also the down-regulation of CD163 and STAB1 scavenger receptors, which are endowed with atheroprotective activity (51, 52), and of various genes involved in the regulation of fatty acid and/or cholesterol biosynthesis/transport and acting as anti-atherogenic factors, e.g., LDLR, ApoE, and CYP27A1 (53, 54). Mn hypoxic profile also showed up-regulation of a number of other pattern recognition receptors critical to host defense, the most relevant of which are the C1qR1, COLEC12, and MARCO. These molecules bind specific Ags on bacteria facilitating their recognition and phagocytosis (55, 56, 57). Interestingly, the TLR family members TLR5 and TLR7, whose function is also to recognize pathogen or their products and hence initiate innate immune responses (58), were down-regulated by hypoxia. Differential modulation of receptors with similar functions is intriguing and is likely to contribute to the fine tuning of Mn antimicrobial activities at sites of infection.

Myeloid cell immune functions are regulated by a balance of inhibitory and activating signals transduced by multiple families of cell surface IRS receptors belonging to the Ig superfamily of inhibitory/stimulatory pairs of molecules (59). These receptors are either characterized by ITIM/ITAM regulatory components in their cytoplasmic domain or pair with ITAM-containing transmembrane adapter proteins (59). Mn exposure to hypoxia resulted in the selective modulation of various members of these receptor families. Of particular interest is the up-regulation of Fc{gamma}RIIA,B and down-regulation of Fc{gamma}RIA. Fc{gamma}RIIA contains a functional ITAM motif, thus triggering cell activation, whereas Fc{gamma}RIIB encodes an ITIM component, leading to repression of cellular responses (60). Fc{gamma}RIA, which associates for signaling with an ITAM-containing {gamma}-chain, also belongs to the activatory Fc{gamma}R subclass (60). Fc{gamma}Rs involvement in various autoimmune inflammatory states was reported (60), and our findings suggest the potential role of hypoxia in influencing the pathogenesis of these diseases through the modulation of distinct Fc{gamma}R genes.

Hypoxic modulation of other ITIM/ITAM Ig family members, specifically up-regulation of FCAR, CMRF-35H, and TREM-1 and down-regulation of LIR9 and LAIR1, was also observed. These molecules mediate a variety of effector functions vital to the adaptative immune response, and their ligation can have both proinflammatory and immunosuppressive consequences by differentially modulating the secretion of pro- or anti-inflammatory mediators (59, 60, 61, 62, 63). The differential expression of inhibitory and activating isoforms of a given receptor family with similar specificities on the same cell is another example of the tight regulatory role of hypoxia on Mn inflammatory responses. In particular, the concomitant down-regulation of the Ig-like inhibitory receptors LILRB1–4 and of their ligand HLA-G, a nonclassical inhibitory HLA class I Ag, is noteworthy given the role of these molecules in immune tolerance and immune escape (64). It is conceivable that inhibition of these molecules may decrease the activation threshold of Mn retained at hypoxic sites. Mn activation under conditions of reduced O2 availability may also be controlled by the selective down- and up-regulation of two C-type lectin receptors, the ITIM-containing inhibitory molecule CLECSF6 (65) and CD69, a member of the NK receptor family and a potent inducer of Mn inflammatory mediator production and cytotoxic activity (66). Although a ligand for CD69 has not been identified, previous studies suggested a pathogenetic role for CD69 in certain inflammatory states characterized by hypoxia, such as rheumatoid arthritis, chronic inflammatory liver diseases, and asthma (66).

Mononuclear phagocyte migratory activity is a highly regulated process which depends on a defined repertoire of chemokines/receptors and adhesion molecules, and dysregulated expression of these proteins may alter their recruitment and activation (67). Various studies have investigated the mechanisms whereby mononuclear phagocytes are retained/concentrated at hypoxic pathological sites (1). One possibility is that hypoxia inhibits their migration in response to chemokines by decreasing the expression of specific chemokine receptors, as demonstrated for CCR5 in Mf (18). In agreement with this hypothesis and with previous findings showing impaired Mn migratory ability to CCL2 under conditions of low pO2 (1), we observed CCR5, CCR1, and CCR2 down-regulation. This study also suggests other potential mechanisms for Mn entrapment in hypoxic areas, such as the up-regulation of RGS1, a member of a new class of G protein-signaling deactivators which inactivates several chemotactic receptors inhibiting chemoattractant-induced Mn migration (68). Furthermore, repression of the secreted cell motility-promoting factor, ENPP2 (69), together with induction of the antichemotactic cytokine, MIF (13), and the GRO family chemokines, CXCL2 and CXCL3, which are specialized Mn-arrest chemokines (70), may also provide a "stop" signal to Mn within hypoxic tissues.

The complexity of the regulation of Mn migratory behavior by hypoxia is further emphasized by the demonstration of dysregulated expression of several other migration-related genes. Mn hypoxic profile was associated with down-regulation of the adhesion molecules CD11C, CD57, and ITGB5,7, which mediate Mn adhesion to the endothelium and/or to the ECM (67), and with up-regulation of both the fractalkine receptor, which binds to the CX3C chemokine fractalkine expressed on endothelial cells functioning as a potent adhesion molecule (71), and the RDC1 receptor, which share with CXCR4 the chemokine CXCL12/SDF-1 as a natural ligand (72). A critical role in the control of mononuclear phagocyte motility is also exerted by MMPs, a group of secreted enzymes that trigger ECM degradation facilitating leukocyte movement in tissues (73). Recent reports have shown up-regulation by hypoxia of MMP-1, MMP-7, and MMP12 in hMDM (20, 21). Interestingly, only MMP1 was induced also in primary Mn that showed specific up-regulation of MMP16 and MMP19 and down-regulation of MMP25. Moreover, the MMP inhibitors, TIMP1 and TFPI2, which reduce ECM degradation inhibiting cell migratory activity (73, 74), were also modulated in hypoxic Mn. Collectively, these studies indicate that regulation of MMPs and their inhibitors is a common denominator of mononuclear phagocyte response to hypoxia, but that different components of these families are controlled depending on the cell differentiation stage. Altered expression of MMPs has been associated with a variety of acute and chronic inflammatory states (73), and hypoxia can probably play a role in the pathogenesis of these diseases by modulating MMP production by infiltrating Mn

Various cytokines/chemokine and/or receptors are modulated by hypoxia in mononuclear phagocytes (1, 43). The Mn hypoxic transcriptome confirmed and extended those findings showing differential expression of other components of the cytokine/chemokine system. Various members of the IL-1 and the TNFR/ligand superfamilies were selectively up- or down-regulated in hypoxic Mn, including molecules associated with and mediating signal transduction from their receptors (e.g., IL1RAP, IRAK3, and the TNFR-associated factor, TRAF). Because of their pleiotropic effects on almost every types of cells, coordinated regulation of the TNF and IL-1 systems is likely to represent an important mechanism to control the amplitude and the duration of inflammatory responses. The demonstration that hypoxia induces CSF1 and CSF3, while inhibiting their receptors, is noteworthy given the role of these factors in the regulation of myeloid cell production, differentiation, and function (75) and is consistent with previous findings suggesting a reciprocal and divergent action of hypoxia on receptor vs ligand expression (16, 18). Accordingly, IL-4 up-regulation and concomitant inhibition of its receptor IL-13RA1 was observed. This interplay is likely to serve as a negative feedback mechanism to control the autocrine activation of producing Mn. However, not all the data presented in this study are consistent with this scenario. Concomitant down-regulation of the Mn chemoattractants CCL2 and CCL8 and of their receptors CCR2 and CCR5 (2) was in fact observed in response to hypoxia. Similarly, we found down-regulation of CCL15, a chemoattractant for neutrophils, monocytes, and lymphocytes (76), and CCL23, a chemokine mediating resting T cell and Mn chemotaxis (77), and of their common receptor CCR1. Collectively, these data indicate that a dynamic change in chemokine/receptor expression profile occurs in Mn within hypoxic tissues. This tight and complex level of control exerted by low O2 tension is clearly of pathophysiologic relevance, representing an important mechanism of regulation of leukocyte trafficking and function at sites of inflammation. Because some of the modulated chemokines have angiogenic activity (1, 2, 76), their altered expression under low pO2 may also influence neoangiogenesis in pathological tissues.

One of the most important findings of this study was the demonstration that hypoxia strongly induced the expression of the CCL20-encoding gene in primary Mn. Marked differences in the basal mRNA expression were detected among individual donors. However, mRNA up-regulation under hypoxia was consistently demonstrated in all Mn preparation analyzed, and gene expression results were associated to a parallel augmentation of protein expression and secretion. Previous reports have shown that the CCL20 gene is markedly up-regulated in PBMC by LPS and inducible in other cell types in response to various mediators of inflammation, including cytokines, growth factors, bacterial, viral and plant products, whereas it is poorly expressed in the absence of inflammatory stimuli (3). This study is the first to identify hypoxia as a new CCL20 inducer. Interestingly, the extent of CCL20 up-regulation by hypoxia exceeded that triggered by LPS, suggesting that hypoxia is a more potent stimulus than LPS for CCL20 production by Mn. Given its role in the recruitment of iDC, effector/memory T lymphocytes, and naive B cells, CCL20 has been proposed as an important mediator for both the initiation and effector phases of the inflammatory reactions, linking innate and acquired immunity (3). Hence, by producing CCL20, Mn may control the kinetics and composition of the cellular infiltrate under various inflammatory conditions and at tumor sites. The identification of CCL20 as a hypoxia-inducible gene may explain, in part, the high levels of this chemokine present in areas of inflammation and in various chronic inflammatory conditions, such as rheumatoid arthritis, inflammatory skin disorders, and tumors (for a review, see Refs. 3 and 78), as these sites are known to be hypoxic and infiltrated by Mn, and is indicative of a pathogenetic role for this molecule in these diseases. Further studies are ongoing in the laboratory to elucidate the molecular mechanisms underlying CCL20 induction by hypoxia.

In summary, we have described the hypoxia transcriptome of primary human Mn and identified a large number of genes not previously known to change as a result of reduced O2 concentrations. Our findings contribute to the definition of the gene cluster commonly induced by hypoxia in cells of different lineage. This study provides novel insights into the molecular responses to the hypoxic stress and the mechanisms linking low pO2 to the regulation of immune and inflammatory responses, leading to new perspectives of the role of hypoxia in programming Mn functions within pathological conditions and identifying potential molecular targets for the development of rational therapeutic approaches.


   Acknowledgments
 
We thank S. Delfino for technical assistance and C. Dabizzi for secretarial work.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Italian Association for Cancer Research, Fondazione Italiana per la Lotta al Neuroblastoma, San Paolo Company, Italian Health Ministry, and Ministero Istruzione Universita’ e Ricerca. Back

2 Address correspondence and reprint requests to Dr. Maria Carla Bosco, Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. E-mail address: mcbosco1{at}virgilio.it Back

3 Abbreviations used in this paper: Mn, monocyte; Mf, macrophage; iDC, immature dendritic 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 receptor nuclear translocator; ECM, extracellular matrix; hMDM, human monocyte-derived macrophage; MMP, matrix metalloproteinase; bHLH, basic helix-loop-helix. Back

Received for publication November 4, 2005. Accepted for publication May 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Murdoch, C., A. Giannoudis, C. E. Lewis. 2004. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood 104: 2224-2234. [Abstract/Free Full Text]
  2. Rossi, D., A. Zlotnik. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18: 217-242. [Medline]
  3. Schutyser, E., S. Struyf, J. Van Damme. 2003. The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev. 14: 409-426. [Medline]
  4. Varesio, L., I. Espinoza-Delgado, G. Gusella, G. Cox, G. Melillo, T. Musso, M. C. Bosco. 1995. Role of cytokines in the activation of monocytes. B. B. Aggarwal, and R. K. Puri, eds. Human Cytokines: Their Role in Disease and Therapy 55-70. Blackwell Scientific Publication, Cambridge.
  5. Paulnock, D. M., K. P. Demick, S. P. Coller. 2000. Analysis of interferon-{gamma}-dependent and-independent pathways of macrophage activation. J. Leukocyte Biol. 67: 677-682. [Abstract]
  6. Kopydlowski, K. M., C. A. Salkowski, M. J. Cody, N. Van Rooijen, J. Major, T. A. Hamilton, S. N. Vogel. 1999. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163: 1537-1544. [Abstract/Free Full Text]
  7. Bosco, M. C., A. Rapisarda, S. Massazza, G. Melillo, H. Young, L. Varesio. 2000. The tryptophan catabolite picolinic acid selectively induces the chemokines macrophage-inflammatory protein-1{alpha} and -1beta in macrophages. J. Immunol. 164: 3283-3291. [Abstract/Free Full Text]
  8. Crowther, M., N. J. Brown, E. T. Bishop, C. E. Lewis. 2001. Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J. Leukocyte Biol. 70: 478-490. [Abstract/Free Full Text]
  9. Semenza, G. L.. 2001. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7: 345-350. [Medline]
  10. Bjornheden, T., M. Levin, M. Evaldsson, O. Wiklund. 1999. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler. Thromb. Vasc. Biol. 19: 870-876. [Abstract/Free Full Text]
  11. Distler, J. H., R. H. Wenger, M. Gassmann, M. Kurowska, A. Hirth, S. Gay, O. Distler. 2004. Physiologic responses to hypoxia and implications for hypoxia-inducible factors in the pathogenesis of rheumatoid arthritis. Arthritis. Rheum. 50: 10-23. [Medline]
  12. Vaupel, P., M. Hockel. 2003. Tumor oxygenation and its relevance to tumor physiology and treatment. Adv. Exp. Med. Biol. 510: 45-49. [Medline]
  13. Harris, A. L.. 2002. Hypoxia—a key regulatory factor in tumour growth. Nat. Rev. Cancer 2: 38-47. [Medline]
  14. Le, Q. T., N. C. Denko, A. J. Giaccia. 2004. Hypoxic gene expression and metastasis. Cancer Metastasis Rev. 23: 293-310. [Medline]
  15. Wenger, R. H., D. P. Stiehl, G. Camenisch. 2005. Integration of oxygen signaling at the consensus HRE. Sci. STKE 2005: re12[Abstract/Free Full Text]
  16. Bosco, M. C., M. Puppo, S. Pastorino, Z. Mi, G. Melillo, S. Massazza, A. Rapisarda, L. Varesio. 2004. Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages. J. Immunol. 172: 1681-1690. [Abstract/Free Full Text]
  17. Schioppa, T., B. Uranchimeg, A. Saccani, S. K. Biswas, A. Doni, A. Rapisarda, S. Bernasconi, S. Saccani, M. Nebuloni, L. Vago, et al 2003. Regulation of the chemokine receptor CXCR4 by hypoxia. J. Exp. Med. 198: 1391-1402. [Abstract/Free Full Text]
  18. Bosco, M. C., G. Reffo, M. Puppo, L. Varesio. 2004. Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages. Cell. Immunol. 228: 1-7. [Medline]
  19. Lockhart, D. J., E. A. Winzeler. 2000. Genomics, gene expression and DNA arrays. Nature 405: 827-836. [Medline]
  20. White, J. R., R. A. Harris, S. R. Lee, M. H. Craigon, K. Binley, T. Price, G. L. Beard, C. R. Mundy, S. Naylor. 2004. Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis. Genomics 83: 1-8. [Medline]
  21. Burke, B., A. Giannoudis, K. P. Corke, D. Gill, M. Wells, L. Ziegler-Heitbrock, C. E. Lewis. 2003. Hypoxia-induced gene expression in human macrophages: implications for ischemic tissues and hypoxia-regulated gene therapy. Am. J. Pathol. 163: 1233-1243. [Abstract/Free Full Text]
  22. Bosco, M. C., I. Espinoza-Delgado, T. K. Rowe, M. G. Malabarba, L. Long, L. Varesio. 1997. Functional role for the myieloid differentiation antigen CD14 in the activation of human monocytes by IL-2. J. Immunol. 159: 2922-2931. [Abstract]
  23. Irizarry, R. A., B. M. Bolstad, F. Collin, L. M. Cope, B. Hobbs, T. P. Speed. 2003. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31: e15[Abstract/Free Full Text]
  24. Quackenbush, J.. 2002. Microarray data normalization and transformation. Nat. Genet. 32: (Suppl.):496-501. [Medline]
  25. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, et al 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25: 25-29. [Medline]
  26. Hosack, D. A., G. Dennis, Jr, B. T. Sherman, H. C. Lane, R. A. Lempicki. 2003. Identifying biological themes within lists of genes with EASE. Genome Biol. 4: R70[Medline]
  27. Dennis, G., Jr, B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H. C. Lane, R. A. Lempicki. 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4: 3
  28. Rozen, R., H. J. Skaletsky. 2000. Primer 3 on the WWW for general users and for biological programmers. S. Krawets, Jr, and S. Misener, Jr, eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology 365 Humana, Totowa.
  29. Pfaffl, M. W., A. Tichopad, C. Prgomet, T. P. Neuvians. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26: 509-515. [Medline]
  30. Muller, P. Y., H. Janovjak, A. R. Miserez, Z. Dobbie. 2002. Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques 32: 1372-1379. [Medline]
  31. Melillo, G., E. A. Sausville, K. Cloud, T. Lahusen, L. Varesio, A. M. Senderovicz. 1999. Flavopiridol, a protein kinase inhibitor, down-regulates hypoxic induction of vascular endothelial growth factor expression in human monocytes. Cancer Res. 59: 5433-5437. [Abstract/Free Full Text]
  32. Louis, C. A., J. S. Reichner, W. L. Henry, Jr, B. Mastrofrancesco, T. Gotoh, M. Mori, J. E. Albina. 1998. Distinct arginase isoforms expressed in primary and transformed macrophages: regulation by oxygen tension. Am. J. Physiol. 274: R775-R782. [Medline]
  33. Jogi, A., J. Vallon-Christersson, L. Holmquist, H. Axelson, A. Borg, S. Pahlman. 2004. Human neuroblastoma cells exposed to hypoxia: induction of genes associated with growth, survival, and aggressive behavior. Exp. Cell Res. 295: 469-487. [Medline]
  34. Denko, N., C. Schindler, A. Koong, K. Laderoute, C. Green, A. Giaccia. 2000. Epigenetic regulation of gene expression in cervical cancer cells by the tumor microenvironment. Clin. Cancer Res. 6: 480-487. [Abstract/Free Full Text]
  35. del Peso, L., M. C. Castellanos, E. Temes, S. Martin-Puig, Y. Cuevas, G. Olmos, M. O. Landazuri. 2003. The von Hippel Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of the HIF-proline hydroxylase genes in response to low oxygen. J. Biol. Chem. 278: 48690-48695. [Abstract/Free Full Text]
  36. Hofbauer, K. H., B. Gess, C. Lohaus, H. E. Meyer, D. Katschinski, A. Kurtz. 2003. Oxygen tension regulates the expression of a group of procollagen hydroxylases. Eur. J. Biochem. 270: 4515-4522. [Medline]
  37. Kitamuro, T., K. Takahashi, K. Ogawa, R. Udono-Fujimori, K. Takeda, K. Furujama, M. Nakayama, J. Sun, H. Fujita, W. Hida, et al 2003. Bach 1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells. J. Biol. Chem. 278: 9125-9133. [Abstract/Free Full Text]
  38. Naldini, A., F. Carraro, S. Silvestri, V. Bocci. 1997. Hypoxia affects cytokine production and proliferative responses by human peripheral mononuclear cells. J. Cell. Physiol. 173: 335-342. [Medline]
  39. Naldini, A., A. Pucci, F. Carraro. 2001. Hypoxia induces the expression and release of interleukin 1 receptor antagonist in mitogen-activated mononuclear cells. Cytokine 13: 334-341. [Medline]
  40. Carta, L., S. Pastorino, G. Melillo, M. C. Bosco, S. Massazza, L. Varesio. 2001. Engineering of macrophages to produce IFN-{gamma} in response to hypoxia. J. Immunol. 166: 5374-5380. [Abstract/Free Full Text]
  41. Nakazato, K., T. Ishibashi, K. Nagata, Y. Seino, Y. Wada, T. Sakamoto, R. Matsuoka, T. Teramoto, M. Sekimata, Y. Homma, Y. Maruyama. 2001. Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia. Atherosclerosis 155: 439-444. [Medline]
  42. Peng, X., C. L. Wood, E. M. Blalock, K. C. Chen, P. W. Landfield, A. J. Stromberg. 2003. Statistical implications of pooling RNA samples for microarray experiments. BMC Bioinformatics 4: 26[Medline]
  43. Lewis, J. S., J. A. Lee, J. C. E. Underwood, A. L. Harris, C. E. Lewis. 1999. Macrophage responses to hypoxia: relevance to disease mechanisms. J. Leukocyte Biol. 66: 889-900. [Abstract]
  44. Qu, X., M. X. Yang, B. H. Kong, L. Qi, Q. L. Lam, S. Yan, P. Li, M. Zhang, L. Lu. 2005. Hypoxia inhibits the migratory capacity of human monocyte-derived dendritic cells. Immunol. Cell Biol. 83: 668-673. [Medline]
  45. Herdegen, T., J. D. Leah. 1998. Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res. Brain Res. Rev. 28: 370-490. [Medline]
  46. Laderoute, K. R., J. M. Calaoagan, C. Gustafson-Brown, A. M. Knapp, G. C. Li, H. L. Mendonca, H. E. Ryan, Z. Wang, R. S. Johnson. 2002. The response of c-jun/AP-1 to chronic hypoxia is hypoxia-inducible factor 1 {alpha} dependent. Mol. Cell. Biol. 22: 2515-2523. [Abstract/Free Full Text]
  47. Tong, J. H., E. Duprez, M. Lanotte. 1999. JEM-1, a novel nuclear co-factor: localisation and functional interaction with AP-1. Leukemia. 13: 1982-1992. [Medline]
  48. Li, Z., D. Wang, X. Na, S. R. Schoen, E. M. Messing, G. Wu. 2003. The VHL protein recruits a novel KRAB-A domain protein to repress HIF-1{alpha} transcriptional activity. EMBO J. 22: 1857-1867. [Medline]
  49. Busca, R., E. Berra, C. Gaggioli, M. Khaled, K. Bille, B. Marchetti, R. Thyss, G. Fitsialos, L. Larribere, C. Bertolotto, et al 2005. Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells. J. Cell Biol. 170: 49-59. [Abstract/Free Full Text]
  50. Fukuhara-Takaki, K., M. Sakai, Y. Sakamoto, M. Takeya, S. Horiuchi. 2005. Expression of class A scavenger receptor is enhanced by high glucose in vitro and under diabetic conditions in vivo: one mechanism for an increased rate of atherosclerosis in diabetes. J. Biol. Chem. 280: 3355-3364. [Abstract/Free Full Text]
  51. Moestrup, S. K., H. J. Moller. 2004. CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response. Ann. Med. 36: 347-354. [Medline]
  52. Prevo, R., S. Banerji, J. Ni, D. G. Jackson. 2004. Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1). J. Biol. Chem. 279: 52580-52592. [Abstract/Free Full Text]
  53. Greenow, K., N. J. Pearce, D. P. Ramji. 2005. The key role of apolipoprotein E in atherosclerosis. J. Mol. Med. 83: 329-342. [Medline]
  54. Vainio, S., E. Ikonen. 2003. Macrophage cholesterol transport: a critical player in foam cell formation. Ann. Med. 35: 146-155. [Medline]
  55. Arredouani, M. S., A. Palecanda, H. Koziel, Y. C. Huang, A. Imrich, T. H. Sulahian, Y. Y. Ning, Z. Yang, T. Pikkarainen, M. Sankala, et al 2005. MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages. J. Immunol. 175: 6058-6064. [Abstract/Free Full Text]
  56. Nakamura, K., H. Funakoshi, K. Miyamoto, F. Tokunaga, T. Nakamura. 2001. Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family. Biochem. Biophys. Res. Commun. 280: 1028-1035. [Medline]
  57. Geertsma, M. F., P. H. Nibbering, H. P. Haagsman, M. R. Daha, R. van Furth. 1994. Binding of surfactant protein A to C1q receptors mediates phagocytosis of Staphylococcus aureus by monocytes. Am. J. Physiol. 267: L578-L584. [Medline]
  58. Pasare, C., R. Medzhitov. 2005. Toll-like receptors: linking innate and adaptive immunity. Adv. Exp. Med. Biol. 560: 11-18. [Medline]
  59. Clark, G. J., B. Cooper, S. Fitzpatrick, B. J. Green, D. N. Hart. 2001. The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family. Tissue Antigens 57: 415-423. [Medline]
  60. Gerber, J. S., D. M. Mosser. 2001. Stimulatory and inhibitory signals originating from the macrophage Fc{gamma} receptors. Microbes Infect. 3: 131-139. [Medline]
  61. Meyaard, L., J. Hurenkamp, H. Clevers, L. L. Lanier, J. H. Phillips. 1999. Leukocyte-associated Ig-like receptor-1 functions as an inhibitory receptor on cytotoxic T cells. J. Immunol. 162: 5800-5804. [Abstract/Free Full Text]
  62. Dietrich, J., H. Nakajima, M. Colonna. 2000. Human inhibitory and activating Ig-like receptors which modulate the function of myeloid cells. Microbes Infect. 2: 323-329. [Medline]
  63. Bleharski, J. R., V. Kiessler, C. Buonsanti, P. A. Sieling, S. Stenger, M. Colonna, R. L. Modlin. 2003. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J. Immunol. 170: 3812-3818. [Abstract/Free Full Text]
  64. LeMaoult, J., K. Zafaranloo, C. Le Danff, E. D. Carosella. 2005. HLA-G up-regulates ILT2, ILT3, ILT4, and KIR2DL4 in antigen presenting cells, NK cells, and T cells. FASEB J. 19: 662-664. [Abstract/Free Full Text]
  65. Bates, E. E., N. Fournier, E. Garcia, J. Valladeau, I. Durand, J. J. Pin, S. M. Zurawski, S. Patel, J. S. Abrams, S. Lebecque, et al 1999. APCs express DCIR, a novel C-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif. J. Immunol. 163: 1973-1983. [Abstract/Free Full Text]
  66. De Maria, R., M. G. Cifone, R. Trotta, M. R. Rippo, C. Festuccia, A. Santoni, R. Testi. 1994. Triggering of human monocyte activation through CD69, a member of the natural killer cell gene complex family of signal transducing receptors. J. Exp. Med. 180: 1999-2004. [Abstract/Free Full Text]
  67. Imhof, B. A., M. Aurrand-Lions. 2004. Adhesion mechanisms regulating the migration of monocytes. Nat. Rev. Immunol. 4: 432-444. [Medline]
  68. Denecke, B., A. Meyerdierks, E. C. Bottger. 1999. RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors. J. Biol. Chem. 274: 26860-26868. [Abstract/Free Full Text]
  69. Black, E. J., T. Clair, J. Delrow, P. Neiman, D. A. Gillespie. 2004. Microarray analysis identifies Autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun. Oncogene 23: 2357-2366. [Medline]
  70. Smith, D. F., E. Galkina, K. Ley, Y. Huo. 2005. GRO family chemokines are specialized for monocyte arrest from flow. Am. J. Physiol. 289: H1976-H1984.
  71. Umehara, H., S. Goda, T. Imai, Y. Nagano, Y. Minami, Y. Tanaka, T. Okazaki, E. T. Bloom, N. Domae. 2001. Fractalkine, a CX3C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1. Immunol. Cell Biol. 79: 298-302. [Medline]
  72. Balabanian, K., B. Lagane, S. Infantino, K. Y. Chow, J. Harriague, B. Moepps, F. Arenzana-Seisdedos, M. Thelen, F. Bachelerie. 2005. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J. Biol. Chem. 280: 35760-35766. [Abstract/Free Full Text]
  73. Shapiro, S. D., R. M. Senior. 1999. Matrix metalloproteinases: matrix degradation and more. Am. J. Respir. Cell Mol. Biol. 20: 1100-1102. [Free Full Text]
  74. Herman, M. P., G. K. Sukhova, W. Kisiel, D. Foster, M. R. Kehry, P. Libby, U. Schonbeck. 2001. Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis. J. Clin. Invest. 107: 1117-1126. [Medline]
  75. Pimentel, E.. 1990. Colony-stimulating factors. Ann. Clin. Lab. Sci. 20: 36-55. [Abstract]
  76. Hwang, J., C. W. Kim, K. N. Son, K. Y. Han, K. H. Lee, H. K. Kleinman, J. Ko, D. S. Na, B. S. Kwon, Y. S. Gho, J. Kim. 2004. Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo. FEBS Lett. 570: 47-51. [Medline]
  77. Forssmann, U., M. B. Delgado, M. Uguccioni, P. Loetscher, G. Garotta, M. Baggiolini. 1997. CKbeta8, a novel CC chemokine that predominantly acts on monocytes. FEBS Lett. 408: 211-216. [Medline]
  78. Ruth, J. H., S. Shahrara, C. C. Park, J. C. Morel, P. Kumar, S. Qin, A. E. Koch. 2003. Role of macrophage inflammatory protein-3{alpha} and its ligand CCR6 in rheumatoid arthritis. Lab. Invest. 83: 579-588. [Medline]
  79. Nikitenko, L. L., D. M. Smith, R. Bicknell, M. C. Rees. 2003. Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia. FASEB J. 17: 1499-1501. [Abstract/Free Full Text]
  80. Eltzschig, H. K., J. C. Ibla, G. T. Furuta, M. O. Leonard, K. A. Jacobson, K. Enjyoji, S. C. Robson, S. P. Colgan. 2003. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J. Exp. Med. 198: 783-796. [Abstract/Free Full Text]
  81. Louis, N. A., K. E. Hamilton, T. Kong, S. P. Colgan. 2005. HIF-dependent induction of apical CD55 coordinates epithelial clearance of neutrophils. FASEB J. 19: 950-959. [Abstract/Free Full Text]
  82. Cosio, G., M. C. Jeziorski, F. Lopez-Barrera, G. M. De La Escalera, C. Clapp. 2003. Hypoxia inhibits expression of prolactin and secretion of cathepsin-D by the GH4C1 pituitary adenoma cell line. Lab. Invest. 83: 1627-1636. [Medline]
  83. May, D., A. Itin, O. Gal, H. Kalinski, E. Feinstein, E. Keshet. 2005. Ero1-L{alpha} plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia: implication for cancer. Oncogene 24: 1011-1020. [Medline]



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