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The Journal of Immunology, 2004, 172: 1681-1690.
Copyright © 2004 by The American Association of Immunologists

Hypoxia Selectively Inhibits Monocyte Chemoattractant Protein-1 Production by Macrophages1

Maria Carla Bosco2,*, Maura Puppo*, Sandra Pastorino{dagger}, Zenghui Mi{ddagger}, Giovanni Melillo§, Stefano Massazza*, Annamaria Rapisarda§ and Luigi Varesio*

* Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy; {dagger} Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and {ddagger} Developmental Therapeutics Program and § Science Applications International Corporation, Tumor Hypoxia Laboratory, National Cancer Institute, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1{beta} (MIP-1{beta}), RANTES, IFN-{gamma}-inducible protein-10, and MIP-2 were not affected, and MIP-1{alpha} was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-{gamma} and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of an inflammatory response requires the coordinated recruitment and activation of different leukocyte populations at sites of injury and infection, driven by the local secretion of appropriate chemotactic signals (1). A critical role in the regulation of leukocyte trafficking in vivo is played by the chemokine superfamily, a group of low-m.w. secreted proteins produced by immune or nonimmune cells and endowed with chemotactic and activating properties for specific leukocyte subsets (2, 3). The chemokine superfamily has been subdivided into four subfamilies, which differ with respect to the number and arrangement of the conserved cysteine residues at the N terminus of the primary amino acid sequence (CXC or {alpha}, CC or {beta}, C or {gamma}, and CX3C or {delta}) (2, 3). Monocyte chemoattractant protein-1 (MCP-1),3 the prototype of the CC-chemokine subfamily, is endowed with chemotactic and activating properties for macrophages (M{phi}), CD4+/CD8+ T lymphocytes, NK cells, and basophils and is critically involved in the regulation of inflammatory processes and antitumor immune responses (4, 5, 6).

A primary source for chemokines is activated M{phi} (1, 2, 4, 7), which are important effector and immunoregulatory cells active against infections and tumors (8, 9, 10). The extent and magnitude of a local M{phi} response are regulated by a complex interplay between stimulatory and inhibitory signals of various natures, which include stimuli derived from the immune system (9, 11, 12), metabolites produced by surrounding cells (9, 13), microbial products (7, 11), and tissue-specific signals such as changes in O2 tension and pH (14, 15). Modulation of chemokine production by M{phi} was observed in response to pro- and anti-inflammatory stimuli, including various cytokines and growth factors, LPS, 12-O-tetradecanoylphorbol-13-acetate, or the tryptophan catabolite picolinic acid (PA) (2, 4, 7, 16, 17, 18). However, the contribution of environmental signals to the control of M{phi}-derived chemokines remains to be elucidated.

M{phi} are ubiquitous cells that reside in the vast majority of tissues under normal physiological conditions but that markedly accumulate in areas of inflammation and tumor growth, and evidence exists that M{phi} reactivity is modulated by stimuli that arise from the pathological microenvironment (10, 19). A common denominator of many pathological processes is represented by low O2 tension (hypoxia). Hypoxia occurs in cardiovascular, hematological, and pulmonary disorders, inflammatory processes, and fibrosis (reviewed in Ref.20). Areas of low O2 concentration are present in solid tumors and are known to contribute to tumor growth, metastatization, and resistance to radio and chemotherapy (21). It is now well recognized that hypoxia is an important environmental stimulus capable of modulating the expression of specific genes involved in energy metabolism (glycolytic and mitochondrial enzymes and glucose transporters), erytropoiesis (erytropoietin), angiogenesis (vascular endothelial growth factor (VEGF) and platelet-derived growth factor), vasomotor control (NO synthases), and iron metabolism (transferrin) (20, 22, 23, 24). Induction of gene expression by hypoxia is mediated mainly by the hypoxia-inducible factor-1 (HIF-1), which binds to and transactivates the hypoxia-responsive element (HRE) present in the promoter or enhancer elements of many hypoxia-responsive genes (reviewed in Refs. 20 and 22). HIF-1 is a heterodimeric complex composed of the basic-helix-loop-helix periodic-aryl hydrocarbon receptor-single-minded proteins, HIF-1{alpha} (or the recently identified homologues HIF-2{alpha}/3{alpha}), and HIF-1{beta}, whose expression is tightly controlled by O2 concentrations (20, 22, 23, 25, 26). The {beta} subunit, also known as the aryl hydrocarbon receptor nuclear translocator, is constitutively expressed in unstimulated cells, whereas the {alpha} molecule is the hypoxia-responsive subunit, which is posttranslationally stabilized against ubiquitination and proteosomal degradation and accumulates under low O2 conditions (20, 22). Signals other than hypoxia can activate HIF-1{alpha} and can induce gene expression through HRE transactivation in normal oxygen conditions (20, 22, 26, 27), including iron chelators such as PA (13) and desferrioxamine (DFX) (20, 28).

Hypoxic conditions have been shown to profoundly impact on M{phi} proinflammatory and immunoregulatory responses (29, 30). Genes coding for various inflammatory cytokines, growth, and angiogenic factors were demonstrated to be modulated in M{phi} exposed to low O2 tension both in vitro and in vivo. Specifically, induction of TNF-{alpha}, VEGF, platelet-derived growth factor, fibroblast growth factor {alpha}{beta}, IL-6, and IL-1 production by human and/or mouse M{phi} (29, 30, 31, 32, 33, 34) and inhibition of GM-CSF expression in LPS-treated human M{phi} were reported (34). Furthermore, we have shown that hypoxia can act as a costimulus with IFN-{gamma} or LPS in triggering the transcriptional activation of the inducible isoform of the NO synthase (iNOS) gene in mouse M{phi}, eliciting NO production (13, 15).4

Recently, up-regulation of IL-8 and macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) chemokines was reported in M{phi} exposed to low O2 concentrations (35, 36). Because the extent and impact of hypoxia on chemokine production by M{phi} are important questions for understanding leukocyte infiltration and activation in pathological tissues, we were interested in further elucidating the regulatory effects of hypoxia on M{phi}-derived chemokines. In this study, we characterize a novel property of hypoxia as a potent and selective inhibitor of both constitutive and IFN-{gamma}- or LPS-induced MCP-1 production, we demonstrate that hypoxia inhibitory activity can be exerted on both mouse and human M{phi}, and we establish the molecular mechanisms accounting for this effect.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions

The mouse M{phi} cell line ANA-1 was established by infecting fresh BM-derived cells from C57BL/6 mice with the J2 retrovirus (carrying the v-raf/v-myc oncogenes) and was shown to display the phenotypic and functional features and the morphology of well-differentiated M{phi} (13, 18, 36). ANA-1 M{phi} were cultured in DMEM (Euroclone; Celbio, Milano, Italy) supplemented with 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT; Celbio), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Celbio). The human THP-1 monocytic cell line was purchased from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Euroclone) supplemented as described for DMEM. Peritoneal M{phi} were obtained from C57BL/6 mice injected i.p. with 1 ml of 3% thioglicollate broth (Sigma-Aldrich, Milano, Italy). After 4 days, the peritoneal exudate cells were collected by lavage of the peritoneal cavity with 10 ml of sterile PBS (Euroclone). Cells were washed, resuspended, and plated in RPMI 1640. M{phi} were isolated by adherence to tissue culture dishes, and their purity was ~94% as assessed by morphology on Giemsa-stained cytocentrifuge slide preparations. Viability, determined by the trypan blue dye exclusion test, was >99%. Cells were maintained at 37°C in a humidified incubator containing 20% O2, 5% CO2, and 75% N2. For experimental purposes, cells were cultured in 10- or 15-cm Costar plates (Celbio) at 0.5 x 106 cells/ml and were stimulated for different time points with the indicated factors. Hypoxic conditions (i.e., 1% O2) were achieved by culturing the cells in a modular incubator chamber flushed with a gas mixture containing 1% O2, 5% CO2, and balanced N2 at 37°C in a humidified atmosphere. Reoxygenation was achieved by transferring the plates to a normoxic humidified incubator.

Reagents

Mouse IFN-{gamma} (specific activity >= 107 IU/mg) and LPS (from Escherichia coli serotype 011:B4) were purchased from Life Technologies (Milano, Italy). Recombinant human IFN-{gamma} (specific activity >= 2.0 x 107 U/mg) was purchased from Roche Diagnostics (Mannheim, Germany). PA and DFX were from Sigma-Aldrich. During the course of experiments, several batches of PA were used, and all of them gave consistent and reproducible results. PA was dissolved in PBS, and the pH was adjusted to 7.4. The stock solution was then passed through a 0.2-µm filter, aliquoted, and stored at -20°C. Actinomycin D (ActD; Calbiochem-Novabiochem, La Jolla, CA) was dissolved in ethanol at 1 mg/ml and used at a final concentration of 5 µg/ml. The endotoxin content, as determined by a chromogenic Limulus amebocyte lysate test (QCL-1000; BioWhittaker, Walkersville, MD), was below the detection limit of 6 pg/ml in all of the reagents used.

Northern blot analysis

Total cellular RNA was purified from ANA-1 M{phi} using the TRIzol reagent (Life Technologies) according to the manufacturer’s instructions (with the addition of one extra chloroform extraction to improve the quality of recovered RNA), resuspended in diethyl pyrocarbonate water, and quantified with a spectrophotometer at 260 nm absorbance. A total of 20 µg of RNA from each sample was electrophoresed under denaturing conditions on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation. Filters were hybridized in Hybrisol I hybridization solution (Oncor, Gaithersburg, MD) at 42°C overnight with 2 x 106 cpm/ml of 32P-labeled probe. Probes were labeled by random priming reaction using a commercial kit (Life Technologies) and 5'-[{alpha}32P]dCTP (3000 Ci/mmol; Amersham, Milano, Italy). For MCP-1 and MIP-1{alpha} detection, the mouse JE/cDNA, the human MCP-1 full-coding sequence from the PUC19 vector, and the mouse MIP-1{alpha} full-length cDNA from the pBR322 vector, kindly provided by Dr. A. Sica (Istituto Mario Negri, Milano, Italy), were used. For iNOS detection, the cDNA probe specific for mouse M{phi}-inducible NOS was used (28). The pEMBL-8 vector containing the {beta}-actin cDNA was kindly provided by Prof. C. Garre’ (IBiG, Facolta’di Medicina e Chirurgia, Universita’ di Genova, Italy). Membranes were then washed three times at 42°C for 10 min in 2x SSC/0.1% SDS and twice at 65°C for 15 min in 0.2x SSC/0.1% SDS before being autoradiographed using Kodak XAR-5 film (Eastman Kodak, Rochester, NY) and intensifying screens at -80°C. To obtain comparable band intensities, blots were exposed for different periods of time depending on the probe used for hybridization. When needed, densitometric analysis of the autoradiographs was performed using the VersaDoc Image Analyzer from Bio-Rad (Hercules, CA), and quantitative assessment of the band intensities was conducted. The significance of mRNA expression differences was determined by the Student t test (significant difference, p < 0.01).

RNase protection assay (RPA)

Total RNA extracted from ANA-1 M{phi} with the TRIzol reagent was subjected to RPA analysis using the RiboQuant MultiProbe RNase Protection Assay System from BD PharMingen (Milano, Italy), as described (18). Briefly, a 32P-labeled antisense RNA probe set specifically for different mouse {alpha}{beta}-chemokines (mCK-5) was hybridized in excess to 10 µg of total RNA from each sample in solution, after which free probe and other single-stranded RNA were digested with RNases. The "RNase-protected" 32P-labeled probes, annealed to homologous sequences in the sample RNA, were resolved on denaturing PAGE and visualized by autoradiography (Kodak XAR-5 films).

Detection of cytokine release

Cell-free supernatants were harvested and assayed for mouse and human MCP-1 content using specific ELISA kits from BioSource International (Milano, Italy) and R&D Systems (Milano, Italy) with a sensitivity of 9 and 5 pg/ml, respectively. The ODs of the plates were determined using a Dynatech MR 5000 plate reader set to 450 nm (Dynatech Laboratories, Chantilly, VA).

Nuclear run on

Run on experiments were performed as described (18). Briefly, nuclei were isolated from 10 x 107 cells/sample by cell lysis in 6 ml of lysis buffer (10 mM Tris-Cl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 150 mM sucrose, and 0.4% Nonidet P-40 (Sigma-Aldrich)) for 5 min on ice. Nuclei were collected after centrifugation at 800 rpm for 5 min at 4°C, resuspended in 150 µl of freezing buffer (50 mM Tris-Cl (pH 8.3), 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA), and stored at -80°C until they were used. In vitro RNA elongation was performed by adding 150 µl of 2x transcription buffer (200 mM KCl, 20 mM Tris-Cl (pH 8), 10 mM MgCl2, 200 mM sucrose, 20% glycerol, 1 mM adenosine triphosphate lithium salt, guanosine triphosphate lithium salt, and cytidine triphosphate lithium salt (Boehringer Mannheim, Indianapolis, IN)) and 100 µCi of 800 Ci/mmol [{alpha}32P]uridine triphosphate (NEN, Boston, MA) to 150 µl of nuclei suspension, and the mixture was incubated at 29°C for 30 min. Twenty microliters of 100 mM CaCl2 and 20 U of RNase free DNase I (Promega Italia, Milano, Italy) were added to the mixture, and the incubation was allowed to continue for 10 min at 30°C with gentle mixing every 2 min. The nuclei were lysed with 1 ml of TRIzol and the RNA was isolated according to the manufacturer’s procedure. Equal amounts of labeled elongated transcripts were added in 4 ml of Hybrisol I to Nytran membranes on which 1 µg of linearized MCP-1/JE and of {beta}-actin cDNAs were immobilized using a dot blot apparatus (Bio-Rad). Hybridization was performed at 42°C for 48 h, and filters were then washed as described for Northern analysis and were autoradiographed. The autoradiographs were then scanned using the VersaDoc Image Analyzer.

Real-time PCR

Total RNA from ANA-1 cells was obtained using an RNA Mini Kit (Qiagen, Valencia, CA). One microgram of total RNA was used to perform RT-PCR using an RT-PCR kit (PE Biosystems, Foster City, CA). The conditions used for RT-PCR were as follows: 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C. To measure murine VEGF and murine AU RNA-binding protein/enoyl-coenzyme A hydratase (AUH) expression, real-time PCR was performed using an ABI-Prism 7700 sequence detector (Applied Biosystems, Foster City, CA), as previously described (37). Typically, 5 ng of reverse transcribed cDNA per sample was used to perform real-time PCR in triplicate. Real-time PCR cycles started with 2 min at 50°C, 10 min at 95°C, and then 40 cycles of the following: 15 s at 95°C and 1 min at 60°C. Primers and specific probes were obtained from Applied Biosystems. The following primers and probes were used: VEGF forward, 5'-GGCTGCACCCACGACAG-3'; reverse, 5'-CGCTGGTAGACGTC CATGAA-3' probe, 5'-FAM-GAGAGCAGAAGTCCCATGAAGTGATCAA-TAMRA-3'; AUH forward, 5'-CGCTACAAGGGAGAATAGGAGG-3'; reverse, 5'-GCTCTGAACCACTTCCAGCAC-3'. Detection of 18S rRNA, used as an internal control, was performed using premixed reagents from Applied Biosystems. Detection of VEGF 18S rRNA was performed using TaqMan Universal PCR Master Mix (Applied Biosystems), whereas AUH detection was performed using Sybr Green PCR Master Mix (Applied Biosystems).

Relative quantitation values were expressed as follow: 2({Delta}Ctr - {Delta}Ctt), where C is the value measured in each well, Ct is the mean of the replicate wells run for each sample, {Delta}Ct is the difference between the mean Ct values of the samples in the target wells and those of the endogenous control for the same wells (18S values), and {Delta}Ctr - {Delta}Ctt represents the difference between {Delta}Ct of the reference sample (medium) and {Delta}Ct of the tested sample (treatment). Values are expressed as fold increases relative to the reference sample (medium).

Preparation of nuclear extracts

Nuclear extracts were prepared by modification of a standard protocol, as previously described (13). Briefly, cells were washed twice with cold PBS and pelletted by centrifugation at 1200 rpm for 5 min at 4°C. The cell pellet was then washed once in a hypotonic buffer (10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM Pefabloc, 2 mM DTT, 2 mM sodium vanadate, and 4 µg/ml leupeptin, aprotinin, and pepstatin) (Roche Diagnostics), resuspended in the same buffer, and incubated on ice for 10 min. The cell suspension was subsequently homogenized with 18–20 strokes in a glass Dounce homogenizer. The nucler pellet was obtained after centrifugation at 1000 x g for 10 min at 4°C and was resuspended in a hypertonic buffer (20 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 0.42 M KCl, 20% glycerol, 1 mM Pefabloc, 2 mM DTT, 2 mM sodium vanadate, and 4 µg/ml leupeptin, aprotinin, and pepstatin) to obtain nuclear extracts. The nuclear suspension was rotated at 4°C for 30 min, and nuclear debris was pelletted by centrifugation at 15,000 x g for 30 min at 4°C. The supernatant was used for Western blot analysis.

Western blot analysis

Fifty micrograms of nuclear protein extracted as described above was separated on a 4–20% Tris-glycine gel (Invitrogen, Carlsbad, CA) and electroblotted on an Immobilon-P membrane (Invitrogen). Membranes were blocked for 1 h at room temperature in blocking buffer containing 5% nonfat dry milk (Bio-Rad) in 1x TTBS (20 mM Tris, 150 mM NaCl (pH 8.2), and 0.1% Tween 20) (Sigma-Aldrich). Membranes were then incubated with anti-HIF-1{alpha} mAb (Novus Biologicals, Littleton, CO) diluted 1/500 in dilution buffer (1x TTBS/1% milk) overnight at 4°C. An anti-{beta}-actin mAb (Sigma-Aldrich) diluted 1/10,000 in 1x TTBS/5% milk was used as an internal control for loading. After washing three times in washing buffer (1x TTBS), membranes were incubated for 30 min at room temperature with a peroxidase-conjugated goat anti-mouse Ab (Sigma-Aldrich) diluted 1/40,000 in dilution buffer. Membranes were then washed three times in washing buffer, and chemiluminescence detection was performed using an ECL kit from Amersham Pharmacia Biotech (Piscataway, NJ), according to the manufacturer’s protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypoxia is a selective inhibitor of MCP-1 production by mouse M{phi}

Initial experiments were performed to compare the expression pattern of several {alpha}- and {beta}-chemokines by MultiProbe RPA in the ANA-1 M{phi} cell line cultured under hypoxic (1% O2) vs normoxic (20% O2) conditions for 18 h. Purified RNA was hybridized with a set of 32P-labeled mouse chemokine-specific antisense RNA probes and was analyzed by PAGE. As depicted in Fig. 1, hypoxia caused a strong down-regulation of the constitutive expression of the mRNA for the {beta}-chemokine, MCP-1/JE, with the extent of inhibition ranging from 2.3- to 2.8-fold in three independent determinations. In contrast, hypoxia increased the mRNA for another {beta}-chemokine, MIP-1{alpha}, by ~2.9-fold without affecting the expression of the {beta}-chemokines, MIP-1{beta} and RANTES, or the {alpha}-chemokines, IFN-{gamma}-inducible protein-10 (IP-10) and MIP-2, suggesting a selective inhibitory activity of hypoxia on MCP-1.



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  		FIGURE 1. Identification by RPA analysis of MCP-1 as a hypoxia-inhibited gene in M{phi}. Total RNA was isolated from ANA-1 M{phi} cultured for 18 h under normoxic (20% O2) or hypoxic conditions (1% O2) and was subjected to RPA analysis, as detailed in Materials and Methods. Quantitation of gene expression changes in hypoxic vs normoxic samples was obtained by densitometric analysis of the band intensity. The mRNA levels for each indicated gene are presented as a bar graph and expressed as fold changes of the hypoxic sample relative to the corresponding normoxic sample. Data shown are the mean ± SD of the results obtained in three independent experiments, with SD < 10% of the mean.

 
To confirm these results, MCP-1 mRNA was assessed by Northern blot analysis. As shown in Fig. 2A, ANA-1 cells constitutively expressed MCP-1 mRNA that was down-regulated by exposure to 1% O2 for 18 h. Although slight fluctuations in the constitutive levels were detected, we consistently observed inhibition of MCP-1 mRNA expression under hypoxic conditions in five independent experiments (data not shown). Consistent with the RPA data, MIP-1{alpha} mRNA was significantly enhanced by hypoxia, excluding toxic effects (Fig. 2A). MCP-1 mRNA inhibition by hypoxia was paralleled by decreased protein release into the supernatant (Fig. 2B). ANA-1 cells constitutively secreted MCP-1, which accumulated in the culture medium, ranging from 350 pg to 390 pg/0.5 x 106 cells/ml in three different experiments after 24 h. The amounts of secreted MCP-1 were decreased by ~57% when cells were exposed to 1% O2 for 24 h (Fig. 2B).



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  		FIGURE 2. Inhibition of constitutive MCP-1 mRNA expression and protein secretion by hypoxia in ANA-1 M{phi}. A, ANA-1 cells were exposed to either normoxia (Med) or hypoxia (Hy) for 18 h, and total RNA was assayed for MCP-1 mRNA expression by Northern blot. Blots were sequentially hybridized with probes specific for MCP-1 and MIP-1{alpha} and were exposed for 48 and 12 h, respectively, to obtain comparable band intensities. Ethidium bromide staining of the 28S and 18S rRNA is shown as a control of RNA loading. B, ANA-1 cells were cultured for 24 h before supernatants were harvested and the amounts of secreted MCP-1 were measured by ELISA. Results from one representative experiment of three performed, expressed as picograms per 0.5 x 106 cells/ml, are shown.

 
We conclude that hypoxia is a selective inhibitor of MCP-1 production by M{phi}.

Hypoxia inhibits MCP-1 induction by IFN-{gamma}

MCP-1 is an IFN-{gamma}-inducible chemokine (18, 38), and we were interested in determining whether hypoxia could inhibit IFN-{gamma} stimulatory activity.

ANA-1 M{phi} were incubated for 18 h under normoxic or hypoxic conditions in the presence or absence of optimal concentrations of IFN-{gamma} (18), and Northern blot analysis was performed to evaluate MCP-1 expression. IFN-{gamma} increased MCP-1 mRNA by ~5-fold, and hypoxia abrogated such response (Fig. 3A). A similar pattern of results was consistently observed in five independent experiments (data not shown). As a control, we show that hypoxia synergized with IFN-{gamma} in inducing iNOS mRNA expression (Fig. 3A), as reported previously (13), excluding the possibility that hypoxia induced a deactivated cellular state. Secretion of MCP-1 protein followed the same pattern of mRNA expression (Fig. 3B).



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  		FIGURE 3. Hypoxia inhibits IFN-{gamma}-dependent up-regulation of MCP-1. A, Total RNA was isolated from ANA-1 M{phi} treated for 18 h with 100 IU/ml murine IFN-{gamma} under normoxic or hypoxic conditions and was analyzed by Northern blot. The blot was sequentially hybridized with the MCP-1/JE and the iNOS cDNAs and was exposed for 36 and 24 h, respectively, to obtain comparable band intensities. Ethidium bromide staining of the 28S and 18S rRNA was assessed to ensure that comparable amounts of RNA were loaded in each lane. B, Supernatants from ANA-1 cells cultured for 24 h with the indicated stimuli were assayed for secreted MCP-1 by ELISA. Results from one representative experiment, expressed as picograms per 0.5 x 106 cells/ml, are shown. C, Total RNA was extracted from ANA-1 cells stimulated for 18 h with medium or murine IFN-{gamma} (100 IU/ml) under normoxic or hypoxic conditions and was subjected to RPA analysis, as described in Materials and Methods. The GAPDH probe was used as a control of RNA loading. Data shown are from one representative experiment of three performed.

 
RPA analysis was then performed to establish whether hypoxia inhibitory effects could be exerted on other IFN-{gamma}-inducible chemokines or were selective for MCP-1. As shown in Fig. 3C, in addition to MCP-1, IFN-{gamma} up-regulated the expression of RANTES and IP-10, but this response was not affected by hypoxia.

Parallel experiments were conducted with the human monocytic cell line THP-1 to evaluate the response of human cells. THP-1 cells did not express constitutive levels of MCP-1 mRNA but responded to IFN-{gamma} stimulation with MCP-1 induction (Fig. 4A). Hypoxia effectively inhibited IFN-{gamma}-triggered MCP-1 mRNA expression (Fig. 4A) and, accordingly, induction of MCP-1 protein secretion (Fig. 4B). Comparable results were observed in three independent experiments (data not shown).



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  		FIGURE 4. Inhibitory effects of hypoxia on MCP-1 expression in human monocytic cells and Thio-M{phi}. THP-1 and Thio-M{phi} were exposed to normoxia or hypoxia in the presence or absence of 500 U/ml human and 100 IU/ml murine IFN-{gamma}, respectively. A and C, Representative MCP-1 Northern blots performed on total RNA isolated from THP-1 cells (A) and Thio-M{phi} (C) after 18 h of culture are shown. B, Supernatants from THP-1 cells cultured for 24 h with the indicated stimuli were assayed for secreted MCP-1 by ELISA. Results from one representative experiment, expressed as picograms per 0.5 x 106 cells/ml, are shown. D, Densitometric analysis of the blot shown in C. Relative MCP-1 mRNA levels normalized to the respective amounts of ethidium bromide-stained 18S rRNA are presented as a bar graph and are expressed in arbitrary units.

 
Thioglycollate-elicited peritoneal M{phi} (Thio-M{phi}) from C57BL/6 mice were tested to investigate the effects of hypoxia on fresh M{phi}. MCP-1 mRNA was constitutively expressed in Thio-M{phi}, and stimulation with IFN-{gamma} for 18 h resulted in increased mRNA accumulation, although to a lesser extent than in ANA-1 cells (Fig. 4, C and D). A similar pattern of results was consistently observed in two independent experiments, with the induction ranging from 2.5- to 3-fold over control. Hypoxia suppressed MCP-1 basal expression and decreased by 72% mRNA induction by IFN-{gamma} (Fig. 4, C and D).

To determine the kinetics of MCP-1 inhibition by hypoxia, ANA-1 M{phi} were cultured under normoxic or hypoxic conditions in the presence or absence of IFN-{gamma} and were tested at different times. As shown in Fig. 5, MCP-1 mRNA up-regulation by IFN-{gamma} occurred within 3 h of stimulation and reached plateau levels at 24 h. Strong inhibition of IFN-{gamma}-induced MCP-1 mRNA by hypoxia was observed as early as 6 h after the onset of the culture, and maximal suppression occurred after 24 h (Fig. 5). Comparable results were observed in three independent experiments (data not shown).



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  		FIGURE 5. Kinetics of MCP-1 mRNA inhibition by hypoxia in IFN-{gamma}-treated ANA-1 M{phi}. ANA-1 M{phi} were cultured for different time points under normoxic or hypoxic conditions in the presence or absence of 100 IU/ml murine IFN-{gamma}, and MCP-1 mRNA expression was then assessed. A, Representative Northern blot of IFN-{gamma}-treated cells is shown. B, Densitometric analysis of the blot shown in A. Relative MCP-1 mRNA levels normalized to the respective amounts of ethidium bromide-stained 18S rRNA are expressed in arbitrary units.

 
These results demonstrate that hypoxia inhibits MCP-1 induction by IFN-{gamma} both in mouse M{phi} and in human monocytic cells.

Hypoxia inhibitory activity is exerted on MCP-1 induction by LPS and reverted by reoxygenation

To establish whether hypoxia antagonized MCP-1 up-regulation by other M{phi} activators, in addition to that elicited by IFN-{gamma}, we studied the response to LPS (Fig. 6). An eightfold increase of MCP-1 mRNA expression over basal levels was observed after 3 h of treatment with LPS, reaching maximal levels after 12 h. Induction by LPS was susceptible to a time-dependent inhibition by hypoxia, although to a lesser extent than the response to IFN-{gamma} (Fig. 6A). Accordingly, MCP-1 release by LPS-treated cells was decreased by ~68% after 24 h of hypoxia (Fig. 6B).



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  		FIGURE 6. Inhibition of LPS-induced MCP-1 production by hypoxia. ANA-1 M{phi} were cultured for different time points under normoxic or hypoxic conditions in the presence or absence of 10 ng/ml LPS before MCP-1 mRNA expression and protein secretion were analyzed. A, Densitometric analysis of MCP-1 mRNA levels assessed by Northern blot. Relative amounts of MCP-1 mRNA normalized to the respective amounts of ethidium bromide-stained 18S rRNA are presented as a bar graph and are expressed in arbitrary units. Data shown are the mean ± SD of the results obtained in two different hybridization experiments, with SD < 10% of the mean. B, Supernatants from ANA-1 cells cultured for 24 h with the indicated stimuli were assayed for secreted MCP-1 by ELISA. Results from one representative experiment of two performed, expressed as picograms per 0.5 x 106 cells/ml, are shown.

 
Previous studies have shown that the exposure of hypoxic M{phi} to a reoxygenation period enhanced the effects of hypoxia on cytokine gene expression (32, 39). To evaluate the effects of cell reoxygenation on MCP-1 expression, ANA-1 M{phi} were treated with medium alone or supplemented with IFN-{gamma} or LPS, and then they were exposed to 1% O2 for a period of 12 h followed by re-exposure to normal oxygen tension for additional 12 h (reoxygenation), or they were cultured under normoxic or hypoxic conditions for the entire length of the experiment. As shown in Fig. 7, reoxygenation reverted the inhibition by hypoxia both in the presence or absence of IFN-{gamma} or LPS, restoring MCP-1 mRNA expression to levels comparable with those detected in normoxic cells.



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  		FIGURE 7. Reoxygenation restores MCP-1 mRNA expression in hypoxic M{phi}. Total RNA from medium-, IFN-{gamma}-, or LPS-treated ANA-1 M{phi}, cultured for 12 h in the presence of 1% O2 (Hy, +) followed by additional 12 h in the presence of normal oxygen levels (Reox, +) or exposed to hypoxic (+) or normoxic (-) conditions for 24 h, was analyzed for MCP-1 expression. A, Representative Northern blot is shown. Ethidium bromide staining of the 28S and 18S rRNA was assessed as a control of RNA loading. B, Densitometric analysis of MCP-1 mRNA expression. MCP-1 mRNA levels in hypoxic (Hy) or reoxygenated (Reox) M{phi} are expressed as a percentage of the amounts detected in cells cultured under normoxic conditions (Normo, 100%). Data shown are the mean ± SD of the results obtained in three separate hybridization experiments, with SD < 10% of the mean.

 
Taken together, these findings indicate that the inhibitory activity of hypoxia on MCP-1 is stimulus independent and reversible.

Hypoxia decreases MCP-1 transcription and mRNA stability

Nuclear run-on assays were then performed to determine whether MCP-1 down-regulation by hypoxia involved changes in gene transcription. Nuclei were isolated from control ANA-1 (Fig. 8, Med) and cells stimulated for 4 and 8 h with hypoxia, IFN-{gamma}, or hypoxia plus IFN-{gamma}. Fig. 8A shows a representative experiment of three performed, which gave comparable results. MCP-1 gene was transcriptionally active in medium-treated M{phi} and susceptible to a 1.5-fold augmentation in response to stimulation with IFN-{gamma}. Incubation of Med- and IFN-{gamma}-treated cells under hypoxia for at least 8 h reduced MCP-1 transcription by ~40 and 60%, respectively, compared with cells cultured under normoxic conditions (Fig. 8A), indicating that gene transcription is one level of control of MCP-1 mRNA expression by hypoxia.



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  		FIGURE 8. Inhibitory activity of hypoxia on MCP-1 gene transcription and mRNA half-life. ANA-1 M{phi} were treated with medium alone or containing murine IFN-{gamma} (100 IU/ml) under normoxic or hypoxic conditions. A, MCP-1 gene transcription was determined by nuclear run-on analysis on nuclei isolated after 4 and 8 h of culture, as detailed in Materials and Methods. Upper panel, Representative run on assay is shown. Lower panel, Quantitative analysis of MCP-1 gene transcription. Relative transcription activities normalized to {beta}-actin are presented as a bar graph and are expressed in arbitrary units. Data shown are the mean ± SD of the results obtained in three independent experiments, with SD < 10% of the mean. B, ActD (5 µg/ml) was added after 12 h of culture. Total RNA was harvested at 1, 2, 4, and 6 h and was examined for MCP-1 expression by Northern blot. Data are presented as the relative amounts of MCP-1 mRNA remaining after addition of ActD and after normalization of the bands to the respective amounts of ethidium bromide-stained 18S rRNA. C, Total RNA was harvested after 6 and 18 h, reverse transcribed, and tested for AUH and VEGF mRNA expression by real-time PCR, as detailed in Materials and Methods. 18S rRNA was tested in parallel as an internal control for input RNA. Results are expressed as fold increase relative to mRNA levels under normoxic (Med) conditions (equal to 1) and are the average of three independent experiments

 
The effects of hypoxia on mRNA stability were next tested by assessing MCP-1 mRNA t1/2. ANA-1 M{phi} were incubated for 12 h with medium alone or supplemented with IFN-{gamma} under normoxic or hypoxic conditions, and the rate of MCP-1 transcript decay was determined by evaluating mRNA expression immediately or at various time points after addition of the transcriptional inhibitor ActD. As depicted in Fig. 8B, MCP-1 mRNA was rapidly degraded in control cells, with calculated t1/2 of 40 min. IFN-{gamma}-treated M{phi} displayed a greater MCP-1 mRNA stability relative to medium-treated cells, with the transcript t1/2 increased to 70 min. In contrast, exposure to hypoxia reduced MCP-1 mRNA stability, with a 50% decrease in transcript levels observed after ~30 min in medium- and 40 min in IFN-{gamma}-treated cells (Fig. 8B). Consistent and reproducible results were obtained in three independent experiments (data not shown), indicating that hypoxia accelerated MCP-1 mRNA decay.

Messenger RNA degradation rate is positively or negatively regulated by trans-acting mRNA-binding proteins in response to various stimuli and in a cell type-specific manner (17, 29, 40). Having established that hypoxia destabilizes MCP-1 mRNA, it was of interest to determine whether this effect was associated with the increased expression of any mRNA-destabilizing factor. We found by real-time PCR that hypoxia induced in ANA-1 M{phi} a 2.1-fold up-regulation of the levels of the mRNA coding for AUH, an RNA-binding protein implicated in the regulation of mRNA decay (41, 42), relative to normoxic conditions (Fig. 8C). Comparable extent of increase was detectable after 6 and 18 h of exposure to hypoxia (Fig. 8C) and was consistently observed in three independent determinations (data not shown). As a control, we show that hypoxia significantly up-regulated VEGF mRNA expression (Fig. 8C), as reported previously in human monocytes (33), causing 4.1- and 2.8-fold increases of constitutive levels at 6 and 18 h, respectively.

We conclude that the inhibitory action of hypoxia on MCP-1 expression is controlled by a combination of transcriptional and posttranscriptional mechanisms.

PA and DFX differentially regulate MCP-1 production by M{phi}

Several stimuli, including PA and DFX, share with hypoxia the ability to induce gene expression under normal O2 tension (13, 20, 26, 27, 28, 43). To determine whether hypoxia inhibitory effects on MCP-1 could be mimicked by hypoxia-like stimuli under normoxic conditions, MCP-1 mRNA levels and protein secretion were assessed in ANA-1 M{phi} (Fig. 9, A and B). We found that PA and DFX exerted opposite effects on MCP-1 (Fig. 9, A and B), despite their common ability to induce iNOS and MIP-1{alpha} (13, 18, 28) (Fig. 9A). DFX reduced both constitutive and IFN-{gamma}-induced MCP-1 mRNA expression (Fig. 9A) and protein release (Fig. 9B), mimicking the effects of hypoxia. Conversely, PA not only did not inhibit, but it even up-regulated MCP-1 mRNA levels in medium-treated cells, slightly enhancing the induction by IFN-{gamma} (Fig. 9A). Messenger RNA up-regulation was paralleled by increased MCP-1 secretion into the supernatant (Fig. 9B). A similar pattern of results was obtained in the human THP-1 cell line (data not shown). Gene induction by hypoxia is associated with increased expression of HIF-1{alpha} (20, 22, 23, 26). To determine whether PA and DFX differential effects on MCP-1 correlated with a different regulation of HIF-1{alpha}, HIF-1{alpha} protein levels were assessed in parallel experiments (Fig. 9C). Western blot analysis revealed barely detectable HIF-1{alpha} basal expression, which was unaffected by treatment with IFN-{gamma} for 6 h (Fig. 9C). Stimulation with PA or DFX up-regulated HIF-1{alpha} protein, although the effects of PA were less pronounced compared with those of DFX (Fig. 9C). Interestingly, PA- or DFX-treated ANA-1 M{phi} expressed significantly higher levels of HIF-1{alpha} in the presence of IFN-{gamma} (Fig. 9C). A similar pattern of results was obtained after 12 h of culture (data not shown), demonstrating that HIF-1{alpha} expression can be stimulated under normoxic conditions by PA or DFX in M{phi} and that IFN-{gamma} can act synergistically with either stimulus in inducing this factor.



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  		FIGURE 9. Differential regulation of MCP-1 by PA and DFX. ANA-1 M{phi} were stimulated with 4 mM PA or 400 µM DFX, in the presence or absence of 100 IU/ml murine IFN-{gamma}. A, Total RNA was isolated after 18 h and was assayed for MCP-1 mRNA expression by Northern blot. The blot was sequentially hybridized with MCP-1/JE, MIP-1{alpha}, and iNOS cDNAs. To obtain comparable band intensities, a 24-h exposure was required after hybridization with MCP-1/JE and iNOS, whereas 8 h of exposure were sufficient for blots hybridized with MIP-1{alpha}. B, Supernatants were assayed after 24 h of treatment for secreted MCP-1 by ELISA. Results from one representative experiment are presented as a bar graph and are expressed as picograms per 0.5 x 106 cells/ml. C, Nuclear extracts (50 µg) isolated after 6 h of treatment were resolved by SDS-PAGE, and HIF-1{alpha} protein levels were assessed by Western blot analysis, as described in Materials and Methods. {beta}-Actin expression was evaluated in parallel to control for protein loading. Results shown are from one representative experiment of two performed.

 
These findings indicate the existence of a differential regulation of MCP-1 by hypoxia-like stimuli, which seems dissociated from their activity on HIF-1{alpha} expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The network of physiopathological stimuli regulating MCP-1 expression in M{phi} is not fully understood and it extends beyond the classical microbial or immune system-derived signals. In this study, we demonstrate that hypoxia is a potent and selective inhibitor of MCP-1 production by both resting and activated M{phi}. Moreover, we show that inhibition of MCP-1 expression by hypoxia is the result of decreased gene transcription and reduced mRNA stability.

The influence of altered O2 concentrations on MCP-1 expression has been previously demonstrated in other cell types, both in vitro and in vivo. Increased MCP-1 mRNA and protein expression was reported in human dermal fibroblasts exposed to hypoxia (44), and human melanoma cell lines were shown to up-regulate MCP-1 mRNA and protein under anoxia/reoxygenation (45). Moreover, stimulatory effects of hypoxia on MCP-1 have been observed in ischemic rat neurons and myocardial cells (46, 47). Conversely, we consistently observed down-regulation of MCP-1 mRNA expression and decreased protein release by hypoxia both in the mouse M{phi} cell line ANA-1 and in THP-1 human monocytic cells, demonstrating that hypoxia is active on different types of mononuclear phagocytes and across species in inhibiting MCP-1 expression. Furthermore, hypoxia inhibitory effects were exerted on primary thioglycollate-induced peritoneal M{phi}, supporting the general validity of the observation. Inhibition of MCP-1 expression has been reported previously in TNF-{alpha}-stimulated human ovarian carcinoma cell lines cultured under hypoxia/anoxia (48), showing that cells other than M{phi} are susceptible to the inhibitory effects of low O2 tension on MCP-1. Therefore, it is conceivable that expression of MCP-1 is differentially regulated by hypoxia depending on the lineage of the target cell, similarly to what has been reported for other hypoxia-regulated genes (34, 49). Interestingly, cell-type-specific modulation of MCP-1 expression has also been observed in response to other stimuli (16).

M{phi} produce several chemokines in addition to MCP-1 (2, 4, 7, 17, 18), and MCP-1 expression can be induced in M{phi} by various proinflammatory stimuli, the prototypes of which are IFN-{gamma} and LPS, as previously shown (4, 7, 16, 18, 38) and confirmed here. Hence, we considered the potential selectivity and stimulus specificity of the response to hypoxia. Interestingly, we found that hypoxia inhibitory effects were selective for MCP-1, because the expression of other {beta}- and {alpha}-chemokines, such as MIP-1{beta}, RANTES, IP-10, and MIP-2, was not affected, and MIP-1{alpha} was induced. Importantly, moreover, hypoxia not only down-regulated MCP-1 constitutive expression, but it also counteracted MCP-1 induction by IFN-{gamma}, without affecting IFN-{gamma}-dependent up-regulation of RANTES and IP-10. MCP-1 stimulation by LPS was similarly susceptible to inhibition by hypoxia, although to a lesser extent than was the response to IFN-{gamma}. These findings suggest that hypoxia can selectively modulate chemokine expression in M{phi} and exert a targeted inhibition of MCP-1 production induced by different M{phi}-activating stimuli. Our recently reported observation that hypoxia stimulatory activity on MIP-1{alpha} in M{phi} is inhibited by IFN-{gamma} (36) further emphasizes the tight regulatory network connecting hypoxia, IFN-{gamma}, and chemokine expression in these cells. We have previously shown that hypoxia acts synergistically with IFN-{gamma} or LPS in triggering iNOS gene expression and enzymatic activity in murine M{phi} (13, 15).4 Thus, it appears that hypoxia can selectively synergize with or antagonize the stimulatory effects of proinflammatory mediators on M{phi} gene expression. The concerted action of hypoxia with immune-derived or microbial factors is likely to play an important role in coordinating M{phi} activity in pathological states, in that these stimuli are concomitantly present at sites of inflammation and/or tumor growth (10, 20, 21).

MCP-1 expression levels were dependent not only on the O2 concentration, but also on the extent and duration of the hypoxic exposure. In fact, we observed a time-dependent inhibition of MCP-1 induction by IFN-{gamma} or LPS in ANA-1 M{phi}, which occurred already within 6 h of exposure to hypoxia and was maximal after 24 h. Furthermore, cell reoxygenation reverted hypoxia inhibitory effects within 24 h, restoring MCP-1 mRNA expression to levels comparable with those detected in normoxic cells. Our results suggest that the pattern of MCP-1 expression in M{phi} in vivo may vary dynamically with the degree of local oxygenation, which is quite heterogeneous and rapidly fluctuating within pathological lesions (50), thus resulting in shifting gradients of secreted MCP-1.

Transcriptional modulation is the primary mechanism of regulation of MCP-1 expression, which is dependent on multiple transcription factors and exhibits both cell type- and stimulus-specific patterns (16, 38, 51, 52). Here, we show that MCP-1 gene was transcriptionally active in the ANA-1 M{phi} cell line and that hypoxia decreased its transcriptional activity. Moreover, the exposure of IFN-{gamma}-treated cells to hypoxia resulted in a strong reduction of IFN-{gamma}-dependent MCP-1 transcriptional increase. These findings suggest that inhibition of transcription represents at least one of the mechanisms by which hypoxia down-regulates MCP-1 expression in M{phi}, thus raising the issue of the regulatory pathway(s) involved. It is reasonable to suggest that one or more hypoxia-inducible transcription repressor(s) might target specific elements in the MCP-1 promoter, thereby decreasing gene transcription, similarly to what has been recently reported for other genes (49, 53). Studies are currently underway to determine whether any of the regulatory elements previously identified in the MCP-1 5'-regulatory region (38, 51, 52) or novel cis-acting sequences are the target of hypoxia inhibitory effects. Interestingly, we found by computer search of the published MCP-1 promoter sequence (GenBank accession number U12470) a region of homology with the HRE (hereafter referred to as MCP-HRE) present in the promoter of many hypoxia-inducible genes (20, 22, 23), comprizing both an HIF-1 binding site (sequence GGCGTGGT; -1639/-1632) and a downstream HIF-1 ancillary sequence (sequence CACGC; -1626/-1622) required for the interaction with additional transcription factors (54). At present, the role of MCP-HRE in the regulation of MCP-1 expression is not clear, because MCP-1 transcriptional induction by hypoxia in human melanoma cell lines and dermal fibroblasts was shown to be dependent on the activation of NF-{kappa}B and Sp-1 binding motifs (44, 45). Experiments are underway in our laboratory to determine whether MCP-1-HRE is active in the control of MCP-1 transcription by hypoxia in M{phi}.

Signals other than hypoxia can activate the HIF-1/HRE pathway under normoxic conditions and can mimic hypoxia in the induction of gene expression (20, 22, 26, 27). We observed increased HIF-1{alpha} protein levels in ANA-1 M{phi} after stimulation with the hypoxia-like stimuli PA and DFX. This effect can probably be ascribed to the iron-chelating property of these agents, because chelation of iron was shown to affect an important biochemical step in the HIF-1{alpha} ubiquitin-proteosomal degradation pathway (26). These findings are consistent with our early reports showing that PA or DFX can induce HRE-binding activity and activate the iNOS-HRE in mouse macrophages (13, 28). Induction of HIF-1{alpha} did not correlate with the regulation of MCP-1 expression, which was inhibited by DFX and increased by PA. These results demonstrate for the first time that PA or DFX can differentially control the expression of selected genes, suggesting that unique molecular events can be triggered by either stimulus in addition to HIF-1{alpha} expression. Recent evidence has been provided that a variety of growth factors and cytokines can induce HIF-1{alpha} accumulation in certain cell types (26, 27). Interestingly, we identified a novel synergism between IFN-{gamma} and PA or DFX in the induction of HIF-1{alpha}, which probably in part accounts for the ability of the stimuli to cooperate in the activation of HRE-dependent iNOS transcription (13, 28), thus providing the first evidence that IFN-{gamma} can affect the expression of this NF. Whether IFN-{gamma} reduces HIF-1{alpha} degradation or increases its transcriptional activation or translation will be addressed experimentally in future studies. The synergism between PA and IFN-{gamma} may be potentially relevant for the expression of various HIF-1-inducible genes under inflammatory conditions in which elevated levels of tryptophan metabolites have been detected (9, 13, 18, 55). In contrast, the cooperation between DFX and IFN-{gamma} may have practical clinical implications, because DFX is used as a therapeutic agent for the treatment of several pathological states in which IFN-{gamma} is produced (18, 28).

Posttranscriptional regulation of mRNA stability is another level of control of gene expression by hypoxia, as shown in the case of a number of hypoxia-regulated genes including VEGF, tyrosine hydroxylase, glucose transporter-1, erytropoietin, and eNOS (29, 56, 57, 58). We found that MCP-1 inhibition by hypoxia was also associated with destabilization of the mRNA, which decayed at a faster rate in hypoxic than in normoxic M{phi}. These results are consistent with the finding that the MCP-1 mRNA contains in its 3' untranslated region an AU-rich destabilizing element (ARE) (59) present in the 3' untranslated region of many labile mammalian transcripts coding for cytokines, inflammatory mediators, and oncoproteins and serving as a signal for rapid mRNA degradation (17, 40). The regulation of mRNA turnover by AREs involves their association with any of a growing number of stabilizing or destabilizing trans-acting mRNA-binding proteins with AU specificity (17, 40, 60). Although a few ARE-binding factors mediating mRNA stabilization by hypoxia have been identified (29, 56, 57), no information is currently available on hypoxia-inducible destabilizing proteins. We demonstrate that hypoxia increases the expression of the mRNA for AUH, an AU-binding factor homologous to a 32-kDa protein suggested to be implicated in ARE-directed mRNA decay (41, 42, 60), providing the first evidence that AUH is a hypoxia-inducible gene. The possibility that AUH may contribute to MCP-1 mRNA degradation in M{phi} exposed to low O2 tension is intriguing and under investigation.

In conclusion, in this study we have characterized a novel property of hypoxia as a selective inhibitor of MCP-1 production by M{phi} and we have demonstrated the existence of a cross-talk among environmental signals (hypoxia), stimuli derived from the immune system (IFN-{gamma}), and microbial factors (LPS) in the regulation of M{phi}-derived MCP-1. Given the central role of MCP-1 in the recruitment of various leukocyte populations, the modulation of its expression is a critical set point for the control of the kinetics and composition of the cellular infiltrate under various inflammatory conditions and at tumor sites (4, 5, 6, 16, 61). Low O2 levels have been described in many pathological conditions characterized by M{phi} infiltration and inflammatory responses (10, 20, 21, 29, 62). Thus, MCP-1 inhibition by hypoxia in M{phi} is likely to be of pathophysiologic relevance and to represent a negative regulatory mechanism to control M{phi}-mediated leukocyte recruitment in pathological tissues. MCP-1 inhibition may be particularly important at sites of inflammation where prolonged or excessive MCP-1 secretion represents a detrimental factor for the control and extinction of an inflammatory response, which could favor the onset of chronic inflammatory disorders (3, 4, 47, 63). In contrast, inhibition of MCP-1 production at sites of tumor growth may potentially contribute to tumor progression. In fact, MCP-1 was reported to differently influence the rate of tumor growth depending on its expression levels, with high levels leading to tumor rejection (4, 61, 64) and low levels supporting tumor growth (65). These and previous findings showing impaired M{phi} migratory ability to MCP-1 under conditions of low O2 tension (66) indicate that hypoxia plays an immunosuppressive role on MCP-1 chemokine/receptor network in M{phi}, which may in part explain the functional impairment of tumor-associated macrophages demonstrated both in tumor-bearing mice and in cancer patients (10, 19, 67). It is important to emphasize, however, that hypoxia can exert a tight regulatory control on M{phi} production of several proinflammatory cytokines, modulating the response to immune-derived and microbial stimuli (13, 15, 29, 30, 32, 34, 35, 36).4 Thus, the activation state of hypoxic M{phi} will be ultimately determined by the balance between hypoxia inhibitory and stimulatory activities, which will establish the temporal and quantitative relationship of the various cytokines in the tissue microenvironment. Ongoing microarray experiments intended to fully characterize the profile of gene expression in hypoxic M{phi} will hopefully lead to a better understanding of the role of hypoxia in shaping the functional profile of these cells.


   Acknowledgments
 
We thank Dr. Howard Young for RPA analysis and Chantal Dabizzi for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the Italian Association for Cancer Research, the San Paolo Company, the Fondazione Italiana per la Lotta al Neuroblastoma, and the Associazione Italiana per la Glicogenosi and has been funded in part with federal funds from the National Cancer Institute (National Institutes of Health) under Contract NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. government. 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: MCP-1, monocyte chemoattractant protein-1; M{phi}, macrophage; PA, picolinic acid; VEGF, vascular endothelial growth factor; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-responsive element; DFX, desferrioxamine; iNOS, inducible NO synthase; MIP, macrophage inflammatory protein; ActD, actinomycin D; RPA, RNase protection assay; AUH, AU RNA-binding protein/enoyl-coenzyme A hydratase; IP-10, IFN-{gamma}-inducible protein-10; Thio-M{phi}, thioglycollate-elicited peritoneal macrophage; ARE, AU-rich destabilizing element. Back

4 A. Rapisarda, L. S. Taylor, A. Brooks, L.Varesio, and G. Melillo. Synergistic activation of hypoxia inducible factor 1 (HIF-1) mediates induction of nitric oxide synthase expression by lipopolysaccharide and hypoxia in macrophages. Submitted for publication. Back

Received for publication June 9, 2003. Accepted for publication November 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ben-Baruk, A., D. Michiel, J. Oppenheim. 1995. Signals and receptor involved in the recruitment of inflammatory cells. J. Biol. Chem. 270:11703.[Free Full Text]
  2. Baggiolini, M., B. Dewald, B. Moser. 1994. Interleukin 8 and related chemotactic cytokines: CXC and CC chemokines. Adv. Immunol. 55:97.[Medline]
  3. Murphy, P.. 1996. Chemokine receptors: structure, functions and role in microbial pathogenesis. Cytokine Growth Factor Rev. 7:47.[Medline]
  4. Matsushima, K., E. T. Baldwin, N. Mukaida. 1992. Interleukin-8 and MCAF: novel leukocyte recruitment and activating cytokines. Chem. Immunol. 51:236.[Medline]
  5. Fuentes, N. E., S. K. Durham, M. R. Swerdel, A. C. Lewin, D. S. Barton, J. R. Megill, R. Bravo, S. A. Lira. 1995. Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1. J. Immunol. 155:5769.[Abstract]
  6. Carr, M. W., S. J. Roth, E. Luther, S. S. Rose, T. A. Springer. 1994. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc. Natl. Acad. Sci. USA 91:3652.[Abstract/Free Full Text]
  7. 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.[Abstract/Free Full Text]
  8. Adams, D. O., T. A. Hamilton. 1992. Macrophages as destructive cells in host defense. J. I. Gallin, and I. M. Goldstein, and R. Snyderman, eds. Inflammation: Basic Principles and Clinical Correlates 637. Raven, New York.
  9. 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. Blackwell Scientific Publications, Cambridge, MA.
  10. Sica, A., A. Saccani, A. Mantovani. 2002. Tumor-associated macrophages: a molecular perspective. Int. Immunopharmacol. 2:1045.[Medline]
  11. 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.[Abstract]
  12. Hamilton, T. A., Y. Ohmori, J. M. Tebo, R. Kishore. 1999. Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines. Pathobiology 67:241.[Medline]
  13. Melillo, G., T. Musso, A. Sica, L. S. Taylor, G. Cox, L. Varesio. 1995. A hypoxia responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med. 182:1683.[Abstract/Free Full Text]
  14. Shi, M. M., J. J. Godleski, J. D. Paulauskis. 1996. Regulation of macrophage inflammatory protein-1{alpha} mRNA by oxidative stress. J. Biol. Chem. 271:5878.[Abstract/Free Full Text]
  15. Melillo, G., L. S. Taylor, A. Brooks, G. Cox, L. Varesio. 1996. Regulation of inducible nitric oxide synthase expression in IFN-{gamma}-treated murine macrophages cultured under hypoxic conditions. J. Immunol. 157:2638.[Abstract]
  16. Colotta, F., A. Borrè, J. M. Wang, M. Tattanelli, F. Maddalena, N. Polentarutti, G. Peri, A. Mantovani. 1992. Expression of a monocyte chemotactic cytokine by human mononuclear phagocytes. J. Immunol. 148:765.
  17. Bosco, M. C., G. Gusella, I. Espinoza-Delgado, D. Longo, L. Varesio. 1994. Interferon-{gamma} upregulates interleukin-8 gene expression in human monocytic cells by a posttranscriptional mechanism. Blood 83:537.[Abstract/Free Full Text]
  18. 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 -1{beta} in macrophages. J. Immunol. 164:3283.[Abstract/Free Full Text]
  19. Elgert, K. D., D. G. Alleva, D. W. Mullins. 1998. Tumor-induced immune dysfunction: the macrophage connection. J. Leukocyte Biol. 64:275.[Abstract]
  20. Semenza, G. L.. 2001. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7:345.[Medline]
  21. Harris, A. L.. 2002. Hypoxia: a key regulatory factor in tumour growth. Nat. Rev. Cancer 2:38.[Medline]
  22. Wenger, R. H.. 2002. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 16:1151.[Abstract/Free Full Text]
  23. Ratcliffe, P. J., J. O’Rourke, P. H. Maxwell, C. W. Pugh. 1998. Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J. Exp. Biol. 201:1153.[Abstract]
  24. Ebert, B. L., J. M. Gleadle, J. F. O’Rourke, S. M. Bartlett, J. Poulton, P. J. Ratcliffe. 1996. Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin. Biochem. J. 313:809.
  25. Talks, K. L., H. Turley, K. C. Gatter, P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe. 2000. The expression and distribution of the hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} in normal human tissues, cancers, and tumor-associated macrophages. Am. J. Pathol. 157:411.[Abstract/Free Full Text]
  26. Huang, L. E., H. F. Bunn. 2003. Hypoxia-inducible factor and its biomedical relevance. J. Biol. Chem. 278:19575.[Free Full Text]
  27. Richard, D. E., E. Berra, J. Pouysségur. 2000. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1{alpha} in vascular smooth muscle cells. J. Biol. Chem. 275:26765.[Abstract/Free Full Text]
  28. Melillo, G., L. S. Taylor, A. Brooks, T. Musso, G. Cox, L. Varesio. 1996. Functional requirement of the hypoxia responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J. Biol. Chem. 272:12236.
  29. 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.[Abstract]
  30. Cramer, T., Y. Yamanishi, B. E. Clausen, I. Förster, R. Pawlinski, N. Mackman, V. H. Haase, R. Jaenisch, M. Corr, V. Nizet, et al 2003. HIF-1{alpha} is essential for myeloid cell-mediated inflammation. Cell 112:645.[Medline]
  31. Kuwabara, K., S. Ogawa, M. Matsumoto, S. Koga, M. Clauss, D. J. Pinsky, P. Lyn, J. Leavy, L. Witte, J. Joseph-Silverstein, et al 1995. Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. Proc. Natl. Acad. Sci. USA 92:4606.[Abstract/Free Full Text]
  32. Koga, S., S. Ogawa, K. Kuwabara, J. Brett, J. Leavy, J. Ryan, Y. Koga, J. Plocinski, W. Benjamin, D. K. Burns. 1992. Synthesis and release of interleukin-1 by reoxygenated human mononuclear phagocytes. J. Clin. Invest. 90:1007.
  33. 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.[Abstract/Free Full Text]
  34. Guida, E., A. Stewart. 1998. Influence of hypoxia and glucose deprivation on tumor necrosis factor-{alpha} and granulocyte-macrophage colony-stimulating factor expression in human cultured monocytes. Cell. Physiol. Biochem. 8:75.[Medline]
  35. Hirani, N., F. Antonicelli, R. M. Strieter, M. S. Wiesener, C. Haslett, S. C. Donnelly. 2001. The regulation of interleukin-8 by hypoxia in human macrophages: a potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS). Mol. Med. 7:685.[Medline]
  36. 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.[Abstract/Free Full Text]
  37. Rapisarda, A., B. Uranchimeg, D. A. Scudiero, M. Selby, E. A. Sausville, R. H. Shoemaker, G. Melillo. 2002. Identification of small molecule inhibitors of hypoxia-inducibile factor 1 transcriptional activation pathway. Cancer Res. 62:4316.[Abstract/Free Full Text]
  38. Zhou, Z.-H. L., P. Chaturvedi, Y. L. Han, S. Aras, Y. S. Li, P. Kolattukudy, D. Ping, J. Boss, R. Ransohoff. 1998. IFN-{gamma} induction of the human monocyte chemoattractant protein (hMCP)-1 gene in astrocytoma cells: functional interaction between an IFN-{gamma}-activated site and a GC-rich element. J. Immunol. 160:3908.[Abstract/Free Full Text]
  39. Maeda, B. Y., M. Matsumoto, O. Hori, K. Kuwabara, S. Ogawa, S. D. Yan, T. Ohtsuki, T. Kinoshita, T. Kamada, D. M. Stern. 1994. Hypoxia/reoxygenation-mediated induction of astrocyte interleukin-6: a paracrine mechanism potentially enhancing neuron survival. J. Exp. Med. 180:2297.[Abstract/Free Full Text]
  40. Chen, C. Y., A. B. Shyu. 1995. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem. Sci. 20:465.[Medline]
  41. Nakagawa, J., H. Waldner, S. Meyer-Monard, J. Hofsteenge, P. Jeno, C. Moroni. 1995. AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity. Proc. Natl. Acad. Sci. USA 92:2051.[Abstract/Free Full Text]
  42. Vakalopoulou, E., J. Schaack, T. Shenk. 1991. A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol. Cell. Biol. 11:3355.[Abstract/Free Full Text]
  43. Rapella, A., A. Negrioli, G. Melillo, S. Pastorino, L. Varesio, M. C. Bosco. 2002. Flavopiridol inhibits vascular endothelial growth factor production induced by hypoxia or picolinic acid in human neuroblastoma. Int. J. Cancer 99:658.[Medline]
  44. Galindo, M., B. Santiago, J. Alcami, M. Rivero, J. Martin-Serrando, J. L. Pablos. 2001. Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts. Clin. Exp. Immunol. 123:36.[Medline]
  45. Kunz, M., G. Bloss, R. Gillitzer, G. Gross, M. Goebeler, U. R. Rapp, S. Ludwig. 2002. Hypoxia/reoxygenation induction of monocyte chemoattractant protein-1 in melanoma cells: involvement of nuclear factor-{kappa}B, stimulatory protein-1 transcription factor and mitogen-activated protein kinase pathways. Biochem. J. 366:299.[Medline]
  46. Ivacko, J., J. Szaflarski, C. Malinak, C. Flory, J. S. Warren, F. S. Silverstein. 1997. Hypoxic-ischemic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain. J. Cereb. Blood Flow Metab. 17:759.[Medline]
  47. Ono, K., A. Matsumori, Y. Furukawa, H. Igata, T. Shioi, K. Matsushima, S. Sasayama. 1999. Prevention of myocardial reperfusion injury in rats by an antibody against monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Lab. Invest. 79:195.[Medline]
  48. Negus, R. P. M., L. Turner, F. Burke, F. R. Balkwill. 1998. Hypoxia down-regulated MCP-1 expression: implications for macrophage distribution in tumors. J. Leukocyte Biol. 63:758.[Abstract]
  49. 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.[Abstract/Free Full Text]
  50. Evans, S. M., S. M. Hahn, D. P. Magarelli, C. J. Koch. 2001. Hypoxic heterogeneity in human tumors: EF5 binding, vasculature, necrosis, and proliferation. Am. J. Clin. Oncol. 24:467.[Medline]
  51. Freter, R. P., J. A. Alberta, G. Y. Hwang, A. L. Wrentmore, C. D. Stiles. 1996. Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-{kappa}B and a 90-KDa phosphoprotein coactivator. J. Biol. Chem. 271:17417.[Abstract/Free Full Text]
  52. Ueda, A., Y. Ishigatsubo, T. Okubo, T. Yoshimura. 1997. Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. J. Biol. Chem. 272:31092.[Abstract/Free Full Text]
  53. Michelotti, G. A., M. J. Bauman, M. P. Smith, D. A. Schwinn. 2003. Cloning and characterization of the rat {alpha}1a-adrenergic receptor gene promoter. J. Biol. Chem. 278:8693.[Abstract/Free Full Text]
  54. Kimura, H., A. Weisz, T. Ogura, Y. Hitomi, Y. Kurashima, K. Hashimoto, F. D’Acquisto, M. Makuuchi, H. Esumi. 2001. Identification of hypoxia-inducible factor-1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide. J. Biol. Chem. 276:2292.[Abstract/Free Full Text]
  55. Rapisarda, A., S. Pastorino, S. Massazza, L. Varesio, M. C. Bosco. 2002. Antagonistic effect of picolinic acid and interferon-{gamma} on macrophage inflammatory protein-1{alpha}/{beta} production. Cell. Immunol. 220:70.[Medline]
  56. Levy, N. S., S. Chung, H. Furneaux, A. P. Levy. 1998. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J. Biol. Chem. 273:6417.[Abstract/Free Full Text]
  57. McGary, E. C., I. J. Rondon, B. S. Beckman. 1997. Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein. J. Biol. Chem. 272:8628.[Abstract/Free Full Text]
  58. McQuillan, L. P., G. K. Leung, P. A. Marsden, S. K. Kostyk, S. Kourembanas. 1994. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am. J. Physiol. 267:1921.
  59. Koerner, T. J., T. Hamilton, M. Introna, C. Tannenbaum, R. Bast, D. Adams. 1987. The early competence genes JE and KC are differentially regulated in murine peritoneal macrophages in respone to lipopolysaccharide. Biochem. Biophys. Res. Commun. 149:969.[Medline]
  60. Myer, V. E., X. C. Fan, J. A. Steitz. 1997. Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay. EMBO J. 16:2130.[Medline]
  61. Zhang, L., A. Khayat, H. Cheng, D. T. Graves. 1997. The pattern of monocyte recruitment in tumors is modulated by MCP-1 expression and influences the rate of tumor growth. Lab. Invest. 76:579.[Medline]
  62. Semenza, G. L.. 2002. HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol. Med. 8:S62.[Medline]
  63. Koch, A. E., S. L. Kunkel, L. A. Harlow, B. Johnson, H. L. Evanoff, G. K. Haines, M. D. Burdick, R. M. Pope, R. M. Strieter. 1992. Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis. J. Clin. Invest. 90:772.
  64. Manome, Y., P. Y. Wen, A. Hershowitz, T. Tanaka, B. D. Rollins, D. W. Kufe, H. A. Fine. 1995. Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors. Cancer Immunol. Immunother. 41:227.[Medline]
  65. Nesbit, A., H. Schaider, T. H. Miller, M. Herlyn. 2001. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in non-tumorigenic melanoma cells. J. Immunol. 166:6483.[Abstract/Free Full Text]
  66. Turner, L., C. Scotton, R. Negus, F. Balkwill. 1999. Hypoxia inhibits macrophage migration. Eur. J. Immunol. 29:2280.[Medline]
  67. Bingle, L., N. J. Brown, C. E. Lewis. 2002. The role of tumor-associated macrophages in tumor progression: implication for new anticancer therapies. J. Pathol. 196:254.[Medline]



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