Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Expression This information is current as of October 1, 2021. Fernando O. Martinez, Siamon Gordon, Massimo Locati and Alberto Mantovani J Immunol 2006; 177:7303-7311; ; doi: 10.4049/jimmunol.177.10.7303

http://www.jimmunol.org/content/177/10/7303 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2006/11/03/177.10.7303.DC1 Material http://www.jimmunol.org/ References This article cites 61 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/177/10/7303.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on October 1, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression1

Fernando O. Martinez,*† Siamon Gordon,‡ Massimo Locati,2*† and Alberto Mantovani*†

Comprehensive analysis of the profiles associated with human monocyte-to-macrophage differentiation and polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage differentiation is associated with activation of cell cycle , substantiating the underestimated proliferation potential of monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature of macrophage differentiation and polarization. 4) Lipid is a main category of modulated transcripts, with expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each Downloaded from step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2- associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology. The Journal of Immunology, 2006, 177: 7303–7311. http://www.jimmunol.org/ onocytes and tissue macrophages provide both imme- ygen and nitrogen intermediates) and inflammatory cytokines (IL- diate defense against foreign agents and assist during 1␤, TNF, IL-6), contribute as inducer and effector cells in the setting off and development of the adaptive im- polarized Th1 responses, and mediate resistance against intracel- M ϩ mune response. Monocytes originally derive from CD34 myeloid lular parasites and tumors (7–11). In contrast, the alternative M2 progenitor cells in the bone marrow, circulate in the bloodstream, form of macrophage activation is a generic name used for various and enter peripheral tissues where they mature into different types forms of nonclassically activated macrophages resulting from cell of resident macrophages, characterized by low oxygen consump- exposure to IL-4 or IL-13, immune complexes, IL-10, glucocorti- tion, low protein synthesis rate, and modest cytokine production coid, or secosteroid (vitamin D3) hormones (3, 9, 12). The various (1, 2). Inflammation due to tissue damage or infection results in forms of M2 macrophages share an IL-12low and IL-23low pheno- by guest on October 1, 2021 resident macrophage activation, which increases the production of type, generally display high levels of scavenger, mannose (13), and cytokines, chemokines, and other inflammatory mediators, as well galactose-type receptors (3), and arginine metabolism is shifted to as monocyte recruitment. In the context of specific immune re- production of ornithine and polyamines via arginase (14). sponse, the cytokine milieu compels mononuclear phagocytes to Previous studies have addressed the issue of profiling gene ex- express specialized and polarized functional properties. Mirroring pression in M1 or M2 macrophage activation in the mouse, leading the Th1/Th2 nomenclature, many refer to polarized macrophages to the identification of new molecules expressed in polarized mu- as M1 and M2 cells (3–6). Classically polarized activated M1 rine macrophages (e.g., Ym1, Fizz1, MRC1) (13, 15, 16). Data on macrophages have long been known to be induced by IFN-␥ alone human mononuclear phagocytes on the contrary are scanty and or in concert with microbial stimuli as LPS, or cytokines as TNF have highlighted important interspecies differences in key mole- and GM-CSF. M1 cells have an IL-12high, IL-23high, IL-10low phe- cules, such as arginase and inducible NO synthase, rendering dif- notype, are proficient producers of effector molecules (reactive ox- ficult extrapolation (17, 18). In this study, we report for the first time a whole genome transcriptional profile analysis of the human monocyte-to-macrophage differentiation and polarized activation *Istituto Clinico Humanitas, Rozzano, Italy; †Institute of General Pathology, Univer- sity of Milan, Milan, Italy; and ‡Sir William Dunn School of Pathology, University processes, describing distinct molecular signatures which shed of Oxford, Oxford, United Kingdom new light on these processes and reveal new candidate markers. Received for publication March 9, 2006. Accepted for publication August 3, 2006. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Reagents with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Italian Association for Cancer Research, Ministero Recombinant human cytokines were obtained from PeproTech. LPS from dell’Istruzione dell’Universita`e della Ricerca (Fondo Investimenti Ricerca di Base, Escherichia coli (serotype 055:B5) was obtained from Sigma-Aldrich. Abs Progetto di Rilevante Interesse Nazionale, and Consiglio Nazionale delle Ricerche were purchased from Serotec, unless specified. Human cytokines were funding), Fondo Interno per la Ricerca Scientifica e Tecnologica (FIRST Project), measured using commercial ELISA kits purchased from R&D Systems, Ministero della Salute, Fondazione Cariplo (NOBEL Project), and the European according to the manufacturer’s instructions. All chemicals were obtained Commission (Innochem Project, FP6-518167; Mugen Project, LSHG-CT-2005- from Sigma-Aldrich, unless specified. 005203). F.O.M. is a recipient of the International PhD program in Cellular and Molecular Biology fellowship from Vita-Salute San Raffaele University. Cell preparation 2 Address correspondence and reprint requests to Dr. Massimo Locati, Istituto Clinico Humanitas, Via Manzoni 56, I-20089 Rozzano, Italy. E-mail address: massimo. Human monocytes were obtained from normal blood donor buffy coats by [email protected] two-step gradient centrifugation followed by an additional step using the

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7304 TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

FIGURE 1. PCA of the transcriptome expressed during macrophage differentiation and polarization. PCA was carried on all genes under in- vestigation to determine expression trends within the data set. Sample trend Downloaded from during maturation and polarization is shown in a scatter plot of the prin- FIGURE 2. K-mean clustering of genes differentially expressed during cipal components 1 and 2, which summarize 98% of the system variance. macrophage differentiation and polarization. Modulated genes were organized Bars represent the SD within the three donors studied. F, monocytes (Mo); by K-means clustering. The x-axis corresponds to the experimental conditions, U, monocytes after 3 days of culture with M-CSF (Md3); E, macrophages the y-axis to expression levels. Each line represents a gene, with red and green after 7 days of culture with M-CSF (M␾); Œ, M1-polarized macrophages for high and low expression levels, respectively. Monocyte differentiation cor- (M1); ‚, M2-polarized macrophages (M2). related with a cluster on early affected genes (A, 478 genes) and a cluster of late http://www.jimmunol.org/ affected genes (B, 390 genes). Two clusters (D, 1108 genes; E, 945 genes) are associated with M1 polarization, and one cluster (F, 104 genes) with M2 po- Monocyte Isolation kit II (Miltenyi Biotec) as previously described (17). larization. C (505 genes) includes genes with a complex behavior not directly ϩ Macrophages were obtained by culturing monocytes (98% CD14 , 13% linked to a specific cell differentiation state. CD16ϩ) for 7 days in RPMI 1640 (Biochrom) supplemented with 20% FCS (HyClone) and 100 ng/ml M-CSF in FCS-coated dishes at a density 5 2 of 1.5 ϫ 10 /cm . Macrophage polarization was obtained by removing the pliant are available at the Gene Expression Omnibus (GEO) website culture medium and culturing cells for an additional 18 h in RPMI 1640 (ͳwww.ncbi.nlm.nih.gov/geoʹ), accession number GSE5099. supplemented with 5% FCS and 100 ng/ml LPS plus 20 ng/ml IFN-␥ (for M1 polarization) or 20 ng/ml IL-4 (for M2 polarization). Five different cell Gene expression analysis by real-time PCR types were generated: freshly isolated monocytes (Mo), cells at interme- by guest on October 1, 2021 diate differentiation (3 days of culture: Md3), resting fully differentiated Real-time PCR was performed using gene-specific primers designed using macrophages (7 days of culture: M␾), classical activated macrophages AutoPrime (ͳwww.autoprime.deʹ). Primer and probe sequences are (M1), alternative activated macrophages (M2). available in the public RTPrimerDB database (ͳhttp://medgen.UGent.be/ rtprimerdb/ʹ) (gene (RTPrimerDB-ID): GPR105 (3478), GPR87 (3479), Transcriptional profile analysis P2RY13 (3480), P2RY12 (3481), GPR171 (3482)) (28). Five replicates per each experimental point were performed, and differences were assessed The transcriptional profile was evaluated in three independent cell prepa- with a two-tailed Student’s t test. Results were normalized using the house- rations, each derived from a different single donor using the Human Ge- keeping gene GAPDH and the ⌬⌬ cycle threshold method (19) and are nome U133 A and B arrays (HG-U133; Affymetrix) containing a total of expressed as relative fold of stimulated over control group, used as ϳ39,000 transcripts. RNA purification and labeling, hybridization, and ar- calibrator. ray scanning were conducted as previously described (19). Scanned images and raw data were processed using robust multiarray average (20, 21). Western blot Principal component analysis (PCA)3 was conducted on all genes analyzed to assign the general variability in the data to a reduced set of variables After removing the medium, cells were washed in PBS and lysed in ice- called principal components (22). Gene expression differences were as- cold lysis buffer (2% Triton X-100, 10 mM Tris-HCl (pH 8), 150 mM sessed by means of Student’s t test, with false discovery rate correction for NaCl, 2 mM NaN3, 2 mM EDTA) containing protease inhibitors (Roche multiple testing (23) (R Bioconductor). Genes with an false discovery rate Molecular Biochemicals) for 45 min at 4°C. Lysates were harvested and Յ0.05 and a fold change Ն2 were considered differentially expressed. centrifuged at 13,400 ϫ g to eliminate nuclei. Protein concentration was After removal of redundant genes, an expression matrix (3530 ϫ 15) was determined using the bicinchoninic acid assay (Pierce) and 30 ␮g of pro- obtained, and figures-of-merit analysis was applied to define the optimal tein was electrophoresed in a 7.5% SDS-PAGE under nonreducing condi- number of clusters (24). Each cluster was then analyzed by K-means and tions and transferred to nitrocellulose using standard procedures. PTGS1 hierarchical clustering algorithms with squared Pearson correlation as sim- and PTGS2 were detected using the specific mAbs CXIII and CX229 ilarity measurement (25). To identify overrepresented biological categories (Alexis). within each cluster, the Expression Analysis Systematic Explorer analysis based on the (GO) database was applied (26), the percent- Results age of genes within each category per total amount of genes in each cluster Global transcriptome analysis was calculated, and hierarchical clustering was conducted grouping clus- ters according to their similarities in gene function representation. In each The transcriptional events associated with M-CSF-dependent cluster, the 50 most down-regulated and the 50 most up-regulated genes monocyte-to-macrophage differentiation and subsequent M1 or were selected for interactome analysis using the ResNet-3.0 literature da- M2 cell polarization induced by LPS plus IFN-␥ or IL-4, respec- tabase (27). The entire data set and technical information requested by Minimum Information about a Microarray Experiment (MIAME) com- tively, were investigated using oligonucleotide microarrays. Re- sults demonstrated the existence of a complex network of gene regulation and clearly identified specific gene expression patterns 3 Abbreviations used in this paper: PCA, principal component analysis; GO, Gene Ontology; GPCR, G protein-coupled receptor; ALOX5, arachidonate 5-lipoxygenase; that characterize each phase. PCA analysis was applied to the com- COX, cyclooxygenase; Mo, monocyte; M␾, macrophage. plete dataset and demonstrated that 98% of the total variance of the The Journal of Immunology 7305 Downloaded from http://www.jimmunol.org/

FIGURE 3. GO analysis of human macrophage differentiation and po- larization. Hierarchical clustering of the percentage of genes associated with a GO category with respect to the total number of genes in the cluster. Columns represent clusters of Fig. 2 (the full graph is present in supple- mental figure 7SM). by guest on October 1, 2021 system lies within the first two components. PCA revealed that monocyte maturation was associated with a significant modifica- tion of the global transcriptome (ϳ35% of the total variance), with larger changes taking place in the early phase of the process (ϳ24% variance in the first 3 days of differentiation), followed by a smaller overhaul (ϳ11% variance in the last 4 days of differen- tiation) in the late phase (Fig. 1). Macrophage polarization was also associated with significant changes at the transcriptional level, although the two polarizing conditions were very different, with M1 polarization profoundly affecting the transcriptional profile (ϳ90% variance in the shift from M␾ to M1), and M2 polarization resulting in only subtle adjustments (ϳ8% variance in the shift from M␾ to M2) (Fig. 1). Differentially expressed genes, selected as described in Materi- als and Methods, were subjected to figures-of-merit analysis, where K-means clustering performed optimally for 12 clusters, with no increase in the predictive value of the algorithm for addi- tional behavioral categories (data not shown). Squared Pearson correlation was used as similarity measurement, cumulating clus- ters with mirror performances and generating a total of 6 gene FIGURE 4. Interactome analysis of representative genes involved in clusters (Fig. 2). To gain insight into the biological processes in- monocyte-to-macrophage differentiation and polarization. A, Prototypical volved, each cluster was then subjected to hierarchical subcluster- macrophage was constructed from the best 100 genes representative of ing (supplemental figures 1–6)4 and GO analysis as included in the each transitional state. A, Composite of genes changed transiently changed Expression Analysis Systematic Explorer (26) (Fig. 3). in maturation. B, Genes stably changed with the maturation and (C) genes Monocyte-to-macrophage differentiation was associated with differentially expressed between M1 and M2 cells (C). For A and B, red and modulation of 868 (2.2%) transcripts in total (Fig. 2, A and B). Of green represent increased and decreased genes, respectively. In C, red rep- resents M1 genes while green stands for M2 genes.

4 The online version of this article contains supplemental material. 7306 TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

Table I. Genes differentially expressed in M1 vs M2 macrophagesa

Gene Gene Name Symbol M1 M2 M1:M2 Ratio

Membrane receptors CCR7 CCR7 5,893 55 107 Interleukin 2 receptor ␣ chain IL2RA 4,062 181 22 Interleukin 15 receptor ␣ chain IL15RA 2,867 178 16 Interleukin 7 receptor IL7R 1,553 115 13 G protein-coupled receptor 86 GPR86 342 1,852 Ϫ5 Purinergic receptor P2Y5 P2RY5 284 2,610 Ϫ9 Transforming growth factor ␤ receptor II TGFBR2 218 2,274 Ϫ10 Histamine receptor H1 HRH1 182 1,918 Ϫ11 Toll-like receptor 5 TLR5 76 915 Ϫ12 C-type lectin receptor DCL-1 DCL-1 179 2,265 Ϫ13 Macrophage scavenger receptor 1 MSR1 7 107 Ϫ14 CXCR4 CXCR4 168 2,678 Ϫ16 C-type lectin superfamily member 12 DECTIN1 130 2,520 Ϫ19 G protein-coupled receptor 105 P2RY14 16 416 Ϫ25 CD209 DCSIGN 315 8,355 Ϫ27 C-type lectin superfamily member 13 CLECSF13 93 2,946 Ϫ32 Membrane-spanning 4-domains, subfamily A, member 6A MS4A6A 247 8,064 Ϫ33 CD36 CD36 73 2,713 Ϫ37 Downloaded from Membrane-spanning 4-domains, subfamily A, member 4 MS4A4A 16 688 Ϫ43 Mannose receptor C type 1 MRC1 173 7,343 Ϫ43 Cytokines and chemokines CXCL11 CXCL11 4,084 19 212 CCL19 CCL19 13,338 103 130 CXCL10 CXCL10 11,659 199 59

CXCL9 CXCL9 15,121 260 58 http://www.jimmunol.org/ Tumor necrosis factor ligand superfamily, member 2 TNF 977 47 21 CCL5 CCL5 6,468 334 19 CCL15 CCL15 2,402 164 15 Interleukin 12B IL12B 198 20 10 Interleukin 15 IL15 259 29 9 Tumor necrosis factor ligand superfamily, member 10 TRAIL 2,354 343 7 Interleukin 6 IL6 591 83 7 CCL20 CCL20 316 47 7 Visfatin PBEF1 2,297 446 5 Endothelial cell growth factor 1 ECGF1 1,636 370 4 Insulin-like growth factor 1 IGF1 217 1,212 Ϫ6 by guest on October 1, 2021 CCL23 CCL23 270 3,542 Ϫ13 CCL18 CCL18 290 5,457 Ϫ19 CCL13 CCL13 133 5,028 Ϫ38 Apoptosis-related genes BCL2-related protein A1 BCL2A1 4,018 117 34 Tumor necrosis factor receptor superfamily, member 6 FAS 2,243 156 14 Baculoviral IAP repeat-containing 3 BIRC3 1,318 162 8 Growth arrest and DNA-damage-inducible, ␥ GADD45G 682 144 5 XIAP associated factor-1 HSXIAPAF1 1,928 445 4 Solute carriers Solute carrier family 7, member 5 SLC7A5 2,509 259 10 Solute carrier family 21, member 15 SLC21A15 371 52 7 Solute carrier family 2, member 6 SLC2A6 2,981 513 6 Solute carrier family 31, member 2 SLC31A2 4,022 850 5 Solute carrier family 2, member 9 SLC21A9 338 2,476 Ϫ7 Solute carrier family 4, member 7 SLC4A7 43 388 Ϫ9 Solute carrier family 38, member 6 SLC38A6 99 2,370 Ϫ24 Indoleamine-pyrrole 2,3 dioxygenase INDO 16,605 142 117 Phospholipase A1 member A PLA1A 1,854 55 33 2Ј–5Ј-oligoadenylate synthetase-like OASL 3,272 237 14 Chitinase 3-like 2 CHI3L2 1,078 89 12 Hydroxysteroid (11-␤) dehydrogenase 1 HSD11B1 4,066 476 9 Adenylate 3 AK3 702 103 7 1 SPHK1 1,974 324 6 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 PFKFB3 2,983 509 6 Proteasome activator subunit 2 PSME2 10,918 1,789 6 PFKP 1,618 310 5 Proteasome subunit ␤ type 9 PSMB9 3,888 817 5 Proteasome subunit ␣ type 2 PSMA2 4,957 1,199 4 2Ј–5Ј-oligoadenylate synthetase 2 OAS2 944 261 4 Cathepsin C CTSC 3,161 13,800 Ϫ4 Hexosaminidase B HEXB 1,582 6,809 Ϫ4 Lipase A cholesterol esterase LIPA 2,252 9,772 Ϫ4

(Table continues) The Journal of Immunology 7307

Table I. (Continued)

Gene Name Gene Symbol M1 M2 M1-M2 Ratio

Adenosine kinase ADK 385 1,810 Ϫ5 Histamine N-methyltransferase HNMT 174 935 Ϫ5 Tyrosylprotein sulfotransferase 2 TPST2 132 824 Ϫ6 CERK 255 1,431 Ϫ6 Heparan sulfate 3-O-sulfotransferase 2 HS3ST2 355 2,025 Ϫ6 Leukotriene A4 hydrolase LTA4H 380 2,556 Ϫ7 Carbonic anhydrase II CA2 118 950 Ϫ8 Arachidonate 15-lipoxygenase ALOX15 53 600 Ϫ11 Heparan sulfate 3-O-sulfotransferase 1 HS3ST1 42 544 Ϫ13 Extracelullar mediators Pentraxin 3 PTX3 914 30 30 Chondroitin sulfate proteoglycan 2 CSPG2 1,712 107 16 Apolipoprotein L3 APOL3 4,453 374 12 Insulin-like growth factor binding protein 4 IGFBP4 3,821 349 11 Apolipoprotein L1 APOL1 3,032 465 7 Platelet-derived growth factor ␣ PDGFA 422 61 7 Endothelin 1 EDN1 539 86 6 Apolipoprotein L2 APOL2 2,027 315 6 Inhibin ␤ A INHBA 688 129 5 Downloaded from Apolipoprotein L6 APOL6 918 202 5 Transforming growth factor ␤-induced protein TGFBI 1,120 8,976 Ϫ8 Selenoprotein P1 SEPP1 811 7,641 Ϫ9 Chimerin 2 CHN2 76 943 Ϫ12 Fibronectin 1 FN1 330 8,382 Ϫ25 Fibrinogen-like 2 FGL2 117 4,569 Ϫ39 DNA-binding factors

Homeobox expressed in ES cells 1 HESX1 1,238 102 12 http://www.jimmunol.org/ Interferon regulatory factor 1 IRF1 4,288 527 8 Activating transcription factor 3 ATF3 4,080 509 8 Interferon regulatory factor 7 IRF7 2,694 394 7 Growth arrest-specific 7 GAS7 257 1,350 Ϫ5 Early growth response 2 EGR2 108 1,238 Ϫ11 v-maf musculoaponeurotic fibrosarcoma oncogene homolog MAF 26 1,187 Ϫ46

a The table shows a selection of genes strictly associated with macrophage polarization grouped into functional categories. M1 and M2 columns report the mean of the expression values. In each category, genes are ranked according to their fold difference between M1 and M2. by guest on October 1, 2021 these, 390 (1.0%) genes were transiently regulated during the first As indicated by PCA, M1 polarization had a major effect on cell stage of the differentiation process and returned to basal levels in transcriptome, affecting 2053 (5.2%) transcripts in total (Fig. 2, C fully differentiated macrophages (Fig. 2A; list of genes in supple- and D). Of these, 1108 (2.8%) genes were exclusively associated mental figure 1SM). In this cluster, GO analysis highlighted an with classical macrophage activation (Fig. 2C; list of genes in sup- overrepresentation of molecules associated with cell cycle (Fig. 3), plemental figure 3SM), while a second cluster of 945 (2.4%) tran- including positive modulators of cell proliferation such as cyclins scripts included a majority of genes associated with classical ac- (A2, that regulates S phase progression; B1 and B2, that regulate tivation and a minor fraction (20 genes, corresponding to ϳ2% of

G2-M phase transition; D1 and D3, which regulate G1 phase pro- the genes) concordantly regulated by IL-4 (Fig. 2D; list of genes in gression; E2, participating in the late G1-S phase transition) and supplemental figure 4SM). GO analysis revealed an overrepresen- cell division-associated proteins 1, 2, 5, 6, 7, and 20 (29–31) (sup- tation of genes related to DNA transcription and protein metabo- plemental figure 1SM). Interactome analysis of transiently modu- lism, such as ribosomal proteins and eukaryotic translation initia- lated genes highlighted three main nodes around the genes CDC2, tion factors (Fig. 3). BCL2L11, and CCL2, as well as the concerted down-regulation of Although less dramatic than M1, M2 polarization exerted a HLA members. Also notable was the high number of nuclear pro- significant effect on macrophage transcriptional profile, modu- teins and the low number of soluble factors among the main reg- lating a total of 104 (ϳ0.3%) transcripts (Fig. 2E; list of genes ulated genes (Fig. 4A). A second cluster contained 478 (1.2%) in supplemental figure 5SM). GO analysis indicated that this set genes rapidly regulated during the differentiation process, main- of genes is particularly rich in immune system-related mole- tained in mature macrophages, and essentially refractory to polar- cules, including cytokines, chemokines, and G protein-coupled izing stimuli (Fig. 2B; list of genes in supplemental figure 2SM). receptors (GPCR) (Fig. 3). These transcripts mostly corresponded to membrane receptors, sig- A peculiar cluster included 505 (1.3%) genes with high expres- nal transducers, and extracellular proteins functionally related to sion in Mo and M1 cells and opposite regulation in M␾ and M2 the immune response (Fig. 3). Interactome analysis of stably M- cells (Fig. 2, cluster F; list of genes in supplemental figure 6SM). CSF-regulated genes showed an inversion in the gene distribution, Unexpectedly, genes with high expression in Mo and M1 macro- with a smaller percentage of nuclear genes and an increase of phages included prototypic M1 polarization markers, such as the membrane receptors and soluble factors. It also highlighted three indoleamine-pyrrole 2,3 dioxygenase (32, 33), the lysosomal-as- main nodes around the down-regulated genes IL-1 and IL-8 and the sociated membrane protein 3, IL-7R (3, 5), and CCR7, though up-regulated gene apolipoprotein E. A concerted down-regulation despite transcript expression, monocytes did not express mem- of chemokine receptors, cystatins, and defensins, and an opposite brane CCR7 (data not shown). Similarly, genes with high expres- increase of complement components was also noticed (Fig. 4B). sion in M␾ and M2 cells included classic M2 polarization markers, 7308 TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION such as the mannose receptor 1 (34, 35), the scavenger receptors the M2 marker arachidonate 15-lipoxygenase and unexpectedly SR-A and M160 (3, 5, 36). GO analysis revealed that a relevant COX-1, here confirmed at the protein level (Fig. 5B). percentage of these genes is involved in cellular metabolic activ- Sphingolipid mediators, such as sphingosine 1-phosphate, cer- ities, such as active transport and oxidoreductase activities (Fig. 3). amide 1-phosphate, and sphingosine are derived by the enzymatic To identify genes strictly associated with macrophage polariza- breakdown of sphingomyelin, and display potent effects on mul- tion, genes included in clusters C to F have been ranked according tiple organ systems. Within this pathway, the most interesting find- to their fold difference between M1 and M2 profiles and further ing is the opposite regulation of the sphingosine and ceramide grouped into functional categories (Table I). The interactome high- , expressed in M1 and M2 macrophages, respectively. lights a central role of a restricted panel of molecules, including CXCR4, TRAIL (TNFSF10/TRAIL), insulin-like growth factor I, GO analysis of functional categories: GPCRs and fibronectin 1, and clearly shows that macrophage polarization A second category highlighted by GO analysis was represented by is mainly associated with regulation of membrane receptors and GPCRs (Fig. 3). From a total of 465 entries of GPCRs represented extracellular proteins with a minor contribution of nuclear factors, in the microarray, 53 were detected as differentially expressed dur- directly opposite to monocyte differentiation (Fig. 4C). ing monocyte differentiation and macrophage polarization (Fig. 6A). The hierarchical clustering demonstrates that each stage is GO analysis of functional categories: lipid metabolism characterized by the expression of a specific group of GPCRs. GO analysis was used to identify functional categories overrepre- Monocytes are characterized by a cluster of 11 highly expressed sented in the panel of genes associated with monocyte differenti- genes, 5 of which correspond to chemotactic receptors: CCR2, ation and macrophage polarization. Consistent with the well-rec- CCR5, CCR7, CX3CR1, and FPR1. The combination of IFN-␥ and Downloaded from ognized capability of macrophages to respond and produce a vast LPS has a broad effect with no clear family overrepresentation, range of lipidic products, one of the most overrepresented catego- while IL-4 activation is characterized by a cluster of 8 genes with ries was lipid metabolism (Fig. 3). In particular, transcriptional high expression, 5 of which are nucleotide receptors: GPR86, analysis revealed a unique regulation profile for different enzymes GPR105, P2Y8, P2Y11, and P2Y12. Interestingly, GPR86, involved in eicosanoid production (Fig. 5A). Monocyte-to-mac- GPR105, and P2Y12 are positioned together in 3. rophage maturation was associated with a gradual loss of PG-en- Real-time PCR confirmed the up-regulation of this M2-associated doperoxide synthases (both PTGS1 and PTGS2), as well as the gene cluster (P2Y12, GPR105, and GPR86), and revealed minor http://www.jimmunol.org/ arachidonate 5-lipoxygenase (ALOX5), and leukotriene A4 hydro- overexpression for the closest genomic neighbors GPR87 and lase. As expected, classical activation was associated with a H963 (Fig. 6B). marked induction of cyclooxygenase (COX)-2 (37), accompanied GO analysis of functional categories: the chemokinome by a significant unexpected further down-regulation of COX-1, leu- kotriene A4 hydrolase, thromboxane A synthase 1, and ALOX5. Macrophage maturation and polarization are characterized by spe- Conversely, alternative activation resulted in the up-regulation of cific patterns of chemokines as suggested by GO ontology analysis (Fig. 7A). In addition to chemokines already known to be differ- entially expressed in polarized macrophages (e.g., CXCL10 for by guest on October 1, 2021 M1; CCL17 for M2), we found new chemokine signatures asso- ciated with cell polarization (Fig. 7A). The profiling results were confirmed by real-time PCR (data not shown) and by measure- ments of released proteins (Fig. 7, B and C).

Discussion The present study was designed to characterize the gene expres- sion profile of human monocytes undergoing differentiation into mature macrophages in the presence of M-CSF and subsequent polarized activation into M1 or M2 cells. These processes were associated with major changes in the global transcriptome. Macrophage polarization to M1 was associated with the most dramatic change in the transcriptome, whereas stimulation with IL-4 of M-CSF-differentiated macrophages caused a relatively mi- nor alteration in gene expression. This apparently minor effect of IL-4 is due to the fact that M-CSF-driven differentiation leads per se to the acquisition of M2 properties, including expression of mannose receptor 1 and scavenger receptors SR-A. This finding is in agreement with previous data showing divergent M1-M2 prop- erties of macrophages differentiated in GM-CSF compared with M-CSF (38, 39). M-CSF is a homeostatic growth factor circulating at high levels in normal blood. Thus, drifting toward M2 may be a default pathway in macrophage differentiation. FIGURE 5. Differential regulation of arachidonate metabolism-related en- In particular, monocyte differentiation in the presence of M-CSF zymes in human macrophage differentiation and polarization. A, Transcrip- was associated with early (day 3) dramatic regulation of cell-cycle tional analysis reveals a unique regulatory network of the PG synthases (PTGS), with M1 polarization resulting in increased expression of PTGS2 genes, including the cyclins A2, B1, B2, D1, D3, E2, and CDCA (COX2) and inhibition of PTGS1 (COX1), and M2 inducing a mirror regu- 1, 2, 5, 6, and 7. Though human mononuclear phagocytes, unlike lation. B, Western blot analysis confirmed selective induction of COX2 in M1 mouse macrophages, are generally considered terminally differen- macrophages and COX1 in M2 macrophages. Results from one experiment are tiated nonproliferating cells, there are reports of human monocyte- shown, representative of three independent experiments. macrophage proliferation (40, 41). Evidence for proliferation in The Journal of Immunology 7309 Downloaded from http://www.jimmunol.org/

FIGURE 7. Differential expression of chemokines during macrophage differentiation and polarization. A, Hierarchical clustering of chemokine by guest on October 1, 2021 expression levels. B and C, ELISA measurement of secreted chemokines in the supernatant of M1- and M2-polarized macrophages. u and f,M1and M2 cells, respectively. All values were statistically significant as assessed by two-tailed paired t test (p Ͻ 0.05), and represent data from five different blood donors. Bars represent the SEM.

donic acid metabolism in M1 cells (37). In contrast, the finding FIGURE 6. Repertoire of GPCRs expressed during macrophage differ- that M-CSF-differentiated macrophages retain high levels of entiation and polarization. A, Hierarchical clustering of differentially ex- COX-1 and that these levels are further augmented by IL-4 is pressed GPCRs, obtained using average linkage and Pearson correlation as novel and unexpected. This induction is of functional relevance for distance. B, Real-time PCR confirmation of microarray results. f and Ⅺ represent M1 and M2 cells, respectively. Statistical analysis was performed eicosanoid production (data not shown) and may contribute to with a two-tailed paired t test in five different blood donors. SE was always pathophysiological reactions, such as toxicity of aspirin and related below 5% of the individual means (data not shown). All genes were sig- drugs in asthma (43, 44). The interconvertible ceramide metabo- nificantly different in M2 vs control macrophages (p Ͻ 0.05). lites sphingosine 1-phosphate and ceramide 1-phosphate have emerged as potent bioactive agents which regulate critical cellular functions including cell proliferation, phagocytosis, differentiation, the culture was also confirmed in the present study (data not angiogenesis, chemotaxis, and cell survival (45). Our results sug- shown). Thus, the proliferative potential of human monocytes gest that these enzymes can aid in distinguishing between polar- should not be underestimated and could be exploited and tailored ized forms of activation, being sphingosine and ceramide kinase for cell expansion. selectively present in M1 and M2 macrophages, respectively Modulation of genes involved in general cellular metabolic ac- (Table I). M1 polarization is associated with the up-regulation of tivities is a prominent feature of macrophage differentiation and ABCA1, the primary gatekeeper for eliminating tissue cholesterol, polarization. In addition to providing tools for macrophage func- and a set of apolipoproteins clustered on chromosome 22q12.3 tion in tissues, these changes may have a more subtle significance. including APOL1, APOL2, APOL3, and APOL6, which play a For instance, macrophages are a major component of adipose tis- central role in cholesterol transport and atherosclerosis (46). sue and play a role in the metabolic syndrome (42). Different stages of monocyte differentiation and polarization are Macrophages are an active source of pro- and anti-inflammatory characterized by different repertoires of GPCR. In agreement with lipid mediators, such as arachidonic acid derivatives and phos- previous reports, CCR2 is rapidly down-regulated during monocyte phosphingolipids. COX-2 has long been associated with arachi- differentiation (47). CX3CR1 is also down-regulated, but at a slower 7310 TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION rate, being still expressed on day 3. LPS and IFN-␥ up-regulate CCR7 not involved in macrophage polarization in the murine system and down-regulate CCR1 in mature macrophages, as they do in den- emerged from this data set as human macrophage alternative ac- dritic cells (48, 49). Presumably, this reciprocal regulation underlies tivation markers, including fibrinoligase (F13A1) and platelet-de- the trafficking of macrophages to lymph nodes, where their disposal rived growth factor C. In contrast, other mouse alternative activa- occurs. Strikingly, of the eight GPCRs highly expressed in the M2 tion markers such as the GPCR cluster discussed above were cells, five are nucleotide receptors (Fig. 6), the UDP-glucose receptor confirmed in our system. Collectively, results indicate that ϳ50% GPR105 being among the most highly regulated genes in human mac- of macrophage polarization markers selectively apply to one spe- rophages in response to IL-4. M2 macrophages are associated with cies and not to the other, cautioning against direct mouse-to-human tissue remodeling. High expression of nucleotide receptors endows translation of polarization markers. A direct comparison based on M2 cells with sensors for tissue damage (50, 51), and preliminary data expression profiling results will be required to fully describe in- suggest that these ligands modulate relevant functions in this cell type terspecies variability. (data not shown). Polarization of mononuclear phagocyte function is a useful The chemokine repertoires of mononuclear phagocytes exposed simplified conceptual framework, describing a continuum of func- to polarizing stimuli are profoundly different (4, 5) and the results tional states. Different forms of M2 polarization have been de- presented here extend this general view. In addition to well-known scribed in vitro and ex vivo (3, 5, 9). The study reported here using polarized chemokines, such as CXCL10 for M1 and CCL17 for a global profiling approach, describes new molecules and signa- M2 cells, we found high levels of CCL8, CCL15, CCL19, CCL20, tures associated with different stages of the human monocyte-to- and CXCL13 in M1 cells, and CCL13, CCL14, CCL17, CCL23, macrophage differentiation and polarization, which may represent and CCL26 in M2 cells. Association of these molecules with po- novel tools and targets in pathophysiology. larized macrophage activation may contribute to pathophysiology. Downloaded from For instance, we found that M2 cells do not produce detectable Disclosures levels of CCL11 but may contribute to the recruitment of CCR3- The authors have no financial conflict of interest. positive leukocytes such as eosinophils, basophils, and some po- larized Th2 cells (4) through the expression of CCL26. References A hallmark of M1 polarization is the synthesis of the proinflam- 1. Grage-Griebenow, E., H. D. Flad, and M. Ernst. 2001. Heterogeneity of human

peripheral blood monocyte subsets. J. Leukocyte Biol. 69: 11–20. http://www.jimmunol.org/ matory cytokines IL-6, IL-12, and IL-15 (5) and receptors for 2. Hume, D. A., I. L. Ross, S. R. Himes, R. T. Sasmono, C. A. Wells, and T. Ravasi. IL-2R ␣-chain, IL-15R ␣-chain, and IL-7R as previously described 2002. The mononuclear phagocyte system revisited. J. Leukocyte Biol. 72: in mice (52, 53). In the other pole, M2 are characterized by the 621–627. 3. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: overexpression of several scavenger receptors able to bind a di- 23–35. verse array of endogenous and foreign molecules (54). Our results 4. Mantovani, A., A. Sica, S. Sozzani, P. Allavena, A. Vecchi, and M. Locati. 2004. The chemokine system in diverse forms of macrophage activation and polariza- confirm the up-regulation by IL-4 of the mannose receptor 1 (13), tion. Trends Immunol. 25: 677–686. the macrophage scavenger receptor 1(36), the C-type lectin-like 5. Mantovani, A., A. Sica, and M. Locati. 2005. Macrophage polarization comes of receptor Dectin-1 (55) and DC-SIGN (CD209) (56) and report for age. Immunity 23: 344–346. 6. Goerdt, S., and C. E. Orfanos. 1999. Other functions, other genes: alternative the first time in mature macrophages the up-regulation of DCIR, activation of antigen-presenting cells. Immunity 10: 137–142. by guest on October 1, 2021 also called CLECSF6 (57), thoroughly studied in dendritic cells 7. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, and and the less described C-type lectin DCL-1 (58) and CLECSF13. T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-␥ genes. Science 259: 1739–1742. Alternatively activated macrophages are also characterized by in- 8. Nathan, C. F., H. W. Murray, M. E. Wiebe, and B. Y. Rubin. 1983. Identification creased expression of fibronectin (59), which is involved in cell of interferon-␥ as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158: 670–689. adhesion and migration processes during embryogenesis, wound 9. Goerdt, S., O. Politz, K. Schledzewski, R. Birk, A. Gratchev, P. Guillot, healing, blood coagulation, and metastasis. N. Hakiy, C. D. Klemke, E. Dippel, V. Kodelja, and C. E. Orfanos. 1999. Al- The solute carrier family of proteins comprises genes whose ternative versus classical activation of macrophages. Pathobiology 67: 222–226. 10. Mills, C. D., K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill. 2000. M-1/M-2 primary role is the transport of divalent cations and small organic macrophages and the Th1/Th2 paradigm. J. Immunol. 164: 6166–6173. molecules. They regulate transcription through DNA-binding pro- 11. Mosser, D. M. 2003. The many faces of macrophage activation. J. Leukocyte teins and metal response elements, the activity of enzymes includ- Biol. 73: 209–212. 12. Mantovani, A., S. Sozzani, M. Locati, P. Allavena, and A. Sica. 2002. Macro- ing metalloproteases, superoxide dismutase, inducible NO syn- phage polarization: tumor-associated macrophages as a paradigm for polarized thase, and functions like endosomal fusion, and metabolism. M2 mononuclear phagocytes. Trends Immunol. 23: 549–555. 13. Stein, M., S. Keshav, N. Harris, and S. Gordon. 1992. Interleukin 4 potently Despite the recognized role of some members in immune disease enhances murine macrophage mannose receptor activity: a marker of alternative susceptibility and infection (60), they have not been associated immunologic macrophage activation. J. Exp. Med. 176: 287–292. with macrophage polarization. We find that classically activated 14. Munder, M., K. Eichmann, and M. Modolell. 1998. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: macrophages are characterized by increased expression of the sol- competitive regulation by CD4ϩ T cells correlates with Th1/Th2 phenotype. ute carrier family members SLC21A15 and SLC31A2, while al- J. Immunol. 160: 5347–5354. ternatively activated macrophages exhibit increased SLC4A7, 15. Zhu, Z., T. Zheng, R. J. Homer, Y. K. Kim, N. Y. Chen, L. Cohn, Q. Hamid, and J. A. Elias. 2004. Acidic mammalian chitinase in asthmatic Th2 inflammation and SLC38A6 expression (Table I). The role of these molecules re- IL-13 pathway activation. Science 304: 1678–1682. mains to be elucidated. 16. Raes, G., P. De Baetselier, W. Noel, A. Beschin, F. Brombacher, and G. Hassanzadeh Gh. 2002. Differential expression of FIZZ1 and Ym1 in alter- Hitherto, expression data related to macrophage polarization pri- natively versus classically activated macrophages. J. Leukocyte Biol. 71: marily concern the murine system (3). Investigation of selected 597–602. markers in the human system have previously highlighted inter- 17. Scotton, C. J., F. O. Martinez, M. J. Smelt, M. Sironi, M. Locati, A. Mantovani, and S. Sozzani. 2005. Transcriptional profiling reveals complex regulation of the species discrepancy (17, 61). This report represents the first com- monocyte IL-1␤ system by IL-13. J. Immunol. 174: 834–845. prehensive description of the human mononuclear phagocyte sys- 18. MacMicking, J., Q. W. Xie, and C. Nathan. 1997. Nitric oxide and macrophage tem, and provides further evidence of relevant interspecies function. Annu. Rev. Immunol. 15: 323–350. 19. Martinez, F. O., M. Sironi, A. Vecchi, F. Colotta, A. Mantovani, and M. Locati. variability. For example, IL-4 in this study, as well as IL-13 in our 2004. IL-8 induces a specific transcriptional profile in human neutrophils: syn- previous expression-profiling experiments (17), did not induce the ergism with LPS for IL-1 production. Eur. J. Immunol. 34: 2286–2292. 20. Bolstad, B. M., F. Collin, K. M. Simpson, R. A. Irizarry, and T. P. Speed. 2004. human homolog of the mouse alternative activation markers argi- Experimental design and low-level analysis of microarray data. Int. Rev. Neuro- nase 1, Fizz1, MMP1 and Ym1. Similarly, a number of molecules biol. 60: 25–58. The Journal of Immunology 7311

21. Irizarry, R. A., B. M. Bolstad, F. Collin, L. M. Cope, B. Hobbs, and T. P. Speed. 42. Lehrke, M., and M. A. Lazar. 2004. Inflamed about obesity. Nat. Med. 10: 2003. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 126–127. 31: e15. 43. Pradalier, A., and D. Vincent. 2000. Aspirin: allergy or intolerance. Rev. Med. 22. Raychaudhuri, S., J. M. Stuart, and R. B. Altman. 2000. Principal components Interne 21(Suppl. 1): 75s–82s. analysis to summarize microarray experiments: application to sporulation time 44. Sanchez-Borges, M., A. Capriles-Hulett, and F. Caballero-Fonseca. 2004. Ad- series. Pac. Symp. Biocomput. 5: 452–463. verse reactions to selective cyclooxygenase-2 inhibitors (coxibs). Am. J. Ther. 11: 23. Reiner, A., D. Yekutieli, and Y. Benjamini. 2003. Identifying differentially ex- 494–500. pressed genes using false discovery rate controlling procedures. Bioinformatics 45. Chalfant, C. E., and S. Spiegel. 2005. Sphingosine 1-phosphate and ceramide 19: 368–375. 1-phosphate: expanding roles in cell signaling. J. Cell Sci. 118: 4605–4612. 24. Yeung, K. Y., D. R. Haynor, and W. L. Ruzzo. 2001. Validating clustering for 46. Duchateau, P. N., C. R. Pullinger, M. H. Cho, C. Eng, and J. P. Kane. 2001. gene expression data. Bioinformatics 17: 309–318. Apolipoprotein L gene family: tissue-specific expression, splicing, promoter re- 25. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein. 1998. Cluster anal- gions; discovery of a new gene. J. Lipid Res. 42: 620–630. ysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 47. Phillips, R. J., M. Lutz, and B. Premack. 2005. Differential signaling mechanisms 95: 14863–14868. regulate expression of CC chemokine receptor-2 during monocyte maturation. 26. Dennis, G., Jr., B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H. C. Lane, and J. Inflamm. 2: 14. R. A. Lempicki. 2003. DAVID: Database for Annotation, Visualization, and In- 48. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, tegrated Discovery. Genome Biol. 4: P3. S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine 27. Nikitin, A., S. Egorov, N. Daraselia, and I. Mazo. 2003. Pathway studio–the receptor expression during dendritic cell maturation. Eur. J. Immunol. 28: analysis and navigation of molecular networks. Bioinformatics 19: 2155–2157. 2760–2769. 28. Pattyn, F., F. Speleman, A. De Paepe, and J. Vandesompele. 2003. RTPrimerDB: 49. Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai, the real-time PCR primer and probe database. Nucleic Acids Res. 31: 122–123. O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regulation of che- 29. Payton, M., and S. Coats. 2002. Cyclin E2, the cycle continues. Int. J. Biochem. mokine receptors during dendritic cell maturation: a model for their trafficking Cell Biol. 34: 315–320. properties. J. Immunol. 161: 1083–1086. 30. Doree, M., and T. Hunt. 2002. From Cdc2 to Cdk1: when did the cell cycle kinase 50. Greenberg, S., F. Di Virgilio, T. H. Steinberg, and S. C. Silverstein. 1988. Ex- join its cyclin partner? J. Cell Sci. 115: 2461–2464. tracellular nucleotides mediate Ca2ϩ fluxes in J774 macrophages by two distinct 31. Vidal, A., and A. Koff. 2000. Cell-cycle inhibitors: three families united by a mechanisms. J. Biol. Chem. 263: 10337–10343. common cause. Gene 247: 1–15. 51. Di Virgilio, F., P. Chiozzi, D. Ferrari, S. Falzoni, J. M. Sanz, A. Morelli, Downloaded from 32. Carlin, J. M., E. C. Borden, and G. I. Byrne. 1989. Interferon-induced indoleam- M. Torboli, G. Bolognesi, and O. R. Baricordi. 2001. Nucleotide receptors: an ine 2,3-dioxygenase activity inhibits Chlamydia psittaci replication in human emerging family of regulatory molecules in blood cells. Blood 97: 587–600. macrophages. J. Interferon Res. 9: 329–337. 52. Murata, Y., T. Ohteki, S. Koyasu, and J. Hamuro. 2002. IFN-␥ and pro-inflam- 33. Hissong, B. D., G. I. Byrne, M. L. Padilla, and J. M. Carlin. 1995. Upregulation matory cytokine production by antigen-presenting cells is dictated by intracellular of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cul- thiol redox status regulated by oxygen tension. Eur. J. Immunol. 32: 2866–2873. tures by lipopolysaccharide, muramyl tripeptide, and interleukin-1. Cell Immunol. 53. Ohteki, T. 2002. Critical role for IL-15 in innate immunity. Curr. Mol. Med. 2: 160: 264–269. 371–380.

34. Martinez-Pomares, L., D. M. Reid, G. D. Brown, P. R. Taylor, R. J. Stillion, 54. Peiser, L., and S. Gordon. 2001. The function of scavenger receptors expressed http://www.jimmunol.org/ S. A. Linehan, S. Zamze, S. Gordon, and S. Y. Wong. 2003. Analysis of mannose by macrophages and their role in the regulation of inflammation. Microbes Infect. receptor regulation by IL-4, IL-10, and proteolytic processing using novel mono- 3: 149–159. clonal antibodies. J. Leukocyte Biol. 73: 604–613. 55. Willment, J. A., A. S. Marshall, D. M. Reid, D. L. Williams, S. Y. Wong, 35. Linehan, S. A., P. S. Coulson, R. A. Wilson, A. P. Mountford, F. Brombacher, S. Gordon, and G. D. Brown. 2005. The human ␤-glucan receptor is widely L. Martinez-Pomares, and S. Gordon. 2003. IL-4 receptor signaling is required expressed and functionally equivalent to murine Dectin-1 on primary cells. Eur. for mannose receptor expression by macrophages recruited to granulomata but J. Immunol. 35: 1539–1547. not resident cells in mice infected with Schistosoma mansoni. Lab. Invest. 83: 56. Relloso, M., A. Puig-Kroger, O. M. Pello, J. L. Rodriguez-Fernandez, G. de la Rosa, 1223–1231. N. Longo, J. Navarro, M. A. Munoz-Fernandez, P. Sanchez-Mateos, and A. L. Corbi. 36. Cornicelli, J. A., D. Butteiger, D. L. Rateri, K. Welch, and A. Daugherty. 2000. 2002. DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated Interleukin-4 augments acetylated LDL-induced cholesterol esterification in mac- by IFN, TGF-␤, and anti-inflammatory agents. J. Immunol. 168: 2634–2643. rophages. J. Lipid Res. 41: 376–383. 57. Kanazawa, N., K. Tashiro, and Y. Miyachi. 2004. Signaling and immune regu-

37. Barrios-Rodiles, M., and K. Chadee. 1998. Novel regulation of cyclooxygenase-2 latory role of the dendritic cell immunoreceptor (DCIR) family lectins: DCIR, by guest on October 1, 2021 ␥ expression and prostaglandin E2 production by IFN- in human macrophages. DCAR, dectin-2 and BDCA-2. Immunobiology 209: 179–190. J. Immunol. 161: 2441–2448. 58. Kato, M., S. Khan, N. Gonzalez, B. P. O’Neill, K. J. McDonald, B. J. Cooper, 38. Hashimoto, S., T. Suzuki, H. Y. Dong, N. Yamazaki, and K. Matsushima. 1999. N. Z. Angel, and D. N. Hart. 2003. Hodgkin’s lymphoma cell lines express a Serial analysis of gene expression in human monocytes and macrophages. Blood fusion protein encoded by intergenically spliced mRNA for the multilectin re- 94: 837–844. ceptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1. J. Biol. 39. Verreck, F. A., T. de Boer, D. M. Langenberg, M. A. Hoeve, M. Kramer, Chem. 278: 34035–34041. E. Vaisberg, R. Kastelein, A. Kolk, R. de Waal-Malefyt, and T. H. Ottenhoff. 59. Gratchev, A., P. Guillot, N. Hakiy, O. Politz, C. E. Orfanos, K. Schledzewski, and 2004. Human IL-23-producing type 1 macrophages promote but IL-10-producing S. Goerdt. 2001. Alternatively activated macrophages differentially express fi- type 2 macrophages subvert immunity to (myco)bacteria. Proc. Natl. Acad. Sci. bronectin and its splice variants and the extracellular matrix protein ␤IG-H3. USA 101: 4560–4565. Scand. J. Immunol. 53: 386–392. 40. Cheung, D. L., and J. A. Hamilton. 1992. Regulation of human monocyte DNA 60. Blackwell, J. M., S. Searle, H. Mohamed, and J. K. White. 2003. Divalent cation synthesis by colony-stimulating factors, cytokines, and cyclic adenosine mono- transport and susceptibility to infectious and autoimmune disease: continuation of phosphate. Blood 79: 1972–1981. the Ity/Lsh/Bcg/Nramp1/Slc11a1 gene story. Immunol. Lett. 85: 197–203. 41. Bischof, R. J., D. Zafiropoulos, J. A. Hamilton, and I. K. Campbell. 2000. Ex- 61. Raes, G., R. Van den Bergh, P. De Baetselier, G. H. Ghassabeh, C. Scotton, acerbation of acute inflammatory arthritis by the colony-stimulating factors M. Locati, A. Mantovani, and S. Sozzani. 2005. Arginase-1 and Ym1 are markers CSF-1 and granulocyte macrophage (GM)-CSF: evidence of macrophage infil- for murine, but not human, alternatively activated myeloid cells. J. Immunol. 174: tration and local proliferation. Clin. Exp. Immunol. 119: 361–367. 6561; author reply 6561–6562.