Stabilin-1 Suppresses the Activation of Th1 Senthil Palani, Kati Elima, Eeva Ekholm, Sirpa Jalkanen and Marko Salmi This information is current as of October 1, 2021. J Immunol published online 25 November 2015 http://www.jimmunol.org/content/early/2015/11/25/jimmun ol.1500257 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/11/25/jimmunol.150025 Material 7.DCSupplemental

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 25, 2015, doi:10.4049/jimmunol.1500257 The Journal of Immunology

Monocyte Stabilin-1 Suppresses the Activation of Th1 Lymphocytes

Senthil Palani,*,†,‡ Kati Elima,*,x Eeva Ekholm,{ Sirpa Jalkanen,*,‖ and Marko Salmi*,‖

In this study, we analyzed the putative functions of stabilin-1 in blood . Microarray analysis revealed downregulation of several proinflammatory in the stabilin-1high monocytes when compared with stabilin-1low monocytes. When cocultured with stabilin-1high monocytes, IFN-g synthesis by T cells was diminished in Ag-recall assays. Knockdown of stabilin-1 in monocytes increased the synthesis of several proinflammatory molecules, including TNF-a, and supported high IFN-g and low IL-4 and IL-5 production by T cells in Ag-specific stimulation assays. Anti–stabilin-1 Ab treatment also led to increased IFN-g synthesis in the recall assays. In clinical settings, the expression of stabilin-1 was diminished on blood monocytes and tissue under proinflammatory conditions. These data define stabilin-1 as a new immunosuppressive molecule and suggest that stabilin-1high monocytes may dampen proinflammatory reactions in vivo. The Journal of Immunology, 2016, 196: 000–000. Downloaded from onocytes are bone marrow–derived subsets of circu- subset of tumor-associated macrophages (11). Stabilin-1 is also lating WBCs, which can differentiate into mac- expressed in monocytes during specific disease conditions, like M rophages and dendritic cells after migration into hypercholesterolemia, but reported to be absent from normal different tissues (1). Monocytes play an important role in tissue monocytes (12). In macrophages, stabilin-1 acts as a scavenging homeostasis, wound healing, and host defense reactions against receptor during endocytosis of selected such as oxidized

microbes, other inflammatory stimuli, and tumor cells. Based on and acetylated low-density lipoprotein (Ac-LDL), OVA, SPARC, http://www.jimmunol.org/ the expression of CD14 and CD16, human monocytes are classi- and placental lactogen (13–16). It has also been reported to be fied into three subsets, which also have different transcriptomes involved in bacterial binding (17), clearance of apoptotic cell (1–3). The majority (80–90%) of blood monocytes belong to the bodies (18), and wound healing (10) and in adhesion and trans- CD14+CD162 subset, which, together with the CD14+CD16+ migration of placental leukocytes through endothelial cells (19). subpopulation, are proinflammatory monocytes analogous to Although stabilin-1 is a useful marker for a subset of type 2 hi hi low mouse Ly6C CCR2 CX3CR1 cells. The nonclassical minor macrophages, nothing is known about its functional role during CD14dimCD16+ human monocyte population, in contrast, resem- the polarization of immune responses. low low hi bles mouse Ly6C CCR2 CX3CR1 cells, which constantly In this study, we unexpectedly found definitive expression of patrol the vessels and are involved in cell maintenance and healing stabilin-1 on the surface of normal blood monocytes when using by guest on October 1, 2021 functions (3, 4). new sensitive Abs. In functional experiments, the stabilin-1low Stabilin-1 (also known as CLEVER-1 or FEEL-1) is a multi- monocyte population and stabilin-1 knockdown monocytes sup- functional type I transmembrane most prominently ex- ported the generation of robust proinflammatory immune re- pressed on selected endothelial cells and alternatively activated sponses. Notably, ligation of monocyte stabilin-1 by an anti– M2 macrophages (5–9). Stabilin-1 synthesis is induced in mac- stabilin-1 Ab also diverted Ag-specific responses into the Th1 rophages during inflammation (10), and it is highly expressed in a direction. These data thus define stabilin-1 as a novel immune modulator.

*MediCity Research Laboratory, University of Turku, Turku 20520, Finland; †Turku Doctoral Programme of Molecular Medicine, University of Turku, Turku Materials and Methods ‡ 20520, Finland; Turku Doctoral Programme of Biomedical Sciences, University Study populations of Turku, Turku 20520, Finland; xDepartment of Medical Biochemistry and Genetics, University of Turku, Turku 20520, Finland; {Department of Obstetrics and Gynecol- ‖ Blood samples from healthy volunteers, tetanus-vaccinated persons (pre- ogy, University of Turku, Turku 20520, Finland; and Department of Medical Mi- viously vaccinated individuals who had received a booster injection within crobiology and Immunology, University of Turku, Turku 20520, Finland past 2 y), and timothy grass–allergic (self-reported) persons were collected ORCID: 0000-0001-7435-5398 (S.P.). using venipuncture. All donors gave their informed consent. Received for publication February 4, 2015. Accepted for publication October 28, Blood samples and samples of placenta and placental bed were taken at 2015. elective caesarean sections performed for defined obstetrical indications from women with normal pregnancies and from those with pre-eclampsia at The sequences presented in this article have been submitted to the Expression Omnibus under accession numbers GSE63519 and GSE63807. the Department of Obstetrics in Turku University Central Hospital. These samples were collected with permission of the Ethical Committee of the Address correspondence and reprint requests to Prof. Marko Salmi, MediCity Re- University of Turku, and a written consent was signed by all patients search Laboratory, University of Turku, Tykisto¨katu 6A, Turku FIN-20520, Finland. participating in this study. Apart from the treating clinician (E.E.), the E-mail address: marko.salmi@utu.fi patients remained anonymous to the other investigators. The online version of this article contains supplemental material. Abbreviations used in this article: Ac-LDL, acetylated low-density lipoprotein; DE, Abs differentially expressed; FMSC, Finnish Microarray and Sequencing Centre; GO, 9-11 (a rat IgG2a) and 3-372 (a mouse IgG1) are against human stabilin-1 ; GSEA, Gene Set Enrichment Analysis; IPA, Ingenuity Pathway Analysis; OSM, oncostatin M; qPCR, quantitative PCR; RNAseq, RNA sequencing; (8, 19). The other mAbs used were: CD14- FITC (mouse IgG2a; Southern RQ, relative quantification; SAA, serum amyloid A; SFC, spot-forming cell; siRNA, Biotechnology Associates), CD16-PerCP–Cy5.5 (mouse IgG1; BD Phar- small interfering RNA. mingen), HLA-DR–allophycocyanin (mouse IgG2a; BD Pharmingen), and mannose receptor (CD206, clone 15-2 MRC-1; Lifespan Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 Biosciences). As isotype controls, IgG2a-FITC, IgG1-PerCP–Cy5.5,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500257 2 IMMUNOSUPPRESSIVE STABILIN-1 IN MONOCYTES

IgG2a-allophycocyanin (all from BD Pharmingen), MEL-14 (rat IgG2a), anti-biotin secondary Ab (1:1000), and spots were developed with BCI- and 3G6 (mouse IgG1) were used. The second-stage reagents were PE- NBT solution. conjugated goat anti-mouse IgG (a whole Ig molecule from Southern The specific spot numbers for each coculture condition were counted Biotechnology Associates) and PE-conjugated goat polyclonal F(ab)2 microscopically by subtracting the number of spots recorded in three against anti-rat F(ab)2 (Abcam). F(ab)2 fragments of 9-11 and MEL-14 controls: 1) the cocultures of the corresponding monocytes and T cells were generated commercially by GenScript. without the Ag; 2) the control cultures of the corresponding monocytes with the Ag only (no T cells); and 3) the control cultures of the T cells and Immunofluorescence stainings and FACS analyses the Ag only (no monocytes) from the number of spots recorded in the cocultures in the presence of the monocytes, T cells, and Ag. To normalize Blood samples were collected in EDTA tubes. RBCs were lysed using a the interindividual variation in the absolute numbers of spots, the specific commercial lysis buffer (BD Pharm Lyse; BD Biosciences). After pre- blocking with human Ig (100 mg/ml; KIOVIG from Baxter) the leukocytes were sequentially incubated with anti–stabilin-1 9-11 F(ab)2 or nega- tive control MEL-14 F(ab)2 (20 mg/ml) and PE-conjugated goat anti-rat F(ab)2–F(ab)2 Ab. In monocyte phenotyping experiments, the cells were further incubated with a mixture of anti-human HLA-DR, CD14, and CD16 mAbs (or with the appropriate isotype controls). In the polarization experiments (see below), the cultured leukocytes were harvested and surface stained for stabilin-1 [as above with 9-11 F(ab)2] or for MRC-1 using the anti–MRC-1 mAb (or an isotype control [10 mg/ml]) followed by PE-conjugated secondary goat anti-mouse Ab. Alternatively, aliquots of the cells were permeabilized (15 s in ice-cold acetone), blocked, and stained for total stabilin-1. The cells were fixed using paraformaldehyde

and analyzed using FACS Aria II or FACSCalibur (BD Biosciences). Downloaded from Cell sortings After RBC lysis, the cells were stained for stabilin-1 and CD14 (see above) for sorting by FACSAria (BD Biosciences). Monocytes were identified using scatter profiles, CD14 and stabilin-1 double-positive monocytes were gated using FACS Diva software (BD Biosciences), and the brightest high low +

(stabilin-1 ) and dimmest (stabilin-1 ) 10% of stabilin-1 monocytes http://www.jimmunol.org/ were collected. Small interfering RNA transfections Purified monocytes (see below) were transfected with stabilin-1 small interfering RNA (siRNA) and negative control siRNA (59-UCAA- GUCGCUGCCUGCAUA-39) (ON-TARGETplus Human STAB-1 and Nontargeting siRNA, respectively; GE Healthcare) as previously described (19). The cells were then cultured for 24–96 h under nonpolarizing culture medium. Stabilin-1 expression was determined by FACS from aliquots of

cells to verify the silencing efficacy. by guest on October 1, 2021 Ac-LDL uptake assay Negative control and stabilin-1 siRNA-transfected monocytes were har- vested on day 2. The cells were treated with Alexa 488–labeled Ac-LDL (Invitrogen; 10 mg/ml in RPMI 1640 containing 10% FCS for 4 h), washed, and analyzed by FACS as described (19). ELISA for quantification of TNF-a Conditioned culture medium from negative control and stabilin-1 siRNA- transfected monocytes were collected on day 2. TNF-a protein levels were quantified using a sensitive ELISA kit (catalog no. KHC3014; Invitrogen) according to the manufacturer’s instructions. Ag-specific recall assays PBMCs from adults recently vaccinated against tetanus and from timothy grass–allergic persons were collected using Ficoll. Monocytes were enriched using MACS negative selection kit (Monocyte isolation Kit II with CD16–MACS; Miltenyi Biotec). Autologous T cells were isolated from the blood samples of the same donors (either on the same day for cocultures with sorted monocytes or on day 21 for cocultures with siRNA- transfected monocytes) using negative selection (T Cell Enrichment Kit, EasySep; Stemcell Technologies). The 96-well ELISPOT plates (Mabtech) were coated with anti-human 2 IFN-g (1 mg/ml; Mabtech), IL-4 (15 mg/ml; Mabtech), or IL-5 (15 mg/ml; FIGURE 1. Human CD14+CD16 and CD14+CD16+ monocytes ex- Mabtech) for 24–48 h at 4˚C, washed with PBS, and blocked (RPMI 1640 press stabilin-1 on the cell surface. (A) Representative FACS analyses of plus 10% FCS). The purified monocytes and T cells were cocultured in the stabilin-1 expression on human whole blood after lysis of erythrocytes. precoated ELISPOT wells as triplicates at a ratio of 1:10 (10,000 mono- The histograms (stabilin-1 [red] and control [gray]) from the monocyte cytes and 100,000 T cells) in a medium (RPMI 1640, 10% FCS, 2 mmol gate (M) are shown. (B) FACS analyses of gated HLA-DR+ monocytes for L-glutamine, 100 mmol 2-ME, and 50 mg/ml gentamicin) containing teta- CD14, CD16, and stabilin-1. Representative histograms and quantification nus toxoid (20 mg/ml; National Public Health Institute, Helsinki, Finland) or timothy extract (100 mg/ml; GREER Laboratories) for 3 d at 37˚C in a of stabilin-1 expression (percentage of positive cells and the level of ex- pression [mean fluorescence intensity (MFI)]) on the indicated populations CO2 incubator. Monocytes and T cells cultured in the same medium without any stimulation served as background controls. On day 3, the wells are shown. All quantitative data are mean 6 SEM (n = 3 individuals). *p , were washed and sequentially incubated with biotinylated anti–IFN-g, 0.05, **p , 0.01. FSC-A, forward light scatter area; SSC-A, side scatter IL-4, or IL-5 (all at 1 mg/ml) Abs and alkaline phosphatase–conjugated area. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Stabilin-1low monocyte population and stabilin-1–silenced monocytes show preferential expression of proinflammatory genes. (A)FACS sorting strategy for separating the 10% of monocytes with the highest and lowest stabilin-1 expression and the purity controls after the sorting. (B) Heat map clustering of the DE genes between stabilin-1high and stabilin-1low monocytes. Hierarchical clustering of the samples and filtered genes has been performed using Pearson’s metrics. The colors represent the relative expression of a given gene in comparison with the median of all samples (red, high expression; green, low expression); the roman numeral denotes each individual sample. (C) Volcano plot of the microarray data depicting the statistical significance as 2log10 p value (y-axis) against the fold change as log2 ratio (x-axis) between stabilin-1high– and stabilin-1low–positive monocytes. Genes with p values #0.01 and log2 fold change (FC) below 21 (green) or .1 (red) are indicated. (D) Differential expression of the 20 most up- and downregulated genes in stabilin-1high monocytes in comparison with stabilin-1low monocytes. Red columns, upregulated genes; green columns, downregulated genes. y-axis, fold change. All data are from four stabilin-1high and three stabilin-1low monocyte populations (each isolated from a different individual). (E) GSEA analyses of enriched gene sets in the stabilin-1low (three samples) and stabilin-1high (four samples) monocyte populations showing the heat (Figure legend continues) 4 IMMUNOSUPPRESSIVE STABILIN-1 IN MONOCYTES spot number in the stabilin-1high and control siRNA-treated wells was then conductor packages. The data were further analyzed using Qiagen’s IPA assigned a value of 100 for each person. platform and GSEA software (http://www.broadinstitute.org/gsea/index. In Ab-blocking experiments, PBMCs (unfractionated) isolated from jsp) (20, 21) and deposited into the Omnibus (acces- tetanus- vaccinated persons were incubated with stabilin-1 (3-372) Ab, a sion number GSE63807). nonbinding negative control Ab 3G6, or a binding, irrelevant control Ab CD14 (all at 20 mg/ml) and plated to IFN-g–coated wells for 3 d. Quantitative PCR In vitro polarizations Total RNA was extracted and reverse-transcribed with the iScript cDNA Synthesis Kit (Bio-Rad) or SuperScript VILO cDNA Synthesis Kit (Life MACS-purified monocytes (1 3 106 cells/well) were cultured in the growth Technologies). TaqMan Gene Expression Assays (Applied Biosystems) for medium (IMDM containing 10% FCS and 2 mmol L-glutamine to obtain ALOX5AP, PADI4, SPP1, ORM2, GNRH2, LHCGR, oncostatin M (OSM), nonpolarized cells. To induce M1 differentiation, TNF-a (50 U/ml) and serum amyloid A (SAA) 2, SCGB3A1, TBP, and B2M were used as primer/ LPS (10 ng/ml) were added, and to induce M2 differentiation, IL-4 (10 probe sets, and the PCR reactions were carried out as suggested by the ng/ml) and M-CSF (10 ng/ml); IL-4 (10 ng/ml), M-CSF (10 ng/ml), and supplier using the Applied Biosystems 7900HT Fast Real-Time PCR dexamethasone (100 nmol); dexamethasone (100 nmol) only; or IL-4 (10 System (Applied Biosystems) in the FMSC. All samples were run as ng/ml) only were added. triplicates, and the expression values were normalized using human TBP or b2-microglobulin as endogenous controls. The results were analyzed with Microarray analysis SDS 2.3 software and DataAssist v3.01. The average mRNA expression of high low each gene was presented as a relative quantification (RQ) values using the RNA was extracted from the sorted stabilin-1 – and stabilin-1 –posi- 2DDCT tive monocytes using the Nucleo-Spin RNAII Total RNA Isolation Kit 2 method, in which RQ = 1 is the value in the control group. (Macherey-Nagel). RNA integrity numbers (Agilent 2100 Bioanalyzer; Immunohistochemistry Agilent Technologies) were .8.00 in all samples. The subsequent microarray analysis using the Agilent Sure Print G3 Human Gene Ex- Small pieces of placental bed were embedded in OCT and stored at 270˚C. pression Microarray 8 3 60K (Agilent Technologies) was performed at the Frozen sections were cut, acetone-fixed, and immunohistochemically Downloaded from Finnish Microarray and Sequencing Centre (FMSC). Fluorescence signals stained for stabilin-1 as described (19). The number of stabilin-1+ cells per were detected using Agilent’s Microarray Scanner System (Agilent field (0.268 mm2) was counted with an Olympus BX-60 microscope Technologies), and the Agilent Feature Extraction Software (Agilent (Olympus). Technologies) was used in further processing of the data. The resulting intensity data were normalized using quantile normali- Statistics zation. The samples were hierarchically clustered and the correlation values Student t test (unpaired, two-sided for the pregnancy samples and paired, determined with Pearson metrics. R package Limma was used for per- two-sided for the rest of the experiments) was used, except for the http://www.jimmunol.org/ forming the statistical testing between the groups. The differentially microarray and RNAseq, in which the R-package Limma was used. The expressed (DE) genes were selected requiring an absolute fold change .2 p values ,0.05 were considered to be statistically significant. and p value ,0.01. Functional enrichment analysis was conducted against the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes databases for both DE gene lists and unfiltered ranked comparison data Results using topGO and GOstats packages in R/Bioconductor. The data were Normal human monocytes express stabilin-1 on the surface further analyzed using Qiagen’s Ingenuity Pathway Analysis (IPA) plat- form and Gene Set Enrichment Analysis (GSEA) software (http://www. Stabilin-1 is reported to be present in M2 macrophages (5–8), but broadinstitute.org/gsea/index.jsp) (20, 21). The data has been deposited not on normal blood monocytes (12). Because monocytes are into the Gene Expression Omnibus with the accession number GSE63519 notoriously difficult to stain due to high expression of FcR, we re- (http://www.ncbi.nlm.nih.gov/genbank). by guest on October 1, 2021 examined this issue by generating a F(ab)2 fragment from our RNA sequencing analysis new specific anti–stabilin-1 mAb 9-11 and isotype-matched nega- RNA was extracted and quality controlled (RNA integrity number .9.00) tive control mAb (19). To further increase the specificity of the from stabilin-1 siRNA-transfected and negative control siRNA-transfected stainings, we preblocked the FcRs with human Igs and took human monocytes from three healthy donors as above. The samples were advantage of a PE-conjugated F(ab)2 fragment of an anti-F(ab)2 sequenced in FMSC with the HiSeq2500 instrument (Illumina) using Ab as the second-stage reagent. Using this protocol, we found low, single-end sequencing chemistry with 50-bp read length. The reads were aligned against the human reference genome (hg19 assembly, downloaded but consistent, stabilin-1 expression on the surface of monocytes from University of California, Santa Cruz) using TopHat version 2.0.10. (identified based on the typical scatter profiles) isolated from The number of uniquely aligned reads was between 10.2 and 14.2 M per normal healthy donors (Fig. 1A). To further characterize stabilin-1 sample. HTSeq (v.0.5.4p3) was used for counting the genewise read counts expression among the heterogeneous monocyte population, we as defined by RefSeq gene annotations. stained PBMCs for HLA-DR, CD14, and CD16 (or the corre- The sample correlation values (Spearman metrics) were between 0.93 + and 0.98 for all sample groups, indicating high reproducibility. The data sponding isotype controls). We then gated for HLA-DR cells were normalized using the trimmed mean of M-values normalization within the monocyte gate, and identified the three monocyte method of the edgeR R/Bioconductor package prior to statistical testing populations based on CD14 and CD16 expression, as reported (3). with Limma R/Bioconductor package. The DE genes were selected re- Stabilin-1 was clearly expressed on CD14+CD16+ and on CD14+ quiring an absolute fold change .2 and p value ,0.05. Functional en- 2 dim + richment analysis was conducted against the GO and Kyoto Encyclopedia CD16 populations, but not on CD14 CD16 cells (Fig. 1B). of Genes and Genomes and Reactome databases for both DE gene lists and Stabilin-1 was absent from the lymphocytes and unfiltered ranked comparison data using topGO and GOstats Gage Bio- (Supplemental Fig. 1). Thus, the two classical CD14+ monocyte

map profiles (red is highest and dark blue is lowest expression) of the top 20 highest ranking genes in the enriched gene sets. (F) Representative examples and quantification of FACS analyses studying stabilin-1 expression (red histograms) in permeabilized monocytes 1 and 2 d after transfection with stabilin-1 or control siRNA. Black histograms are isotype controls. Stabilin-1 expression in negative control (Neg co) siRNA-treated monocytes was set to 100 in each experiment (n = 3). Heat map clustering of the DE genes (G), volcano plot (H, genes with p values #0.05 and log2 fold change (FC) below 21 (green) or .1 (red) are indicated), and differential gene expression (I) in RNAseq analyses between stabilin-1 siRNA-transfected monocytes and negative control siRNA-transfected monocytes after 1 d. (J) IPA upstream regulator analysis showing the predicted upstream regulator (the central square, TNF) and the known target genes of the upstream regulator, which were found among the DE genes in the RNAseq experiments (symbols surrounding the central square). (K) ELISA analyses of secreted TNF-a protein concentrations in the conditioned medium from negative control (Neg co) and stabilin-1 siRNA-silenced monocytes on day 2. The TNF level in the negative control siRNA-treated monocytes was set to 100 in each experiment (n = 4). All quantitative data are mean 6 SEM. *p , 0.05. FSC-A, forward light scatter area; FSC-H, forward light scatter height; MFI, mean fluorescence intensity; NES, normalized enrichment score; ROS, reactive oxygen species; SSC-H, side scatter height. The Journal of Immunology 5 subpopulations in human blood express stabilin-1 on their surface under normal conditions. Stabilin-1high and stabilin-1low monocyte subpopulations have distinct gene expression signatures We sorted CD14+ monocytes into stabilin-1high– and stabilin-1low– positive cells (Fig. 2A). Reanalyses of the sorted cells verified that the purity of each subpopulation was .95% (Fig. 2A). Heat map and volcano plot analyses of the cDNA microarray data revealed many DE genes between the two populations (Fig. 2B–D, Supplemental Table I). The maximally differently up- and downregulated genes (with fold change .2) in the stabilin-1high monocytes compared with stabilin-1low are shown in Fig. 2D. Because protein–protein interactions and signaling pathways related to stabilin-1 are relatively poorly understood, we used manual inspection of the top downregulated hits and performed IPA to assign these DE genes into functional categories and pathways. These analyses revealed that several of the downregu- lated genes in the stabilin-1high population (Supplemental Table I) exhibit immune-related activities. For instance, the three top hits Downloaded from are, in general, known to support proinflammatory reactions (GO: 0006954 [inflammatory response, 16 genes; p value 2.5 3 1029], GO: 0006952 [defense response, 19 genes; p value 6.3 3 1029], and GO: 0009611 [response to wounding, 18 genes; p value 6.8 3 1029] and data not shown). Furthermore, the upstream regulator analysis of IPA predicted that TNF-a, a prototype proin- http://www.jimmunol.org/ flammatory cytokine, is inhibited in the stabilin-1high population (data not shown). Further GSEA analysis revealed four signifi- cantly enriched gene sets (normalized enrichment scores with false discovery rates ,25%), including proinflammatory IL-2/ STAT5 and TNF/NF-kB gene sets, in the stabilin-1low pop- ulation (Fig. 2E). Therefore, we then chose three of the proin- flammatory genes, ALOX5AP, PADI4, and SPP1 (22–24), which were found to be downregulated in stabilin-1high cells in the microarray, and further verified their expression by quantitative by guest on October 1, 2021 PCR (qPCR). The results showed that the expression of all three were indeed lower in the stabilin-1high population (RQs for ALOX5AP, PADI4, and SPP1 were 0.23, 0.23, and 0.29, respec- tively) when compared with stabilin-1low population. Collectively, these data imply that a population of CD14+ monocytes charac- terized by high stabilin-1 expression has a transcriptome sugges- tive of a reduced proinflammatory potential.

Downregulation of stabilin-1 is linked to upregulation of proinflammatory genes To study the effects of stabilin-1 knockdown on global gene ex- pression, we aimed at silencing stabilin-1 in monocytes using siRNA. Initial FACS analyses of stabilin-1 expression in per- meabilized cells (detecting both surface and intracellular protein) showed that stabilin-1 knockdown led to a 44.8 6 9.0% (mean 6 SEM; n = 3) decrease in stabilin-1 protein expression on day 1 and 64.1 6 9.8% (mean 6 SEM; n = 3) decrease on day 2 (Fig. 2F). FIGURE 3. Monocyte stabilin-1 suppresses proinflammatory Ag-specific The reduction in the stabilin-1 expression after the knockdown Th1 responses. (A) Schematic diagram showing the experimental set up also correlated to diminished binding of Alexa 488–labeled Ac- for ELISPOT assays with stabilin-1low– and stabilin-1high–sorted monocyte LDL (24.2 6 7.2% [mean 6 SEM; n =6;p value = 0.0001]), populations and stabilin-1 siRNA-silenced monocytes. The Ag was tetanus which is one of the known functions of stabilin-1 (13, 17, 19). toxoid (TT) or timothy grass extract. IFN-g production by TT-specific T cells high low B After confirming the efficacy and functional consequences of cocultured with stabilin-1 and stabilin-1 monocytes (n =4)( )and stabilin-1 and negative control (Neg co) siRNA-silenced monocytes for 3 d stabilin-1 silencing in monocytes, we then subjected stabilin-1 and (C)(n = 3). (D) Representative ELISPOT well micrographs from (C). (E)IL- negative control siRNA-treated monocytes to RNA sequencing 4 and IL-5 production on day 3 by T cells cocultured with negative control (RNAseq) analyses after 1 and 2 d culture under nonpolarizing (Neg co) and stabilin-1 siRNA-transfected monocytes (n = 3). (F)IFN-g conditions. Already on day 1, several genes (such as OSM, production after TT stimulation at day 3 in total PBMCs culture of tetanus- CXCL13, and SAA2) involved in the activation of the proin- vaccinated persons treated with anti–stabilin-1 or nonbinding (3G6) and flammatory pathway were upregulated in stabilin-1–silenced cells binding (CD14) negative control mAbs (n = 4). Quantitative data are mean 6 when compared with negative control siRNA-transfected mono- SEM. *p , 0.05, **p , 0.01, ***p , 0.001. No Mono, no monocytes. 6 IMMUNOSUPPRESSIVE STABILIN-1 IN MONOCYTES cytes (Fig. 2G–I, Supplemental Table II). Our qPCR results ver- To study whether stabilin-1 itself has an impact on cytokine ified the altered gene expression of the selected hits in stabilin-1– production, we silenced stabilin-1 in the total pool of monocytes silenced cells (RQs for CXCL13, OSM, SAA2, and SCGB3A1 using siRNA during the coculture experiments (Fig. 3A). Stabilin-1 were 2.71, 2.22, 1.26, and 0.69, respectively, in stabilin-1 siRNA- knockdown specifically led to a 48.5 6 9.4% (mean 6 SEM; n = treated cells when compared with negative control siRNA-treated 3) decrease in stabilin-1 protein expression in monocytes at the cells). GSEA analysis did not reveal any significantly enriched beginning of the coculture and to a 65.4 6 4.9% (mean 6 SEM; gene sets in stabilin-1–silenced cells. However, further analysis n = 3) decrease at the end of the coculture. The ELISPOT assays with IPA on DE genes from stabilin-1–silenced monocytes pre- revealed that the numbers of IFN-g SFC produced in the pres- dicted that TNF-a is involved in the regulation of several of these ence of stabilin-1 siRNA-transfected monocytes were higher induced genes (Fig. 2J). To verify this prediction, we analyzed than those produced in the presence of negative control siRNA- TNF-a levels from the conditioned culture medium of control transfected monocytes (Fig. 3C). The controls verified the and stabilin-1 siRNA-treated monocytes by ELISA. The results specificity of the assays, because no spots were detectable in the showed that stabilin-1–silenced monocytes produced significantly coculture in the absence of the specific Ag or in the absence of more TNF-a when compared with control transfected cells (Fig. T cells (Fig. 3D, Supplemental Fig. 2). These results thus show 2K). These data indicate that stabilin-1 in monocytes directly or that stabilin-1 itself, directly or indirectly, is involved in the indirectly controls the activation of several proinflammatory genes regulation of the immune responsiveness and that low levels of in human monocytes. stabilin-1 in monocytes during Ag presentation favor strong IFN-g production by T cells. Monocyte stabilin-1 regulates cytokine production during Tetanus toxoid stimulation did not lead to the generation of immune responses quantifiable numbers of type 2 cytokine-forming spots (data not Downloaded from To study the functional difference between stabilin-1high and sta- shown). Therefore, we chose timothy grass extract as a potential bilin-1low monocytes, we evaluated cytokine production in Ag- allergen inducing Th2-type cytokine production (Fig. 3A). To restimulation experiments. We isolated blood monocytes from enhance the responsiveness, we isolated both monocytes and the tetanus-vaccinated persons, separated stabilin-1high and stabilin- autologous T cells for the coculture experiments from persons 1low monocytes by sorting and cocultured them with T cells iso- with known allergy against timothy. In these ELISPOT assays, we lated from the same persons in the presence of tetanus toxoid found that T cells in the presence of stabilin-1 siRNA-transfected http://www.jimmunol.org/ (Fig. 3A). In these experiments, T cells cocultured with stabilin- monocytes produced fewer IL-4 and IL-5 spots compared with 1high monocytes produced lower numbers of IFN-g spot-forming those cultured with negative control siRNA-transfected monocytes cells (SFC) compared with those cultured with stabilin-1low–pos- (Fig. 3E). Collectively, these data show that stabilin-1 in mono- itive monocytes (Fig. 3B). These data show that when the Ag cytes favors the generation of Th2/immunosuppressive responses presentation takes place via the stabilin-1high monocyte pop- and impairs the formation of Th1/proinflammatory immune re- ulation, the outcome is an impaired Th1 cytokine response. sponsiveness in humans. by guest on October 1, 2021 FIGURE 4. M1 polarization sup- presses stabilin-1 expression in monocytes/macrophages. (A) Rep- resentative FACS analyses of stabi- lin-1 and macrophage mannose receptor (MRC-1) expression in freshly isolated monocytes and in monocytes cultured for 2 d under the indicated nonpolarizing (neutral medium), M1-polarizing (LPS and TNF-a), or M2-polarizing (M-CSF and IL-4, or M-CSF, IL-4 and dexamethasone, IL-4 only, or dexa- methasone only) conditions without or with permeabilization. Black histograms are isotype controls. Quantification of surface stabilin-1 (n =4)(B), surface mannose re- ceptor (MRC-1) (n =4)(C), and total stabilin-1 (permeabilized cells; n =3)(D) expression (mean fluo- rescence intensity [MFI]) in mono- cytes after the 2 d culture under the indicated polarization conditions. Under each condition the MFI of a negative control Ab has been assigned value of 100, and the bars show the MFI of the specific stainings. All quantitative data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. DEX, dexametha- sone. The Journal of Immunology 7

Anti–stabilin-1 Abs modulate Ag responses to a proinflammatory direction To study whether stabilin-1 would represent a new target for therapeutic manipulation of immune responses, we took advantage of anti–stabilin-1 Abs. We treated unfractionated PBMCs (only monocytes express stabilin-1 in this cell population; Supplemental Fig. 1), isolated from tetanus-vaccinated persons with anti– stabilin-1 and control Abs. We found that PBMCs blocked with anti–stabilin-1 Ab produced high numbers of IFN-g SFC com- pared with PBMCs treated with irrelevant nonbinding (3G6) or binding (anti-CD14) control Abs (Fig. 3F). Anti–stabilin-1 Ab also specifically increased the number of IFN-g SFC when a lower dose (1 and 10 mg/ml) of the Ag was used (data not shown). Thus, ligation of stabilin-1 on monocytes with a function blocking Ab tilts the Ag-specific recall response to a proinflammatory Th1 direction. Stabilin-1 is downregulated in proinflammatory monocytes/ macrophages Downloaded from To verify the induction of stabilin-1 during in vitro differentiation of monocytes into M2 macrophages (7), we cultured the purified monocytes isolated from human blood under nonpolarizing, M1- polarizing (LPS and TNF-a), or M2-polarizing (M-CSF and IL-4; M-CSF, IL-4, and dexamethasone; IL-4 only; or dexamethasone only) conditions. Stainings after 2 d showed clear stabilin-1 pos- itivity with our new anti–stabilin-1 Ab on the surface of mono- http://www.jimmunol.org/ cytes cultured under nonpolarizing and all M2-inducing conditions FIGURE 5. High expression of stabilin-1 on the monocyte and macro- (Fig. 4A, 4B). In contrast, stabilin-1 expression was strongly phage surfaces correlates with immunosuppression in vivo. FACS analyses + A + downregulated under M1-polarizing (LPS and TNF-a) conditions. of stabilin-1 expression in CD14 blood monocytes ( ) and CD14 pla- cental macrophages (B) in normal pregnancy and pre-eclampsia (n = 6). Macrophage mannose receptor, a prototype marker for M2 cells, Stabilin-1 staining is shown in red histograms, and black histograms are was also induced, as expected, under all M2-inducing conditions, isotype controls. (C) Immunohistological analyses of stabilin-1 expression but not under nonpolarizing or M1 (LPS and TNF-a) (Fig. 4C). in placental bed in normal pregnancy and pre-eclampsia. Representative Notably, M2 stimulation not only prevented the loss of stabilin-1 micrographs (arrows point to representative brown [stabilin-1–expressing] 2 expression on the cell surface when compared with non- cells in a pre-eclampsia sample) and quantification (field is 0.268 mm )of by guest on October 1, 2021 polarizing conditions, but also induced intracellular stabilin-1 the numbers of stabilin-1–positive macrophages are shown (n = 6). Orig- expression (Fig. 4D). Collectively, these data show that human inal magnification 3400. All quantitative data are mean 6 SEM. *p , monocytes retain stabilin-1 surface expression when the cells are 0.05, ***p , 0.001. MFI, mean fluorescence intensity. polarizing to M2 direction, whereas they lose it upon M1 induc- tion, which is consistent with the functional immunosuppressive role of stabilin-1. Discussion We report in this study that human stabilin-1 is expressed on CD14+ Stabilin-1 is downregulated in a proinflammatory condition CD162 and CD14+CD16+ monocytes in healthy individuals. Si- in vivo in humans lencing of stabilin-1 in monocytes results in activation of several To study whether stabilin-1 expression would be different in proinflammatory genes. When compared with stabilin-1low proinflammatory and immunosuppressive conditions in vivo, we monocytes, stabilin-1high monocytes supported more Th2-type and studied blood monocytes, placental macrophages, and placental less Th1-type cytokine production in Ag-specific T cell recall bed macrophages during pregnancy. Strong immunosuppression assays. Also under pathophysiological conditions in vivo, down- and Th2 deviation is characteristic to normal pregnancy (25), regulation of stabilin-1 expression on monocytes correlated with whereas an abnormal proinflammatory reaction takes place in the an enhanced proinflammatory response in pre-eclampsia. Impor- placenta in pre-eclampsia (25, 26). Interestingly, we found that tantly, ligation of stabilin-1 on monocytes by anti–stabilin-1 Abs in patients with pre-eclampsia, stabilin-1 expression was sig- shifted the Ag-specific recall responses to the proinflammatory nificantly lower on CD14+ blood monocytes compared with direction. Collectively, our findings identify stabilin-1 as a new normal pregnant women (Fig. 5A). Monocyte-derived macro- immunosuppressive molecule on monocytes. phages also infiltrate the placenta in large numbers during the Stabilin-1 has been reported to be absent from normal mono- pregnancy (27). When the leukocytes were isolated from pla- cytes, even though it is found on monocytes in hypercholesterol- centas and stained for FACS, the level of stabilin-1 expression emic patients (12). Our phenotypical analysis with refined reagents was significantly lower on CD14+ macrophages in patients with and protocols unambiguously showed that both CD14+CD162 and pre-eclampsia than in patients with normal uncomplicated CD14+CD16+ monocytes do express stabilin-1 on their surface, pregnancy (Fig. 5B). Significantly fewer stabilin-1+ macro- whereas CD14dimCD16+ cells do not. Collectively, these data phages were found in the placental bed samples in patients with imply that stabilin-1 may serve previously unnoticed functions in pre-eclampsia than in normal pregnancy (Fig. 5C). These data normal human monocytes. Indeed, our microarray data showed show that there is a link between the numbers of stabilin-1+ that stabilin-1high and stabilin-1low monocytes have different blood monocytes and macrophages and the level of immuno- transcriptomes, and RNAseq analyses further demonstrated that suppression in vivo. stabilin-1 is, directly or indirectly, involved in the upregulation of 8 IMMUNOSUPPRESSIVE STABILIN-1 IN MONOCYTES several proinflammatory genes such as OSM, CXCL13, and 1high and stabilin-1low monocyte subpopulations aimed at ana- SAA2. Interestingly, among other functions these molecules have lyzing whether the monocyte subpopulation expressing the highest been reported to control cytokine activity, including pathways level of stabilin-1 displays diminished proinflammatory properties regulating IFN-g, and may therefore be linked to the stabilin-1– in general (stabilin-1 was used as a phenotypic surface marker for dependent shift of polarization. OSM treatment of dendritic cells subpopulation isolation). The analyses of DE genes in control and induces IFN-g secretion by T cells (28). Similarly, SAA–derived stabilin-1 siRNA-treated monocytes, in contrast, aimed at identi- peptides, stimulate IFN-g secretion by CD4 T lymphocytes in fying target genes in the total monocyte pool, for which expres- human synovial fluid (29) and boost the T cell–stimulating ca- sion could be directly or indirectly affected by the stabilin-1 pacity of APCs (30). Moreover, IPA predicted that TNF could molecule itself. Therefore, as expected, when comparing the list of serve as a common upstream regulator of several genes, for which the DE genes between stabilin-1high and stabilin-1low monocytes to expression is regulated by stabilin-1. Notably, analyses of TNF that between control and siRNA-silenced monocytes we did not protein concentration in conditioned monocyte medium indeed find practically any overlap (only TPST1 and HTRA1 were showed increased TNF production in stabilin-1–silenced cells. common). For similar reasons, it was not surprising to see that In addition to the correlative data, we demonstrate an immu- certain genes (ORM2 and SCGB3A1) DE in the two arrays nosuppressive function for stabilin-1 in functional assays. In showed corresponding differences in qPCR analyses of the total ELISPOT assays, T cells cocultured with stabilin-1low or stabilin- pool of blood monocytes or placental macrophages in pre- 1–silenced monocytes in the presence of tetanus toxoid produced eclampsia, whereas others (GNHRH2, LHCGR) did not (data more IFN-g spots compared with those cultured with stabilin-1high not shown). monocytes or negative control siRNA-treated monocytes, respec- In conclusion, we define stabilin-1 as a novel immunosup- tively. Notably, also blocking of stabilin-1 with a mAb led to pressive molecule. The stabilin-1low monocyte population, sta- Downloaded from generation of increased numbers of IFN-g spots. In line with these bilin-1–silenced monocytes, and anti–stabilin-1 Ab-treated observations, ELISPOT assays revealed reduced numbers of IL- monocytes all support enhanced generation of Th1-dominant 4– and IL-5–secreting T cells in the presence of stabilin-1 siRNA- immune responses. Thus, monocyte stabilin-1 is a novel im- treated monocytes. These data not only show that the stabilin-1high mune modulator that augments formation of Th2-type Ag-specific monocyte population is functionally different from stabilin-1low T cell responses. Interestingly, anti–stabilin-1 Abs can be used to monocytes, but also that the stabilin-1 molecule itself, directly or promote Th1-dependent inflammatory responses, which may be http://www.jimmunol.org/ indirectly, regulates the capacity of monocytes to polarize T cells useful, for instance, for counteracting tumor-induced immunosup- into Th1 versus Th2 direction upon Ag stimulation. Moreover, pression. therapeutic ligation of stabilin-1 with Abs can be used to change the direction of T cell polarization during Ag activation. Acknowledgments These data are consistent with the earlier reported correlations + We thank Maritta Pohjansalo, Etta-Liisa Va¨a¨na¨nen, and Sari Ma¨ki for of stabilin-1 macrophages with immunosuppressive conditions. technical help; the obstetricians in the Turku University Hospital for In macrophages, stabilin-1 is known to be a good marker for im- the blood samples, placentas, and placental beds; and Anne Sovikoski- munosuppressive M2 cells both in vitro and in vivo (13, 16, 18, 19, Georgieva for secretarial help. by guest on October 1, 2021 31–34). Our current results show that M2-polarizing conditions (31) (M [M-CSF and IL-4], M [M-CSF, IL-4, and dexametha- Disclosures sone], M [dexamethasone] and M [IL-4]) augmented stabilin-1 The authors have no financial conflicts of interest. expression on monocyte-derived macrophages, whereas M1- polarizing (M [LPS and TNF-a]) induction led to the loss of stabilin-1 from the macrophages. 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