Folate β Regulates Integrin CD11b/CD18 Adhesion of a Subset to Collagen

This information is current as Christian Machacek, Verena Supper, Vladimir Leksa, Goran of October 2, 2021. Mitulovic, Andreas Spittler, Karel Drbal, Miloslav Suchanek, Anna Ohradanova-Repic and Hannes Stockinger J Immunol published online 17 August 2016 http://www.jimmunol.org/content/early/2016/08/17/jimmun ol.1501878 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2016/08/17/jimmunol.150187 Material 8.DCSupplemental http://www.jimmunol.org/

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 17, 2016, doi:10.4049/jimmunol.1501878 The Journal of Immunology

Folate Receptor b Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen

Christian Machacek,* Verena Supper,* Vladimir Leksa,*,† Goran Mitulovic,‡ Andreas Spittler,x Karel Drbal,{,1 Miloslav Suchanek,{ Anna Ohradanova-Repic,* and Hannes Stockinger*

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) b, a GPI-anchored belonging to the folate receptor family. As FRb shows a restricted expression to cells of myeloid origin and only a subset of activated and placental cells have been shown to express functional FRb, it represents a promising target for future thera- peutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenviron- b b+ b ment of FR in the plasma membrane of human FR macrophages and FR -transduced monocytic THP-1 cells. In this manner, Downloaded from we identified a novel role of FRb: that is, we report functional interactions of FRb with receptors mediating cellular adhesion, in

particular the CD11b/CD18 b2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRb+ human primary macrophages and THP-1 cells to collagen in comparison with their FRb2 counterparts. We further show that FRb is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRb as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen. The Journal of

Immunology, 2016, 197: 000–000. http://www.jimmunol.org/

olate receptor (FR) b, a GPI-anchored protein belonging to Unlike the broadly expressed reduced folate carrier (SLC19A1) the folate receptor family (1), constitutes one of the pathways and proton-coupled folate transporter (SLC46A1) (6), FRb shows F providing folic acid necessary for essential cellular functions a restricted expression to cells of the myeloid origin, and only a such as DNA synthesis, repair, and methylation (2, 3). Contrary to the subset of activated macrophages and placental cells has been embryonically lethal FRa knockout mice (4), genomic ablation of shown to express functional FRb (7). Macrophages show remark- FRb has no reported phenotype except for an increased risk of em- able plasticity, and their phenotype can vary greatly depending on bryonic lethality through arsenate or valproic acid exposure (5). the stimulus. On the one hand, classically activated (e.g., LPS plus by guest on October 2, 2021 However, a comprehensive functional analysis of this receptor, as IFN-g) macrophages (M1 type) drive Th1 responses and mediate well as an identification of its interacting , is still missing. host defense from bacteria as well as viruses and propagate anti- tumor functions. On the other hand, alternatively activated (e.g., by *Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infecti- Th2 IL-4) macrophages (M2 type) induce a Th2-mediated ology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; immune response against parasites and fungi and display an anti- † Laboratory of Molecular Immunology, Institute of Molecular Biology, Slovak Acad- inflammatory as well as wound healing phenotype (8). Reports that emy of Sciences, 845 51 Bratislava, Slovakia; ‡Department of Clinical Chemistry and Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; xDepart- FRb is expressed on subpopulations of and macro- ment of Surgery and Core Facility Flow Cytometry, Medical University of Vienna, { phages in rheumatoid arthritis (9, 10), on M2 macrophages in 1090 Vienna, Austria; and EXBIO Praha, 252 42 Vestec, Czech Republic cancer (11, 12) as well as on myeloid leukemia cells (13, 14), have 1 Current address: Department of Cell Biology, Faculty of Science, Charles University placed attention on FRb as a tool for targeting pathogenic and in Prague, Prague, Czech Republic. malignant myeloid cells. ORCIDs: 0000-0003-2831-600X (V.S.); 0000-0003-1964-3965 (G.M.); 0000-0003- 2657-6836 (A.S.); 0000-0003-4450-4625 (K.D.); 0000-0002-8005-8522 (A.O.-R.); As GPI-anchored proteins are inserted into the outer leaflet of the 0000-0001-6404-4430 (H.S.). cell membrane, they require interaction partners in the form of Received for publication August 20, 2015. Accepted for publication July 14, 2016. transmembrane proteins to transmit outside–in signals. Therefore, This work was supported by the European Union Seventh Framework Programme characterization of the protein microenvironment of FRb is one (FP7/2007-2013) under Grant Agreement NMP4-LA-2009-228827 NANOFOL. V.L. possible route to investigate its function. Recently, a functional was supported by Austrian Science Fund Grant P22908 and Slovak Scientific Grant interaction between FRa and gp130 was reported (15), explaining Agency Grant 2/0063/14. how FRa is able to convey a proliferative advantage to cancer Address correspondence and reprint requests to Prof. Hannes Stockinger and Dr. Anna Ohradanova-Repic, Institute for Hygiene and Applied Immunology, cells. As FRb exhibits restricted expression to cells of myeloid Center for Pathophysiology, Infectiology and Immunology, Medical University origin, we investigated whether FRb is involved in the regulation of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria. E-mail addresses: hannes. of immune cell function. [email protected] (H.S.) and [email protected] (A.O.-R.) One central function is the ability of immune cells to locate in The online version of this article contains supplemental material. areas with active inflammation. This, however, can drive disease Abbreviations used in this article: AF, Alexa Fluor; ATRA, all-trans retinoic acid; CR3, complement receptor type 3; ECM, extracellular matrix; FR, folate receptor; progression in instances of chronic inflammation (e.g., rheumatoid HA, hemagglutinin; IP, immunoprecipitation; LC-MS/MS, liquid chromatography– arthritis) (16). The integrin family of proteins constitutes a major MS/MS; MFI, mean fluorescence intensity; MS, mass spectrometry; MS/MS, tandem factor in the regulation of leukocyte trafficking (17), and its MS; TFA, trifluoroacetic acid; vD , 1,25-dihydroxyvitamin D . 3 3 members were shown to associate with various GPI-anchored Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 proteins that modulate their activity (18–20). In this study, we

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501878 2 CD11b/CD18 ADHESION TO COLLAGEN REGULATED BY FRb report a functional interaction of FRb with the CD11b/CD18 (A-11070) and AF 680–conjugated goat anti-mouse IgG were purchased integrin heterodimer Mac-1 resulting in modulated adhesion to from Invitrogen. Anti-hemagglutinin (HA) mAb (clone 12CA5) and the extracellular matrix (ECM) component collagen. peroxidase-conjugated anti-mouse IgG (A9044) were purchased from Sigma-Aldrich. IRDye 800CW–conjugated donkey anti-rabbit IgG was purchased from LI-COR Biosciences (Lincoln, NE). AF 488–conjugated Materials and Methods goat-anti-mouse IgG (H+L) was purchased from Thermo Fischer Sci- Cell isolation and cultivation entific (Waltham, MA). Human PBMCs were isolated from leukocyte reduction chambers of Flow cytometry healthy adult blood donors by density gradient centrifugation using Cells were detached using 1.5 mM EDTA in HBSS. Cells were washed with Lymphoprep (Axis-Shield, Oslo, Norway). CD14+ monocytes were purified staining buffer (PBS supplemented with 1% BSA [Carl Roth, Karlsruhe, from PBMCs by MACS using CD14 microbeads (Miltenyi Biotec, Ber- Germany] and 0.02% NaN ), and nonspecific binding of the mAbs to Fc gisch Gladbach, Germany) with a minimal purity of 98%. THP-1 cells 3 receptors was prevented by blocking with 2.4 mg/ml human IgG (Beri- were a gift of Alexander M. Carmo (Instituto de Biologia Molecular e globin P; CSL Behring, King of Prussia, PA) on ice for 30 min. Then, Ab/ Celular, Porto, Portugal). All cells were maintained in complete culture fluorochrome conjugates or appropriate isotype controls were added. Cells medium consisting of standard RPMI 1640 medium (containing 2.27 mM were incubated for 30 min on ice and then washed twice with staining folate) supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, buffer. Nonconjugated Abs were detected by an additional incubation of 100 U/ml penicillin, and 10% heat-inactivated FCS unless stated otherwise 30 min with fluorochrome-conjugated secondary Ab. Samples were ana- (all from Invitrogen, Carlsbad, CA). Folate starvation conditions were lyzed on an LSR II flow cytometer (BD Biosciences) and the data were established by maintaining cells in folate-free RPMI 1640 medium (Invi- further processed with FlowJo software (Tree Star, Ashland, OR). Living trogen) supplemented as described above. Primary monocytes were dif- cells were gated according to their forward and side scatter characteristics ferentiated to macrophages using 25 ng/ml GM-CSF (Novartis, Basel, and exclusion of dead cells using DAPI (Sigma-Aldrich). In graphs, mean Switzerland) or 50 ng/ml M-CSF (PeproTech, Rocky Hill, NJ) for 7 d.

fluorescence intensity (MFI) corrected for background staining using Downloaded from THP-1 cells were differentiated with 60 nM calcitriol (1,25-dihydroxy- matched isotype control mAbs is shown. vitamin D3 [vD3]; Santa Cruz Biotechnology, Dallas, TX) and 100 nM all-trans retinoic acid (ATRA; Sigma-Aldrich, St. Louis, MO) for 6 d. Adhesion assay Classical activation was achieved using 100 ng/ml LPS (Escherichia coli serotype O55:B5; Sigma-Aldrich) plus 25 ng/ml IFN-g (PeproTech) for Flat-bottom 96-well plates were coated with 10 mg/ml of the ECM proteins 2 d, whereas 25 ng/ml IL-4 (Novartis AG) was used for alternative activa- human fibronectin, human vitronectin, rat type I collagen, and human type tion. For inside–out activation of b2 integrins, THP-1 cells were activated I or human type IV collagen (all purchased from Sigma-Aldrich) for 16 h at 4˚C. To ensure the proper coating, MaxiSorp 96-well plates (Sigma- with 10 ng/ml PMA (Sigma-Aldrich) for 3 d. http://www.jimmunol.org/ Aldrich) were used in assays involving C3b, iC3b (EMD Millipore, Bill- Abs erica, MA), and the human rICAM-1 (gift of Johannes Huppa, Medical University of Vienna, Vienna, Austria). Plates were subsequently blocked AF (Alexa Fluor ) 647–, AF 488– and biotin-conjugated anti-FRb mAb with 10 mg/ml BSA for 1 h at 37˚C and washed with PBS. Cells were (clone EM-35 and EM-36), AF 647-conjugated CD11a mAb (clone detached by incubation in HBSS supplemented with 1.5 mM EDTA, MEM-25), purified CD11b mAb (clone ICRF44), purified CD11c mAb washed with PBS, and resuspended in RPMI 1640 medium supplemented (clone BU15), AF 647– and biotin-conjugated CD18 mAb (clone MEM- with 10 mg/ml BSA, 2 mM CaCl , 2 mM MgCl , 0.5 mM MnCl and 48), AF 488–conjugated CD29 mAb (clone MEM-101A), AF 647–con- 2 2 2 20 mM HEPES at pH 7. For assays involving function-modifying mAbs, jugated CD222 mAb (clone MEM-238), purified CD305 mAb (clone cells were incubated for 30 min at 4˚C in medium supplemented with NKTA255), and isotype control mAb (clone MOPC-21) were provided 0.8 mg/ml human IgG and subsequently incubated with 10 mg/ml dialyzed by EXBIO Praha (Vestec, Czech Republic). CD14 mAb (clone MEM- mAbs for 30 min prior to seeding on the ECM-coated plates. by guest on October 2, 2021 18), CD11b mAb (clone MEM-170), CD18 mAb (clone MEM-148), and Per well, 1 3 105 cells were seeded and incubated for 30 min at 37˚C. an isotype control mAb (clone PPV-06) were a gift of Vaclav Horejsi Then the plates were washed three times with PBS and the cells were fixed (Institute of Molecular Genetics, Academy of Sciences of the Czech with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 15 min at room Republic, Prague, Czech Republic). Brilliant Violet 510–conjugated temperature, stained with 0.1% crystal violet (Sigma-Aldrich) in H O for CD11b mAb (clone ICRF44) was purchased from BD Biosciences 2 30 min, washed three times with H O, and finally lysed in 1% Triton (Franklin Lakes, NJ). Purified CD11b mAb (clone VIM12) was a gift of 2 X-100 (Sigma-Aldrich) in PBS. Absorbance at 595 nm was measured with Johannes Sto¨ckl (Institute of Immunology, Medical University of Vienna, a Mithras LB 940 multimode microplate reader (Berthold, Bad Wildbad, Vienna, Austria). AF 647–conjugated CD49a mAb (clone TS2/7), AF 488– Germany). The data were corrected for background absorbance of the conjugated CD49b mAb (clone AK-7), allophycocyanin-Cy7–conjugated empty wells and values were normalized to basal adhesion measured from CD206 mAb (clone 15-2), AF 488– and AF 647–conjugated isotype control simultaneous adhesion assays of the same cells seeded onto BSA-blocked mAb (clone MOPC-21) were purchased from BioLegend (San Diego, CA). wells. Purified rabbit polyclonal CD280 Ab (ab70132) was purchased from Abcam (Cambridge, U.K.). Rabbit anti-GAPDH mAb 14C10 was purchased from ECM uptake assay Cell Signaling Technology (Danvers, MA). Allophycocyanin-conjugated F(ab9)2 fragment of goat anti-mouse IgG plus IgM (H+L) (115-136-068) Culture plates (48 wells) were coated with 10 mg/ml BSA, human type I was purchased from Jackson ImmunoResearch Laboratories (West Grove, or type IV collagen, labeled with amine-reactive AF 647 dye (Invitrogen) PA). AF 488–conjugated F(ab9)2 fragment of goat anti-rabbit IgG (H+L) for 2 h at 37˚C, and subsequently blocked with 10 mg/ml unlabeled BSA

FIGURE 1. Ectopically expressed FRb mediates folate uptake of THP-1 cells and imposes a proliferative advantage under low-folate culture conditions. (A) Expression of FRb on the surface of control- and FRb-transduced THP-1 cells as measured by flow cytometry. (B) Control- and FRb-transduced THP-1 cells were cultivated under folate-reduced (-folate) or high-folate conditions for 24 h. Specific uptake of folate-coupled or control Qdots was assessed as described in Materials and Methods. MFI 6 SEM of two independent experiments with two-way ANOVA and subsequent Bonferroni posttest is shown. (C) Proliferation of control- and FRb-transduced THP-1 cells cultivated under folate-reduced or high-folate conditions. One representative of three independent experiments is shown. **p , 0.01. The Journal of Immunology 3 for 1 h at 37˚C. Cells (2.5 3 105 per well), treated as described for the Acclaim C18 trap column (300 mm inner diameter 3 5 mm) was operated at adhesion assay, were seeded, incubated for 1 h at 37˚C, treated with trypsin the ambient temperature, and the Acclaim C18 nano separation column for 3 min at 37˚C, washed twice with staining buffer (PBS supplemented (75 mm inner diameter 3 250 mm) was mounted in the column oven and with 1% BSA and 0.02% NaN3), and fluorescence was analyzed with an operated at 45˚C. Samples were loaded onto the trap column using 0.1% LSR II flow cytometer as described in the Flow cytometry section. TFA at 30 ml/min, and the nano separation was performed in gradient mode at 300 nl/min. A user defined injection program was used for sample in- Invasion assay jection and additional injector and trap column wash. Every sample injection Growth factor reduced BD Matrigel invasion chambers (no. 354483; BD was followed by two blank runs with injections of 2,2,2-trifluoroethanol for Biosciences) were rehydrated according to the manufacturer’s instructions and removal of possible sample remains in the injector or on the trap column and cells (1 3 105 in 200 ml complete RPMI 1640 medium containing 2 mM prevention of carryover in the separation system (22). Mass spectrometry L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% heat- (MS) analysis was performed using the LTQVelos ion trap mass spectrom- inactivated FCS) were loaded in the upper chamber of the Transwell insert, eter (Thermo Fisher Scientific) with the “top 20” method; that is, the 20 most which was subsequently placed into a 24-well containing 750 mlcomplete intensive ions from the MS scan were selected for tandem MS (MS/MS). RPMI 1640 medium supplemented when indicated with 50 ng/ml M-CSF to Single-charged ions were excluded from fragmentation, and detected ions provide a chemotactic gradient. Invading cells were measured after 21 h of were excluded for further fragmentation for 3 min after initial MS/MS cultivation by removing cells present in the upper chamber and treatment of fragmentation had been performed. Additionally, prior to the MS, UV adherent cells with 4% paraformaldehyde for 15 min and subsequent staining peptide detection at 214 nm was also performed. Data analysis was per- formed using Mascot 2.4.1 (Matrix Science, London, U.K.) and searching with 0.1% crystal violet in H2OfollowedbywashingwithH2O. Cells were quantified by counting stained cells in four random fields. the most recent version of the SwissProt database. All search results were refined and researched using Scaffold 3.6.2 (Proteome Software, Portland, Cloning OR). Statistical evaluation of MS data were performed using an unpaired cDNA from the human myeloid cell line KG-1 was used as a template for the amplification of FRb open reading frame utilizing the primers FRb Downloaded from forward (59-AGTGAATTCGGACAGAAAGACATGGTCTGGA-39) and FRb reverse (59-GCAGGTAGTCTGGGAGGACTGA-39) with the Phu- sion polymerase (Finnzymes, Espoo, Finland). The PCR product was subcloned into pcDNA3.1(+) using EcoRI and EcoRV, and then the open reading frame of FRb was cloned in the correct orientation into the in- house–modified retroviral expression vector pBMN-I-GFP (plasmid 1736; Addgene, Cambridge, MA) devoid of the IRES-GFP sequence, resulting in the pBMN-FRb construct. For HA-tagged FRb, we recloned FRb into http://www.jimmunol.org/ vector pfMSCV-N-Strep3xHA-IRES-GFP (gift of Giulio Superti-Furga, Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria). The tandem Strep3xHA-tag was introduced between the signal peptide and the coding sequence of FRb, resulting in the plasmid pfMSCV-TAP-FRb-I-GFP, which we verified by sequencing. Retroviral transduction

HEK293T cells, maintained in DMEM supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% heat-inactivated FCS were cultivated to ∼70% confluency and subsequently transfected with the by guest on October 2, 2021 plasmid of interest (pBMN-FRb, pBMN-GFP-GPI, or pfMSCV-TAP-FRb- IRES-GFP or the respective control plasmids pBMN or pfMSCV-TAP-IRES- GFP) along with the helper vectors pGagPol (Addgene) and pMD2.G (constructed by D. Trono, E´ cole Polytechnique Fe´de´rale de Lausanne; obtained via Addgene). Viral supernatants were harvested after 48 h, filtered, and used for the transduction of THP-1 cells. THP-1 cells were transduced overnight in the presence of 4 mg/ml Polybrene. Immunoprecipitation After washing with PBS, 1 3 107 cells/ml were lysed for 30 min on ice in lysis buffer (0.5% n-dodecyl-b-d-maltoside [Thermo Fisher Scientific], 140 mM NaCl, 50 mM NaF, 0.2 mM Na3VO4,50mMHEPES[pH7.5][Sigma- Aldrich], 1 3 complete EDTA-free protease inhibitor cocktail [Roche, Basel, Switzerland]). The insoluble material was removed by centrifuga- tion at 15,000 3 g for 20 s at 4˚C. Lysates were incubated with anti-HA mAb- coupled agarose (Sigma-Aldrich), isotype-, CD18-, or anti-FRb mAb–coupled protein A/G-Plus beads (Santa Cruz Biotechnology) for 1 h at 4˚C with constant rotation. Beads were washed three times with complete lysis buffer for 10 min at 4˚C and eluted with 6 M urea, 2 M thiourea, and 50 mM HEPES (ph 7.5) (all purchased from Sigma-Aldrich) for 15 min at 50˚C for mass FIGURE 2. FRb predominantly coprecipitates proteins mediating cellular spectrometric analysis. Alternatively, proteins were eluted with 1.53 nonre- adhesion. (A) Lysates from HA-tagged FRb and control-transduced THP-1 ducing Laemmli buffer at 95˚C for 5 min for subsequent use in SDS-PAGE. cells were subjected to IP with anti-HA mAb–conjugated beads, followed by LC-MS/MS detection. The volcano plot calculated by the Perseus software Mass spectrometry displays the enrichment of proteins coprecipitated with HA-tagged FRb Prior to digestion, immunoprecipitated proteins were precipitated using the compared with the negative control depicted as the natural logarithm from methanol-chloroform procedure according to Wessel and Flugge€ (21). Pre- the ratio of normalized intensity values (x-axis) with significance given as the cipitated proteins were dissolved in 50 mM triethylammonium bicarbonate, negative decimal logarithm of the p value (y-axis). FRb and the highly and protein concentration was determined using the Bradford assay. Proteins significant interaction partners (false discovery rate , 0.05, S0 = 2, as de- were tryptically digested overnight at 37˚C using a trypsin/protein ratio of termined by Perseus) described to affect cell adhesion and interactions with 1:50. Digestion was stopped by acidification with 10 ml of 1% trifluoroacetic acid (TFA), and 20 ml of digested peptides was further diluted with 30 mlof ECM components are highlighted. Data represent two independent experi- B 0.1% TFA for injection onto the separation column. Peptides were separated ments. ( ) Representative Western blot of anti-HA IPs from the HA-tagged using an UltiMate Plus nano HPLC (LC Packings, Amsterdam, The Neth- FRb- or control-transduced THP-1 cells probed with anti-FRb or CD18 erlands) separation system, consisting of Famos autosampler, Switchos mAbs. The bands were visualized by peroxidase-conjugated secondary Abs column switching unit, the UltiMate nano pump, and the UV detector. The and chemiluminescence. 4 CD11b/CD18 ADHESION TO COLLAGEN REGULATED BY FRb

Student t test with equal variances (Perseus software package 1.5.2.6; Max and cocapping was quantified by the IDEAS software package as described in Planck Institute) (parameters: false discovery rate # 0.05, S0 = 2). Further Beum et al. (23). analysis and visualization of the results were performed using Ingenuity Pathway Analysis (Qiagen, Hilden, Germany). silencing + Western blotting Human primary CD14 monocytes were differentiated with M-CSF for 4 d as described above. Cells were transfected with 50 nM FOLR2 (sense Lysates were prepared as described in the “Immunoprecipitation” (IP) 59-GCUGCUUCUGGUCUGUGUAGC-39, antisense 59-UACACAGACCA- section. Equal amounts of proteins were separated by nonreducing GAAGCAGCAG-39)orcontrol(sense59-AGGUAGUGUAAUCGC- SDS-PAGE, blotted onto a polyvinylidene difluoride membrane CUUGGU-39, antisense 59-CAAGGCGAUUACACUACCUAG-39) small (Immobilon P; Millipore), blocked with 3% BSA in TBST, probed with interfering RNA using Viromer Blue (Lipocalyx; Saale, Germany) according primary Ab in TBST supplemented with 3% BSA and 0.02% NaN3, to the manufacturer’s protocol. After 4 h of incubation, the medium was and subsequently washed thee times with TBST followed by incubation replaced by fresh culture medium supplemented with 50 ng/ml M-CSF and with peroxidase-conjugated secondary Ab in TBST supplemented with cells were differentiated for another 3 d. 3% BSA. After washing, the membrane was developed using ECL (ECL-Prime; GE Healthcare, Little Chalfont, U.K.) followed by detection Statistical analysis using an LAS4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Alternatively, for fluorescence detection, proteins were blotted onto a low- All experiments were performed at least three times unless otherwise specified. Data are presented as the mean 6 SEM of the values from all fluorescent polyvinylidene difluoride membrane (Immobilon FL; Millipore), , and the binding of primary Abs was detected with fluorescently labeled experiments. A p value 0.05 was considered significant. Analysis and secondary Abs using an Odyssey LI-COR imager (LI-COR Biosciences). graphing were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Folate binding assay

THP-1 cells were cultivated in either standard of folate-free RPMI 1640 me- Results Downloaded from dium, both supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, Ectopically transduced FRb increases folate binding and 100 U/ml penicillin, and 10% heat-inactivated FCS, representing high folate or folate-reduced conditions, respectively, for 24 h. Cells were then harvested, proliferation of THP-1 cells under low-folate conditions resuspended in either standard or folate-free RPMI 1640 medium, and incu- To find a suitable model for the investigation of the function of FRb in bated with 16 nM control- or folate-labeled Qdot 655 nanocrystals (gift of immune cells, we screened human myeloid cell lines for expression of Georges Bismuth, Institut Cochin, Paris, France) for 4 h at 37˚C. After washing FRb. Among all cell lines screened, only KG1 (24) and THP-1 with staining buffer (1% BSA and 0.02% NaN3 in PBS), the Qdot binding and uptake were measured with an LSR II flow cytometer. (Supplemental Fig. 1A) cells showed low expression of FRb.This http://www.jimmunol.org/ basal expression on THP-1 cells was not significantly increased after Proliferation assay differentiation with either vD3 (25) plus ATRA or by the subsequent Control- and FRb-transduced THP-1 cells were cultivated under either stimulation with LPS plus IFN-g or alternatively with IL-4. Differ- high folate or folate-reduced conditions for 14 d. Cell number was entiation with PMA even decreased the surface levels of endogenously assessed with a CASY cell counter (Roche) in technical triplicates once every 24 h. Cultures were supplemented with equal amounts of fresh expressed FRb (Supplemental Fig. 1B). For the detailed character- media every 48 h. ization of the receptor, we therefore decided to establish a stably transduced, FRb-expressing THP-1 cell line (Fig. 1A). To test whether Cocapping assay the ectopically expressed FRb was functionally active, we investi- by guest on October 2, 2021 CellswerewashedwithRPMI1640medium,blockedincompleteculture gated its ability to bind folate (26). When cultivated under folate- medium supplemented with 2.4 mg/ml human IgG on ice for 30 min, and reduced conditions (∼1nM),FRb-transduced THP-1 cells exhibited subsequently stained with 10 mg/ml dialyzed CD11b mAb MEM-170 on ice significantly increased binding of folate-labeled Qdots compared with for 30 min. Cells were washed and capping was induced by incubation with dialyzed AF 488–conjugated goat anti-mouse IgG at 37˚C for 8 min. Capping control-transduced THP-1 cells, whereas cultivation in standard cul- was stopped by washing the cells with ice-cold staining buffer (PBS supple- ture medium, containing high amounts of folate (∼2.2 mM), mented with 1% BSA and 0.02% NaN3) and cells were again blocked with abrogated this effect (Fig. 1B). We further investigated the 250 mg/ml isotype control mAb PPV-06 and 2.4 mg/ml human IgG in staining functionality of FRb by monitoring the proliferation of control- and buffer. Cocapping was assessed by staining with AF 647–conjugated mAbs (MEM-48toCD18,EM-35andEM-36toFRb, MEM-238 to CD222) and FRb-transduced THP-1 cells. Although no significant change in measured using an ImageStream flow cytometry (Amnis ImageStreamX MKII; proliferation was detected in standard culture media, FRb-transduced EMD Millipore). Data were acquired using the INSPIRE software package, cells exhibited a clear proliferative advantage when only a minimal

FIGURE 3. FRb-transduced THP-1 cells exhibit lower adhesion to collagen. (A) Adhesion assay of vD3 plus ATRA–differentiated control- and FRb- transduced THP-1 cells to plates coated with various ECM proteins. Relative adhesion of FRb-transduced cells versus control THP-1 is displayed. Data represent ratios 6 SEM of more than four independent experiments performed in technical quadruplicates and significance was calculated with a one- sample t test. (B) ECM protein uptake assay: vD3 plus ATRA–differentiated THP-1 cells were seeded on cell culture plates coated with fluorescently labeled human type I or type IV collagen as well as BSA as control and incubated for 1 h at 37˚C. Cells were trypsinized, washed, and protein uptake was determined by flow cytometry. Data of three experiments with significance calculation via t test are shown. (C) Invasion assay of vD3 plus ATRA– differentiated FRb-transduced versus control THP-1 cells through Matrigel-coated Transwell inserts with 8-mm pore size. Unspecific- as well as M-CSF– directed invasion 6 SEM of two independent experiments performed in technical duplicates are displayed. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5 amount of folate originating from FCS was present in the culture purification/mass spectrometry (MS) analysis of FRb immuno- medium (Fig. 1C). We therefore concluded that the ectopically complexes. For this purpose, we overexpressed HA-tagged FRb in expressed FRb was functional in THP-1 cells. THP-1 cells (Supplemental Fig. 1C, 1D), which exhibited simi- lar capacity to bind folate-coupled Qdots as untagged FRb b FR is coprecipitating with proteins mediating cellular (Supplemental Fig. 1E). We pulled down FRb-containing protein adhesion complexes using anti-HA mAb–coupled agarose and detected To unravel putative interaction partners of FRb that could shed copurified proteins via in-solution digestion followed by liquid on macrophage-specific functions of FRb, we performed affinity chromatography–MS/MS (LC-MS/MS). We detected a wide range Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. FRb-transduced THP-1 cells display altered conformation of CD11b/CD18 and reduced adhesion to collagen. Cell surface staining of the indicated proteins on vD3 plus ATRA–differentiated control- and FRb-transduced THP-1 cells was measured by flow cytometry. When indicated, cells were treated with acidic buffer (pH 3.5) prior to Ab staining. (A and B) Representative histograms and (C) relative MFI of FRb-transduced versus control THP-1 cells 6 SEM of more than three experiments with significance calculation via a one-sample t test are shown. (D) Western blotting of CD18 expression in lysates of vD3 plus ATRA–differentiated control- and FRb-transduced THP-1 cells. GAPDH was used as the loading control. One representative blot of two independent experiments is displayed. (E)FRb-transduced THP-1 cells, differentiated with vD3 plus ATRA, were subjected to CD11b capping, and cocapping was assessed using AF 647–labeled CD18, CD222, and anti-FRb mAbs and ImageStream flow cytometry (for details, see Supplemental Fig. 2). Bright detail similarity score of median values from three independent experiments 6 SEM with significance calculation via one-way ANOVA with a Bonferroni posttest is shown. (F and G) Relative adhesion of vD3 plus ATRA–differentiated FRb-transduced versus control THP-1 cells to type I or type IV collagen-coated plates. Cells were treated with the indicated integrin-blocking mAbs as stated in the Materials and Methods section. Data represent ratios 6 SEM of more than three independent experiments performed in technical quadruplicates with significance calculation via one-way ANOVAwith a

Bonferroni posttest. (H) Relative adhesion of vD3 plus ATRA–differentiated control and FRb-transduced THP-1 cells to high adsorption plates coated with fibronectin or different CD11b/CD18 ligands. Data represent ratios 6 SEM of more than four independent experiments performed in technical quadru- plicates with significance calculation via a one-sample t test. *p , 0.05, **p , 0.01, ***p , 0.001. 6 CD11b/CD18 ADHESION TO COLLAGEN REGULATED BY FRb of proteins known to affect cell–cell as well as cell–matrix inter- (Fig. 3B), pointing to FRb impairing direct collagen binding actions (highlighted in Fig. 2A, Supplemental Table IA). To confirm rather than intracellular degradation. To investigate the functional the MS identification of coprecipitated proteins, we analyzed consequence of this reduction of adhesion to collagen, we the presence of selected candidate proteins, including the top assessed the migratory properties of THP-1 cells. In the absence of candidate CD18, in the HA pulldowns by Western blotting a chemotactic gradient, FRb- and control-transduced THP-1 cells (Fig. 2B, Supplemental Fig. 1F). We further explored the data displayed equal capacity to invade the lower compartment of an with the Ingenuity Pathway Analysis to functionally categorize invasion chamber coated with Matrigel consisting of laminin the protein microenvironment of FRb (Supplemental Fig. 1G). and type IV collagen. However, when an M-CSF gradient was We detected a highly significant enrichment of proteins mediating present, control-transduced THP-1 cells showed a significant cell to cell signaling and interactions (p =73 10219, 12 proteins) M-CSF–directed migration toward the chemotactic gradient whereas and cellular movement (p = 4.65 3 10218, 14 proteins) of mono- FRb-transduced cells did not (Fig. 3C). nuclear leukocytes. FRb expression alters adhesiveness of b integrin CD11b/ The use of HA-tagged FRb detects proteins interacting with 2 CD18 both immature and intracellular FRb during the processing and transport of the molecule (such as calnexin) as well as with the To elucidate the mechanism underlying the collagen adhesion mature molecule on the cell surface. To get a clearer picture of the regulation of FRb, we analyzed cell surface expression of known microenvironment of FRb on the cell surface, we surface labeled collagen-binding proteins that we identified as interaction partners living cells with an anti-FRb mAb raised against the mature FRb. of FRb (Supplemental Table I): heterodimers of CD29 (ITGB1) Afterwards, we lysed the cells and immunoprecipitated FRb. with its a-chains CD49a (ITGA1) and CD49b (ITGA2) (27), as Comparison of the data showed that proteins associated with ad- well as CD305 (LAIR1, identified as a receptor for collagen with Downloaded from hesion and migration (e.g., b2 integrin CD11b/CD18, b1 integrin immunomodulatory properties) (28) and the mannose receptors CD49e/CD29, or the CD87) were indeed as- CD280 (MRC2/UPARAP) and CD206 (MRC1), which have re- sociated with the mature FRb on the cell surface (Supplemental cently been described to affect collagen uptake in alternatively Table IB). activated murine macrophages (29). However, cell surface staining of these receptors was not affected by overexpression of FRb b FR expression reduces cell adhesion to collagen (Fig. 4A). http://www.jimmunol.org/ Based on the results of the MS and pathway analysis, we inves- Recently, b2 integrins have been implicated to mediate leuko- tigated the impact of FRb on cellular adhesion. To obtain a cyte adhesion to collagen as well (30). Because we identified macrophage-like phenotype, we differentiated THP-1 cells with CD18 as the most abundant protein coprecipitating with FRb in vD3 plus ATRA. FRb-expressing cells showed a significant re- both the HA-pulldown and the surface-labeling IP, we investigated duction in their ability to adhere to fibril-forming type I and CD18 and its associated a-chains CD11a (ITGAL), CD11b network-forming type IV collagen but not to other ECM compo- (ITGAM), and CD11c (ITGAX). Although no difference in the nents (Fig. 3A). As the FRb-dependent reduction of adhesion to CD18 surface expression was evident in FRb-expressing versus collagen could be caused by an increase in collagen degradation or control THP-1 cells when we used the CD18 mAb MEM-48, internalization, we next determined the capacity of the cells to CD18 mAb MEM-148, which specifically recognized a confor- by guest on October 2, 2021 internalize fluorescently labeled collagen. FRb-expressing cells mational epitope arising from either activation-dependent opening exhibited a significantly reduced ability to internalize collagen of the integrin heterodimer (31) or from proteolytically cleaved

FIGURE 5. PMA activation abrogates FRb-mediated alterations in CD11b/ CD18 staining and adhesion to collagen. Cell surface expression of the indicated proteins on vD3 plus ATRA–differentiated (upper panel) or PMA-differentiated (lower panel) control- or FRb-transduced THP-1 cells. (A) Representative histograms and (B) relative MFI of FRb-transduced ver- sus control THP-1 cells 6 SEM of more than three experiments with significance calculation via a one-sample t test are shown. (C) Relative adhesion of FRb- transduced- versus control THP-1 cells after vD3 plus ATRA or PMA differentiation. Data represent ratios 6 SEM of more than three independent experiments performed in technical quadruplicates with a one-sample t test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7

(32) or a-chain-free CD18 (33), yielded significantly higher conformational-dependent mAb MEM-148 epitope. Indeed, PMA staining on FRb-expressing cells (Fig. 4B, 4C). Based on this treatment significantly increased CD18 staining detected with information, we dissociated the integrin heterodimers through a conventional CD18 mAb MEM-48 as well as with the activation- brief treatment of the cells with pH 3.5. This procedure increased sensitive mAb MEM-148 (Fig. 5A, 5B). Interestingly, the increase drastically the binding of mAb MEM-148. Notably, under this of MEM-148 staining observed in FRb-transduced THP-1 cells condition, both the FRb+ and FRb2 THP-1 cells displayed a was not seen any longer after PMA treatment; similarly, we did comparable staining with MEM-148, suggesting that FRb induces not see the increased staining of CD11b mAb ICRF44, recog- either a conformational change or a proteolytic cleavage of CD18. nizing the ligand-binding domain of CD11b. Furthermore, the Therefore, we probed for the presence of a 65- to 70-kDa pro- CD11b mAb VIM-12, which binds the conformational-insensitive teolytic fragment of CD18 (32). As we could neither detect any membrane proximal part of the extracellular domain of CD11b quantitative difference in total CD18 nor the presence of a trun- (37), did not show a FRb-mediated alteration in staining in neither cated form of CD18 in the FRb-expressing cells (Fig. 4D), we condition (Fig. 5A, 5B), which substantiated our assumption that concluded that a fraction of CD18 underwent a conformational FRb affected the CD11b/CD18 conformation. Activation of THP-1 change in FRb-expressing THP-1 cells. In line with this inter- cells with PMA also abrogated the FRb-dependent reduction in pretation, FRb-expressing THP-1 cells showed increased staining adhesion to collagen (Fig. 5C), highlighting that the adhesion with CD11b mAb ICRF44, which recognizes the ligand-binding phenotype coincided with the conformational change of the CD11b/ domain of CD11b (34), and which was also equalized upon low CD18 heterodimer, as revealed by the increased binding of the pH treatment (Fig. 4B, 4C). The other CD18-associated a-chains conformation-dependent CD18 mAb MEM-148 and the ligand- CD11a and CD11c remained unaffected by the presence of FRb. binding domain–specific CD11b mAb ICRF44 in the FRb- These results indicate that either the conformation or the integrity transduced cells compared with the control THP-1 cells. Downloaded from of the integrin heterodimer CD11b/CD18 (Mac-1/CR3) is affected Conformation-modulating CD18 mAb MEM-148 reconstitutes by the presence of FRb. cellular adhesion of FRb-transduced THP-1 cells to collagen To verify the interaction of FRb with the CD11b/CD18 hetero- dimer in the living cell without the influence of a detergent, we CD18 mAb MEM-148, which shows an enhanced binding to FRb- performed a cocapping experiment. By using an ImageStream expressing THP-1 cells (Fig. 4B, 4C), was described to induce a flow cytometer we compared the degree of CD11b cocapping with high-affinity conformation in b2 integrins (31). In contrast, mAb http://www.jimmunol.org/ CD18, FRb, and the non–FRb- or CD11b-associated membrane MEM-48 was described as a blocking CD18 mAb (38). When we protein CD222 (the cation-independent mannose-6-phosphate measured cell adhesion to type I and type IV collagen in the receptor/insulin like growth factor 2 receptor). FRb showed a comparable cocap with CD11b as did CD11b with its b-chain CD18, whereas CD222 was not specifically enriched in the CD11b caps (Fig. 4E, Supplemental Fig. 2). To address whether CD11b/CD18 is indeed mediating collagen binding, we performed an adhesion assay in the presence or ab- by guest on October 2, 2021 sence of blocking mAbs to CD11a, CD11b, and CD11c. Only blocking of CD11b (Fig. 4F, 4G) led to a reduction in adhesion to both the type I and the type IV collagen. This indicates that the CD11b/CD18 heterodimer (Mac-1/CR3) is predominantly re- sponsible for adhesion of vD3 plus ATRA–differentiated THP-1 cells to collagen. The curtailed adhesion to collagen by FRb overexpression could also be an unspecific effect resulting from the overexpression of a GPI-anchored protein. Therefore, we compared control- and FRb-transduced THP-1 cells to THP-1 cells overexpressing GPI-anchored GFP. The GFP-GPI THP-1 cells neither exhibited an increased exposure of conformationally open CD18 (Supplemental Fig. 3A, 3B) nor showed these cells reduced adhesion to collagen (Supplemental Fig. 3C). Therefore, we concluded that the di- minished adhesion to collagen as well as the conformational change of CD18 was caused by the direct interaction of FRb with integrin CD11b/CD18. CD11b/CD18 is also described as a receptor for C3b, iC3b, and ICAM-1 (35, 36). Therefore, we investigated whether FRb re- duced also the adhesive property of CD11b/CD18 to these ligands. FRb-transduced THP-1 cells showed only a moderate decrease of adhesion to C3b and binding to iC3b and ICAM-1 was not changed (Fig. 4H). Taken together, these findings indicate that FRb FIGURE 6. Reconstitution of FRb-dependent reduction of cell adhesion tunes specifically the adhesive function of Mac-1/CR3 for to collagen by function-modulating CD18 mAb MEM-148. Relative ad- hesion of vD plus ATRA–differentiated control versus FRb-transduced collagen. 3 THP-1 cells to type I (upper panel) or type IV (lower panel) collagen- PMA stimulation abrogates FRb-dependent reduction of coated plates normalized as described in Fig. 3. Cells were treated with the 6 adhesion to collagen indicated mAbs as stated in Materials and Methods. Data represent ratios SEM of more than three independent experiments performed in technical Drbal et al. (32) described that cell stimulation by PMA led quadruplicates with significance calculation via one-way ANOVA with a to inside–out activation of b2 integrins and exposure of the Bonferroni posttest. *p , 0.05, **p , 0.01, ***p , 0.001. 8 CD11b/CD18 ADHESION TO COLLAGEN REGULATED BY FRb presence of these mAbs, MEM-48 was able to abrogate binding of reduction of adhesion of FRb+ M-CSF–differentiated macro- control- and FRb-expressing THP-1 cells to type I collagen and phages to rat type I collagen whereas no such effect was evident severely reduced adhesion to type IV collagen (Fig. 6). Alterna- for other ECM components (Fig. 7D). Finally, silencing of en- tively, mAb MEM-148 treatment increased adhesion of FRb- dogenous FRb in M-CSF–differentiated macrophages (Fig. 7E) transduced THP-1 cells to type I and type IV collagen, and in the increased selectively their adhesion to collagen (Fig. 7F), con- case of type IV collagen, mAb MEM-148 treatment even resulted firming the functional consequence of FRb on the adhesiveness of in a reconstitution of the adhesion to the level comparable to CD11b/CD18 also in primary cells. Therefore, we conclude that control cells. Because MS revealed CD18 as the top interaction FRb modulates the ability of the human macrophage subset ex- partner of FRb (Fig. 2A, Supplemental Table I), we therefore posed in vivo to M-CSF to adhere to collagen. conclude that the protein–protein interaction of FRb with CD18 mediates a conformational change in the integrin heterodimer Discussion CD11b/CD18, which reduces the cell’s capacity to adhere to By MS of proteins coprecipitating with FRb, we identified collagen. multiple cell surface receptors described to be involved in in- ternalization (CD280), degradation of (MMP14, CD13, CD44, b A subset of primary human macrophages expressing FR CD147), and adhesion to (CD18, CD11a, CD11b, CD11c, CD29, shows reduced adhesion to collagen CD49d, CD49e) the ECM component collagen (Supplemental To assess whether this interaction of FRb and CD18 and its func- Table I). Among those, we found CD18 as the most abundant tional consequence on cell adhesion can be recapitulated in cells protein coprecipitating with FRb from lysates of myeloid THP-1 naturally expressing FRb, we analyzed the protein microenviron- cells and of a human -derived macrophage subset. Downloaded from ment of FRb in primary human macrophages and investigated their CD18 is the common b-chain of b2 integrins (exclusively adhesion properties to ECM components. We isolated CD14+ expressed by immune cells and thus also called leukocyte monocytes from peripheral blood of healthy donors and differen- integrins) and noncovalently assembles to one of four a-chains tiated them with either GM-CSF or M-CSF. We found that FRb is resulting in LFA-1 (CD11a), Mac-1/CR3 (CD11b), CR4 (CD11c), moderately expressed already on blood monocytes and completely and adb2 (CD11d) (17). By mass spectrometric analysis, we also absent on GM-CSF–differentiated macrophages, but it is highly found known functional interaction partners of CD11b/CD18 to upregulated upon differentiation with M-CSF (Fig. 7A). Similarly coprecipitate with FRb: the GPI-anchored proteins CD14 and http://www.jimmunol.org/ to FRb-transduced THP-1 cells, we detected a coprecipitation of CD87, as well as galectin-1 (39) (Supplemental Table I). As all of CD18 with FRb with the M-CSF–differentiated macrophages these proteins associate with FRb in the presence of the detergent (Fig. 7B, 7C). Vice versa, we confirmed the coprecipitation of lauryl maltoside, which does not preserve lipid-mediated interac- FRb in a CD18-specific pulldown (Fig. 7C). Additionally, we tions, we conclude that FRb is directly associating with CD11b/ investigated whether GM-CSF- and M-CSF-differentiated mac- CD18 and several of its functional interaction partners via protein– rophages adhere differently to collagen. We detected a significant protein interactions. In addition to the physical interaction, we show by guest on October 2, 2021 FIGURE 7. FRb associates with CD18 also in a human macrophage subset differentiated with M-CSF and mediates its reduced adhesion to collagen. (A) Primary human CD14+ mono- cytes were analyzed either immediately after isolation or were differentiated to macrophages with either GM-CSF or M-CSF for 7 d. FRb expression was assessed by flow cytometry. (B) Proteins, enriched in FRb versus isotype control IPs of M-CSF–differentiated macrophages, detected via LC-MS/MS of four independent experiments. Significance calculation was per- formed using an unpaired Student t test with equal variances. The five most abundant pro- teins detected are displayed. (C) Western blots of FRb (upper panel) and CD18 (lower panel) IPs of lysates of M-CSF–differentiated macro- phages probed with anti-FRb and CD18 mAbs. (D) Relative adhesion of 7 d GM-CSF– versus M-CSF–differentiated macrophages to ECM- coated plates. Data represent ratios 6 SEM of five independent experiments performed in technical quadruplicates with significance cal- culation via a one-sample t test. (E and F) FOLR2- and control small interfering RNA– treated, M-CSF–differentiated macrophages were tested for (E)FRb expression by flow cytometry and (F) adhesion to fibronectin- and type I collagen– coated plates. Data represent ratios 6 SEM of three independent experiments with significance cal- culation via a one-sample t test. **p , 0.01, ***p , 0.001. The Journal of Immunology 9 in the present study that the GPI-anchored FRb regulates the ad- Acknowledgments hesiveness of CD11b/CD18 to collagen. We thank Vaclav Horejsi and Johannes Sto¨ckl for providing mAbs, FRb is not the first described regulator of CD11b/CD18. In Johannes Huppa for recombinant human ICAM-1, Georges Bismuth and particular, other GPI-anchored proteins, such as the LPS co- Delphine Le Roux for control- and folate-labeled Qdot-655 nanocrystals, receptor CD14 (40, 41) and the urokinase receptor CD87 (18, 20), Giulio Superti-Furga for the pfMSCV-N-Strep3xHA-IRES-GFP plasmid, both coprecipitate with FRb and CD11b/CD18 as described and Alexander M. Carmo for the THP-1 cell line. above. Further, the Fcg receptor CD16 (42, 43) and the CD31 counterreceptor CD177 on (44) were shown to mod- Disclosures ulate CD11b/CD18–mediated phagocytosis, migration, as well as M.S. is employed by EXBIO Praha. The other authors have no financial cell–cell and cell–matrix adhesion of monocytes, macrophages, conflicts of interest. and neutrophils. The lectin domain of CD11b was identified to be essential for these interactions, as GPI-anchored proteins could References be displaced through competition with b-glucans or N-acetyl-D- 1. 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