OSCAR Is a Receptor for Surfactant Protein D That Activates TNF- Α Release from Human CCR2 + Inflammatory Monocytes

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OSCAR Is a Receptor for Surfactant Protein D That Activates TNF- α Release from Human CCR2 + Inflammatory Monocytes

This information is current as Alexander D. Barrow, Yaseelan Palarasah, Mattia Bugatti, of October 1, 2021. Alex S. Holehouse, Derek E. Byers, Michael J. Holtzman, William Vermi, Karsten Skjødt, Erika Crouch and Marco Colonna J Immunol 2015; 194:3317-3326; Prepublished online 25 February 2015; doi: 10.4049/jimmunol.1402289 Downloaded from http://www.jimmunol.org/content/194/7/3317

Supplementary http://www.jimmunol.org/content/suppl/2015/02/24/jimmunol.140228 http://www.jimmunol.org/ Material 9.DCSupplemental References This article cites 40 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/194/7/3317.full#ref-list-1

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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. The Journal of Immunology

OSCAR Is a Receptor for Surfactant Protein D That Activates TNF-a Release from Human CCR2+ Inﬂammatory Monocytes

Alexander D. Barrow,* Yaseelan Palarasah,† Mattia Bugatti,‡ Alex S. Holehouse,x,{ Derek E. Byers,‖ Michael J. Holtzman,‖ William Vermi,*,‡ Karsten Skjødt,† Erika Crouch,* and Marco Colonna*

Surfactant protein D (SP-D) is critical for maintenance of lung homeostasis and provides a first line of defense to pathogens at mucosal surfaces. Polymorphisms in the SP-D–encoding gene SFTPD have been associated with chronic obstructive pulmonary disease and ulcerative colitis. Identification of the immunoreceptors that bind SP-D is essential for understanding its contribution to lung homeostasis and mucosal defense. We located a putative binding motif for the osteoclast-associated receptor (OSCAR) Downloaded from within the SP-D collagenous domain. An OSCAR-Fc fusion protein specifically bound to the collagenous region of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2+ inflammatory monocytes, which secreted TNF-a when exposed to SP-D in an OSCAR-dependent fashion. OSCAR and SP-D did not exclusively colocalize in lung, as they were also highly expressed in atherosclerotic plaques of human aorta, supporting a role for this interaction in atherosclerosis. Our results identify the OSCAR:SP-D interaction as a potential therapeutic target in http://www.jimmunol.org/ chronic inflammatory diseases of the lung as well as other diseases involving tissue accumulation of SP-D, infiltration of inflammatory monocytes, and release of TNF-a. The Journal of Immunology, 2015, 194: 3317–3326.

urfactant protein D (SP-D) is a member of the collagenous dues (Cys15 and Cys20). Multimeric SP-D dodecamers can be lectins (collectins), which provide a first line of humoral formed through N-terminal disulfide bonding of trimeric SP-D S innate immune defense (1–3). The collectin family also monomers. Within the collectin family, the formation of dodeca- includes, but is not limited to, mannan-binding lectin (MBL) and mers is unique to SP-D, which can be observed as the charac- surfactant protein A (SP-A). Collectins are soluble proteins that teristic cruciform structures by electron microscopy (5). The by guest on October 1, 2021 are structurally characterized by an N-terminal collagenous re- collectin family is expressed in different mucosae and plays an gion, a flexible coiled coil neck region, and a C-terminal carbo- important tissue-specific role in the innate immune response (4). hydrate recognition domain (CRD), which binds various sugars in SP-D is predominantly secreted by alveolar type II epithelial a calcium-dependent fashion (4). The hydrophobic N-terminal cells, but is also produced outside of the lung, in the gastrointestinal region of the SP-D polypeptide encodes two cysteine (Cys) resi- and genital mucosae, salivary glands, prostate, kidney, pancreas, skin, and endothelial cells (6). SP-D can act as a pattern recognition receptor through binding of the CRD to evolutionary con- *Department of Pathology and Immunology, Washington University School of Med- icine, St. Louis, MO 63110; †Department of Cancer and Inflammation, University of served glycolipids and glycoproteins associated with infectious Southern Denmark, 5000 Odense, Denmark; ‡Department of Molecular and Trans- agents, such as LPS from certain bacterial species or viral enve- lational Medicine, Section of Pathology, School of Medicine, University of Brescia, lope glycoproteins. SP-D can thus opsonize, neutralize, and ag- Brescia 25123, Italy; xDivision of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63110; {Center for Biological Systems Engineering, Wash- glutinate infectious microorganisms predisposing to elimination ington University, St. Louis, MO 63130; and ‖Division of Pulmonary and Critical by phagocytes. In the lung, SP-D also plays an important ho- Care Medicine, Department of Medicine, Washington University School of Medi- meostatic role through CRD-dependent scavenging of surfactant cine, St. Louis, MO 63110 phospholipids by alveolar macrophages (7, 8). SP-D–deficient ORCID: 0000-0002-4155-5729 (A.S.H.). (Sftpd2/2) mice developed accumulation of surfactant phospho- Received for publication September 10, 2014. Accepted for publication January 24, 2015. lipids in the lungs, as well as infiltration of monocytes and the proinflammatory activation of alveolar macrophages, leading to This work was supported by National Institutes of Health Grant 5R01HL097805 (to M.C.) and a Marie Curie International Outgoing Fellowship (to A.D.B.). chronic inflammation, emphysema, and fibrosis (9, 10). Correction 2/2 Address correspondence and reprint requests to Prof. Marco Colonna at the current of some, but not all, of the pulmonary abnormalities in Sftpd address: BJC Institute of Health at Washington University, 425 South Euclid, Campus mice required the transgenic expression of SP-D with an intact Box 8118, St. Louis, MO 63110. E-mail address: [email protected] collagenous domain (11). SP-D–deficient children were suscepti- The online version of this article contains supplemental material. ble to more frequent pneumonias, and long-term outcome was Abbreviations used in this article: BAL, bronchoalveolar lavage; COPD, chronic worse than SP-D–sufficient control children (12). obstructive pulmonary disease; C1q, complement component 1q; CRD, carbohy- drate recognition domain; Cys, cysteine; ILT, Ig-like transcript; LAIR, leukocyte- Human SFTPD genotype can influence the assembly, concen- associated Ig-like receptor; MBL, mannan-binding lectin; NCRD, neck CRD; tration, and biological function of SP-D in vivo (13). Interestingly, OSCAR, osteoclast-associated receptor; SP-A, surfactant protein A; SP-D, surfac- polymorphisms in SFTPD have been associated with susceptibil- tant protein D; TREM, triggering receptor expressed on myeloid cells. ity to chronic and infectious lung diseases, such as chronic ob- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 structive pulmonary disease (COPD) (14, 15), emphysema (16), www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402289 3318 OSCAR IS A NOVEL MONOCYTE SP-D RECEPTOR pneumococcal lung disease (17), and tuberculosis (18), and may cDNA with Platinum Pfx DNA Polymerase (Invitrogen) using the fol- even influence clinical outcome following lung transplantation lowing forward, 59-CATCCTCGAGCAGGAGGAAGATCTGCCCAG-39 (19). Serum SP-D levels have been associated with lung function and reverse, 59-GAATTCTAGAATGCTCAGCTTTCAGGCCTTG-39 primers (XhoI and XbaI restriction sites underlined). XhoI- and XbaI- or health status in patients with severe COPD (20). SFTPD ge- restricted PCR products were cloned into the Signal Ig plus vector, notype has also been associated with inflammatory bowel dis- whichencodedtheLAIR-1ectodomaininframewithhumanIgG1Fc. eases, such as Crohn’s disease and ulcerative colitis (21). SP-D is Cloning and expression of Ig-like transcript (ILT)-1, -3, and -7 and trig- also produced by vascular endothelial cells and has been impli- gering receptor expressed on myeloid cells (TREM)-1 and -2 Fc-fusion 2 2 proteins were as described (28). cated in lipid homeostasis and vascular lipid deposition. Sftpd / mice were protected from diet-induced atherosclerosis (22), Cell culture, expression, and purification of recombinant whereas a polymorphism of SFTPD has been associated with Fc-fusion proteins coronary artery disease (23). These data suggest an important role The 293T were transiently transfected with 48 mg plasmid DNA/10-cm dish for SP-D in regulating pulmonary homeostasis in addition to roles using Lipofectamine 2000 diluted in Optimem (both Invitrogen), according in the gut and vascular system, although the molecular basis for to the manufacturer’s instructions. Fc-fusion proteins were purified from this remains ill defined. serum-free culture supernatants by protein A affinity chromatography. Identification of the immunoreceptors that capture SP-D and Recombinant collectin and collectin-like molecules transduce intracellular signaling is therefore essential for under- The cloning and expression of recombinant full-length human SP-D and the standing how SP-D contributes to lung homeostasis and in- full-length and mutant isoforms of rat SP-D have been described in detail nate mucosal defense and SFTPD disease associations (3, 24). before (5, 29). Briefly, S15, 20 is a recombinant rat SP-D mutant with Osteoclast-associated receptor (OSCAR) is an activating receptor a substitution of serine for Cys15 and Cys20 that is assembled exclusively for collagen expressed by osteoclasts that costimulates osteo- as SP-D trimers (29). Mini SP-D is a mutant form of rat SP-D that lacks Downloaded from two internal exons (3, 4) encoding the SP-D collagenous domain. The neck clastogenesis (25). OSCAR transmits intracellular signals through CRD (NCRD) (trimeric neck + CRD) is a rat SP-D truncation mutant the associated adapter FcRg (26), which contains an ITAM that lacking the N-terminal peptide and collagen domains that retains lectin recruits the protein tyrosine kinase Syk. Although in mouse OS- activity (5). Recombinant human MBL was purchased from Sino Bio- CAR is exclusively expressed in osteoclasts, human OSCAR was logical (Beijing, China), and complement component 1q (C1q) purified reported to be also expressed on monocytes, macrophages, neu- from human serum was purchased from Complement Technology. The endotoxin level in our stock (112 mg/ml) human SP-D reagent was de- http://www.jimmunol.org/ trophils, and dendritic cells (26). Human OSCAR was shown to termined at 120 EU/ml by Limulus amebocyte lysate assay (Lonza). enhance the proinflammatory response of monocytes, although the Immobilization of Abs, proteins, and peptides monocyte subset that specifically expressed OSCAR was not described (26, 27). The wider expression of OSCAR by human Collagen I from rat tails and BSAwere purchased from Sigma-Aldrich. BSA myeloid immune cells suggested to us that OSCAR might play (2%), collagens (2.5 mg/ml), recombinant SP-D, and isoforms (2.5 mg/ml) a role in innate immunity in addition to the reported role in were immobilized onto either tissue culture or ELISA plates overnight in 100 ml of 0.01 M acetic acid at 4˚C. Fc-fusion proteins (0.25 mg/ml) were osteoclastogenesis. In this study, we identified SP-D as a candidate immobilized onto plates in 100 ml coating buffer (15 mM Na2CO3, 35 ligand for OSCAR by a bioinformatics search for proteins en- mM NaHCO3 [pH 9.6]) overnight at 4˚C. All peptides were certified LPS coding OSCAR-binding motifs (25). We further demonstrated that free by Limulus amebocyte lysate assay (Lonza). All wells were washed by guest on October 1, 2021 OSCAR and SP-D colocalized in alveolar macrophages and that three times in TBS to remove excess proteins before blocking in 5% BSA in TBS for 1 h at 37˚C prior to performing binding assays. the OSCAR:SP-D interaction triggered TNF-a production by inflammatory monocytes. Solid- and soluble-phase SP-D–binding assays For solid-phase SP-D–binding assays, 5 mg/ml each Fc-fusion protein in Materials and Methods 100 ml TBS-binding buffer (10 mM Tris.HCl [pH 7.5], 150 mM NaCl, 6 Bioinformatics 0.1% BSA, 0.05% Tween 20 5 mM CaCl2) was incubated at room temperature for 1 h in ELISA plates. Wells were then washed five times in To initially identify putative OSCAR-binding candidate proteins for further TBS-binding buffer 6 5mMCa2+ before incubating with 1:5000 goat anti- scrutiny, an exhaustive protein BLAST search was carried out using all human Ig-HRP conjugate (Southern Biotech) for 1 h in TBS-binding buffer permutations of the 9-aa minimum OSCAR-binding motif and side chain– 6 5mMCa2+. Wells were then washed an additional five times in TBS- binding variants defined previously (25): G[A|P|G]PG[P|A][A|S]G[F| binding buffer 6 5mMCa2+ before developing with O-phenylenediamine D|S|Y][A|R|P|Q]. In addition, a less stringent but longer 12-aa con- dihydrochloride, and the absorbance at 490 nM was recorded. For soluble- sensus OSCAR-binding sequence, derived from the same data and used phase SP-D–binding assays, 2-fold dilutions of either recombinant human to generate the minimum OSCAR-binding sequence, was also used: SP-D dodecamer (starting at 125 ng/ml) or human bronchoalveolar lavage GXPGPX9GFX9GXP (where X is any amino acid). BLAST searches were (BAL) (starting at 1/4 dilution) were made in TBS-binding buffer and done with an expected threshold of 20,000, a word size of 2, and using the incubated in Fc-fusion–coated wells for 3 h. Captured SP-D was detected PAM30 matrix, as recommended by National Center for Biotechnology using 2 mg/ml anti-human SP-D mAb (Hyb-246-04) for 1 h at room Information when searching for short, nearly exact matches. Candidate temperature, followed by rabbit anti-mouse IgG-HRP (Dako). motifs in both collagen and noncollagen proteins were identified. For noncollagen proteins, putative motifs were selected in which protein Generation and purification of mAbs BLAST provided an alignment of 80% or better, although one or two To generate blocking mAbs to the OSCAR ectodomain, NMRI mice were substitutions between amino acids of similar physiochemical properties immunized s.c. three times with 20 mg human OSCAR-Fc. For generation were treated as matching. As an additional crucial criterion, putative motifs of mAbs to the OSCAR cytoplasmic tail, mice were immunized with were also required to lie within a collagenous domain, as had previously been a synthetic peptide, representing the last 14 C-terminal residues (Cys- identified either experimentally or through computational approaches. For DWRSQNRAPAGIRP) of human OSCAR. The synthetic peptides were collagens, an 8/9-aa sequence alignment with one of the minimum OSCAR- coupled to diphtheria toxoid via the cysteine using amine-to-sulfhydryl binding motif variant sequences was required for a motif to be recognized, cross-linker (Thermo Scientific). Three days prior to the fusion, the mice although for the vast majority a perfect alignment was obtained. In the non- received an i.v. injection with 50 mg immunogen administered together collagen proteins, the biological roles of each protein could be broadly clas- with adrenalin. The fusion and selection were done essentially as described sified into receptor proteins, secreted proteins, or extracellular matrix proteins. (30). The SP2/0-AG14 myeloma cell line was used as fusion partner. Plasmids Positive clones for the OSCAR ectodomain were selected by differential screening of OSCAR-Fc versus an irrelevant Fc fusion by ELISA. Positive The human OSCAR-Fc construct has been described before (25). The clones for the OSCAR C-terminal cytoplasmic tail were identified by extracellular domain of human leukocyte-associated Ig-like receptor screening to the peptide used for immunization, described above. Cloning (LAIR)-1 was amplified from plasmid DNA containing the human LAIR-1 was performed by limited dilution. Single clones were grown in culture The Journal of Immunology 3319

flasks in RPMI 1640 plus 10% FCS, and mAbs were purified from culture Monocyte isolation supernatant by protein A affinity chromatography. Human monocytes were isolated from 50 ml blood using the Monocyte Abs Negative Isolation Kit (Invitrogen), according to the manufacturer’s instructions. Goat anti-human OSCAR polyclonal Ab was purchased from Santa Cruz Biotechnologies (25). The SP-D mAb 245-01 was purchased from Enzo Cytokine analysis Life Sciences. The anti–SP-D mAb 246-04 was purchased from Bioporto Diagnostics (Gentofte, Denmark). The mAb KP1, which recognizes human A total of 100,000 monocytes in flat-bottom 96-well plates was stimulated CD68, was purchased from Serotec. Donkey anti-goat IgG Alexa 568 with 2.5 mg/ml recombinant human SP-D in the presence or absence of 5 and donkey anti-mouse IgG Alexa 488 were purchased from Invitrogen. mg/ml purified mAbs. TNF-a in tissue culture supernatants was deter- Purified and PE-conjugated anti-human OSCAR mAb 11.1CN5 was pur- mined using the BD CBA human inflammatory cytokine kit (BD Bio- chased from Beckman Coulter. The HLA-DR-allophycocyanin, CD16- sciences). FITC, and CD56-PE-Cy7 anti-human Abs, used to define human monocytes, granulocytes, and NK cells, respectively, were purchased from BD Statistical analysis Pharmingen, and the CD14-Pacific Blue, CD45-AF700, and CCR2-PerCP- Statistical significance was determined using GraphPad Prism 6.0. Statis- Cy5.5 anti-human Abs were all purchased from BioLegend. Mouse mAb tical differences were determined by two-tailed Student t test. A p value to thyroid transcription factor 1, clone SPT24, was from Novocastra ,0.05 was considered significant. Laboratories. Mouse mAb to human CD163, which recognizes cells of the monocyte/macrophage lineage, was from Neomarkers. mAb clone 13-9-17 Study approval was used in immunostainings for the OSCAR C terminus. Rabbit anti–SP- D clone H-120 was from Santa Cruz. Rabbit isotype control Ab was from All studies were reviewed and approved by institutional human and animal Cell Signaling, and mouse IgG1 isotype control was from BD Biosciences. studies committees, and all experiments were carried out according to For immunohistochemistry, primary Abs were revealed with Envision national and regional legislation and regulations. Informed consent was Dako Polymer HRP. obtained from all subjects included in this study. Downloaded from Microscopy Results Paraffin-embedded human lung sections from COPD and normal control OSCAR is a receptor for SP-D subjects were deparaffinized using citrisolve (Vector Laboratories). Sections were then rehydrated in a graded ethanol series before Ag retrieval by We hypothesized that OSCAR may interact with collagenous boiling in Ag retrieval solution (Vector Laboratories) in a microwave, molecules other than extracellular matrix collagens, which may according to the manufacturer’s instructions. Sections were blocked in fish have immunological roles. We conducted a bioinformatics search http://www.jimmunol.org/ gel (Sigma-Aldrich) for 1 h before incubating with primary Abs diluted in using two partially overlapping linear amino acid sequences (see fish gel overnight at 4˚C. Slides were then washed three times in large volumes of PBS before incubating individually with secondary Abs, re- Materials and Methods) derived from the previously defined spectively. Tiramide enhancement of fluorescent immunostaining was triple-helical OSCAR-binding motif (25). The search yielded 22 performed after each individual secondary Ab step, according to the noncollagen-based sequences (Table I) and 16 putative collagen- manufacturer’s instructions (Invitrogen). For confocal microscopy, lung based OSCAR-binding motifs (Supplemental Table I). The col- sections were mounted with vectashield containing DAPI, which stained lectins SP-A, SP-D, and MBL, and collectin-like molecules, such nuclei blue (Vector Laboratories), and analyzed on an Olympus Fluoview FV1000 confocal microscope. For light microscopy, sections stained as C1q (Table I), represented good candidates for interacting with immunohistochemically were photographed using the DP-70 Olympus OSCAR-expressing leukocytes because they play central roles in by guest on October 1, 2021 digital camera mounted on the Olympus BX60 microscope. tissue-specific immunity (1). Focusing on SP-D, the minimum Human subjects and BAL isolation 9-aa OSCAR-binding motif used in the bioinformatics search aligned with 77.8% direct amino acid sequence identity to the SP- Human clinical samples were obtained from lung transplant donors and recipients, as described previously (31). In brief, lung explants were har- D sequence 106–114 (GPPGPPGVP) (Supplemental Fig. 1A). The vested at the time of lung transplantation from COPD recipients (n =2) 12-aa consensus OSCAR-binding motif used in the bioinformatics with very severe disease (GOLD Stage 4). Control samples were obtained search aligned with 83.3% direct amino acid identity with the from normal donors (n = 3) using lung explants that were otherwise not SP-D sequence 106–117 (GPPGPPGVPGPA) (Supplemental Fig. useable for transplantation. BAL was performed under general anesthesia using standard procedures: briefly, the bronchoscope was placed in the 1B). The putative OSCAR-binding motifs are encoded by exon 3 right middle lobe and the lavage was performed using 3 3 50 ml sterile of human SFTPD, which encodes part of the SP-D collagenous saline and aspirated under low pressure. The concentration of SP-D in domain (Supplemental Fig. 1A). Because the 9- and 12-aa search BAL was determined at 3495 ng/ml, as previously described (32). motifs are largely overlapping, we conclude that each trimeric Flow cytometry subunit of the SP-D monomer encodes one triple-helical OSCAR- binding sequence. Dodecameric SP-D, comprised of four trimeric Cells were prepared from human lung explant tissue using protease digestion, as described previously (31). Single-cell suspensions were gen- subunits, would thus be expected to encode four triple-helical erated from minced lung tissue that was subjected to collagenase (Liberase OSCAR-binding motifs. Blendzyme III; Roche), hyaluronidase (Sigma-Aldrich), and DNase I We next determined whether OSCAR could bind to some of the (grade II; Roche) digestion for 45 min at 37˚C. For flow cytometry, frozen collectin or collectin-like molecules identified from the motif aliquots of cell preparations were thawed and washed in complete medium before analysis. Following FcR blockade, cell suspensions were incubated search (Table I). We assayed for binding of a human OSCAR Fc- with labeled Abs on ice prior to flow cytometric analysis. For peripheral fusion protein (OSCAR-Fc) to recombinant forms of SP-D, in blood leukocytes, granulocytes were defined by electronic gating as side addition to C1q and MBL proteins (Fig. 1). OSCAR-Fc bound to 2 scatterhighCD45+CD56 CD16+ cells and monocytes as side scatterlow full-length dodecameric human and rat SP-D and trimeric subunits + + 2 CD45 HLA-DR CD56 . Monocytes were further distinguished based on of rat SP-D (Fig. 1A). OSCAR-Fc did not bind to Mini SP-D, expression of CD14, CD16, and CCR2 (27). a recombinant rat SP-D lacking the SP-D collagenous domain OSCAR internalization assays encoded by exons 2 and 3 of the SP-D gene (5), or a recombinant Purified monocytes were left untreated (0 min) or treated with 2.5 mg/ml protein encoding the NCRD region of rat SP-D (Fig. 1A). recombinant human SP-D at 37˚C for 5, 15, 30, and 60 min, respectively. OSCAR-Fc binding was specific for SP-D because OSCAR-Fc did Monocytes were then placed on ice and washed twice with ice-cold PBS not bind to either C1q or MBL (Fig. 1A), even though these before fixing with 2% paraformaldehyde for 10 min on ice. After washing three times in PBS, OSCAR internalization was determined by flow proteins were predicted to encode putative OSCAR-binding motifs cytometry as a reduction in monocyte cell surface staining with non- (Table I). OSCAR-Fc binding to full-length dodecameric or tri- blocking anti-OSCAR mAb, 11.1CN5. meric SP-D or collagen I was significantly increased by, but not 3320 OSCAR IS A NOVEL MONOCYTE SP-D RECEPTOR

Table I. Amino acid sequences and start positions of putative OSCAR-binding motifs within collagenous proteins (noncollagens)

Biological Class Protein Name Uniprot Sequence Position Notes Extracellular EMILIN-1 Q9Y6C2 GPPGPPGLQGPP 820 Secreted adhesive protein, involved in matrix and GPPGPAGPPGSP 829 elastic ﬁber anchoring adhesion EMILIN-2 Q9BXX0 TVPGAEGFAGAP 876 Secreted adhesive protein, involved in elastic ﬁber anchoring/vessel assembly Protein sidekick-2 Q58EX2 GAPGPPGVP 1823 Adhesion protein (neuronal development) Receptors Collectin-12 (scavenger Q5KU26 GPPGPAGER 476 Pathogen recognition/phagocytosis receptor with C-type lectin GVPGPRGLPGLP 557 type I) Macrophage receptor with Q9UEW3 GPPGLAGFP 286 Pattern recognition receptor—binds collagenous structure bacteria (MARCO) Scavenger receptor class A Q6AZY7 GAPGPPGPR 457 Scavenges oxidative species member 3 (SCARA3) GPPGPPGSQ 595 Scavenger receptor class A Q6ZMJ2 GPPGPKGDQ 306 Ferritin receptor—nontransferrin- member 5 (SCARA5) dependent delivery of iron Secreted C1q-related factor O75973 GPPGPPGDP 79 Implicated in excitatory synapse regulation on hippocampus neurons Cell proliferation-inducing A1KY36 GLPGPPGFPGIG 523 — Downloaded from protein 41 Collagen and calcium- Q6UXH8 GAPGPRGSPGPP 321 Angiogenic and lymphangioblastic binding EGF domain- growth during embryogenesis containing protein 1 Complement C1q-like Q86Z23 GPPGPPGPRGPP 70 Implicated in excitatory synapse protein 4 regulation on hippocampus neurons

Complement C1q P02746 GGPGAPGAP 99 Part of the complement cascade http://www.jimmunol.org/ subcomponent subunit B GAPGAPGPK 102 Complement C1q P02747 GVPGPMGIPGEP 97 Part of the complement cascade subcomponent subunit C Complement C1q TNF- Q9BXJ5 GAPGPSGMMGRM 49 — related protein 2 GPPGRTGNRGKP 82 Ectodysplasin-A (EDA) Q92838 GPPGPPGPQGPP 189 Epithelial-mesenchymal signaling during morphogenesis Ficolin-2 Q15485 GPPGPNGAP 84 Liver-secreted, innate immunity protein Ficolin-3 O75636 GAPGPQGPP 57 May activate lectin complement pathway by guest on October 1, 2021 Mannose-binding protein C P11226 GNPGPSGSPGPK 80 Innate immune calcium-dependent lectin Surfactant protein A Q8IWL2 GPPGPMGPP 55 Collectin, found in the lungs, many roles Surfactant protein D P35247 GPPGPPGVP 106 Collectin, found in the lungs, many GPPGPPGVPGPA 106 roles Other Gliomedin Q6ZMI3 GPPGPPGPPGPP 236 Membrane protein involved in formation of the nodes of Ranvier premRNA 39 end processing Q9C0J8 GPPGPRGMQGPP 742 Cleavage and polyadenylation of pre- protein WDR33 mRNA 39 ends (nuclear protein)

wholly dependent on, the presence of 5 mM Ca2+ (Fig. 1A). Be- soluble SP-D, as might reflect conditions in vivo. We incubated cause collagen lacks lectin activity and neither recombinant lectin dilutions of human BAL (Fig. 1B) in wells coated with OSCAR-Fc domains (NCRD) nor Mini SP-D, which encodes an intact lectin or LAIR1-Fc. The amount of protein captured by the immobilized domain, bind to OSCAR (Fig. 1A), we conclude the small in- Fc-fusion proteins was then detected using the Hyb-246-04 mAb, crease in calcium-dependent binding of OSCAR-Fc to SP-D or which is specific for the SP-D CRD (6). Dose-dependent binding collagen must be mediated through OSCAR binding to collagen of a protein reactive with the Hyb-246-04 mAb was observed to domains and not the SP-D lectin domain. SP-D binding was not immobilized OSCAR-Fc, but not to LAIR1-Fc (Fig. 1B). These observed for Fc-fusion proteins that encoded the extracellular results suggest OSCAR-Fc can capture native SP-D from human domains of human LAIR1 or ILT3, which are known to bind BAL. However, because we have detected a putative OSCAR-binding collagen (33, 34), or TREM1, TREM2, ILT1, and ILT7 (Fig. 1A motif in SP-A (Table I) and SP-A has been shown to bind to Igs (36), and data not shown). Although SP-D was recently shown to bind it may be possible that solid-phase OSCAR-Fc could also be cap- LAIR1 (35), we only detected SP-D binding to mouse LAIR1, but turing SP-A from BAL. Captured SP-A may, in turn, bind to the Fc not human LAIR1 (data not shown). This discrepancy may depend portion of the secondary Abs used to detect bound SP-D. We there- on the sensitivity of our assay and the relative affinities of mouse fore determined whether immobilized OSCAR-Fc could capture and human LAIR1 for SP-D. In conclusion, our results show that soluble recombinant human SP-D dodecamer in the absence of SP-A. OSCAR can bind specifically to the SP-D collagenous domain. Solid-phase OSCAR-Fc, but not LAIR1-Fc, captured soluble recom- Because SP-D is secreted as a soluble protein associated with binant human SP-D dodecamer (Fig. 1C). These results show that lung surfactant, we next determined whether OSCAR could bind to OSCAR can bind to soluble SP-D dodecamer. The Journal of Immunology 3321 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. OSCAR is a novel SP-D receptor. (A) OSCAR-Fc, LAIR1-Fc, ILT3-Fc, and TREM1-Fc binding to different human (h) and rat (r) SP-D isoforms and collagen I (65mMCa2+) in solid phase. (B) Capture of Hyb-246-04 mAb reactive protein from human BAL (n = 1 donor) or (C) soluble recombinant human SP-D dodecamer by plate-immobilized OSCAR-Fc (circles) or LAIR1-Fc (triangles). (D) GFP expression from OSCAR-CD3z NFAT- GFP reporter cells incubated with recombinant SP-D isoforms in solid or (E) soluble phase. Data were performed in triplicate and are representative of three independent experiments. *p , 0.05.

To test whether OSCAR could act as a receptor for SP-D, we secreted in lung surfactant where it is readily taken up by alveo- performed a sensitive GFP-inducible assay using NFAT-GFP re- lar macrophages and monocytes exhibit greater infiltration of porter cells, which have been successfully used to determine inflamed lung airways (38), we next determined OSCAR and SP- immunoreceptor:ligand interactions (37) (Fig. 1D). In this assay, D expression in lungs from control and COPD subjects. OSCAR clustering of the OSCAR ectodomain fused to the TCR CD3z was expressed in alveolar macrophages with a seemingly intra- signaling chain will result in ITAM signaling and transactivation cellular granular pattern, whereas SP-D was predominantly ex- of a transgene encoding GFP (25, 37). GFP was induced by OSCAR- pressed in alveolar type II epithelial cells (Fig. 2A). Immunofluo- CD3z/NFAT-GFP reporter cells cultured on tissue culture plates rescence analysis for OSCAR and the monocyte/macrophage coated with human SP-D, collagen I, recombinant rat dodecameric marker CD68 confirmed the localization of OSCAR in intracel- or trimeric (S15, 20) SP-D, but not BSA, Mini SP-D, or NCRD lular compartments of CD68+ alveolar macrophages in both nor- (Fig. 1D). OSCAR-CD3z/NFAT-GFP reporter cells also expressed mal and COPD lung sections (Fig. 2B). A few infiltrating CD68+ GFP when cultured with soluble human SP-D dodecamer, but not interstitial monocytic cells displayed a membrane pattern of OS- soluble collagen I (Fig. 1E). These results show that OSCAR is CAR staining in COPD sections (Fig. 2B). Because immunohis- a receptor for dodecameric SP-D either as a plate-bound aggre- tochemical and immunocytochemical electron microscopy studies gated ligand or as soluble SP-D dodecamer. had found SP-D localized to endocytic compartments and associated with phagolysosomal structures in human and rat alveolar OSCAR is expressed in intracellular compartments of human macrophages (6, 39), we hypothesized that OSCAR and SP-D alveolar macrophages that contain SP-D may colocalize in the same intracellular compartments of alveo- Human OSCAR has previously been located by immunohisto- lar macrophages. Accordingly, double immunofluorescence for chemistry in osteoclasts and their precursors but not in monocytes OSCAR and SP-D in lung sections demonstrated intracellular or macrophages in tissues outside of bone (25). Because SP-D is colocalization of OSCAR and SP-D in alveolar macrophages 3322 OSCAR IS A NOVEL MONOCYTE SP-D RECEPTOR Downloaded from http://www.jimmunol.org/

FIGURE 2. OSCAR expression in lung-resident immune cells. (A) Representative light microscopy sections of normal lung immunostained with the following: (Ai) rabbit (brown) and mouse IgG1 (blue; original magnification 3200) control Abs; (Aii) rabbit anti–SP-D (brown; original magnification 3200); (Aiii) rabbit anti–SP-D and thyroid transcription factor 1 mAb (blue; original magnification 3600); or (Aiv) OSCAR C terminus mAb (brown; original magnification 3200). (B) Representative confocal sections of normal (Bi–Biii) or COPD (Biv–Bvi) lungs immunostained with the following: (Bi) goat (red) and mouse IgG2a (green) control Abs; (Bii–Bvi) goat anti-OSCAR (red) and CD68 mAb (green; colocalization, yellow). Scale bar, 20 mM. (Biii by guest on October 1, 2021 and Bv) Higher magnification confocal microscopy of areas demarcated by white squares in (Bii) and (Biv), respectively. Scale bars, 5 mM. (Bvi) Alveolar macrophage (arrow) and smaller interstitial lung monocyte (arrowhead). Scale bar, 5 mM. (C) Representative confocal sections of normal lungs immunostained with (Ci) SP-D mAb 245-01 (green) and goat anti-OSCAR (red). Scale bar, 20 mM. (Cii) Higher magnification confocal microscopy of area demarcated by white square in (Ci). Scale bar, 2 mM.

(Fig. 2C). These data show that OSCAR interacts with SP-D in alveolar macrophages, OSCAR is predominantly found in an in- alveolar macrophages and OSCAR is expressed by tissue-infiltrating tracellular compartment; in lung interstitial myeloid cells and monocytes in COPD. blood inflammatory monocytes, OSCAR is mainly expressed on the cell surface. Lung interstitial myeloid cells and blood monocytes express OSCAR on the cell surface OSCAR stimulates the secretion of TNF-a by CCR2+ Immunofluorescence analysis of lung tissues showed that OSCAR inflammatory monocytes was expressed not only in alveolar macrophages but also in few To study the impact of OSCAR in monocyte functions, we im- CD68+ interstitial myeloid cells (Fig. 2B). Interestingly, in these munized mice with our human OSCAR-Fc and generated a panel cells OSCAR displayed a membrane pattern. Flow cytometric of novel mAbs to human OSCAR (Supplemental Fig. 2). Among analysis revealed a marked OSCAR surface expression also on several mAb that stained OSCAR-CD3z/NFAT-GFP reporter monocytes (Fig. 3A, upper histograms), whereas alveolar mac- cells (Supplemental Fig. 2A) and human blood monocytes rophages showed poor OSCAR surface expression, consistent with (Supplemental Fig. 2B), we selected mAb that could block a predominant intracellular distribution (Fig. 3A, lower histo- binding of OSCAR-Fc to collagen I (Supplemental Fig. 3A). grams). Although OSCAR had been previously found on blood Using this approach, we identified four blocking mAbs (11.20.02, monocytes and neutrophils (26), it was unclear whether it was 11.20.06, 11.20.08, and 11.20.25), which were subsequently pu- expressed on a specialized monocyte subset. A recent mRNA rified and titrated based on their ability to block OSCAR-CD3z/ profiling of human monocyte subsets suggested that OSCAR may NFAT-GFP reporter cell signaling on plates coated with either be expressed on the CCR2+ inflammatory monocytes, but not collagen I (Supplemental Fig. 3B), rat SP-D (Supplemental Fig. CCR22 patrolling monocytes (40). We conclusively demonstrated 3C), or human SP-D (Supplemental Fig. 3D). Two mAbs, 11.20. that OSCAR was preferentially expressed on CCR2+ inflamma- 08 and 11.20.25, were most effective in blocking GFP expression tory monocytes by flow cytometry (Fig. 3B). In contrast to a pre- in reporter cells with peak blocking activities observed at 5 mg/ml. vious report using the anti-human OSCAR mAb 11.1CN5 (26), We next determined whether SP-D could stimulate the release of we did not detect OSCAR staining on neutrophils (Fig. 3B). We proinflammatory cytokines by human blood CCR2+ monocytes conclude that OSCAR has two distinct patterns of expression. In through engagement of OSCAR. A recombinant human SP-D The Journal of Immunology 3323

FIGURE 3. Lung and blood monocytes express cell surface OSCAR. (A) Representative histogram plots for lung cells triple stained with mAbs to CD14, OSCAR, and HLA-DR (open histograms) compared with isotype control mAb (gray histograms) for low (Lo) forward scatter (FSC)/side scatter (SSC) lung tissue monocytes (gate R1, upper histogram panels) or high (Hi) FSC/SSC alveolar macrophages (gate R2, lower histogram panels). (B) Representative plots for OSCAR immunostaining of peripheral blood Downloaded from SSChighCD45+CD562CD16+ granulocytes (upper panels). Within the SSClowCD45+CD562 HLA-DR+ monocytic population, CCR2+CD14high CD162 inﬂammatory monocytes express OSCAR (bottom panels), but not CCR22CD14lowCD16high noninﬂammatory monocytes (center panels).

Staining is representative of three different donors. http://www.jimmunol.org/ by guest on October 1, 2021 dodecamer stimulated TNF-a release by blood CCR2+ monocytes tionally expressed on monocytes, macrophages, and dendritic cells from two different donors, which was blocked by the anti-human (26), suggesting that OSCAR may play a role in the immune OSCAR mAbs 11.20.08 and 11.20.25, respectively (Fig. 4A). response. Motivated by this hypothesis, we designed a bio- OSCAR was not internalized by monocytes exposed to SP-D informatics search for OSCAR-binding motifs in collagenous (Supplemental Fig. 3E). We conclude the SP-D/OSCAR interac- molecules that might provide insight into OSCAR immunobiol- tion triggers a proinflammatory response in CCR2+ monocytes. ogy. We identified putative OSCAR-binding motifs in the collectins, MBL, SP-A, and SP-D, and collectin-like molecules, such OSCAR is expressed in lipid-laden macrophages infiltrating as C1q, and ficolins, that all play important roles in innate im- atherosclerotic plaques munity (1). Of the putative ligands tested, we detected binding of Although SP-D has been implicated in atherosclerosis (22, 23), the OSCAR-Fc to SP-D, but not to MBL or C1q. OSCAR may not mechanism is poorly understood. We hypothesized that SP-D may bind to C1q because we only detected OSCAR-binding motifs in engage OSCAR in the atherosclerotic plaque, promoting inflam- the B and C chains of C1q and none in the A chain (Table I). mation. To test this hypothesis, we investigated OSCAR expres- Because OSCAR only binds to triple-helical motifs in collagenous sion in atherosclerotic lesions of human aorta. OSCAR was molecules and not to linear motifs (25), C1q therefore lacks strongly expressed in numerous lipid-laden macrophages infil- a motif in the A chain to form a triple-helical sequence capable of trating the tunica intima and in fewer macrophages penetrating the binding OSCAR. Further structural and biophysical studies are tunica media. SP-D was found in the vascular endothelium as well required to determine the precise molecular binding specificities as in many smooth muscular cells of the tunica media, consistent of OSCAR for collectin and collectin-like molecules. with previous reports (22, 41) (Fig. 4B). These data suggest that Our study demonstrates that human OSCAR is a novel myeloid SP-D may contribute to atherosclerosis by binding OSCAR on immunoreceptor for SP-D expressed by alveolar macrophages and inflammatory monocytes that infiltrate the atherosclerotic plaque inflammatory CCR2+ monocytes, which triggers the release of and stimulating the release of TNF-a, which will exacerbate the TNF-a in inflammatory CCR2+ monocytes exposed to SP-D. The vascular lesion. identification of SP-D receptors is essential to fully understand the function of SP-D in different tissues and organs in the context of Discussion infections and inflammation. Previous studies have shown that SP- In humans and mice, OSCAR is expressed by osteoclasts and their D or the closely related collectin SP-A can interact with distinct precursors and can bind to collagens exposed on bone surfaces to immunoreceptors, resulting in different functional outcomes. costimulate osteoclastogenesis (25). In humans, OSCAR is addi- When not bound to a pathogen, SP-D and SP-A bind through their 3324 OSCAR IS A NOVEL MONOCYTE SP-D RECEPTOR Downloaded from http://www.jimmunol.org/

FIGURE 4. SP-D functionally interacts with OSCAR. (A)TNF-a release by human CCR2+ monocytes from two different donors treated with soluble SP-D in the presence of either IgG1 or anti-OSCAR blocking mAbs, 11.20.08 or 11.20.25. (B) Representative serial sections of atherosclerotic lesions (i–viii) from human aorta immunostained with the following: (i and v) rabbit anti–SP-D (brown; original magnification 3100); (ii and vi) anti-OSCAR C-terminal mAb 13-9-17 (brown; original magnification 3100); (iii and vii) high power views (original magnification 3200) of areas defined by black rectangles in (ii)and(vi), representing intima and tunica media, respectively; (iv) OSCAR C-terminal mAb 13-9-17 (brown) and anti-CD163 mAb (blue, original magnification 3600); (viii) rabbit (brown) and mouse IgG1 (blue) control Abs (original magnification 3200). by guest on October 1, 2021 lectin domains to SIRP-a, delivering an inhibitory signal, which distribution of OSCAR between the cell membrane and the prevents activation of mononuclear phagocytes and secretion of endosomal compartment in different myeloid cells might also inflammatory cytokines (24). In contrast, when microorganisms or depend on the relative abundance of SP-D to which these cells are cell debris are bound to the C-type lectin domain, SP-D and SP-A exposed in their microenvironment. Alternatively, the different interact through their free collagen-like domain to CD91/ subcellular localizations of OSCAR in alveolar macrophages and calreticulin, promoting cell activation (24). SP-D also binds to monocytes could be caused by the alternative splicing of OSCAR CD14, interfering with the binding of LPS to the CD14-TLR4- transcripts encoding OSCAR isoforms with different subcellular MD2 complex, thereby reducing macrophage inflammatory re- trafficking properties (45). Future studies to determine the role, if sponses elicited by LPS (42, 43). Finally, a recent study showed any, OSCAR isoforms may play in the cell biology and Ag pro- that the Ig-superfamily receptor LAIR1, which is known to bind cessing of SP-D for alveolar macrophages and dendritic cells are collagen, also binds to SP-D, delivering an inhibitory signal that therefore merited. reduces inflammation (35). Because the OSCAR:SP-D interaction Previous studies have implicated SP-D in atherosclerosis and promotes activation of inflammatory monocytes, we conclude that coronary heart disease (22, 23, 41). We found that OSCAR is the ultimate functional effect of SP-D depends on the balance highly expressed in CD163+ monocytes and macrophages infil- between inhibitory and activating receptors engaged at any one trating the atherosclerotic plaques in aorta. We envision that ex- time in a given tissue. posure of these OSCAR+ monocytes and macrophages to SP-D Our study also demonstrates that, in alveolar macrophages, produced by endothelial cells in the tunica intima and by smooth OSCAR is predominantly found in an intracellular compartment muscular cells in the tunica media may lead to the production of where it colocalizes with SP-D. This result suggests that OSCAR TNF-a, exacerbating the degenerative process. Similar mecha- may capture SP-D in the alveolar space and drive SP-D inter- nisms may operate in lung tissue. For example, monocytes can nalization into a phagolysosomal compartment in alveolar mac- traffic into the lung during the steady state (46), but do not pref- rophages where SP-D and its cargo are processed and degraded. erentially enter airway mucosal surfaces, unless there is a proin- The internalization of SP-D may be crucial for the phagocytosis flammatory or chemotactic stimulus (38). Therefore, CCR2+ and killing of pathogens or the removal of necrotic and apoptotic monocytes that patrol lung interstitial tissues in the steady state cells in the lung as well as in various other mucosae and organs may not become exposed to SP-D secreted in airway lung sur- (2, 44). However, we did not detect any OSCAR internalization factant. In contrast, monocytes increasingly infiltrate the airways in monocytes exposed to SP-D. It is possible that alveolar mac- in response to a proinflammatory or chemotactic stimuli (38). rophages are capable of internalizing OSCAR:SP-D complexes, Thus, upon tissue damage or the induction of proinflammatory because macrophages are more phagocytic than monocytes. The stimuli in the lung airways, as might be operating in lung pa- The Journal of Immunology 3325 thologies like COPD, CCR2+ monocytes will traffic from blood to 19. Aramini, B., C. Kim, S. Diangelo, E. Petersen, D. J. Lederer, L. 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