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Ligation of the FcRγ Chain-Associated Human Osteoclast-Associated Receptor Enhances the Proinflammatory Responses of Human Monocytes and Neutrophils This information is current as of September 24, 2021. Estelle Merck, Claude Gaillard, Mathieu Scuiller, Patrizia Scapini, Marco A. Cassatella, Giorgio Trinchieri and Elizabeth E. M. Bates J Immunol 2006; 176:3149-3156; ; doi: 10.4049/jimmunol.176.5.3149 Downloaded from http://www.jimmunol.org/content/176/5/3149

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ligation of the FcR␥ Chain-Associated Human Osteoclast-Associated Receptor Enhances the Proinﬂammatory Responses of Human Monocytes and Neutrophils1

Estelle Merck,2* Claude Gaillard,* Mathieu Scuiller,* Patrizia Scapini,† Marco A. Cassatella,† Giorgio Trinchieri,3* and Elizabeth E. M. Bates4*

We have previously described the human osteoclast associated receptor (hOSCAR), expressed in all cells of the myeloid lineage, and its immune functions. This receptor, which associates with the FcR␥ chain to transduce an activating signal, induces calcium flux in monocytes and dendritic cells, and modulates specific responses of dendritic cells. In this study, we have examined the effects of hOSCAR ligation on various proinflammatory responses of monocytes and neutrophils. Monocytes stimulated via hOSCAR ligation released IL-8/CXCL8 and other chemokines such as epithelial neutrophil-activating peptide-78/CXCL5, macrophage- Downloaded from derived chemokine/CCL22, and MCP-1/CCL2 and up-regulated markers involved in cell adhesion and costimulatory functions. Monocytes stimulated via hOSCAR in the absence of survival factors had an increased life span. Although the life span of neutrophils was unaffected, these cells, when stimulated via hOSCAR, rapidly released reactive oxygen intermediates, degranu- lated lactoferrin, myeloperoxidase, and matrix metalloproteinase-9 and also secreted IL-8/CXCL8. Neutrophils also underwent changes in cell surface molecule expression with the cleavage of CD62L and increased expression of CD11b and CD66b after 2-h stimulations.

Finally, we demonstrated synergy between hOSCAR and TLR ligands on both monocytes and neutrophils, with up to 8-fold http://www.jimmunol.org/ increases in cytokine secretion when hOSCAR was cross-linked in the presence of LPS or R-848. Overall, our data demonstrate that hOSCAR is a functional receptor on monocytes and neutrophils, involved in the induction of the primary proinﬂammatory cascade and the initiation of downstream immune responses. The Journal of Immunology, 2006, 176: 3149–3156.

onocytes, monocyte-derived macrophages, and neu- The ability of monocytes and neutrophils to respond to envi- trophils are phagocytic cells, the activation of which ronmental signals depends on the repertoire of plasma membrane M greatly contributes to the triggering and development receptors that they express and that allows them to recognize all of proinflammatory responses and the containment of infections. classes of macromolecules (4, 5). In this regard, a large number of These cells rapidly migrate through the endothelial barrier into receptors of the innate immune system use the ITAM signaling by guest on September 24, 2021 inflamed tissues, where they act as a first line of defense. Accord- pathway, which induces cellular activation via a phosphorylation ingly, monocytes and neutrophils, upon appropriate stimulation, cascade triggered by protein tyrosine kinases (6). ITAMs are release a number of proinflammatory mediators, including reactive mainly found in the cytoplasmic tail of transmembrane adaptors, oxygen intermediates and antimicrobial products, produce cyto- such as CD3␨, FcR␥, or DAP12, molecules that provide the role of kines and chemokines, modify the expression of surface mole- signaling subunits when associated with cell surface receptors. For cules, and efficiently phagocytose and kill invading pathogens (1, instance, the triggering receptor expressed on myeloid cells 2). The secretion of cytokines and chemokines by phagocytes at (TREM)5 family has recently attracted particular attention due to the infection site not only favors the recruitment of other effector the capacity of these DAP12-associated members to activate sev- cells (3), but also prepares and amplifies the subsequent immune eral myeloid cell types (7–9). Of interest, TREM-1 plays a crucial response. role in innate immunity, particularly during septic shock (10). In addition, TREM-1 engagement is able to enhance the responses of monocytes and neutrophils upon stimulation with TLR ligands (7, 11, 12). *Laboratory for Immunological Research, Schering-Plough Research Institute, Dar- Among the ITAM-linked receptors, the osteoclast-associated re- dilly, France; and †Department of Pathology, Division of General Pathology, Uni- versity of Verona, Verona, Italy ceptor (OSCAR), a myeloid cell receptor associated with the ␥ Received for publication August 19, 2005. Accepted for publication December ITAM-bearing chain FcR (13–15), has an expression pattern that 12, 2005. overlaps that of the TREM family. We have recently demonstrated The costs of publication of this article were defrayed in part by the payment of page that human OSCAR (hOSCAR) is expressed by myeloid dendritic charges. This article must therefore be hereby marked advertisement in accordance cells (DC), is involved in Ag internalization and presentation, and with 18 U.S.C. Section 1734 solely to indicate this fact. causes cellular activation and partial maturation in this cell type 1 E.M. was a recipient of a grant from the Fondation Marcel Me´rieux (Lyon, France). M.A.C. was supported by grants from Ministero dell’Istruzione, dell’Universitaè della Ricerca (Programmi di Ricerca di Interesse Nazionale 2003 and Fondo per gli Investimenti delle Ricerca di Base), from Associazione Italiana per la Ricerca sul 4 Current address: Molecular and Biochemical Analytical Services, Bayer BioScience Cancro, and Fondazione Cassa di Risparmio di Verona-Vicenza-Ancona e Belluno. N.V., Technologiepark 38, 9052 Gent, Belgium. 2 Address correspondence and reprint requests to Dr. Estelle Merck at the current 5 Abbreviations used in this paper: TREM, triggering receptor expressed on myeloid address: Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 cells; OSCAR, osteoclast-associated receptor; hOSCAR, human OSCAR; DC, den- Epalinges, Switzerland. E-mail address: [email protected] dritic cell; GAM, goat anti-mouse; MMP-9, matrix metalloproteinase-9; MPO, my- 3 Current address: Laboratory of Parasitic Disease, National Institute of Allergy and eloperoxidase; ROI, reactive oxygen intermediate; MDC, macrophage-derived Infectious Diseases, National Institutes of Health, Bethesda, MD. chemokine.

(14, 16). In addition, we have shown that numerous DC functions maintain cellular integrity in culture. After the indicated incubation times, triggered by hOSCAR stimulation are negatively regulated by the cells were harvested and apoptotic cells were detected using the FITC- ITIM-signaling receptor CD85j (leukocyte Ig-like receptor-1/Ig- Annexin V kit (BD Pharmingen) followed by flow cytometer analysis. like transcript 2), confirming that hOSCAR is a classical ITAM- Measurement of soluble mediator release linked receptor that plays a role in maintaining the equilibrium of Supernatants of cells stimulated for the times indicated in the results were the immune response (17). Some of those studies (14, 16) also collected and tested by ELISA for production of IL-1␤, IL-6, IL-8/CXCL8, described the role of hOSCAR in adaptive immunity and under- IL-10, IL-12 p40, TNF, MCP-1/CCL2, GM-CSF (OptEIA kits; BD Pharm- lined differences between FcR␥- and DAP12-associated receptors. ingen), epithelial neutrophil-activating peptide-78/CXCL5, macrophage- Despite the increasing understanding of the role of hOSCAR in derived chemokine (MDC)/CCL22, M-CSF, matrix metalloproteinase-9 (MMP-9) (DuoSet kit; R&D Systems), and myeloperoxidase (MPO) DC biology, no information has been reported on the potential role (Merck Calbiochem). The detection of lactoferrin was performed as pre- of hOSCAR in modulating the effector functions of phagocytic viously described (20). Briefly, supernatants were diluted in 0.05 M car- cells. bonate buffer (pH 9.6) and proteins were immobilized in Nunc-Immuno In the present work, we have investigated the capacity of 96-well plates (Nunc) overnight at 4°C followed by blocking with 0.5% hOSCAR to induce cellular stimulation in human monocytes and BSA in PBS 0.1% Tween 20 (Sigma-Aldrich) for 1 h. After the wells were washed, anti-lactoferrin (dilution 1/2000, Sigma-Aldrich) was added and neutrophils. We show that hOSCAR engagement triggered several allowed to react for 1 h, followed by incubation with anti-rabbit peroxidase proinflammatory responses, including the production of soluble (dilution 1/5000; Amersham Biosciences) for 1 h. After a final washing, mediators and the acquisition of an activated phenotype in mono- tetramethylbenzidine solution (Sigma-Aldrich) was used to read the absor- cytes and degranulation in neutrophils. Interestingly, we also show bance of each well. Standard curves were interpreted with respect to known dilutions of human lactoferrin (Sigma-Aldrich). that ligation of hOSCAR acted synergistically with TLR ligands to Downloaded from enhance proinflammatory responses in both monocytes and neu- Production of reactive oxygen intermediates (ROI) trophils. Finally, hOSCAR ligation promoted survival of mono- Hydrogen peroxide (H O ) and superoxide anion (O.) release were mea- cytes but not of neutrophils. Taken together, our data demonstrate 2 2 2 sured as previously described (21). Briefly, the production of H2O2 was that OSCAR is a functional receptor on monocytes and neutrophils assayed in neutrophil suspensions (106/ml) stirred in HBSS at 37°C, as the that is probably involved in the primary inflammatory response. H2O2-HRP-dependent oxidation of homovanillic acid by using a spec- trofluorometer (model LS 50B; Applied Biosystems). In these assays, neu- http://www.jimmunol.org/ Materials and Methods trophils were stimulated for up to 30 min with soluble primary anti- hOSCAR mAbs at 10 ␮g/ml, cross-linked with GAM (H ϩ L)-specific Cell cultures Ј ␮ . F(ab )2 (Immunotech) at 20 g/ml. O2 production was assayed as the re- 5 ␮ Human peripheral blood samples were obtained according to institutional duction of ferricytochrome C by neutrophils (10 /100 l) stimulated in guidelines. PBMC were purified by Ficoll-Hypaque centrifugation. After plate-bound anti-hOSCAR or isotype-matched control mAbs for 30 min. Percoll gradient, monocytes were purified by immunomagnetic depletion Stimulation via 100 nM fMLP (Sigma-Aldrich) was used as the positive of low density PBMC isolated on a 52% Percoll gradient as described (18). control. Ͼ Ͻ Highly purified granulocytes (neutrophils, 96.5%; eosinophils, 3%) Statistical analyses were isolated as previously described (19). Briefly, PBMC and platelets were removed by centrifugation through a Ficoll-Hypaque gradient. Neu- Statistical analyses were performed in Microsoft Excel 5.0 (Microsoft) trophils were then separated from erythrocytes by sedimentation with 4% using two-tailed Student’s t tests. A p value Ͻ0.05 was considered statis- by guest on September 24, 2021 dextran, and residual RBC were removed by hypotonic lysis. tically significant. Cells were cultured in RPMI 1640 (Invitrogen Life Technologies) sup- plemented with 10% (v/v) heat-inactivated FBS (Eurobio), 2 mM L-glu- Results tamine, and 100 ␮g/ml gentamicin (Schering-Plough). hOSCAR expression by monocytes and neutrophils In vitro stimulation of cells We analyzed the expression of hOSCAR on monocytes and neu- Ј Anti-hOSCAR mAb and F(ab )2 mAb were produced as previously de- trophils. Fig. 1 shows the expression of hOSCAR on freshly iso- scribed (14). Anti-hOSCAR mAb and F(abЈ) (clone 11.1CN5), and irrel- 2 lated monocytes (Fig. 1A) and neutrophils (Fig. 1B). The experi- evant mAb MOPC21 (Sigma-Aldrich) or anti-CD13 (Immunotech) isotype controls were coated for4hat37°C on flat-bottom plates with a final ment shown is representative of five donors studied. All monocytes concentration of 20 ␮g/ml in PBS. Monocytes and neutrophils were plated and neutrophils expressed hOSCAR, although the level of expres- at a concentration of 1 ϫ 106 and 3 ϫ 106 cells/ml, respectively. sion was higher on monocytes. Stimulation of these cells with Activating factors were used at the following final concentrations: 100 Pam Cys, LPS, and R-848, ligands for TLR2, TLR4, and TLR7/8, ng/ml Pam Cys (synthetic palmitoylated mimic of bacterial lipopeptides; 3 3 respectively, did not significantly alter the cell surface expression InvivoGen); 100 ng/ml Escherichia coli LPS (Sigma-Aldrich); and 10 ␮M R-848 (imidazoquinoline resiquimod synthesized in our laboratory). of hOSCAR (Fig 1, C and D). Monocytes and neutrophils were collected after 24 and 2 h, respectively, and cell surface markers were tested by flow cytometry. Supernatants were hOSCAR ligation provides a survival signal for monocytes, but removed after 24-h incubation and tested by ELISA. All mAb used for has no effect on neutrophil life span in culture tissue culture were shown to be endotoxin-free, as determined by Limulus- Amebocyte Assay (BioWhittaker). To block an eventual effect of trace Our previous study showed that hOSCAR ligation promotes cell amounts of LPS, polymyxin B (Sigma-Aldrich) was added to certain wells survival of monocyte-derived DCs in the absence of survival fac- at 10 ␮g/ml. tors, such as GM-CSF (16). This activity is a long-term effect, Flow cytometry allowing cell survival up to 10 days or more. We thus investigated the ability of hOSCAR engagement to modify the life span of ex ␮ For hOSCAR staining, cells were incubated with 10 g/ml anti-hOSCAR vivo monocytes and neutrophils cultured in the absence of survival mAb 11.1CN5, then labeled with PE-conjugated goat anti-mouse (GAM; DakoCytomation). For phenotypical analysis, cell staining was performed factors for 48 and 24 h, respectively. Cellular stimulation by plas- using PE-conjugated mouse mAb anti-CD54 (eBioscience), anti-CD40, tic-coated Abs is known to induce receptor aggregation, required -CD63, -CD83 (Immunotech), anti-CD25, -CD62L, -CD86 (BD Pharmin- for the triggering of the ITAM-signaling pathway (22). This gen), and FITC-conjugated mouse mAb anti-CD11b and -CD66b method was previously shown to be efficient in the triggering of (Immunotech). DC maturation through hOSCAR ligation and was therefore also Detection of apoptosis used to engage hOSCAR at the surface of monocytes and neutro- Monocytes and neutrophils were stimulated with coated mAb for 48 and phils. Approximately 80% of monocytes treated with either whole Ј 24 h, respectively. As a positive control, 20 ng/ml GM-CSF was used to Ab or F(ab )2 specific for hOSCAR survived in the absence of The Journal of Immunology 3151

coated or not on tissue culture plates, or by TLR ligands known to stimulate monocytes. Ј After 24 h of stimulation, both coated F(ab )2 and whole anti- hOSCAR mAb were able to trigger the activation of monocytes, as shown by up-regulation of the cell surface costimulatory molecule CD86 (Fig. 3A). The two isotype-matched control Abs, MOPC21 (which did not bind an Ag on these cells) or anti-CD13, had no signiﬁcant effect on monocytes. Thus, as we had previously observed on DCs, the activation observed by using anti-hOSCAR was not due to the engagement of FcR. Cross-linking of hOSCAR was required for cellular activation, because no effect was observed with noncoated (soluble) anti-hOSCAR. No endotoxin con- tamination was detected in any Ab preparation used in these experiments as measured with the Limulus assay. In addition, the activating effect of anti-hOSCAR was maintained in the presence of polymyxin B and was absent when the same Ab was used in soluble form, excluding the role of trace levels of endotoxins. In- terestingly, the up-regulation of CD86 in the case of hOSCAR

stimulation was stronger than that observed with TLR ligands. Downloaded from FIGURE 1. hOSCAR is expressed by blood monocytes and neutrophils hOSCAR stimulation also triggered up-regulation at the monocyte and its expression is maintained upon cellular activation by TLR ligands. surface of CD40, CD54, and CD83 costimulatory molecules (Fig. Monocytes (A and C) and neutrophils (B and D) were labeled and analyzed 3B). Unlike TLR ligands, hOSCAR engagement was not able to by flow cytometer for cell surface expression of hOSCAR, using anti- induce the expression of the activation marker CD25 by mono- hOSCAR and a PE-conjugated GAM Ab. In histograms A (freshly isolated cytes, however, it induced the expression of CD83 which was monocytes) and B (freshly isolated neutrophils), dashed profiles indicate background staining with a control IgG1 mAb, shaded profiles hOSCAR poorly induced by Pam3Cys and LPS, but strongly induced by http://www.jimmunol.org/ specific staining, and numbers in the corners correspond to the mean flu- R-848. These results point to an activation of circulating blood orescence intensity of hOSCAR staining. Histograms C and D show monocytes via hOSCAR, similar to the activation observed via hOSCAR expression upon cellular activation. Monocytes (C) and neutro- other stimuli, but with several significant differences. phils (D) were cultured with 100 ng/ml LPS, 10 ␮M R-848, 100 ng/ml Immune responses during the early steps of inflammation are Pam3Cys or 20 ng/ml GM-CSF, and after 24 h culture, cells were analyzed mediated by soluble factors capable of recruiting and activating by flow cytometry for hOSCAR expression. The data are expressed as other immune cells which also may have a toxic effect on the ⌬ MFI, the mean fluorescence intensity minus the fluorescence detected invading pathogens. In monocytes, cross-linking of hOSCAR by with isotype control. The results presented are representative of five inde- Ј whole Ab or F(ab )2 induced a strong secretion of IL-8/CXCL8,

pendent experiments. by guest on September 24, 2021 which was not observed in response to the isotype controls (MOPC21 and anti-CD13) or soluble anti-hOSCAR (Fig. 3C). Al- endogenous survival factors for 2 days (Fig. 2A), similar to the though the amount of IL-8/CXCL8 secreted was low in compari- level of survival induced by GM-CSF treatment. In control cul- son to that induced by LPS or R-848, it was statistically significant tures, in the presence of plastic-coated MOPC21 isotype control and very similar to the amount produced by activation via Pam3Cys. Ab or anti-CD13, a high proportion of apoptotic monocytes (an- The presence of polymyxin B did not block the ability of anti- nexin V-positive cells) was observed. Unlike the effect of hOSCAR to induce IL-8/CXCL8 secretion, demonstrating that the hOSCAR Abs on survival of monocytes, neither control Ab nor activation observed was not mediated by contaminating endotoxin. In anti-hOSCAR decreased the proportion of apoptotic neutrophils, monocytes, hOSCAR ligation also induced the secretion of the whereas GM-CSF had the ability to promote neutrophil survival chemokines epithelial neutrophil-activating peptide-78/CXCL5, (Fig. 2B). Altogether, these results suggest that hOSCAR is able to MDC/CCL22, and MCP-1/CCL2, but no release of proinflamma- promote monocyte survival, but is incapable of extending the du- tory cytokines such as IL-1␤, TNF, or IL-12 p40 (Table I). ration of neutrophil responses by attenuating apoptosis. hOSCAR engagement induces an activated phenotype and hOSCAR cross-linking on freshly isolated neutrophils leads to soluble mediator release by monocytes degranulation and rapid changes in cell phenotype To investigate the role of hOSCAR in primary inflammatory re- Although hOSCAR engagement had no effect on neutrophil sur- Ј sponses, freshly isolated blood monocytes were stimulated by Abs vival, cross-linking with either coated F(ab )2 or whole mAb anti-

FIGURE 2. Engagement of hOSCAR promotes monocyte survival, but does not rescue neutrophils from apoptosis. Freshly isolated monocytes (A) and neutrophils (B) were stimulated with plastic-coated mAb (MOPC21, anti-CD13, anti-hOSCAR, and anti-hOSCAR Ј F(ab )2) or 20 ng/ml GM-CSF as indicated. After stimulation of monocytes for 2 days and neutrophils for 24 h, cells were harvested and analyzed for annexin V binding, as a marker of apoptotic cells. Numbers in the corners correspond to the percentage of positive cells for annexin V. Data shown are representative of three independent experiments. 3152 ACTIVATION VIA hOSCAR ENHANCES PROINFLAMMATORY RESPONSES

hOSCAR triggered phenotypical changes. Anti-hOSCAR stimulation also triggered the release of lactoferrin, MPO, and MMP-9 (Table II). This release of soluble mediators is indicative of degranulation. Equally, anti-hOSCAR-treated neutrophils produced IL-8/CXCL8, although in a lower concentration than that produced by monocytes (Fig. 4A). An absence of IL-6 in the supernatant of TLR ligand-treated neutrophils (data not shown) demonstrated the absence of contaminating monocytes in the cell preparation and indicated that the hOSCAR-induced IL-8/CXCL8 and other soluble mediators were neutrophil-derived (Fig. 4A). We also detected cleavage of CD62L at the neutrophil surface, an early marker of cell activation (Fig. 4B) analyzed here 2 h after hOSCAR cross-linking. In addition, hOSCAR engagement triggered a slight up-regulation of the expression of CD11b and CD66b (Fig. 4C). hOSCAR stimulation did not increase the expression of CD63, which was, however, slightly up-regulated by the TLR ligand R-848 (Fig. 4C). Cross-linking of hOSCAR was required for these phenotypic changes observed after 2 h stimulation, because no effect was observed with soluble mAb. Isotype Downloaded from controls (irrelevant MOPC21 mAb and anti-CD13 recognizing a molecule expressed on neutrophil surface) had no signiﬁcant effect on neutrophil phenotype. In addition to the release of hydrolytic enzymes, production of proinﬂammatory mediators, and modulation of molecules involved in neutrophil extravasation, we also detected a stimulation of respiratory burst activity by hOSCAR- http://www.jimmunol.org/ treated neutrophils, as measured by both hydrogen peroxide . (H2O2) and superoxide anion (O2) production (Fig. 4D). Stimula- tion of ROI production over control (medium or isotype control mAbs) was detectable within a few minutes after hOSCAR ligation and linearly increased for 30 min (Fig. 4D).

hOSCAR ligation enhances the proinﬂammatory responses by guest on September 24, 2021 induced by TLR ligands Cells of the myeloid lineage have a large number and variety of receptors regulating their activity, and the combination of different signals may regulate the amplitude and the duration of the immune response. To investigate the role of hOSCAR as a coreceptor in proinﬂammatory responses triggered by pathogenic stimuli, we stimulated monocytes and neutrophils with anti-hOSCAR in the presence of different doses of TLR ligands. We used the TLR

ligands Pam3Cys, LPS, and R-848 that target TLR expressed by both monocytes and neutrophils and that have no effect on hOSCAR expression on these cells (Fig. 1, C and D). As shown in Fig. 5A, we observed a 2- to 10-fold increase in secretion of proinflammatory factors (IL-8/CXCL8, IL-1␤, TNF) by monocytes cultured in both anti-hOSCAR mAb and TLR ligands as compared with those cultured with individual stimuli. Strikingly, although FIGURE 3. hOSCAR ligation induces phenotypic changes and secre- the effect of single TLR ligands dropped rapidly over 10-fold dim- tion of IL-8/CXCL8 by monocytes. Monocytes were stimulated by coated inutions in concentration, the costimulatory effects of hOSCAR control IgG (MOPC21, anti-CD13), coated anti-hOSCAR whole mAb or ligation showed a slower decline, indicating that hOSCAR aug- Ј ␮ F(ab )2, 10 ng/ml Pam3Cys, 100 ng/ml LPS, 10 M R-848, or soluble ments the response to suboptimal doses of pathogen-derived TLR anti-hOSCAR. After 24 h, cells were analyzed by flow cytometry for the ligands. These data suggest that signals produced by hOSCAR indicated markers (A and B) and supernatants were tested by ELISA for ligation synergize with various TLR to up-regulate production of IL-8/CXCL8 secretion (C). In A and B, the numerical values indicate the specific mean fluorescence intensity of the staining (for shaded histo- proinflammatory cytokines and chemokines by monocytes. grams), and the dotted line shows the binding of an isotype control mAb to We also detected a significant increase in IL-8/CXCL8, lacto- the cells. Data are representative of four independent experiments. In C, ferrin, and MMP-9 by neutrophils costimulated with both anti- IL-8/CXCL8 secretion data are mean Ϯ SD of triplicate samples from one hOSCAR and TLR ligands (Fig. 5B). Although the effect is less representative experiment of three performed with similar results. Statis- striking than that seen for monocytes, ligation of hOSCAR main- p Ͻ 0.001 are given by comparison to values tained a strong inflammatory response, even in the presence of ,ءء tical significances of obtained with the negative control anti-CD13. suboptimal doses of TLR ligands. This enhancement in the proinflammatory response of neutrophils was consistent and could be due, at least in part, to enhanced degranulation. We also analyzed The Journal of Immunology 3153

a Table I. Panel of cytokine and chemokine secretion by monocytes upon 24 h of stimulation by anti-CD13, anti-hOSCAR, Pam3Cys, LPS, and R-848

Medium MOPC21 Anti-hOSCAR Pam3Cys LPS R-848 IL-1␤ ND ND ND 0.319 Ϯ 0.016 2.08 Ϯ 0.039 36.59 Ϯ 6.34 IL-10 ND ND ND ND 0.244 Ϯ 0.060 0.342 Ϯ 0.027 IL-12 p40 ND ND ND ND 1.29 Ϯ 0.059 6.51 Ϯ 0.069 TNF ND ND ND 0.144 Ϯ 0.009 0.806 Ϯ 0.053 12.16 Ϯ 1.540 IL-6 0.850 Ϯ 0.048 1.525 Ϯ 0.028 2.012 Ϯ 0.009 1.690 Ϯ 0.074 62.54 Ϯ 1.15 149.47 Ϯ 13.5 ENA-78/CXCL5 0.279 Ϯ 0.016 0.721 Ϯ 0.091 18.291 Ϯ 0.51 22.26 Ϯ 6.34 63.45 Ϯ 1.54 113.38 Ϯ 4.32 MDC/CCL22 0.097 Ϯ 0.014 0.382 Ϯ 0.116 19.34 Ϯ 0.54 12.26 Ϯ 3.45 85.99 Ϯ 6.73 243.59 Ϯ 8.66 MCP-1/CCL2 0.556 Ϯ 0.079 0.274 Ϯ 0.047 26.8 Ϯ 4.08 33.5 Ϯ 3.7 59.9 Ϯ 2.5 92.5 Ϯ 6.17 GM-CSF ND ND ND ND 0.041 Ϯ 0.010 0.154 Ϯ 0.019

the expression of CD11b and CD66b at the cell surface of neutro- OSCAR ligation promoted survival of monocytes, but did not phils after 2 h of stimulation with anti-OSCAR Abs and TLR li- affect the differentiation of monocytes into DC (data not shown). gands. These experiments revealed an additional up-regulation of Interestingly, cross-linking of hOSCAR did not induce secretion of Downloaded from these two markers of release of speciﬁc and/or gelatinase granules, GM-CSF, excluding a role of this cytokine in the survival of however, the increase was minimal, not statistically signiﬁcant in monocytes induced by hOSCAR Abs. However, similarly to all cases and resembled an additive effect (data not shown). TREM-1 (12), hOSCAR did not promote survival of neutrophils, but rapidly stimulated granule secretion and even IL-8/CXCL8 Discussion release. The latter observations indicate that hOSCAR is functional

Growing evidence suggests that ITAM-associated receptors on in neutrophils but its activities are in part different from those http://www.jimmunol.org/ myeloid cells are involved in the regulation of innate resistance elicited in monocytes. It remains to be investigated whether the and adaptive immunity. We have previously shown that hOSCAR, differences between neutrophils and monocytes are caused by a a receptor associated with the FcR␥ chain, can activate human DCs defective capacity of hOSCAR-stimulated neutrophils to appropri- and modulate the adaptive response in the presence of TLR ligands ately transduce the signals that control cell viability or simply by (16). We reported that hOSCAR is expressed on monocytes and the lack of neutrophil expression of crucial proteins controlling neutrophils (14) and our preliminary results showed the induction survival. The importance of receptor density in reaching the nec- ofaCa2ϩ flux on monocytes, indicative of early cellular activation essary threshold to trigger strong signals is well-known for the (14). In this study, we further examined the effects of hOSCAR receptors signaling through ITAM, and especially the FcR. The stimulation on these cells. One of our early observations on expression level of hOSCAR is lower on neutrophils than on the by guest on September 24, 2021 hOSCAR was that the expression of this molecule, unlike that of monocyte cell surface. Thus, one possible hypothesis is that the other FcR␥-associated molecules, was not modulated by activation number of receptors cross-linked on the neutrophil cell surface of DCs with various physiological activators (14). We now show may be insufficient to reach the threshold required for induction of that this is also true for monocytes and neutrophils. In our hands, survival and a synergistic OSCAR-TLR effect in the up-regulation all neutrophils and monocytes expressed hOSCAR and no signif- of degranulation markers. In addition, neutrophils have been re- icant difference in expression was seen after 24 h of activation with ported to express relatively low FcR␥ levels, compared with either TLR ligands or GM-CSF. In contrast, the DAP12-associated monocytes (26). A stoichiometric relationship between hOSCAR receptor TREM-1 is up-regulated upon monocyte and neutrophil and the FcR␥ chain would be hypothesized, as is the case for the activation by fungus and extracellular bacteria, and especially by other receptors associated with FcR␥: one hOSCAR molecule LPS and lipoteichoic acid which are microbial components, but not would thus associate with a covalently linked dimer of the FcR␥ nonbacterial TLR ligands (7, 10, 23). TREM-1 is also up-regulated chain through ionic interaction. A single positively charged amino in neutrophils of patients with pneumonia (24) and in monocytes acid (arginine) is found in the transmembrane domain of OSCAR of patients suffering from septic shock (23, 25) indicating a role in which can interact with the negatively charged amino acid (aspar- the detection and response to microbial infection. In contrast, tic acid) of FcR␥. It is therefore possible that a limited availability hOSCAR expression did not change in the conditions of neutrophil of FcR␥ in neutrophils results in weaker effects of hOSCAR liga- activation analyzed in this study, suggesting that the physiologic tion compared with monocytes. regulation of hOSCAR reactivity during inflammatory responses Activation of monocytes via hOSCAR led to a differential ex- may depend on availability or regulation of the expression of the pression of cell surface markers than those induced by TLR li- counter-receptor. gands. CD83 was only induced by hOSCAR and R-848, CD25 was

a Table II. Panel of soluble mediators released by neutrophils upon 24 h of stimulation by anti-CD13, anti-hOSCAR, Pam3Cys, LPS, and R-848

Medium MOPC21 Anti-hOSCAR Pam3Cys LPS R-848 Lactoferrin 18.27 Ϯ 1.05 14.88 Ϯ 1.06 266.12 Ϯ 35.9 93.24 Ϯ 7.16 451.45 Ϯ 19.9 962.51 Ϯ 92.2 MPO 192.51 Ϯ 14.5 189.59 Ϯ 26.9 653.65 Ϯ 34.1 325.16 Ϯ 38.9 338.95 Ϯ 34.5 632.47 Ϯ 58.7 MMP-9 49 Ϯ 3.64 32 Ϯ 1.57 214 Ϯ 8.76 124 Ϯ 7.33 152 Ϯ 27.16 275 Ϯ 16.62

a Values are shown in nanograms per milliliter per 106 cells. Data are mean Ϯ SD of triplicate samples from one representative experiment of three performed with similar results. MPO, myeloperoxidase. 3154 ACTIVATION VIA hOSCAR ENHANCES PROINFLAMMATORY RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. hOSCAR ligation on neutrophils induces phenotypical changes, IL-8/CXCL8 release, and respiratory burst activity. Neutrophils were Ј stimulated with plastic-coated isotype control (MOPC21, anti-CD13), anti-hOSCAR whole mAb or F(ab )2, soluble anti-hOSCAR, 10 ng/ml Pam3Cys, 100 ng/ml LPS, and 10 ␮M R-848. After 24 h, supernatant fluids were harvested and tested by ELISA for IL-8/CXCL8 secretion (A) and cells were analyzed for the indicated markers after a 2-h incubation (B and C). A, Data are mean Ϯ SD of triplicate samples from one representative experiment of three p Ͻ 0.001 are given by comparison to values obtained with the negative ,ءء p Ͻ 0.01 and ,ء performed with similar results. Statistical significances of control anti-CD13. B and C, Numerical values indicate the specific mean fluorescence intensity of the staining (for shaded histograms). The dotted line shows the binding of an isotype control mAb to the cells. Data shown are representative of four independent experiments. D, Hydrogen peroxide (H O ) . 2 2 and superoxide anion (O2) production were measured as described in Materials and Methods. fMLP (100 nM) was used as a positive control stimulus. Data p Ͻ 0.01 are given by comparison to values obtained with the mouse ,ء are mean Ϯ SD of three experiments (three donors). Statistical significances of isotype-matched control. induced by TLR ligands but not hOSCAR, and hOSCAR induced high endothelial venules of peripheral lymphoid tissue (30). Thus, a higher expression of CD86 than TLR ligands. hOSCAR cross- activation of myeloid cells via hOSCAR interaction with its ligand linking alone did not induce secretion of proinflammatory cyto- may by itself be sufficient to set up a gradient of recruitment of kines such as TNF and IL-1␤ by monocytes, but secretion of che- immune cells to the site of interaction. In our hands, LPS and mokines was observed. Among the chemokine induced by R-848 and at a lower extent hOSCAR cross-linking, but not hOSCAR, MCP-1 recruits of monocytes to sites of injury and in- Pam3Cys, induced in neutrophils cell surface translocation of fection (27, 28) and MDC is chemoattractant for monocytes, CD11b from specific granules, gelatinase granules, and secretory monocyte-derived DCs, and NK cells (29). On neutrophils, stim- vesicles. Similarly, the appearance of CD66b at the cell surface ulation via hOSCAR led to the secretion of IL-8/CXCL8, another was induced, although only at low level, by hOSCAR ligation. chemokine that promotes recruitment of immune cells, including Ligation of hOSCAR thus appears to selectively cause degranula- neutrophils and subsets of monocytes and T lymphocytes. Neutro- tion of specific (POϪ) granules and at a lower level, of azurophil phils treated with anti-hOSCAR also cleaved surface CD62L. This (POϩ) granules, as suggested by the analysis of the granule mark- cleavage was also strongly induced by LPS and R-848 but only ers MPO and CD63 (31). Degranulation of neutrophils is accom- weakly by Pam3Cys. CD62L mediates leukocyte rolling on acti- panied by the secretion of MPO, MMP-9, and lactoferrin, all mol- vated endothelium in inflamed tissues and lymphocyte homing to ecules that are implicated in the antibacterial response (31). The Journal of Immunology 3155 Downloaded from http://www.jimmunol.org/

FIGURE 5. hOSCAR ligation synergistically up-regulates soluble mediator release by monocytes and neutrophils upon TLR stimulation. Monocytes (A) by guest on September 24, 2021 and neutrophils (B) were stimulated for 24 h as previously described, in the presence of varying concentrations of Pam3Cys, LPS, and R-848. Supernatants were tested by ELISA for IL-8/CXCL8, IL-1␤, TNF, lactoferrin, and MMP-9 secretion. Data are mean Ϯ SD of duplicate samples from one representative experiment of three performed with similar results.

MMP-9 is contained in specific and gelatinase granules and its tion induced by suboptimal doses of TLR ligands was significantly secretion is instrumental for the degradation of extracellular matrix augmented by hOSCAR ligation. Thus, coactivation of monocytes and the mobilization of stem cells (32). Interestingly, MMP-9- and, in part, neutrophils with hOSCAR in the presence of patho- mediated N-terminal cleavage of IL-8/CXCL8 is known to aug- gen-derived molecules activates a strong proinflammatory ment IL-8/CXCL8-mediated activation of neutrophils, as mea- pathway. sured by increased intracellular calcium, chemotaxis, and secretion Altogether our results show that triggering of monocytes and of MMP-9 itself (33), suggesting an autoamplification circuit for neutrophils via hOSCAR, acting through the FcR␥ chain, led to IL-8/CXCL8. In addition, hOSCAR ligation contributes to the activation of proinflammatory and innate responses, similar to that early antibacterial response of neutrophils by rapidly mediating the observed with members of the TREM family via DAP12. production of reactive oxygen species within 30 min after receptor TREM-2, a similar myeloid receptor, was recently shown to be cross-linking. important in both osteoclast biology and triggering of the immune We had previously reported that hOSCAR ligation modulated response (9). TREM-2 expressed by both osteoclasts (34, 35) and the responses of DCs to TLR ligands. In this paper, we investi- DC (8) has potentially multiple ligands, represented by microbial gated the effect of costimulation of phagocytes with pathogen- products (36), molecules expressed on the NK cell surface (37) or derived TLR ligands and hOSCAR. The secretion of IL-8/CXCL8 present in the bone environment (38). Data from different groups by monocytes after incubation with optimal doses of TLR ligands strongly suggests that in vivo ligation of mouse OSCAR on oste- together with hOSCAR ligation was doubled. This effect was more oclasts is essential for differentiation of these cells (13, 38). An marked at suboptimal doses of TLR ligands, because coincubation endogenous ligand for OSCAR on osteoblasts has been inferred with hOSCAR with doses of TLR ligands that alone showed no from these studies by using a soluble form of the mouse OSCAR effect allowed us to detect IL-8/CXCL8 and other cytokines. In the extracellular domain. However, the putative ligand has not yet case of IL-1␤, a dramatic effect of hOSCAR costimulation was been isolated and molecularly characterized. These results have led seen with Pam3Cys that alone induced no secretion, but a marked us and other groups to propose that hOSCAR can be added to the 6-fold increase was also seen with LPS at 10 ng/ml and R-848 at receptors known to play a role in both bone homeostasis and ac- 1 ␮M. hOSCAR ligation also had a strong enhancing effect on the tivation of immune responses. The presence of an hOSCAR ligand secretion of TNF in combination with 10 ng/ml R-848 (5-fold) and in the immune system environment awaits discovery, and supposes 1 ␮M LPS (8-fold). In neutrophils, lactoferrin and MMP-9 secre- that this molecule will regulate the precarious balance between 3156 ACTIVATION VIA hOSCAR ENHANCES PROINFLAMMATORY RESPONSES immune activation and suppression, roles well-defined for recep- 18. Bates, E. M., N. Fournier, E. Garcia, J. Valladeau, I. Durand, J. J. Pin, tors linked to ITAM/ITIM signaling. Because hOSCAR is a stably S. M. Zurawski, S. Patel, J. S. Abrams, S. Lebecque, et al. 1999. Antigen-pre- senting cells express DCIR, a novel C-type lectin surface receptor containing an expressed molecule widely present on myeloid cells, the presence immunoreceptor tyrosine-based inhibitory motif. J. Immunol. 163: 1973–1983. of the hOSCAR ligand will be determinant in defining where and 19. Cassatella, M. A., F. Bazzoni, R. M. Flynn, S. Dusi, G. Trinchieri, and F. Rossi. 1990. Molecular basis of interferon-␥ and lipopolysaccharide enhancement of when hOSCAR is engaged, and active. hOSCAR could recognize phagocyte respiratory burst capability: studies on the gene expression of several pathogen components, similar to TREM-2, or be involved in the NADPH oxidase components. J. Biol. Chem. 265: 20241–20246. sensing of endogenous infection- or stress-induced molecules. In- 20. Mocsai, A., E. Ligeti, C. A. Lowell, and G. Berton. 1999. Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck. J. 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