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The Journal of Immunology, 2006, 176: 3149-3156.
Copyright © 2006 by The American Association of Immunologists

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

Estelle Merck2,*, Claude Gaillard*, Mathieu Scuiller*, Patrizia Scapini{dagger}, Marco A. Cassatella{dagger}, Giorgio Trinchieri3,* and Elizabeth E. M. Bates5,*

* Laboratory for Immunological Research, Schering-Plough Research Institute, Dardilly, France; and {dagger} Department of Pathology, Division of General Pathology, University of Verona, Verona, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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{gamma} 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-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, degranulated 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Monocytes, monocyte-derived macrophages, and neutrophils are phagocytic cells, the activation of which greatly contributes to the triggering and development of proinflammatory responses and the containment of infections. These cells rapidly migrate through the endothelial barrier into inflamed tissues, where they act as a first line of defense. Accordingly, monocytes and neutrophils, upon appropriate stimulation, release a number of proinflammatory mediators, including reactive oxygen intermediates and antimicrobial products, produce cytokines and chemokines, modify the expression of surface molecules, and efficiently phagocytose and kill invading pathogens (1, 2). The secretion of cytokines and chemokines by phagocytes at the infection site not only favors the recruitment of other effector cells (3), but also prepares and amplifies the subsequent immune response.

The ability of monocytes and neutrophils to respond to environmental signals depends on the repertoire of plasma membrane receptors that they express and that allows them to recognize all classes of macromolecules (4, 5). In this regard, a large number of receptors of the innate immune system use the ITAM signaling pathway, which induces cellular activation via a phosphorylation cascade triggered by protein tyrosine kinases (6). ITAMs are mainly found in the cytoplasmic tail of transmembrane adaptors, such as CD3{zeta}, FcR{gamma}, or DAP12, molecules that provide the role of signaling subunits when associated with cell surface receptors. For instance, the triggering receptor expressed on myeloid cells (TREM)4 family has recently attracted particular attention due to the capacity of these DAP12-associated members to activate several myeloid cell types (7, 8, 9). Of interest, TREM-1 plays a crucial 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).

Among the ITAM-linked receptors, the osteoclast-associated receptor (OSCAR), a myeloid cell receptor associated with the ITAM-bearing chain FcR{gamma} (13, 14, 15), has an expression pattern that overlaps that of the TREM family. We have recently demonstrated that human OSCAR (hOSCAR) is expressed by myeloid dendritic cells (DC), is involved in Ag internalization and presentation, and causes cellular activation and partial maturation in this cell type (14, 16). In addition, we have shown that numerous DC functions triggered by hOSCAR stimulation are negatively regulated by the ITIM-signaling receptor CD85j (leukocyte Ig-like receptor-1/Ig-like transcript 2), confirming that hOSCAR is a classical ITAM-linked receptor that plays a role in maintaining the equilibrium of the immune response (17). Some of those studies (14, 16) also described the role of hOSCAR in adaptive immunity and underlined differences between FcR{gamma}- and DAP12-associated receptors. Despite the increasing understanding of the role of hOSCAR in DC biology, no information has been reported on the potential role of hOSCAR in modulating the effector functions of phagocytic cells.

In the present work, we have investigated the capacity of hOSCAR to induce cellular stimulation in human monocytes and neutrophils. We show that hOSCAR engagement triggered several proinflammatory responses, including the production of soluble mediators and the acquisition of an activated phenotype in monocytes and degranulation in neutrophils. Interestingly, we also show that ligation of hOSCAR acted synergistically with TLR ligands to enhance proinflammatory responses in both monocytes and neutrophils. Finally, hOSCAR ligation promoted survival of monocytes but not of neutrophils. Taken together, our data demonstrate that OSCAR is a functional receptor on monocytes and neutrophils that is probably involved in the primary inflammatory response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell cultures

Human peripheral blood samples were obtained according to institutional guidelines. PBMC were purified by Ficoll-Hypaque centrifugation. After Percoll gradient, monocytes were purified by immunomagnetic depletion of low density PBMC isolated on a 52% Percoll gradient as described (18). Highly purified granulocytes (neutrophils, >96.5%; eosinophils, <3%) were isolated as previously described (19). Briefly, PBMC and platelets were removed by centrifugation through a Ficoll-Hypaque gradient. Neutrophils were then separated from erythrocytes by sedimentation with 4% dextran, and residual RBC were removed by hypotonic lysis.

Cells were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% (v/v) heat-inactivated FBS (Eurobio), 2 mM L-glutamine, and 100 µg/ml gentamicin (Schering-Plough).

In vitro stimulation of cells

Anti-hOSCAR mAb and F(ab')2 mAb were produced as previously described (14). Anti-hOSCAR mAb and F(ab')2 (clone 11.1CN5), and irrelevant mAb MOPC21 (Sigma-Aldrich) or anti-CD13 (Immunotech) isotype controls were coated for 4 h at 37°C on flat-bottom plates with a final concentration of 20 µg/ml in PBS. Monocytes and neutrophils were plated at a concentration of 1 x 106 and 3 x 106 cells/ml, respectively.

Activating factors were used at the following final concentrations: 100 ng/ml Pam3Cys (synthetic palmitoylated mimic of bacterial lipopeptides; InvivoGen); 100 ng/ml Escherichia coli LPS (Sigma-Aldrich); and 10 µM R-848 (imidazoquinoline resiquimod synthesized in our laboratory). Monocytes and neutrophils were collected after 24 and 2 h, respectively, and cell surface markers were tested by flow cytometry. Supernatants were removed after 24-h incubation and tested by ELISA. All mAb used for tissue culture were shown to be endotoxin-free, as determined by Limulus-Amebocyte Assay (BioWhittaker). To block an eventual effect of trace amounts of LPS, polymyxin B (Sigma-Aldrich) was added to certain wells at 10 µg/ml.

Flow cytometry

For hOSCAR staining, cells were incubated with 10 µg/ml anti-hOSCAR mAb 11.1CN5, then labeled with PE-conjugated goat anti-mouse (GAM; DakoCytomation). For phenotypical analysis, cell staining was performed using PE-conjugated mouse mAb anti-CD54 (eBioscience), anti-CD40, -CD63, -CD83 (Immunotech), anti-CD25, -CD62L, -CD86 (BD Pharmingen), and FITC-conjugated mouse mAb anti-CD11b and -CD66b (Immunotech).

Detection of apoptosis

Monocytes and neutrophils were stimulated with coated mAb for 48 and 24 h, respectively. As a positive control, 20 ng/ml GM-CSF was used to maintain cellular integrity in culture. After the indicated incubation times, cells were harvested and apoptotic cells were detected using the FITC-Annexin V kit (BD Pharmingen) followed by flow cytometer analysis.

Measurement of soluble mediator release

Supernatants of cells stimulated for the times indicated in the results were collected and tested by ELISA for production of IL-1beta, IL-6, IL-8/CXCL8, IL-10, IL-12 p40, TNF, MCP-1/CCL2, GM-CSF (OptEIA kits; BD Pharmingen), epithelial neutrophil-activating peptide-78/CXCL5, macrophage-derived chemokine (MDC)/CCL22, M-CSF, matrix metalloproteinase-9 (MMP-9) (DuoSet kit; R&D Systems), and myeloperoxidase (MPO) (Merck Calbiochem). The detection of lactoferrin was performed as previously described (20). Briefly, supernatants were diluted in 0.05 M carbonate buffer (pH 9.6) and proteins were immobilized in Nunc-Immuno 96-well plates (Nunc) overnight at 4°C followed by blocking with 0.5% 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 allowed to react for 1 h, followed by incubation with anti-rabbit peroxidase (dilution 1/5000; Amersham Biosciences) for 1 h. After a final washing, tetramethylbenzidine solution (Sigma-Aldrich) was used to read the absorbance of each well. Standard curves were interpreted with respect to known dilutions of human lactoferrin (Sigma-Aldrich).

Production of reactive oxygen intermediates (ROI)

Hydrogen peroxide (H2O2) and superoxide anion (O

{cjs1138}

2) release were measured as previously described (21). Briefly, the production of H2O2 was assayed in neutrophil suspensions (106/ml) stirred in HBSS at 37°C, as the H2O2-HRP-dependent oxidation of homovanillic acid by using a spectrofluorometer (model LS 50B; Applied Biosystems). In these assays, neutrophils were stimulated for up to 30 min with soluble primary anti-hOSCAR mAbs at 10 µg/ml, cross-linked with GAM (H + L)-specific F(ab')2 (Immunotech) at 20 µg/ml. O

{cjs1138}

2 production was assayed as the reduction of ferricytochrome C by neutrophils (105/100 µl) stimulated in plate-bound anti-hOSCAR or isotype-matched control mAbs for 30 min. Stimulation via 100 nM fMLP (Sigma-Aldrich) was used as the positive control.

Statistical analyses

Statistical analyses were performed in Microsoft Excel 5.0 (Microsoft) using two-tailed Student’s t tests. A p value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
hOSCAR expression by monocytes and neutrophils

We analyzed the expression of hOSCAR on monocytes and neutrophils. Fig. 1 shows the expression of hOSCAR on freshly isolated monocytes (Fig. 1A) and neutrophils (Fig. 1B). The experiment shown is representative of five donors studied. All monocytes and neutrophils expressed hOSCAR, although the level of expression was higher on monocytes. Stimulation of these cells with Pam3Cys, LPS, and R-848, ligands for TLR2, TLR4, and TLR7/8, respectively, did not significantly alter the cell surface expression of hOSCAR (Fig 1, C and D).


Figure 1
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  		FIGURE 1. hOSCAR is expressed by blood monocytes and neutrophils and its expression is maintained upon cellular activation by TLR ligands. Monocytes (A and C) and neutrophils (B and D) were labeled and analyzed by flow cytometer for cell surface expression of hOSCAR, using anti-hOSCAR and a PE-conjugated GAM Ab. In histograms A (freshly isolated monocytes) and B (freshly isolated neutrophils), dashed profiles indicate background staining with a control IgG1 mAb, shaded profiles hOSCAR specific staining, and numbers in the corners correspond to the mean fluorescence intensity of hOSCAR staining. Histograms C and D show hOSCAR expression upon cellular activation. Monocytes (C) and neutrophils (D) were cultured with 100 ng/ml LPS, 10 µM R-848, 100 ng/ml Pam3Cys or 20 ng/ml GM-CSF, and after 24 h culture, cells were analyzed by flow cytometry for hOSCAR expression. The data are expressed as {Delta}MFI, the mean fluorescence intensity minus the fluorescence detected with isotype control. The results presented are representative of five independent experiments.

 
hOSCAR ligation provides a survival signal for monocytes, but has no effect on neutrophil life span in culture

Our previous study showed that hOSCAR ligation promotes cell survival of monocyte-derived DCs in the absence of survival factors, such as GM-CSF (16). This activity is a long-term effect, allowing cell survival up to 10 days or more. We thus investigated the ability of hOSCAR engagement to modify the life span of ex vivo monocytes and neutrophils cultured in the absence of survival factors for 48 and 24 h, respectively. Cellular stimulation by plastic-coated Abs is known to induce receptor aggregation, required for the triggering of the ITAM-signaling pathway (22). This method was previously shown to be efficient in the triggering of DC maturation through hOSCAR ligation and was therefore also used to engage hOSCAR at the surface of monocytes and neutrophils. Approximately 80% of monocytes treated with either whole Ab or F(ab')2 specific for hOSCAR survived in the absence of endogenous survival factors for 2 days (Fig. 2A), similar to the level of survival induced by GM-CSF treatment. In control cultures, in the presence of plastic-coated MOPC21 isotype control Ab or anti-CD13, a high proportion of apoptotic monocytes (annexin V-positive cells) was observed. Unlike the effect of hOSCAR Abs on survival of monocytes, neither control Ab nor anti-hOSCAR decreased the proportion of apoptotic neutrophils, whereas GM-CSF had the ability to promote neutrophil survival (Fig. 2B). Altogether, these results suggest that hOSCAR is able to promote monocyte survival, but is incapable of extending the duration of neutrophil responses by attenuating apoptosis.


Figure 2
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  		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 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.

 
hOSCAR engagement induces an activated phenotype and soluble mediator release by monocytes

To investigate the role of hOSCAR in primary inflammatory responses, freshly isolated blood monocytes were stimulated by Abs coated or not on tissue culture plates, or by TLR ligands known to stimulate monocytes.

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 contamination 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. Interestingly, the up-regulation of CD86 in the case of hOSCAR stimulation was stronger than that observed with TLR ligands. hOSCAR stimulation also triggered up-regulation at the monocyte surface of CD40, CD54, and CD83 costimulatory molecules (Fig. 3B). Unlike TLR ligands, hOSCAR engagement was not able to induce the expression of the activation marker CD25 by monocytes, however, it induced the expression of CD83 which was poorly induced by Pam3Cys and LPS, but strongly induced by R-848. These results point to an activation of circulating blood monocytes via hOSCAR, similar to the activation observed via other stimuli, but with several significant differences.


Figure 3
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  		FIGURE 3. hOSCAR ligation induces phenotypic changes and secretion of IL-8/CXCL8 by monocytes. Monocytes were stimulated by coated control IgG (MOPC21, anti-CD13), coated anti-hOSCAR whole mAb or F(ab')2, 10 ng/ml Pam3Cys, 100 ng/ml LPS, 10 µM R-848, or soluble anti-hOSCAR. After 24 h, cells were analyzed by flow cytometry for the indicated markers (A and B) and supernatants were tested by ELISA for IL-8/CXCL8 secretion (C). In A and B, the numerical values indicate the specific mean fluorescence intensity of the staining (for shaded histograms), and the dotted line shows the binding of an isotype control mAb to the cells. Data are representative of four independent experiments. In C, IL-8/CXCL8 secretion data are mean ± SD of triplicate samples from one representative experiment of three performed with similar results. Statistical significances of **, p < 0.001 are given by comparison to values obtained with the negative control anti-CD13.

 
Immune responses during the early steps of inflammation are mediated by soluble factors capable of recruiting and activating other immune cells which also may have a toxic effect on the invading pathogens. In monocytes, cross-linking of hOSCAR by whole Ab or F(ab')2 induced a strong secretion of IL-8/CXCL8, which was not observed in response to the isotype controls (MOPC21 and anti-CD13) or soluble anti-hOSCAR (Fig. 3C). Although the amount of IL-8/CXCL8 secreted was low in comparison to that induced by LPS or R-848, it was statistically significant and very similar to the amount produced by activation via Pam3Cys. The presence of polymyxin B did not block the ability of anti-hOSCAR to induce IL-8/CXCL8 secretion, demonstrating that the activation observed was not mediated by contaminating endotoxin. In monocytes, hOSCAR ligation also induced the secretion of the chemokines epithelial neutrophil-activating peptide-78/CXCL5, MDC/CCL22, and MCP-1/CCL2, but no release of proinflammatory cytokines such as IL-1beta, TNF, or IL-12 p40 (Table I).


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

 
hOSCAR cross-linking on freshly isolated neutrophils leads to degranulation and rapid changes in cell phenotype

Although hOSCAR engagement had no effect on neutrophil survival, cross-linking with either coated F(ab')2 or whole mAb anti-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).


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

 

Figure 4
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  		FIGURE 4. hOSCAR ligation on neutrophils induces phenotypical changes, IL-8/CXCL8 release, and respiratory burst activity. Neutrophils were 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 µM 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 ± SD of triplicate samples from one representative experiment of three performed with similar results. Statistical significances of *, p < 0.01 and **, p < 0.001 are given by comparison to values obtained with the negative 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 (H2O2) and superoxide anion (O{cjs1138}2) production were measured as described in Materials and Methods. fMLP (100 nM) was used as a positive control stimulus. Data are mean ± SD of three experiments (three donors). Statistical significances of *, p < 0.01 are given by comparison to values obtained with the mouse isotype-matched control.

 
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 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-treated neutrophils, as measured by both hydrogen peroxide (H2O2) and superoxide anion (O

{cjs1138}

2) production (Fig. 4D). Stimulation 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 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-1beta, TNF) by monocytes cultured in both anti-hOSCAR mAb and TLR ligands as compared with those cultured with individual stimuli. Strikingly, although the effect of single TLR ligands dropped rapidly over 10-fold diminutions in concentration, the costimulatory effects of hOSCAR ligation showed a slower decline, indicating that hOSCAR augments the response to suboptimal doses of pathogen-derived TLR ligands. These data suggest that signals produced by hOSCAR ligation synergize with various TLR to up-regulate production of proinflammatory cytokines and chemokines by monocytes.


Figure 5
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  		FIGURE 5. hOSCAR ligation synergistically up-regulates soluble mediator release by monocytes and neutrophils upon TLR stimulation. Monocytes (A) 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-1beta, TNF, lactoferrin, and MMP-9 secretion. Data are mean ± SD of duplicate samples from one representative experiment of three performed with similar results.

 
We also detected a significant increase in IL-8/CXCL8, lactoferrin, and MMP-9 by neutrophils costimulated with both anti-hOSCAR and TLR ligands (Fig. 5B). Although the effect is less striking than that seen for monocytes, ligation of hOSCAR maintained a strong inflammatory response, even in the presence of 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 expression of CD11b and CD66b at the cell surface of neutrophils after 2 h of stimulation with anti-OSCAR Abs and TLR ligands. These experiments revealed an additional up-regulation of these two markers of release of specific and/or gelatinase granules, however, the increase was minimal, not statistically significant in all cases and resembled an additive effect (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Growing evidence suggests that ITAM-associated receptors on myeloid cells are involved in the regulation of innate resistance and adaptive immunity. We have previously shown that hOSCAR, a receptor associated with the FcR{gamma} chain, can activate human DCs and modulate the adaptive response in the presence of TLR ligands (16). We reported that hOSCAR is expressed on monocytes and neutrophils (14) and our preliminary results showed the induction of a Ca2+ flux on monocytes, indicative of early cellular activation (14). In this study, we further examined the effects of hOSCAR stimulation on these cells. One of our early observations on hOSCAR was that the expression of this molecule, unlike that of other FcR{gamma}-associated molecules, was not modulated by activation of DCs with various physiological activators (14). We now show that this is also true for monocytes and neutrophils. In our hands, all neutrophils and monocytes expressed hOSCAR and no significant difference in expression was seen after 24 h of activation with either TLR ligands or GM-CSF. In contrast, the DAP12-associated receptor TREM-1 is up-regulated upon monocyte and neutrophil activation by fungus and extracellular bacteria, and especially by LPS and lipoteichoic acid which are microbial components, but not nonbacterial TLR ligands (7, 10, 23). TREM-1 is also up-regulated in neutrophils of patients with pneumonia (24) and in monocytes of patients suffering from septic shock (23, 25) indicating a role in the detection and response to microbial infection. In contrast, hOSCAR expression did not change in the conditions of neutrophil activation analyzed in this study, suggesting that the physiologic regulation of hOSCAR reactivity during inflammatory responses may depend on availability or regulation of the expression of the counter-receptor.

OSCAR ligation promoted survival of monocytes, but did not affect the differentiation of monocytes into DC (data not shown). Interestingly, cross-linking of hOSCAR did not induce secretion of GM-CSF, excluding a role of this cytokine in the survival of monocytes induced by hOSCAR Abs. However, similarly to TREM-1 (12), hOSCAR did not promote survival of neutrophils, but rapidly stimulated granule secretion and even IL-8/CXCL8 release. The latter observations indicate that hOSCAR is functional in neutrophils but its activities are in part different from those elicited in monocytes. It remains to be investigated whether the differences between neutrophils and monocytes are caused by a defective capacity of hOSCAR-stimulated neutrophils to appropriately transduce the signals that control cell viability or simply by the lack of neutrophil expression of crucial proteins controlling survival. The importance of receptor density in reaching the necessary threshold to trigger strong signals is well-known for the receptors signaling through ITAM, and especially the FcR. The expression level of hOSCAR is lower on neutrophils than on the monocyte cell surface. Thus, one possible hypothesis is that the number of receptors cross-linked on the neutrophil cell surface may be insufficient to reach the threshold required for induction of survival and a synergistic OSCAR-TLR effect in the up-regulation of degranulation markers. In addition, neutrophils have been reported to express relatively low FcR{gamma} levels, compared with monocytes (26). A stoichiometric relationship between hOSCAR and the FcR{gamma} chain would be hypothesized, as is the case for the other receptors associated with FcR{gamma}: one hOSCAR molecule would thus associate with a covalently linked dimer of the FcR{gamma} chain through ionic interaction. A single positively charged amino acid (arginine) is found in the transmembrane domain of OSCAR which can interact with the negatively charged amino acid (aspartic acid) of FcR{gamma}. It is therefore possible that a limited availability of FcR{gamma} in neutrophils results in weaker effects of hOSCAR ligation compared with monocytes.

Activation of monocytes via hOSCAR led to a differential expression of cell surface markers than those induced by TLR ligands. CD83 was only induced by hOSCAR and R-848, CD25 was induced by TLR ligands but not hOSCAR, and hOSCAR induced a higher expression of CD86 than TLR ligands. hOSCAR cross-linking alone did not induce secretion of proinflammatory cytokines such as TNF and IL-1beta by monocytes, but secretion of chemokines was observed. Among the chemokine induced by hOSCAR, MCP-1 recruits of monocytes to sites of injury and infection (27, 28) and MDC is chemoattractant for monocytes, monocyte-derived DCs, and NK cells (29). On neutrophils, stimulation via hOSCAR led to the secretion of IL-8/CXCL8, another chemokine that promotes recruitment of immune cells, including neutrophils and subsets of monocytes and T lymphocytes. Neutrophils treated with anti-hOSCAR also cleaved surface CD62L. This cleavage was also strongly induced by LPS and R-848 but only weakly by Pam3Cys. CD62L mediates leukocyte rolling on activated endothelium in inflamed tissues and lymphocyte homing to high endothelial venules of peripheral lymphoid tissue (30). Thus, activation of myeloid cells via hOSCAR interaction with its ligand may by itself be sufficient to set up a gradient of recruitment of immune cells to the site of interaction. In our hands, LPS and R-848 and at a lower extent hOSCAR cross-linking, but not Pam3Cys, induced in neutrophils cell surface translocation of CD11b from specific granules, gelatinase granules, and secretory vesicles. Similarly, the appearance of CD66b at the cell surface was induced, although only at low level, by hOSCAR ligation. Ligation of hOSCAR thus appears to selectively cause degranulation of specific (PO–) granules and at a lower level, of azurophil (PO+) granules, as suggested by the analysis of the granule markers MPO and CD63 (31). Degranulation of neutrophils is accompanied by the secretion of MPO, MMP-9, and lactoferrin, all molecules that are implicated in the antibacterial response (31). MMP-9 is contained in specific and gelatinase granules and its secretion is instrumental for the degradation of extracellular matrix and the mobilization of stem cells (32). Interestingly, MMP-9-mediated N-terminal cleavage of IL-8/CXCL8 is known to augment IL-8/CXCL8-mediated activation of neutrophils, as measured by increased intracellular calcium, chemotaxis, and secretion of MMP-9 itself (33), suggesting an autoamplification circuit for IL-8/CXCL8. In addition, hOSCAR ligation contributes to the early antibacterial response of neutrophils by rapidly mediating the production of reactive oxygen species within 30 min after receptor cross-linking.

We had previously reported that hOSCAR ligation modulated the responses of DCs to TLR ligands. In this paper, we investigated the effect of costimulation of phagocytes with pathogen-derived TLR ligands and hOSCAR. The secretion of IL-8/CXCL8 by monocytes after incubation with optimal doses of TLR ligands together with hOSCAR ligation was doubled. This effect was more marked at suboptimal doses of TLR ligands, because coincubation with hOSCAR with doses of TLR ligands that alone showed no effect allowed us to detect IL-8/CXCL8 and other cytokines. In the case of IL-1beta, a dramatic effect of hOSCAR costimulation was seen with Pam3Cys that alone induced no secretion, but a marked 6-fold increase was also seen with LPS at 10 ng/ml and R-848 at 1 µM. hOSCAR ligation also had a strong enhancing effect on the secretion of TNF in combination with 10 ng/ml R-848 (5-fold) and 1 µM LPS (8-fold). In neutrophils, lactoferrin and MMP-9 secretion induced by suboptimal doses of TLR ligands was significantly augmented by hOSCAR ligation. Thus, coactivation of monocytes and, in part, neutrophils with hOSCAR in the presence of pathogen-derived molecules activates a strong proinflammatory pathway.

Altogether our results show that triggering of monocytes and neutrophils via hOSCAR, acting through the FcR{gamma} chain, led to activation of proinflammatory and innate responses, similar to that observed with members of the TREM family via DAP12. TREM-2, a similar myeloid receptor, was recently shown to be important in both osteoclast biology and triggering of the immune response (9). TREM-2 expressed by both osteoclasts (34, 35) and DC (8) has potentially multiple ligands, represented by microbial products (36), molecules expressed on the NK cell surface (37) or present in the bone environment (38). Data from different groups strongly suggests that in vivo ligation of mouse OSCAR on osteoclasts is essential for differentiation of these cells (13, 38). An endogenous ligand for OSCAR on osteoblasts has been inferred from these studies by using a soluble form of the mouse OSCAR extracellular domain. However, the putative ligand has not yet been isolated and molecularly characterized. These results have led us and other groups to propose that hOSCAR can be added to the receptors known to play a role in both bone homeostasis and activation of immune responses. The presence of an hOSCAR ligand in the immune system environment awaits discovery, and supposes that this molecule will regulate the precarious balance between immune activation and suppression, roles well-defined for receptors linked to ITAM/ITIM signaling. Because hOSCAR is a stably expressed molecule widely present on myeloid cells, the presence of the hOSCAR ligand will be determinant in defining where and when hOSCAR is engaged, and active. hOSCAR could recognize pathogen components, similar to TREM-2, or be involved in the sensing of endogenous infection- or stress-induced molecules. Interestingly, binding to endogenous molecules and to pathogen-derived components has been described for other dual receptors such as DC-SIGN or dectin-1 (39). Clearly, a better understanding of the physiologic role of these families of ITAM-signaling receptors expressed on myeloid cells awaits the precise identification of the nature, origin, expression, and distribution of their ligands.


   Acknowledgments
 
We thank colleagues from Etablissement Français du Sang-Lyon who provided us with blood samples. We are grateful to Federica Calzetti for technical help on neutrophil experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 E.M. was a recipient of a grant from the Fondation Marcel Mérieux (Lyon, France). M.A.C. was supported by grants from Ministero dell’Istruzione, dell’Università e 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 Cancro, and Fondazione Cassa di Risparmio di Verona-Vicenza-Ancona e Belluno. Back

2 Address correspondence and reprint requests to Dr. Estelle Merck at the current address: Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address: Estelle.Merck{at}isrec.unil.ch Back

3 Current address: Laboratory of Parasitic Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. Back

4 Abbreviations used in this paper: TREM, triggering receptor expressed on myeloid cells; OSCAR, osteoclast-associated receptor; hOSCAR, human OSCAR; DC, dendritic cell; GAM, goat anti-mouse; MMP-9, matrix metalloproteinase-9; MPO, myeloperoxidase; ROI, reactive oxygen intermediate; MDC, macrophage-derived chemokine. Back

5 Current address: Molecular and Biochemical Analytical Services, Bayer BioScience N.V., Technologiepark 38, 9052 Gent, Belgium. Back

Received for publication August 19, 2005. Accepted for publication December 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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