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The Journal of Immunology, 2007, 178: 4250-4259.
Copyright © 2007 by The American Association of Immunologists, Inc.

Paired Ig-Like Receptors Bind to Bacteria and Shape TLR-Mediated Cytokine Production1

Masafumi Nakayama*, David M. Underhill{dagger}, Timothy W. Petersen2,*, Bin Li*, Toshio Kitamura{ddagger}, Toshiyuki Takai§ and Alan Aderem3,*

* Institute for Systems Biology, Seattle, WA 98103; {dagger} Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048; {ddagger} Divisions of Cellular Therapy and Hematopoietic Factors, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and § Department of Experimental Immunology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system uses a wide variety of pattern recognition receptors including TLRs, scavenger receptors, and lectins to identify potential pathogens. A carefully regulated balance between activation and inhibition must be kept to avoid detrimental and inappropriate inflammatory responses. In this study, we identify murine-paired Ig-like receptor (PIR)-B, and its human orthologs Ig-like transcript 2 and Ig-like transcript 5 as novel receptors for Staphylococcus aureus. PIR-B contains four ITIM motifs and is thought to be an inhibitory receptor. Expression of these receptors enables NIH3T3 cells to bind S. aureus. In mouse bone marrow-derived macrophages, masking of PIR-B by anti-PIR mAb or genetic deletion of PIR-B shows significantly impaired recognition of S. aureus and enhanced TLR-mediated inflammatory responses to the bacteria. These data suggest a novel mechanism for innate immune regulation by paired Ig-like receptor family members.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages initiate innate immune responses by recognizing pathogens, phagocytosing them, and secreting inflammatory mediators. An effective response requires that macrophages recognize pathogen-associated molecular patterns that distinguish the infectious agents from self, and, in addition, discriminate among pathogens (1, 2, 3, 4). Recently, the TLRs have emerged as key receptors for effective innate immunity (5, 6, 7).

The TLRs are a family of 13 innate immune recognition receptors that recognize a broad range of microbial products including LPS, flagellin, lipoproteins, and bacterial DNA, and that play a vital role in the transcriptional responses of cells of the innate immune system (6, 7). Each TLR has multiple extracellular leucine-rich repeats as well as an intracellular signaling domain that is homologous to the cytoplasmic tail of the IL-1R. Stimulation of TLRs direct activation of NF-{kappa}B and production of proinflammatory cytokines (6, 7). Along with the TLRs, many additional innate immune receptors participate in the recognition of microbes, thus fine-tuning the innate immune system’s unique responses to each microbe (4, 8). Activation of these TLR-mediated inflammatory responses must be tightly regulated; too little response leaves the host susceptible to infection, and too much response may lead to lethal systemic inflammation or autoimmunity.

Staphylococcus aureus, an extracellular Gram-positive bacteria, is a major source of mortality in medical facilities (9, 10). The primary site of infection is often a breach in the skin that may lead to minor skin and wound infections, but S. aureus can also infect any other tissue of the body, causing life-threatening diseases such as osteomyelitis, endocarditis, pneumonia, and septicemia (9, 10). We have previously observed that S. aureus-induced inflammatory cytokine production by macrophages is mediated by TLR2 (11, 12), and Takeuchi et al. have reported that TLR2- or MyD88-deficient mice are highly susceptible to S. aureus infection (13). Recently, CD36, a class B scavenger receptor, has been reported to facilitate TLR2 activation for effective innate immune responses to S. aureus, indicating that accessory receptors can be important for certain TLR-mediated inflammatory responses (14, 15). Compared with infection by Gram-negative bacteria, S. aureus triggers lower quantities of inflammatory cytokines (16, 17). The mechanisms by which different types of bacteria elicit different types of inflammatory responses are poorly understood, but they are likely to be controlled by interactions between the different repertoires of pattern recognition receptors that distinguish between various bacteria.

The inhibitory and activating Ig-like receptors, such as FcR and NK receptor, provide negative and positive regulation of immune cells upon recognition of various ligands, thus enabling those cells to elicit a balanced response to extrinsic stimuli (18, 19). Murine-paired Ig-like receptors (PIR4)-B (also known as Lilrb3) and PIR-A are expressed on a wide variety of immune cells including B cells, mast cells, macrophages, and dendritic cells (DCs), mostly in a pair-wise fashion (20, 21, 22). PIR-B and PIR-A are encoded by a gene cluster, wherein PIR-B is encoded by a single gene and PIR-A is encoded by multiple genes (20, 21, 22). Whereas the ectodomains of PIR-B and PIR-A are very similar (>92% aa identity), the cytoplasmic domains differ significantly (22, 23). PIR-B has four ITIMs in the cytoplasmic region and inhibits activating signals by surface receptors such as B cell Ag receptors and chemokine receptors (22, 23, 24). In contrast, PIR-A contains a short cytoplasmic tail lacking any signal transduction motif. Instead, PIR-A has a basic arginine residue within the transmembrane domain and associates with FcR{gamma} common chain, which contains an ITAM. Cross-linking of the PIR-A/FcR{gamma} complex results in mast cell activation through an ITAM-dependent manner (22, 23). Recently, it has been reported that MHC class I (H-2) molecules are ligands for both PIR-B and PIR-A (25). Therefore, PIR-B and PIR-A appear to provide a homeostatic balance of inhibitory and activating signals to the immune system by interaction with MHC class I. Indeed, in a mouse model of graft-vs-host disease, in which allogeneic splenocytes were transferred into PIR-B-deficient mice, enhanced PIR-A signaling in the host DCs augmented the activation of the Pirb+/+ donor T cells, resulting in enhanced IFN-{gamma} production by DCs, CD4+ T cells, and CTLs (25).

In this study, we used a retrovirus-mediated expression-cloning strategy to identify PIR-B as a receptor for S. aureus. Subsequent analysis suggests that at least one specific form of PIR-A (PIR-A1), as well as the PIR-B human orthologs Ig-like transcript (ILT)2 and ILT5 also recognize the pathogen. We propose that these PIRs help fine-tune TLR-mediated inflammatory response to S. aureus, thus providing a novel entry point for therapeutic intervention.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Anti-PIR mAb (6C1) and recombinant mouse IL-10 were purchased from BD Pharmingen. Anti-mouse IL-10-neutralizing mAb (JES-2A5) and control rat IgG1 mAb were purchased from eBioscience. Tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin was purchased from Molecular Probes. Dextran sulfate was purchased from Amersham. Polyinosinic acid and polycytidylic acid were purchased from Sigma-Aldrich. LPS and the synthetic lipopeptide PAM3CSK4 were purchased from List and EMC Microcollections, respectively. Peptidoglycan and lipoteichoic acid were purchased from Fluka. Murine IL-6, TNF-{alpha}, and IL-10 ELISA kits were purchased from R&D Systems.

Mice and bone marrow-derived macrophages

C57BL/6 mice were obtained from The Jackson Laboratory. Pirb–/– mice (129/SvJ/C57BL/6 background) (26) were backcrossed for at least 10 generations with C57BL/6 mice. Tlr2–/– mice and Myd88–/– mice (129/SvJ/C57BL/6 background (27); provided by S. Akira, Osaka University, Osaka, Japan), were backcrossed for eight generations with C57BL/6 mice. All mice were used according to the guidelines of the institutional animal care and use committee established at the Institute for Systems Biology. Mouse bone marrow-derived macrophages were grown in complete RPMI 1640 (RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine) with 50 ng/ml recombinant human M-CSF (Chiron). For cytokine measurement, the cells (2 x 105/well) were seeded onto 24-well plates and cultured overnight. Then, cells were treated with the indicated stimuli for 12 h at 37°C. For some experiments, the cells were pretreated with the indicated mAb for 1 h at 4°C. The amounts of IL-6, TNF-{alpha}, and IL-10 in supernatants were measured by ELISA.

Construction of cDNA library and retroviral expression cloning

Construction of retroviral cDNA library was performed as described previously (28). Briefly, complementary cDNAs were synthesized from poly(A)+RNA of C57BL/6 mouse bone marrow-derived macrophages with oligo(dT) primer using the SuperScript Choice System (Invitrogen Life Technologies) according to the manufacturer’s instructions. The synthesized cDNAs were cloned between the BstXI sites of pMX (29) using BstXI adaptors (Invitrogen Life Technologies), generating a retroviral cDNA expression library. The cDNA library contained ~1 x 106 individual clones. For production of retroviruses, culture supernatants of Phoenix-Ampho cells (provided by G. Nolan, Stanford University, Stanford, CA) were harvested 3 days after transfection with the retroviral cDNA library. NIH3T3 cells (4.8 x 106) were infected with retroviruses expressing this cDNA library in the presence of 8 µg/ml polybrene. Two days after infection, these cells were cultured with TRITC-labeled S. aureus (Wood strain without protein A; Molecular Probes; 10 bacteria per cell) for 30 min at 37°C. After washing with PBS twice, cells were harvested by trypsinization and subjected to cell sorting. After four rounds of sorting, 40 individual clones obtained by limiting dilution were examined on the binding to TRITC-S. aureus. From the positive cell clones that are able to bind TRITC-S. aureus, genomic DNAs were isolated. The insert cDNAs were amplified by PCR from the genomic DNA, and sequenced. The PIR-B cDNA was subcloned into pMXs-IRES-puro (pMXs-IP) (29), generating pMX-IP-PIR-B.

Cell lines

NIH3T3 (American Type Culture Collection (ATCC) no. CRL-1658) cells were maintained in complete DMEM (DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine). For construction of expression vectors, the coding region for PIR-A1 and PIR-A2 were amplified from RAW264.7 cDNAs, and the coding region for PIR-A2h1 and PIR-A2h2 were amplified from C57BL/6 mouse bone marrow-derived macrophage cDNAs. These fragments were inserted into pMXs-IP. Several mutant forms of PIR-B were prepared by PCR-based mutagenesis using PIR-B/pMXs-IP as a template. The coding region for ILT2 (leukocyte Ig-like receptor (LILR)B1) and ILT5 (LILRB3) were amplified from human peripheral mononuclear cell cDNAs, and then inserted into pMXs-IP and tagged at the C terminus, which includes an epitope from protein C recognized by the HPC4 mAb (Amersham) and the streptavidin-binding peptide (30). NIH3T3 cells were infected with these retroviruses in the presence of 8 µg/ml polybrene. Two days after infection, these cells were selected by puromycin (10 µg/ml; Calbiochem). The expressions of PIR and ILT were confirmed by flow cytometry using 6C1 and HPC4 mAbs, respectively.

Homology modeling of PIR-B structure

The three-dimensional structure of PIR-B was predicted through the SWISS-MODEL server (http://swissmodel.expasy.org/) (31). Crystallographic structures of human LIR-1 and Fc{alpha}RI proteins (1ugn, 1g0x, 1p7q, 1uct, and 1ovz in Research Collaboratory for Structural Bioinformatics protein data bank (www.rcsb.org)) were chosen to be the template structures based on pair-wise sequence alignments (PIR-B and 1ugn shared 49.33% sequence identity).

Fluorescent labeling of bacteria

S. aureus (Wood 46 strain; ATCC), Bacillus subtilis (Marburg strain, ATCC), Listeria monocytogenese (strain 10403S; provided by D. Portnoy, University of California, Berkely, CA), Helicobacter pylori (provided by N. Salama, Fred Hutchinson Cancer Research Center, Seattle, WA), Pseudomonas aeruginosa (provided by D. Speert, Child and Family Institute, Vancouver, Canada), and Escherichia coli (K-12 strain; ATCC) were grown to saturation, then washed three times with PBS. After heat-killing (65°C, 20 min), these bacteria were incubated with PBS containing FITC (50 µg/ml; Molecular Probes) or TRITC (50 µg/ml; Molecular Probes) for 30 min at room temperature. The reaction was stopped by hydroxylamine (150 mM; Sigma-Aldrich), followed by washing five times with PBS. Labeling efficiency was confirmed by flow cytometry. For labeling of live bacteria, the bacterial viability was checked by plating serial dilutions and was typically >90%.

Measurement of cells recognizing bacteria

NIH3T3 cells and bone marrow macrophages (2 x 105/well) were seeded on 24-well plates and cultured overnight. These cells were incubated with the indicated dose of the fluorescently labeled bacteria for 30 min at 4 or 37°C. In some assay, cells were pretreated with the indicated inhibitor for 30 min at 37°C. After incubation with bacteria, cells were washed with PBS twice, and then harvested and analyzed on a FACSCalibur (BD Biosciences).

Immunofluorescence microscopy

This assay was performed as described previously (12). Briefly, cells grown on glass coverslips were treated with FITC-S. aureus for 30 min at 37°C, then washed with PBS, and fixed with 10% formalin in PBS for 15 min. Cells were permeabilized and stained with TRITC-phalloidin. Images were acquired using a Leica SP2 laser scanning confocal microscope equipped with a 63x/1.4 objective lens (Leica). Images were cropped and placed on pages using Adobe Photoshop version 6.0 (Adobe Systems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of PIR-B as a receptor for S. aureus

We previously observed that TLR2 is responsible for inflammatory responses to S. aureus, but not for direct binding to the bacteria (11, 12). Recently, we and others have reported that non-TLRs binding whole microbes functionally interact with TLR2 (14, 15, 32). To identify novel receptors that directly bind S. aureus and functionally interact with TLR, we used an expression cloning strategy. Bone marrow-derived macrophages from C57BL/6 mice efficiently recognized TRITC-labeled heat-killed S. aureus, whereas the binding to mouse fibroblast NIH3T3 cells was minimal, suggesting that bone marrow macrophages, but not NIH3T3 cells, express the receptors that directly bind the bacteria. Thus, we generated a retroviral cDNA expression library from the macrophages, infected NIH3T3 cells, and screened these cells by FACS to select cells that acquired the ability to bind fluorescently labeled S. aureus. After four rounds of sorting and amplification, clones were isolated by limiting dilution, resulting in the identification of three single-cell clones, 4S2C37, 4S2C43, and 4S2C46, that bind S. aureus (Fig. 1A and data not shown). We sequenced the retrovirus inserts and found that all three clones harbor PIR-B cDNA in the sense orientation. Based on cell surface staining by anti-PIR mAb 6C1 (33), parental NIH3T3 cells lacked PIR-B expression, whereas 4S2C37, 4S2C43, and 4S2C46 cells expressed PIR-B on their cell surface (Fig. 1B and data not shown). To verify that the PIR-B gene, and not a secondary mutation, was responsible for the recognition of S. aureus in these clones, we specifically regenerated NIH3T3 cells stably expressing PIR-B (PIR-B/NIH3T3). These cells expressed PIR-B on their surface and gained the ability to bind S. aureus (Fig. 1A and B). Immunofluorescence microscopy showed that PIR-B/NIH3T3 cells, but not NIH3T3 cells, bound the bacteria efficiently (Fig. 1C). To rule out the possibility that ectopic expression of PIR-B in NIH3T3 cells may be inducing the expression of some other S. aureus-binding molecules (i.e., via its ITIMs), we generated NIH3T3 cells expressing a mutant form of PIR-B lacking all cytoplasmic signaling ITIMs (PIR-B{Delta} 711-841/NIH3T3) and examined whether these cells still bind S. aureus. As shown in Fig. 1A, truncation of the functional cytoplasmic region did not alter S. aureus binding. Furthermore, we found that anti-PIR mAb 6C1 could abrogate the binding of S. aureus to PIR-B/NIH3T3 cells (Fig. 1D). These results demonstrate that PIR-B is a receptor for S. aureus. In addition, we observed that PIR-B could bind not only heat-killed S. aureus, but also paraformaldehyde-fixed and live S. aureus efficiently (data not shown).


Figure 1
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  		FIGURE 1. Identification of PIR-B as a receptor for S. aureus. A, Parental NIH3T3 cells, clone 4S2C37 cells, and NIH3T3 cells infected with retrovirus expressing the cloned PIR-B or PIR-B{Delta} 711-841 cDNA were cultured with (shaded histogram) or without (open histogram) TRITC-labeled S. aureus (10 bacteria/cell) for 30 min at 37°C. Cell surface binding was quantified by flow cytometry. B, Cells were stained with PE-labeled rat IgG1 (thin histogram) or PE-labeled anti-PIR mAb 6C1 (thick histogram), and analyzed by flow cytometry. C, NIH3T3 cells and PIR-B/NIH3T3 cells were incubated with FITC-labeled S. aureus (10 bacteria/cell) for 30 min at 37°C, and then stained with TRITC-phalloidin. Cells were visualized by immunofluorescence microscopy. D, PIR-B/NIH3T3 cells were preincubated with 5 µg/ml control rat IgG1 or 6C1 mAb, then cultured with (shaded histogram) or without (open histogram) TRITC-S. aureus (10 bacteria/cell) for 30 min at 37°C, and analyzed by flow cytometry. Data are representative of three independent experiments.

 
Recognition of S. aureus by PIR through the Ig-like domain D2

The ectodomain of PIR-A shares >92% aa identity with that of PIR-B (20, 21). This result prompted us to investigate whether PIR-A could also bind S. aureus. It is known that PIR-A is encoded by multiple genes. We therefore isolated four forms of PIR-A by PCR from the RAW264.7 mouse macrophage-like cell line and C57BL/6 bone marrow-derived macrophages, and generated stable NIH3T3 transfectants for each. Based on the amino acid identity (Fig. 2), we call these PIR-A1, PIR-A2, PIR-A2 homologue 1 (PIR-A2h1), and PIR-A2 homologue 2 (PIR-A2h2). Association of PIR-A with FcR{gamma} chain is required for efficient expression of PIR-A on the surface of cells such as the LTK fibroblast cell line, macrophages, and splenocytes (33). In contrast, PIR-A presents on the surface of COS7 cells even in absence of FcR{gamma} (34). All forms of PIR-A used in this study were highly expressed on the surface of NIH3T3 cells in the absence of the FcR{gamma} chain (Fig. 3A). Although expression levels of the four PIR-A isoforms were largely equivalent (Fig. 3A), unexpectedly only PIR-A1/NIH3T3 cells could bind S. aureus efficiently, and the binding was abrogated by 6C1 mAb (Fig. 3, B and C).


Figure 2
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  		FIGURE 2. Alignment of PIR used in this study. Amino acids are numbered with reference to the start of the signal sequence (SS) of each sequence, and one Ig-like domain is aligned per block of amino acid sequence.

 

Figure 3
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  		FIGURE 3. Recognition of S. aureus by PIR. A, Parental NIH3T3 cells and NIH3T3 cells stably expressing the indicated form of PIR were stained with PE-labeled rat IgG1 (thin histogram) or PE-labeled anti-PIR mAb 6C1 (thick histogram), then analyzed by flow cytometry. B, The cells were cultured with TRITC-labeled S. aureus (10 bacteria/cell) for 30 min at 37°C, then analyzed by flow cytometry. C, The cells were preincubated with 5 µg/ml control rat IgG1 or 6C1, then cultured with TRITC-labeled S. aureus (10 bacteria/cell) for 30 min at 37°C. Percentage of recognition (% recognition) was quantified by flow cytometry. Data are represented as mean ± SD of triplicates. D, PIR-B/NIH3T3 cells were pretreated with the indicated dose of inhibitors for 30 min, and then incubated with TRITC-labeled S. aureus (10 bacteria/cell) for 30 min at 37°C. The recognition (relative to recognition without inhibitor) was quantified by flow cytometry. Data are represented as the mean of triplicates (SD were <10% of the mean; data not shown). Data are representative of two to three independent experiments.

 
To further characterize the binding of S. aureus by PIR-B, we tried to compete the interaction with various putative bacterial cell wall ligands. As shown in Fig. 3D, the Gram-positive bacterial cell wall components lipoteichoic and peptidoglycan, and the Gram-negative bacterial cell wall component LPS failed to inhibit the binding. However, the polyanionic reagents dextran sulfate and polyinosinic acid, which interfere with binding of some scavenger receptors to bacteria, blocked PIR-B binding to S. aureus, whereas the polycationic polycytidylic acid did not. The data suggest that PIR-B has scavenger receptor-like binding activity toward S. aureus.

We next addressed the PIR-B recognition site. PIR-B and PIR-A1, which share a higher degree of conservation across Ig-like domain D2 as compared with the other PIR-As (Figs. 2 and 4A), bind S. aureus efficiently (Fig. 3B), and the binding was abrogated by polyanionic reagents (Fig. 3D). We hypothesized that charged amino acids in D2 might be important for the binding. We therefore specifically mutated some of the unique amino acid residues in D2 of PIR-B to residues corresponding to the sequence of PIR-A2, and generated stable NIH3T3 cell lines for each resulting mutant. After confirmation of equivalent expression levels of each mutant (Fig. 4B), we examined their binding activities. In the first mutant, we replaced a short segment HNDHK by PSYDR based on the sequence information (Fig. 4A), and also reduced the positive charged amino acids from three to one. In addition, this segment locates on a surface loop in the three-dimensional structural model of PIR-B, which makes it possible to be involved in molecular recognition (Fig. 4C). The substitution, however, did not alter the recognition of S. aureus (Fig. 4D). We next generated mutants G119E, P210A, and the double mutant G119E/P210A. These two residues are close to each other and located near the linking area of the D1 and D2 domains, therefore they might help to stabilize the overall structure of PIR-B (Fig. 4C). Although each single substitution did not alter the binding to S. aureus, the double mutant did show a slight reduction in binding activity (Fig. 4D). Finally, we tested a construct containing all three substitutions (PSYDR-G119E, P210A-PIR-B/NIH3T3) and found that the binding activity was reduced to a level equivalent to that of PIR-A2 (Fig. 4D). Overall, these mutants provided evidence in support of our hypothesis that the domain D2 of PIR-B is important for the recognition of S. aureus.


Figure 4
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  		FIGURE 4. PIR-B recognizes S. aureus through the Ig-like domain D2. A, Amino acid sequences of Ig-like domain D2 from the different PIRs were aligned with ClustalW. Arrows indicate PIR-B residues that were mutated to those of PIR-A2. B, Indicated cells were stained with PE-labeled rat IgG1 (thin histogram) or PE-labeled anti-PIR mAb 6C1 (thick histogram), then analyzed by flow cytometry. C, Three-dimensional structure of Ig-like domain D1 and D2 of PIR-B was built using the SWISS-MODEL program. The color of the protein main-chain was gradually changed along the sequence from blue (N-terminal) to red (C-terminal). The arrows indicate the positions of residues that were mutated, and the side-chain atoms of these residues are shown (G119 does not have a side-chain). D, Indicated cells were cultured with fluorescently labeled S. aureus (10 bacteria/cell) for 30 min at 37°C, then analyzed by flow cytometry. Data are represented as mean ± SD of triplicates. Data are representative of two to three independent experiments.

 
PIR-B-deficient macrophages show impaired binding of S. aureus

We next examined whether PIR-B is involved in the binding of S. aureus by macrophages. As shown in Fig. 5, Pirb–/– bone marrow-derived macrophages showed significantly impaired recognition of S. aureus, suggesting that although macrophages express several scavenger receptors such as SR-AI, SR-AII, macrophage receptor with collagenous structure, and CD36, all of which bind S. aureus (4), PIR-B also plays a role in the recognition. TLR2 senses S. aureus and triggers inflammatory responses, but there was no significant difference in S. aureus binding between wild-type (WT) and Tlr2–/– macrophages (Fig. 5), consistent with our previous results (11, 12). It has been reported that macrophages express both PIR-A and PIR-B (26, 33), which we verified through the use of anti-PIR mAb 6C1, which recognizes both PIR-A and PIR-B. We found that the 6C1 staining level was reduced >6-fold on Pirb–/– macrophages but was still detectable, indicating that although PIR-B is expressed at a much higher level, PIR-A is also expressed on the surface of macrophages (Fig. 6, A and B). To examine the involvement of PIR-A in the binding of S. aureus to macrophages, we used 6C1 to simultaneously block binding to PIR-A and PIR-B (Fig. 3C). S. aureus binding to Pirb–/– macrophages was roughly equivalent to that seen in WT macrophages pretreated with 6C1 (Fig. 6C). The fact that pretreatment of the Pirb–/– cells with 6C1 did not result in a further suppression of bacterial binding suggests that PIR-A does not contribute to the recognition of S. aureus by macrophages (Fig. 6C).


Figure 5
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  		FIGURE 5. Involvement of PIR-B in the recognition of S. aureus by bone marrow-derived macrophages. WT, Pirb–/–, or Tlr2–/– bone marrow-derived macrophages were incubated with fluorescently labeled S. aureus (1 or 10 bacteria/cell) for 30 min at 4°C. Then these cells were analyzed by flow cytometry. Data are representative of three independent experiments.

 

Figure 6
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  		FIGURE 6. PIR-B, but not PIR-A, contributes to the recognition of S. aureus by bone marrow-derived macrophages. A, Expression of PIR on bone marrow-derived macrophages. WT, Pirb–/–, or Tlr2–/– bone marrow-derived macrophages were stained with PE-rat IgG1 (thin histogram) or PE-anti-PIR mAb 6C1 (thick histogram), then analyzed by flow cytometry. B, Each mean fluorescence intensity (MFI) is represented. C, WT or Pirb–/– bone marrow macrophages were preincubated with 5 µg/ml control rat IgG1 (rIgG1) or 6C1 for 30 min at 4°C, and then were incubated with fluorescently labeled S. aureus (1 or 10 bacteria/cell) for 30 min at 4°C. Cells were then analyzed by flow cytometry. Data are representative of three independent experiments.

 
PIR-B suppresses TLR-mediated inflammatory response to S. aureus

It has been reported that PIR-B inhibits activation signals by B cell Ag receptor and chemokine receptors such as CCR1 and CXCR2 (22, 24). Thus, we next examined whether PIR-B negatively regulates the macrophage inflammatory response to S. aureus. As shown in Fig. 7, A and B, Pirb–/– bone marrow-derived macrophages produced ~2-fold higher levels of the proinflammatory cytokines such as IL-6 and TNF-{alpha} in response to S. aureus. In contrast, levels of the anti-inflammatory cytokine IL-10 were suppressed to a similar degree in Pirb–/– macrophages (Fig. 7C).


Figure 7
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  		FIGURE 7. PIR-B-deficient macrophages show enhanced inflammatory responses to S. aureus. WT, Tlr2–/–, or Pirb–/– bone marrow macrophages were incubated with the indicated dose of S. aureus, PAM3CSK4 lipopeptide, or LPS for 12 h at 37°C. Induction of IL-6 (A), TNF-{alpha} (B), and IL-10 (C) was measured by ELISA. Data are represented as mean ± SD of triplicates. Data are representative of three independent experiments. n.d., Not detected.

 
Because it has been reported that PIR binds various MHC class I molecules (25), we considered whether PIR-B exerts a general suppression on TLR signaling in macrophages through its constitutive interaction with MHC class I. However, when we treated WT and Pirb–/– macrophages with pure TLR ligands such as PAM3CSK4 lipopeptides (TLR2 ligand) or LPS (TLR4 ligand), we observed no differences in pro- or anti-inflammatory cytokine production (Fig. 7). Also, it has been demonstrated that mAb 6C1 does not block the interaction of PIR-B with MHC class I (25), whereas we have observed that the Ab blocks the interaction of PIR-B with S. aureus (Figs. 1D and 3C). As shown in Fig. 8, A and B, IL-6 production was enhanced and IL-10 production was suppressed in 6C1-pretreated WT macrophages, which is consistent with the results from Pirb–/– macrophages. These data demonstrate that the interaction between S. aureus and PIR directly suppresses proinflammatory activation of macrophages.


Figure 8
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  		FIGURE 8. PIR-B suppresses TLR-mediated inflammatory responses. A and B, WT, Tlr2–/–, Pirb–/–, or Myd88–/– bone marrow macrophages were pretreated with 5 µg/ml control rat IgG1 or 6C1 for 1 h at 4°C, and then cultured with or without S. aureus (10 bacteria/cell) for 12 h at 37°C. Induction of IL-6 (A) and IL-10 (B) was measured by ELISA. C, WT or Pirb–/– macrophages were cultured with S. aureus (10 bacteria/cell) and the indicated dose of recombinant mouse IL-10 for 12 h at 37°C. Induction of IL-6 was measured by ELISA. D, WT or Pirb–/– macrophages were cultured with or without S. aureus (10 bacteria/cell) in the presence of 5 µg/ml control rat IgG1 or anti-IL-10-neutralizing mAb JES-2A5 for 12 h at 37°C, and induction of IL-6 was measured by ELISA. Data are represented as mean ± SD of triplicates. Data are representative of three independent experiments. n.d., Not detected.

 
TLR2-deficient bone marrow-derived macrophages showed impaired IL-6 production (Fig. 7A), indicating that S. aureus-induced IL-6 production is mainly TLR2-dependent, as described previously (11, 12). We next addressed whether the enhancement of IL-6 production by PIR-B deficiency is also TLR2-dependent. As shown in Fig. 8A, 6C1 mAb pretreatment enhanced IL-6 production in WT, but not Tlr2–/– or Myd88–/– macrophages, suggesting that PIR-B negatively regulates TLR2-mediated inflammatory responses. In contrast, Tlr2–/– macrophages produced an equivalent amount of IL-10 as WT macrophages in response to S. aureus (Fig. 7C), and 6C1 suppressed IL-10 production to the same extent in both WT and Tlr2–/– macrophages (Fig. 8B). In Myd88–/– macrophages, IL-10 production was neither induced by S. aureus nor was it affected by 6C1 treatment (Fig. 8B). These results suggest that S. aureus-induced IL-10 production is TLR2 independent, but is probably dependent on some other TLR, and is enhanced by PIR-B signaling.

Because IL-10 suppresses several proinflammatory cytokines in macrophages (35), we next asked whether enhancement of proinflammatory cytokines is caused by IL-10 suppression in Pirb–/– macrophages. Addition of exogenous IL-10 suppressed IL-6 production from Pirb–/– macrophages stimulated with S. aureus in a dose-dependent manner, and from WT macrophages to a much lesser extent (Fig. 8C). Furthermore, anti-IL-10-neutralizing mAb JES-2A5 enhanced IL-6 production from WT macrophages stimulated with S. aureus to the equivalent level of that from similarly treated Pirb–/– macrophages (Fig. 8D). Taken together, these results suggest that the S. aureus-induced enhancement of inflammatory responses observed in Pirb–/– macrophages is largely due to suppression of IL-10 production.

PIRs discriminate between bacteria

We have demonstrated that PIR-B is a novel receptor for S. aureus. We next examined whether PIR-B recognizes other types of bacteria. Although PIR-B/NIH3T3 cells bind S. aureus, they did not efficiently bind other Gram-positive bacteria such as B. subtilis or L. monocytogenese. PIR-B did, however, recognize the Gram-negative bacteria H. pylori and E. coli, but not P. aeruginosa. PIR-A2h1 could not bind any of these bacteria efficiently (Fig. 9A). We also examined IL-6 production from Pirb–/– macrophages in response to these Gram-negative bacteria. As shown in Fig. 9B, Pirb–/– macrophages produced enhanced IL-6 in response to H. pylori, but not P. aeruginosa, suggesting that PIR-B discriminates between bacteria.


Figure 9
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  		FIGURE 9. PIR family members discriminate between bacteria. A, Parental NIH3T3 cells and NIH3T3 cells stably expressing PIR were incubated with the indicated fluorescently labeled bacteria (10 bacteria/cell) for 30 min at 37°C. The percentage of recognition (% recognition) was quantified by flow cytometry. B, WT or Pirb–/– bone marrow-derived macrophages were cultured with the indicated bacteria (5 or 10 bacteria/cell) for 12 h at 37°C. Induction of IL-6 was measured by ELISA. C, Parental NIH3T3 cells and NIH3T3 cells stably expressing ILT2 or ILT5 were incubated with the indicated fluorescently labeled bacteria (10 bacteria/cell) for 30 min at 37°C. The recognition was quantified by flow cytometry. Data are represented as mean ± SD of triplicates. Data are representative of two to three independent experiments.

 
Human ILTs recognize bacteria

Based on their similarities in structure, expression profiles, and genomic localization, murine PIRs are considered to be the orthologs of human ILT receptors (also called LILRs, or LIRs, and monocyte/macrophage Ig-related receptors (22, 36, 37). Therefore, we asked whether any ILTs recognize a similar array of bacteria as PIR-B. We generated NIH3T3 cells stably expressing ILT2 or ILT5, each of which contain ITIMs like PIR-B, and tested the ability of these cells to recognize various bacteria. As shown in Fig. 9C, both ILT2/NIH3T3 and ILT5/NIH3T3 cells bind S. aureus. It is of interest that PIR-B/NIH3T3 cells bind H. pylori, whereas ILT2/NIH3T3 and ILT5/NIH3T3 cells do not. ILT2/NIH3T3 cells also bind E. coli, whereas ILT5/NIH3T3 cells do not. These data suggest that, in humans, PIR family members may also play an important role in innate immune response to bacterial infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Paired inhibitory and activating receptors, including FcRs and NK receptors, tightly regulate immune responses (18, 19). In this study, we identify PIR-B as a novel macrophage receptor for S. aureus, demonstrating for the first time that the members of the PIR family act as pattern-recognition receptors for bacteria. Furthermore, we have shown that S. aureus recognition by PIR-B, in conjunction with TLR, directs anti-inflammatory responses to the bacteria.

The inhibitory PIR-B protein is encoded by single gene, whereas the activating PIR-A proteins are encoded by multiple genes. All of the ectodomains, each containing six Ig-like domains, are >92% identical at the amino acid level (20, 21). Therefore, it had been thought that both PIR-B and PIR-A recognize the same ligands. Indeed, both receptors have been recently reported to bind various mouse MHC class I molecules (25). In this study, we show that PIR-B and PIR-A1, but not other forms of PIR-A used in this study, bind S. aureus efficiently. Furthermore, mutagenesis analysis of PIR-B showed that the Ig-like domain D2 is important for the recognition. Anti-PIR mAb 6C1 inhibits the binding of PIR to S. aureus, but not to MHC class I (25), suggesting that the respective binding sites are different. Although the bacterial ligand remains to be determined, the binding was abrogated by negatively charged reagents, which interfere with binding some scavenger receptors to bacteria, suggesting that the PIRs have scavenger receptor-like binding activity toward S. aureus.

Although macrophages express scavenger receptors such as SR-AI, SR-AII, and MARCO, which recognize both Gram-positive and Gram-negative bacteria (4), these scavenger receptors have only a short cytoplasmic tail and are thought to contribute mainly to microbial binding (3); it remains unknown whether scavenger receptors per se transmit intracellular signals and regulate macrophage transcriptional responses. The fact that PIR-B has ITIMs in the cytoplasmic region (20, 21) suggests that PIR-B does transmit an inhibitory signal in macrophages upon microbial recognition. We demonstrate in this study that upon binding to S. aureus, PIR-B suppresses the TLR-mediated inflammatory response via the anti-inflammatory cytokine IL-10. Overexpression of full-length PIR-B, but not an ITIM-truncated mutant in RAW264.7 cells (macrophage-like cell line), enhanced IL-10 production in response to S. aureus (our unpublished data), highlighting the importance of the ITIMs for the immunosuppressive effect. We have not been able to demonstrate a direct interaction between PIR-B and TLR2, suggesting that the immunosuppressive effect is a consequence of independent activation of these receptors by S. aureus. It is well known that different types of bacteria elicit different types of inflammatory responses, although the mechanisms are poorly understood. For example, S. aureus infection produces lower amounts of inflammatory cytokines than Gram-negative bacteria infection (16, 17). This mild response may be perfectly effective for the clearance in the vast majority of cases where a person/mouse is exposed to "normally encountered" S. aureus. Alternatively, S. aureus might have evolved to bind to the inhibitory PIR-B receptor to suppress TLR activation in macrophages, thereby blunting the innate immune response. We are currently investigating the in vivo role of PIR-B in host defense to infection by S. aureus.

In addition to PIR-B, we found that the activating receptor PIR-A1 recognizes S. aureus. The physiological role of PIR-A in innate immunity remains unknown. However, the fact that PIR-A associates with FcR{gamma}, which has ITAMs (22), coupled with our finding that the PIR-A/FcR{gamma} complex enhanced TLR2-mediated signaling in HEK293 cells (our unpublished data), suggest that PIR-A1 positively regulates TLR-mediated inflammatory responses. Given that PIR-B may regulate inflammatory responses to the bacteria for host defense, multiple PIR-As might have evolved from a single ancestral PIR-B to further buffer/control this response. It is noteworthy that, although PIR-A1 has only a short cytoplasmic region, three ITIM-like motifs are preserved as relics in the 3'-untranslated region, suggesting that the inhibitory receptor was the original (our unpublished observation). Recently, Arase et al. have reported that NK-activating Ly49H and inhibitory Ly49I receptors directly recognize mouse CMV-encoded MHC-like protein m157 (38), and they have proposed that viruses might provide the evolutionary pressure to cause diversification of NK cell receptors (39). Likewise, we support the hypothesis that bacteria provide evolutionary pressure for PIR diversification.

It is noteworthy that S. aureus infection can cause septic arthritis, which gives rise to a pronounced polyclonal B cell response with elevated serum Ig levels (40, 41). By stimulating murine PIR or human ILT, S. aureus might cause unbalanced activation of these receptor-expressing immune cells, resulting in an increased risk for autoimmune diseases. ILTs are encoded by multiple genes with many polymorphisms (22, 36, 37), some of which may affect individual susceptibility to bacteria infection. Interestingly, several recent reports suggest that ILT polymorphisms are associated with some autoimmune diseases including rheumatoid arthritis (42, 43, 44). Thus, alteration of immune homeostasis by disruption of one member of an ILT pair may contribute to an increased risk for autoimmune disease.

In conclusion, we have shown in this study that PIRs recognize bacteria and that activation of these receptors influences innate inflammatory responses triggered by TLRs. The novel function thus ascribed to these surface receptors opens the door to new therapeutic approaches to combat infections and autoimmune diseases.


   Acknowledgments
 
We thank Dr. S. Akira for Tlr2–/– mice and Myd88–/– mice; Dr. C. Lowell for Pirb–/– mouse bones; Dr. G. Nolan for Phoenix Ampho cells; Drs. N. Salama, D. Speert, and D. Portnoy for bacteria strains; Dr. T. Saitoh for advice for generation of the cDNA library; Aderem laboratroy members for helpful discussions; and Drs. M. Brunkow, E. Miao, and C. Rosenberger for critical reading of the manuscript.


   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 This work was supported by the National Institutes of Health. M.N. was supported by a postdoctoral fellowship from Uehara Memorial Foundation. Back

2 Current address: Cytopeia Incorporated, 12730 28th Avenue Northeast, Seattle, WA 98125. Back

3 Address correspondence and reprint requests to Dr. Alan Aderem, Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103. E-mail address: aderem{at}systemsbiology.org Back

4 Abbreviations used in this paper: PIR, paired Ig-like receptor; DC, dendritic cell; ILT, Ig-like transcript; LILR, leukocyte Ig-like receptor; TRITC, tetramethylrhodamine isothiocyanate; WT, wild type. Back

Received for publication December 7, 2006. Accepted for publication January 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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