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Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
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Abstract |
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Introduction |
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The leukocyte-associated Ig-like receptor 1 (LAIR-1) is a member of the Ig superfamily (IgSF) that is expressed on the majority of PBMCs, including NK cells, T cells, B cells, monocytes, and dendritic cells, as well as the majority of thymocytes (3). Cross-linking of LAIR-1 by mAb in vitro delivers a potent inhibitory signal that is capable of inhibiting cellular functions of NK cells, effector T cells, B cells, and dendritic cell precursors (3, 4, 5, 6). In agreement with the observed inhibitory capacity of LAIR-1, the molecule bears two ITIMs in its cytoplasmic tail and selectively recruits the tyrosine phosphatases SHP-1 (3, 7, 8, 9, 10) and SHP-2 (3, 8, 10) upon activation.
LAIR-1 is structurally related to several other inhibitory IgSF members, including human killer cell Ig-like receptors (KIRs), human Fc
R, human leukocyte Ig-like receptors (LILRs; also known as Ig-like transcripts, leukocyte Ig-like receptors, monocyte-macrophage inhibitory receptors, and CD85), mouse gp91 or paired Ig-like receptors (PIRs) and mouse gp49 (3). Interestingly, LAIRs, KIRs, LILRs, and FcR are all localized to the leukocyte-receptor cluster (LRC) on human chromosome 19q13.4, suggesting that these molecules have evolved from a common ancestral gene (reviewed in Refs. 11 and 12). It is generally believed that the LRC is only partially conserved between humans and mice, as illustrated by the absence of some family members in rodents, such as the KIRs and FcR. As homologues of the LRC-encoded LAIR genes have not yet been identified, they were long believed not to be present in mice.
In this study we identified the first LAIR family member in mice, which we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the LRC is located. The protein is broadly expressed on various immune cells and is capable of inhibiting immune responses. The mLAIR-1 cytoplasmic tail can become phosphorylated, thereby recruiting SH2-containing tyrosine phosphatase-2 (SHP-2). Interestingly, unlike human LAIR-1, SHP-1 is not recruited to the mLAIR-1 cytoplasmic tail. Soluble human (h) LAIR-1 and mLAIR-1 fusion proteins bind to both human and mouse cell lines, indicating that they might bind the same ligand(s) on these cells. Identification of a mouse homologue of LAIR-1 allows for in vivo studies of the function of LAIR molecules in the regulation of different immune responses.
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Materials and Methods |
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Cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured using standard techniques. Mouse B cell lines IIA1.6 (BALB/cAnN), Sp2/0 (BALB/c), and NS-1 (BALB/c); mouse T cell lines D011.10 (BALB/c) and EL4 (C57BL/6); mouse melanoma B16 (C57BL/6); mouse sarcoma CMS7 (BALB/c); mouse fibroblasts: L929 (C3H/An) and 3T6 (Swiss albino), mouse brain-derived endothelioma bEND3; human colon carcinomas HT29 and LS174; human embryonic kidney 293T cells; Jurkat human T cells; and rat basophilic leukemia cell line RBL-2H3 were used (13). The human NK-like tumor cell line YT.2C2 was provided by Dr. K. Smith (Cornell University, Ithaca, NY) (14). YT.2C2 stably transfected with hLAIR-1 and the EBV-transformed human B cell line 721.221 stably transfected with human Fc
RIIa (CD32) (15) were generated at the DNAX Research Institute (Palo Alto, CA) and have been described previously (3, 5). The Armenian hamster fibroblast line ARHO12 was provided by Dr. J. Hamann (Academic Medical Center, Amsterdam, The Netherlands).
Bone marrow-derived dendritic cells were obtained as described by Inaba et al. (16). Briefly, bone marrow was flushed from mouse femurs (BALB/c), erythrocytes were lysed, and cells were grown at 1 x 106/ml RPMI 1640 medium supplemented with 10% FBS, 50 IU/ml penicillin, and 50 µg/ml streptomycin in the presence of 10 ng/ml GM-CSF (Immunex, Seattle, WA). Nonadherent cells were replated on day 1, and nonadherent cells were removed on days 2 and 4 from the cultures, with concomitant refreshment of culture medium. Nonadherent and loosely adherent DC were harvested on day 7.
Antibodies
The DX26 IgG1 mAb directed against hLAIR-1 has been described previously (3). The 8A8 (IgG1)-producing hybridoma was generated by fusing the SP2/0 myeloma cell line with splenocytes from a BALB/c mouse immunized with purified hLAIR-1 protein. Rat and mouse IgG isotype controls and PE-conjugated streptavidin were purchased from BD Biosciences (San Diego, CA). Rat anti-mouse CD16/CD32 (Mouse Fc Block; BD PharMingen, San Diego, CA). Anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal rabbit anti-SHP-1 (C19) and anti-SHP-2 (C18) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotin-conjugated, goat anti-human IgG1 mAbs were obtained from Caltag Laboratories (Burlingame, CA). Biotin-conjugated, goat anti-rabbit Abs were purchased from Vector Laboratories (Burlingame, CA). Goat anti-mouse F(ab)2 were purchased from Southern Biotechnology Associates (Birmingham, AL). For Western blot analysis, HRP-conjugated rabbit anti-mouse Ab (DAKO, Glostrup, Denmark) and HRP-conjugated goat anti-rabbit Ab (Pierce, Rockford, IL) were used. Monoclonal IgE anti-2,4,6-trinitrophenyl (TNP) was provided by Prof. Dr. L. Aarden (Sanquin Research, Amsterdam, The Netherlands). For mouse fusion protein FACS stainings, cells were pretreated with 10% BSA, 20% FCS, 10% normal mouse serum, and 20 µg/ml anti-mouse CD32/16 (FcR) mAbs to block mouse FcRs.
Generation of mLAIR-1 polyclonal antibodies
The extracellular domain of mLAIR-1 was fused to a six-histidine tag in a pET21a expression vector (Novagen, Madison, WI) and subsequently overexpressed in Escherichia coli BL21(DE3) cells. The histidine-tagged protein was purified by Ni2+-chelate affinity chromatography in the presence of 8 M urea according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Antisera were produced in two rabbits using a standard immunization protocol (Eurogentec, Seraing, Belgium).
cDNA constructs and transfectants
cDNA encoding hLAIR-1a was cloned into the pcDNA3.1/zeo+ vector (Invitrogen, Breda, The Netherlands) and the pMX puro retroviral vector. Chimeric hLAIR-1/mLAIR-1 proteins were constructed in the same vectors by fusing the extracellular part of hLAIR-1 (hLAIR-1a; aa. 1–160) to the transmembrane region and cytoplasmic domains of mLAIR-1 (mLAIR-1a; aa. 140–263) by means of a linker sequence encoding the amino acids leucine and glutamic acid. The chimeric protein allows detection and triggering using anti-hLAIR-1 antibodies. Myc-tagged mLAIR-1 was constructed by fusing a Myc epitope (EQKLISEEDL) to the C-terminal part of mLAIR-1 and subsequently cloning in the pcDNA3.1/zeo+ vector. The DNA sequences were confirmed by automated DNA sequencing. To generate stable transfectants expressing either hLAIR-1 or hLAIR-1/mLAIR-1 chimeric proteins, RBL-2H3 cells were transfected by electroporation. Stable transfectants were selected in 50 µg/ml zeocin (Invitrogen) and subsequently cloned by the limiting dilution method. Stable YT.2C2 transfectants were generated by retroviral transfection as previously described (5). Stable ARHO12 transfectants of Myc-tagged mLAIR-1 were generated by the FuGENE 6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions and subsequent cloning by the limiting dilution method in presence of 500 µg/ml zeocin (Invitrogen). Expression levels of the various LAIR-expressing clones were assessed by standard flow cytometry methods.
RT-PCR
Total RNA was isolated from several mouse cell lines and mouse (C57BL/6) organs using the RNeasy method (Qiagen). Total RNA was converted to first-strand cDNA with oligo(dT)18 primers and murine leukemia virus reverse transcriptase using the GeneAmp RNA PCR kit (PE Applied Biosystems, Foster City, CA). The cDNA mixtures were amplified by PCR using mLAIR-1-specific forward (5'-GCTCTGACCAGACCTGGTAAGG-3') and reverse (5'-CCATGTGTGTCTCCAGGTGTGC-3') primers and the AmpliTaq Gold DNA polymerase system (PE Applied Biosystems). These primers correspond to the 5'- and 3'-untranslated regions adjacent to the mLAIR-1-coding region. Each amplification reaction underwent 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s at 64°C, and elongation at 72°C for 50 s. As a control, GAPDH transcripts were amplified using GAPDH-specific primers (5'-ATCAACGACCCCTTCAT-3' and 5'-CACACCCATCACAAACAT-3'). Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
The mLAIR-1 isoforms present in the bone marrow RT-PCR sample were cloned into pGEM-T Easy vectors using the pGEM-T Easy vector system (Promega, Madison, WI) and subsequently sequenced on an ABI 3100 sequencer (PE Applied Biosystems) using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems). The sequences obtained were analyzed by Lasergene software (DNASTAR, London, U.K.)
Northern blot analysis
Total RNA from several mouse cell lines and organs was separated on a 1.6% formaldehyde agarose gel and blotted onto a 
-Probe GT blotting membrane (Bio-Rad, Hercules, CA) by capillary transfer in 10x SSC as described by Sambrook et al. (17). Radiolabeled DNA probes were generated using the RadPrime DNA Labeling System (Invitrogen) of a DNA probe of the intracellular domain of mLAIR-1 and subsequently used for hybridization under stringent conditions using the ExpressHyb Hybridization Solution (Clontech Laboratories, Palo Alto, CA), according to the manufacturer’s prescriptions. After washing the blots, they were exposed to phosphor screens and analyzed on a STORM PhosphorImager (Molecular Dynamics, Wokingham, U.K.).
Degranulation assay
The degranulation assay of RBL-2H3 clones has previously been described (10). Measurements were performed using triplicate cultures. The percentage of inhibition of degranulation by hLAIR-1- and chimeric hLAIR-1/mLAIR-1-transfected RBL-2H3 clones was calculated as: percent inhibition = 100 x [(OD405 without LAIR-1 cross-linking – OD405 with LAIR-1 cross-linking)/(OD405 without LAIR-1 cross-linking – OD405 spontaneous release)].
Cytotoxicity assay
721.221 target cells stably expressing FcRIIa were labeled with 51Cr and used in a 4-h cytotoxicity assay using transfected YT.2C2 cells as effector cells as described previously (18). To engage hLAIR-1 and the hLAIR-1/mLAIR-1 chimeric molecules, 10 µg/ml 8A8 anti-LAIR Ab was added. The maximum release was determined by lysing target cells with 5% Triton X-100. The percentage of specific lysis was calculated as: [(cpm specific 51Cr release – cpm spontaneous 51Cr release)/(cpm maximum 51Cr release – cpm spontaneous 51Cr release)] x 100. Data are expressed as the mean of triplicate cultures.
Tyrosine phosphorylation and phosphatase recruitment
RBL-2H3 cells stably transfected with hLAIR-1 and the hLAIR-1/mLAIR-1 chimeric proteins were treated with pervanadate and subsequently subjected to immunoprecipitation using DX26 anti-human LAIR-1 Ab as previously described (10).
Detection of mLAIR-1 and hLAIR-1 ligand(s)
Chimeric proteins composed of the leader sequence and the extracellular parts of mLAIR-1 (aa 1–139) or hLAIR-1 (aa 1–162) fused to the Fc region of human IgG1 were inserted into the pcDNA3.1/zeo+ vector (Invitrogen). The proteins, designated mLAIR-1-hIg and hLAIR-1-hIg, respectively, were produced by stable expression in 293T cells and subsequently purified by affinity chromatography on protein A-Sepharose columns (Amersham, Freiburg, Germany). Cell lines were screened for the presence of putative mLAIR-1 and hLAIR-1 ligand(s) by assaying for binding of the fusion proteins. Approximately 2.5 x 105 cells were incubated at room temperature for 30 min with 20 µl of PBS containing 
1 µg of mLAIR-1-hIg or hLAIR-1-hIg, 5% normal mouse serum, 5% BSA, 10% FCS, and 20 µg/ml Mouse Fc Block. After washing, 10 µg/ml (15 µl) biotin-conjugated goat anti-human-IgG1 was added for 30 min at room temperature, followed by washing and 30-min incubation with 10 µg/ml PE-conjugated streptavidin. Cells were assayed on a FACSCalibur with the addition of propidium iodide to exclude dead cells. As isotype controls, 1 µg of hIgG1 or irrelevant hIgG1 fusion protein was used. For fusion protein blocking studies, hLAIR-1-hIgs or mLAIR-1-hIgs were incubated for 30 min at room temperature with anti hLAIR-1 Abs (8A8) or polyclonal anti mLAIR-1 Abs, respectively, before the above-described procedure. As a control, mLAIR-1-hIgs were incubated with preimmune serum from the same rabbits that served to generate polyclonal Abs against mLAIR-1.
Computer-assisted analysis
Identification of mLAIR-1 was achieved by homology search on the Celera mouse genome database (www.celera.com). Comparison of the human and mouse LRC was performed by comparing both regions on the genomic databases of Celera and the National Center of Biotechnology Information (www.ncbi.nih.gov/Genomes/). The protein sequence alignment was generated by the Clustal method, using Lasergene analysis software (DNASTAR, Madison, WI).
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Results |
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To identify a mouse homologue of human LAIR-1, the Celera mouse genome database was searched with a BLASTN algorithm for sequences bearing homology with the human LAIR-1 cDNA sequence. As a result, a mouse LAIR locus was identified on chromosome 7, the syntenic chromosome of human chromosome 19q13.4 where both LAIR-1 and LAIR-2 loci are located. Specific primers were generated to PCR-amplify putative LAIR homologue transcripts using C57BL/6J mouse bone marrow-derived cDNA as template, resulting in amplification of at least six different LAIR transcripts (see Fig. 2A). One transcript contained the full-length open reading frame of 792 nt with the first ATG start codon contained in a consensus Kozak sequence. The deduced polypeptide conformed to a type I transmembrane protein composed of 263 aa, including a 21-aa signal peptide, a 121-aa extracellular domain, a hydrophobic transmembrane segment of 22 aa, and a 99-aa cytoplasmic tail (Fig. 1, A and B). The putative hLAIR-1 homologue had a predicted relative molecular mass of 29.8 kDa and two potential sites for N-linked glycosylation at positions N34 and N90, indicated by a circle in Fig. 1A.
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Aligning the protein sequence of hLAIR-1a to its mouse homologue (Fig. 1A) showed that the molecules are moderately conserved, with overall sequence identity and homology of 40 and 50%, respectively. In particular, the cytoplasmic domains of both LAIR molecules were more conserved than their extracellular domains. The N-terminal ITIM sequences were highly similar between both receptors; only a single amino acid substitution was observed, whereas the C-terminal ITIM sequences were less alike. Based on the sequence identity between hLAIR-1 and the newly identified mouse LAIR molecule, the protein was named mLAIR-1.
The mLAIR-1 gene maps to a LRC-like region on chromosome 7
The mLAIR-1 gene spans an area of 53.6 kb of genomic sequence (Fig. 1B) and closely resembles the human LAIR-1 gene. Although hLAIR-1 contains 10 exons, and its mouse homologue only eight (mLAIR-1 misses human exons 4 and 5), the shared exons are highly similar in size, have almost identical intron-exon boundaries (data not shown), and encode similar regions of the protein (Fig. 1B).
The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the LRC is located (Fig. 1C). Based on sequence similarity, the region located near the mLAIR-1 gene resembles that of the hLRC, indicating that it is conserved through evolution. Genes homologous to the Ig-like protein platelet gp-VI, lymphocyte Ag 94 (or natural cytotoxicity-triggering receptor 1), and LILRs appear to be conserved between the species. Furthermore, several non-Ig molecules, such as the LRC-encoded novel genes, ribosomal protein S9, Tweety homologue 1, and binder of Rho GTPase 3, seem conserved. Significantly, the mouse LRC lacks genes encoding LAIR-2, KIRs, and FcR. Furthermore, there appear to be fewer PIR genes present compared with genes encoding its human homologues (LILRs). Only mLRC contained putative genes encoding proteins with homology to a protease, 40S ribosomal protein and MO-25. MO25 (CAB39) is a gene transcribed during early mouse development encoding a putative Ca2+ binding protein (19).
Mouse LAIR-1 is expressed in hemopoietic cells
To asses the cellular distribution of mLAIR-1, RT-PCR analysis of various mouse tissues (C57BL/6) and cell lines using mLAIR-1-specific primers was performed (Fig. 2A). Transcripts were detected in lymphoid organs (bone marrow, spleen, lymph nodes, and thymus), but not in nonlymphoid organs such as muscle and skin. Furthermore, cell lines of hemopoietic origin of various mouse strains contained mLAIR-1 transcripts (IIA1.6, Sp2/0, NS-1, D011.10, and EL4), whereas nonhemopoietic cell lines (CT26, B16, CMS7, L929, and 3T6) did not.
Several mLAIR-1 transcripts were detected by RT-PCR (Fig. 2A). Cloning, sequencing, and subsequent sequence alignment indicated that the different transcripts were all transcribed from the same gene and were characteristic of alternative RNA splicing (data not shown). The two major transcripts encoded the full-length mLAIR-1a and a splice variant missing the entire Ig-like domain, which is encoded by exon 3 (mLAIR-1b). Two minor forms, which we designated mLAIR-1d and mLAIR-1e, encoded isoforms missing exons 3 and 4 or exon 4 alone, respectively. Furthermore, two minor transcripts were detected similar to mLAIR-1a and mLAIR-1b, with a 115-nt intronic sequence adjacent to exon 2 resulting in a transcript encoding a nonsense protein. The DNA sequences of mLAIR-1a, -b, -d, and -e were deposited in the GenBank database under accession numbers AY392763, AY392764, AY392765, and AY392766, respectively.
Northern blot analysis of bone marrow-derived DCs (BALB/c) showed two predominant transcripts of 1.7 and 3.3 kb (Fig. 2B). Hybridization transcripts were not detected in other RNA samples (CMS7, Fig. 2B; EL4, SP2/0, NS-1, CT26, IIA1.6, and D011.10, data not shown) and only faintly in lymphoid tissues (spleen, bone marrow, and lymph nodes; data not shown), indicating that mLAIR-1 transcripts are relatively low abundant transcripts in immune-associated tissues other than DCs. Taken together, these data indicate that, like human LAIR-1, mLAIR-1 is exclusively expressed by cells of hemopoietic origin.
Mouse LAIR-1 is able to inhibit degranulation of RBL-2H3 cells and cytotoxic activity of NK cells
To investigate the potential inhibitory capacity of mLAIR-1, we generated a chimeric molecule composed of the extracellular domain of human LAIR-1 fused to the transmembrane and cytoplasmic domains of mLAIR-1. Stable transfectants of the rat basophilic leukemia cell line (RBL-2H3) and the human NK cell line YT.2C2 were generated using this chimeric protein and hLAIR-1 as a control. Cell surface immunofluorescence analysis (Fig. 3A) indicated that the transfected RBL cells expressed both proteins at comparable levels in two independent transfectants. We investigated whether these chimeric molecules could inhibit signaling mediated by the endogenous ITAM-bearing IgE receptor Fc
RI expressed on RBL cells in a 
-glucuronidase release assay (degranulation assay). Incubation of the transfectants with TNP-specific IgE and subsequent triggering with TNP-conjugated BSA led to degranulation of the cells and release of -glucuronidase. Simultaneous cross-linking of the stably transfected chimeric LAIR molecules or hLAIR-1 with anti-hLAIR-1 mAb (8A8) and anti-mIg led to an inhibition of IgE-induced -glucuronidase release for the LAIR molecules, whereas no effect was observed for nontransfected RBL cells (Fig. 3B). Furthermore, triggering of the chimeric protein by mAbs on transfectants of the human NK cell line YT.2C2 (Fig. 4A) inhibited its spontaneous cytotoxic activity toward 721.221 FcRIIa-bearing target cells (Fig. 4B). The inhibitory effect observed in the cytotoxicity assay was completely due to triggering of the chimeric LAIR molecules alone, as previous experiments showed that mutating both tyrosines of hLAIR-1 to phenylalanine abolished the inhibition of cytotoxicity of the NK cell line toward its target cells in an identical experimental set-up (10). Taken together, these data indicate that, like hLAIR-1, cross-linking of the hLAIR-1/mLAIR-1 chimeric protein results in inhibition of ITAM-dependent signals initiated via the FcRI complex on RBL-2H3 cells and of the cytotoxic activity of NK cells toward FcR-bearing target cells.
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The existence of two potential ITIM sequences within the cytoplasmic domain of mLAIR-1 (Fig. 1A) suggested that the generation of inhibitory signals in the degranulation and cytotoxicity assay (Figs. 3B and 4B) was manifested by the recruitment of SHP-1 and/or SHP-2. To determine whether the chimeric proteins were capable of binding protein tyrosine phosphatases, the previously described RBL-2H3 transfectants were stimulated with pervanadate (an inhibitor of protein tyrosine phosphatases inducing tyrosine phosphorylation) (20), lysed, and immunoprecipitated with anti-hLAIR-1 mAbs. Immunoprecipitates were subsequently analyzed by Western blotting using Abs specific for phosphorylated tyrosine residues, human LAIR-1, SHP-1, and SHP-2. As shown in Fig. 5A, the chimeric proteins became phosphorylated upon pervanadate treatment. Unexpectedly, unlike hLAIR-1, the chimeric protein did not recruit SHP-1 upon phosphorylation. Analogous to hLAIR-1, SHP-2 was recruited after pervanadate stimulation (Fig. 5B), albeit to a much lesser extent. These results suggest that the negative signal transduced via engagement of the mLAIR-1 molecule might be mediated through recruitment of SHP-2, whereas SHP-1 does not seem to play a role.
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To identify the natural ligand(s) for mLAIR-1, we constructed a fusion protein consisting of the extracellular domain of mLAIR-1 fused to the Fc portion of hIgG1 (mLAIR-1-hIg). The protein was used as a staining reagent to screen mouse cell lines for the presence of putative ligands. The mouse fibroblast cell lines 3T6 and L929 and the mouse brain-derived endothelial cell line bEND3 bound the protein, whereas several other cell lines did not (NS-1, IIA1.6, Sp2/0, D011.10, and EL4 cells; Fig. 6A and data not shown). To examine whether hLAIR-1 also binds ligand(s) on these cells, a hLAIR-1-hIg fusion protein was generated. This fusion protein stained the same cells, indicating that hLAIR-1 reacts with a ligand on mouse cells. This observation led us to expand the screening for putative ligands to human cell lines. Both mLAIR-1 and hLAIR-1 fusion proteins stained the same human cells (human embryonic kidney 293T and colon carcinoma cell lines HT29 and LS174), whereas the human Jurkat T cell line appeared not to express a ligand for these receptors. This could indicate that both LAIR molecules recognize the same ligand(s) on these cells, confirming that the proteins are true homologues. However, the receptors might also bind to different cell surface molecules expressed on these cells. Binding of hLAIR-1-hIg and mLAIR-1-hIg to all cell lines was abolished by prior incubation with anti hLAIR-1 Abs (8A8) or polyclonal anti-mLAIR-1 Abs, respectively, demonstrating the specificity of these interactions (Fig. 6B). The mLAIR-1-hIg binding could not be abolished by prior incubation of the human LAIR-1 recognizing Ab (8A8; data not shown). Interestingly, hLAIR-1-hIg binding was not abolished by prior incubation of another anti-hLAIR-1 Ab (DX26; Fig. 6B), indicating that 8A8 Abs recognize an hLAIR-1 epitope that is involved in ligand binding, whereas DX26 Abs do not. Specific binding of the polyclonal antiserum to mLAIR-1 was confirmed using flow cytometry by staining ARHO12 cells stably transfected with or without mLAIR-1 (Fig. 6C).
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Discussion |
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The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region similar to the hLRC (Fig. 1C). Like hLAIR-1, the mLAIR-1 molecule is structurally related to several other inhibitory IgSF members located on the same region of the chromosome, suggesting that these molecules could have evolved from a common ancestral gene. Comparison of the hLRC to its syntenic region in mice reveals remarkable similarities as well as some striking differences. Besides encoding many members of the IgSF, both regions appear to show a considerable degree of genetic polymorphism and are characterized by extensive gene duplications (12, 23). Furthermore, the orientation of several homologous genes in the human LRC indicate that this region arose as a result of a large inverse duplication (23); this same pattern is observed in the mouse. The mouse LRC, however, is smaller in size than the human variant (530 vs 900 kb, respectively) and appears to encode fewer IgSF members. A direct homologue for the hFcR (CD89), for instance, is absent in the mLRC. Furthermore, whereas humans encode 13 KIR family members (24), mice completely lack KIR genes on mouse chromosome 7. Recently, however, a KIR-like locus was identified outside the LRC on mouse chromosome X, suggesting that the KIR family did evolve from a primordial gene already present in the common rodent/primate ancestor (25, 26). The mouse PIR gene family, comprised of both activating and inhibitory receptors with six Ig domains, is the closest in sequence homology and gene structure to the human LRC-encoded LILRs (reviewed in Ref. 27). Although both gene families are encoded in the LRC and share a moderate level of sequence identity, their relatedness was long a subject of debate, as the PIRs possess six Ig-like domains, whereas the LILRs contain only two or four. Recently, however, studies suggested that PIR-B interacts with the HLA class Ib molecule HLA-G (28), a molecule that also serves as a binding partner of several members of the LILR family (reviewed in Ref. 29).
In contrast to the hLAIR family, which consists of the transmembrane protein LAIR-1 and the putatively secreted protein LAIR-2, which shares 84% sequence homology to hLAIR-1 (3), the mouse genome appears to encode only the membrane-bound variant. Searching the mouse EST and genome databases did not retrieve a LAIR-2 homologue. It seems that the LAIR-1 gene appeared before the rodent/primate split in mammalian evolution, and that hLAIR-2 might originate as a result of a LAIR-1 gene duplication event in primates. The similar architecture and high sequence identity between the hLAIR-1 and hLAIR-2 genes and the absence of a mouse LAIR-2 gene supports this hypothesis. Although the human and mouse LRC show obvious similarities, it is apparent that the region is highly dynamic and that its members have probably evolved by a series of duplications, deletions, and rearrangements from an ancient common gene.
Like hLAIR-1 (3), its mouse homologue is broadly expressed on immune cells. Interestingly, several different mLAIR-1 splice variants were detected by RT-PCR analysis, among these a high abundant splice variant lacking the entire Ig domain encoded by exon 3 (mLAIR-1b). To our knowledge, there are no other immune receptors with similar splice variants, it would therefore be interesting to determine whether this protein is also expressed on mouse immune cells and to explore what function it might have.
The presence of two ITIM-like structures in the mLAIR-1 cytoplasmic tail corresponds with the observed inhibition of immune responses by YT.2C2 and RBL cells. However, although the N-terminal sequence fits the consensus sequence for ITIMs, the C-terminal sequence does not completely. This raises the question of whether the C-terminal ITIM-like structure of mLAIR-1 can function as such. Studies using human KIR molecules have indicated that two intact ITIMs are required for SHP-1 recruitment (30, 31). However, whereas a KIR mutant containing only the C-terminal ITIM is no longer effective, a KIR mutant containing only the N-terminal ITIM still recruits SHP-2 (32) and has inhibitory capacity (30, 31). These studies are in agreement with our recent study (10) in which we showed that mutating either ITIM of hLAIR-1 resulted in loss of SHP-1 recruitment upon Ab triggering, whereas mutating the C-terminal ITIM still allowed SHP-2 recruitment. It appears that mutating any ITIM of inhibitory receptors bearing two ITIM sequences abolishes SHP-1 recruitment (10, 30, 31). This suggests that receptors bearing a single ITIM are not able to recruit SHP-1. C-type lectin inhibitory receptors such as LILR and lymphocyte Ag 49 (Ly49), however, often bear a single ITIM in their cytoplasmic tail and do recruit SHP-1 upon tyrosine phosphorylation (33, 34). The latter class of molecules, however, can be expressed as a disulfide-linked homodimer (35, 36), allowing one receptor to carry two identical ITIMs. The mast cell function-associated Ag is an exception to this; the receptor bears a single ITIM and can be expressed on the cell membrane as a dimer, but does not recruit SHP-1, whereas SHP-2 and SH2-containing inositol 5'-phosphatase are recruited to this molecule upon phosphorylation (reviewed in Ref. 37). As mLAIR-1 does not recruit SHP-1 upon pervanadate treatment, and the C-terminal ITIM-like sequence does not completely match the ITIM consensus, it is possible that this motif might not function as such. This would correspond with the hypothesis that two bona fide ITIMs are required for SHP-1 recruitment. However, mutational studies are required to determine whether this motif completely lacks inhibitory signaling capacities or if other, as yet unknown, factors play a role in the inhibitory capacities of mLAIR-1.
Studies determining the basis for specific binding of SHP-2 to tyrosine motifs have indicated that position +1 (relative to pY) of the ITIM contributes to specificity. The N-terminal SH2 domain of SHP-2 has a preference for I/V/T at this position (37, 38). The presence of an I at position pY +1 in the N-terminal ITIM of mLAIR-1 suggests that SHP-2 is recruited to this ITIM. Although hLAIR-1 contains an alanine at this position, this N-terminal ITIM proved responsible for SHP-2 recruitment (10).
In contrast to previous reports, hLAIR-1 does not interact with the epithelial cell adhesion molecule (Ep-CAM) as a ligand (38); therefore we used mLAIR-1-hIg and hLAIR-1-hIg fusion proteins to screen a panel of human and mouse cell lines for the presence of putative LAIR ligands. As both hLAIR-1-hIg and mLAIR-1-hIg fusion proteins bound to the same human and mouse cell lines, it is tempting to speculate that both receptors cross-react with the same ligand(s) expressed on these cells. However, both receptors could potentially bind to different cell surface molecules expressed on these cells. It is imperative for understanding LAIR biology to identify the molecules that bind these fusion proteins and whether ligation can stimulate the inhibitory function of both human and mouse LAIR-1.
The data presented in this report show that mLAIR-1 is a genuine homologue of hLAIR-1. Despite the moderate level of sequence identity, the genes encoding the receptors are both located in the LRC, their exons are very similar in size, and they have almost identical intron-exon boundaries. Furthermore, both proteins have similar expression patterns, share a potent inhibitory capacity, and potentially bind the same ligand(s) on both human and mouse cells. Although both receptors recruit SHP-2 upon phosphorylation of their cytoplasmic tails, their difference in SHP-1 recruitment is intriguing.
To conclude, identification of the mouse homologue of LAIR-1 could facilitate in vivo studies on the role of this receptor in the regulation of immune responses and broaden the general knowledge of the function of inhibitory receptors in immune surveillance.
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2 Address correspondence and reprint requests to Dr. Linde Meyaard, Department of Immunology, University Medical Center Utrecht, Room KC02.085.2, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail address: l.meyaard{at}lab.azu.nl
3 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; h, human; IgSF, Ig superfamily; KIR, killer cell Ig-like receptor; LAIR, leukocyte-associated Ig-like receptor; LILR, leukocyte Ig-like receptor; LRC, leukocyte-receptor cluster; m, mouse; PIR, paired Ig-like receptor; SH2, Src homology region 2; SHP, SH2-containing protein tyrosine phosphatase; TNP, 2,4,6-trinitrophenyl.
Received for publication September 26, 2003. Accepted for publication February 20, 2004.
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) and noncoding sequences (
). The protein structure is subdivided into the leader peptide (LP), the extracellular domain (EC), the transmembrane domain (TM), and cytoplasmic domains (CP). The two cysteines forming the putative disulfide bonds (C-C) in the extracellular domain are shown. Putative ITIM sequences are indicated by asterisks. Introns 1, 2, and 3 of the mLAIR-1 gene are shown at 15% of their relative size. C, Schematic organization of the human LRC on chromosome 19q13.4 (top panel) compared with its syntenic region on mouse chromosome 7 (bottom panel). The genes include platelet gpVI (GP-VI), lymphocyte Ag 94 (Ly-94 or natural cytotoxicity triggering receptor 1 (NCR-1)), Fc
) of 10 µg/ml 8A8 anti-LAIR-1 antibody. Data are representative of four independent experiments.
