Talitha de Ruiter

The Soluble Leukocyte-Associated Ig-Like Receptor (LAIR)-2 Antagonizes the Collagen/LAIR-1 Inhibitory Immune Interaction

Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human PBMC, mirroring the expression profile of LAIR-1. LAIR-2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline-dependent manner. In vitro stimulation of PBMC induces secretion of LAIR-2. We detect high amounts of LAIR-2 in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR-2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared with osteoarthritis patients. We hypothesize that soluble LAIR-2 may function as a natural competitor for LAIR-1, thereby regulating its inhibitory potential. Indeed, LAIR-2 prevents binding of human LAIR-1 to collagens and LAIR-1 cross-linking in vitro, suggesting that the protein has an immunoregulatory function in vivo. Hence, we reveal a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane-bound LAIR-1 via competition for ligands.

Lack of CD200 enhances pathological T cell responses during influenza infection

Influenza virus infection can be accompanied by life-threatening immune pathology most likely due to excessive antiviral responses. Inhibitory immune receptors may restrain such overactive immune responses. To study the role of the inhibitory immune receptor CD200R and its ligand CD200 during influenza infection, we challenged wild-type and CD200−/− mice with influenza virus. We found that CD200−/− mice in comparison to wild-type controls when inoculated with influenza virus developed more severe disease, associated with increased lung infiltration and lung endothelium damage. CD200−/− mice did develop adequate adaptive immune responses and were able to control viral load, suggesting that the severe disease was caused by a lack of control of the immune response. Interestingly, development of disease was completely prevented by depletion of T cells before infection, despite dramatically increased viral load, indicating that T cells are essential for the development of disease symptoms. Our data show that lack of CD200-CD200R signaling increases immune pathology during influenza infection, which can be reduced by T cell depletion.

The Mouse Homologue of the Leukocyte-Associated Ig-Like Receptor-1 Is an Inhibitory Receptor That Recruits Src Homology Region 2-Containing Protein Tyrosine Phosphatase (SHP)-2, but Not SHP-1

We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that 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 leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.

Leukocyte‐associated Ig‐like receptor‐1 has SH2 domain‐containing phosphatase‐independent function and recruits C‐terminal Src kinase

Most inhibitory receptors in the immune system contain one or several immunoreceptor tyrosine‐based inhibitory motifs (ITIM) and recruit the SH2 domain‐containing phosphatases SHP‐1, SHP‐2 and/or SHIP, which are generally believed to be essential for the inhibitory function. However, it has not been systematically investigated whether ITIM‐bearing receptors exert their function through alternative interactions. Here we describe that leukocyte‐associated Ig‐like receptor (LAIR)‐1 has inhibitory function in DT40 chicken B cells that lack both SHP‐1 and SHP‐2. In addition, we found that LAIR‐1 did not recruit SHIP upon phosphorylation. Thus, LAIR‐1 can function independently from SH2 domain‐containing phosphatases and must recruit at least one other signaling molecule. Using a yeast‐tri‐hybrid system, we found that phosphorylated LAIR‐1 bound the C‐terminal Src kinase (Csk). The interaction required the SH2 domain of Csk and phosphorylation of the tyrosine in the N‐terminal ITIM of LAIR‐1. We propose that Csk is an additional player in the regulation of the immune system by ITIM‐bearing receptors.

Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III.

Immune responses are tightly controlled by the opposing actions of activating and inhibitory immune receptors. Previously we identified collagens as ligands for the inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1), revealing a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. This interaction can be blocked by LAIR-2, a secreted member of the LAIR-1 family. LAIR-1 specifically interacts with synthetic trimeric peptides containing 10 repeats of glycine-proline-hydroxyproline (GPO) residues which can directly inhibit immune cell activation in vitro. Here we studied the interaction of human LAIR-1 and LAIR-2 with collagen in more detail by using novel overlapping synthetic trimeric peptides (Toolkits) encompassing the entire triple-helical domain of human collagens II and III. LAIR-1 and LAIR-2 bind several of these collagen-like peptides, with LAIR-2 being able to bind more than LAIR-1. LAIR binding to trimeric collagen peptides was influenced by GPO content of the peptide, although additional non-GPO triplets contributed to the interaction. Furthermore, we identified several trimeric peptides that were potent LAIR-1 ligands and could efficiently induce inhibition of T cell activation and FceRI-induced degranulation of RBL-2H3 cells through binding to LAIR-1. A detailed understanding of the LAIR recognition motifs within collagen may lead to the development of potent reagents that can be used in in vitro, ex vivo, and in vivo functional studies to dissect the biology and function of the collagen/LAIR-1 interaction.

Differential expression of leukocyte‐associated Ig‐like receptor‐1 during neutrophil differentiation and activation

Inhibitory receptors containing immunoreceptor tyrosine‐based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte‐associated immunoglobulin‐like receptor‐1 (LAIR‐1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR‐1 was expressed on peripheral blood eosinophils, cell‐surface expression on mature neutrophils was low, suggesting that LAIR‐1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL‐60 expressed LAIR‐1, but the expression decreased during chemical‐induced differentiation toward neutrophils. Similarly, in bone marrow‐derived neutrophil precursors, the most immature cells expressed LAIR‐1, and loss of LAIR‐1 expression was associated with neutrophil maturation. LAIR‐1 was re‐expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor α, granulocyte macrophage‐colony stimulating factor, or N‐formyl‐methionyl‐leucyl‐phenylalanine, indicating that LAIR‐1 may also regulate neutrophil effector function. Our studies suggest that LAIR‐1 may play a regulatory role in differentiation and function of human granulocytes.

Regulated expression of the inhibitory receptor LAIR‐1 on human peripheral T cells during T cell activation and differentiation

The leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1) is capable of inhibiting immune cell function through interaction with collagens. LAIR is expressed on the majority of peripheral blood mononuclear cells. The abundant expression of both receptor and ligand calls for regulatory mechanisms to relieve the continuous interaction between collagens and LAIR‐1. This regulation may occur at the expression level of the receptor. Here, we report that LAIR‐1 is indeed differentially expressed during human T cell differentiation. Naive CD4+ and CD8+ T cells as well as CD8+ T cells of the effector phenotype express higher levels of LAIR‐1 compared to memory T cells. In vitro stimulation revealed a decrease in LAIR‐1 expression upon activation, and the lower LAIR‐1 expression on CD127– T cells suggests that activation‐induced down‐modulation of LAIR‐1 may also occur in vivo. Furthermore, crosslinking of LAIR‐1 on primary T cells results in an inhibition of T cell function. Our data suggest that regulated expression of LAIR‐1 and the subsequent change in the threshold for activation may be a mechanism to modulate inhibition of the immune system.

Enhanced Secretion of Leukocyte-Associated Immunoglobulin-like Receptor 2 (LAIR-2) and Soluble LAIR-1 in Rheumatoid Arthritis LAIR-2 Is a More Efficient Antagonist of the LAIR-1-Collagen Inhibitory Interaction Than Is Soluble LAIR-1

OBJECTIVE Human leukocyte-associated immunoglobulin-like receptor 1 (hLAIR-1) is an immune inhibitory receptor for collagen that is expressed on most immune cells. We previously showed that the LAIR-1-collagen interaction could be antagonized by the secreted homolog hLAIR-2, which can be detected in the synovial fluid of rheumatoid arthritis (RA) patients. In addition, the extracellular part of hLAIR-1 is a putative antagonist upon shedding from the cell membrane. The purpose of this study was to determine the relative roles of hLAIR-2 and soluble hLAIR-1 (shLAIR-1) in the regulation of the LAIR-1-collagen interaction. METHODS The ability of recombinant LAIR proteins to abrogate LAIR-1-collagen binding was tested by flow cytometry and adhesion assays. Collagen binding capacity was analyzed by surface plasmon resonance. Plasma, urine, and synovial fluid were screened for the presence of sLAIR-1 and LAIR-2 by enzyme-linked immunosorbent assay. RESULTS Recombinant LAIR-2 proteins abrogated the binding of collagen to LAIR-1 more efficiently than did recombinant sLAIR-1. Consistent with these findings, surface plasmon resonance analysis showed that LAIR-2 had a higher affinity for collagen than did LAIR-1. Activated CD4+ T cells were the main producers of LAIR-2, whereas the source of sLAIR-1 remains elusive. Both soluble LAIR-1 and LAIR-2 could be detected in the plasma and urine of healthy control subjects and patients with RA. Urinary levels of both proteins were significantly increased in RA patients, and LAIR-2 levels in urine were significantly correlated with markers of inflammation. CONCLUSION Our data suggest that LAIR-2 is a more potent antagonist of LAIR-1 function in vivo, while both sLAIR-1 and LAIR-2 are potential biomarkers that may be used to monitor urine samples for evidence of systemic inflammation.

Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1.

Many tumor types over-express collagens, what correlates with enhanced metastatic capacity and unfavorable clinical outcome. This is generally explained by the importance of collagens in creating a microenvironment that supports tumor cell survival and enhances cell migration. Importantly, collagens act as ligands for the inhibitory receptor LAIR-1, which inhibits the function of multiple types of immune cells. Here we propose a new role for tumor expressed collagens and show that these structural proteins can be exploited by tumor cells to inhibit immune responses through an interaction with LAIR-1. We show that both LAIR-1-Fc fusion proteins and LAIR-1 expressing cells bind to transmembrane collagens expressed by tumor cells. Interference with collagen expression by specific knock-down of prolyl 4-hydroxylase diminishes LAIR-1 binding to tumor cells, demonstrating the specificity of the interaction. Consistently, both transmembrane collagens and extracellular collagens produced by multiple tumor cell types can activate LAIR-1. Furthermore, overexpression of collagen XVII on target cells results in diminished NK cell cytotoxic activity. Thus tumor-expressed collagens can bind and trigger immune inhibitory signaling via LAIR-1, suggesting that collagens indeed may affect tumor immune evasion.

Identification and characterization of the rat homologue of LAIR-1

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a cell-surface molecule that functions as an inhibitory receptor on various immune cells in both humans and mice. We have cloned a LAIR-1 homologue from the rat that we have named rat LAIR-1. The LAIR-1 gene maps to rat chromosome 1q12 in a region showing conserved synteny with human chromosome 19q13.4 and mouse chromosome 7, where the leukocyte receptor cluster is located. Rat LAIR-1 shows 40 and 71% protein sequence identity with human LAIR-1 and mouse LAIR-1, respectively, has a single Ig-like domain and contains two immunoreceptor tyrosine-based inhibitory motif-like sequences in its cytoplasmic tail. Soluble rat LAIR-1 fusion proteins bind to the same adherent cell lines as human LAIR-1 and mouse LAIR-1, indicating that a putative ligand for all the LAIR-1 molecules is expressed on these cells. Furthermore, we show that rat and mouse LAIR-1 bind the same molecule expressed on human HT29 cells. Since many autoimmune diseases are studied in rat models, identification of rat LAIR-1 allows for in vivo studies on the function of LAIR molecules in these systems.