Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes.

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50-70%.

Chicken immunoregulatory Ig-like receptor families: an overview and expression details on ggTREM-A1.

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.

Characterization of the chicken CD200 receptor family.

The modulation of myeloid cells via inhibitory and activating immunoglobulin superfamily members has been a subject of intense study in mammals. One such example is the inhibitory receptor for CD200, which is shown to regulate the activation threshold of myeloid cells by interaction with the broadly distributed CD200 molecule. By looking at sequence homology and synteny conservation in the chicken genome, we identified two members of the CD200 receptor family in chicken on chromosome one. Cloning and further characterization of the protein sequence yielded a potentially inhibitory ggCD200R-B1 with a splice variant lacking a transmembrane region and a potentially soluble ggCD200R-S1. Both showed a typical V/C2-set Ig domain arrangement and we present evidence that these two genes have evolved by gene duplication. The inhibitory receptor displayed an uncharged transmembrane region and a long cytoplasmic tail encoding four tyrosine residues, one of them embedded in a motif similar to the mammalian NPxY motif. Further characterization of ggCD200R-B1 showed that it is expressed as a highly glycosylated protein and that its cytoplasmic tyrosine residues can be phosphorylated. Real-time RT-PCR analysis of various tissues and primary cells showed that ggCD200R-B1 is predominantly expressed in macrophages, whereas ggCD200R-S1 is highly expressed in peripheral blood mononuclear cells, but not macrophages. In summary, we showed that there is a homologue of mammalian CD200R conserved in chicken suggesting a similar function in avian species. Furthermore, the presence of potentially soluble CD200R molecules implies an important role for these in the regulation of myeloid cells in chicken.

The chicken leukocyte receptor complex encodes a primordial, activating, high-affinity IgY Fc receptor.

Fc receptors are key players of the immune system that link the fine specificity of immunoglobulins and innate effector responses. Here, we describe a nonmammalian Fcgamma receptor, CHIR-AB1, a member of the leukocyte receptor complex, that binds IgY with high affinity with its single Ig domain. It is expressed on immature and mature B lymphocytes, monocytes, macrophages, and natural killer cells and harbors motifs of activating and inhibitory Fc receptors. In the absence of FcepsilonRIgamma, CHIR-AB1 can be expressed on B cells but cross-linking does not induce intracellular calcium release. In contrast, cells expressing CHIR-AB1 and FcepsilonRIgamma are triggered to release intracellular calcium upon stimulation with heat-aggregated IgY. CHIR-AB1 thus represents a primordial Fc receptor that combines features of different mammalian counterparts.