38 research outputs found
The Turkey Ig-like receptor family: identification, expression and function.
No full textThe chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes
Das Hühner CLEC-2 Homolog
No full textDas Hühner CLEC-2 Homolog: Ein Thrombozytenrezeptor mit aktivierender Funktion
Der Natürliche Killer Gen Komplex (NKC) des Huhnes ist auf dem Chromosom 1 lokalisiert und weist zwei C-typ Lektine auf, von denen das eine als Homolog zu CD69 und das andere zu CD94 und NKG2 beschrieben worden ist. Diese Studie trägt durch die Generierung eines spezifischen monoklonalen Antikörpers zur Charakterisierung des Hühner C-typ Lektin ähnlichen Proteins CD94/NKG2 als potentiellen NK Zellrezeptor bei. Durchflußzytometrische Messung von lymphatischem Gewebe des Huhnes ergab, dass das C-typ Lektin von einer Vielzahl von Zellen des PBMC, wenigen Milzzellen und keinerlei Bursa- oder Thymuszellen exprimiert wird. In PBMC von Huhn und Pute konnte der monoklonale Antikörper auf Thrombozyten nachgewiesen werden. Die biochemische Analyse konnte zeigen, dass das Molekül als ein glykosyliertes Homodimer auf der Zelloberfläche exprimiert wird und durch Kreuzvernetzung Thrombozyten aktiviert werden, was über CD107 Expression gemessen wurde. Die Sequenzanalyse deutete auf ein Motiv im Zytoplasma hin, welches als hem Immunrezeptor Tyrosin-basierendes aktivierendes Motiv bei C-typ Lekinen wie DECTIN1 und CLEC-2 vorkommt, aber nicht für CD94/NKG2 beschrieben ist. In der Sequenz konnte ein zusätzliches Cystein im extrazellulären Bereich gefunden werden. Zusammengenommen geben diese Ergebnisse Hinweise darauf, dass das Gen nicht einem CD94/NKG2 Hybrid ähnlich ist, sondern ein CLEC-2 Homolog darstellt.Chicken CLEC-2 homologue: a thrombocyte receptor with activation function
The chicken NKC was described to consist of only two C-type lectins, CD69 and a CD94/NKG2 hybrid, both encoded on chromosome 1. This study contributes to the characterisation of the chicken C-type lectin CD94/NKG2 by production of a specific monoclonal mab which was used to analyse the potential function on NK cells. Immunofluorescent analysis of lymphoid chicken tissues revealed in expression of the C-type lectin on a large percentage of PBMC, a small percentage of splenocytes and no reactivity in bursa and thymus. In chicken and turkey PBMC, the mab reacted with virtually all thrombocytes. The biochemical analyses demonstrated that CLEC-2 is presented on the cell surface as a highly glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. Sequence analysis indicated a cytoplasmic motif known as hem immunoreceptor tyrosine-based activation motif (hemITAM), which was found to be presented in C-type lectins like DECTIN1 and CLEC-2 but not CD94/NKG2. In addition the sequence exhibits a further extracellular cysteine residue. These findings indicate that the gene may not resemble CD94/NKG2 but rather CLEC-2 homologue
Characterization of the chicken C-type lectin-like receptors B-NK and B-lec suggests that the NK complex and the MHC share a common ancestral region.
No full textThe sequencing of the chicken MHC led to the identification of two open reading frames, designated B-NK and B-lec, that were predicted to encode C-type lectin domains. C-type lectin domains are not encoded in the MHC of any animal described to date; therefore, this observation was completely unexpected, particularly given that the chicken has a "minimal essential MHC." In this study, we describe the initial characterization of the B-NK and B-lec genes, and show that they share greatest homology with C-type lectin-like receptors encoded in the human NK complex (NKC), in particular NKR-P1 and lectin-like transcript 1 (LLT1), respectively. In common with NKR-P1 and LLT1, B-NK and B-lec are located next to each other and transcribed in opposite orientation. Like human NKR-P1, B-NK has a functional inhibitory signaling motif in the cytoplasmic tail and is expressed in NK cells. In contrast, B-lec contains an endocytosis motif in the cytoplasmic tail, and like LLT1, is an early activation Ag. Further analysis leads us to propose that there are four subgroups of C-type lectin-like receptors in the NKC, which arose as a result of duplication events. Moreover, this analysis suggests that the NKC may be considered a fifth paralogous region, and therefore shares an ancient common origin with the MHC. This provides evidence that C-type lectin-like receptors were present in the preduplication, primordial MHC region, and suggests that an original function of MHC molecules was for recognition by NK cell receptors encoded nearby
A striking example of convergent evolution observed for the ggFcR:IgY interaction closely resembling that of mammalian FcR:IgG
No full text
Double fluorescence analyses of turkey PBMC.
No full text<p>Cells were labeled with either CHIR-AB1 specific mab 8D12 (A) or CHIR-A2 specific mab 13E2 (B) in combination with the T-cell specific mab CD4 and CD8, the class II specific mab 2G11 and the αVβ3 specific mab 23C6. (C) Double staining of blood leukocytes with 8D12 and 13E2. Gates were set according to forward and sideward scatter features on the lymphocyte gate in A, B and C. (D) Combined immunfluorescence of 8D12 or 13E2 in combination with the MHC class II specific mab 2G11 as in (A) or (B), but gated on the larger monocytes based on forward/side scatter characteristics. Frequencies of positive cells are indicated in the quadrants. One representative of three experiments is shown.</p
Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes.
No full textTriggering 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%
Avian NK activities, cells and receptors.
No full textNatural killer (NK) activity has been examined in birds for over 30 years, but evidence that avian NK activity plays crucial roles in disease is only suggestive. In chickens, NK activity is mediated by TCR0 cells in the intestinal epithelium, but elsewhere subsets of alphabeta and gammadelta T cells (NKT cells) may be more important. There are few lectin-like NK receptor genes, located in the genomic region syntenic with the natural killer complex (NKC) as well as the major histocompatibility complex (MHC). In contrast, a huge number of Ig-like receptor genes are located in a region syntenic with the leukocyte receptor complex (LRC)
