Molecular cloning of canine IL-13 receptor alpha chain (alpha1 and alpha2) cDNAs and detection of corresponding mRNAs in canine tissues

This communication reports the cloning of cDNAs encoding two canine IL-13 receptor alpha chains (caIL-13Ralpha1 and caIL-13Ralpha2). As described for the members of type-I cytokine receptors, both caIL-13Ralpha1 and caIL-13Ralpha2 were found to contain the highly conserved motifs, such as cysteine and tryptophan residues in their N-terminal portion and the WSXWS at C-terminus. The isolated caIL-13Ralpha1 cDNA contains 1547 nucleotides with an open reading frame that encodes 405 amino acid residues. Canine IL-13Ralpha1 is 82.0 and 69.3% identical to human and mouse IL-13Ralpha1s, respectively, at the amino acid level. Canine IL-13Ralpha1 has an almost identical cytoplasmic domain to its human and mouse counterparts. The isolated caIL-13Ralpha2 cDNA contains 1454 nucleotides and encodes an open reading frame of 386 amino acid residues. Canine IL-13Ralpha2 is 62.6 and 47.5% identical to its human and mouse counterparts, respectively, at the amino acid level. Using RT-PCR with caIL-13Ralpha1 and caIL-13Ralpha2 specific primers, mRNAs of caIL-13Ralpha1 and caIL-13Ralpha2 were detected in most dog tissues. In addition, RT-PCR detected caIL-13Ralpha1 mRNA in one of two canine mastocytoma (C2 but not Br) cell lines and in a canine macrophage-derived cell line (DH82). CaIL-13Ralpha2 mRNA was detected in all three canine cell lines.     

Genomic characterization of equine interleukin-4 receptor alpha-chain (IL4R)

Three overlapping fragments of the equine interleukin-4 receptor alpha chain gene (IL4R) were cloned and sequenced. The resulting 3553 bp cDNA sequence exhibited homology to human, murine and bovine IL4R. The equine IL4R exhibits many conserved features when compared to other species, including intron-exon boundary positions and amino acid sequence motifs characteristic of type I cytokine receptors. The IL4R gene was localized to horse chromosome ECA13 by synteny mapping on a somatic cell hybrid panel. Evidence for an alternative splice variant of IL4R was found in the genomic sequence and subsequently verified using RT-PCR on equine monocyte RNA. A polymorphism screen of the largest exon, homologous to exon 12 of the human IL4R gene, was performed using DNA from 60 horses of various breeds which yielded 11 coding-region single nucleotide polymorphisms (SNPs), 7 synonymous and 4 non-synonymous. Three of the four non-synonymous SNPs occur at high frequencies and are found very near a conserved tyrosine residue.     

Production and characterization of monoclonal antibodies reactive with the chicken interleukin-15 receptor alpha chain

We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor.     

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.     

Expression of interleukin-18 receptor mRNA in the mouse endometrium

Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) alpha subunit was analyzed. IL-18Ralpha mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Ralpha riboprobe demonstrated that IL-18Ralpha mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Ralpha mRNA expression level changed with the estrous cycle. The uterine IL-18Ralpha mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Ralpha mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17beta or progesterone. These findings suggest that IL-18Ralpha gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.     

GFRα-1 is a reliable marker of bovine gonocytes/undifferentiated spermatogonia: A mini-review

Identification of the specific biomarkers is of great importance to enrich and expand the gonocytes or spermatogonial stem cells (SSCs) in livestock. The glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFRα-1) is a conserved marker of the gonocytes/SSCs in multiple species including rodents, primates and human; however, its expression in bovine gonocytes/SSCs is debated. Recently, we and other teams clearly demonstrated the expression of GFRα-1 in bovine gonocytes/SSCs. This is useful for bovine gonocytes/SSCs-related research or application. Nonetheless, new methods still need to be developed to identify the undifferentiated spermatogonial subsets in large livestock and elucidate their spermatogenic potency.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

Production and characterization of monoclonal bovine immunoglobulins G1, G2 and M from bovine × murine hybridomas

Three bovine × murine hybridomas, which secrete bovine IgG₁, IgG₂ and IgM, respectively, were derived by the fusion of normal bovine B lymphocytes to the murine cell line SP-2/0. The hybridomas were initially selected by testing the culture supernatant of individual clones with rabbit anti-bovine light chain antibody. The bovine nature of Ig secreted by the bovine × murine hybridomas was confirmed by the ability of bovine serum but not murine serum to inhibit specific adsorption by murine anti-bovine Ig coupled to Sepharose 4B and the inability of sheep anti-mouse Ig (all isotypes) to bind biosynthetically labelled Ig from the respective bovine × murine hybridomas or to react with bovine × murine hybridoma Ig in Ouchterlony analysis. The isotype of the bovine Ig produced by each hybridoma was determined by cRIA using bovine Ig isotype-specific murine mcab, Ouchterlony analysis with guinea pig anti-bovine Ig isotypes and molecular weight analysis of unreduced and reduced affinity purified Ig from the respective bovine × murine hybridomas. Even though the hybridomas in this study showed chromosome loss, they have continued to secrete bovine Ig in culture for over 16 months, indicating that it is possible to isolate and stabilize interspecific hybridomas retaining the chromosome, or at least the genes, encoding an Ig.These hybridomas will provide monoclonal bovine Ig for; 1) serological standards, 2) production of polyclonal and monoclonal antisera to bovine Ig isotypes, 3) sequencing studies, 4) serological and structural studies of bovine Ig classes and subclasses, and messenger RNA for the production of cDNA probes for the cloning of bovine Ig genes and for determining the organization of Ig genes in the bovine genome.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.     

V(D)J recombinational signal sequence DNA binding activities expressed by fetal bovine thymus

V(D)J recombination, or immunoglobulin gene rearrangement is a developmentally regulated, cell type specific, site directed recombination event that brings either immunoglobulin or T-cell receptor gene segments together to form mature, expressible Ig or TCT genes. This DNA recombination is directed by the recombinational signal sequences or RSS elements present adjacent to Ig and TCR gene segments. The RSS element is composed of a conserved nonamer element and a conserved heptamer element separated by a conserved length spacer region. In this report, we examine the expression of DNA binding proteins that interact with the RSS element in the bovine fetal thymus using EMSA assays. Our data indicates that the nonamer portion of the RSS element is the primary site of recognition for RSS binding proteins expressed in the bovine fetal thymus. We also show that these proteins are expressed from early stages of bovine fetal development through to full term development.     

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

ABSTRACT: The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.     

Bovine natural cell mediated cytotoxicity (NCMC): activation by cytokines

Incubation of bovine peripheral blood mononuclear leukocytes (PBML) with the cytokines (CK) IL-2, alpha-IFN, gamma-IFN or IL-4 resulted in significant increases in natural cell mediated cytotoxicity (NCMC) over endogenous levels, as determined in an 18 h 51Cr-release assay using the human K562 or mouse Yac-1 target cell lines. Endogenous cytotoxic activity of bovine natural effector cells (NEC) using K562 or Yac-1 target cells was minimal (killing less than 8%). After 18 h of incubation with the CK hurIL-2, alpha-bovrIFN, gamma-bovrIFN or hurIL-4, NEC had significant increases in cytotoxic activity for both K562 and Yac-1 target cells. Significant increases in cytotoxic activity were not found after incubation of NEC with IL-1 or beta-IFN. Specific killing varied with CK concentration in a dose dependent manner and was proportional to effector:target cell ratio. Activation of the bovine NEC by CK was rapid, occurring within 6-12 h of incubation with alpha-IFN or gamma-IFN and within 12-18 h of incubation with IL-2. Incubation of bovine PBML with IL-2 and alpha- or gamma-IFN or with alpha-IFN and gamma-IFN showed that these CK do not act in a synergistic manner to increase NCMC in the bovine NEC.     

Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.