Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

猪圆环病毒2型感染猪皮肤源树突状细胞炎性反应能力的变化

用PCV2 B1株经鼻腔接种40日龄SPF仔猪,于接种后3、7、14 d宰杀,收集皮肤源树突状细胞(DC).利用实时荧光定量PCR技术对感染仔猪皮肤源DC的IL-10、TNF-α、IFN-α、IL-8、趋化因子受体1(CCR1)、CCR5在mRNA转录水平的变化进行定量分析.结果表明,IFN-α在接种后3 d(3DPI)显著下调(P＜0.05),TNF-α、IL-10在7DPI时显著上调(P＜0.05);趋化因子IL-8在3、7、14 DPI时均下调,差异接近显著;MCP-1在感染后3、14DPI下调,7DPI均上调,但不显著;MIP-1β在3、7DPI明显上调,14DPI恢复正常;趋化因子受体CCR1、CCR5在3、7和14DPI均上调,且7DPI显著上调(P＜0.05).以上结果表明PCV2在感染早期可抑制DC炎性反应的能力,免疫应答失调,影响了动物机体的细胞和体液免疫功能的发挥.     

Production of a monoclonal antibody to canine thymus and activation-regulated chemokine (TARC) and detection of TARC in lesional skin from dogs with atopic dermatitis

A monoclonal antibody to canine thymus and activation-regulated chemokine (TARC/CCL17) was developed to examine the association of TARC with the immunopathogenesis of canine atopic dermatitis (AD). Recombinant canine TARC was prepared using an E. coli expression system. Results of transwell chemotaxis assay demonstrated that the recombinant canine TARC showed chemotactic activity for canine lymphoid cells expressing CC chemokine receptor 4 (CCR4). Mice were then immunized with the recombinant canine TARC to obtain monoclonal antibodies. Among the monoclonal antibodies thereby obtained, one monoclonal antibody (CTA-1) was found to react with both recombinant and authentic canine TARC in ELISA and flowcytometric assays, respectively. Immunohistochemical analysis using the monoclonal antibody CTA-1 demonstrated that keratinocytes were major TARC producing cells in lesional skin of dogs with AD.     

Role of beta1 integrins in adhesion of canine mastocytoma cells to extracellular matrix proteins

The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with alpha and beta subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin beta1 and alpha1 through alpha5 subunits and that they moderately expressed the alpha6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-beta1 and alpha5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-beta1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-alpha integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.     

Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

Monocyte subsets have been shown to differ in the pattern of chemokine receptor expression and their migratory properties, both in human and mouse. Previously we have characterized in the swine several monocyte subpopulations, based on the expression of CD163, Tük4 and SLA-II, which share features with the populations described in human and mouse. Here, we have analysed the expression of different chemokine receptors in the CD163⁻Tük4⁺SLA-II⁻ and CD163⁺Tük4⁻SLA-II⁺ populations of porcine monocytes. CD163⁺Tük4⁻SLA-II⁺ monocytes expressed higher CX₃CR1 but lower CCR2 and CXCR4 mRNA levels than CD163⁻Tük4⁺SLA-II⁻ monocytes. Moreover, porcine CCL2 binding on Tük4⁺SLA-II⁻ but not on Tük4⁻SLA-II⁺ cells was detected by using a CCL2-green fluorescence protein (pCCL2-GFP) fusion protein. Finally, flow cytometric analyses of monocytes recovered after chemotaxis assays show a clear increase in the proportion of Tük4⁺SLA-II⁻ cells in the fraction migrating toward CCL2, consistent with the polarized CCR2 expression in this monocyte population. The pattern of expression of these chemokine receptors reinforces the similarities of these porcine subsets with their human and mouse counterparts.     

Expression analysis of CCL27 and CCL28 mRNA in lesional and non-lesional skin of dogs with atopic dermatitis

Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD, patterns of chemokine production in dogs may also be similar with those in humans. The aim of this study was to examine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrate similarity of chemokine production with human counterparts. RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4, CCL19, CCL20, CCL21, CCL24, CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. Seven types of chemokines examined were constitutively expressed in both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different between lesional and non-lesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-lesional skin. On the other hand, CCL28 mRNA expression in lesional skin was found to be higher than that in non-lesional skin. These results suggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD, indicating that experimental canine study may provide additional information that can be extrapolated to human AD.     

Diazepam ameliorated myocardial ischemia-reperfusion injury via inhibition of C-C chemokine receptor type 2/tumor necrosis factor-alpha/interleukins and Bcl-2-associated X protein/caspase-3 pathways in experimental rats

Myocardial ischemia-reperfusion injury (IRI) is one of the most leading concerns for public health globally. Diazepam, a local anesthetic, has been reported for its cardioprotective potential. The present investigation aimed to evaluate the possible mechanism of action of diazepam against left anterior descending ligation-induced myocardial IRI in experimental rats. IRI was induced in healthy male rats by ligating coronary artery for 30 min and then reperfused for 60 min. The animals were pre-treated with either vehicle or diltiazem (10 mg/kg) or diazepam (1, 2.5, and 5 mg/kg) for 14 days. Compared to the IRI group, diazepam (2.5 and 5 mg/kg) markedly (P<0.05) attenuated IRI-induced alterations in cardiac function and oxido-nitrosative stress. In addition, diazepam prominently (P<0.05) improved cardiac Na⁺K⁺ATPase, Ca²⁺ATPase levels and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA expression. It also significantly (P<0.05) down-regulated cardiac mRNA expressions of cardiac troponin I (cTn-I), C-C chemokine receptor type 2 (CCR2), tumor necrosis factor-alpha (TNF-α), interleukins (IL)-1β, and IL-6. In western blot analysis, IRI-induced myocardial apoptosis was reduced by diazepam treatment reflected by a marked (P<0.05) decreased in Bcl-2-associated X protein (Bax) and Caspase-3 protein expression. Diazepam also efficiently (P<0.05) improved IRI-induced histological aberration in cardiac tissue. In conclusion, diazepam exerts cardioprotective effect by inhibiting inflammatory release (CCR2, TNF-α, and ILs), oxido-nitrosative stress, and apoptosis (Bax and Caspase-3) pathway during myocardial IRI in experimental rats.     

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18⁺ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18⁺ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.     

The porcine skin associated T-cell homing chemokine CCL27: molecular cloning and mRNA expression in piglets infected experimentally with Staphylococcus hyicus

CCL27 (also named CTACK, ALP, ILC and ESkine) is a CC chemokine primarily expressed by keratinocytes of the skin. The cognate receptor of CCL27 named CCR10 (GPR-2), is also expressed in skin-derived cells, and in addition by a subset of peripheral blood T-cells and in a variety of other tissues. In this paper, we report the cloning of porcine CCL27 cDNA and investigation of CCL27 mRNA expression in Staphylococcus hyicus infected piglets. At the protein level, 77 and 74% homology was found to human and mouse CCL27 sequences, respectively. The results of the expression analyses show that CCL27 mRNA is upregulated in the skin of infected piglets and to a lesser extent in piglets recovered from disease and without clinical signs of infection, indicating a role for CCL27 both during inflammation and after recovery from an infection.     

Isolation and characterization of canine tumor endothelial cells

The present study involved the isolation and characterization of canine tumor endothelial cells (TECs) from 2 malignancies. TECs were isolated using magnetic cell sorting following FITC labeling with UEA1 lectin, and they were characterized by measuring genetic and histopathological endothelial markers. Isolated TECs exhibited a cobblestone-like morphology and expressed both vascular endothelial growth factor receptor 2 (VEGFR2) and Von Willebrand factor (vWF). Further, both TECs and tumor cells derived from a seminoma exhibited increased C-X-C chemokine receptor type 7 (CXCR7) expression. However, CXCR7 expression was not detected in TECs and tumor cells derived from a hepatocellular carcinoma. Understanding TEC specific traits may be important in the development of more efficacious anti-angiogenic therapies that do not induce adverse effects.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.