ABSTRACT
CD45 is one of the most abundant molecules expressed on the white blood cell surface in various mammals. In this study, we investigated the differential expression of CD45 isoforms in normal canine white blood cells. It has been shown that all canine nucleated blood cells express CD45. We characterized two major isoforms of canine CD45 derived from alternative splicing: a higher molecular weight isoform, CD45RA, and a lower molecular weight isoform, CD45RO. The nucleotide sequences of the two isoforms were identical, except for the region corresponding to a part in the extracellular domain. Flow cytometry analysis using an antibody that recognizes CD45RA, but not CD45RO, revealed that granulocytes did not express CD45RA, and monocytes express low levels of CD45RA. We further analyzed the expression levels of CD45RA in each lymphocyte subpopulation and found that the expression of CD45RA on CD21+ B cells was uniform. On the other hand, expression of CD45RA on CD3+ T cells was variable. Upon stimulation of lymphocytes with Con A, the CD45RA+ fraction increased, indicating that not only the phenotypes but also the activation status influences the isoform expression pattern of CD45. Our finding provides a basic knowledge of the expression of canine CD45, which could be a tool to study lymphocytes with various phenotypes, developmental stages, and activation status.
Subject(s)
Dogs/metabolism , Gene Expression Regulation/immunology , Leukocyte Common Antigens/metabolism , Leukocytes/metabolism , Animals , Leukocyte Common Antigens/genetics , Protein IsoformsABSTRACT
Fractalkine, also known as CX(3)CL1, is a unique chemokine that mediates inflammatory responses and is involved in the pathogenesis of several inflammatory disorders, including inflammatory bowel disease (IBD) in humans. In this study, we isolated cDNAs encoding canine fractalkine and its receptor CX(3)CR1, and assessed the biological activity of these molecules. The deduced amino acid sequence of the canine fractalkine cDNA showed 66% and 57% identity to human and mouse homologs, respectively. The N-terminal chemokine domain of the canine fractalkine showed 68% and 65% identity to human and mouse counterparts, respectively. The canine CX(3)CR1 amino acid sequence showed close homology to its human (83% identity) and mouse (81% identity) counterparts. Fractalkine and CX(3)CR1 mRNA were detected in all tissues in this study. Relatively higher expression levels of fractalkine mRNA were observed in the brain, medulla spinalis, small intestine, and mesenteric lymph nodes (MLNs), whereas higher expression levels of CX(3)CR1 mRNA were observed in the medulla spinalis, brain, liver, small intestine, and MLNs. The cross-reactivities of anti-human fractalkine antibody and anti-rat CX(3)CR1 antibody to canine proteins were confirmed using recombinant canine fractalkine and a cell line overexpressing canine CX(3)CR1, respectively. A transwell chemotaxis assay showed that the recombinant canine fractalkine induced migration in canine lymphoid cells expressing CX(3)CR1. The present study will be useful in understanding the canine immune system and the immunopathogenesis of canine inflammatory diseases.
