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Canine idiopathic polyarthritis (IPA) is characterized by increased numbers of polymorphonuclear leukocytes (PMNs) in the synovial fluid (SF). In humans, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 8 (CXCL8) recruit monocytes and neutrophils, respectively, and are involved in various inflammatory disorders. The aim of this study was to assess the roles of these chemokines in driving PMNs infiltration into the joints of dogs with IPA. SF samples were collected from dogs with IPA (n = 19) and healthy controls (n = 8), and the concentrations of SF CCL2 and CXCL8 were determined by ELISA. Dogs with IPA had significantly higher concentrations of CCL2 (3316 ± 2452 pg/ml, mean ± SD) and CXCL8 (3668 ± 3879 pg/ml) compared with the healthy controls (235 ± 45 pg/ml and <15.6 pg/ml, respectively). Then, an in vitro chemotaxis assay was performed using a modified Boyden chamber (pore size: 3 μm). SF from IPA dogs had a chemoattractant activity for PMNs that purified from the peripheral blood of a healthy dog. We subsequently found that combination treatment with MK-0812 (an antagonist of CCL2 receptor) and repertaxin (an antagonist of CXCL8 receptors) significantly inhibited the migration of PMNs to SF from IPA dogs. Thus, expression of the CCL2 receptor (chemokine (CC motif) receptor 2 (CCR2)) was examined using polymerase chain reaction and immunocytochemistry. Canine peripheral blood PMNs exhibited significantly higher CCR2 mRNA expression levels than those in monocytes. In addition, we observed strong CCR2 expression on PMNs obtained from healthy controls and IPA dogs, although mononuclear cells did not express CCR2. Taken together, the data suggest that CCL2 acts as a canine PMNs chemotactic factor as well as CXCL8 and both CCL2 and CXCL8 facilitate the infiltration of PMNs into the joints of dogs with IPA. [ABSTRACT FROM AUTHOR]

Abstract: Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca2+ concentration was measured using the Ca2+-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P <0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P <0.01), thymus and activation regulated chemokine (TARC/CCL17, P <0.01), and interleukin 8 (IL-8/CXCL8, P <0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P <0.05), GM-CSF (P <0.05), TARC/CCL17 (P <0.05), and IL-8/CXCL8 (P <0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca2+ concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis. [Copyright &y& Elsevier]

Abstract: Keratinocytes produce inflammatory mediators that are involved in the pathogenesis of skin disorders such as atopic dermatitis (AD). In particular, the CC chemokines, thymus and activation regulated chemokine (TARC)/CCL17 and mucosae-associated epithelial chemokine (MEC)/CCL28 are considered to play an important role in the lesional infiltration of lymphocytes in canine AD. However, there have been no reports on the regulatory mechanisms of CCL17 and CCL28 transcription in canine keratinocytes. In this study, we investigated whether CCL17 and CCL28 transcription in cultured keratinocytes is induced by TNF-α, IL-1β, or IFN-γ. It was found that CCL17 mRNA transcription is augmented by TNF-α only, whereas the CCL28 mRNA level could be increased by TNF-α, IL-1β, or IFN-γ. The present study suggests that pro-inflammatory cytokines are important inducing factors for the production of CCL17 and CCL28 in the lesional skin of dogs with AD. [Copyright &y& Elsevier]