The Inhibitory Effect of S-777469, a Cannabinoid Type 2 Receptor Agonist, on Skin Inflammation in Mice.

We investigated the effects of S-777469 (1-[[6-Ethyl-1-[4-fluorobenzyl]-5-methyl-2-oxo-1, 2-dihydropyridine-3-carbonyl]amino]-cyclohexanecarboxylic acid), a novel cannabinoid type 2 receptor (CB2) agonist, on 1-fluoro-2,4-dinitrobenzene (DNFB)-induced ear inflammation and mite antigen-induced dermatitis in mice. The oral administration of S-777469 significantly suppressed DNFB-induced ear swelling in a dose-dependent manner. In addition, S-777469 significantly alleviated mite antigen-induced atopic dermatitis-like skin lesions in NC/Nga mice. A histological analysis revealed that S-777469 significantly reduced the epidermal thickness and the number of mast cells infiltrating skin lesions. We demonstrated that S-777469 inhibited mite antigen-induced eosinophil accumulation in skin lesions and an endogenous CB2 ligand, 2-arachidonoylglycerol (2-AG)-induced eosinophil migration in vitro. Moreover, we confirmed that 2-AG levels significantly increased in skin lesions of mite antigen-induced dermatitis model. Together, these results suggest that S-777469 inhibits skin inflammation in mice by blocking the activities of 2-AG.

S-777469, a novel cannabinoid type 2 receptor agonist, suppresses itch-associated scratching behavior in rodents through inhibition of itch signal transmission.

We have previously reported that S-777469 [1-([6-ethyl-1-(4-fluorobenzyl)-5-methyl-2-oxo-1,2-dihydropyridine-3-carbonyl]amino)-cyclohexanecarboxylic acid], a novel cannabinoid type 2 receptor (CB2) agonist, significantly suppressed compound 48/80-induced scratching behavior in mice in a dose-dependent manner when it was administered orally. Here, we demonstrated that the inhibitory effects of S-777469 on compound 48/80-induced scratching behavior are reversed by pretreatment with SR144528, a CB2-selective antagonist. In addition, we investigated the effects of S-777469 on itch-associated scratching behavior induced by several pruritogenic agents in mice and rats. S-777469 significantly suppressed scratching behavior induced by histamine or substance P in mice or by serotonin in rats. In contrast, the H1-antihistamine fexofenadine clearly inhibited histamine-induced scratching behavior but did not affect scratching behavior induced by substance P or serotonin. Moreover, S-777469 significantly inhibited histamine-induced peripheral nerve firing in mice. In conclusion, these results suggest that S-777469 produces its antipruritic effects by inhibiting itch signal transmission through CB2 agonism.

Inhibitory effect of a potent and selective cytosolic phospholipase A2alpha inhibitor RSC-3388 on skin inflammation in mice.

Cytosolic phospholipase A2alpha (cPLA2alpha) preferentially hydrolyzes membrane phospholipids containing arachidonic acid, resulting in the biosynthesis of eicosanoids such as prostaglandins and leukotrienes. To examine the contribution of cPLA2alpha to skin inflammation, we evaluated the effect of (E)-N-[(2S,4R)-4-[N-(biphenyl-2-ylmethyl)-N-2-methylpropylamino]-1-[2-(2,4-difluorobenzoyl)benzoyl]pyrrolidin- 2-yl]methyl-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl) phenyl]acrylamide (RSC-3388), a potent and selective cPLA2alpha inhibitor, on 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced ear inflammation and mite antigen-induced dermatitis in mice. Topical application of RSC-3388 showed a significant inhibitory activity against TNCB-induced ear swelling and eicosanoid production in mice. Comprehensive expression analysis using Gene-Chip technology and subsequent experiments concerning mRNA and protein expression demonstrated that RSC-3388 clearly reduced the levels of interleukin-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta in a TNCB-induced mouse model. In addition, RSC-3388 ointment significantly alleviated atopic dermatitis-like skin lesions induced by repeated application of mite antigen. Furthermore, increased expression of cPLA(2)alpha, assessed by anti-phospho-cPLA2alpha antibody, was observed in the skin lesions of mite-antigen-induced dermatitis. These results indicate that cPLA2alpha is involved in the development of skin inflammation in mice, and RSC-3388 is expected to be useful for the treatment of inflammatory skin disorders such as atopic dermatitis.

Prostanoid DP receptor antagonists suppress symptomatic asthma-like manifestation by distinct actions from a glucocorticoid in rats.

While inhaled glucocorticoids are the best treatment for the majority of chronic asthmatics, there is a small group who do not respond to these drugs or whose disease can only be controlled by high doses of oral glucocorticoids with risks of severe side effects. Therefore, a safe novel anti-asthmatic agent which has a different mechanism from that of glucocorticoids is needed for the management of asthma. We have previously shown that an orally active prostanoid DP receptor antagonist, S-5751, had potent anti-inflammatory effects in guinea pig and sheep asthma models. In this study, using a rat asthma like model, we found that lung neutrophilia and proinflammatory cytokine secretion as well as bronchial hyperresponsiveness and lung eosinophilia were induced by repeated antigen-inhalations after antigen-sensitization. These symptoms are similar to the pathogenesis of symptomatic asthma. Orally-administered prostanoid DP receptor antagonists S-5751 and pinagladin significantly suppressed not only bronchial hyperresponsiveness and lung eosinophilia but also neutrophilia and mucus secretion in the lung, while oral prednisolone inhibited only bronchial hyperresponsiveness and eosinophil infiltration. In addition, prostanoid DP receptor antagonists significantly suppressed interleukin (IL)-1ß, IL-6 and CXCL1 mRNA in contrast to suppression of IL-4 and CCL11 mRNA by prednisolone. The majority of prostanoid DP receptor-expressing cells in both rat and human asthmatic lungs are infiltrative macrophages and/or monocytes. These results suggest that prostanoid DP receptor antagonists utilize different mechanisms from glucocorticoids, and that they would be a novel alternative and/or combination drug for asthma therapy.

Role of TRPV3 in immune response to development of dermatitis.

BACKGROUND: Recently, it has been reported that the Gly573Ser substitution of transient receptor potential V3 (TRPV3) leads to increased ion-channel activity in keratinocytes. Our previous studies have indicated that the spontaneous hairless and dermatitis phenotypes of DS-Nh mice, which were newly established as an animal model of atopic dermatitis (AD), are caused by TRPV3Gly573Ser. Although this substitution causes hairlessness in several kinds of rodents, in our investigations, dermatitis developed in only a few animals. Here, we generated NC/Nga-Nh mice to elucidate the role of TRPV3Gly573Ser in NC/Nga mice, which is one of the most studied animal models of AD. METHODS: To establish and validate the new AD animal model, NC/Nga-Nh mice were generated using NC/Nga and DS-Nh mice, and their clinical features were compared. Next, T-cell receptor (TCR) Vbeta usage in splenocytes, evaluation of bacterial colonization, and serological and histological analyses were carried out. Finally, repeated-hapten-application dermatitis was induced in these mice. RESULTS: NC/Nga-Nh mice did not develop spontaneous dermatitis, whereas DS-Nh mice displayed this phenotype when maintained under the same conditions. Serological analysis indicated that there really was a phenotypic difference between these mice, and TCR repertoire analysis indicated that TCRVbeta haplotypes played an important role in the development of dermatitis. Artificial dermatitis developed in DS and NC/Nga-Nh mice, but not in DS-Nh and NC/Nga mice. Histological and serological analyses indicated that mouse strains were listed in descending order of number of skin mast cells: DS-Nh > DS approximately NC/Nga-Nh > NC/Nga, and serum IgE levels were increased after 2,4,6 trinitrochlorobenzene application in these mice. Serum IgE level in DS-Nh mice was lower than that mesured in other strains. CONCLUSION: Our results confirm the contribution of the TRPV3Gly573Ser gene to the development of repeated hapten dermatitis, but not spontaneous dermatitis in NC/Nga mice.

Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice.

We reported that the Gly573Ser substitution in transient receptor potential vanilloid 3 (TRPV3) led to increased ion channel activity in keratinocytes and caused spontaneous hairlessness in DS-Nh mice. DS-Nh mice also develop allergic and pruritic dermatitis. As the hairless and dermatitis phenotypes were both inherited in an autosomal dominant fashion and could not be segregated from each other, we speculated that TRPV3(Gly573Ser) might be responsible for the dermatitis. Here, we constructed TRPV3(Gly573Ser) transgenic mice, with a putative promoter sequence in the 5' region of TRPV3, to investigate the involvement of TRPV3 in the development of specific types of dermatitis. These transgenic mice spontaneously developed dermatitis, whereas wild-type mice did not display this phenotype when maintained under the same conditions. Histological and serological analyses were carried out to better understand the clinical features of TRPV3(Gly573Ser) transgenic mice. A physiological study revealed that TRPV3(Gly573Ser) induced a higher nerve growth factor response to heat. Finally, C57BL-Nh mice were used to investigate the penetrance of the TRPV3(Gly573Ser) gene for dermatitis. Interestingly, C57BL-Nh mice developed spontaneous scratching behavior, separately from the development of dermatitis. We propose that TRPV3(Gly573Ser) is a cause of pruritus and/or dermatitis associated with scratching, and suggest that TRPV3 may represent a therapeutic target in pruritic dermatitis.

Impact of T-cell receptor Vbeta haplotypes on the development of dermatitis in DS-Nh mice: synergistic production of interleukin-13 caused by staphylococcal enterotoxin C and peptide glycans from Staphylococcus aureus.

Although the pathogenic role of interleukin-13 (IL-13) is a key for atopic dermatitis (AD), the mechanism of IL-13 production in AD remains unclear. To investigate the role of the T-cell receptor Vbeta (TCR Vbeta) haplotype in the development of dermatitis and the production of IL-13 in the naturally occurring dermatitis model by staphylococcal enterotoxin C (SEC)-producing Staphylococcus aureus, we raised DS-Nh mice harbouring the TCR Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, including TCR Vbeta8S2. Observation and histopathological analysis of the two mouse substrains with spontaneous dermatitis indicated that later onset and weaker severity of AD-like dermatitis were identified in mice with TCR Vbeta(a) compared to those with TCR Vbeta(b). Immunohistochemical examination revealed the infiltration of a large number of CD4-bearing T cells in the skin lesions in mice with TCR Vbeta(b) but not in those with TCR Vbeta(a). Interestingly, much lower levels of serum IL-13 were detected in mice with the TCR Vbeta(a) than in those with the TCR Vbeta(b) haplotype. In vitro, synthetic ligands (Pam(2)CSK4) of toll-like receptor 2 (TLR2) synergistically produced IL-13 with SEC in splenocytes of mice with TCR Vbeta(b) but not of those with TCR Vbeta(a), and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vbeta-haplotype-dependent synergism with TLR2 plays an important role in the development of AD-like dermatitis in DS-Nh mice.

Influence of TRPV3 mutation on hair growth cycle in mice.

We recently reported that Gly573Ser substitution of the transient receptor potential cation channel, subfamily V member 3 (TRPV3) caused hair loss in DS-Nh mice. To further elucidate the effects of this mutation on the development of the spontaneous hairless phenotype, we examined the temperature-response to epidermal sheets from DS-Nh and DS mice. It was indicated that the mutation was gain-of-function. We also performed genetic and histological analyses with both strain skins. DNA microarray data revealed that the levels of keratin-associated protein 16-1, 16-3, and 16-9 genes related to the anagen phase were decreased in the skins of DS-Nh mice compared with those of three days old DS mice. Histological analysis revealed that the anagen phase persisted in DS-Nh mice, and that the telogen phase was seen in DS but not DS-Nh mice at 21 days of age. Regulation of TRPV3 appears to be important for appropriate hair development in rodents.

The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with alpha-catenin.

Enterohaemorrhagic Escherichia coli (EHEC) belongs to a family of pathogens that cause attaching and effacing (A/E) lesion on target cells. The EspB protein of EHEC is translocated both to the host cell cytoplasm and to the membrane, and is essential for the signalling events leading to A/E lesion. To determine the actual role of EspB in this process, we tried to identify the EspB binding partner of the host cell protein, using a yeast two-hybrid assay, and obtained a cytoskeletal-associated protein, alpha-catenin. The alpha-catenin bound directly to the N-terminal region of EspB, both in solid (overlay assay) and solution (pull-down assay) phases, and it was recruited to the EHEC adherence site, dependent on EspB. Expression of the N-terminal region of EspB, as well as the whole EspB in host cells, inhibited F-actin accumulation on the adherence site. We conclude that EspB recruits alpha-catenin at the EHEC adherence site by direct interaction, and that the recruitment of alpha-catenin is essential for EHEC-induced A/E lesion formation.

Cortactin is necessary for F-actin accumulation in pedestal structures induced by enteropathogenic Escherichia coli infection.

Cortactin and the translocated intimin receptor, Tir, interacted with each other in pedestal formation in HeLa cells infected with enteropathogenic Escherichia coli (EPEC). Cortactin is shown to be necessary for organizing actin pedestals in response to EPEC, based on the expression of green fluorescent protein-fused cortactin derivatives in HeLa cells.