Ichiro Hikita

Antifibrotic action of pirfenidone and prednisolone : Different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis

Pirfenidone, a broad-spectrum antifibrotic agent, is known to have efficacy in certain fibrotic disease models, and is under clinical trials in patients with idiopathic pulmonary fibrosis. We investigated the antifibrotic effect of pirfenidone, and its regulatory effect on various pulmonary cytokines, in bleomycin-induced lung fibrosis in mice at the protein level, using prednisolone as a reference agent. Pirfenidone attenuated the bleomycin-induced pulmonary fibrosis at a minimum effective dose of 30 mg/kg/day t.i.d. from the analysis of lung hydroxyproline content. Both pirfenidone (30, 100 mg/kg/day t.i.d) and prednisolone (3, 15 mg/kg/day q.d.) suppressed lung inflammatory edema; however, prednisolone failed to suppress pulmonary fibrosis, which was significantly suppressed only by pirfenidone. Both pirfenidone and prednisolone suppressed the increase in lung interleukin (IL)-1beta, IL-6, IL-12p40 and monocyte chemoattractant protein (MCP)-1 levels induced by bleomycin. On the other hand, pirfenidone prevented the bleomycin-induced decrease in lung interferon (IFN)-gamma levels, while prednisolone had no such effect. Furthermore, pirfenidone suppressed elevation of lung basic-fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1 levels, but prednisolone had no such effect. The increases in lung stroma cell derived factor (SDF)-1alpha and IL-18 were also suppressed. These findings suggest that pirfenidone exerts its antifibrotic effect through regulation of lung IFN-gamma, bFGF and TGF-beta1 levels during the development of bleomycin-induced pulmonary fibrosis in mice. The effect on SDF-1alpha and IL-18 levels may also be related to the antifibrotic effects of pirfenidone.

Characterization of dermatitis arising spontaneously in DS-Nh mice maintained under conventional conditions: another possible model for atopic dermatitis

DS-Nh (DS Nh/+) mice spontaneously develop dermatitis when they are housed in a conventional environment. In this study, we analyzed the clinical and histopathological features of dermatitis in DS-Nh mice, which is characterized by erythema, edema, and erosion on the face, neck, chest and flexor surfaces of their forelegs with marked scratching behavior. Histopathological examination, including immunohistochemistry, revealed that inflammatory cells consisting of mast cells, eosinophils, CD4-positive T cell-dominant lymphocytes and CD11b-positive macrophages infiltrated the skin lesions. The cytokine production pattern of inflammatory cells in a lesional skin tissue was shifted to the Th2-type (IL-4) rather than the Th1 type (IFN-gamma). Serum IgE levels were elevated and correlated with the severity of the clinical skin conditions. These skin symptoms were observed in association with a colonization of Staphylococcus aureus. Similar clinical and histopathological symptoms were inducible with repeated percutaneous immunization of heat-killed S. aureus on the back of SPF DS-Nh mice. These results suggest that the spontaneous dermatitis that occurs in conventionally raised DS-Nh mice is comparable to a certain type of human atopic dermatitis (AD), which is associated with S. aureus, a recognized environmental factor. Thus, we consider that DS-Nh mice offer a useful model for investigating the pathogenesis of AD and for developing new therapeutic approaches or drugs for treating AD.

Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis.

Itching is one of the major clinical symptoms in atopic dermatitis (AD) and complicates the management of this pathological condition. An animal model of AD‐like pruritus would contribute to a better understanding of AD and could lead to the development of safe and effective antipruritic agents. DS non‐hair (DS‐Nh) mice raised under conventional conditions spontaneously develop pruritus, which is associated with a dermatitis similar to human AD. There is a significant positive correlation between disease severity and the period of scratching behaviour in DS‐Nh mice. In the present study, we found that levels of histamine and nerve growth factor (NGF) in serum and/or skin tissue were higher in DS‐Nh mice with AD‐like dermatitis than in age‐matched mice without dermatitis. The histopathological data indicated that nerve fibres extend into and mast cells infiltrate the surrounding area of the skin lesion. NGF production by XB‐2 cells, which was derived from mouse keratinocytes, was enhanced by histamine via the H1 receptor. We also found that prolonged treatment with an H1‐antagonist was effective against pruritus through depression of the production of NGF, which is thought to be generated by keratinocytes. We conclude that DS‐Nh mice can serve as a suitable model for gaining a better understanding of pruritus in AD, and that prolonged treatment with an H1‐antagonist may be beneficial in patients with AD‐associated pruritus.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS–Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS–Nh mice with AD‐like dermatitis and that the serum levels of anti‐SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC‐producing S. aureus to DS–Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL‐4 and IFN‐γ from spleen mononuclear cells (MNC) from DS–Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL‐4 responses and lower IFN‐γ responses to SEC showed correlation with TCRBV8S2‐positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC‐producing S. aureus was able to survive in DS–Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host–S. aureus relationships.

Inhibitory Effect of a Potent and Selective Cytosolic Phospholipase A2α Inhibitor RSC-3388 on Skin Inflammation in Mice

Cytosolic phospholipase A2α (cPLA2α) preferentially hydrolyzes membrane phospholipids containing arachidonic acid, resulting in the biosynthesis of eicosanoids such as prostaglandins and leukotrienes. To examine the contribution of cPLA2α 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 cPLA2α 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-1β, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β 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 cPLA2α, assessed by anti-phospho-cPLA2α antibody, was observed in the skin lesions of mite-antigen-induced dermatitis. These results indicate that cPLA2α 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.

Low-dose ultraviolet B radiation synergizes with TNF-α to induce apoptosis of keratinocytes

High-dose ultraviolet B (UVB) irradiation is known to induce apoptosis of keratinocytes, but low-dose UVB dose not. In this paper we present evidence that low-dose UVB can induce TNF-alpha-dependent apoptosis of keratinocytes. In our study, 5 mJ/cm(2) doses of UVB were not sufficient by themselves to induce apoptosis of cultured human keratinocytes, but 20 mJ/cm(2) doses of UVB were. The combination of 5 mJ/cm(2) doses of UVB and exogenous TNF-alpha (15 ng/ml) induced significant apoptosis of keratinocytes, although exogenous TNF-alpha without UVB did not. This phenomenon was accompanied by enhanced clustering of tumor necrosis factor receptor 1 (TNFR1). TNF-alphas promotion of the induction of apoptosis by low-dose UVB was seen until 30 min after irradiation but not at 1 h. We confirmed this finding using a skin organ culture system. UVB (20 mJ/cm(2)), which did not induce transformation of epidermal keratinocytes into sunburn cells, induced apoptosis when TNF-alpha was added to the culture medium. These results suggest that one of the possible mechanisms of inducing keratinocyte apoptosis by low-dose UVB and TNF-alpha is that low-dose UVB augments ligand-binding-induced TNFR1 clustering, resulting in increased apoptotic cell death.

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.

Impact of T‐cell receptor Vβ 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 Vβ (TCR Vβ) 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 Vβa haplotype with a central deletion in the TCRBV gene segments, including TCR Vβ8S2. 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 Vβa compared to those with TCR Vβb. Immunohistochemical examination revealed the infiltration of a large number of CD4‐bearing T cells in the skin lesions in mice with TCR Vβb but not in those with TCR Vβa. Interestingly, much lower levels of serum IL‐13 were detected in mice with the TCR Vβa than in those with the TCR Vβb haplotype. In vitro, synthetic ligands (Pam2CSK4) of toll‐like receptor 2 (TLR2) synergistically produced IL‐13 with SEC in splenocytes of mice with TCR Vβb but not of those with TCR Vβa, and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vβ‐haplotype‐dependent synergism with TLR2 plays an important role in the development of AD‐like dermatitis in DS‐Nh mice.