Noriko Shiraishi

Possible Pathogenic Role of Th17 Cells for Atopic Dermatitis

The critical role of IL-17 has recently been reported in a variety of conditions. Since IL-17 deeply participates in the pathogenesis of psoriasis and keratinocyte production of certain cytokines, the involvement of T helper cell 17 (Th17) in atopic dermatitis (AD) is an issue to be elucidated. To evaluate the participation of Th17 cells in AD, we successfully detected circulating lymphocytes intracellularly positive for IL-17 by flow cytometry, and the IL-17+ cell population was found exclusively in CD3+CD4+ T cells. The percentage of Th17 cells was increased in peripheral blood of AD patients and associated with severity of AD. There was a significant correlation between the percentages of IL-17+ and IFN-gamma+ cells, although percentage of Th17 cells was not closely related to Th1/Th2 balance. Immunohistochemically, IL-17+ cells infiltrated in the papillary dermis of atopic eczema more markedly in the acute than chronic lesions. Finally, IL-17 stimulated keratinocytes to produce GM-CSF, TNF-alpha, IL-8, CXCL10, and VEGF. A marked synergistic effect between IL-17 and IL-22 was observed on IL-8 production. The number of Th17 cells is increased in the peripheral blood and acute lesional skin of AD. Th17 cells may exaggerate atopic eczema.

Cutaneous Hypersensitivities to Hapten Are Controlled by IFN-γ-Upregulated Keratinocyte Th1 Chemokines and IFN-γ-Downregulated Langerhans Cell Th2 Chemokines

There are immediate, late-phase, and delayed-type reactions to exogenous agents. In IFN-gamma-knockout (IFN-gamma(-/-)) and wild-type B6 mice, we examined the response to picryl chloride (PCl) for assessing delayed-type reactions, and the responses to repeatedly challenged FITC for immediate and late-phase reactions. The delayed-type hypersensitivity was depressed in IFN-gamma(-/-) mice, and the immediate and late-phase reactions were enhanced in IFN-gamma(-/-) mice. As skin-infiltrating lymphocytes were scarce at the PCl-challenged site of IFN-gamma(-/-) mice, we investigated chemokine production by keratinocytes and Langerhans cells (LCs). A real-time PCR analysis demonstrated that Th1 chemokines (CXCL9 and CXCL10) and Th2 chemokines (CCL17 and CCL22) were derived mainly from keratinocytes and LCs, respectively. Challenge with PCl or FITC augmented keratinocyte expression of Th1 chemokines in wild-type but not in IFN-gamma(-/-) mice, and Th2 chemokine production by LCs was induced by repeated FITC in IFN-gamma(-/-) mice. Finally, transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled draining lymph node cells from hapten-sensitized B6 mice or lymph node cells from sensitized green fluorescent protein (GFP) mice to naive IFN-gamma(-/-) mice revealed less infiltration of CFSE(+) or GFP(+) lymphocytes at the challenged site. Our study suggests that one of the crucial actions of IFN-gamma is upregulation of keratinocyte production of Th1 chemokines and downregulation of LC production of Th2 chemokines.

Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.

Modulation of semaphorin 3A expression by calcium concentration and histamine in human keratinocytes and fibroblasts

BACKGROUND Both neurotrophins and chemorepellents are involved in the elongation and sprouting of itch-associated C-fibers in the skin. Nerve growth factor (NGF) and semaphorin 3A (Sema3A) are representatives of these two types of axon-guidance factors, respectively. OBJECTIVE We investigated the effects of calcium concentration and histamine on the expression of NGF and Sema3A in normal human epidermal keratinocytes (NHEK) and normal human fibroblasts (NHFb). METHODS NHEK and NHFb were cultured under different calcium concentrations (0.15-0.9 mM) with or without histamine, and the expression of mRNA for NGF and SEMA3A was assessed by real-time PCR analysis. An immunohistochemical study was performed for Sema3A using normal skin and skin cancer specimens. RESULTS In NHEK, SEMA3A expression was elevated by high calcium concentration and reduced by low calcium condition, while NGF expression was not dependent on calcium. Their expressions were unchanged by calcium in NHFb. Immunohistochemically, keratinocytes in the prickle layer of normal epidermis and squamous cell carcinoma cells were positive for Sema3A, sparing basal cells and suprabasal cells. The addition of histamine to NHEK at 10 μg/ml enhanced SEMA3A expression but depressed NGF expression. In NHFb, however, histamine decreased both NGF and SEMA3A levels. CONCLUSIONS Sema3A inhibits C-fiber elongation/sprouting in the upper layers of the epidermis, where calcium concentration is high, thereby determining the nerve endings. Histamine reduces Sema3A production by fibroblasts, allowing C-fibers to elongate in the dermis. In contrast, the histamine-augmented keratinocyte production of Sema3A might suppress C-fiber elongation and exaggerated pruritus.

Prostaglandin E2-EP3 axis in fine-tuning excessive skin inflammation by restricting dendritic cell functions.

Prostaglandin E2 (PGE2) is produced in the skin and is suggested to play a role in the regulation of cutaneous immune homeostasis and responses. However, the multifaceted functions of PGE2 continue to elude our understanding, especially because of the multiplicity of PGE2 receptors—EP1, EP2, EP3, and EP4. While cAMP-elevating EP4 is known to activate the functions of cutaneous dendritic cells (DCs), including Langerhans cells (LCs) and dermal DCs, the role of cAMP-suppressing EP3 in this process remains unknown. Here we demonstrated that an EP3 receptor selective agonist, ONO-AE-248, inhibited chemotaxis and co-stimulatory molecule expressions of DCs in vitro. A suboptimal dose of antigen was sufficient to induce contact hypersensitivity in EP3-deficient mice. Intriguingly, EP3 deficiency did not impair skin inflammation at all when the antigen dose was sufficiently high. EP3 limited the functions of cutaneous DCs only when the antigen dose was low. In contrast to EP4, the observed unappreciated function of EP3 may stabilize the cutaneous DCs to halt the impetuous response to a suboptimal dose of antigen. Taken together, PGE2-EP3 signaling is essential for fine-tuning excessive skin inflammation by restricting DC functions.

Topical Cholecystokinin Depresses Itch-Associated Scratching Behavior in Mice

Cholecystokinin (CCK) serves as a gastrointestinal hormone and also functions as a neuropeptide in the central nervous system (CNS). CCK may be a downregulator in the CNS, as represented by its anti-opioid properties. The existence of CCK in the peripheral nervous system has also been reported. We investigated the suppressive effects of various CCKs on peripheral pruritus in mice. The clipped backs of ICR mice were painted with CCK synthetic peptides and injected intradermally with substance P (SP). The frequency of SP-induced scratching was reduced significantly by topical application of sulfated CCK8 (CCK8S) and CCK7 (CCK7S), but not by nonsulfated CCK8, CCK7, or CCK6. Dermal injection of CCK8S also suppressed the scratching frequency, suggesting that dermal cells as well as epidermal keratinocytes (KCs) are the targets of CCKs. As determined using real-time PCR, mRNA for CCK2R, one of the two types of CCK receptors, was expressed highly in mouse fetal skin-derived mast cells (FSMCs) and moderately in ICR mouse KCs. CCK8S decreased in vitro compound 48/80-promoted degranulation of FSMCs with a transient elevation of the intracellular calcium concentration. These findings suggest that CCK may exert an antipruritic effect via mast cells and that topical CCK may be clinically useful for pruritic skin disorders.