Shoso Yamamoto

Characterization of T cells specific for an epitope of human 60-kD heat shock protein (hsp) in patients with Behcet's disease (BD) in Japan

BD is prevalent in the area of the Silk Route. It has been shown that hsp are involved in the T cell activation in patients with BD in the UK, where this disease has developed sporadically. We have thus examined whether the T cell response to the hsp‐derived peptides may be induced in patients with BD in Japan, an east pole of the Silk Route. As with patients in the UK, the human 60‐kD hsp peptide 336–351 also yielded vigorous proliferation of T cells in Japanese patients with BD, but neither in normal subjects nor in patients with rheumatoid arthritis (RA); there was significant association between proliferation by this peptide and the presence of ocular lesion, but not any other symptoms of BD. To clarify whether the peptide stimulates T cells as a polyclonal activator, a specific antigen or a superantigen‐like substance, we analysed T cell receptor (TCR) usage of responding T cells by means of MoAbs specific for TCR Vβ subfamily and polymerase chain reaction (PCR)‐single‐strand conformation polymorphism (SSCP)‐based technique. We found that T cells with certain TCR Vβ subfamilies (including Vβ5.2–3, 8, 13.6, 18, 21.3) were increased in circulation and responded to the hsp peptide in an antigen‐specific fashion. In addition, TCR Vβ gene‐amplified products of freshly isolated T cells of patients with BD formed several bands in the PCR‐SSCP analysis; some of them became prominent after stimulation with the peptide. This suggests that T cells in patients with this disease have already been expanded oligoclonally in vivo, which may be a result of stimulation by triggering antigens, including the hsp peptide. In addition, hsp peptide stimulation induced proinflammatory cytokine mRNA expression in peripheral blood mononuclear cells, including IL‐8, tumour necrosis factor‐alpha (TNF‐α) and TNF‐β in eight out of eight patients studied. Taken together, the results suggest that hsp antigen may play a role in the pathogenesis of BD, not only in the area of the Silk Route, but also outside the Silk Route area.

Fur mites induce dermatitis associated with IgE hyperproduction in an inbred strain of mice, NC/Kuj

An inbred strain of mice, NC, has been introduced as an animal model for atopic dermatitis because the mice develop dermatitis associated with severe scratch preceded by elevated serum IgE level when kept in conventional conditions. Although hypersensitivity to some environmental factors is suggested to cause dermatitis, the precise factor remains unclear. As the mice maintained under conventional conditions were often infected with fur mites, we investigated whether an infection of fur mites induces skin lesions in NC. Infection with the fur mites induced NC to develop skin lesions associated with highly elevated serum IgE, whereas no obvious skin lesions were observed in BALB/c and C57BL/6, and the elevation of serum IgE level was minimal in these two strains of mice. The role of the fur mites in the manifestation of skin lesions and IgE hyperproduction was confirmed by eliminating the fur mites by treatment with ivermectin. In addition, the existence of specific IgE antibody to Myocoptes musculinus antigen in the sera of mite-infested NC was detected by the antigen-induced histamine release from bone marrow-derived cultured mast cells after sensitization with the serum. These results suggest that continuous exposure to fur mite antigen is a potential factor in the development of dermatitis in NC. We provide a new model system of antigen-induced dermatitis for investigating the role of IgE in eliciting dermatitis.

Chemical mediators in atopic dermatitis: Involvement of leukotriene B4 released by a type I allergic reaction in the pathogenesis of atopic dermatitis

BACKGROUND The mediators produced from a type I allergic reaction have not yet been able to explain the pathogenesis of atopic dermatitis (AD). OBJECTIVE The purpose of this study was to elucidate the involvement of leukotriene (LT) B4 produced from a type I allergic reaction in the pathogenesis of AD. METHOD The release of LTB4 was measured both in vitro, in passively sensitized and antigen-challenged human skin slices, as well as in vivo, in skin chambers on patients with AD. RESULTS LTB4 was released from in vitro human skin by stimulation of the antigen (54.9 +/- 14.6 pg/g wet weight of skin by antigen challenge and 28.0 +/- 11.1 pg/g in control skin, P <.002). Antigen-specific release of LTB4 and histamine was also observed in vivo in nonlesional skin from the patients with AD by using the skin chamber technique. CONCLUSION LTB4 release during type I allergic reaction in human skin has been determined in vitro. The released LTB4 possibly contributes to cellular response at the acute inflammatory lesion of AD.

Rescue by cytokines of apoptotic cell death induced by IL-2 deprivation of human antigen-specific T cell clones

The control of cell survival and cell death is of central importance in tissues with high cell turnover such as the lymphoid system. We have examined the effect of cytokines on IL‐2 deprivation‐induced apoptosis of human antigen‐specific T helper clones with different cytokine production profiles. We found that IL‐2, interferon‐alpha (IFN‐α), and IFN‐β inhibited IL‐2 deprivation apoptosis in Th0, Th1, and Th2 clones. We also found that IL‐2 protects T cell clones from IL‐2 deprivation apoptosis accompanying active proliferation and enhanced expression of P53, Rb and Bcl‐xL proteins. In contrast, IFN‐α/β rescued T cell clones from apoptosis without active proliferation, and expression of apoptosis‐associated proteins tested so far was unaffected. This may be due to the fact that T cells treated with IL‐2 contained those located in S+G2/M phases of the cell cycle, whereas the vast majority of T cells treated with IFN‐α/β were located in G0/G1 phase. IFN‐α/β specifically induced tyrosine phosphorylation and translocation into nucleus of signal transducers and activators of transcription (STAT) 2 protein in the T cell clones. In addition, over‐expression of STAT2 by transfection of the cDNA prevented apoptosis of the T cell clones. Our present study shows that IFN‐α and ‐β mediate anti‐apoptotic effect through other pathways than that of IL‐2 in growth factor deprivation apoptosis.

In-vitro Permeability to Salicylic Acid of Human, Rodent, and Shed Snake Skin

Abstract— In‐vitro permeability to salicylic acid of human, rodent, and shed snake skin has been examined for the purpose of selecting model membranes for human skin corresponding to different anatomic sites, since a marked regional variation is suggested among the different sites. The greatest permeability to salicylic acid was observed in the scrotum, that of the sole was negligible. The cheek, neck, and inguinal skin seemed more permeable than the breast, back, thigh, lower leg, or foot skin. Shed snake and skin of hairless rat were found to show similar permeability to human breast and thigh skin. Wistar rat and nude mouse skin showed similar permeability to human cheek, neck, and inguinal skin.

IgE-mediated Hypersensitivity Against Human Sweat Antigen in Patients with Atopic Dermatitis

Sweating aggravates itch in atopic dermatitis, but the mechanism is unclear. In this study, we examined the involvement of type I hypersensitivity in the aggravation of atopic dermatitis by sweating. Skin tests with autologous sweat were positive in 56 of 66 patients (84.4%) with atopic dermatitis, but only in 3 of 27 healthy volunteers (11.1%). Sweat samples from both patients and healthy volunteers induced varying degrees of histamine release from basophils of patients with atopic dermatitis. However, the histamine release was impaired by removal of IgE on the basophils. Incubation of basophils with myeloma IgE before sensitization with serum of patients blocked the ability to release histamine-induced sweat. IgE antibody against antigen(s) in sweat may be present in serum of patients with atopic dermatitis. Key words:

Expression of c-kit ligand in human keratinocytes.

The c-kit ligand is expressed on tissue-anchored stromal cells. It plays an important role in the development of c-kit-bearing cells, such as haematopoietic cells, germ cells, mast cells and melanocytes. In the present study, we used the reverse transcriptase-mediated polymerase chain reaction (PCR) technique to investigate whether human keratinocytes are able to express c-kit ligand mRNA. Two sets of primers were designed to distinguish two types of c-kit ligand mRNA (full-length type and spliced type). One set was used to amplify an 882-bp DNA fragment from the full-length type, and a 798-bp DNA fragment from the spliced type. Another set was used to amplify a 375-bp DNA fragment from the full-length type only. A cDNA fragment corresponding to the full-length type mRNA was amplified from a cDNA preparation of cultured human keratinocytes as well as from epidermis obtained by the suction blister technique. This result indicates the spontaneous transcription of full-length type mRNA of the c-kit ligand in human keratinocytes.

Food-dependent exercise-induced anaphylaxis: a report of two cases and determination of wheat-γ-gliadin as the presumptive allergen

Water/salt‐insoluble wheat proteins have been identified as the most frequent allergenic foodstuffs in patients with food‐dependent exercise‐induced anaphylaxis (FDEIA) in Japan. However, the specific allergenic proteins in wheat‐dependent exercise‐induced anaphylaxis have not been well defined. Challenge testing, skin testing and a fluoroenzyme immunoassay were used for diagnosis in two patients suspected by history of having wheat‐dependent exercise‐induced anaphylaxis. Gel chromatography and IgE immunoblotting followed by N‐terminal amino acid sequencing were used to identify the allergenic wheat protein. The challenge test revealed that both patients had FDEIA. The skin tests and the immunoassay results suggested that wheat gluten was the allergen in both patients. Gel chromatography of wheat gluten revealed that the antigens had molecular weights ranging from 40 to 250 kDa. IgE immunoblotting and subsequent N‐terminal amino acid sequencing revealed that wheat‐γ‐gliadin was the antigen predominantly bound by IgE in the two patients.

Histamine H1-receptor in endothelial and smooth muscle cells of guinea-pig aorta

The location of histamine H1-receptors in the thoracic aorta of guinea-pigs was studied with a [3H]mepyramine binding assay. [3H]Mepyramine binding studies of whole and rubbed aortas, and of cultured endothelial and smooth muscle cells showed that the Kd values were all in the range 0.53-0.76 nM, but that the Bmax values were 19.1, 10.1, 63.3 and 11.6 fmol/mg protein, respectively. Thus, the whole aorta contained more H1-receptors than the rubbed one (free of endothelium), and cultured endothelial cells contained more H1-receptors than smooth muscle cells. These results indicate that more histamine H1-receptors were concentrated in the endothelial cells than in the smooth muscle cells in guinea-pig aorta.

The IL-6 family cytokines, interleukin-6, interleukin-11, oncostatin M, and leukemia inhibitory factor, enhance mast cell growth through fibroblast-dependent pathway in mice

Abstract Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the mechanisms involved in the pathogenesis of these conditions remains largely uninvestigated, it is speculated that mediators produced in the lesional skin provide a favorable microenvironment for mast cell growth. Among the proinflammatory mediators, leukemia inhibitory factor (LIF), which shares a receptor component (gp130 subunit) with interleukin-6 (IL-6), has been identified as a mast cell growth-enhancing factor produced by cells of the keratinocyte-derived cell line (KCMH-1). In this study, we investigated the effect of four IL-6 family cytokines, IL-6, IL-11, oncostatin M (OSM) and LIF on mast cell growth in a mast cell/fibroblast coculture system. When mouse bone marrow-derived cultured mast cells (BMMC) were maintained on a NIH/3T3 fibroblast monolayer, these cytokines induced proliferation of the mast cells, but none of the cytokines had any effect on mast cell proliferation in the absence of fibroblasts. mRNA for gp130 and receptors for the four IL-6 family cytokines were detected in NIH/3T3 fibroblasts by reverse transcriptase-mediated polymerase chain reaction. In contrast, only mRNA for the IL-11 receptor and gp130 were detected in BMMC. Tyrosine phosphorylation of gp130 was observed in NIH/3T3 fibroblasts after stimulation with all the cytokines. Some IL-6 family cytokines enhanced the production of stem cell factor (SCF), a potent mast cell growth factor, from NIH/3T3 fibroblasts, but the amount of SCF produced by NIH/3T3 fibroblasts was not paralleled by the mast cell growth-enhancement induced by the IL-6 family cytokines. When anti-SCF antibody was added with the IL-6 family cytokines in the BMMC/fibroblast coculture system, a significant effect of these cytokines remained, although the growth-enhancing activity was markedly reduced. A similar result was obtained when BMMC were prepared from W/W V -mice, which lack functional c-kit , in the BMMC/fibroblast coculture system. These results suggest that IL-6 family cytokines stimulate mast cell growth by a fibroblast-dependent mechanism, and also suggest the existence of another pathway between BMMC and NIH/3T3 fibroblasts cooperating with the SCF/ c-kit pathway. IL-6 family cytokines may thus contribute to mast cell hyperplasia in skin diseases.