Eishin Morita

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.

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.

Safe and Effective Treatment of Refractory Facial Lesions in Atopic Dermatitis Using Topical Tacrolimus following Corticosteroid Discontinuation

Background: Topical corticosteroids are commonly applied in atopic dermatitis (AD) treatment. However, their chronic use may be associated with significant side effects at the application site. Skin atrophy and other undesirable effects are frequently seen after long-term corticosteroid treatment. In addition, when application of corticosteroids is discontinued, a rebound phenomenon in the facial lesions can occur within several days. Topical tacrolimus, an immunosuppressant currently used to prevent rejection after solid-organ transplantation, presents a potential alternative therapeutic agent for AD. Objective: The present study is the first trial designed to evaluate the efficacy and safety of topically applied tacrolimus ointment after corticosteroid discontinuation without a washout phase in severe, long-term facial AD. Patients/Methods: Forty-seven patients with facial refractory AD were recruited, of whom 38 had undergone topical corticosteroid treatment for at least 4 weeks before enrollment (group 1) and the other 9 had not received steroid treatment (group 2). All 47 patients received 0.1% tacrolimus ointment, and the severity index and pruritus score were assessed as an AD clinical activity index every week and compared with baseline data. Results: Both the severity index and pruritus score improved significantly in group 1 after 1 and 2 weeks of application (p < 0.01, respectively). Group 2 showed the greatest improvement at 4 weeks (p < 0.05). In this trial, none of the patients experienced a rebound phenomenon associated with tacrolimus treatment. A transient sensation of burning at the application site was the only adverse event in 31 of the 47 (66%) enrolled patients, but this condition improved after several days. Spectrophotometric assessment of the facial lesion following treatment revealed significant improvement in group 1 (p < 0.05). Conclusion: The present results indicate that topical tacrolimus treatment following corticosteroid discontinuation is safe and effective in refractory facial AD.

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.

A Case of Acquired Autoimmune Bullous Disease Associated with IgM Macroglobulinaemia

A variety of autoimmune bullous dermatoses have been reported to develop in association with lymphoproliferative disorders. We report a patient with IgM macroglobulinaemia, who presented with a skin fragility similar to but somewhat milder than that seen in epidermolysis bullosa acquisita. Immunofluorescence detected circulating IgM autoantibodies reacting with the basement membrane zone, which reacted predominantly with dermal side of 1 M NaCl‐split skin. Immunoblotting of the epidermal and dermal extracts with the patients serum showed no specific reactivity. Further studies are needed to identify the antigenic molecules responsible for the IgM deposition.

Enhancement of fibroblast-dependent mast cell growth in mice by a conditioned medium of keratinocyte-derived squamous cell carcinoma cells

We investigated the increase in mast cell numbers at the sites of inoculation of keratinocyte-derived squamous cell carcinoma cell line (KCMH-1) cells in mice. A significant increase in the number of mast cells was observed at the sites of tumours developed at the sites of inoculation of the KCMH-1 cells. Enhancement of mast cell growth was observed by culturing bone marrow-derived mast cells (BMMC) on NIH/3T3 fibroblast monolayers in the presence of conditioned medium (TCM) obtained from KCMH-1. Activities of known factors for mast cell growth, such as interleukin-3 (IL-3), IL-4, IL-9, IL-10 and stem cell factor (SCF), were not detected in the TCM. Nerve growth factor (NGF) did not induce mast cell growth. Mast cell growth induced by the TCM needed 3T3 fibroblasts. These results suggest that KCMH-1 cells may produce a factor which induces mast cell growth with 3T3 fibroblasts, other than the already known mast cell growth factors. This may be the mechanism of mast cell accumulation at sites of tumours.

Characteristics of T cell lines established from skin lesions of Behcet's disease

Hypersensitivity to a streptococcal antigen is postulated to be the pathogenesis of Behçets disease. We analyzed T lymphocyte-phenotypes infiltrated in cutaneous pustular lesions in Behçets disease and found that CD4+ T cells were predominant components although CD8+ T cells were also present in the lesion. In addition, we established T cell lines from pustular lesions of the four patients with a streptococcal antigen, KTH-1. Two of the cell lines showed the cell surface markers of CD8+TCR alpha beta +, and expressed mRNAs for interleukin (IL)-8, tumor necrosis factor (TNF) alpha, and perforin. Two other T cell lines expressed the cell surface markers for CD4+TCR alpha beta +. Cytokine expression pattern of the two CD4+ T cell lines revealed that one is Th1 type and the other is Th2 type. The Th2 type cell line showed marked proliferation with autologous peripheral blood mononuclear cells, suggesting that the self-reactive T cells play some role on the pathogenesis of Behçets disease.

Interleukin-1α enhances mast cell growth by a fibroblast-dependent mechanism

Abstract Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the pathogenesis of these conditions remains largely uninvestigated, it has been speculated that lesional mediators provide a favorable microenvironment for mast cell growth. We investigated the effect of an inflammatory cytokine, IL-1α, on mast cell growth in a mast cell/fibroblast coculture system. When mouse bone marrow-derived cultured mast cells (BMMC) were cultured on a NIH/3T3 fibroblast monolayer, IL-1α stimulated mast cell proliferation. However, IL-1α did not stimulate 3 H-thymidine incorporation in BMMC in the absence of fibroblasts. Separation of BMMC from fibroblasts by a permeable micropore membrane reduced the effect of IL-1α. When BMMC were prepared from W/W v mice, which lack a functional c- kit , or when NIH/3T3 fibroblasts were substituted with Sl/Sl d -derived fibroblasts, which lack membrane-bound stem cell factor (SCF), a lower, but significant, effect of IL-1α was observed. Flow cytometric analysis revealed no enhancement of SCF expression on fibroblasts following stimulation with IL-1α. Neutralizing antibodies against IL-3, IL-4, IL-10, and nerve growth factor (NGF) showed no inhibition. On the other hand, indomethacin inhibited the effect of IL-1α, and prostaglandin E 2 induced mast cell growth in the cocultures. These results indicate that IL-1α stimulates mast cell growth by a fibroblast-dependent mechanism, in which SCF/c- kit interaction may participate in a major way. The mast cell growth activity induced by this cytokine can, at least in part, be attributed to prostaglandins. Inflammatory cytokines may thus contribute to mast cell hyperplasia in skin diseases.

Leukemia inhibitory factor enhances mast cell growth in a mast cell/fibroblast co-culture system through Stat3 signaling pathway of fibroblasts

Leukemia inhibitory factor (LIF) enhanced mast cell growth in a mast cell/3T3 fibroblast co‐culture system, however the precise mechanisms have not been defined. Western blot analysis showed that bone marrow‐derived mast cells failed to express both LIF receptor (LIFR) and gp130, whereas 3T3 fibroblasts expressed both LIFR and gp130. This result indicates that the activity of LIF for mast cell growth is mediated by 3T3 fibroblasts. Signal transducer and activator of transcription (Stat) 3‐transfected 3T3 fibroblasts enhanced mast cell growth. In addition, dominant‐negative Stat3‐transfected fibroblasts blocked LIF‐mediated mast cell growth in the co‐culture system. In conclusion, LIF‐induced mast cell growth in the co‐culture system is mediated by an indirect pathway via 3T3 fibroblasts through activating Stat3 signaling pathway in 3T3 fibroblasts.