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Introduction: Epicutaneous (e.c.) allergen exposure is an important route of sensitization toward allergic diseases in the atopic march. Allergen sources such as house dust mites contain proteases that involve in the pathogenesis of allergy. Prostanoids produced via pathways downstream of cyclooxygenases (COXs) regulate immune responses. Here, we demonstrate effects of COX inhibition with nonsteroidal anti-inflammatory drugs (NSAIDs) on e.c. sensitization to protease allergen and subsequent airway inflammation in mice. Methods: Mice were treated with NSAIDs during e.c. sensitization to a model protease allergen, papain, and/or subsequent intranasal challenge with low-dose papain. Serum antibodies, cytokine production in antigen-restimulated skin or bronchial draining lymph node (DLN) cells, and airway inflammation were analyzed. Results: In e.c. sensitization, treatment with a nonspecific COX inhibitor, indomethacin, promoted serum total and papain-specific IgE response and Th2 and Th17 cytokine production in skin DLN cells. After intranasal challenge, treatment with indomethacin promoted allergic airway inflammation and Th2 and Th17 cytokine production in bronchial DLN cells, which depended modestly or largely on COX inhibition during e.c. sensitization or intranasal challenge, respectively. Co-treatment with COX-1-selective and COX-2-selective inhibitors promoted the skin and bronchial DLN cell Th cytokine responses and airway inflammation more efficiently than treatment with either selective inhibitor. Conclusion: The results suggest that the overall effects of COX downstream prostanoids are suppressive for development and expansion of not only Th2 but also, unexpectedly, Th17 upon exposure to protease allergens via skin or airways and allergic airway inflammation.

Introduction: Repeated skin contact to detergents causes chronic irritant contact dermatitis (ICD) associated with itch sensation and eczema. However, the mechanisms of detergent-induced ICD are poorly understood. Here, we established a new murine model of detergent-induced ICD with H1-antihistamine-refractory itch. Methods: Ear skin of wild-type and mast cell-deficient mice on the C57BL/6 genetic background was treated with a detergent, sodium dodecyl/lauryl sulfate (SDS), daily for approximately 2 weeks with or without administration of an H1-antihistamine, fexofenadine. Skin inflammation, barrier dysfunction, and itching were analyzed. Quantitative PCR for earlobe gene expression and flow cytometry analysis for draining lymph node cells were conducted. Results: SDS treatment induced skin inflammation with ear swelling, increased transepidermal water loss, and hind-paw scratching behaviors in the wild-type and mast cell-deficient mice. The peak value of scratching bouts was retained for at least 48 h after the last SDS treatment. H1-antihistamine administration showed no or little reduction in the responses. SDS treatment upregulated gene expression for a Th2 cytokine IL-4 and Th17/Th22 cytokines, IL-17A, IL-17F, and IL-22, and increased cell numbers in draining lymph nodes of CD4+ T, CD8+ T, and γδT cells with enhanced expression of GATA3, RORγt, T-bet, or FOXP3 compared with untreated mice. Conclusions: The present study showed that SDS treatment of ear skin in C57BL/6 mice induces mast cell-independent skin inflammation with H1-antihistamine-refractory itch and suggested a possible Th cytokine- and/or lymphocyte-mediated regulation of the model. The model would be useful for elucidation of mechanisms for inflammation with H1-antihistamine-refractory itch in detergent-induced ICD.

Background: Pollen is an important trigger of seasonal rhinitis, conjunctivitis, and/or allergic asthma, and an exacerbating factor in atopic dermatitis. Pollen grains contain allergen proteins, enzymes, and bioactive lipid mediators, the latter two possibly involved in the pathogenesis of allergic diseases through IgE-independent mechanisms. Methods: We analyzed the patterns of release of endopeptidases from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and birch, ragweed, and two grasses, Kentucky blue and cultivated rye, using synthetic substrates, class-specific inhibitors, and zymography. The proteins released were analyzed by gel electrophoresis. Eicosanoid-like substances were measured by enzyme-linked immunosorbent assays for prostaglandin E2 and leukotriene B4. Results: Major fractions of proteins, eicosanoid-like substances, and at least one molecular species of serine endopeptidase were released into phosphate-buffered saline from the pollen grains at 37°C within 25 min or 60 min without sonication. In the Cupressaceae/Taxodiaceae family, sonication was necessary for the release of other proteins and another serine endopeptidase. In birch, ragweed, and the grasses, most of the serine and cysteine endopeptidases were released without sonication. Proteases released within 25 min digested gelatin and/or casein differently among plant species. Conclusions: Grains of allergenic pollen release proteases, which can digest not only short synthetic substrates but also protein substrates, along with eicosanoid-like substances and proteins. The release of these components could contribute to the formation of a microenvironment optimum for initiation of the sensitization or the exacerbation of pollen allergy in tissues exposed to pollen grains.

Background: Although exposure to mite allergens is an important risk factor for the production of IgE and is associated with various allergic diseases, there has been uncertainty as to the route of exposure by which sensitization occurs. Cystatin A is a skin-derived dominant inhibitor against proteolytic activity of major mite allergens, Der f 1 and Der p 1, and blocks the upregulation of IL-8 release from human keratinocytes stimulated with the allergens. We analyzed whether the stimulation of keratinocytes with the allergens upregulates the release of granulocyte-macrophage colony-stimulating factor (GM-CSF), which has many actions relevant to allergic diseases including atopic dermatitis, and if so, whether cystatin A can block this process. Methods: Normal human keratinocytes and the human keratinocyte cell line HaCaT were stimulated with recombinant group 1 allergens in the absence or presence of cystatin A. Results: Stimulation with the recombinant allergens upregulated the release of GM-CSF from normal human keratinocytes in a culture with high calcium concentration and HaCaT cells, which could be inhibited by the addition of cystatin A. The allergens exhibiting proteolytic activity did not digest cystatin A. Proteolytic activity of recombinant Der f 1 was partially regenerated after incubation with keratinocytes even without preactivation by L-cysteine. Conclusion: Proteolytic activity of recombinant Der f 1 and Der p 1 upregulates GM-CSF and IL-8 release from keratinocytes in vitro, suggesting possible contributions to sensitization through the skin and the perpetuation of atopic dermatitis, as well as a homeostatic role for cystatin A against inflammation of the skin.

Background: Weak signals for allergen-specific IgE are a problem in murine models for the study of allergies. It has been reported that the removal of IgG from murine sera enhances signal intensity. Very recently, buffer solutions designed to enhance signals in immunoassays have been developed and made commercially available. Methods: Sera from mice immunized either with a recombinant form of one of the major mite allergens Der p 1, Der f 1 and Der f 2, or with ovalbumin adsorbed to alum were used for the assays. Total IgE was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Allergen-specific IgE was assayed using plates coated with the allergens after the removal of IgG from sera with protein G-coupled sepharose beads in wells of other plates or with the use of commercially available enhancer solutions without the removal of IgG. IgE binding was detected with horseradish peroxidase-conjugated anti-murine IgE monoclonal antibody as the secondary antibody. Results: Significant levels of total IgE were produced after the immunizations. The in-well pretreatment of diluted sera (1/10 dilution) with protein G-coupled beads enhanced the signals for allergen-specific IgE. The use of the enhancer solutions for dilution of the sera and secondary antibody and prolonged incubation remarkably enhanced the signals at a more extensive dilution of sera (1/200 or less) without the removal of IgG. Conclusions: An ELISA simply modified with the use of immunoreaction enhancer solutions has advantages in terms of signal intensity and ease of handling for the detection of allergen-specific murine IgE and would be useful for the study of allergies with murine models.

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcΕRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α1-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α1-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α1-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.

A cell line, termed NCJ, was established from the bone marrow-derived mast cells (BMMCs) of NC/Nga mice that are mouse models for atopic dermatitis. NCJ cells expressed FcΕRI and c-kit and showed a metachromasia of the granules with a toluidine blue-positive and safranin-negative staining pattern that is characteristic for immature-type mast cells. Interestingly, NCJ cells showed proliferation independent of IL-3, which was associated with constitutive phosphorylation of Raf-1 and Erk kinases. Although NCJ cells had several characteristics of mast cells, we failed to detect FcΕRI-mediated β-hexosaminidase release and its histamine content. These findings indicated that NCJ cells represented a mast cell line with an immature phenotype and the ability to proliferate in the absence of mast cell growth factors. NCJ cells might thus be useful to study the molecular basis of mast cell proliferation.

Atopic dermatitis (AD) is a common pruritic disease that occurs primarily in infancy and childhood. AD is characterized by itching and the patient having an individual or family history of atopic diseases. Although AD is also frequently associated with elevated serum IgE levels and with common environmental factors contributing to its pathogenesis, the etiology of AD is still unknown. We examined NC/Nga mice (NC mice) that showed AD-like skin lesions with aging as a possible mouse model for AD. NC mice were maintained under conventional (Conv) or specific pathogen-free (SPF) conditions. Clinical symptoms, serum IgE levels and histopathology of the skin were compared between these 2 groups, and we explored their application as a model of human AD. It was found that the skin lesions of inbred NC mice were clinically and histologically very similar to human AD when the mice were raised under Conv conditions, but not under SPF conditions, and we assumed that some kinds of environmental factors might trigger AD-like signs and symptoms in NC mice. To further investigate the pathophysiology and treatment of AD, a suitable animal model is absolutely required, and NC mice are very useful for this purpose.

Background: Thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes in skin lesions of atopic dermatitis patients and bronchial epithelial cells in asthma, plays a key role in allergic diseases. Information on triggers for the release of TSLP in keratinocytes is still limited. Keratinocytes express Toll-like receptor (TLR) 5, the ligand for which is flagellin, the major structural protein of the flagella of Gram-negative bacteria. IL-4, IL-13 and TNF-α (Th2/TNF) are associated with allergic diseases. TGF-α, one of the ligands for the epidermal growth factor receptor, is overexpressed in keratinocytes in atopic dermatitis. We investigated the induction of TSLP expression in keratinocytes stimulated with flagellin and its modulation by the Th2/TNF cytokines and TGF-α. Methods: Primary human keratinocytes were stimulated with flagellin with or without cytokines. The TSLP released was measured by ELISA. Gene expression was analyzed by quantitative real-time PCR. Results: Stimulation of keratinocytes with flagellin induced the release of TSLP protein and upregulation of the gene expression of TSLP and other pro-inflammatory molecules. The flagellin-induced release of TSLP was enhanced by the Th2/TNF cytokines or TGF-α. Small interfering RNA-mediated knockdown of TLR5 expression suppressed the flagellin-induced TSLP gene expression. Conclusions: Flagellin induces TSLP expression in keratinocytes via TLR5 and the expression can be upregulated by a cytokine milieu with Th2/TNF or TGF-α, suggesting that exposure of barrier-defective skin to Gram-negative bacteria or environmental flagellin contributes to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis through the induction of TSLP expression in keratinocytes.

Background: To elucidate the usefulness of antigen-specific immunotherapy based on oral vaccination with an edible part of the plant, we examined the effect of transgenic (Tg) rice seeds expressing an immunodominant fragment of the group 1 antigen of Dermatophagoides pteronyssinus (Der p 1) on a murine model of asthma. Methods: Mice were orally vaccinated with the Tg or non-Tg rice seeds for 2 weeks, then they were immunized with recombinant Der p 1 (rDer p 1) and alum intraperitoneally. Antigen-induced immune responses, such as proliferation and cytokine production of CD4+ T cells, antigen-specific serum IgE and IgG, and infiltration of inflammatory cells into the airways were investigated in those mice. Results: The proliferation and Th2 cytokine production of CD4+ T cells in vitro, antigen-specific IgE and IgG synthesis as well as accumulation of eosinophils and lymphocytes into the airways in vivo were significantly inhibited by administration of the Tg rice. Conclusions: These results suggest that the edible vaccines using Tg rice seeds are useful for the treatment of allergic disorders including bronchial asthma.

Background: Thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes in skin lesions in atopic dermatitis and bronchial epithelial cells in asthma, plays a key role in allergic diseases. Double-stranded RNA (dsRNA) stimulates keratinocytes to release TSLP in vitro. Objective: To examine the potential of glucocorticoids and calcineurin inhibitors to suppress dsRNA-induced release of TSLP from keratinocytes. Methods: Primary human kerarinocytes were stimulated with dsRNA in the presence of IL-4, IL-13 and TNF-α. TSLP release was measured by ELISA. The effects of glucocorticoids and 2 calcineurin inhibitors, cyclosporin A and FK506/tacrolimus, were analyzed. Results: The glucocorticoids inhibited dsRNA-induced release of TSLP. The inhibitory effect became saturated (50–70% reduction) at concentrations higher than 10–10M. Cyclosporin A inhibited the release of TSLP by 50–60% at 10–5 and 10–4M. FK506 had no effect at 10–5M or less, but almost completely inhibited the release of TSLP at 10–4M. No synergistic effect was obtained with a glucocorticoid plus either of the calcineurin inhibitors. An additive inhibitory effect was obtained with a glucocorticoid plus 10–5M cyclosporin A. Glucocorticoid inhibited dsRNA-induced TSLP transcription in the absence of Th2/TNF cytokines. Conclusions: Glucocorticoids inhibited the dsRNA-induced release of TSLP in the atopic cytokine milieu at much lower concentrations than calcineurin inhibitors, suggesting that they could be effective in the treatment of atopic dermatitis when exogenous or endogenous dsRNA is involved in the pathogenesis. In addition, the in vitro system established in this study would be useful for screening of therapeutic reagents which target TSLP expression.

Background: Polyphenol-enriched fractions, which are extracted from unripe apples (Rosaceae, Malus spp.), consisting of procyanidins (polymers of catechins) are known to have an anti-allergenic effect on patients with various allergic diseases. Although it has been reported that apple extracts inhibit histamine release from mast cells, the molecular mechanisms for this anti-allergenic effect are not well understood. To elucidate the molecular mechanisms by which apple extracts induce their anti-allergenic effects, the effects of purified apple extract components on high-affinity receptors for IgE (FcΕRI)-mediated mast cell activation were investigated. Methods: The anti-allergic effect of oral administration of apple procyanidin extracts on passive cutaneous anaphylactic responses of BALB/c mice was assessed. We evaluated the effects of procyanidin C1 (PC1) [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin], a component of the procyanidin fraction, on mouse bone-marrow-derived mast cell degranulation, cytokine production, protein tyrosine phosphorylation and on the generation of intracellular reactive oxygen species (ROS) of cells stimulated by FcΕRI cross-linking in vitro. Results: In an in vivo study, oral administration of the procyanidin fraction suppressed the mast-cell-dependent allergic reaction. In in vitro studies, PC1 dose-dependently decreased FcΕRI-mediated degranulation and cytokine production of mast cells. Furthermore, PC1 inhibited tyrosine phosphorylation of Syk and linker for activation of T cells, and the ROS generation in stimulated mast cells. Conclusions: PC1 suppresses FcΕRI-mediated mast cell activation by inhibiting intracellular signaling pathways. These observations provide evidence for the anti-allergenic effects of the procyanidin-enriched apple extract.

Background: There is a paradoxical finding that eosinophils are frequently accumulated at the sites of allergic inflammation where transforming growth factor (TGF)-β, a negative regulator of eosinophil survival, is upregulated; however, eosinophil accumulation is persistent. We thus hypothesized that eosinophils might have aberrant TGF-β signaling and be unresponsive to TGF-β. To test the hypothesis, we examined the expression and function of Smad proteins, which are central mediators for TGF-β signaling, in human eosinophils. Methods: Eosinophils were isolated from the peripheral blood of normal donors, and the expression and activation of endogenous Smad proteins were examined by reverse transcription polymerase chain reaction and Western blotting. The Smad function in the transcription of the major TGF-β target gene Smad7 was investigated using a dominant negative form of Smad3. The effect of TGF-β on eosinophil survival was then evaluated by a cell viability assay using normal and asthmatic eosinophils. Results: Human eosinophils expressed mRNAs and proteins of TGF-β typeI and type II receptors, Smad2, Smad3 and Smad4. TGF-β induced the phosphorylation of Smad2 in eosinophils, which was blocked by SB431542, an inhibitor of TGF-β type I receptor kinase. A dominant negative Smad3 protein suppressed TGF-β-induced Smad7 mRNA expression in eosinophils. Finally, TGF-β prevented granulocyte macrophage colony-stimulating factor- or interferon-γ-mediated survival of eosinophils obtained from asthmatic patients as well as normal subjects. Conclusion: Human eosinophils have an intact Smad signaling pathway leading to a major TGF-β target gene expression. Thus, eosinophils might become resistant to TGF-β only in in vivo circumstances.

Background: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. Methods: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. Results: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. Conclusions: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.

The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcepsilonRII), the alpha-subunit of the IL-2 receptor (CD25), and a protease inhibitor alpha1-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice.Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human alpha1-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA.Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human alpha1-antitrypsin, and elicited significant serum IgE levels in immunized mice.The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.

Background: Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost. Methods: The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively. Results: Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture. Conclusions: The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.