Modulation of allergenicity of major house dust mite allergens Der f 1 and Der p 1 by interaction with an endogenous ligand.

Although many allergens bind endogenous molecules other than Abs in the human body, whether the interaction can modulate allergenicity has been unknown. Here, we investigated the effect of the interaction of recombinant major mite group 1 allergens (Der f 1 and Der p 1), which belong to the papain-like cysteine protease family, with an endogenous protease inhibitor, cystatin A, on their allergenicity. Cystatin A bound reduced forms of the allergens, in which the cysteine residue at the catalytic center of the protease activity was reduced by treatment with L-cysteine, but did not bind oxidized forms. Cystatin A partially inhibited the binding of IgE in mite-allergic volunteers' sera to the reduced forms, but unexpectedly enhanced the basophil histamine-releasing activity. A catalytic site-mutant of Der f 1 behaved in terms of histamine release, similarly to the reduced form. Molecular modeling showed that cystatin A interacts with the allergens within a narrow area. The results indicate that interaction with cystatin A reduces the limited number of IgE epitopes of the allergens but enhances their biological activity to release histamine, suggesting a new concept, that interaction between allergens and their endogenous ligands modulates the allergenicity even toward enhancement in the effector phase. On the other hand, i.p. immunization without alum of mice with cystatin A-treated reduced Der f 1 induced less serum Der f 1-specific IgE than immunization with reduced Der f 1 alone, suggesting that endogenous protease inhibitors suppress the induction of allergen-specific IgE, which is dependent on the enzymatic activity of cysteine protease-allergens, in the sensitization process.

A case report of steroid and immunosuppressant-resistant pyoderma gangrenosum successfully treated by granulocytapheresis.

Granulocytapheresis (GCAP) therapy is a newly developed therapeutic modality for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Pyoderma gangrenosum (PG) is a chronic inflammatory skin disease characterized by the appearance of erythematous macules and plaques with pustules or nodules that rapidly progress to ragged, undermined multiple ulcers. We attempted GCAP therapy in a patient with PG resistant to prednisolone and various other immunosuppressants. GCAP therapy was initiated at three- to four-day intervals and a good response from all skin lesions, with eventual total epithelialization, was observed after 10 sessions of this therapy. Furthermore, circulating levels of inflammatory cytokines such as interleukin-8 (IL-8) and granulocyte colony stimulating factor (G-CSF) also decreased after the GCAP therapy. Our results suggest that GCAP is a safe and useful tool for the treatment of intractable PG, and that IL-8 and G-CSF are likely to be involved in the pathogenesis of PG.

Expression of DNMT-1 in patients with atopic dermatitis.

DNA methylation is known to play an important role in gene transcription and alterations of methylation that contribute to the development of certain disorders such as cancer, immunodeficiency, and autoimmune diseases. We investigated the DNA methylation profiles in patients with atopic dermatitis (AD). Messenger RNA (mRNA) levels for DNA methyltransferase-1 (DNMT-1) in peripheral blood mononuclear cells (PBMC) were examined using a real-time quantitative polymerase chain reaction method. The levels of DNMT-1 mRNA were significantly lower in PBMC from the AD patients who had higher serum IgE levels compared with normal controls. Our observations suggest that suppression of DNMT-1 might be related to the pathogenesis of AD, especially in whom serum IgE level is high. This is the first report of DNMT-1 expression in AD patients.

Application of immunoreaction enhancer solutions to an enzyme-linked immunosorbent assay for antigen-specific IgE in mice immunized with recombinant major mite allergens or ovalbumin.

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.

Crucial commitment of proteolytic activity of a purified recombinant major house dust mite allergen Der p1 to sensitization toward IgE and IgG responses.

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-gamma. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

Tenascin-C is upregulated in the skin lesions of patients with atopic dermatitis.

BACKGROUND: Tenascin-C is a large, hexameric extracellular matrix glycoprotein that is expressed during embryogenesis, carcinogenesis and wound healing. In normal adult human skin the expression level of tenascin-C is low, but levels are elevated in skin tumors and rise significantly in the dermal compartment during wound healing. Although the expression of tenascin-C could be upregulated by inflammatory cytokines, the role of tenascin-C in atopic dermatitis (AD) is still unclear. OBJECTIVE: To identify genes that plays a role in AD. METHODS: We screened for differentially expressed genes in lesional and non-lesional skin of AD patients using DNA microarray. Then we monitored with quantitative PCR the expression of the novel disease related genes in human keratinocytes or pinnae from NC/Nga mice. RESULTS: We found that tenascin-C gene expression was expressed at higher levels in lesional skin compared to non-lesional skin of the patients, whereas it was not upregulated in the skin of psoriatic patients or healthy controls. In human cultured keratinocytes, tenascin-C was markedly upregulated by IL-4 and IL-13, and moderately upregulated by IFN-gamma. Tenascin-C expression was also upregulated in the AD-like skin lesions induced in NC/Nga mice ears by intradermal injection of mite antigen, and this upregulation was inhibited by prednisolone. CONCLUSION: These results suggest that upregulation of the tenascin-C expression is specific to AD lesions, and that tenascin-C may therefore play a critical role in regulating the underlining inflammatory processes, which are involved in the pathology of AD.

Cystatin A inhibits IL-8 production by keratinocytes stimulated with Der p 1 and Der f 1: biochemical skin barrier against mite cysteine proteases.

BACKGROUND: Der p 1 and Der f 1 are the most immunodominant allergens produced by house dust mites and are suspected to be involved in the pathogenesis of allergy through their cysteine protease activity. However, stimulation of keratinocytes by these protease allergens and protective systems in the skin against them have not been well investigated. OBJECTIVE: We purified and identified the dominant skin-derived inhibitor against the proteolytic activities of these allergens and analyzed its effect on keratinocyte activation. METHODS: Recombinant allergens were used for the experiments. We analyzed whether human sweat inhibits the enzymatic activities of Der p 1 and Der f 1 and used sweat as the skin-derived material to isolate the inhibitor. The inhibitor was purified by means of column chromatography and subsequently identified by means of protein sequencing and immunoblotting. Keratinocytes were stimulated with the allergens in the absence or presence of the inhibitor, and the concentration of secreted IL-8 was measured. RESULTS: Sweat inhibited the proteolytic activities of Der p 1 and Der f 1. The sweat inhibitor was identified as cystatin A. The stimulation of normal human keratinocytes and the human keratinocyte cell line HaCaT with these protease allergens upregulated IL-8 secretion, and addition of cystatin A blocked this upregulation. Normal human keratinocytes secreted cystatin A into the medium. CONCLUSIONS: The proteolytic activity of Der p 1 and Der f 1 stimulates human keratinocytes in vitro. Cystatin A produced by keratinocytes is the dominant biochemical skin barrier that eliminates the enzymatic activity of these mite cysteine proteases and prevents them from stimulating keratinocytes.

Polymorphisms in the Fc epsilon RI beta promoter region affecting transcription activity: a possible promoter-dependent mechanism for association between Fc epsilon RI beta and atopy.

The beta subunit of the high-affinity IgE receptor (FcepsilonRI) plays an important role in IgE-mediated allergic reactions as an amplifier for cell surface expression and signal transduction of FcepsilonRI. FcepsilonRIbeta is presumed to be one of the genes linked with atopic diseases. However, the validity of the associations previously found between single nucleotide polymorphisms (SNPs) in FcepsilonRIbeta and atopic diseases is questionable. In the present study, we found correlation between the SNP of FcepsilonRIbeta at +6960A/G, resulting in a Glu237Gly amino acid substitution, and the cell surface expression level of FcepsilonRI on blood basophils, although it has been shown that the Glu237Gly mutation itself does not affect the surface expression or function of FcepsilonRI. We additionally found four SNPs in the promoter region of FcepsilonRIbeta, among which -426T/C and -654C/T were tightly linked with +6960A/G. Reporter plasmids carrying the -426C and -654T promoter displayed higher transcriptional activity than those carrying the -426T and -654C promoter. We found that transcription factor YY1 preferentially bound and transactivated the -654T promoter. Furthermore, expression of FcepsilonRI beta-chain mRNA in basophils from individuals who have the minor heterozygous genotype was significantly higher than that of the major homozygous genotype. These results suggest that the SNPs in the FcepsilonRIbeta promoter are causally linked with atopy via regulation of FcepsilonRI expression.

Gene expression of enzymes for tryptophan degradation pathway is upregulated in the skin lesions of patients with atopic dermatitis or psoriasis.

BACKGROUND: Atopic dermatitis (AD) and psoriasis are common inflammatory skin diseases. Although many reports implicate Th2 cytokines in the pathophysiology of AD and Th1 cytokines in psoriasis, the precise etiology of these diseases remains elusive. OBJECTIVE: We investigated novel AD- or psoriasis-related genes to further understand the pathogenesis of these diseases. METHODS: We performed a comprehensive analysis of mRNA expression in skin biopsies from AD or psoriasis patients using DNA microarrays. Quantitative PCR was then used to monitor the expression of novel disease-related genes in human keratinocytes or pinnae from NC/Nga mice. RESULTS: Levels of mRNA for IDO (indoleamine 2,3-dioxygenase) and kynureninase, enzymes constituting the tryptophan degradation pathway, were found to be upregulated in the skin lesions as compared to the uninvolved skin of patients with AD or psoriasis. Expression of these two genes was induced in human epidermal keratinocytes stimulated with IFN-gamma in vitro. Moreover, in NC/Nga mice, the expression of kynureninase mRNA in the ear skin was induced following development of AD-like skin lesions. CONCLUSION: The tryptophan degradation pathway may play an important role in the pathophysiology of AD and psoriasis.

A novel -66T/C polymorphism in Fc epsilon RI alpha-chain promoter affecting the transcription activity: possible relationship to allergic diseases.

We found a novel polymorphism, -66T/C, in the promoter region of human FcepsilonRIalpha, the specific component of the high affinity receptor for IgE (FcepsilonRI), which is essential for the cell surface expression of FcepsilonRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at -66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (-80/-59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous -66T/C genotype, while most of allergic individuals have homozygous -66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcepsilonRIalpha polymorphism related to the allergic diseases.