RESUMO
How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1-specific IgG1 and eosinophilia. Although papain-, Der f 1-, and Der p 1-stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor-treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33-deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33-deficient mice. We demonstrated IL-33 release, subsequent IL-33-dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage(-)CD25(+)CD44(+) innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33-type 2 innate lymphoid cell-IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33-responsive innate cells in protease-dependent allergic airway inflammation.
Assuntos
Imunidade Adaptativa/imunologia , Alérgenos/imunologia , Cisteína Proteases/imunologia , Hipersensibilidade/imunologia , Imunidade Inata/imunologia , Interleucinas/imunologia , Pulmão/imunologia , Animais , Formação de Anticorpos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Basófilos/imunologia , Cisteína Endopeptidases/imunologia , Feminino , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Inflamação/imunologia , Interleucina-13/imunologia , Interleucina-33 , Interleucina-5/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Papaína/imunologia , Eosinofilia Pulmonar/imunologiaRESUMO
PURPOSE: To investigate the effect of tacrolimus on chemokine production by corneal myofibroblasts compared with that of cyclosporine or dexamethasone. METHODS: We investigated the expression of FK506-binding protein 12, calcineurin, and nuclear factor of activated T cells in corneal myofibroblasts by immunocytochemistry and flow cytometry. Next, we investigated whether toll-like receptor 9 ligand, CpG-DNA, or toll-like receptor 3 ligand, poly (I:C), induced the production of chemokines such as interleukin (IL) 8, Gro-α, and IL-6 by corneal myofibroblasts using enzyme-linked immunosorbent assay. Finally, we assessed the effect of tacrolimus on the production of these chemokines compared with that of cyclosporine or dexamethasone. RESULTS: FK506-binding protein 12, calcineurin, and nuclear factor of activated T cells were constantly expressed by cultured corneal myofibroblasts. CpG DNA and poly (I:C) enhanced the production of IL-8, Gro-α, and IL-6 by corneal myofibroblasts. At a concentration 10 nM/L or higher, dexamethasone strongly inhibited the production of these chemokines. In contrast, cyclosporine concentrations of 10 ng/mL or more enhanced the production of these chemokines by cells stimulated with poly (I:C). On the other hand, tacrolimus (at 10 ng/mL or more) inhibited the production of these chemokines by corneal myofibroblasts stimulated with poly (I:C) but not with CpG-DNA. CONCLUSIONS: These results suggest that tacrolimus and dexamethasone, but not cyclosporine, have a direct immunosuppressive effect on corneal myofibroblasts. Therefore, when inflammatory diseases of the ocular surface are treated by tacrolimus, especially in combination with steroids, caution with regard to the risk of corneal infection may be needed.
Assuntos
Quimiocinas/metabolismo , Córnea/efeitos dos fármacos , Imunossupressores/farmacologia , Miofibroblastos/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Idoso , Calcineurina/metabolismo , Células Cultivadas , Córnea/metabolismo , Ilhas de CpG/genética , Ciclosporina/farmacologia , Citocinas/metabolismo , Dexametasona/farmacologia , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Poli I-C/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/farmacologiaRESUMO
To determine the role of TGF-ß(1) in the tissue eosinophilia associated with vernal keratoconjunctivitis (VKC), we investigated the immunohistochemical expression of TGF-ß(1) and TGF-ß(1)-related proteins in giant papillae obtained from VKC patients. We also investigated the effect of TGF-ß(1) on production of eotaxin by cultured conjunctival and corneal fibroblasts using ELISA. Finally, the effects of glucocorticoids, cyclosporine, and tacrolimus on eotaxin production by corneal fibroblasts were assessed. Our investigations revealed that eosinophils expressing TGF-ß(1) and TGF-ß(1)-related proteins (such as phosphorylated Smad2, integrin αvß(6), α-smooth muscle actin, type I procollagen, and tenascin-C) were expressed in the giant papillae. TGF-ß(1) and IL-4/IL-13 caused a synergistic increase of eotaxin production in cultured conjunctival and corneal fibroblasts. This effect of TGF-ß(1) and IL-4/IL-13 was inhibited by glucocorticoids, but neither by cyclosporine nor by tacrolimus. In conclusion, TGF-ß(1) has an important role in the tissue eosinophilia associated with VKC.
Assuntos
Conjuntivite Alérgica/metabolismo , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Actinas/metabolismo , Adolescente , Adulto , Idoso , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Quimiocina CCL11/biossíntese , Criança , Colágeno Tipo I/metabolismo , Túnica Conjuntiva/citologia , Córnea/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glucocorticoides/farmacologia , Humanos , Técnicas Imunoenzimáticas , Imunossupressores/farmacologia , Integrinas/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Proteína Smad2/metabolismo , Tenascina/metabolismo , Adulto JovemRESUMO
To evaluate the effects of the transcription factor GATA-1 on determining cell fate between dendritic cell (DC) and mast cell (MC) lineages, GATA-1 was exogenously expressed in bone marrow-derived (BM) DCs. Exogenous expression of GATA-1 by a retrovirus in BMDCs inhibited expression of CD11c, CD80, CD86, and major histocompatibility complex class II with suppression of antigen-presenting ability and morphological changes toward granulocyte-like cells. Transcription of MC proteases and c-kit was markedly upregulated by GATA-1. Expression of IRF-4 and -8 was markedly suppressed, whereas PU.1 mRNA level was not affected by GATA-1. Chromatin immunoprecipitation assay showed that recruitment of PU.1 on the IRF-8 promoter was reduced in GATA-1-expressing DCs. These results indicate that GATA-1 suppresses PU.1 function but not PU.1 transcription. Thus, GATA-1 appears to determine cell fate by regulating several cell-specific transcription factors.
Assuntos
Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica/fisiologia , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Western Blotting , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Imunoprecipitação da Cromatina , Citometria de Fluxo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To investigate the expression and function of fibroblast growth factor-inducible 14 (Fn14) in human retinal pigment epithelial cells. METHODS: A human retinal pigment epithelial cell line (RPE cells: ARPE-19) was used. Expression of Fn14 protein was assessed by flow cytometry. An antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of RPE cells cultured with or without stimulation by TWEAK and/or TGF-beta(1). To explore the mechanism by which TWEAK stimulates RPE cells, we investigated phosphorylation of MAP kinase in TWEAK-stimulated cells. We also investigated whether TWEAK induced the migration of RPE cells by performing an in vitro wound assay. RESULTS: RPE cells showed constitutive surface expression of Fn14 protein. FGF, VEGF, and TGF-beta(1) did not induce Fn14 expression by RPE cells. TWEAK increased the production of IL-8 and MCP-1 by RPE cells via Fn14, and TGF-beta(1) augmented TWEAK-induced production of these chemokines. TWEAK induced the phosphorylation of MAP kinase in RPE cells and promoted the migration of these cells via MAP kinase. CONCLUSION: TWEAK/Fn14 interaction may have proinflammatory effects in RPE cells.
Assuntos
Inflamação , Receptores do Fator de Necrose Tumoral/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Fatores de Necrose Tumoral/metabolismo , Linhagem Celular , Movimento Celular , Quimiocina CCL2/biossíntese , Citocina TWEAK , Fatores de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Interleucina-8/biossíntese , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia , Receptor de TWEAK , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.
Assuntos
Adjuvantes Imunológicos , Cupressaceae/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Imunoglobulina E/imunologia , Pólen/imunologia , Administração Intranasal , Alérgenos/imunologia , Alérgenos/farmacologia , Ambrosia/imunologia , Animais , Betula/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Feminino , Imunoglobulina E/sangue , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Poaceae/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
PU.1 is a myeloid- and lymphoid-specific transcription factor that serves many important roles in the development and specific gene regulation of hematopoietic lineages. Mast cells (MC) and dendritic cells (DC) express PU.1 at low and high levels, respectively. Previously, we found that enforced expression of PU.1 in MC resulted in acquisition of DC-like characteristics, including repression of several IgE-mediated responses due to reduced expression of IgE-signaling related molecules. In contrast, PU.1 overexpression in MC up-regulated TNF-alpha production in response to IgE- and LPS-stimulation suggesting that PU.1 positively regulates TNF-alpha expression. However, the role of PU.1 in the expression of TNF-alpha is largely unknown. In the present study, the effects of PU.1 on the TNF-alpha promoter in mouse bone marrow-derived (BM) MC and DC were studied. Real-time PCR, ELISA, and chromatin immunoprecipitation assays indicated that the kinetics and magnitude of TNF-alpha expression levels following LPS- or IgE-stimulation are related to the amount of PU.1 binding to the promoter. In brief, higher and delayed up-regulation of TNF-alpha promoter function was observed in DC, whereas there were lower and rapid responses in MC. When PU.1-overexpressing retrovirus vector was introduced into MC, the amount of PU.1 recruited to the TNF-alpha promoter markedly increased. The knockdown of PU.1 in BMDC by siRNA resulted in a reduction of TNF-alpha protein produced from LPS-stimulated BMDC. These observations indicate that PU.1 transactivates the TNF-alpha promoter and that the amount of PU.1 binding on the promoter is associated with promoter activity.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , Técnicas de Silenciamento de Genes , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Transativadores/genética , Transcrição GênicaRESUMO
Three frequent genetic polymorphisms in the human high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) were shown to be associated with allergic disorders and/or total serum IgE levels in allergic patients. Two of these were previously demonstrated to affect FcepsilonRIalpha expression while the third -18483A>C (rs2494262) has not yet been subjected to functional studies. We hypothesized that the -18483A>C variant affects transcriptional activity of the FcepsilonRIalpha distal promoter in monocytes in which FcepsilonRIalpha transcription is driven through that regulatory region. Indeed, we confirmed preferential binding of the YY1 transcription factor to the -18483C allele, resulting in lower transcriptional activity when compared with the -18483A allele.
Assuntos
Polimorfismo de Nucleotídeo Único , Receptores de IgE/genética , Transcrição Gênica , Fator de Transcrição YY1/metabolismo , Povo Asiático/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , População Branca/genéticaRESUMO
Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN-gamma was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr(-/-) BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN-gamma stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBPalpha in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.
Assuntos
Interferon gama/imunologia , Mastócitos/imunologia , Monócitos/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Meios de Cultivo Condicionados/farmacologia , Genes MHC da Classe II , Humanos , Interferon gama/farmacologia , Interleucina-3/farmacologia , Interleucina-4/farmacologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interferon/metabolismo , Fator de Células-Tronco/farmacologia , Transativadores/genética , Fator de Necrose Tumoral alfa/farmacologia , Tirosina Quinase 3 Semelhante a fms/farmacologia , Receptor de Interferon gamaRESUMO
The interaction of fibroblast growth factor-inducible 14 (Fn14) and, its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to be important in wound healing of tissues. However, to our knowledge, expression and function of Fn14 in corneal myofibroblasts, which have a crucial role in wound healing of corneal stroma, has not been investigated. In this study, we investigated the expression and function of Fn14 in corneal myofibroblasts. Expression of Fn14 protein was assessed by flow cytometry. Corneal myofibroblasts showed strong expression of Fn14 protein, while keratocytes did not. TGF-beta(1) promoted the differentiation of keratocytes into corneal myofibroblasts, and induced Fn14 expression. These data reveal that keratocytes phenotype determines the level of Fn14 expression. ELISA was used to detect chemokines and matrix metalloproteinases in the supernatant of corneal myofibroblasts cultured with or without stimulation by TWEAK and/or TGF-beta(1). TWEAK increased the production of IL-8, MCP-1, and RANTES by corneal myofibroblasts via Fn14. TGF-beta(1) augmented the TWEAK-induced production of these chemokines. TWEAK also increased the production of MMP-1 and -3 by corneal myofibroblasts via Fn14, while TGF-beta(1) inhibited this effect of TWEAK on MMP production. TWEAK-induced phosphorylation of NF-kappaB and MAP kinase in corneal myofibroblasts. Furthermore, TWEAK partially inhibited the differentiation of keratocytes into corneal myofibroblasts promoted by TGF-beta(1). These data suggest that the Fn14/TWEAK system may have several roles in wound healing by corneal myofibroblasts. In the future, modulation of the TWEAK/Fn14 system may become a novel approach for control corneal wound healing.
Assuntos
Córnea/metabolismo , Proteínas do Olho/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Fibroblastos/metabolismo , Actinas/metabolismo , Idoso , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Quimiocinas/biossíntese , Córnea/citologia , Córnea/efeitos dos fármacos , Citocina TWEAK , Interações Medicamentosas , Proteínas do Olho/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Fator de Crescimento Transformador beta1/farmacologia , Fatores de Necrose Tumoral/farmacologia , Cicatrização/fisiologiaRESUMO
It was shown previously that bee-collected pollen (bee pollen, BP), inhibited in vitro murine mast cell activation. This study further analysed the antiallergic effect of BP in vivo by measuring cutaneous mast cell activation using a passive cutaneous anaphylaxis reaction. Daily oral administration of BP to mice, dose-dependently reduced the cutaneous mast cell activation elicited by IgE and specific antigens. Administration of BP also reduced the plasma concentration of malondialdehyde (MDA), an indicator of lipid peroxidation. The inhibitory effect of BP was mostly in a lipid- but not in water-soluble fraction. The HPLC analysis of isoflavones in BP revealed that genistein was a major isoflavone. However, administration of genistein alone at the concentration found in BP, did not show an inhibitory effect as observed in whole BP, suggesting that component(s) other than genistein would be responsible for the inhibitory effect of BP. These results first reveal that lipid-soluble components of BP exert an antiallergic action by inhibiting the FcåRI-mediated cutaneous mast cell activation.
Assuntos
Antialérgicos/farmacologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Pólen/imunologia , Animais , Abelhas , Genisteína/farmacologia , Peroxidação de Lipídeos , Masculino , Malondialdeído/sangue , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: In studies on allergies in mouse models, IgE production is an essential parameter to be evaluated. Here, we examine the effect of commercially available immunoreaction enhancer solutions and different blocking reagents in enzyme-linked immunosorbent assay (ELISA) for total or antigen-specific murine IgE in order to improve the assays. METHODS: Sera from mice immunized with recombinant house dust mite major allergens, Der f 1 and Der p 1, were used for the assays. Total IgE was measured by sandwich ELISA using monoclonal antibodies against murine IgE. Antigen-specific IgE was assayed using allergen-coated plates. Sensitivity or signal intensity in ELISA was compared among conditions differing in the use of enhancer solutions, blocking reagents, or monoclonal antibodies, and incubation time. RESULTS: Use of enhancer solutions improved the sensitivity of ELISA for total IgE by approximately 30-fold of that using a conventional buffer. A blocking reagent caused more unwanted enhancement of the background signal in blank wells in ELISA for total IgE compared with another blocking reagent, however, improved signal intensity in ELISA for antigen-specific ELISA without significant enhancement of the background signal. Optimal assay conditions were determined. CONCLUSIONS: Enhancer solutions are effective in improving ELISAs for total and antigen-specific murine IgE. Selection of blocking reagents was important to decrease unwanted enhancement of background signals and was effective in enhancing signals for positive samples. The ELISAs improved in this study are useful for the study of allergies in mouse models.
Assuntos
Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Hipersensibilidade/imunologia , Imunoglobulina E/análise , Imunoglobulina E/imunologia , Alérgenos/genética , Alérgenos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos/genética , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes , Cisteína Endopeptidases , Feminino , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Imunoglobulina E/isolamento & purificação , Indicadores e Reagentes/química , Camundongos , Camundongos Endogâmicos CBA , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , VacinaçãoRESUMO
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 (Fc epsilon RI)-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-(4beta-->8)-epicatechin-(4beta-->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 Fc epsilon RI 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 Fc epsilon RI-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 Fc epsilon RI-mediated mast cell activation by inhibiting intracellular signaling pathways. These observations provide evidence for the anti-allergenic effects of the procyanidin-enriched apple extract.
Assuntos
Biflavonoides/farmacologia , Catequina/farmacologia , Malus/química , Mastócitos/efeitos dos fármacos , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Receptores de IgE/metabolismo , Animais , Biflavonoides/administração & dosagem , Catequina/administração & dosagem , Degranulação Celular , Citocinas/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/imunologia , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Extratos Vegetais/química , Proantocianidinas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Tirosina/metabolismoRESUMO
Bee-collected pollen (bee pollen [BP]) has been used as a folk medicine for centuries against various diseases, including allergy. There is no study elucidating how BP exerts such an anti-allergic effect. Since mast cells play a central role in the pathogenesis of various allergic diseases, we investigated the effect of BP on mast cell activation elicited by the Fc immunoglobulin E (IgE) receptor (Fc epsilon RI)-mediated pathways. The in vivo effect of orally administered BP on cutaneous mast cell activation was examined by passive cutaneous anaphylaxis reaction. In vitro mast cell degranulation and IgE binding to mast cells and the status of protein tyrosine phosphorylation were examined using bone marrow-derived mast cells. Daily oral administration of BP to mice significantly reduced the cutaneous mast cell activation elicited by IgE and specific antigens. BP also reduced in vitro mast cell degranulation and tumor necrosis factor-alpha production by inhibiting IgE binding to Fc epsilon RI on mast cells. The inhibitory effect of BP on mast cell degranulation by preventing IgE binding was confirmed by the reduced levels of protein tyrosine phosphorylation, which occurred as downstream events in activated mast cells via Fc epsilon RI. These results first revealed that the anti-allergic action of BP was exerted by inhibiting the Fc epsilon RI-mediated activation of mast cells, which plays important roles, not only in the early phase, but also in the late phase of allergic reactions.
Assuntos
Abelhas , Degranulação Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Pólen/química , Animais , Células da Medula Óssea , Citocinas/biossíntese , Imunoglobulina E/efeitos dos fármacos , Imunoglobulina E/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/fisiologiaRESUMO
The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.
Assuntos
Queratinócitos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fator de Transcrição Sp1/fisiologia , Transcrição Gênica , Cálcio/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Doença de Darier/etiologia , Doença de Darier/terapia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Pênfigo Familiar Benigno/terapia , Regiões Promotoras Genéticas , RNA Interferente Pequeno/farmacologia , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genéticaRESUMO
PURPOSE: To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts. METHODS: Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis. RESULTS: Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9. CONCLUSIONS: This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.
Assuntos
Córnea/metabolismo , Expressão Gênica/fisiologia , Receptor 3 Toll-Like/genética , Receptor Toll-Like 9/genética , Idoso , Antígenos CD34 , Biomarcadores , Diferenciação Celular , Células Cultivadas , Quimiocinas/metabolismo , Córnea/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Fagocitose , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases , Proteoglicanas/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doadores de Tecidos , Receptor 3 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Retinal pigment epithelial cells (RPE cells) are key players in the first-line defense against invading organisms such as viruses and bacteria. The interaction between RPE cells and viral or bacterial components is very important for clearance of these organisms. Toll-like receptors are a family of recognition receptors involved in innate immunity. Each TLR acts as a primary sensor of conserved microbial components and drives the induction of specific biological responses. TLR 3 is involved in the recognition of viral components, such as double-stranded RNA (dsRNA) and poly(I:C), while TLR 9 recognizes viral or bacterial DNA without methylation at CpG motifs. In the present study, we investigated the expression and function of TLR 3 and 9 in RPE cells. PCR analysis revealed expression of genes for TLR 3 and 9 in RPE cells. Expression of TLR 3 and 9 protein was detected in RPE cells by flow cytometry. TLR 3 and 9 showed strong intracellular expression. To detect angiogenetic factors produced by RPE cells, culture supernatant was examined with the Human Angiogenesis Antibody Array, which can simultaneously detect 20 different angiogenetic factors including cytokines, chemokines, soluble cytokine receptors, and growth factors. RPE cells showed high production of interleukin-8 (IL-8) and monocyte chemotactic protein-I (MCP-I). Furthermore, stimulation of RPE cells with the dsRNA analogue poly(I:C) enhanced the secretion of IL-8 and MCP-I, as well as enhancing the expression of junctional adhesion molecule-I (Jam-I) and intracellular adhesion molecule-I (ICAM-I), and promoted the adhesion of monocyte to these cells. In contrast, stimulation with the CpG-DNA motif only enhanced the secretion of IL-8. However, CpG-DNA motif enhanced phagocytosis in RPE cells. These results may indicate that TLR 3 and 9 play a distinct role in the inflammatory response that clears viruses from the retina.
Assuntos
Epitélio Pigmentado Ocular/metabolismo , RNA/genética , Receptor 3 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Adesão Celular , Linhagem Celular , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Humanos , Interleucina-8/metabolismo , Fagocitose , Epitélio Pigmentado Ocular/citologia , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Cisteína Endopeptidases/metabolismo , Dermatophagoides pteronyssinus/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Animais , Antígenos de Dermatophagoides/administração & dosagem , Proteínas de Artrópodes , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Temperatura Alta , Hidrólise , Imunização/métodos , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Desnaturação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/isolamento & purificação , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.
Assuntos
Fator de Transcrição GATA1/antagonistas & inibidores , Regulação da Expressão Gênica/genética , Mastócitos/imunologia , Proteínas Nucleares/farmacologia , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/genética , Fatores de Transcrição/farmacologia , Transcrição Gênica , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/imunologia , Receptores de IgE/imunologia , Relação Estrutura-Atividade , Fatores de Transcrição/biossíntese , Fatores de Transcrição/imunologiaRESUMO
Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.