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2.
Front Allergy ; 5: 1373511, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38601026

RESUMO

Mast cells are activated through a variety of different receptors to release preformed granules and mediators synthesized de novo. However, the physiology and function of mast cells are not fully understood. Traditional studies of mast cell activation in humans have utilized cultures of tissue-derived mast cells including CD34+ progenitor cells or well-characterized commercially available cell lines. One limitation of these methods is that mast cells are no longer in a natural state. Therefore, their applicability to human skin disorders may be limited. Human skin explant models have been utilized to investigate the short-term effects of cell mediators, drugs, and irritants on skin while avoiding the ethical concerns surrounding in vivo stimulation studies with non-approved agents. Nonetheless, few studies have utilized intact human tissue to study mast cell degranulation. This "Methods" paper describes the development and application of an intact skin explant model to study human mast cell activation. In this manuscript, we share our protocol for setting up ex vivo human skin explants and describe the results of stimulation experiments and techniques to minimize trauma-induced histamine release. Skin explants were generated using de-identified, full-thickness, non-diseased skin specimens from plastic and reconstructive surgeries. Results were reproducible and demonstrated FcɛRI- and MRGPRX2-induced mediator release which was inhibited with the use of a BTK inhibitor and QWF, respectively. Thus, this explant model provides a quick and accessible method of assessing human skin mast cell activation and inhibition.

3.
Allergy Asthma Proc ; 44(3): 208-211, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37160754

RESUMO

A 56-year-old man with well-controlled human immunodeficiency virus, anxiety, depression, and hypercholesterolemia developed acute urticaria, lip angioedema, and respiratory distress after consumption of a cheeseburger, French fries, lemonade, and ibuprofen. He was evaluated in the emergency department and, during admission, developed asystole, diaphoresis, pallor, and a brief episode of posturing that was treated with two doses of epinephrine. Results of the initial workup with electrocardiogram, troponin, complete blood cell count, and comprehensive metabolic panel were normal. He was subsequently evaluated by an allergist for further recommendations.


Assuntos
Infecções por HIV , Parada Cardíaca , Masculino , Humanos , Pessoa de Meia-Idade , Parada Cardíaca/diagnóstico , Parada Cardíaca/etiologia , Morte Súbita Cardíaca/etiologia , Epinefrina , Alergistas , Infecções por HIV/complicações , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico
4.
J Allergy Clin Immunol ; 147(6): 2271-2280.e8, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33713769

RESUMO

BACKGROUND: The mechanisms underlying disease pathogenesis in chronic spontaneous urticaria (CSU) and improvement with omalizumab are incompletely understood. OBJECTIVES: This study sought to examine whether the rate of clinical remission is concordant with baseline basophil features or the rate of change of IgE-dependent functions of basophils and/or plasmacytoid dendritic cells during omalizumab therapy. METHODS: Adults (n = 18) with refractory CSU were treated with omalizumab 300 mg monthly for 90 days. Subjects recorded daily urticaria activity scores, and clinical assessments with blood sampling occurred at baseline and on days 1, 3, 6, 10, 20, 30, 60, and 90 following omalizumab. At baseline, subjects were categorized by basophil functional phenotypes, determined by in vitro histamine release (HR) responses to anti-IgE antibody, as CSU-responder (CSU-R) or CSU-non-responder (CSU-NR), as well as basopenic (B) or nonbasopenic (NB). RESULTS: CSU-R/NB subjects demonstrated the most rapid and complete symptom improvement. By day 6, CSU-R/NB and CSU-NR/NB had increased anti-IgE-mediated basophil HR relative to baseline, and these shifts did not correlate with symptom improvement. In contrast, CSU-NR/B basophil HR did not change during therapy. The kinetics of the decrease in surface IgE/FcεRI was similar in all 3 phenotypic groups and independent of the timing of the clinical response. Likewise, plasmacytoid dendritic cells' surface IgE/FcεRI decline and TLR9-induced IFN-α responses did not reflect clinical change. CONCLUSIONS: Changes in basophil IgE-based HR, surface IgE, or FcεRI bear no relationship to the kinetics in the change in clinical symptoms. Baseline basophil count and basophil functional phenotype, as determined by HR, may be predictive of responsiveness to omalizumab.


Assuntos
Antialérgicos/uso terapêutico , Basófilos/imunologia , Urticária Crônica/tratamento farmacológico , Urticária Crônica/etiologia , Omalizumab/uso terapêutico , Antialérgicos/administração & dosagem , Antialérgicos/efeitos adversos , Basófilos/metabolismo , Biomarcadores , Doença Crônica , Urticária Crônica/diagnóstico , Urticária Crônica/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Liberação de Histamina , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Omalizumab/administração & dosagem , Omalizumab/efeitos adversos , Fenótipo , Fatores de Tempo , Resultado do Tratamento
7.
J Allergy Clin Immunol ; 135(5): 1283-92, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25542883

RESUMO

BACKGROUND: Studies suggest that oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) for food allergy hold promise; however, the immunologic mechanisms underlying these therapies are not well understood. OBJECTIVE: We sought to generate insights into the mechanisms and duration of suppression of immune responses to peanut during immunotherapy. METHODS: Blood was obtained from subjects at baseline and at multiple time points during a placebo-controlled trial of peanut OIT and SLIT. Immunologic outcomes included measurement of spontaneous and stimulated basophil activity by using automated fluorometry (histamine) and flow cytometry (activation markers and IL-4), measurement of allergen-induced cytokine expression in dendritic cell (DC)-T-cell cocultures by using multiplexing technology, and measurement of MHC II and costimulatory molecule expression on DCs by using flow cytometry. RESULTS: Spontaneous and allergen-induced basophil reactivity (histamine release, CD63 expression, and IL-4 production) were suppressed during dose escalation and after 6 months of maintenance dosing. Peanut- and dust mite-induced expression of TH2 cytokines was reduced in DC-T-cell cocultures during immunotherapy. This was associated with decreased levels of CD40, HLA-DR, and CD86 expression on DCs and increased expression of CD80. These effects were most striking in myeloid DC-T-cell cocultures from subjects receiving OIT. Many markers of immunologic suppression reversed after withdrawal from immunotherapy and in some cases during ongoing maintenance therapy. CONCLUSION: OIT and SLIT for peanut allergy induce rapid suppression of basophil effector functions, DC activation, and TH2 cytokine responses during the initial phases of immunotherapy in an antigen-nonspecific manner. Although there was some interindividual variation, in many patients suppression appeared to be temporary.


Assuntos
Dessensibilização Imunológica , Hipersensibilidade a Amendoim/imunologia , Hipersensibilidade a Amendoim/terapia , Administração Oral , Administração Sublingual , Alérgenos/administração & dosagem , Alérgenos/imunologia , Arachis/efeitos adversos , Basófilos/imunologia , Basófilos/metabolismo , Biomarcadores , Citocinas/metabolismo , Células Dendríticas/imunologia , Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Interleucina-4/metabolismo , Hipersensibilidade a Amendoim/genética , Projetos Piloto , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tetraspanina 30/metabolismo , Resultado do Tratamento
8.
Clin Immunol ; 155(1): 47-59, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25173802

RESUMO

Sublingual (SLIT) and oral immunotherapy (OIT) are promising treatments for food allergy, but underlying mechanisms are poorly understood. Dendritic cells (DCs) induce and maintain Th2-type allergen-specific T cells, and also regulate innate immunity through their expression of Toll-like receptors (TLRs). We examined how SLIT and OIT influenced DC innate and adaptive immune responses in children with IgE-mediated cow's milk (CM) allergy. SLIT, but not OIT, decreased TLR-induced IL-6 secretion by myeloid DCs (mDCs). SLIT and OIT altered mDC IL-10 secretion, a potent inhibitor of FcεRI-dependent pro-inflammatory responses. OIT uniquely augmented IFN-α and decreased IL-6 secretion by plasmacytoid DCs (pDCs), which was associated with reduced TLR-induced IL-13 release in pDC-T cell co-cultures. Both SLIT and OIT decreased Th2 cytokine secretion to CM in pDC-T, but not mDC-T, co-cultures. Therefore, SLIT and OIT exert unique effects on DC-driven innate and adaptive immune responses, which may inhibit allergic inflammation and promote tolerance.


Assuntos
Alérgenos/administração & dosagem , Alérgenos/uso terapêutico , Hipersensibilidade a Leite/terapia , Administração Oral , Administração Sublingual , Adolescente , Células Cultivadas , Criança , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas , Método Duplo-Cego , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Peptidoglicano/farmacologia , Receptores de IgE/genética , Receptores de IgE/metabolismo , Linfócitos T/imunologia , Linfócitos T/fisiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo
10.
Sci Transl Med ; 5(195): 195ra94, 2013 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-23884466

RESUMO

Transforming growth factor-ß (TGFß) is a multifunctional cytokine that plays diverse roles in physiologic processes as well as human disease, including cancer, heart disease, and fibrotic disorders. In the immune system, TGFß regulates regulatory T cell (Treg) maturation and immune homeostasis. Although genetic manipulation of the TGFß pathway modulates immune tolerance in mouse models, the contribution of this pathway to human allergic phenotypes is not well understood. We demonstrate that patients with Loeys-Dietz syndrome (LDS), an autosomal dominant disorder caused by mutations in the genes encoding receptor subunits for TGFß, TGFBR1 and TGFBR2, are strongly predisposed to develop allergic disease, including asthma, food allergy, eczema, allergic rhinitis, and eosinophilic gastrointestinal disease. LDS patients exhibited elevated immunoglobulin E levels, eosinophil counts, and T helper 2 (TH2) cytokines in their plasma. They had an increased frequency of CD4(+) T cells that expressed both Foxp3 and interleukin-13, but retained the ability to suppress effector T cell proliferation. TH2 cytokine-producing cells accumulated in cultures of naïve CD4(+) T cells from LDS subjects, but not controls, after stimulation with TGFß, suggesting that LDS mutations support TH2 skewing in naïve lymphocytes in a cell-autonomous manner. The monogenic nature of LDS demonstrates that altered TGFß signaling can predispose to allergic phenotypes in humans and underscores a prominent role for TGFß in directing immune responses to antigens present in the environment and foods. This paradigm may be relevant to nonsyndromic presentations of allergic disease and highlights the potential therapeutic benefit of strategies that inhibit TGFß signaling.


Assuntos
Predisposição Genética para Doença/genética , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Linfócitos T CD4-Positivos/metabolismo , Citocinas/sangue , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Interleucina-13/metabolismo , Síndrome de Loeys-Dietz/genética , Síndrome de Loeys-Dietz/imunologia , Síndrome de Loeys-Dietz/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Linfócitos T Reguladores/metabolismo
11.
J Allergy Clin Immunol ; 125(4): 896-901.e6, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20132969

RESUMO

BACKGROUND: Dendritic cells (DCs) and other professional antigen-presenting cells express a variant of the high-affinity IgE receptor known as alphagamma(2), which, on the basis of in vitro findings, has long been implicated to function in facilitating allergen uptake and presentation to T(H) cells. OBJECTIVES: To use omalizumab as an in vivo tool to neutralize IgE binding to circulating dendritic cells and to assess whether this results in altered DC-dependent T-cell responsiveness to allergen ex vivo. METHODS: Subjects with cat allergy were enrolled in a 3.5-month, double blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing for allergic asthma. Blood plasmacytoid and myeloid DCs were assessed at baseline and posttreatment for expression of surface IgE, FcepsilonRIalpha, and induction of CD4(+)T-cell proliferation and cytokine responses to cat allergen. RESULTS: IgE expression on plasmacytoid and myeloid DCs from omalizumab-treated subjects (n = 12) decreased by > or =95% posttreatment (P = .0005), whereas FcepsilonRIalpha expression decreased by 66% and 48%, respectively (P = .0005). Cat allergen-induced proliferation in DC/T-cell cocultures observed at baseline was suppressed approximately 20% to 40% postomalizumab treatment (P = .001). Multiplexing for cytokines in plasmacytoid DC/T-cell cocultures also showed decreases in IL-5, IL-13, and IL-10 (P < .05), whereas IL-2 and IFN-gamma were unaltered or slightly increased. These changes were not evident in placebo-control subjects (n = 4). CONCLUSION: IgE likely facilitates allergen presentation by dendritic cells in vivo and is also important in regulating DC-dependent T-cell cytokines during effector phases of allergic disease.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Células Dendríticas/imunologia , Imunoglobulina E/metabolismo , Células Th2/imunologia , Adolescente , Adulto , Alérgenos/imunologia , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/administração & dosagem , Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Gatos , Técnicas de Cocultura , Citocinas/metabolismo , Método Duplo-Cego , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Pessoa de Meia-Idade , Omalizumab , Receptores de IgE/metabolismo , Adulto Jovem
12.
J Immunol ; 182(4): 2432-8, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19201898

RESUMO

Although IL-3 is commonly recognized for its growth factor-like activity, in vitro studies have long demonstrated a unique capacity for this cytokine to also augment the proinflammatory properties and phenotype of human basophils. In particular, basophils secrete mediators that are hallmarks in allergic disease, including vasoactive amines (e.g., histamine), lipid metabolites (e.g., leukotriene C(4)), and cytokines (e.g., IL-4/IL-13), which are all markedly enhanced with IL-3 pretreatment. This priming phenomenon is observed in response to both IgE-dependent and IgE-independent stimulation. Additionally, IL-3 directly activates basophils for IL-13 secretion and enhanced CD69 expression, two markers that are elevated in allergic subjects. Lymphocytes are commonly thought to be the source of the IL-3 that primes for these basophil responses. However, we demonstrate herein for the first time that basophils themselves rapidly produce IL-3 (within 4 h) in response to IgE-dependent activation. More importantly, our findings definitively show that basophils rapidly bind and utilize the IL-3 they produce, as evidenced by functional and phenotypic activity that is inhibited in the presence of neutralizing anti-IL-3 receptor (CD123) Abs. We predict that autocrine IL-3 activity resulting from low-level IgE/FcepsilonRI cross-linking by specific allergen represents an important mechanism behind the hyperreactive nature of basophils that has long been observed in allergic disease.


Assuntos
Comunicação Autócrina/imunologia , Basófilos/imunologia , Hipersensibilidade/imunologia , Interleucina-3/imunologia , Adulto , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Basófilos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Interleucina-3/metabolismo , Lectinas Tipo C , Pessoa de Meia-Idade , Fenótipo , RNA Mensageiro/análise , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
J Allergy Clin Immunol ; 123(1): 217-23, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18845324

RESUMO

BACKGROUND: Immature human blood monocytoid dendritic cells (mDCs) express high-affinity receptors for IgE (Fc epsilon RI), yet their exact function and regulation remain poorly understood. OBJECTIVE: We sought to characterize Fc epsilon RI-dependent cytokine responses and their regulation in circulating human blood mDCs. METHODS: Fc epsilon RI-dependent cytokine responses of circulating mDCs were studied by using anti-Fc epsilon RI alpha stimulation. Plasmacytoid dendritic cell (pDC) cross-regulation through Toll-like receptor 9 on these responses was investigated by examining the effects of exogenous IFN-alpha pretreatment and by coculturing pDCs and mDCs stimulated with CpG. Culture supernatants were analyzed by means of ELISA to determine cytokine levels. Cell markers were determined by means of flow cytometry. RESULTS: mDCs express marked levels of Fc epsilon RI (net mean fluorescence intensity, 196 +/- 49; n = 4). After Fc epsilon RI-dependent activation in mDCs, TNF-alpha (2189 +/- 864 pg/10(6) mDCs, n = 3) levels were upregulated within 4 hours, whereas IL-10 (112 +/- 47 pg/10(6) mDCs, n = 3) levels were detectable only after 24 hours of incubation. After adding IL-10-neutralizing antibody, TNF-alpha Fc epsilon RI-dependent responses were significantly augmented (3903 +/- 197 pg/10(6) mDCs, P < .01, n = 3). Conversely, recombinant IL-10 dose-dependently inhibited Fc epsilon RI-mediated TNF-alpha responses up to 86% +/- 3% (n = 3, P < .001). Pretreatment of mDCs with IFN-alpha (100 U/mL) enhanced Fc epsilon RI-dependent secretion of IL-10 by 3.2-fold (183 +/- 11 pg/10(6) mDCs, n = 4) compared with that seen in untreated cells (57 +/- 33 pg/10(6) mDCs, P < .001, n = 4). In pDC/mDC cocultures pretreated with CpG, Fc epsilon RI-dependent IL-10 secretion by mDCs was similarly augmented by 3-fold. CONCLUSIONS: Autocrine secretion of IL-10, a critical autoregulator of Fc epsilon RI-dependent proinflammatory responses in mDCs, is cross-regulated by IFN-alpha, a major product of Toll-like receptor 9 responses in pDCs that normally promotes T(H)1 immunity.


Assuntos
Células Dendríticas/imunologia , Interleucina-10/imunologia , Monócitos/imunologia , Receptores de IgE/imunologia , Adjuvantes Imunológicos/farmacologia , Adulto , Antivirais/imunologia , Antivirais/farmacologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Feminino , Humanos , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Interleucina-10/biossíntese , Interleucina-10/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptores de IgE/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
14.
J Leukoc Biol ; 84(4): 1151-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18625911

RESUMO

Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6-8 weeks and basophils with IL-3 (6.7 ng/ml) for 2-3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 +/- 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 +/- 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.


Assuntos
Basófilos/fisiologia , Biomarcadores Tumorais/farmacologia , Liberação de Histamina/efeitos dos fármacos , Mastócitos/fisiologia , Toxina Pertussis/farmacologia , Monoéster Fosfórico Hidrolases/deficiência , Basófilos/citologia , Basófilos/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Humanos , Imunoglobulina E/farmacologia , Inositol Polifosfato 5-Fosfatases , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/genética , Proteína Tumoral 1 Controlada por Tradução
15.
J Allergy Clin Immunol ; 121(2): 486-91, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18036648

RESUMO

BACKGROUND: Functional significance for the alphagamma(2) variant of the high-affinity IgE receptor (FcepsilonRI) reportedly expressed on human dendritic cell subtypes remains poorly understood. Studies show that immature plasmacytoid dendritic cells (pDCs) secrete large quantities of TNF-alpha and IL-6 when directly stimulated with anti-IgE antibody. This mode of activation, however, reduces Toll-like receptor 9 (TLR9) expression in pDCs and their ability to mount an IFN-alpha response when subsequently activated with oligodeoxynucleotide containing CpG. OBJECTIVE: To investigate the mechanisms underlying this IgE-dependent suppression of TLR9 and innate immune responsiveness in pDCs by focusing on autocrine cytokine responses. METHODS: pDCs were isolated from blood by using blood dendritic cell antigen 4 selection. Cytokine responses to anti-IgE antibody-dependent and/or CpG-dependent stimulation were measured by using ELISA. TLR9 expression was determined by using quantitative RT-PCR and Western blotting. RESULTS: The time required for downregulating TLR9 expression in pDCs after anti-IgE stimulation correlated with the induction and duration of TNF-alpha secreted by these cells. Pretreatment of pDCs with recombinant TNF-alpha (but not IL-6 or IL-10) markedly suppressed TLR9 expression. Functional response to CpG (ie, IFN-alpha induction) was also inhibited with TNF-alpha pretreatment (inhibitory concentration(50) = approximately 200 pg/mL). Finally, an antibody that neutralizes TNF-alpha activity completely restored TLR9 expression during anti-IgE stimulation and significantly improved IFN-alpha secretion on subsequent activation with CpG. CONCLUSION: Autocrine TNF-alpha secretion resulting from IgE/FcepsilonRI-dependent activation plays a critical role in suppressing TLR9-dependent responses in pDCs that normally promote T(H)1 activity.


Assuntos
Comunicação Autócrina/fisiologia , Células Dendríticas/metabolismo , Imunoglobulina E/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Células Cultivadas , Ilhas de CpG , Citocinas/metabolismo , Regulação para Baixo , Humanos , Tolerância Imunológica , Imunidade Inata , Cinética , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Receptor Toll-Like 9/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
16.
J Allergy Clin Immunol ; 118(2): 504-10, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16890778

RESUMO

BACKGROUND: Immunostimulatory DNA sequences (ISS) are potent immunomodulators that can drive T(H)1 responses to antigens or allergens. This effect can be dramatically enhanced by direct linkage of ISS to the protein. OBJECTIVE: Evaluate the effects of the number of ISS bound to the major ragweed allergen Amb a 1 on immunogenicity and allergenicity. METHODS: Immunogenicity in mice and allergenicity using PBMC or sera from subjects with ragweed allergy were assayed. RESULTS: Both antibody induction in vivo and antibody recognition in vitro were highly sensitive to the number of ISSs linked. IgE recognition of Amb a 1 in competitive ELISA or histamine release assays was inhibited by ISS linkage and showed an inverse relationship to the number of ISSs bound. Type and magnitude of antibody induction in mice was also highly dependent on the number of ISS bound. At the highest ISS to protein ratios, antibody induction was very low. Moderate ISS to protein ratios induced high antibody responses in which IgG(2a) generally predominated. Low ISS to protein ratios produced the highest overall antibody responses in which IgG(1) predominated. In contrast, varied ISS to protein ratios did not affect T-cell responses. In both in vivo mouse studies and in vitro human PBMC studies, all ISS to protein ratios evaluated induced similar responses represented by high levels of IFN-gamma and low levels of T(H)2 cytokines. CONCLUSION: Controlling the number of ISS bound to a protein allows manipulation of antibody recognition and induction while retaining the potent T(H)1 properties of an ISS-linked protein. CLINICAL IMPLICATIONS: Immunostimulatory DNA sequence-linked Amb a 1 conjugate represents a safe, novel therapeutic approach for treating ragweed allergy.


Assuntos
Alérgenos/farmacologia , Anticorpos/imunologia , Oligonucleotídeos/farmacologia , Proteínas de Plantas/farmacologia , Adjuvantes Imunológicos/farmacologia , Alérgenos/imunologia , Animais , Antígenos de Plantas , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Liberação de Histamina , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Imunização , Imunoglobulina E/imunologia , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/imunologia
17.
J Immunol ; 175(9): 5724-31, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16237063

RESUMO

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.


Assuntos
Células Dendríticas/fisiologia , Receptores de IgE/fisiologia , Receptor Toll-Like 9/fisiologia , Adulto , Regulação para Baixo , Humanos , Imunidade Inata , Imunoglobulina E/farmacologia , Interleucina-3/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/farmacologia , Fenótipo , Plasmócitos/citologia , Receptores de IgE/análise , Receptor Toll-Like 9/análise
18.
J Immunol ; 175(7): 4543-54, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16177098

RESUMO

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.


Assuntos
Mastócitos/metabolismo , Receptores de IgE/metabolismo , Quinases da Família src/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Precursores Enzimáticos/fisiologia , Histamina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Leucemia Basofílica Aguda/metabolismo , Mastócitos/enzimologia , Camundongos , Fosfoproteínas/fisiologia , Fosforilação , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/fisiologia , Ratos , Transdução de Sinais/fisiologia , Quinase Syk , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Tirosina/metabolismo , Quinases da Família src/genética
19.
J Allergy Clin Immunol ; 115(2): 295-301, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15696084

RESUMO

BACKGROUND: Toll-like receptor (TLR) molecules play a critical role in directing the course of acquired immunity, including that associated with allergic disease, by recognizing specific microbial products that activate immune cells for effector functions. OBJECTIVE: We investigated whether human basophils express 2 such molecules (TLR2 and TLR4), and assessed whether putative ligands for these receptors activate nuclear factor kappaB (NFkappaB) and modulate mediator release and cytokine secretion either alone or in response to stimulation. METHODS: Toll-like receptor expression was assessed by using RT-PCR and flow cytometry. Immunoblotting detected nuclear NFkappaB. Automated fluorometry, RIA, and ELISA detected concurrent changes in histamine, leukotriene C 4 , and cytokine, respectively, after culture with specific ligands. RESULTS: mRNA and protein for TLR2 and TLR4 were detected in basophils. However, in assessing nuclear localization of NFkappaB as a measure of functional receptor responses, basophils selectively reacted only to peptidoglycan, a TLR2 ligand, and not to LPS, a TLR4 ligand. Likewise, basophils secreted both IL-4 and IL-13 in direct response to peptidoglycan but not to LPS. Although neither ligand induced histamine or leukotriene C 4 release, several TLR2-specific ligands augmented the secretion of these mediators (and cytokine) in response to IgE-dependent activation and of IL-13 in response to IgE-independent stimulation. Finally, a selective inhibitor of NFkappaB did not prevent these enhancing effects mediated by TLR2 ligands. CONCLUSION: These data suggest that innate immune responses mediated through TLR2 play a role in augmenting allergic reactions, in part by modulating basophil cytokine secretion and mediator release independently of NFkappaB activation.


Assuntos
Basófilos/metabolismo , Citocinas/metabolismo , Imunoglobulina E/fisiologia , Glicoproteínas de Membrana/metabolismo , Peptidoglicano/farmacologia , Receptores de Superfície Celular/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Interleucina-13/metabolismo , Interleucina-4/biossíntese , Ligantes , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like
20.
J Allergy Clin Immunol ; 112(5): 944-50, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14610485

RESUMO

BACKGROUND: Innate immune responses play a critical role in determining the course of acquired immunity, including that associated with allergic disease. Type I interferons, which are generated early in these reactions, are important soluble factors that prime for TH1-like activity. OBJECTIVE: Because human basophils secrete IL-4 and IL-13 in response to both IgE-dependent and IgE-independent stimuli, we tested whether IFN-alpha, a major type I IFN, affects the production of these TH2 cytokines and/or mediator release from these cells. METHODS: Basophils isolated from blood were treated with IFN-alpha in the presence and absence of IL-3 priming before stimulating through the IgE receptor to release histamine, leukotriene C4, and IL-4. Effects of IFN-alpha on IL-3-mediated IL-13 secretion and basophil survival were also tested. IFN-alpha receptor expression was determined by RT-PCR. RESULTS: IFN-alpha specifically inhibited the effects IL-3 has on basophil cytokine secretion. Enhanced secretion of IL-4 resulting from IL-3 priming was significantly inhibited in cells concurrently cultured with IFN-alpha. This effect was specific for cytokine generation, because histamine and leukotriene C4 were unaffected. Furthermore, IFN-alpha blocked IL-13 secretion directly induced by IL-3. Although IFN-beta also possessed some inhibitory activity, IFN-gamma (a type II IFN) had no effect on basophil cytokine secretion. Basophils constitutively expressed mRNA for the type I IFN receptor, and IFN-alpha did not affect basophil viability with regard to inhibition of cytokine secretion. CONCLUSIONS: These results support the belief that early innate immune responses resulting in IFN-alpha production negatively regulate allergic responses by also inhibiting priming of basophil cytokine release.


Assuntos
Basófilos/metabolismo , Citocinas/metabolismo , Histamina/metabolismo , Interferon-alfa/farmacologia , Interleucina-3/farmacologia , Adulto , Basófilos/efeitos dos fármacos , Basófilos/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/antagonistas & inibidores , Humanos , Imunoglobulina E/fisiologia , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Interleucina-13/biossíntese , Leucotrieno C4/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/genética
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