RESUMO
The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.
Assuntos
Linfócitos B/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Memória Imunológica/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptor Toll-Like 9/imunologia , Adolescente , Animais , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Citometria de Fluxo , Quinase 2 de Adesão Focal/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Fosforilação , Fator de Transcrição STAT3/imunologia , Quinases da Família src/imunologiaRESUMO
Atopic dermatitis (AD) is the most common inflammatory skin disease. It is characterized by a defective skin barrier and a Th2 dominated skin inflammation. The TNF family member a proliferation-inducing ligand (APRIL) and its receptors transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) are expressed by immune cells and epithelial cells including keratinocytes. We demonstrate that APRIL expression is upregulated in the epidermis of skin lesions from patients with AD as well as in mouse skin undergoing allergic inflammation elicited by epicutaneous (EC) sensitization with the antigen ovalbumin. We show that APRIL from OVA sensitized mouse skin causes keratinocytes to upregulate the expression of IL-6, an inflammatory cytokine implicated in AD pathogenesis. These results suggest a role for APRIL in allergic skin inflammation and a potential role for APRIL blockade in treating AD.
Assuntos
Dermatite Atópica/imunologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Alérgenos/imunologia , Animais , Antígenos/imunologia , Antígeno de Maturação de Linfócitos B/imunologia , Linfócitos B/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Queratinócitos/imunologia , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Pele/imunologia , Regulação para CimaRESUMO
BACKGROUND: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. OBJECTIVE: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. METHODS: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. RESULTS: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. CONCLUSION: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.
Assuntos
Pneumonia Pneumocócica/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnicas de Introdução de Genes , Células HEK293 , Haploinsuficiência , Heterozigoto , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/imunologia , Reação em Cadeia da Polimerase , Proteína Transmembrana Ativadora e Interagente do CAML/imunologiaRESUMO
The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Immunoblotting , Switching de Imunoglobulina/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias épsilon de Imunoglobulina/genética , Cadeias épsilon de Imunoglobulina/imunologia , Cadeias épsilon de Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/metabolismo , Interleucina-4/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fenilenodiaminas/imunologia , Fenilenodiaminas/farmacologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismoRESUMO
Immunoglobulin E (IgE) isotype antibodies are associated with atopic disease, namely allergic rhinitis, asthma and atopic dermatitis, but are also involved in host immune defence mechanisms against parasitic infection. The commitment of a B cell to isotype class switch to an IgE-producing cell is a tightly regulated process, and our understanding of the regulation of IgE-antibody production is central to the prevention and treatment of atopic disease. Both those that are presently in use and potential future therapies to prevent IgE-mediated disease take advantage of our existing knowledge of the specific mechanisms that are required for IgE class switching.
Assuntos
Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Isotipos de Imunoglobulinas/imunologia , Animais , Humanos , Switching de Imunoglobulina/genética , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Isotipos de Imunoglobulinas/genética , CamundongosRESUMO
The autosomal recessive form of the Hyper IgE syndrome (AR-HIES) with dedicator of cytokinesis 8 (DOCK8) deficiency is associated with difficult to treat persistent viral skin infections, including papilloma virus infection. Type I interferons play an important role in the defense against viruses. We examined the effect of therapy with IFN-α 2b in an 11-year old boy with DOCK8 deficiency due to a homozygous splice donor site mutation in DOCK8 intron 40. His unremitting warts showed dramatic response to IFN-α 2b therapy. Immunological studies revealed decreased circulating plasmacytoid dendritic cells (pDCs) and profound deficiency of IFN-α production by his peripheral blood mononuclear cells in response to treatment with CpG oligonucleotides. These findings indicate that underlying pDC deficiency and impaired IFN-α production may predispose to chronic viral infections in DOCK8 deficiency. IFN-α 2b therapy maybe useful in controlling recalcitrant viral infections in these patients.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/deficiência , Interferon-alfa/uso terapêutico , Síndrome de Job/complicações , Síndrome de Job/genética , Verrugas/tratamento farmacológico , Verrugas/etiologia , Criança , Análise Mutacional de DNA , Humanos , Fatores Imunológicos/uso terapêutico , Imunofenotipagem , Interferon alfa-2 , Síndrome de Job/diagnóstico , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Mutação , Linhagem , Proteínas Recombinantes/uso terapêutico , Pele/patologiaRESUMO
BACKGROUND: Combined immunodeficiency (CID) is characterized by severe recurrent infections with normal numbers of T and B lymphocytes but with deficient cellular and humoral immunity. Most cases are sporadic, but autosomal recessive inheritance has been described. In most cases, the cause of CID remains unknown. OBJECTIVE: We wanted to identify the genetic cause of CID in 2 siblings, the products of a first-cousin marriage, who experienced recurrent bacterial and candidal infections with bronchiectasis, growth delay, and early death. METHODS: We performed immunologic, genetic, and biochemical studies in the 2 siblings, their family members, and healthy controls. Reconstitution studies were performed with T cells from mucosa-associated lymphoid tissue lymphoma-translocation gene 1-deficient (Malt1(-/-)) mice. RESULTS: The numbers of circulating T and B lymphocytes were normal, but T-cell proliferation to antigens and antibody responses to vaccination were severely impaired in both patients. Whole genome sequencing of 1 patient and her parents, followed by DNA sequencing of family members and healthy controls, showed the presence in both patients of a homozygous missense mutation in MALT1 that resulted in loss of protein expression. Analysis of T cells that were available on one of the patients showed severely impaired IκBα degradation and IL-2 production after activation, 2 events that depend on MALT1. In contrast to wild-type human MALT1, the patients' MALT1 mutant failed to correct defective nuclear factor-κB activation and IL-2 production in MALT1-deficient mouse T cells. CONCLUSIONS: An autosomal recessive form of CID is associated with homozygous mutations in MALT1. If future patients are found to be similarly affected, they should be considered as candidates for allogeneic hematopoietic cell transplantation.
Assuntos
Caspases/genética , Mutação , Proteínas de Neoplasias/genética , Imunodeficiência Combinada Severa/genética , Sequência de Aminoácidos , Animais , Caspases/análise , Células Cultivadas , Criança , Pré-Escolar , Humanos , Quinase I-kappa B/metabolismo , Ativação Linfocitária , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/análise , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/metabolismoRESUMO
CD40 induces B cells to switch to IgE in the presence of IL-4 and up-regulates their expression of the low-affinity receptor for IgE, CD23, which promotes the immune response to allergen complexed with IgE antibody. CD40 binds to CD40L and to the C4b-binding protein (C4BP) using distinct sites. CD46 is a receptor for the product of activated complement C4b. Some microbial antigens bind both C4BP and CD46, potentially bridging CD40 to CD46. In addition, immune complexes containing both C4b and C4BP may cross-link CD40 to CD46. We demonstrate that cross-linking CD46 to CD40 on B cells inhibits CD40-mediated up-regulation of surface CD23 expression and induction of IL-4-dependent IgE isotype switching. This was associated with inhibition of induction of Cε germ line transcripts and of activation-induced cytidine deaminase mRNA expression. Furthermore, co-ligation of CD46 to CD40 blocked CD40-mediated NF-κB activation. These observations suggest that complement components may play an important role in regulating CD40 activation of B cells and the allergic response.
Assuntos
Linfócitos B/fisiologia , Antígenos CD40/metabolismo , Reagentes de Ligações Cruzadas , Proteína Cofatora de Membrana/metabolismo , Transdução de Sinais , Células Cultivadas , Reagentes de Ligações Cruzadas/química , Regulação da Expressão Gênica/imunologia , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Ligação ProteicaRESUMO
BACKGROUND: TNFRSF13B, which encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), is mutated in 10% of patients with common variable immunodeficiency. One of the 2 most common TACI mutations in common variable immunodeficiency, C104R, abolishes ligand binding and is found predominantly in the heterozygous state. The murine TACI mutant C76R is the equivalent of the human TACI mutant C104R. OBJECTIVE: We sought to define the consequence of the C76R mutation on TACI function in mice that express both wild-type TACI and the murine C76R mutant. METHODS: Transgenic mice that express murine TACI C76R, the counterpart of human TACI C104R, on the TACI(+/-) B6/129 background (C76R/TACI(+/-) mice) were constructed. Serum immunoglobulins and antibody responses to the type II T-independent antigen trinitrophenylated (TNP)-Ficoll were determined by means of ELISA. B-cell proliferation in response to a proliferation-inducing ligand was determined based on tritiated thymidine incorporation into DNA. IgG1 secretion by B cells in response to a proliferation-inducing ligand plus IL-4 was determined by means of ELISA. RESULTS: C76R/TACI(+/-) mice had significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll compared with TACI(+/+) B6/129 control animals, and their B cells were impaired in their capacity to proliferate and secrete IgG1 in response to TACI ligation. Unexpectedly, TACI(+/-) mice had similarly impaired B-cell function as C76R/TACI(+/-) littermates. Impaired TACI function caused by haploinsufficiency was confirmed in TACI(+/-) mice on the C57BL/6 background. CONCLUSION: These results suggest that the human TACI mutant C104R might impair TACI function in heterozygotes through haploinsufficiency.
Assuntos
Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/imunologia , Haploinsuficiência/imunologia , Proteínas Mutantes/metabolismo , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Animais , Formação de Anticorpos/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Proliferação de Células , Células Cultivadas , Imunodeficiência de Variável Comum/genética , Ficoll/análogos & derivados , Ficoll/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Mutação/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Trinitrobenzenos/imunologiaRESUMO
The isotype switch defect in CD40(-/-) mice is corrected by wild-type (WT) CD40 transgene, but not by a mutant CD40 transgene that does not bind tumor necrosis factor receptor-associated factors (TRAF) 2 and 3. To define the individual roles of TRAF2 and TRAF3 in CD40 activation of B cells, we introduced mutant CD40 transgenes that selectively lack the ability to bind TRAF2 (DeltaTR2), TRAF3 (DeltaTR3) or both (DeltaTR2,3) into B cells of CD40(-/-) mice. Serum IgG1 and IgE levels, IgG1 antibody response to sub-optimal doses of the T cell-dependent antigen keyhole limpet hemocyanin, germinal center formation, CD40-mediated proliferation, isotype switching and activation of the non-canonical NF-kappaB pathway were partially diminished in DeltaTR2 and DeltaTR3 mice and virtually absent in DeltaTR2,3 mice. These results suggest that TRAF2 and TRAF3 can each independently mediate class switch recombination (CSR) driven by CD40, but both are required for optimal CD40-driven isotype switching.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/imunologia , Switching de Imunoglobulina/imunologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Animais , Linfócitos B/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Hemocianinas/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Transdução de Sinais/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/imunologiaAssuntos
Células Dendríticas/imunologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Antivirais/uso terapêutico , Criança , Pré-Escolar , Células Dendríticas/metabolismo , Feminino , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/imunologia , Homozigoto , Humanos , Imunofenotipagem , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Mutação , Proteínas Recombinantes/uso terapêutico , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Resultado do TratamentoAssuntos
Linfócitos B/patologia , Linfócitos T CD8-Positivos/patologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Síndrome de Job/diagnóstico , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Consanguinidade , Feminino , Citometria de Fluxo , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Síndrome de Job/genética , Síndrome de Job/imunologia , Síndrome de Job/terapia , Masculino , LinhagemRESUMO
Measuring lymphocyte response to mitogens and antigens is a mainstay of screening for cellular immunodeficiency. Few reports analyze performance as a screening tool in diverse patient cohorts. We studied proliferation assays performed at Children's Hospital Boston from 1996 to 2003 using mitogens phytohemagglutinin (PHA), concanavalin A (CONA) and pokeweed mitogen, and antigens tetanus (TT) and diphtheria (DT) toxoids, and compared a subset of patients with T cell dysfunction with adult controls using receiver operating characteristic analysis. Results were correlated with clinical data. CONA was superior to PHA in identifying patients with immunodeficiency. TT was second best. Interpretation based on raw CPM, a stimulation index, or reference to simultaneous controls all performed equally. Combining data from multiple mitogens and/or antigens did not enhance performance. Proliferation testing is a useful component of screening for cellular immunodeficiency, but is not a sensitive predictor of cellular immune compromise or risk of opportunistic infection.
Assuntos
Síndromes de Imunodeficiência/diagnóstico , Mitógenos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Concanavalina A/farmacologia , Toxoide Diftérico/farmacologia , Feminino , Humanos , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/imunologia , Recém-Nascido , Ativação Linfocitária/efeitos dos fármacos , Masculino , Fito-Hemaglutininas/farmacologia , Mitógenos de Phytolacca americana/farmacologia , Toxoide Tetânico/farmacologia , Adulto JovemRESUMO
BACKGROUND: During differentiation, B cells receive signals by antigen through the B-cell receptor (BCR) and signals that induce isotype switching. OBJECTIVE: We sought to investigate the effects of BCR ligation on isotype switching. METHODS: Naive B cells from BALB/c mice were stimulated with LPS plus IL-4 alone or plus anti-IgM (0.1-10 mug/mL). IgE and IgG1 levels in supernatants were measured by means of ELISA on day 6. Cmu or Cvarepsilon germline transcripts, activation-induced cytidine deaminase (AID), and Imu-Cvarepsilon postswitch transcripts were measured by means of RT-PCR. Deletional switch recombination was assessed by means of digestion circularization PCR of Smu-Svarepsilon products. RESULTS: BCR cross-linking inhibited IgE and IgG1 switching in a dose-dependent fashion. This was not due to inhibition of proliferation, increased apoptosis, or cell death. BCR cross-linking had no effect on Cmu or Cvarepsilon germline transcripts but suppressed the generation of Smu-Svarepsilon switch products and Imu-Cvarepsilon postswitch transcripts and caused a delay in the expression of AID mRNA, with decreased expression on days 2 and 3 after stimulation. Concomitantly, the number of DNA repair foci at the IgH locus on day 3 was significantly decreased. AID expression and activity became normal on day 4, but isotype switching remained profoundly diminished 8 days after stimulation. CONCLUSION: BCR cross-linking delays AID expression. This might interfere with class-switch recombination by disrupting the temporal coordination of signals that lead to class-switch recombination.
Assuntos
Citidina Desaminase/biossíntese , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina E/biossíntese , Receptores de Antígenos de Linfócitos B/metabolismo , Recombinação Genética , Animais , Linfócitos B/metabolismo , Células Cultivadas , Indução Enzimática , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Boys with X-linked ectodermal dysplasia and immunodeficiency caused by mutations of nuclear factor-kappaB essential modulator have defects in innate and adaptive immunity, and some have colitis. OBJECTIVE: We sought to determine whether curing the immune defect in such patients by means of allogeneic hematopoietic stem cell transplantation abolishes the susceptibility to colitis. METHODS: A boy with X-linked hypohydrotic ectodermal dysplasia with immunodeficiency underwent allogeneic transplantation from a matched unaffected sibling identified by means of preimplantation genetic diagnosis. Toll-like receptor (TLR) function was assessed by measuring TLR agonist-induced cytokine production in whole blood tested in vitro. B-cell proliferation was measured by means of tritiated thymidine incorporation. Natural killer cell function was examined in PBMCs by means of K562 target cell lysis. Colitis severity was assessed clinically based on corticosteroid requirement and histology of large intestinal biopsy specimens. RESULTS: Defects in cytokine production in response to TLR agonists, CD40-mediated proliferation, and natural killer cell cytotoxicity were all corrected after hematopoietic stem cell transplantation. Despite successful hematopoietic and immune reconstitution, the patient continued to have flares of colitis, often associated with bacterial infection. CONCLUSIONS: Our findings strongly suggest that nuclear factor-kappaB essential modulator deficiency intrinsic to the intestinal epithelium is sufficient to predispose to colitis, despite robust correction of immune defects.
Assuntos
Colite/genética , Displasia Ectodérmica/genética , Displasia Ectodérmica/terapia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Adolescente , Linfócitos B/imunologia , Linfócitos B/patologia , Proliferação de Células , Criança , Pré-Escolar , Colite/diagnóstico , Colite/imunologia , Colite/patologia , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/imunologia , Displasia Ectodérmica/patologia , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Células K562 , Masculino , Irmãos , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Transplante HomólogoAssuntos
Imunodeficiência de Variável Comum/genética , Transdução de Sinais/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Sequência de Aminoácidos , Separação Celular , Imunodeficiência de Variável Comum/metabolismo , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Mutação , Transfecção , Proteína Transmembrana Ativadora e Interagente do CAML/químicaRESUMO
Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.
Assuntos
Antígenos CD/genética , Antígenos CD/imunologia , Síndromes de Imunodeficiência/genética , Mutação de Sentido Incorreto , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Imunidade Adaptativa/genética , Anemia/genética , Animais , Antígenos CD/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Endocitose , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oxirredutases , Linhagem , Receptores da Transferrina/metabolismoRESUMO
BACKGROUND: CD40 ligation activates nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases p38 and C-Jun N-terminal kinase (JNK) and causes immunoglobulin class-switch recombination (CSR) in B cells. Both NF-kappaB and p38 are important for CD40-mediated CSR. The role of JNK activation in CD40-mediated isotype switching is unknown. OBJECTIVE: We sought to determine the role of JNK activation in CD40-mediated isotype switching. METHODS: Splenic B cells from BALB/c mice were stimulated with anti-CD40 mAb and IL-4 or with soluble CD40 ligand in the presence or absence of SP600125, an anthrapyrazolone inhibitor of JNK. The following events were examined: IgE production by means of ELISA; S(mu)-S(epsilon) deletional switch recombination by means of digestion circularization PCR; Cepsilon germline, mature epsilon, and activation-induced deaminase (AID) transcription by means of RT-PCR; and proliferation by tritiated thymidine incorporation and surface expression of CD23, CD54, and CD86 by means of FACS analysis. RESULTS: SP600125 at 10 microM drastically inhibited JNK phosphorylation but had little effect on CD40-mediated p38 phosphorylation and expression of the NF-kappaB dependent genes c-Myc and bcl-xL. SP600125 inhibited IgE synthesis by approximately 88% but had no effect on B-cell proliferation and survival in response to anti-CD40 + IL-4 or on upregulation of CD23, CD54, and CD86 in response to CD40 ligation. Analysis of molecular events involved in IgE class switching revealed that SP600125 had no effect on the expression of C(epsilon) germline and AID transcripts. In contrast, SP600125 severely reduced S(mu)-S(epsilon) switch recombination and expression of mature epsilon transcripts. CONCLUSION: These results demonstrate that JNK activation is essential for CD40-mediated CSR to IgE and suggest that JNK is important for AID activity in B cells.