RESUMO
Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.
Assuntos
Proteínas de Transporte/metabolismo , Espinhas Dendríticas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Esquizofrenia/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fatores Etários , Animais , Western Blotting , Proteínas de Transporte/genética , Células Cultivadas , Disbindina , Proteínas Associadas à Distrofina , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Neurônios/ultraestrutura , RNA Interferente Pequeno , Ratos , Esquizofrenia/fisiopatologia , Sinapses/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
We have previously shown that antigen-induced eosinophil recruitment into the tissue of sensitized mice is mediated by CD4+ T cells and interleukin 5. To determine whether interferon gamma (IFN-gamma) regulates antigen-induced eosinophil recruitment into the tissue, we studied the effect of recombinant (r) murine IFN-gamma and of anti-IFN-gamma monoclonal antibody (mAb) on the eosinophil infiltration of the trachea induced by antigen inhalation in mice. The intraperitoneal administration of rIFN-gamma prevented antigen-induced eosinophil infiltration in the trachea of sensitized mice. The administration of rIFN-gamma also decreased antigen-induced CD4+ T cell but not CD8+ T cell infiltration in the trachea. On the other hand, pretreatment with anti-IFN-gamma mAb enhanced antigen-induced eosinophil and CD4+ T cell infiltration in the trachea. These results indicate that IFN-gamma regulates antigen-induced eosinophil recruitment into the tissue by inhibiting CD4+ T cell infiltration.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Eosinófilos/imunologia , Interferon gama/farmacologia , Animais , Antígenos/imunologia , Testes de Provocação Brônquica , Antígenos CD8/análise , Movimento Celular , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Proteínas Recombinantes , Traqueia/imunologiaRESUMO
To determine the role of vascular cell adhesion molecule 1 (VCAM-1)/very late activation antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions in causing antigen-induced eosinophil and T cell recruitment into the tissue, we studied the effect of the in vivo blocking of VCAM-1, ICAM-1, VLA-4, and LFA-1 by pretreatment with monoclonal antibodies (mAb) to these four adhesion molecules on the eosinophil and T cell infiltration of the trachea induced by antigen inhalation in mice. The in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, prevented antigen-induced eosinophil infiltration into the mouse trachea. On the contrary, the in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, increased blood eosinophil counts after antigen challenge, but did not affect blood eosinophil counts without antigen challenge in sensitized mice. Furthermore, the expression of VCAM-1 but not ICAM-1 was strongly induced on the endothelium of the trachea after antigen challenge. In addition, pretreatment with anti-IL-4 mAb decreased the antigen-induced VCAM-1 expression only by 27% and had no significant effect on antigen-induced eosinophil infiltration into the trachea. The in vivo blocking of VCAM-1 and VLA-4 inhibited antigen-induced CD4+ and CD8+ T cell infiltration into the trachea more potently than that of ICAM-1 and LFA-1. In contrast, regardless of antigen challenge, the in vivo blocking of LFA-1, but not of ICAM-1, increased blood lymphocyte counts more than that of VCAM-1 and VLA-4. These results indicate that VCAM-1/VLA-4 interaction plays a predominant role in controlling antigen-induced eosinophil and T cell recruitment into the tissue and that the induction of VCAM-1 expression on the endothelium at the site of allergic inflammation regulates this eosinophil and T cell recruitment.
Assuntos
Moléculas de Adesão Celular/fisiologia , Eosinófilos/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Receptores de Antígeno muito Tardio/fisiologia , Linfócitos T/fisiologia , Administração por Inalação , Animais , Anticorpos Monoclonais/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Quimiotaxia de Leucócito , Feminino , Molécula 1 de Adesão Intercelular , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Traqueia/imunologia , Molécula 1 de Adesão de Célula VascularRESUMO
Transforming growth factor (TGF)-beta has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-beta contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-beta/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-beta in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-beta/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-beta was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-beta/Smad signaling in mature T cells as a regulatory component of allergic asthma.
Assuntos
Asma/etiologia , Hiper-Reatividade Brônquica/etiologia , Proteínas de Ligação a DNA/fisiologia , Linfócitos T/fisiologia , Traqueia/patologia , Transativadores/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Linfócitos B/fisiologia , Citocinas/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Ovalbumina/imunologia , Proteína Smad7RESUMO
A novel subset of T cells characterized by the expression of an invariant T cell antigen receptor (TCR) encoded by V alpha 24J alpha Q gene segments was investigated in patients with systemic sclerosis (SSc). Polymerase chain reaction analysis demonstrated that the V alpha 24 TCR repertoire was selectively used in CD4-CD8- double-negative T cells both in patients and in healthy individuals, while almost all families of TCR V alpha were expressed in single-positive T cell fractions. The V alpha 24+ double-negative T cells were increased by approximately fivefold in patients. However, sequence analysis clearly showed significant differences in the V alpha 24 TCR repertoire dominating in patients and healthy donors. In healthy individuals, the invariant V alpha 24J alpha Q was expanded and comprised 20-50% of the total TCR-alpha, while their selective reduction was observed in SSc patients who also showed expansion of invariant V alpha 24 TCR other than V alpha 24J alpha Q. Analogous to murine invariant V alpha 14J alpha 281 TCR, these results suggest that T cells with invariant V alpha 24J alpha Q TCR would function as regulatory T cells, whereas T cells bearing other invariant V alpha 24 TCR in SSc patients could be autoaggressive T cells in nature.
Assuntos
Doenças Autoimunes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Escleroderma Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Povo Asiático , Sequência de Bases , Antígenos CD4/análise , Antígenos CD8/análise , Separação Celular , Clonagem Molecular , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Seleção Genética , Análise de Sequência de DNARESUMO
Vinexin is an adaptor protein supposed to play pivotal roles in various cellular events such as cell adhesion, cytoskeletal organization, signaling and gene expression. Despite the possible importance, physiological functions and regulatory mechanisms of vinexin are largely unknown. In addition, although vinexin was reported to be phosphorylated by extracellular signal-regulated kinase (ERK), physiological significance of the phosphorylation remains to be elucidated. Here we carried out characterization of endogenous vinexin and found that it was enriched at the leading edge of migrating cells and focal adhesions of spread cells. In the analyses using ERK-phosphorylated vinexin-specific antibody, the phosphorylation signal was also detected at the leading edges of migrating cells and at cell periphery of spreading cells, whereas only faint signal was observed at focal adhesions of well-spread cells. We then established LNCaP cell lines stably expressing GFP-fused vinexinbeta or two mutants at Ser189 that mimic the ERK-phosphorylated or -unphosphorylated vinexin beta. Based on the analyses using the lines, the phosphorylation was likely to inhibit the cell spreading and migration. On the other hand, anchorage-independent cell growth was inhibited by unphosphorylated vinexinbeta. Taken together, ERK-mediated phosphorylation of vinexinbeta is strongly suggested to occur in a spatio-temporally regulated manner and play important roles in cell spreading, migration and anchorage-independent growth.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Proteínas Musculares/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Pseudópodes/metabolismoRESUMO
TGF-beta plays an important role in lung fibrosis, which is a major cause of suffering and death seen in pulmonary disease. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of lung fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant human type 5 adenovirus vector, on bleomycin-induced lung fibrosis in mice. C57BL/6 mice with bleomycin-induced lungs received an intratracheal injection of a recombinant adenovirus carrying mice Smad7 cDNA. These mice demonstrated suppression of type I precollagen mRNA, reduced hydroxyproline content, and no morphological fibrotic responses in the lungs when compared with mice administered adenovirus carrying Smad6 cDNA. In addition, we found that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs. These data indicated that gene transfer of Smad7 (but not Smad6) prevented bleomycin-induced lung fibrosis, suggesting that Smad7 may have applicability in the treatment of pulmonary fibrosis.
Assuntos
Bleomicina/toxicidade , Proteínas de Ligação a DNA/fisiologia , Terapia Genética , Fibrose Pulmonar/prevenção & controle , Transativadores/fisiologia , Transfecção , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adenoviridae/genética , Animais , Líquido da Lavagem Broncoalveolar/química , Colágeno/biossíntese , Colágeno/genética , Citomegalovirus/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/fisiologia , Proteína Smad6 , Proteína Smad7 , Transativadores/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS.
Assuntos
Aparelho Lacrimal/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Glândulas Salivares/imunologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Autoantígenos , Sequência de Bases , Biópsia , Southern Blotting , Células Clonais , Antígenos HLA-DQ/análise , Antígenos HLA-DR/análise , Humanos , Aparelho Lacrimal/patologia , Dados de Sequência Molecular , Família Multigênica , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Glândulas Salivares/patologia , Síndrome de Sjogren/patologiaRESUMO
We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.
Assuntos
Membrana Celular/metabolismo , Traqueia/metabolismo , Animais , Membrana Celular/enzimologia , Células Cultivadas , Cães , Células Epiteliais , Epitélio/enzimologia , Epitélio/metabolismo , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Inibidores de Proteases , Serina/metabolismo , Sulfatos/metabolismo , Radioisótopos de Enxofre , Temperatura , Traqueia/citologia , Traqueia/enzimologia , TripsinaRESUMO
We determined whether platelet-activating factor (PAF) activates mitogen-activated protein (MAP) kinases in human eosinophils, and if so, which signaling pathways are utilized for the MAP kinase activation. PAF activated 42-and 44-kDa MAP kinases (ERK1/ERK2) in eosinophils, which became maximal at 1 min after stimulation. The PAF receptor antagonist E6123 and pertussis toxin inhibited the PAF-induced MAP kinase activation in eosinophils. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, tyrosine kinase inhibitors herbimycin A and genistein, and an intracellular Ca2+ chelator BAPTA/AM inhibited PAF-induced MAP kinase activation in eosinophils, whereas protein kinase C inhibitors staurosporine and calphostin C had no effect. Furthermore, wortmannin as well as herbimycin A and genistein, but not BAPTA/AM, prevented PAF-induced tyrosine phosphorylation of Shc adapter protein in eosinophils. Finally, the specific MEK inhibitor PD98059 inhibited PAF-induced chemotaxis in eosinophils. Taken together, these results indicate that PAF activates MAP kinases in eosinophils through the activation of PI 3-kinase and a tyrosine kinase and the increase in intracellular Ca2+ and that PAF-induced MAP kinase activation mediates chemotaxis in eosinophils.
Assuntos
Eosinófilos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Fator de Ativação de Plaquetas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Previous studies suggested that the eosinophil recruitment into the site of cutaneous late-phase reaction (LPR) was dependent on IgE antibody and mast cells. In this study, we determined the role of CD4+ T cells and CD8+ T cells in causing antigen-induced eosinophil recruitment of LPR in mouse skin. Eosinophil infiltration into the subcutaneous tissue of ovalbumin (OVA)-sensitized BALB/c mice was biphasic, reaching the first peak at 6 h after the subcutaneous challenge with OVA and the second peak at 24 to 48 h. The in vivo depletion of CD4+ T cells by pretreatment with anti-L3T4 monoclonal antibody (mAb) significantly decreased the second peak (at 24 h and 48 h), but not the first peak (at 6 h), of OVA-induced eosinophil infiltration into the skin of OVA-sensitized mice. However, the depletion of CD8+ T cells by pretreatment with anti-Lyt-2 mAb had no significant effect on either the first peak or second peak of OVA-induced cutaneous eosinophilia. Pretreatment with anti-murine interleukin-5 (IL-5) mAb also decreased the second peak, but not the first peak, of OVA-induced cutaneous eosinophilia. In contrast to the inhibitory effects of depletion of CD4+ T cells and of anti-IL-5 mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate and a selective antagonist for platelet activating factor (PAF) CV-6209 decreased the first peak of OVA-induced cutaneous eosinophilia in the mouse. These results indicate that CD4+ T cells, but not CD8+ T cells, cause the second peak of antigen-induced eosinophil recruitment of cutaneous LPR and that IL-5 mediates this eosinophil recruitment. In contrast, the first peak of antigen-induced eosinophil recruitment of cutaneous LPR is mediated by mast cells and PAF.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Eosinófilos/fisiologia , Hipersensibilidade/imunologia , Interleucina-5/fisiologia , Pele/imunologia , Animais , Degranulação Celular , Movimento Celular , Cromolina Sódica/farmacologia , Depleção Linfocítica , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/fisiologia , Fatores de TempoRESUMO
We investigated the distribution of neutral endopeptidase (NEP; EC 3.4.24.11) activity, a possible regulatory enzyme for neuropeptide-induced leukocyte activations, in each cell type of human blood leukocytes. The NEP activity assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils and the common acute lymphoblastic leukemia antigen (CALLA)-positive leukemic cells (59 pmol/min/10(6) cells and 62 pmol/min/10(6) cells, respectively); however, the NEP activity was virtually absent in lymphocytes, monocytes, eosinophils, basophils, CALLA-negative leukemic cells, or a promyelocytic cell line HL-60. The enzymatic activity was characterized as NEP on the basis of the values of kinetic parameters (Km = 61 microM, Kcat = 1,692 min-1, and Kcat/Km = 28 min-1 microM-1) and the values of IC50 of two NEP inhibitors phosphoramidon and thiorphan (7.4 nM and 8.4 nM, respectively). The distribution of NEP detected immunocytochemically using anti-NEP monoclonal antibodies was also found to be parallel with the distribution of NEP activity among peripheral blood leukocytes.
Assuntos
Leucócitos/enzimologia , Neprilisina/sangue , Basófilos/enzimologia , Linhagem Celular , Membrana Celular/enzimologia , Separação Celular/métodos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Eosinófilos/enzimologia , Humanos , Técnicas In Vitro , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucócitos/citologia , Neprilisina/isolamento & purificação , Neutrófilos/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologiaRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) transmits anti-apoptotic signals in eosinophils and is involved in tissue eosinophilia at the site of allergic inflammation. We determined whether phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAP kinase) are involved in anti-apoptotic signals of GM-CSF in eosinophils. GM-CSF phosphorylated Akt, a downstream component of PI 3-kinase, and MAP kinases (ERK1 and ERK2) at 10 min after stimulation in eosinophils. GM-CSF prevented eosinophil apoptosis and sustained its survival during the 5-day culture. However, neither two PI-3 kinase inhibitors, wortmannin and LY294002, nor MEK inhibitor PD98059 inhibited GM-CSF-induced survival of eosinophils, although wortmannin and PD98059 inhibited GM-CSF-induced Akt phosphorylation and MAP kinase activation in eosinophils, respectively. In contrast, JAK2 inhibitor AG-490 inhibited both GM-CSF-induced JAK2 phosphorylation and cell survival in eosinophils. These results indicate that activation of JAK2, but not activation of PI 3-kinase/Akt and MAP kinase pathways, is critical for anti-apoptotic signals of GM-CSF in human eosinophils. Our findings suggest that manipulation of JAK2 activation would be useful for the treatment of allergic disorders.
Assuntos
Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Eosinófilos/enzimologia , Eosinófilos/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Janus Quinase 2 , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Proteína Oncogênica v-akt , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Proteínas Oncogênicas de Retroviridae/fisiologia , Tirfostinas/farmacologia , WortmaninaRESUMO
We have reported that the three serine residues in alphaB-crystallin are phosphorylated under various stress conditions. We prepared affinity-purified antibodies recognizing each of the phosphorylated serine residues (Ser-19, Ser-45, and Ser-59, respectively) in alphaB-crystallin with peptides (p19S, p45S, or p59S) that contained the corresponding phosphorylated serine residue. Immunocytochemically anti-p45S antibodies stained the cytoplasm of mitotic cells (J. Biol. Chem. 273, 28,346-28,354). We have now found that the anti-p59S antibodies recognize centrosomes and midbodies of dividing cells. alphaB-Crystallin was the only protein recognized by the anti-p59S antibodies in Western blot analyses of isolated centrosome fractions. alphaB-Crystallin phosphorylated at Ser-59 was localized at the microtubule organizing centers by means of double staining with anti-beta-tubulin antibody in aster formation analysis and was co-localized with gamma-tubulin in centrosomes. Gamma-Tubulin was co-immunoprecipitated with alphaB-crystallin in U373 glioma cell extracts. On the other hand, the location of the phosphorylated alphaB-crystallin deviated from that of alpha-tubulin or gamma-tubulin in the midbody region. Taken together with the evidences that several chaperones are distributed to centrosomes, these results suggest that alphaB-crystallin as a chaperone might be also involved in the quality control of proteins.
Assuntos
Centrossomo/metabolismo , Cristalinas/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Epitopos , Células HeLa , Humanos , Imunoquímica , Camundongos , Mitose/fisiologia , Fosforilação , Serina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Systemic lupus erythematosus is an autoimmune disease characterized by the presence of autoantibodies and by lymphocytic infiltration into lesions at several sites such as skin, kidney, and other organs. Immunohistologic studies have clarified that the majority of lymphocytes in the skin are CD4+ alphabeta T cells. In the present work, to clarify the pathologic role of T cells in the skin of systemic lupus erythematosus patients, we analyzed T cell receptor (TCR) clonotypes of T cells infiltrating into skin lesions. TCR Vbeta gene transcripts from T cells from discoid lesions of the skin and peripheral blood lymphocytes of four systemic lupus erythematosus patients were amplified by reverse transcriptase polymerase chain reaction. Southern blot analysis of polymerase chain reaction product demonstrated the heterogeneous TCR Vbeta repertoire of T cells in the skin of systemic lupus erythematosus. Single-strand conformation polymorphism analysis showed several distinct bands for smears of most TCR Vbeta genes from T cells infiltrating the skin, whereas smears with few bands were found for all TCR Vbeta genes from peripheral blood lymphocytes of the same patients. The number of bands encoding each TCR Vbeta gene from the skin was significantly higher compared with peripheral blood lymphocytes. Sequencing analysis showed a Leucine-X-Glycine amino acid motif at position 96-98 in the CDR3 region at the frequency of 23-24% in skin-accumulated T cells from two patients, whereas the frequency of this motif in peripheral T cells was only 0-3%, indicating limited T cell epitopes. In conclusion, T cells infiltrating into the skin of systemic lupus erythematosus patients might recognize restricted T cell epitopes on autoantigens and trigger the autoimmune reaction in skin lesions.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Pele/química , Sequência de Aminoácidos , Biópsia , Linfócitos T CD4-Positivos/fisiologia , Células Clonais , Sequência Conservada , Epitopos de Linfócito T/genética , Humanos , Lúpus Eritematoso Sistêmico/sangue , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Pele/patologiaRESUMO
A missense mutation of Smad2 identified in cancer cells was reconstructed on the corresponding residue of Smad3. This mutant, Smad3D407E, was not phosphorylated by the constitutively active form of type I receptor for transforming growth factor-beta (TGF-beta), and inhibited the phosphorylation of co-expressed wild-type Smad2 and Smad3. This mutant also had a dominant negative effect on the growth inhibition of HaCaT cells and on the expression of p3TP-lux reporter gene induced by TGF-beta. However, it did not alter the phosphorylation of Smad1 induced by the constitutively active form of the bone morphogenetic protein type IA receptor. These findings showed that a single missense mutation in Smad3 could specifically block TGF-beta signals by preventing activation of both Smad2 and Smad3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais/fisiologia , Transativadores , Fator de Crescimento Transformador beta/farmacologia , Sequência de Aminoácidos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Células COS , Linhagem Celular , Proteínas de Ligação a DNA/genética , Queratinócitos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento/química , Proteínas Recombinantes de Fusão , Proteína Smad2 , Proteína Smad3 , Ativação Transcricional , TransfecçãoRESUMO
We determined the developmental changes in the phosphorylation state of alphaB-crystallin in lenses from rats at various post-natal ages by isoelectric focusing gel electrophoresis or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of lenses using antibodies that recognized the carboxy-terminal sequence or each of the three phosphorylated serine residues (Ser-19, Ser-45 and Ser-59) in alphaB-crystallin. Phosphorylated forms of alphaB-crystallin were barely detected at birth but they became detectable at 3 weeks of age and reached plateau levels at 8 weeks of age. The phosphorylation of alphaB-crystallin at Ser-45 was observed preferentially. The active form of p44/42 MAP kinase, which is responsible for the phosphorylation of Ser-45 in alphaB-crystallin, also increased in a development-dependent manner. Thus we found that the developmental increase of the phosphorylation at Ser-45 of alphaB-crystallin in the rat lens was due to the developmental activation of p44/42 MAP kinase.
Assuntos
Cristalinas/metabolismo , Cristalino/metabolismo , Animais , Animais Recém-Nascidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Humanos , Masculino , Fosforilação , Proteínas Quinases/metabolismo , Ratos , Ratos WistarRESUMO
In this article, we show that induction of Th2 cell tolerance prevents antigen-induced eosinophil recruitment into the tissue and IgE antibody production, and that ICAM-1/LFA-1 interaction is involved as a costimulatory signal in inducing T cell tolerance to a soluble antigen. In vivo pretreatment with anti-ICAM-1 monoclonal antibody (mAb), anti-LFA-1 mAb, and a soluble antigen inhibited antigen-induced eosinophil recruitment into the airways and IgE antibody production in mice in an antigen-specific manner. In vitro antigen-induced IL-2, IL-4 and IL-5 production were decreased in spleen cells of the mice pretreated with the two mAbs and the antigen, indicating the induction of both Th1 and Th2 cell tolerance in vivo. These results suggest that the induction of antigen-specific Th2 cell tolerance by allergen immunotherapy with blockade of the ICAM-/LFA-1 interaction would be a rational therapeutic approach to allergic inflammation such as asthma.
Assuntos
Tolerância Imunológica , Células Th2/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Imunoglobulina E/biossíntese , Molécula 1 de Adesão Intercelular/imunologia , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucina-5/biossíntese , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Transdução de SinaisRESUMO
We have previously shown that neutral endopeptidase (NEP; EC 3.4.24.11) regulates neuropeptide-induced responses. Recently, Pierart et al. reported that NEP degraded purified interleukin-1 (IL-1) using thymocyte proliferation assay. Since IL-1 is an important cytokine in the immune response and inflammation, we have assessed whether NEP hydrolyzes recombinant human IL-1 beta using three assay systems (bioassay, immunoassay, and HPLC analysis). NEP on the NALM-6 cells (both intact cells and the solubilized plasma membrane fraction) efficiently hydrolyzed Met5-enkephalin and substance P. However, NEP did not significantly decrease the amount of rhIL-1 beta assessed by the growth inhibitory activity of a human melanoma, by the immunoassay, or by the direct analysis on HPLC. Therefore, we conclude that NEP does not significantly hydrolyze rhIL-1 beta. Our results suggest that, in contrast to the regulatory role of NEP in neuropeptide-induced responses, NEP is not a regulatory enzyme for IL-1-induced responses.