RESUMO
Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (tumeric). It was previously described that curcumin had a potent anti-inflammatory effect and inhibited the proliferation of a variety of tumor cells. In the present study, we investigated the inhibitory effects of curcumin on the response of normal murine splenic B cells. Curcumin inhibited the proliferative response of purified splenic B cells from BALB/c mice stimulated with the Toll-like receptor ligands LPS and CpG oligodeoxynucleotides. LPS-induced IgM secretion was also inhibited by curcumin. The proliferative response induced by either the T-independent type 2 stimuli anti-delta-dextran or anti-IgM antibodies was relatively resistant to the effect of curcumin. We investigated the intracellular signaling events involved in the inhibitory effects of curcumin on murine B cells. Curcumin did not inhibit the increase in calcium levels induced by anti-IgM antibody. Western blotting analysis showed that curcumin inhibited TLR ligands and anti-IgM-induced phosphorylation of ERK, IkappaB and p38. Curcumin also decreased the nuclear levels of NFkappaB. Our results suggested that curcumin is an important inhibitor of signaling pathways activated upon B cell stimulation by TLR ligands. These data indicate that curcumin could be a potent pharmacological inhibitor of B cell activation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Linfócitos B/efeitos dos fármacos , Curcumina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Animais , Anticorpos Anti-Idiotípicos , Linfócitos B/metabolismo , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Curcuma , Feminino , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The B cell antigen receptor complex (BCR) is composed of membrane Ig and heterodimers of Ig-alpha and Ig-beta/gamma. Recent findings indicate that Ig-alpha associates with Src-family kinases, including Fyn and Lyn, via an approximately 26 amino acid motif termed ARH1. Studies reported here (i) define two mechanisms whereby this motif binds Fyn and (ii) reveal an important functional consequence of binding, i.e. kinase activation. Mutational analysis indicates that specific low-affinity binding is determined by a short sequence, -DCSM-, in the motif and is not dependent on motif tyrosine residues. In contrast, the doubly tyrosine phosphorylated motif binds independently of DCSM and with high affinity. Importantly, this binding leads to Fyn activation. Taken together with studies which map low-affinity binding of Fyn or Lyn to the kinase's N-terminal unique region and high-affinity binding to the kinase's SH2 domain, these results suggest a mechanism of BCR activation in which the non-phosphorylated resting receptor is associated with Src-family kinases and, upon stimulation, tyrosine phosphorylation of Ig-alpha leads to reorientation and activation of receptor-associated kinases.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Análise Mutacional de DNA , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos B/genética , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
T-cell tolerance to the minor lymphocyte-stimulating antigen Mls-1a in a T-cell receptor (TcR) V beta 8.1 transgenic line of mice is maintained by both clonal deletion and clonal anergy. Approximately 20-50% of peripheral CD4+ (but not CD8+) T cells isolated from these mice are anergic and fail to proliferate following TcR ligation. We have examined key events in T-cell signaling in peripheral T cells isolated from these mice. In this report, we show that the anergic CD4+ T cells did not mobilize calcium or express receptors for interleukin 2 (IL-2) following TcR ligation. However, the cells retained viability and functional potential because stimulation with phorbol 12-myristate 13-acetate and ionomycin bypassed the block in receptor-mediated signaling and induced IL-2 receptor expression and proliferation of the anergic cells.
Assuntos
Tolerância Imunológica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Antígenos de Superfície/genética , Antígenos CD4/análise , Antígenos CD4/imunologia , Cálcio/análise , Deleção Cromossômica , Cruzamentos Genéticos , Expressão Gênica/efeitos dos fármacos , Antígenos H-2/genética , Ionomicina/farmacologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Antígenos Secundários de Estimulação de Linfócitos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina-2/análise , Receptores de Interleucina-2/genética , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
CD45 is a member of a family of membrane proteins that possess phosphotyrosine phosphatase activity, and is the source of much of the tyrosine phosphatase activity in lymphocytes. In view of its enzymatic activity and high copy number, it seems likely that CD45 functions in transmembrane signal transduction by lymphocyte receptors that are coupled to activation of tyrosine kinases. The B cell antigen receptor was found to transduce a Ca(2+)-mobilizing signal only if cells expressed CD45. Also, both membrane immunoglobulin M (mIgM) and CD45 were lost from the surface of cells treated with antibody to CD45, suggesting a physical interaction between these proteins. Finally, CD45 dephosphorylated a complex of mIg-associated proteins that appears to function in signal transduction by the antigen receptor. These data indicate that CD45 occurs as a component of a complex of proteins associated with the antigen receptor, and that CD45 may regulate signal transduction by modulating the phosphorylation state of the antigen receptor subunits.
Assuntos
Antígenos de Diferenciação/fisiologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , Animais , Antígenos de Diferenciação/genética , Cálcio/fisiologia , Linhagem Celular , Membrana Celular/fisiologia , Células Cultivadas , Células Clonais , Antígenos de Histocompatibilidade/genética , Imunoglobulina M/fisiologia , Antígenos Comuns de Leucócito , Camundongos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Plasmocitoma , Proteínas Tirosina Fosfatases , RNA Mensageiro/genética , Baço/imunologia , TransfecçãoRESUMO
We have previously shown that ligation of murine B cell membrane IgM or IgD can lead to inactivation of the signal transducing ability of unligated Ag receptors. We describe further studies of the molecular basis of this desensitization. Consistent with the possibility that ligand induced desensitization is mediated by protein kinase C (PKC) are findings that demonstrate that both Ig binding ligands and PKC activators (DIC8 or PMA) induce desensitization in virtually all resting B cells. However, ligand-induced desensitization is longer lived than PMA- or DIC8-induced desensitization and insensitive to the PKC inhibitor staurosporine. Further, biochemical studies indicate that insufficient PKC activation is induced by ligation of membrane Ig to mediate the observed desensitization. Thus data indicate that PKC must play only a minor role in ligand-induced membrane Ig desensitization. Further studies explored the molecular source and target of effectors that mediate ligand-induced desensitization. Data indicate that phosphoinositide hydrolysis is neither necessary nor sufficient for ligand induction of desensitization. Finally, ligand-induced desensitization appears to be mediated by uncoupling of membrane Ig from G proteins that regulate phospholipase C because ligand desensitized cells are hyperresponsive to agents including ALF4- and mastoparan which activate G proteins leading to mobilization of Ca2+. Thus, the function of G proteins and further downstream elements that mediate Ca2+ mobilization is intact. Taken together, these data are most consistent with ligand-induced membrane Ig desensitization being mediated by a non-PKC, non phosphatidylinositol 4,5-bisphosphate hydrolysis involving mechanism that has as its target a structure that is very proximal to the receptor, such as the receptor itself or a transducer complex analogous to CD3.
Assuntos
Linfócitos B/fisiologia , Regulação para Baixo , Receptores de Antígenos de Linfócitos B/fisiologia , Sequência de Aminoácidos , Animais , Diglicerídeos/farmacologia , Ativação Enzimática , Proteínas de Ligação ao GTP/fisiologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/fisiologia , Transdução de Sinais , Fatores de TempoRESUMO
Binding of ligand to B-cell membrane immunoglobulin (mIg) can lead to activation of a number of distinct biologic responses, including altered expression of genes encoding c-fos, c-myc, and Ia, as well as proliferation and immunologic tolerance. Tolerance could reflect a functional uncoupling of receptors from systems that generate intracellular second messengers (i.e., receptor desensitization). To better understand the molecular basis of immune regulation, we examined the ability of mIg to function as a signal transducer after the cell's initial contact with mIg-binding ligand. The results show that ligand binding to as little as 2-10% of mIgM or mIgD renders the cell unresponsive to ligand binding to the reciprocal isotype as judged by Ca2+ mobilization and protein kinase C translocation responses. This heterologous receptor desensitization lasts longer than 24 hr and does not reflect loss of receptor from the cell surface. Studies with the calcium ionophore ionomycin, 1,2-dioctanoyl-sn-glycerol, and the protein kinase inhibitor staurosporine indicate that both protein kinase C-dependent and protein kinase C-independent (staurosporine-insensitive) mechanisms mediate heterologous desensitization after mIg crosslinking.
Assuntos
Linfócitos B/imunologia , Cálcio/metabolismo , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo , Linfócitos B/metabolismo , Membrana Celular/imunologia , Imunofluorescência , Imunoglobulina D/fisiologia , Imunoglobulina G/fisiologia , Ligantes , Camundongos , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos B/imunologia , Baço/imunologiaRESUMO
Clinical and pathological findings in three adults with toxoplasmosis of the central nervous system are reported. Symptoms and signs in the first patient, a Haitian woman who had lived in France for 2 years, were suggestive of a right hemispheric space-occupying process. The second case was a woman treated for Hodgkin's disease who showed symptoms and signs of a meningoencephalitis. The presenting lesion in case 3 mimicked a tumor of the posterior fossa. Analysis of these 3 cases and of those previously reported underlines: 1) diagnostic difficulties, particularly in immunodepressed patients; 2) the effectiveness of immunoperoxidase for pathological diagnosis; 3) the poor prognosis due to absence of a specific treatment.