RESUMO
Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)-specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.
Assuntos
Apoptose , Linfócitos B/imunologia , Tolerância Imunológica , Interleucina-4/farmacologia , Receptores de Antígenos de Linfócitos B/fisiologia , Receptor fas/fisiologia , Animais , Ligante de CD40 , Masculino , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muramidase/imunologiaRESUMO
The inability of B and T lymphocytes from mice expressing the lpr mutation to express functional Fas on their cell surface leads to an immunoregulatory defect associated with excessive autoantibody production. Nevertheless, T-dependent antibody response to foreign antigens in these mice appears relatively normal. To better understand exactly how Fas/FasL interactions control autoantibody production, studies were undertaken to determine (1) what kind(s) of B cells are sensitive to Fas-mediated apoptosis and (2) where the autoantibody-producing cells in lpr mice are located. We found that B cells activated by CD40L are extremely sensitive as targets in assays of Th1 CMC. However, B cells that receive a complete signal through their sIgM antigen receptor acquire a FasL-resistant phenotype. In situ analysis of splenic sections from lpr mice demonstrated that autoantibody-producing cells were uniquely localized to the T cell-rich inner PALS. A similar distribution pattern of IgG AFC was found in mice with chronic GVH disease. These data are consistent with the premise that the inner PALS, and not the germinal center, is the major site of FasL regulation of B cell activity and that, as a result of genetic or inducible loss of sensitivity to Fas-mediated apoptosis, autoreactive B cells may survive and differentiate in this location to cause serological autoimmunity.
Assuntos
Antígenos de Superfície/fisiologia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Glicoproteínas de Membrana/fisiologia , Receptor fas/fisiologia , Animais , Apoptose/imunologia , Linfócitos B/citologia , Proteína Ligante Fas , Doença Enxerto-Hospedeiro/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Baço/citologia , Linfócitos T/imunologiaRESUMO
The Addi-Chek Quality Control System (Millipore Corporation) and Ivex-2 Filterset (Abbott Laboratories) were evaluated to determine their effectiveness, applicability, and cost as part of a pharmacy quality-control program. Each method was tested using 50 solutions, 25 of which had been contaminated by inoculation with one of five micro-organisms; the other 25 solutions were used as controls. Aseptic technique was used, and procedures were carried out in a laminar air flow hood. Contaminated solutions were blinded from the person performing the tests. Addi-Chek detected contamination in all the inoculated solutions and in three of the uninoculated solutions. The latter may have been a result of adventitious contamination during the testing procedure. Ivex-2 detected contamination in 24 of the 25 inoculated solutions; no other contamination was found. The effectiveness of the methods in detecting low-level microbial contamination appears comparable. Both methods have been shown to be useful in the pharmacy setting, but Ivex-2 could be used to test for contamination when used as an in-line filter at the patient level. Ivex-2 is less expensive and warrants further evaluation in monitoring for microbial contamination during preparation and administration of intravenous solutions.
Assuntos
Infecção Hospitalar/prevenção & controle , Contaminação de Medicamentos/prevenção & controle , Infusões Parenterais/normas , Controle de Qualidade , Boston , Composição de Medicamentos/normas , Estudos de Avaliação como Assunto , Hospitais com 300 a 499 Leitos , Humanos , Métodos , Serviço de Farmácia Hospitalar/normasAssuntos
Apoptose/imunologia , Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-bcl-2 , Transdução de Sinais/imunologia , Receptor fas/imunologia , Animais , Linfócitos B/citologia , Cálcio/imunologia , Células Cultivadas , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Biossíntese de Proteínas , Proteína Quinase C/imunologia , Proteínas Proto-Oncogênicas/imunologia , Células Th1/imunologia , Proteína bcl-XRESUMO
Activated B cells express Fas (CD95) and are targets for apoptosis induced by CD4+ Th1 effector cells that kill in a Fas-dependent fashion. We report here that IL-4 reverses the susceptibility to Fas-mediated apoptosis that characterizes CD40-stimulated primary B cells. IL-4-induced Fas resistance is not associated with an alteration in the elevated level of Fas expression produced by CD40 ligand and does not depend on additional receptor-mediated signals. However, IL-4-induced resistance to Th1 cell-mediated cytotoxicity (Th1-CMC) develops more slowly than resistance mediated by surface Ig and is not affected by protein kinase C depletion, unlike anti-Ig-induced Fas resistance. By these two criteria, IL-4-and anti-Ig-induced resistance to Th1-CMC appear to be driven through distinct mechanisms; in keeping with this, suboptimal doses of IL-4 and anti-Ig act in synergy to induce marked protection against Th1-CMC. An important role for IL-4-induced Fas resistance is suggested by the observation that sera from IL-4-overexpressing transgenic mice contain autoreactive Abs.
Assuntos
Linfócitos B/efeitos dos fármacos , Interleucina-4/farmacologia , Receptor fas/fisiologia , Animais , Apoptose , Linfócitos B/metabolismo , Ligante de CD40 , Citotoxicidade Imunológica , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
CD40 ligand (CD40L) has been shown to increase surface Fas expression and induce B cell sensitivity to Fas-dependent CD4+ Th1 cell-mediated cytotoxicity (Th1-CMC). We investigated the role of unmethylated mitogenic CpG motifs in regulating Fas-mediated apoptosis in primary murine B cells. Unmethylated CpG motifs protected CD40L-stimulated B cells from Th1-CMC and apoptosis mediated by Fas-specific antibody. Mitogenic CpG motifs downregulated Fas expression on CD40L-stimulated B cells in a time-dependent fashion. These observations suggest that Fas-mediated apoptosis requires minimum upregulation of surface Fas expression and that CpG motifs protect B cells from Fas-mediated apoptosis by decreasing surface Fas expression. Thus, these results suggest a novel mechanism for induction of Fas resistance in B cells.
Assuntos
Apoptose , Linfócitos B/citologia , Glicoproteínas de Membrana/farmacologia , Receptor fas/fisiologia , Animais , Ligante de CD40 , Metilação de DNA , Fosfatos de Dinucleosídeos , Regulação para Baixo , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de TempoRESUMO
Primary murine splenic B cells are rendered sensitive or resistant to Fas-mediated apoptosis in a receptor-specific fashion. B cells stimulated though CD40 are Fas sensitive unless they also receive a signal though surface Ig that produces a state of resistance to Fas killing. Protection from Fas-mediated apoptosis takes time to develop and requires ongoing macromolecular synthesis; therefore, it appears to involve the induction and accumulation of one or more gene products. The role of Bcl-x was evaluated by examining the expression and function of this gene in primary B cells. bcl-x mRNA was induced by anti-IgM treatment of otherwise sensitive (CD40 ligand-treated) B cells. Bcl-x protein expression was induced by anti-IgM and appeared in a time frame that correlates well with the onset of anti-IgM-induced Fas resistance. Further, B cells from Bcl-x Tg mice were found to be resistant to Fas-mediated apoptosis. These results strongly suggest that the protection against Fas killing afforded by cross-linking surface Ig is mediated, at least in part, by an increase in Bcl-x.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptor fas/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos CD40/fisiologia , Interleucina-4/farmacologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , Regulação para Cima/imunologia , Proteína bcl-X , Receptor fas/imunologiaRESUMO
The susceptibility of primary B cells to Fas (APO-1, CD95)-mediated apoptosis is modulated by signals derived from additional surface receptors: CD40 engagement produces upregulation of Fas expression and marked sensitivity to Fas-induced cell death, whereas antigen receptor engagement, or interleukin-4 receptor (IL-4R) engagement, inhibits Fas killing and thereby produces Fas resistance, even in otherwise susceptible, CD40-stimulated targets. Surface immunoglobulin (sIg) and IL-4R utilize distinct signaling pathways to produce Fas resistance that rely on protein kinase C and signal transducer and activator of transcription 6, respectively sIg signaling for inducible Fas resistance requires nuclear factor-kappaB and depends on new macromolecular synthesis. Proximate mediators for Fas resistance include the known anti-apoptotic gene products Bcl-xL and FLIP (but not Btk), and a novel anti-apoptotic gene that encodes Fas apoptosis inhibitory molecule (FAIM). FAIM was identified by differential display and was cloned as two alternatively spliced forms: FAIM-S is broadly expressed, whereas faim-L expression is tissue specific. faim is highly evolutionarily conserved, suggesting an important function throughout phylogeny. Inducible resistance to Fas-mediated apoptosis is speculated to protect antigen-specific B cells during potentially dangerous interactions with FasL-bearing T cells; the elevated sIg-signaling threshold for inducible Fas resistance in autoreactive, tolerant B cells would insure against autoimmunity. However, aberrant acquisition of Fas resistance may allow autoreactive B cells to escape Fas deletion and malignant lymphocytes to thwart antitumor immunity.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas/imunologia , Receptores de Superfície Celular/metabolismo , Receptor fas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Linfócitos B/metabolismo , Humanos , Tolerância Imunológica , Dados de Sequência Molecular , Proteínas/genética , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Cytotoxic CD4+ Th1-cells induce cell death by triggering a Fas-dependent apoptotic pathway. Potential targets include activated B cells, but it is not known whether the mode of B-cell stimulation influences susceptibility to Th1-mediated cytotoxicity. Here we report that CD40-ligand-stimulated B cells were extremely sensitive, whereas anti-IgM-stimulated B cells were resistant, to Fas-mediated apoptosis. B cells stimulated by both CD40L and anti-IgM were not susceptible to cytolysis, demonstrating that anti-IgM-mediated protection is an active, dominant process. Resistance to Th1-mediated cytotoxicity was similarly observed in CD40L-stimulated 3-83 (anti-H-2Kk,b) transgenic B cells co-cultured with H-2Kk or H-2Kb (but not H-2Kd) splenocytes. These results indicate that B cells can participate in regulating their own destruction. Protection against Fas-dependent apoptosis afforded by immunoglobulin-receptor engagement may constitute a fail-safe mechanism that eliminates bystander B cells activated by CD40L-expressing T cells, but ensures survival of antigen-specific B cells.
Assuntos
Antígenos de Superfície/imunologia , Apoptose , Linfócitos B/imunologia , Citotoxicidade Imunológica , Células Th1/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Sequência de Bases , Antígenos CD40 , Ligante de CD40 , Primers do DNA , Imunoglobulina M/imunologia , Técnicas In Vitro , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Baço/citologia , Receptor fasRESUMO
The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity.
Assuntos
Linfócitos B/imunologia , Epitopos/imunologia , Ativação Linfocitária , Cooperação Linfocítica , Complexo Principal de Histocompatibilidade/imunologia , Glicoproteínas de Membrana/farmacologia , Células Th1/imunologia , Receptor fas/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Comunicação Celular/imunologia , Células Cultivadas , Regulação para Baixo/imunologia , Epitopos/efeitos dos fármacos , Proteína Ligante Fas , Imunoglobulinas/metabolismo , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Cooperação Linfocítica/genética , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Complexo Principal de Histocompatibilidade/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologiaRESUMO
CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.