RESUMEN
Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso/metabolismo , Seudópodos/metabolismo , Células 3T3 , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Animales , Western Blotting , Bradiquinina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glutatión Transferasa/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Shigella/metabolismo , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Antigenic stimulation is associated with enhanced adhesion between T cells and antigen-presenting cells (APC). Binding of ligands to the T cell antigen receptor activates the adhesion function of lymphocyte function-associated molecule 1 (LFA-1; CD11a/CD18). We demonstrate here that ligand binding to major histocompatibility complex class II (Ia) molecules also activates LFA-1 function, providing a reciprocal mechanism for the induction of adhesion between T cells and Ia+ APC. Adhesion was affected by a qualitative change in LFA-1 molecules and was reversed by the protein kinase C inhibitor sphingosine. These results define a novel role for Ia molecules as signal transducing receptors that regulate LFA-1-dependent adhesion via a putative, Ia-coupled protein kinase(s).
Asunto(s)
Antígenos de Histocompatibilidad Clase II/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Linfocitos/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Linfocitos B/ultraestructura , Adhesión Celular/efectos de los fármacos , Activación Enzimática/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Linfocitos/citología , Linfocitos/fisiología , Proteína Quinasa C/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Linfocitos T/ultraestructura , Regulación hacia Arriba/fisiologíaRESUMEN
Supernates of human T cells stimulated with TT antigen contain a factor that induces mitogenesis and immunoglobulin synthesis in autologous as well as allogeneic B cells. A fraction of the IgG produced has specificity against TT. The T-cell-derived LMF-TT eluted after albumin on Sephadex G 200 and did not contain immunoglobulin heavy chain determinants. LMF-TT was active on B cells from TT immune as well as TT- nonimmune individuals but in the latter instance the IgG secreted had no specificity against TT. B cells incubated with LMF-TT in the presence of a second antigen (DT) made IgG with specifity to that antigen provided the B-cell donor was immune to that second antigen. LMF-TT-containing supernates were depleted of TT antigen by Sephadex G 200 chromatography followed by passage over anti-TT immunosorbent columns. The antigen-free supernates were able to induce mitogenesis and IgG synthesis in B cells but the IgG produced failed to exhibit specificity against TT unless the TT antigen was readded to the B-cell cultures. The optimal concentration of LMF-TT (50 percent) inducing B-cell mitogenesis was different from the optimal concentration (20 percent) causing IgG synthesis by B cells. At low LMF concentrations (less than or equal 10 percent) addition of a second antigen to which the cell donor was immune caused an increase in the degree of B-cell mitogenesis. Submitogenic concentrations of LMF-TT (less than or equal to 5 percent) were still capable of inducingimmunoglobulin synthesis in B cells At these low concentrations of LMF-TT the proportion of anti-TT IgG over total IgG increased sharply. B cells from TT immune donors were separated on TT immunosorbent columns. Cells that bound to the column were more sensitive to the mitogenic and IgG synthetic effects of LMF-TT than unfractionated B cells. Thus, LMF is a nonspecific human T-cell helper factor which behaves like a polyclonal B-cell activator. However, in the presence of specific antigen (TT) the antigen-specific B cell is preferentially triggered by LMF. The experimental design of the present study does not rule out the additional presence of an antigen-specific helper factor in the supernates of TT-stimulated human T cells.
Asunto(s)
Linfocitos T/inmunología , Formación de Anticuerpos , Células Productoras de Anticuerpos/inmunología , Antígenos , Linfocitos B/inmunología , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Inmunidad , Inmunoglobulina G/biosíntesis , Inmunoadsorbentes/farmacología , Mitosis , Toxoide TetánicoRESUMEN
Two human alloreactive T cell clones were established from a one-way mixed lymphocyte culture involving two nonatopic donors, and were assessed for their capacity to induce IgE synthesis by B cells obtained from the original stimulator. The two alloreactive T cell clones studied induced IgG but not IgE synthesis in normal B cells. However, one of the two clones, clone 2H6, induced IgE synthesis in the presence of supernatants from T cell lines derived from patients with the hyper-IgE syndrome (HIE), and enriched for T cells bearing receptors for IgE. These supernatants by themselves caused no IgE synthesis in nonatopic B cells. The potentiating factors in these supernatants were shown to bind to IgE. Time sequence experiments indicated that interaction of the B cells with the alloreactive clone 2H6 renders them responsive to the action of the IgE-potentiating factors. These results indicate that induction of IgE synthesis in normal B cells involves at least two sequential T cell derived signals. Furthermore, T cell clones are heterogenous in their capacity to provide these signals.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Proteínas de Secreción Prostática , Linfocitos T/inmunología , Células Clonales/inmunología , Humanos , Inmunoglobulina G/biosíntesis , Activación de Linfocitos , Linfocinas/inmunología , Receptores Fc/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The induction of IgE synthesis by IL-4 requires T cells and monocytes, as well as T cell- and monocyte-derived cytokines. Optimal cytokine combinations, however, fail to induce highly purified B cells to secrete IgE, indicating that additional signals are required. We show herein that the induction of human IgE synthesis by rIL-4 requires cognate interaction between the T cell receptor/CD3 complex on T cells and MHC class II antigens on B cells: mAbs directed against these molecules completely blocked IL-4-dependent IgE induction. mAbs against cell adhesion molecules (CD2, CD4, LFA-1) also inhibited IgE synthesis induced by IL-4, confirming that cell-cell contact is necessary for IgE induction. The requirement for cognate T/B cell interaction was further shown by comparing the IgE-inducing ability of two human IL-4-producing alloreactive T cell clones: F6, which recognizes MHC class II antigens on both B cells and monocytes, and A1, which recognizes an HLA-DP-associated epitope expressed on monocytes, but not on B cells. When incubated with B cells and monocytes from a normal donor bearing the appropriate alloantigen, clone F6, but not clone A1, induced vigorous IgE synthesis, although both clones proliferated and secreted IL-4. Taken together, our results suggest that at least two, possibly synergizing, signals are required for the T cell-dependent induction of IgE synthesis by B cells: one signal is delivered by cognate T/B cell interaction, the other by T cell-derived IL-4.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Linfocitos B/inmunología , Antígenos HLA-D/fisiología , Inmunoglobulina E/biosíntesis , Interleucinas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Anticuerpos Monoclonales/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos de Superficie/fisiología , Complejo CD3 , Moléculas de Adhesión Celular , Humanos , Técnicas In Vitro , Interleucina-4 , Cooperación LinfocíticaRESUMEN
IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4-treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti-CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper-IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG inhibits switching to IgE in B cells and suggest a novel potential mechanism for the prevention of allergic disease by DSCG.
Asunto(s)
Linfocitos B/inmunología , Cromolin Sódico/farmacología , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Inmunoglobulina E/biosíntesis , Recombinación Genética , Linfocitos B/efectos de los fármacos , Secuencia de Bases , ADN , Humanos , Cambio de Clase de Inmunoglobulina/genética , Interleucina-4/fisiología , Activación de Linfocitos , Datos de Secuencia Molecular , Monocitos/inmunología , Eliminación de Secuencia , Linfocitos T/inmunología , Transcripción GenéticaRESUMEN
The high affinity immunoglobulin G (IgG) receptor Fc gamma RI (CD64) is expressed constitutively on monocytes and macrophages, and is inducible on neutrophils. Fc gamma RI has recently been shown to be associated with the signal transducing gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma). Induction of cytoplasmic protein tyrosine phosphorylation by Fc gamma RI cross-linking is known to be important in mediating Fc gamma RI-coupled effector functions. Recently, syk has been implicated in this role. We now report that the src-type kinases hck and lyn are physically and functionally associated with Fc gamma RI. Hck and lyn coimmunoprecipitated with Fc gamma RI from detergent lysates of normal human monocytes and of the monocytic line THP-1. Hck and lyn showed rapidly increased phosphorylation and increased exogenous substrate kinase activity after cross-linking of Fc gamma RI. These results demonstrate both physical and functional association of the Fc gamma RI/Fc epsilon RI gamma receptor complex with hck and lyn, and suggest a potential signal transducing role for these kinases in monocyte/macrophage activation.
Asunto(s)
Proteínas Tirosina Quinasas/análisis , Proteínas Proto-Oncogénicas/análisis , Receptores de IgG/análisis , Familia-src Quinasas , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-hck , Receptores de IgG/fisiología , Células Tumorales CultivadasRESUMEN
CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti-CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3-kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Isoenzimas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Familia-src Quinasas , 1-Fosfatidilinositol 4-Quinasa , Anticuerpos Monoclonales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Western Blotting , Antígenos CD40 , Línea Celular , Activación Enzimática , Humanos , FosforilaciónRESUMEN
A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Interleucina-4/farmacología , Antígenos CD/inmunología , Linfocitos B/efectos de los fármacos , Antígenos CD40 , Células Cultivadas , Citometría de Flujo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Interleucina-6/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.
Asunto(s)
Linfocitos B/inmunología , Cambio de Clase de Inmunoglobulina , Glicoproteínas de Membrana/análisis , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Linfocitos T/fisiología , Adulto , Animales , Ligando de CD40 , Células Cultivadas , Humanos , Inmunoglobulina E/biosíntesis , Interferón gamma/biosíntesis , Ionomicina/farmacología , Glicoproteínas de Membrana/genética , Ratones , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Relatively pure populations of human T and B lymphocytes were obtained from blood and tonsils using density gradient centrifugation in bovine serum albumin. Antigen alone was incapable of triggering the B lymphocyte into blast transformation or to secrete antibody. However, supernatants from tetanus toxoid-stimulated T cells obtained from immune donors contained a factor mitogenic for B lymphocytes. 50-60% of B cells responded to this lymphocyte mitogenic factor (LMF) by proliferation, loss of C3 reactivity, and change to a secretory state. LMF-stimulated B cells exhibited a three- to fivefold increase in protein secretion and a six- to eightfold increase in gamma G globulin secretion. De novo secreted IgG had specificity directed to the tetanus toxoid present in the LMF containing T-cell supernatants. This was confirmed by an increase in the number of indirect plaque-forming cells to tetanus toxoid-coated sheep red blood cells after stimulation of B cells with LMF. It is proposed that in the course of the response to a previously encountered protein antigen, sensitized human T cells emit a signal in the form of a soluble product that, together with antigen, triggers B cells into division and antibody secretion. The experimental model utilized can be adapted to study human T-B cell cooperation under various conditions in normal individuals and in individuals with immunodeficiency diseases.
Asunto(s)
Formación de Anticuerpos , Reacciones Antígeno-Anticuerpo , Linfocitos B/inmunología , División Celular , Linfocitos T/inmunología , Animales , Células Productoras de Anticuerpos , Autorradiografía , Linfocitos B/metabolismo , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/biosíntesis , Células Cultivadas , ADN/biosíntesis , Eritrocitos/inmunología , Humanos , Inmunodifusión , Inmunoelectroforesis , Mitógenos , Tonsila Palatina/inmunología , Albúmina Sérica Bovina , Ovinos/inmunologíaRESUMEN
Supernates of tetanus toxoid (TT) antigen-stimulated human T cells were studied for the presence of an antigen-specific T-cell helper factor (ASF). Supernates were circulated over an immunosorbent column consisting of insolubilized TT antigen. The material which bound to the column was eluted with 3 M NaCNS and was shown to contain a factor which in the presence of TT-induced specific IgG anti-TT antibody synthesis in autologous B cells without causing readily detectable proliferation. ASF activity was partially inhibited by antisera directed against the B-cell alloantigens of the ASF donor. Immunosorbent columns containing such antisera removed ASF activity. Immunosorbent columns containing antisera to human immunoglobulin heavy chain determinants did not remove ASF activity; whereas immunosorbent columns containing rabbit idiotypic antiserum directed against anti-TT antibodies completely removed ASF activity. ASF was destroyed by treatment with proteolytic enzymes; its molecular weight was estimated by Sephadex G-100 gel column chromatography to be between 25,000 and 75,000 daltons.
Asunto(s)
Formación de Anticuerpos , Cooperación Linfocítica , Linfocitos T/inmunología , Toxoide Tetánico , Linfocitos B/inmunología , Humanos , Inmunoglobulina G/biosíntesis , Idiotipos de Inmunoglobulinas , Técnicas de Inmunoadsorción , Linfocinas/aislamiento & purificación , Peso Molecular , Antitoxina TetánicaRESUMEN
Human T cell helper/inducer clones were used to induce IgE synthesis in B cells from both allergic and nonallergic donors. An alloreactive T cell clone, activated by recognition of specific HLA-DR antigens, stimulated peripheral blood B cells from both allergic and nonallergic donors to synthesize IgE antibody. B cells of allergic donors differed from those of nonallergic donors in their requirements for induction of IgE synthesis. Induction of IgE synthesis in B cells from nonallergic individuals occurred only under conditions of cognate interaction, in which the B cells expressed the alloantigen recognized by the T cells. In contrast, IgE synthesis in B cells from allergic donors occurred under conditions of cognate interaction with T cells as well as bystander conditions where the B cells did not express the alloantigen recognized by the T cell clones and where the T cell clones were stimulated by third-party monocytes bearing the relevant alloantigens. Furthermore, bystander stimulation of IgE synthesis in allergic donors occurred in the presence of tetanus toxoid (TT) antigen-specific T cell clones activated by the appropriate TT-pulsed monocytes. In contrast to the differing requirements of B cells from normal vs. allergic subjects for the induction of IgE synthesis, these B cells did not differ in their requirements for the induction of IgG synthesis. IgG synthesis was induced in all B cells under conditions of cognate interaction with the T cells as well as under conditions of bystander stimulation. These results suggest that cognate T-B cell interactions may be important in the development of IgE immune responses in the normal host.
Asunto(s)
Linfocitos B/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/biosíntesis , Linfocitos T/inmunología , Adulto , Células Clonales/inmunología , Humanos , Isoantígenos/inmunología , Activación de Linfocitos , Cooperación LinfocíticaRESUMEN
T cells respond in a V beta-restricted fashion to bacterial enterotoxins bound to major histocompatibility complex (MHC) class II molecules. The requirement for CD4 in MHC class II-restricted T cell responses is very well established. We have assessed the role of CD4 in the T cell response to the bacterial enterotoxins Staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1. Three CD4- murine T cell hybridomas were transfected with the human CD4 molecule and assayed for interleukin 2 production in the presence of accessory cells bearing human MHC class II molecules and of the appropriate enterotoxin. The results clearly indicate that CD4- cells responded even to suboptimal concentrations of enterotoxin(s) equally well as CD4+ cells. Furthermore, expression of CD4 did not result in the acquisition of previously undetectable reactivity to enterotoxins. These results suggest that unlike the case with antigen-specific responses, formation of a T cell receptor-CD3/CD4 supramolecular complex is not always essential for T cell activation by bacterial enterotoxins.
Asunto(s)
Toxinas Bacterianas/inmunología , Antígenos CD4/fisiología , Enterotoxinas/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD4/genética , Línea Celular , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Hibridomas/inmunología , Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Staphylococcus , TransfecciónRESUMEN
The molecular events leading to immunoglobulin E (IgE) synthesis in human sIgE- B cells stimulated with interleukin 4 (IL-4) and anti-CD40 monoclonal antibody (mAb) 626.1 were analyzed. Anti-CD40 mAb increased the levels of IL-4-induced germline C epsilon transcripts and induced the production of mature C epsilon mRNA. These effects were dependent on the presence of IL-4. Nested primer PCR revealed deletional switch recombination occurring only in B cell stimulated with both IL-4 and anti-CD40 mAb. DNA sequence analysis of switch fragments showed direct S mu/S epsilon joining, without the deletions or duplications within S mu often found in B cells stimulated with IL-4 and Epstein-Barr virus. Analysis of the switch junction map sites showed "hot spots" for recombination within S mu, but not within S epsilon. These findings indicate that IL-4 provides a signal to B cells to induce germline C epsilon transcription and concurrent CD40 engagement induces S mu/S epsilon deletional switch recombination, production of mature C epsilon mRNA, and IgE synthesis.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Linfocitos B/inmunología , Inmunoglobulina E/genética , Interleucina-4/farmacología , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Antígenos CD40 , Clonación Molecular , Humanos , Inmunoglobulina E/biosíntesis , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
rIL-4 (B cell stimulatory factor 1) induces the expression of Fc epsilon R2/CD23 on normal human monocytes (Mo). Fc epsilon R2/CD23 induction was detectable both by flow cytometry using anti-CD23 mAbs as well as soluble IgE, and by the immunoprecipitation with CD23-specific mAb or IgE of a 45-kD band from 125I-lactoperoxidase-labeled Mo. Fc epsilon R2/CD23 was fully expressed after a 24-h incubation with rIL-4, and was still detectable after 72 h from the addition of IL-4. This effect was specific, because none of the other rILs tested (IL-1, IL-2, IL-3, IL-5, B cell stimulatory factor 2, granulocyte-macrophage colony stimulating factor, and IFN-gamma) could induce FC epsilon R2/CD23, either alone or in various combinations. No synergism was observed between IL-4 and other ILs. IFN-gamma was not able to inhibit the IL-4-induced expression of Fc epsilon R2/CD23 on Mo, neither when added to the culture together with IL-4, nor when added 36 h earlier.
Asunto(s)
Interleucinas/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Receptores Fc/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina E/inmunología , Interleucina-4 , Leucocitos Mononucleares/metabolismo , Linfocinas/farmacología , Receptores de IgE , Proteínas Recombinantes/farmacología , Estimulación QuímicaRESUMEN
Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.
Asunto(s)
Anticuerpos/inmunología , Glicoproteínas/farmacología , Interleucina-1/farmacología , Síndrome Mucocutáneo Linfonodular/inmunología , Venas/efectos de los fármacos , Antígenos de Superficie/inmunología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Preescolar , Endotelio/efectos de los fármacos , Femenino , Humanos , Lactante , Masculino , Factor de Necrosis Tumoral alfa , Venas/citologíaRESUMEN
Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.
Asunto(s)
Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales , Alelos , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Línea Celular , Citometría de Flujo , Regulación de la Expresión Génica , Reordenamiento Génico , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Transducción de Señal/inmunologíaRESUMEN
Wiskott-Aldrich syndrome (WAS) is an inherited immune deficiency that is marked by eczema, bleeding and recurrent infections. The lymphocytes and platelets of WAS patients display cytoskeletal abnormalities, and their T lymphocytes show a diminished proliferative response to stimulation through the T-cell receptor-CD3 complex (TCR-CD3). The product of the WAS gene, WAS protein (WASP), binds to the small GTPase Cdc42. Small GTPases of the Rho family are crucial for the regulation of the actin-based cytoskeleton. WASP and its relative NWASP might play an important role in regulating the actin cytoskeleton. Since both WASP and NWASP have the potential to bind to multiple proteins, they might serve as a hub to coordinate the redistribution of many cellular signals to the actin cytoskeleton. In this review, the authors discuss the possible role of WASP/NWASP and of the newly described protein WIP, which interacts with WASP and NWASP, in coupling signals from the T-cell receptor to the actin-based cytoskeleton.
Asunto(s)
Proteínas/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Actinas , Animales , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Linfocitos , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich , Proteínas de Unión al GTP rhoRESUMEN
The leukocyte CD11/CD18 adhesion molecules (beta 2 integrins) are a family of three heterodimeric glycoproteins each with a distinct alpha subunit (CD11a, b, or c) and a common beta subunit (CD18). CD11/CD18 mediate crucial leukocyte adhesion functions such as chemotaxis, phagocytosis, adhesion to endothelium, aggregation, and cell-mediated cytotoxicity. The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation of CD11/CD18 functions. To elucidate the nature of the qualitative modifications that occur, we examined the phosphorylation status of these molecules in resting human leukocytes and upon activation with PMA or with the chemotactic peptide F-met-leu-phe (FMLP). In unstimulated cells, all three CD11 subunits were found to be constitutively phosphorylated. In contrast, phosphorylation of the common CD18 subunit was minimal. PMA induced rapid and sustained phosphorylation of CD18 that occurred at high stoichiometry, but had only minimal effects on phosphorylation of the associated CD11 subunits. FMLP also induced rapid phosphorylation of CD18, but the effect was of short duration. FMLP-induced phosphorylation of CD18 was not related to its Ca++-mobilizing effect, as CD18 phosphorylation was not observed upon treatment of leukocytes with the Ca++ ionophore, ionomycin. Phosphoamino acid analysis of CD11/CD18 in PMA- or FMLP-treated monocytes revealed a predominance of phosphoserine residues in all CD11/CD18 subunits. A small component of phosphothreonine was present in CD11c and CD18 and a minor component of phosphotyrosine was also detected in CD18 upon leukocyte activation may regulate the adhesion functions mediated by the CD11/CD18 family of molecules.