RESUMEN
Carbonic anhydrase (CA) IV facilitates HCO(3) reabsorption in the renal proximal tubule by catalyzing the reversible hydration of CO(2). CAIV is tethered to cell membranes via a glycosylphosphatidylinositol (GPI) lipid anchor. As there is basolateral as well as apical CAIV staining in proximal tubule, the molecular identity of basolateral CAIV was examined. Biotinylation of confluent monolayers of rat inner medullary collecting duct cells stably transfected with rabbit CAIV showed apical and basolateral CAIV, and in the cell transfectants expressing high levels of CAIV, a transmembrane form was targeted to the basolateral membrane. Basolateral expression of CAIV ( approximately 46 kDa) was confirmed in normal kidney tissue by Western blotting of vesicle fractions enriched for basolateral membranes by Percoll density fractionation. We examined the mode of membrane linkage of basolaterally expressed CAIV in the kidney cortex. CAIV detected in basolateral or apical membrane vesicles exhibited similar molecular size by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis following deglycosylation, and was equally sensitive to phosphatidylinositol-specific phospholipase C digestion, indicating that CAIV is expressed on the basolateral membrane as a GPI-anchored protein. Half of the hydratase activity of basolateral vesicles was resistant to SDS denaturation, compatible with being CAIV. Thus, GPI-anchored CAIV resides in the basolateral membrane of proximal tubule epithelia where it may facilitate HCO(3) reabsorption via association with kNBC1.
Asunto(s)
Anhidrasa Carbónica IV/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Túbulos Renales Proximales/enzimología , Animales , Anticuerpos/inmunología , Bicarbonatos/metabolismo , Anhidrasa Carbónica IV/análisis , Anhidrasa Carbónica IV/genética , Membrana Celular/enzimología , Corteza Renal/enzimología , Peso Molecular , Transporte de Proteínas , Conejos , Ratas , TransfecciónRESUMEN
Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2). CA is expressed in most segments of the kidney. CAII and CAIV predominate in human and rabbit kidneys; in rodent kidneys, CAXII, and CAXIV are also present. CAIX is expressed by renal cell carcinoma (RCC). Most of these isoforms, except for rodent CAIV, have high turnover rates. CAII is a cytoplasmic enzyme, whereas the others are membrane-associated; CAIV is anchored by glycosylphosphatidylinositol linkage. Membrane polarity is apical for CAXIV, basolateral for CAXII, and apical and basolateral for CAIV. Luminal membrane CAs facilitate the dehydration of carbonic acid (H(2)CO(3)) that is formed when secreted protons combine with filtered bicarbonate. Basolateral CA enhances the efflux of bicarbonate via dehydration of H(2)CO(3). CAII and CAIV can associate with bicarbonate transporters (e.g., AE1, kNBC1, NBC3, and SCL26A6), and proton antiporter, NHE1 in a membrane protein complex called a transport metabolon. CAXII and CAXIV may also be associated with transporters in normal kidney and CAIX in RCCs. The multiplicity of CAs implicates their importance in acid-base and other solute transport along the nephron. For example, CAII on the cytoplasmic face and CAIV on the extracellular surface provide the 'push' and 'pull' for bicarbonate transport by supplying and dissipating substrate respectively.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Anhidrasas Carbónicas/fisiología , Riñón/enzimología , Animales , Anhidrasas Carbónicas/análisis , Anhidrasas Carbónicas/metabolismo , Carcinoma de Células Renales/enzimología , Humanos , Riñón/fisiología , Neoplasias Renales/enzimología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , ConejosRESUMEN
Although resting B cells as APC are tolerogenic for naive T cells in vivo, we show here that they can provide all the costimulatory signals necessary for naive T cell proliferation in vivo and in vitro. In the absence of an activating signal through the B cell Ag receptor, T cell proliferation after Ag recognition on resting B cells depends on CD40 expression on the B cells, implying that naive T cells use the membrane-bound cytokine, CD40 ligand (CD154), to induce the costimulatory signals that they need. Induction of B7-1 (CD80) and increased or sustained expression of CD44H, ICAM-1 (CD54), and B7-2 (CD86) are dependent on the interaction of CD40 ligand with CD40. Transient expression (12 h) of B7-2 is T cell- and peptide Ag-dependent, but CD40-independent. Only sustained (>/=24 h) expression of B7-2 and perhaps increased expression of ICAM-1 could be shown to be functionally important in this system. T cells cultured with CD40-deficient B cells and peptide remain about as responsive as fresh naive cells upon secondary culture with whole splenic APC. Therefore, B cells, and perhaps other APC, may be tolerogenic not because they fail to provide sufficient costimulation for T cell proliferation, but because they are deficient in some later functions necessary for a productive T cell response.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Antígenos CD40/fisiología , Glicoproteínas de Membrana/fisiología , Subgrupos de Linfocitos T/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Antígeno B7-2 , Antígenos CD28/fisiología , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligando de CD40 , Células Cultivadas , Anergia Clonal , Interfase/inmunología , Ligandos , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Streptococcus pneumoniae is a significant pathogen of young children and the elderly. Systemic infection by pneumococci is a complex process involving several bacterial and host factors. We have investigated the role of CD40L in host defense against pneumococcal infection. Treatment of mice with MR-1 antibody (anti-CD154/CD40L) markedly reduced antibody responses to the pneumococcal protein PspA, elicited by immunization of purified protein or whole bacteria. In mice immunized with whole bacteria, MR-1 treatment reduced antibody responses to capsular polysaccharides but not cell wall polysaccharides. MR-1 did not suppress antibody responses to isolated capsular polysaccharides but did reduce the production of antibody to a capsular polysaccharide-protein conjugate, indicating that when presented in the context of whole bacteria, the humoral response to capsular polysaccharides is partially T-cell dependent. Despite the reduction of the protective humoral responses to pneumococcal infection, administration of MR-1 had no effect on sepsis, lung infection, or nasal carriage in nonimmune mice inoculated with virulent pneumococci. Thus, short-term neutralization of CD40L does not compromise innate host defenses against pneumococcal invasion.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Streptococcus pneumoniae/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40 , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Polisacáridos Bacterianos/inmunología , Receptores del Factor de Necrosis Tumoral/fisiologíaRESUMEN
Coupling of membrane Ig (mIg) and CD40 to the extracellularly regulated kinase (ERK) signal transduction pathway was examined in the WEHI-231 B lymphoma and normal mouse B cells. Cross-linking mIg induces ERK activation in both WEHI-231 and normal B cells. In contrast, CD40 cross-linking failed to induce ERK activation in WEHI-231, but signals through CD40 were more effective than mIg as a stimulus for ERK activation in normal B cells. However, several lines of evidence suggest that CD40 and the B cell Ag regulate ERK through distinct pathways that converge at the level of MEK-1, mitogen-activated protein kinase kinase. Abs to mIg or CD40 induced MEK-1 activation with different kinetics. Cross-linking of mIg, but not CD40, induced tyrosine phosphorylation of the SHC adapter molecule that couples receptors to Ras-dependent signaling pathways. Finally, agents that elevate cAMP, causing protein kinase A-mediated inhibition of Raf-1, inhibited activation of ERK in response to mIg cross-linking, but had no affect on ERK activation in response to anti-CD40 or Jun N-terminal kinase activation by signals through either receptor. Thus, CD40 uses an unidentified protein kinase A-insensitive MEK kinase, rather than Raf-1, to regulate ERK activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Antígenos CD40/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/inmunología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Antígenos CD40/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , AMP Cíclico/metabolismo , Activación Enzimática/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos , Linfoma de Células B , MAP Quinasa Quinasa 1 , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/fisiología , Proteínas Adaptadoras de la Señalización Shc , Bazo/citología , Bazo/inmunología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Células Tumorales Cultivadas , Tirosina/metabolismo , Dominios Homologos src/inmunologíaRESUMEN
Interleukin-5 plays a pivotal role in the regulation of Ig secretion from murine B cells. Resting B cells express few, if any, IL-5 receptors and do not respond to the lymphokine. Culture of resting B cells with IL-4 induced expression of the IL-5R alpha-chain, while signaling through membrane Ig stimulated expression of the IL-5R beta-chain. Surprisingly, IL-4 suppressed expression of the beta-chain on activated B cells and inhibited responsiveness to IL-5 in subsequent cultures. Simultaneous culture of B lymphoblasts with IL-4 and IL-5 elicited sustained expression of the beta-chain and promoted secretion of IgM. Thus, expression of IL-5 receptors and induction of Ig secretion requires coordination of signals provided by membrane Ig, IL-4, and IL-5.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Interleucina-4/fisiología , Interleucina-5/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Interleucina/biosíntesis , Transducción de Señal/inmunología , Animales , Linfocitos B/efectos de los fármacos , Células Cultivadas , Femenino , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/análisis , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/genética , Receptores de Interleucina-5RESUMEN
Induction of switch recombination to the gamma 1 and epsilon immunoglobulin (Ig) heavy chain loci was examined in B cells preactivated with anti-Ig (B lymphoblasts). In B lymphoblasts cultured with interleukin 4 (IL-4), IL-5 induced the accumulation of S micro-S gamma 1 rearrangements, but not epsilon recombination. Thus, IL-5 facilitates switch recombination directed to the gamma 1 heavy chain locus by IL-4, but additional signals are required to drive rearrangements to epsilon. Lipopolysaccharide (LPS), in the presence of IL-4, induced the accumulation of both S micro-S gamma 1 and S micro-S epsilon rearrangements, and cells treated with LPS exhibited 40-50-fold more S micro-S gamma 1 rearrangements than cells cultured with IL-5. Induction of switch recombination was not always associated with secretion of the respective Ig isotype, since concentrations of IL-4 that were sufficient to direct switch recombination to gamma 1 and epsilon in blasts treated with LPS failed to elicit secretion of IgG1 and IgE. These results demonstrate differential requirements for switch recombination to the gamma 1 and epsilon loci, as well as independent regulation of Ig gene rearrangement and secretion of each isotype.
Asunto(s)
Linfocitos B/inmunología , Regulación de la Expresión Génica , Genes de Inmunoglobulinas , Genes de Cambio , Inmunoglobulina E/genética , Inmunoglobulina G/genética , Isotipos de Inmunoglobulinas/genética , Recombinación Genética , Animales , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Células Cultivadas , ADN/aislamiento & purificación , ADN/metabolismo , Cartilla de ADN , Femenino , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , Isotipos de Inmunoglobulinas/biosíntesis , Interleucina-4/farmacología , Interleucina-5/farmacología , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Bazo/inmunologíaRESUMEN
We have examined the contributions of Interleukin 4 (IL-4), IL-5, and other stimuli to the expression of Immunoglobulin G1 (IgG1) and IgE in murine B lymphoblasts activated with anti-Ig. The combination of IL-4 and -5 induced B lymphoblasts to proliferate and to secrete IgM and IgG1. However, an additional stimulus was required along with IL-4 and -5 for induction of IgE secretion. This stimulus was provided by lipopolysaccharides (LPS) or cytokines produced by TC-1 or EL4 cells. In the absence of IL-5, exceptionally high concentrations of IL-4 (greater than 1,000 U/ml) were required to elicit IgG1 and IgE secretion from B lymphoblasts cultured with either LPS or TC-1-conditioned media (CM). To investigate regulation of expression of gamma 1 and epsilon genes by IL-4, -5, and LPS, the requirements for induction of gamma 1 and epsilon germline and productive transcripts were examined. Germline gamma 1, but not epsilon, transcripts were detected in RNA from B lymphoblasts treated with IL-4 and -5 for 48 h. In contrast, both germline gamma 1 and epsilon transcripts could be detected in B lymphoblasts cultured with IL-4 and LPS, and steady state levels of germline gamma 1 transcripts were four- to sevenfold higher in blasts cultured with LPS and IL-4, compared with blasts cultured with IL-4 and -5. LPS enhanced steady state levels of germline transcripts induced by IL-4, but LPS did not promote substantial accumulation of productive gamma 1 and epsilon transcripts. In contrast, IL-5 did not affect steady state levels of germline transcripts stimulated by IL-4, but did markedly increase levels of productive gamma 1 and epsilon transcripts. Thus, lymphokines regulate two distinct events in isotype switching: induction of germline transcripts (IL-4), and production of VDJ-C gamma 1 and VDJ-C epsilon mRNA (IL-5), which leads to secretion of IgG1 and IgE.
Asunto(s)
Linfocitos B/fisiología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Interleucina-4/fisiología , Interleucina-5/fisiología , Animales , Células Productoras de Anticuerpos/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia de Células B/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas/genética , Animales , Linfocitos B/fisiología , Ciclo Celular , Separación Celular , Genes , Genes myc , Histonas/genética , Ratones , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-myb , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Anti-immunoglobulin (Ig)-activated B lymphoblasts, prepared by culturing high-density B cells with anti-Ig-Sepharose for 48 h, can be induced to secrete IgM and IgG1 by a mixture of T cell-derived lymphokines containing interleukin (IL) 4. In this study we have examined the conditions required for lymphokine-mediated induction of IgG1 secretion and membrane (m)IgG1 expression in B lymphoblasts. Resting B cells exposed to IL 4 (10-100 U/ml) during anti-Ig-mediated blast transformation did not secrete IgG1 upon subsequent culture with lipopolysaccharide (LPS) regardless of whether IL 4 was present or absent during the secondary culture. In contrast, B lymphoblasts previously exposed to IL 4 did secrete IgG1 in response to T cell-derived lymphokines [EL 4 supernatant depleted of IL 4; (D)EL 4 SN]. However, optimal IgG1 secretion was obtained when B lymphoblasts were simultaneously exposed to IL 4 and other lymphokines. Pre-exposure to (D)EL 4 SN, which contains IL 5 and IL 2, failed to prepare anti-Ig blasts to secrete IgG1 in response to LPS and IL 4. Inhibition of IL 5 and IL 2 activity in EL 4 SN suppressed IL 4-mediated IgG1 secretion. Together, these data indicate that B lymphoblasts require IL 5 and IL 2 in addition to IL 4 to secrete IgG1, and that the IL 4 signal(s) must precede or accompany those provided by the other lymphokines. As a measure of the fraction of cells capable of switching to IgG1, we assessed expression of mIgG1 on B lymphoblasts by fluorescence flow cytometry. B lymphoblasts cultured for 3 days with (D)EL 4 SN and IL 4 (10-100 U/ml) were 8% to 20% mIgG1+; in the absence of IL 4 blasts did not express detectable mIgG1. Although anti-Ig blasts treated with LPS and IL 4 did not secrete appreciable IgG1, a substantial fraction of B lymphoblasts (4% - 19%) cultured with LPS and IL 4, but not LPS alone, expressed mIgG1. These results suggest that LPS and IL 4 are sufficient to commit B lymphoblasts to mIgG1 expression, but that additional signals provided by T cell-derived lymphokines are required to elicit IgG1 secretion.
Asunto(s)
Linfocitos B/fisiología , Inmunoglobulina G/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Interleucina-4/farmacología , Linfocinas/farmacología , Animales , Anticuerpos Antiidiotipos , Esquema de Medicación , Interleucina-2/farmacología , Interleucina-5/farmacología , Lipopolisacáridos/farmacología , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
We have analyzed requirements for IL-4-induced secretion of IgG1 from anti-Ig-activated B cells. Activated B cell blasts prepared by culture of high density B cells with anti-Ig failed to secrete IgG1 upon subsequent culture with LPS and IL-4. However, IL-4 markedly suppressed IgM secretion in the same cultures. Addition of a mixture of T cell-derived lymphokines or rIL-5 to LPS-stimulated anti-Ig blasts restored IL-4-stimulated IgG1 secretion; rIL-2 further enhanced the response to IL-4 + rIL-5. These results suggest that IL-4, IL-5, and IL-2 cooperate in the regulation of B lymphocyte Ig isotype expression.