RESUMEN
Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Calostro/química , Proteínas de Drosophila , Receptores de Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Leche Humana/química , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Bovinos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Cruzamientos Genéticos , Medio de Cultivo Libre de Suero , Humanos , Isotipos de Inmunoglobulinas/sangre , Inmunoglobulina M/sangre , Cadenas kappa de Inmunoglobulina/sangre , Receptores de Lipopolisacáridos/química , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal , Solubilidad , Bazo/citología , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.
Asunto(s)
Antígenos CD11/fisiología , Antígenos CD18/fisiología , Proteínas de Drosophila , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , Paclitaxel/farmacología , Receptores de Superficie Celular/fisiología , Transporte Activo de Núcleo Celular/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD11/biosíntesis , Antígenos CD11/genética , Antígenos CD18/biosíntesis , Antígenos CD18/genética , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Factores Reguladores del Interferón , Interferón gamma/metabolismo , Interferón gamma/fisiología , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-12/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Drosophila Toll protein is shown to activate the innate immune system in adult and regulate the dorsoventral patterning in the developing embryo. Recently, five human homologs of Drosophila Toll, designated as Toll-like receptors (TLRs), have been identified and shown to play a role in the innate immune response. We report here the molecular cloning and characterization of a new member of Toll-like receptor family, Toll-like receptor 6 (TLR6). Human and murine TLR6 are type-I transmembrane receptors that contain both an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic Toll/IL-1 receptor (IL-1R)-like region. The amino acid sequence of human TLR6 (hTLR6) is most similar to that of hTLR1 with 69% identity. RT-PCR analysis revealed that murine TLR6 is expressed predominantly in spleen, thymus, ovary and lung. Like other TLR family members, constitutively active TLR6 activates both NF-kappaB and c-Jun N-terminal kinase (JNK). The TLR6 gene, as well as the TLR1 gene, mapped to the proximal region of murine chromosome 5 within 1.7cM of each other. These results suggest that TLR6 is a novel member of an expanding TLR family.