RESUMEN
Staphylococcus aureus invades osteoblasts and can persist in the intracellular environment. The present study examined the role of osteoblast mitogen-activated protein kinase (MAPK) pathways in bacterial invasion. S. aureus infection of normal human and mouse osteoblasts resulted in an increase in the phosphorylation of the extracellular signal-regulated protein kinases (ERK 1 and 2). This stimulation of ERK 1 and 2 correlated with the time course of S. aureus invasion, and bacterial adherence induced the MAPK pathway. ERK 1 and 2 phosphorylation was time and dose dependent and required active S. aureus gene expression for maximal induction. The nonpathogenic Staphylococcus carnosus was also able to induce ERK 1 and 2 phosphorylation, albeit at lower levels than S. aureus. Phosphorylation of the stress-activated protein kinases was increased in both infected human and mouse osteoblasts; however, the p38 MAPK pathway was not activated in response to S. aureus. Finally, the transcription factor c-Jun, but not Elk-1 or ATF-2, was phosphorylated in response to S. aureus infection.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/microbiología , Staphylococcus aureus/patogenicidad , Animales , Adhesión Bacteriana , Células Cultivadas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Staphylococcus aureus/crecimiento & desarrollo , Rayos UltravioletaRESUMEN
Expression of high affinity IL-12 receptors is required for IL-12-mediated IFN-gamma production. Activation of this pathway has been shown to be critical in generating optimal cell-mediated immunity. Therefore, increased IL-12 receptor expression might be expected in the host response after infection by an intracellular bacterial pathogen. In the present study, we have made the surprising discovery that infection with Salmonella results in an early reduction of high affinity IL-12 receptor expression and activation. After oral inoculation with Salmonella, the level of mRNA expression encoding IL-12 receptor beta2 (IL-12Rbeta2) subunit was diminished 12 h postinfection in the mesenteric lymph nodes and subsequently in the spleen. Furthermore, decreased IL-12Rbeta2 mRNA expression was observed in CD4+ T lymphocytes isolated from the mesenteric lymph nodes and spleens of infected mice. Attenuated IL-12Rbeta2 mRNA expression correlated with reduced receptor signaling, as demonstrated by reduced IL-12-induced STAT4 phosphorylation in enriched T lymphocytes isolated from the mesenteric lymph nodes and spleens of Salmonella-infected mice. These in vivo results were substantiated with an in vitro model system. In this model system, T lymphocytes cocultured with Salmonella-infected macrophages expressed less IL-12Rbeta2 mRNA. The cocultured T cells were also less responsive to IL-12 as assessed by reduced phosphorylation of STAT4 and limited IFN-gamma secretion. Together, these studies suggest that Salmonella can limit an optimal host immune response by reducing the expression and activity of high affinity IL-12 receptors.
Asunto(s)
Receptores de Interleucina/biosíntesis , Salmonelosis Animal/inmunología , Salmonelosis Animal/metabolismo , Animales , Relación CD4-CD8 , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Interleucina-12/antagonistas & inhibidores , Interleucina-12/farmacología , Intubación Gastrointestinal , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Macrófagos/inmunología , Macrófagos/microbiología , Mesenterio , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , ARN Mensajero/biosíntesis , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Factor de Transcripción STAT4 , Salmonella/inmunología , Bazo/inmunología , Bazo/microbiología , Bazo/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/microbiología , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismoRESUMEN
Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-kappaB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.
Asunto(s)
Calcio/metabolismo , Células Dendríticas/metabolismo , Membranas Intracelulares/metabolismo , Macrófagos Peritoneales/metabolismo , FN-kappa B/fisiología , Sustancia P/farmacología , Animales , Citosol/metabolismo , Células Dendríticas/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB CRESUMEN
Optimal immune responses against an intracellular bacterial pathogen, such as Salmonella, involve the production of gamma interferon (IFN-gamma), which activates macrophages. It has recently been suggested that, interleukin-18 (IL-18), in addition to IL-12, contributes to the induction of IFN-gamma following infection. Given this hypothesis, an optimal host immune response against intracellular bacterial pathogens would include the induction of IL-18 secretion by macrophages due to Salmonella infection. We questioned whether Salmonella could induce macrophages to upregulate their expression of IL-18 mRNA and secretion of IL-18. With cultures of murine macrophages, we were surprised to find that infection by wild-type Salmonella dublin resulted in decreased expression of IL-18 mRNA and IL-18 secretion rather than an increase. Reduction of macrophage-derived IL-18 expression by wild-type Salmonella occurred early in the response, suggesting a direct effect. Furthermore, mice orally inoculated with wild-type Salmonella were shown to have reduced IL-18 mRNA expression at mucosal sites within hours postinoculation. Together these studies demonstrate Salmonella-induced reductions in IL-18 expression, suggesting that this intracellular pathogen may be capable of limiting a potentially protective immune response.
Asunto(s)
Interleucina-18/biosíntesis , Macrófagos/metabolismo , Salmonelosis Animal/inmunología , Secuencia de Aminoácidos , Animales , Sueros Inmunes/inmunología , Interferón gamma/biosíntesis , Interleucina-18/genética , Interleucina-18/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed. While significant amounts of recombinant IL-18 were present in E. coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody. Surprisingly, the majority of recombinant IL-18 present in E. coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody. However, we found that the BL21 strain of E. coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18. Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.
Asunto(s)
Interleucina-18/aislamiento & purificación , Péptidos/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión/genética , Endopeptidasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Hidrólisis , Interleucina-18/genética , Interleucina-18/inmunología , Ratones , Datos de Secuencia Molecular , Oligopéptidos , Péptidos/inmunología , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range.