RESUMEN
BACKGROUND: Clinical and experimental analyses have identified a central role for IgE/FcεRI/mast cells in promoting IgE-mediated anaphylaxis. Recent data from human studies suggest that bacterial infections can alter susceptibility to anaphylaxis. OBJECTIVE: We examined the effect of LPS exposure on the induction of IgE-mast cell (MC) mediated reactions in mice. METHODS: C57BL/6 WT, tlr4-/- and IL10-/- mice were exposed to LPS, and serum cytokines (TNF and IL-10) were measured. Mice were subsequently treated with anti-IgE, and the symptoms of passive IgE-mediated anaphylaxis, MC activation, Ca2+ -mobilization and the expression of FcεRI on peritoneal MCs were quantitated. RESULTS: We show that LPS exposure of C57BL/6 WT mice constraints IgE-MC-mediated reactions. LPS-induced suppression of IgE-MC-mediated responses was TLR-4-dependent and associated with increased systemic IL-10 levels, decreased surface expression of FcεRI on MCs and loss of sensitivity to IgE activation. Notably, LPS-induced desensitization of MCs was short term with MC sensitivity to IgE reconstituted within 48 hours, which was associated with recapitulation of FcεRI expression on the MCs. Mechanistic analyses revealed a requirement for IL-10 in LPS-mediated decrease in MC FcεRI surface expression. CONCLUSIONS & CLINICAL RELEVANCE: Collectively, these studies suggest that LPS-induced IL-10 promotes the down-regulation of MC surface FcεRI expression and leads to desensitization of mice to IgE-mediated reactions. These studies indicate that targeting of the LPS-TLR-4-IL-10 pathway may be used as a therapeutic approach to prevent adverse IgE-mediated reactions.
Asunto(s)
Inmunoglobulina E/inmunología , Lipopolisacáridos/inmunología , Mastocitos/inmunología , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Calcio/metabolismo , Degranulación de la Célula/inmunología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hematócrito , Interleucina-10/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Noqueados , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
The identification of the TLRs as key sensors of microbial infection has presented a series of new targets for drug development. The TLRs are linked to the most powerful inflammatory pathways in mammals. The question arises from the start: do we wish to stimulate TLR signaling in order to eradicate specific infections and/or neoplastic diseases? Or do we wish to block TLR signaling to treat inflammatory diseases? If we accept that it would be useful to modulate TLR signaling, the next step is to identify the correct molecular target(s) for the task. Perhaps it might even be possible to exercise selectivity, modulating some aspects of TLR signaling and not others. Classical and reverse genetic analyses offer insight into the possibilities that exist, and point to specific checkpoints within signaling pathways at which modulation might normally be imposed.
Asunto(s)
Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Transducción de Señal/efectos de los fármacos , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inhibidores , Infecciones Bacterianas/tratamiento farmacológico , Receptores Toll-Like/inmunologíaRESUMEN
Microbial components, such as lipopolysaccharides, augment immune responses by activating Toll-like receptors (TLRs). Some have interpreted this to mean that TLR signalling might not only help to initiate the adaptive immune response, but may also be required for it. The expanded view is shared by Pasare and Medzhitov, who conclude from an analysis of mice deficient in MyD88 (a TLR-signalling adaptor protein) that the generation of T-dependent antigen-specific antibody responses requires activation of TLRs in B cells. However, we show here that robust antibody responses can be elicited even in the absence of TLR signals. This appreciable TLR-independence of immune responses should be taken into account in the rational design of immunogenic and toleragenic vaccines.
Asunto(s)
Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Receptores Toll-Like/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Linfocitos B/inmunología , Humanos , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Inmunológicos , Factor 88 de Diferenciación Mieloide , Reproducibilidad de los Resultados , Transducción de Señal , Linfocitos T/inmunología , Vacunas/inmunologíaRESUMEN
With the mouse genome almost entirely sequenced and readily accessible to all who wish to examine it, the challenge across most biological disciplines now lies in the decipherment of gene and protein function rather than in the realm of gene identification per se. In the field of innate immunity, forward genetic methods have repeatedly been applied to identify key sensors, adapters, and effector molecules. However, most spontaneous mutations that affect innate immune function have been mapped and cloned, and the need for new monogenic phenotypes has been felt evermore keenly. N-Ethyl-N-nitrosourea (ENU) mutagenesis is an efficient tool for the creation of aberrant monogenic innate immune response phenotypes. In this review, we will discuss the potential of the forward genetic approach and ENU mutagenesis to identify new genes and new functions of known genes related to innate immunity.
Asunto(s)
Técnicas Genéticas , Inmunidad Innata/genética , Mutagénesis , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/fisiología , Antígenos CD36/genética , Antígenos CD36/fisiología , Etilnitrosourea/farmacología , Marcación de Gen , Genes Recesivos , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Modelos Inmunológicos , Mutágenos/farmacología , Factor 88 de Diferenciación Mieloide , Fenotipo , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiología , Receptores Toll-LikeRESUMEN
OBJECTIVE AND DESIGN: To investigate the effects of beta(2)-adrenoceptor (beta(2)-AR) stimulation on endotoxin-induced liver damage and systemic cytokine levels in rats. SUBJECTS: Standard male Wistar rats. TREATMENT: A disease-model of lipopolysaccharide (LPS)-induced acute systemic inflammation was used. The beta(2)-selective AR agonist clenbuterol was administered before, during, and after LPS-challenge to investigate its effects on the acute inflammatory response and associated liver-failure. METHODS: The following parameters have been measured in plasma: TNF alpha, IL-1 beta, IL-6, IL-10, AST, ALT, and Bilirubin. Liver histological examination was performed to look for changes in tissue morphology. RESULTS: Administration of clenbuterol (p.o.) one hour before, or intravenous at the same time as LPS-challenge resulted in a marked reduction of plasma levels of TNF alpha, IL-1 beta, and IL-6. A change both in plasma-level and in time-concentration profile of the anti-inflammatory cytokine IL-10 was found. Clenbuterol minimized LPS-induced liver damage, as represented by significantly lowered concentrations of several parameters for liver-failure (AST, ALT, Bilirubin), and improved hepatic tissue morphology. Clenbuterol administration after LPS challenge failed to inhibit TNF alpha-release but reduced liver-damage. Simultaneous use of the beta(2)-AR antagonist propranolol augmented LPS-induced liver failure, suggesting a role of endogenous adrenoceptor-agonists in prevention of organ-failure during systemic inflammation. CONCLUSIONS: The results indicate that a selective beta(2)-AR agonist might be used as an additional therapeutic agent in the clinic for the treatment of (acute) systemic inflammatory disorders in order to reduce or prevent subsequent liver failure.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Clenbuterol/farmacología , Endotoxinas/farmacología , Fallo Hepático/prevención & control , Hígado/efectos de los fármacos , Hígado/patología , Antagonistas Adrenérgicos beta/farmacología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Clenbuterol/antagonistas & inhibidores , Endotoxinas/antagonistas & inhibidores , Inflamación/prevención & control , Interleucina-1/antagonistas & inhibidores , Interleucina-1/sangre , Interleucina-10/antagonistas & inhibidores , Interleucina-10/sangre , Interleucina-6/antagonistas & inhibidores , Interleucina-6/sangre , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Masculino , Concentración Osmolar , Propranolol/farmacología , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Macrophages and dendritic cells are in the front line of host defense. When they sense host invasion, they produce cytokines that alert other innate immune cells and also abet the development of an adaptive immune response. Although lipolysaccharide (LPS), peptidoglycan, unmethylated DNA, and other microbial products were long known to be the primary targets of innate immune recognition, there was puzzlement as to how each molecule triggered a response. It is now known that the Toll-like receptors (TLRs) are the principal signaling molecules through which mammals sense infection. Each TLR recognizes a restricted subset of molecules produced by microbes, and in some circumstances, only a single type of molecule is sensed (e.g., only LPS is sensed by TLR4). TLRs direct the activation of immune cells near to and far from the site of infection, mobilizing the comparatively vast immune resources of the host to confine and defeat an invasive organism before it has become widespread. The biochemical details of TLR signaling have been analyzed through forward and reverse genetic methods, and full elucidation of the molecular interactions that transpire within the first minutes following contact between host and pathogen will soon be at hand.
Asunto(s)
Inmunidad Innata , Infecciones/inmunología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/fisiología , Humanos , Inflamación/inmunología , Lipopolisacáridos/toxicidad , Factor 88 de Diferenciación Mieloide , Receptores Inmunológicos/fisiología , Sepsis/inmunología , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Antígenos de Diferenciación/fisiología , Lipopolisacáridos/farmacología , Receptores Inmunológicos/fisiología , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Antígenos de Diferenciación/genética , Escherichia coli/fisiología , Homocigoto , Interferón Tipo I/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/virología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Factor 88 de Diferenciación Mieloide , Fenotipo , Mapeo Físico de Cromosoma , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/genética , Análisis de Secuencia de ADN , Especificidad por Sustrato , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismo , Virus Vaccinia/fisiologíaRESUMEN
Pentoxifylline (PTX) has been shown to exert hepatoprotective effects in various liver injury models. However, little information is available about the effect of PTX on the hepatic acute phase response. In the present study, the effect of PTX on a lipopolysaccharide (LPS)-induced acute phase response in primary porcine liver cell cultures was examined. During 72 hr of incubation with or without LPS, the ability of PTX to influence the secretion of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), acute phase proteins, and nitric oxide (NO) was assessed. PTX completely inhibited LPS-induced TNF-alpha production and attenuated IL-6 only after 48 hr of incubation. In contrast, PTX potentiated NO production and the expression of inducible nitric oxide synthase (iNOS) in hepatocytes after stimulation with LPS. The increased expression of iNOS and concurrent production of NO was also observed when liver cell cultures were incubated with dibutyryl cyclic adenosine monophosphate. No effect of PTX on acute phase protein secretion was observed during 72 hr of incubation. The present results show that PTX differentially affects the endotoxin-induced inflammatory response in primary porcine liver cell cultures by suppressing TNF-alpha and IL-6 while potentiating NO production.
Asunto(s)
Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Pentoxifilina/farmacología , Sustancias Protectoras/farmacología , Proteínas de Fase Aguda/efectos de los fármacos , Proteínas de Fase Aguda/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Citoprotección , Hígado/enzimología , Hígado/fisiología , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , PorcinosRESUMEN
This study focuses on the importance of direct contact between Kupffer cells (KCs) and hepatocytes (HCs) during the hepatic inflammatory response using an in vitro approach. The lipopolysaccharide (LPS)-induced inflammatory response in monocultures of porcine HCs and KCs were compared with cocultures prepared either with direct contact between KCs and HCs (DC cocultures) or without direct contact using cell culture membrane inserts. Our data show that DC cocultures exhibited the highest production of tumor necrosis factor (TNF)-alpha, interleukin-6, and nitric oxide (NO) compared with the other cultures. Immunohistochemical studies revealed that TNF-alpha was exclusively produced by KCs, whereas HCs were responsible for NO production after LPS stimulation. Biotransformation capacity, as determined by cytochrome P-450 and UDP glucuronosyl transferase enzyme activities, was most significantly decreased in DC cocultures. These results provide evidence that direct contact between KCs and HCs favors the extensive TNF-alpha production by KCs but in turn affects HC functionality and viability. These findings suggest that direct contact between KCs and HCs plays a key role in the development of a fulminating hepatic inflammatory response.
Asunto(s)
Comunicación Celular/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Endotoxinas/farmacología , Hepatocitos/fisiología , Macrófagos del Hígado/fisiología , Animales , Biotransformación , Western Blotting , Permeabilidad de la Membrana Celular/fisiología , Supervivencia Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Técnicas de Cocultivo , Hepatocitos/enzimología , Inmunohistoquímica , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Macrófagos del Hígado/enzimología , Masculino , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Especies Reactivas de Oxígeno , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.
Asunto(s)
Proteínas de Fase Aguda/biosíntesis , Reacción de Fase Aguda/genética , Regulación de la Expresión Génica/efectos de los fármacos , Haptoglobinas/biosíntesis , Hígado/metabolismo , Proteínas de Fase Aguda/genética , Animales , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Haptoglobinas/genética , Humanos , Interleucina-1/farmacología , Interleucina-6/farmacología , Macrófagos del Hígado/metabolismo , Lipopolisacáridos/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In this study primary hepatocyte cultures (HC cultures) and cocultures comprised of hepatocytes and Kupffer cells (HC/KC cocultures) were compared to investigate the inflammatory response induced by lipopolysaccharide (LPS). In addition both culture types were compared to study the hepatotoxic effects of two frequently used drugs: tiamulin and chlorpromazine. The inflammatory response in both culture types was determined by measurement of tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and nitric oxide (NO). The drug-induced hepatotoxic effects were determined by measuring production of intracellular reactive oxygen species (ROS) and cytotoxicity. Exposure of both cultures to LPS resulted in a significantly increased production of TNF-alpha, IL-6 and NO. However, the production of TNF-alpha, IL-6 and NO was substantially increased in culture supernatant of cocultures, compared to single HC-cultures. Both tiamulin and chlorpromazine were potent inducers of intracellular ROS production at concentrations > or = 50 microM. High ROS production was paralleled by increased cytotoxicity as observed in both culture types. Incubation of cocultures with chlorpromazine resulted in a significant increased ROS production as compared to HC cultures. In contrast, no significant differences between HC-cultures and HC/KC cocultures were observed for tiamulin induced ROS production or cytotoxicity. The present study demonstrates that cocultures between Kupffer cells and hepatocytes provide an excellent model for the study of hepatotoxic compounds which exert (part) of their toxic effects via the activation of Kupffer cells. Furthermore they offer a valuable tool to study increased susceptibility to intoxication from xenobiotic agents in case of a concurrent or pre-existing inflammation.
Asunto(s)
Macrófagos del Hígado/fisiología , Hígado/citología , Hígado/efectos de los fármacos , Drogas Veterinarias/toxicidad , Animales , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Modelos Animales de Enfermedad , Macrófagos del Hígado/efectos de los fármacos , Hígado/patología , Porcinos , Xenobióticos/toxicidadRESUMEN
Exposure of cultured neonatal rat heart cells to simulated ischaemia results in a cessation of the spontaneous contractile activity and changes at both the level of sarcolemmal phospholipid topology and the ultrastructural level. Reperfusion at a timepoint before irreversible cell damage develops leads to a recovery of contractile activity. Furthermore, the shift in transbilayer distribution of sarcolemmal phosphatidylethanolamine in favour of the outer membrane leaflet, due to the ischaemic period, is reversed during subsequent reperfusion. Also the morphological changes (mitochondrial oedema, reorganization of the mitochondrial cristae and the formation of extrusions at the sarcolemma) are reversible. At the same time total intracellular ATP levels are restored to 80% of control. The role of cellular ATP content on sarcolemmal phospholipid topology was further studied by the use of the calcium antagonist verapamil (10 microM), which preserved cellular ATP content by inhibiting cell contractility before the onset of ischaemia. After 120 min of ischaemia, cell ATP content was still 63% of control in the presence of verapamil, versus 20% of control in untreated cells. Verapamil treatment also prevented the loss of the asymmetrical distribution of phosphatidylethanolamine and sarcolemmal disruption, the latter occurring during 120 min of ischaemia in untreated cells. It is proposed that maintenance of phospholipid asymmetry of the sarcolemma of the myocytes depends on the cellular ATP concentrations, indicating the involvement of an ATP dependent aminophospholipid translocase.