RESUMEN
Due to their capacity to skew T cell responses towards Th1 oriented immunity, oligonucleotides containing unmethylated CpG motifs (CpG) have emerged as interesting adjuvants for vaccination. Whereas the signalling pathways in response to CpG mediated TLR9 activation have been extensively documented at the level of the individual cell, little is however known on the precise identity of the innate immune cells that govern T cell priming and polarisation to CpG adjuvanted protein antigens in vivo. In this study, we demonstrate that optimal induction of Th1 oriented immunity to CpG adjuvanted protein vaccines requires the coordinated actions of conventional DCs and of monocytes. Whilst conventional DCs were required for antigen presentation and initial T cell priming, monocytes constitute the main source of the Th1 polarising cytokine IL-12.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Inmunidad Celular , Inflamación/patología , Interleucina-12/biosíntesis , Monocitos/patología , Oligodesoxirribonucleótidos/farmacología , Células TH1/inmunología , Vacunas/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Antígenos Ly/metabolismo , Movimiento Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Inmunidad Celular/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fenotipo , Receptores CCR2/metabolismo , VacunaciónRESUMEN
Asthma is a heterogeneous disorder, evidenced by distinct types of inflammation resulting in different responsiveness to therapy with glucocorticoids (GCs). Tumor necrosis factor α (TNFα) is involved in asthma pathogenesis, but anti-TNFα therapies have not proven broadly effective. The effects of anti-TNFα treatment on steroid resistance have never been assessed. We investigated the role of TNFα blockade using etanercept in the responsiveness to GCs in two ovalbumin-based mouse models of airway hyperinflammation. The first model is GC sensitive and T helper type 2 (Th2)/eosinophil driven, whereas the second reflects GC-insensitive, Th1/neutrophil-predominant asthma subphenotypes. We found that TNFα blockade restores the therapeutic effects of GCs in the GC-insensitive model. An adoptive transfer indicated that the TNFα-induced GC insensitivity occurs in the non-myeloid compartment. Early during airway hyperinflammation, mice are GC insensitive specifically at the level of thymic stromal lymphopoietin (Tslp) transcriptional repression, and this insensitivity is reverted when TNFα is neutralized. Interestingly, TSLP knockout mice displayed increased inflammation in the GC-insensitive model, suggesting a limited therapeutic application of TSLP-neutralizing antibodies in subsets of patients suffering from Th2-mediated asthma. In conclusion, we demonstrate that TNFα reduces the responsiveness to GCs in a mouse model of neutrophilic airway inflammation. Thus antagonizing TNFα may offer a new strategy for therapeutic intervention in GC-resistant asthma.
Asunto(s)
Asma/inmunología , Resistencia a Medicamentos/efectos de los fármacos , Etanercept/farmacología , Hipersensibilidad/inmunología , Inmunosupresores/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiasmáticos/farmacología , Modelos Animales de Enfermedad , Femenino , Glucocorticoides/farmacología , Inmunoensayo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
Non-specific anti-inflammatory medication is actually the treatment of choice for controlling the T-helper type 2 (Th-2) cell-driven airway inflammation in asthma. The induction of counterbalancing Th-1 cell clones, long considered a promising approach for immunotherapy, has failed to fulfil its promise because of potentially detrimental side-effects. This is therefore probably not a valid option for the treatment of asthma. With the increasing awareness that active immune mechanisms exist to control inflammatory responses, interest rises to investigate whether these can be exploited to control allergen-induced airway disease. The induction of antigen-specific T cells with suppressive characteristics (regulatory T cells) is therefore a potentially interesting approach. These regulatory T cells mediate tolerance in healthy, non-atopic individuals and have the potential of becoming an effective means of preventing allergen-induced airway inflammation and possibly of suppressing ongoing allergic immune responses. Here we review the available knowledge about allergen-induced suppressive immunity obtained from animal models taking into account the different developmental stages of allergic airway disease.
Asunto(s)
Traslado Adoptivo/métodos , Asma/terapia , Modelos Animales de Enfermedad , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Alérgenos/administración & dosificación , Animales , Asma/inmunología , Humanos , Tolerancia Inmunológica , RatonesRESUMEN
The vertebrate globin family has been extended with two members: neuroglobin and cytoglobin. We here investigate the changes of expression levels upon hypoxia of cytoglobin in parallel with neuroglobin, in vivo and in vitro, by using real-time quantitative PCR. Our data prove that cytoglobin is upregulated upon hypoxia in all tissues. The mechanism of induction of cytoglobin is regulated by the hypoxia-inducible factor 1, a posttranscriptionally regulated transcription factor controlling several hypoxia-inducible genes. The latter is argumented by: (1) cytoglobin is significantly upregulated upon hypoxia and this is dependent on the tissue and severity of hypoxia; (2) the regulation of cytoglobin expression in HIF-1 (+/-) knockout mice is affected; (3) the variations of the expression regulation are in the same manner as seen in the expression of our control gene VEGF, that is proven to be regulated by the HIF-1-pathway; and (4) cytoglobin promoter region contains HRE sites.
Asunto(s)
Globinas/fisiología , Hipoxia/fisiopatología , Regulación hacia Arriba , Animales , Globinas/genética , Técnicas In Vitro , Ratones , Ratones Noqueados , Reacción en Cadena de la PolimerasaRESUMEN
TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.
Asunto(s)
Interferón gamma/inmunología , Interleucina-15/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Autotolerancia , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Apoptosis , Complejo CD3/inmunología , Citometría de Flujo , Interferón gamma/genética , Activación de Linfocitos , Ratones , Ratones NoqueadosRESUMEN
In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Mitocondrias/metabolismo , Necrosis , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasas/efectos de los fármacos , Grupo Citocromo c/metabolismo , Humanos , Cinética , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Factores de Tiempo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/efectos de los fármacos , Receptor fas/metabolismoRESUMEN
Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrow's medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Proteínas Recombinantes de Fusión/biosíntesis , Fosfatasa Alcalina/inmunología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/metabolismo , Especificidad de Anticuerpos , Western Blotting , División Celular , Dimerización , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Ratones , Peso Molecular , Proteínas de Mieloma/genética , Proteínas de Mieloma/metabolismo , Placenta/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Fragmentos Fab de Inmunoglobulinas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/farmacología , Sitios de Unión de Anticuerpos/genética , Línea Celular , Citotoxicidad Inmunológica/genética , Dimerización , Estabilidad de Medicamentos , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrow's medicine. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerization in mammalian cells was obtained and a novel intermediate sized, disulfide stabilized BsAb could be efficiently produced. This type of antibody derivative easily allows for the production of trispecific antibodies, BsAb with bivalent binding for one antigen, or immunoconjugates.
Asunto(s)
Anticuerpos Biespecíficos , Especificidad de Anticuerpos , Proteínas Recombinantes/inmunología , Anticuerpos Biespecíficos/genética , Especificidad de Anticuerpos/genética , Dimerización , Humanos , Fragmentos de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Región Variable de Inmunoglobulina , Tecnología Farmacéutica , Células Tumorales CultivadasRESUMEN
Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.
Asunto(s)
Presentación de Antígeno , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Pinocitosis/inmunología , Receptores de Antígenos/fisiología , Animales , Presentación de Antígeno/efectos de los fármacos , Compartimento Celular/efectos de los fármacos , Compartimento Celular/inmunología , Línea Celular Transformada , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Virus de la Influenza A/inmunología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuraminidasa/farmacología , Péptidos/inmunología , Péptidos/metabolismo , Pinocitosis/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.
Asunto(s)
Proteínas de Microtúbulos , Microtúbulos/efectos de los fármacos , Fosfoproteínas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Muerte Celular/efectos de los fármacos , Imidazoles/farmacología , Ratones , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación , Piridinas/farmacología , Estatmina , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Clonal deletion and anergy are 2 mechanisms used by the immune system to establish peripheral tolerance. In vitro, these mechanisms are induced in T lymphocytes by triggering the T-cell receptor (signal 1) in the absence of costimulation (signal 2). T-cell clones have been shown either to become anergic or to die in response to signal 1 alone; yet the factors that govern this choice remain unknown. This study evaluated the influence of the cytokines interleukin (IL)-2 and IL-15 on the response of the Th1 clone hemagglutinin (T-HA) to signal 1, delivered by stimulation with immobilized anti-CD3 monoclonal antibody (mAb). The response induced by immobilized anti-CD3 mAb was dependent on the cytokine milieu; in the presence of IL-2, T-HA cells were subject to apoptosis, whereas in the presence of IL-15 the cells remained viable but showed proliferative unresponsiveness. After release from the anti-CD3 stimulus, the IL-15-rescued T-HA cells regained responsiveness to IL-2 and IL-15 growth factor activity. However, they were unable to proliferate when stimulated with their cognate antigen presented by professional antigen-presenting cells (signal 1 plus 2) and thus had acquired an anergic phenotype. These data assign a novel function to the previously reported antiapoptotic activity of IL-15, namely, the capacity to redirect the T-cell response to partial stimulation from clonal deletion to anergy. Furthermore, they emphasize that the cytokine environment can critically influence the outcome of a tolerizing stimulus.
Asunto(s)
Apoptosis/inmunología , Interleucina-15/inmunología , Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/patología , Linfocitos T/fisiología , Animales , Complejo CD3/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Femenino , Interleucina-15/farmacología , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BLRESUMEN
The molecular motor kinesin is an ATPase that mediates plus end-directed transport of organelles along microtubules. Although the biochemical properties of kinesin are extensively studied, conclusive data on regulation of kinesin-mediated transport are largely lacking. Previously, we showed that the proinflammatory cytokine tumor necrosis factor induces perinuclear clustering of mitochondria. Here, we show that tumor necrosis factor impairs kinesin motor activity and hyperphosphorylates kinesin light chain through activation of two putative kinesin light chain kinases. Inactivation of kinesin, hyperphosphorylation of kinesin light chain, and perinuclear clustering of mitochondria exhibit the same p38 mitogen-activated kinase dependence, indicating their functional relationship. These data provide evidence for direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways.
Asunto(s)
Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Fosforilación , Piridinas/farmacología , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.
Asunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Mitocondrias/metabolismo , Fosfatidilserinas/metabolismo , Animales , Línea Celular , Etopósido , Sustancias de Crecimiento/deficiencia , Humanos , Potenciales de la Membrana , Ratones , Estaurosporina , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa , Receptor fas/genética , Receptor fas/farmacologíaRESUMEN
BACKGROUND: Hyperresponsiveness to histamine is a key feature of a variety of pathological conditions, including bronchial asthma, food allergy, colitis ulcerosa, and topical allergic disorders. Cells isolated from hyperresponsive individuals do not display exaggerated histamine responses ex vivo and thus the molecular mechanisms underlying histamine responsiveness remain obscure. Importantly, several in vivo observations implicate cysteinyl leukotrienes as possible mediators of increased histamine responses. We decided to investigate whether cysteinyl leukotrienes enhance the cellular reaction to histamine in cell types involved in pathological and immunological histamine hyperresponsiveness, as this might provide an in vitro system for studying histamine responsiveness and could shed light on the underlying molecular mechanisms. MATERIALS AND METHODS: Histamine responsiveness was determined by measuring histamine-induced prostaglandin E(2) production. Scatchard analysis was performed to determine the number of histamine H(1) receptors. Mouse macrophages, primary isolated human peripheral blood monocytes, and human umbilical smooth muscle cells were investigated before and after cysteinyl leukotriene stimulation. RESULTS: In all three cell types tested, cysteinyl leukotrienes instantaneously enhanced histamine-induced prostaglandin E(2) production. This increase in prostaglandin E(2) production coincided with the immediate and transient appearance of additional H(1) receptors on the plasma membrane. CONCLUSIONS: Cysteinyl leukotrienes prime histamine responses by recruiting additional histamine receptors in immunologically relevant cell types in vitro.
Asunto(s)
Cisteína/química , Antagonistas de los Receptores Histamínicos/farmacología , Histamina/farmacología , Leucotrienos/farmacología , Receptores Histamínicos H1/metabolismo , Animales , Sangre , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo , Dinoprostona/biosíntesis , Histamina/metabolismo , Humanos , Cinética , Leucotrienos/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Receptores Histamínicos H1/biosíntesisRESUMEN
TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.
Asunto(s)
Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Fibrosarcoma , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/fisiología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Lipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the biological effects of LPS in these cell types are only partially understood. We decided, therefore, to investigate the influence of LPS on macrophage pinocytosis and Fc receptor-mediated endocytosis, two prominent and related macrophage effector functions. We observed that LPS did not greatly influence endocytosis in either macrophages or monocytes, but did exert a dual action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS caused a decrease in pinocytosis in both macrophages and monocytes, whereas at higher LPS concentrations, enhanced pinocytosis in macrophages was observed. Detoxified LPS was two orders of magnitude less potent in producing these effects. After inhibition of the LPS receptor CD14, the LPS-induced decrease in pinocytosis was absent, and stimulation of pinocytosis at lower LPS concentrations was unmasked. We conclude that LPS can influence pinocytosis via CD14-dependent and CD14-independent signaling pathways. Furthermore, as addition of LPS to macrophages effected pinocytosis but not Fc receptor-mediated endocytosis, these two processes are independently regulated in macrophages.
Asunto(s)
Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Pinocitosis/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Transducción de Señal/efectos de los fármacosRESUMEN
TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mphi cytoskeleton, and that the ability of TNF to inhibit Mphi chemotaxis may be due to decreased filopodium formation downstream of Cdc42.
Asunto(s)
Actinas/fisiología , Proteínas de Ciclo Celular/fisiología , Inhibición de Migración Celular , Proteínas de Unión al GTP/fisiología , Macrófagos/fisiología , Proteínas Quinasas Activadas por Mitógenos , Seudópodos/fisiología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Actinas/antagonistas & inhibidores , Actinas/metabolismo , Animales , Antígenos CD/química , Antígenos CD/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Membrana Celular/fisiología , Células Cultivadas , Proteínas de Unión al GTP/antagonistas & inhibidores , Leucemia P388 , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Fragmentos de Péptidos/fisiología , Pinocitosis/efectos de los fármacos , Seudópodos/enzimología , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Acetato de Tetradecanoilforbol/farmacología , Proteína de Unión al GTP cdc42 , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR)-based subtraction suppression hybridization (SSH) to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.
Asunto(s)
Regulación de la Expresión Génica , Neoplasias Experimentales/genética , Proteínas/genética , ARN Mensajero/análisis , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/patología , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Tumor necrosis factor (TNF) induces a caspase-independent but mitochondria-dependent cell death process in the mouse fibrosarcoma cell line L929. Mitochondria actively participate in this TNF-induced necrotic cell death by the generation of mitochondrial reactive oxygen species (ROS). The aim of this study was to identify the mitochondrial components involved in TNF-induced production of ROS and their regulation by bioenergetic pathways. Therefore, we analyzed the bioenergetic characteristics in two metabolic L929 variants that exhibit different sensitivities to TNF. L929gln cells use glutamine as respiratory substrate and are far more susceptible to TNF-induced ROS generation and cell death as L929glc cells that use glucose as respiratory substrate. We show that the higher levels of reducing NAD(P)H equivalents, detected in the desensitized L929glc cells, do not cause diminished ROS generation. To the contrary, TNF increases the levels of NAD(P)H, probably altering complex I activity. A multiparameter analysis of electron flux through the mitochondrial electron transport chain, TNF-induced ROS levels, and cell death convincingly demonstrates a dependence of TNF signaling on complex I activity. Also, the sensitizing effect of glutamine metabolism correlates with an enhanced contribution of complex I to the overall electron flux. This participation of complex I activity in TNF-induced cell death is regulated by substrate availability rather than by a direct modification of complex I proteins. From the results presented in this paper we conclude that TNF-induced ROS generation and cell death are strongly regulated by bioenergetic pathways that define electron flux through complex I of the electron transport chain.