RESUMEN
Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.
Asunto(s)
Citometría de Flujo , Inhibidor NF-kappaB alfa , FN-kappa B , Humanos , Citometría de Flujo/métodos , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Proteolisis , Células THP-1 , Macrófagos/metabolismo , Macrófagos/inmunologíaRESUMEN
OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Isoquinolinas/uso terapéutico , Lactamas/uso terapéutico , Macrófagos/efectos de los fármacos , Enfermedades Reumáticas/tratamiento farmacológico , Sinoviocitos/efectos de los fármacos , Animales , Artritis Experimental/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Isoquinolinas/farmacología , Lactamas/farmacología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Ratones , Ratas , Enfermedades Reumáticas/inmunología , Sinoviocitos/inmunologíaRESUMEN
In this Letter, we describe the synthesis of several nonamidine analogs of biaryl acid factor VIIa inhibitor 1 containing weakly basic or nonbasic P1 groups. 2-Aminoisoquinoline was found to be an excellent surrogate for the benzamidine group (compound 2) wherein potent inhibition of factor VIIa is maintained relative to most other related serine proteases. In an unanticipated result, the m-benzamide P1 (compounds 21a and 21b) proved to be a viable benzamidine replacement, albeit with a 20-40 fold loss in potency against factor VIIa.
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Ácidos Carboxílicos/química , Descubrimiento de Drogas , Factor VIIa/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Benzamidinas , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Factor VIIa/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Serina Proteinasa/síntesis química , Relación Estructura-ActividadRESUMEN
H37Rv and H37Ra have been widely used as models of virulent and avirulent strains, respectively, of Mycobacterium tuberculosis. Since the sequencing of H37Rv, microarrays have been used to investigate gene expression of M. tuberculosis strains under various conditions, and to compare gene expression of specific isolates of the organism. Because differences in the virulence of these organisms could also be manifest via their differential induction of host genes, we used Affymetrix Human Genome Arrays U133A and U133B to evaluate human alveolar macrophage (AM) responses to infection with H37Rv and H37Ra. H37Rv altered expression of far more genes than did H37Ra. Moreover, the genes induced by H37Rv to a greater extent than by H37Ra were predominantly associated with the development of effective immunity. H37Rv markedly increased expression of IL-23 p19, whereas neither organism significantly induced IL-12 p35 expression. Quantitative PCR confirmed that H37Rv induced significantly more AM p19 expression than did H37Ra. After low-level infection of both AM and peripheral blood monocytes (MN) with H37Rv, neither cell type produced IL-12 (by ELISA). In contrast, AM displayed significant IL-23 production in response to H37Rv, whereas MN did not. Our findings thus suggest an important role for IL-23 in human host responses to pulmonary infection with M. tuberculosis, and are consistent with epidemiologic and genetic studies that imply that H37Rv may not have unusual capacity to cause human disease.
Asunto(s)
Regulación de la Expresión Génica , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Mycobacterium tuberculosis/fisiología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/microbiología , Regulación hacia Abajo/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-23/biosíntesis , Espacio Intracelular/microbiología , Monocitos/metabolismo , Monocitos/microbiología , Mycobacterium tuberculosis/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Tuberculosis/genética , Tuberculosis/microbiología , Regulación hacia Arriba/genéticaRESUMEN
Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.
Asunto(s)
Adipocitos/efectos de los fármacos , Azoles/antagonistas & inhibidores , Compuestos de Bifenilo/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Adipocitos/metabolismo , Animales , Sitios de Unión , Modelos Animales de Enfermedad , Epidermis/metabolismo , Hipoglucemiantes/síntesis química , Hipolipemiantes/síntesis química , Ratones , Modelos Químicos , Ensayo de Unión RadioliganteRESUMEN
BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ascariasis/inmunología , Interleucina-13/antagonistas & inhibidores , Neumonía/inmunología , Neumonía/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antígenos Helmínticos/inmunología , Ascaris suum , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Neumonía/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The structure of the complex formed between bovine beta-trypsin and the highly potent synthetic inhibitor 2-{3'-[5'-methoxy-2'-(N-p-diaminomethylphenyl)amido]-1'-pyrido}-5-(N-2''-t-butylethanol)amidobenzoic acid (C(28)H(32)N(5)O(6)) has been determined at 0.97 A resolution. X-ray intensity data were collected to 0.97 A under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R(cryst) factors of 13.4 and 12.6% and R(free) factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F(o) - F(c)| difference map, which accounts for an estimated 35% of all H atoms at the 2.5sigma level. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195-His57-Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N(delta1) and Asp102 O(delta1).
Asunto(s)
Inhibidores de Tripsina/química , Tripsina/química , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Tripsina/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
A novel class of 1H-(benzimidazol-2-yl)-1H-pyridin-2-one inhibitors of insulin-like growth factor I (IGF-1R) kinase is described. This report discusses the SAR of 4-(2-hydroxy-2-phenylethylamino)-substituted pyridones with improved IGF-1R potency.
Asunto(s)
Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Fosfotransferasas/antagonistas & inhibidores , Animales , Baculoviridae/enzimología , Línea Celular , Proliferación Celular/efectos de los fármacos , Indicadores y Reactivos , Ratones , Modelos Moleculares , Piridonas/farmacología , Relación Estructura-Actividad , Timidina/metabolismoRESUMEN
IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.
Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Modelos Animales de Enfermedad , Interleucina-13/antagonistas & inhibidores , Interleucina-13/inmunología , Oveja Doméstica/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Ascaris suum/fisiología , Asma/inducido químicamente , Asma/fisiopatología , Secuencia de Bases , Hiperreactividad Bronquial/parasitología , Hiperreactividad Bronquial/patología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Carbacol/farmacología , Femenino , Células HT29 , Humanos , Interleucina-13/química , Interleucina-13/genética , Cinética , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores de Interleucina-13/metabolismo , Oveja Doméstica/parasitología , Solubilidad/efectos de los fármacos , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Transparent substrates having heterogeneities ranging from nanometer to micrometer lateral length scale were fabricated to study cell migration. The surfaces were generated using thin films of block copolymers and homopolymer blends on ultra smooth transparent polyethylene terephthalate films. Results show that the lateral size scale of the surface heterogeneities affects fibroblast (NIH-3T3) adhesion, spreading and motility. More specifically, fibroblasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology on the micron patterned surfaces resemble cells cultured in a 3D environment. These surfaces, therefore, can potentially be utilized as models to study cell behavior in physiologically relevant conditions which can add to our fundamental understanding of cell-substrate interactions and facilitate development of surfaces for medical devices.
Asunto(s)
Movimiento Celular , Fibroblastos/citología , Polímeros/farmacología , Células 3T3 , Animales , Materiales Biocompatibles/farmacología , Adhesión Celular , Forma de la Célula , Ratones , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Compound 3 (BMS-536924), a novel small-molecule inhibitor of the insulin-like growth factor receptor kinase with equal potency against the insulin receptor is described. The in vitro and in vivo biological activity of this interesting compound is also reported.
Asunto(s)
Antineoplásicos/síntesis química , Bencimidazoles/síntesis química , Piridinas/síntesis química , Piridonas/síntesis química , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Haplorrinos , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Trasplante de Neoplasias , Piridinas/química , Piridinas/farmacología , Piridonas/química , Piridonas/farmacología , Ratas , Receptor de Insulina/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 microm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features.
Asunto(s)
Fibroblastos/citología , Fibroblastos/metabolismo , Nanoestructuras/química , Polímeros/química , Actinas/metabolismo , Animales , Adhesión Celular , Forma de la Célula , Tamaño de la Célula , Ratones , Microscopía Electrónica de Rastreo , Células 3T3 NIH , Seudópodos/metabolismo , Fibras de Estrés/metabolismoRESUMEN
IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.
Asunto(s)
Fibrosarcoma/inmunología , Interleucina-13/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Receptores de Interleucina/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral/trasplante , Fibrosarcoma/terapia , Terapia Genética , Subunidad alfa1 del Receptor de Interleucina-13 , Macrófagos/inmunología , Melanoma/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/inmunología , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/uso terapéutico , Receptores de Interleucina-13RESUMEN
Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B(4) (LTB(4)) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E(2) (PGE(2)) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0-120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB(4) signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB(4). Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE(2) reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro.
Asunto(s)
Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Cicatrización de Heridas/fisiología , Animales , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Regulación Enzimológica de la Expresión Génica , Indometacina/farmacología , Leucotrieno B4/metabolismo , Ratones , Células 3T3 NIHRESUMEN
A series of nonguanidine N1-activated C4-carboxy azetidinone tryptase inhibitors was prepared by solid-phase methodology to quickly assess the SAR associated with distal functionality on the N1-activating group. From these studies, potent inhibitors with improved specificity were discovered.
Asunto(s)
Azetidinas/síntesis química , Azetidinas/farmacología , Serina Endopeptidasas/efectos de los fármacos , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Azetidinas/química , Cristalografía por Rayos X , Modelos Moleculares , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/química , Relación Estructura-Actividad , TriptasasRESUMEN
The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility.
Asunto(s)
Actinas/efectos de los fármacos , Actinas/ultraestructura , Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Inhibidores de la Ciclooxigenasa/farmacología , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente Indirecta , Adhesiones Focales , Células HeLa , Humanos , Ratones , Microscopía Confocal , Miosina Tipo II/metabolismo , Células 3T3 NIH , Fibras de Estrés/metabolismoRESUMEN
Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.
Asunto(s)
Ácido Araquidónico/metabolismo , Proteínas Bacterianas , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Toxinas Bacterianas/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Matriz Extracelular/metabolismo , Células HeLa , Humanos , Transducción de Señal , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)alpha2 is a critical down-regulatory factor of IL-13-mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Ralpha2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Ralpha2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Ralpha2-deficient mice were treated with a soluble IL-13Ralpha2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Ralpha2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.
Asunto(s)
Interleucina-13/fisiología , Cirrosis Hepática/inmunología , Receptores de Interleucina/metabolismo , Esquistosomiasis mansoni/inmunología , Células Th2/inmunología , Animales , Regulación hacia Abajo , Femenino , Humanos , Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13 , Hígado/inmunología , Hígado/metabolismo , Hígado/parasitología , Hígado/patología , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina-13 , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/inmunología , Receptores de Interleucina-4/metabolismo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/patologíaRESUMEN
Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)alpha and IL-13Ralpha1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Ralpha2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Ralpha2, mice deficient in IL-13Ralpha2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Ralpha2-/- mice despite the fact that serum IL-13 was absent and immune interferon gamma production increased compared with wild-type mice. IL-13Ralpha2-deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Ralpha2 in regulating immune responses in wild-type mice.