RESUMO
Mast cells (MCs) are classically associated with allergic asthma but their role in antiviral immunity is unclear. Human rhinoviruses (HRVs) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of HRV infection is the airway epithelium and MCs localise to this site with increasing asthma severity. The asthma susceptibility gene, IL-33, encodes an epithelial-derived cytokine released following HRV infection but its impact on MC antiviral responses has yet to be determined. In this study we investigated the global response of LAD2 MCs to IL-33 stimulation using RNA sequencing and identified genes involved in antiviral immunity. In spite of this, IL-33 treatment increased permissiveness of MCs to HRV16 infection which, from the RNA-Seq data, we attributed to upregulation of ICAM1. Flow cytometric analysis confirmed an IL-33-dependent increase in ICAM1 surface expression as well as LDLR, the receptors used by major and minor group HRVs for cellular entry. Neutralisation of ICAM1 reduced the IL-33-dependent enhancement in HRV16 replication and release in both LAD2 MCs and cord blood derived MCs. These findings demonstrate that although IL-33 induces an antiviral signature in MCs, it also upregulates the receptors for HRV entry to enhance infection. This highlights the potential for a gene-environment interaction involving IL33 and HRV in MCs to contribute to virus-induced asthma exacerbations.
Assuntos
Asma , Infecções por Picornaviridae , Humanos , Rhinovirus/fisiologia , Interleucina-33/farmacologia , Mastócitos/metabolismo , Antivirais/farmacologia , Permissividade , Replicação Viral , Células EpiteliaisRESUMO
A multichannel microfluidic platform for real-time monitoring of epithelial barrier integrity by electrical impedance has been developed. Growth and polarization of human epithelial cells from the airway or gastrointestinal tract was continuously monitored over 5 days in 8 parallel, individually perfused microfluidic chips. Electrical impedance data were continuously recorded to monitor cell barrier formation using a low-cost bespoke impedance analyser. Data was analysed using an electric circuit model to extract the equivalent transepithelial electrical resistance and epithelial cell layer capacitance. The cell barrier integrity steadily increased overtime, achieving an average resistance of 418 ± 121 Ω cm2 (airway cells) or 207 ± 59 Ω cm2 (gastrointestinal cells) by day 5. The utility of the polarized airway epithelial barrier was demonstrated using a 24 hour challenge with double stranded RNA to mimic viral infection. This caused a rapid decrease in barrier integrity in association with disruption of tight junctions, whereas simultaneous treatment with a corticosteroid reduced this effect. The platform is able to measure barrier integrity in real-time and is scalable, thus has the potential to be used for drug development and testing.
Assuntos
Espectroscopia Dielétrica , Microfluídica , Impedância Elétrica , Células Epiteliais , Humanos , Junções ÍntimasRESUMO
Mouse bone marrow-derived mast cells (mBMMCs) are an invaluable tool for the study of mast cell function as they represent a primary source of mature mast cells. They can be sourced from wild-type, knockout, and transgenic mice and are used to repopulate mast cell-deficient mice. This method describes the isolation of mast cell hematopoietic progenitors from the bone marrow of mouse femurs and their subsequent culture in an IL-3-rich culture medium. After 4 weeks in culture, mBMMCs are obtained in high number and are of high purity. Assessment of their granularity by toluidine staining and IgE receptor expression by flow cytometry is also described. These cells are a useful tool in the determination of in vitro and in vivo mast cell function in innate and adaptive immunity.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Mastócitos/citologia , Células-Tronco Mesenquimais/citologia , Cultura Primária de Células/métodos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Interleucina-3/análise , Interleucina-3/farmacologia , Mastócitos/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Receptores de IgE/genética , Receptores de IgE/metabolismoRESUMO
Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3â¯L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2â¯weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4â¯weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1ß and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Cultura Primária de Células/métodos , Adjuvantes Imunológicos/farmacologia , Antígenos CD34/metabolismo , Infecções Bacterianas/imunologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cesárea , Meios de Cultura/metabolismo , Citocinas/metabolismo , Células Dendríticas , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Recém-Nascido , Lipopolissacarídeos/imunologia , Poli I-C/imunologia , Viroses/imunologiaRESUMO
Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.
Assuntos
Engenharia Tecidual , Acústica , Animais , Biomarcadores , Reatores Biológicos , Adesão Celular , Impedância Elétrica , Células Epiteliais , Fibroblastos , HumanosRESUMO
INTRODUCTION: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood. METHODS: In a close-contact co-culture model of human airway epithelial and endothelial cells, cellular crosstalk was analyzed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy, and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system, the temporal release of mediators was analyzed in the co-culture model. RESULTS: Within 4 h of challenge, double-stranded RNA induced the release of TNF-α by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8 h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-α. CONCLUSION: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signaling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation.
Assuntos
Comunicação Celular , Células Epiteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Brônquios/citologia , Linhagem Celular , Células Cultivadas , Quimiocina CX3CL1/metabolismo , Técnicas de Cocultura , Selectina E/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Microfluídica , Poli I-C/farmacologia , RNA de Cadeia Dupla/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection.
Assuntos
Comunicação Celular , Microambiente Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Interleucina-1alfa/metabolismo , Mucosa Respiratória/metabolismo , Permeabilidade Capilar , Linhagem Celular , Células Cultivadas , Quimiocina CXCL10/metabolismo , Células Epiteliais/virologia , Fibroblastos/virologia , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Rhinovirus/patogenicidade , Transdução de SinaisRESUMO
The bronchial epithelium plays a key role in providing a protective barrier against many environmental substances of anthropogenic or natural origin which enter the lungs during breathing. Appropriate responses to these agents are critical for regulation of tissue homeostasis, while inappropriate responses may contribute to disease pathogenesis. Here, we compared epithelial barrier responses to different pollen species, characterized the active pollen components and the signaling pathways leading to epithelial activation. Polarized bronchial cells were exposed to extracts of timothy grass (Phleum pratense), ragweed (Ambrosia artemisifolia), mugwort (Artemisia vulgaris), birch (Betula alba) and pine (Pinus sylvestris) pollens. All pollen species caused a decrease in ionic permeability as monitored trans-epithelial electrical resistance (TER) and induced polarized release of mediators analyzed by ELISA, with grass pollen showing the highest activity. Ultrafiltration showed that the responses were due to components <3kDa. However, lipid mediators, including phytoprostane E1, had no effect on TER, and caused only modest induction of mediator release. Reverse-phase chromatography separated 2 active fractions: the most hydrophilic maximally affected cytokine release whereas the other only affected TER. Inhibitor studies revealed that JNK played a more dominant role in regulation of barrier permeability in response to grass pollen exposure, whereas ERK and p38 controlled cytokine release. Adenosine and the flavonoid isorhamnetin present in grass pollen contributed to the overall effect on airway epithelial barrier responses. In conclusion, bronchial epithelial barrier functions are differentially affected by several low molecular weight components released by pollen. Furthermore, ionic permeability and innate cytokine production are differentially regulated.
RESUMO
The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.
Assuntos
Células Epiteliais/citologia , Microfluídica , Sistema Respiratório/citologia , Humanos , Técnicas In VitroRESUMO
Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon-γ (IFN-γ) enhances FcγR-dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN-γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN-γ. IFN-γ also promoted bacteria killing, ß-hexosaminidase release and eicosanoid production. Interferon-γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-α and CXCL8 in S. aureus-stimulated huMCs. The ability of IFN-γ to increase CXCL8 and GM-CSF protein levels was confirmed by ELISA. Fibronectin or a ß1 integrin blocking antibody completely abrogated IFN-γ-dependent S. aureus binding and reduced S. aureus-dependent CXCL8 secretion. These data demonstrate that IFN-γ primes huMCs for enhanced anti-bacterial and pro-inflammatory responses to S. aureus, partially mediated by ß1 integrin.
Assuntos
Interferon gama/imunologia , Mastócitos/imunologia , Mastócitos/microbiologia , Staphylococcus aureus/imunologia , Imunidade Adaptativa , Animais , Células Cultivadas , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Citocinas/genética , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Integrina beta1/metabolismo , Interferon gama/farmacologia , Interleucina-8/genética , Interleucina-8/metabolismo , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/patogenicidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have previously shown that underground railway particulate matter (PM) is rich in iron and other transition metals across coarse (PM10-2.5), fine (PM2.5), and quasi-ultrafine (PM0.18) fractions and is able to generate reactive oxygen species (ROS). However, there is little knowledge of whether the metal-rich nature of such particles exerts toxic effects in mucus-covered airway epithelial cell cultures or whether there is an increased risk posed by the ultrafine fraction. Monolayer and mucociliary air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) were exposed to size-fractionated underground railway PM (1.1-11.1 µg/cm(2)) and release of lactate dehydrogenase and IL-8 was assayed. ROS generation was measured, and the mechanism of generation studied using desferrioxamine (DFX) and N-acetylcysteine (NAC). Expression of heme oxygenase-1 (HO-1) was determined by RT-qPCR. Particle uptake was studied by transmission electron microscopy. Underground PM increased IL-8 release from PBECs, but this was diminished in mucus-secreting ALI cultures. Fine and ultrafine PM generated a greater level of ROS than coarse PM. ROS generation by ultrafine PM was ameliorated by DFX and NAC, suggesting an iron-dependent mechanism. Despite the presence of mucus, ALI cultures displayed increased HO-1 expression. Intracellular PM was observed within vesicles, mitochondria, and free in the cytosol. The results indicate that, although the mucous layer appears to confer some protection against underground PM, ALI PBECs nonetheless detect PM and mount an antioxidant response. The combination of increased ROS-generating ability of the metal-rich ultrafine fraction and ability of PM to penetrate the mucous layer merits further research.
Assuntos
Brônquios/efeitos dos fármacos , Cílios/efeitos dos fármacos , Material Particulado/toxicidade , Meios de Transporte , Brônquios/citologia , Células Cultivadas , Células Epiteliais/citologia , Humanos , Tamanho da PartículaRESUMO
Mouse bone marrow-derived mast cells (mBMMCs) are an invaluable tool for the study of mast cell function from wild-type, knockout, and transgenic mice. This method describes the isolation of mast cell progenitors from the bone marrow of mouse femurs and their subsequent culture in an IL-3-rich culture medium. After 4 weeks, mBMMCs are obtained in high number and are of high purity. Assessment of their granularity by toluidine staining and IgE receptor expression by flow cytometry are also described. These cells are a useful tool in the determination of mast cell function in innate and adaptive immunity.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Hematopoéticas/fisiologia , Mastócitos/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular/imunologia , Separação Celular/métodos , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Mastócitos/citologia , CamundongosRESUMO
BACKGROUND: Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. OBJECTIVES: We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. METHODS: Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. RESULTS: TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1ß, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. CONCLUSIONS: Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.
Assuntos
Asma/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Quinases da Família src/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Brônquios/citologia , Caderinas/metabolismo , Cateninas/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Quinases da Família src/antagonistas & inibidores , delta CateninaRESUMO
The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3α, CCL22/monocyte-derived chemokine and tumour necrosis factor-α was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-γ-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.
Assuntos
Asma/imunologia , Brônquios/patologia , Células Epiteliais/patologia , Extratos Vegetais/química , Pólen/química , Alérgenos/química , Asma/metabolismo , Broncoscopia , Células Cultivadas , Quimiocinas/imunologia , Humanos , Inflamação , Interleucina-8/imunologia , Ligantes , PoaceaeRESUMO
This review will focus on human cell-based experimental models to study respiratory diseases, in particular models of the large airways relevant to asthma and chronic obstructive pulmonary disease. Such models have the advantage of incorporating cells that can be derived from disease-relevant tissue and so have retained important genetic and epigenetic features that contribute to the human disease. These models can be used for mechanistic studies, target identification and validation and toxicological testing. While many models have been developed to varying degrees of sophistication, the challenge remains to develop an integrated system that recapitulates the complex cell-cell and cell-matrix interactions that occur in vivo and to provide these with a 'circulation' to study the dynamics of immune and inflammatory cell influx and efflux.
Assuntos
Pneumopatias/patologia , Pulmão/patologia , Engenharia Tecidual , Comunicação Celular , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/patologia , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Fibroblastos/patologia , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Pneumopatias/metabolismo , Pneumopatias/fisiopatologia , Técnicas Analíticas Microfluídicas , Técnicas de Cultura de Órgãos , Engenharia Tecidual/métodosRESUMO
A bio-impedance chip has been developed for real-time monitoring of the kinetics of epithelial cell monolayers in vitro. The human bronchial epithelial cell line (16-HBE 14o-) was cultured in Transwells creating a sustainable and interactive model of the airway epithelium. Conducting polymer polypyrrole (PPy) doped with polystyrene sulfonate (PSS) was electrochemically deposited onto the surface of gold-plated electrodes to reduce the influence of the electrical double layer on the impedance measurements. Finite element and equivalent circuit models were used to model and determine the electrical properties of the epithelial cell monolayer from the impedance spectra. Electrically tight, confluent monolayers of 16 HBE 14o- cells were treated with increasing concentrations of either Triton X-100 to solubilize cell membranes or ethylene glycol-bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid (EGTA) to disrupt cell-cell adhesion. Experimental impedance data showed that disruption of epithelial barrier function in response to Triton X-100 and EGTA can be successfully measured by the bio-impedance chip. The results were consistent with the conventional hand-held trans-epithelial electrical resistance measurements. Immunofluorescent staining of the ZO-1 tight junction protein in the untreated and treated 16HBEs was performed to verify the disruption of the tight junctions by EGTA.
Assuntos
Células Epiteliais/citologia , Procedimentos Analíticos em Microchip , Linhagem Celular , Impedância Elétrica , Eletrodos , Humanos , Cinética , Polímeros/química , Poliestirenos/química , Pirróis/química , Fatores de TempoRESUMO
Activated mast cells are a major source of the eicosanoids PGD(2) and leukotriene C(4) (LTC(4)), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A(2), thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC(4) and PGD(2), respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcepsilonRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcepsilonRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcepsilonRI-dependent PGD(2), LTC(4), and ROS production in bone marrow-derived mast cells of Btk(-/-) mice. The FcepsilonRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A(2) phosphorylation but rather appears to regulate the generation of ROS, LTC(4), and PGD(2) by contributing to the necessary Ca(2+) signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.
Assuntos
Antígenos/farmacologia , Leucotrieno C4/imunologia , Mastócitos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Prostaglandina D2/imunologia , Proteínas Tirosina Quinases/imunologia , Espécies Reativas de Oxigênio/imunologia , Tirosina Quinase da Agamaglobulinemia , Androstadienos/farmacologia , Animais , Antígenos/genética , Antígenos/imunologia , Antígenos/metabolismo , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/genética , Ácido Araquidônico/imunologia , Ácido Araquidônico/metabolismo , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Humanos , Hipersensibilidade/enzimologia , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Leucotrieno C4/biossíntese , Leucotrieno C4/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Mastócitos/enzimologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipases A2 Citosólicas , Fosforilação/efeitos dos fármacos , Prostaglandina D2/biossíntese , Prostaglandina D2/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
We investigated the enzymes responsible for FcepsilonRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcepsilonRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcepsilonRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91(phox)- or p47(phox)-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcepsilonRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcepsilonRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcepsilonRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcepsilonRI-dependent degranulation and cytokine production.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ciclo-Oxigenase 1/metabolismo , Mastócitos/enzimologia , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgE/metabolismo , Receptores de IgG/metabolismo , Animais , Araquidonato 5-Lipoxigenase/deficiência , Araquidonato 5-Lipoxigenase/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Células Cultivadas , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Imunoglobulina G/farmacologia , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Inibidores de Lipoxigenase , Mastócitos/citologia , Mastócitos/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/imunologia , Camundongos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/imunologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Receptores de IgE/imunologia , Receptores de IgG/imunologiaRESUMO
Inhalation of crystalline silica results in pulmonary fibrosis and silicosis. It has been suggested that mast cells play a role in these conditions. How mast cells would influence pathology is unknown. We thus explored mast cell interactions with silica in vitro and in B6.Cg-kit(W-sh) mast cell-deficient mice. B6.Cg-kit(W-sh) mice did not develop inflammation or significant collagen deposition after instillation of silica, while C57Bl/6 wild-type mice did have these findings. Given this supporting evidence of a role for mast cells in the development of silicosis, we examined the ability of silica to activate mouse bone marrow-derived mast cells (BMMC), including degranulation (beta-hexosaminidase release); production of reactive oxygen species (ROS) and inflammatory mediators; and the effects of silica on Fc epsilon RI-dependent activation. Silica did not induce mast cell degranulation. However, TNF-alpha, IL-13, monocyte chemotactic protein-1, protease activity, and production of ROS were dose-dependently increased after silica exposure, and production was enhanced after Fc epsilon RI stimulation. This mast cell activation was inhibited by anti-inflammatory compounds. As silica mediates some effects in macrophages through scavenger receptors (SRs), we first determined that mast cells express scavenger receptors; then explored the involvement of SR-A and macrophage receptor with colleagenous structure (MARCO). Silica-induced ROS formation, apoptosis, and TNF-alpha production were reduced in BMMC obtained from SR-A, MARCO, and SR-A/MARCO knockout mice. These findings demonstrate that silica directs mast cell production of inflammatory mediators, in part through SRs, providing insight into critical events in the pathogenesis and potential therapeutic targets in silicosis.
Assuntos
Células da Medula Óssea/fisiologia , Mastócitos/fisiologia , Receptores Depuradores Classe A/metabolismo , Dióxido de Silício/toxicidade , Silicose/patologia , Animais , Apoptose , Células da Medula Óssea/metabolismo , Degranulação Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Colágeno/metabolismo , Interleucina-13/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgE/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Depuradores Classe A/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe the protocols used for the isolation and/or culture of these cells and discuss the relative merits of their use.