RESUMO
Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.
Assuntos
Nicotina/toxicidade , Receptores Nicotínicos/fisiologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/fisiopatologia , Animais , Bungarotoxinas/toxicidade , Cálcio/metabolismo , Células Cultivadas , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Transporte de Íons , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nicotina/administração & dosagem , Nicotina/metabolismo , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The spatial organization of proteins in a cell population or in tissues is an important parameter to study the functionality of biological specimens. In this article, we have focused on tight junctions which form network-like features in immunofluorescence microscopy images. Usually, the organization or disorganization of tight junctions is noticed qualitatively. The aim of this article is to present a simple method to quantify the organization level of tight junction network using image analysis with a dedicated macro developed with Image J software. The method has been validated with simulated images displaying regular decrease of network organization. Then, the macro has been applied to immunofluorescence microscopy images of cells in culture and of tissue sections.
Assuntos
Células Epiteliais/metabolismo , Processamento de Imagem Assistida por Computador , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Simulação por Computador , Humanos , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Elastase Pancreática , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
In different physiopathological situations such as embryogenesis, wound repair and tumor invasion, isolated cells, or cell populations exhibit changes to their normal behavior and may acquire different migratory phenotypes. Live-cell imaging associated with the use of appropriate in vitro models in culture has become a powerful analytical tool for studying factors involved in cell migration and in cell-to-cell interactions. The scope of this chapter is to give an overview of in vitro models of cell migration and the technical advances permitting multiparameter quantification. The chapter hereby provides a detailed description of two-dimensional and three-dimensional approaches of cell dispersion and migration and finally gives a brief insight on computational quantification of the cell behavior.
Assuntos
Rastreamento de Células/métodos , Células Epiteliais/citologia , Imageamento Tridimensional/métodos , Microscopia de Vídeo/métodos , Técnicas de Cultura de Tecidos/métodos , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Humanos , Microscopia de Vídeo/instrumentação , Invasividade Neoplásica/patologia , CicatrizaçãoRESUMO
The lubricating abilities and the protective functions of hyaluronan, a structural component of interstitial and connective tissues, were assessed in in vitro models of airway mucus transport and epithelial barrier. We found that hyaluronan enhanced the transport of airway mucus by cilia and by cough: the lower the hyaluronan molecular weight, the higher the increase. By immunofluorescence and western blot, we observed a significant dose-dependent (0.1, 1, 5 and 10 mg/ml) increase by low molecular weight hyaluronan (40 kDa) in the expression of tight junction proteins such as ZO-1, as well as an increase in the trans-epithelial resistance. Incubation of airway epithelial cells with hyaluronan 40 kDa also significantly increased the gap junction functionality. Finally, we demonstrated that hyaluronan 40 kDa protects the airway epithelium against injury induced by bacterial products during infection. These results demonstrate that the expression and functionality of intercellular adhesion molecules are increased by hyaluronan which can also act as a lubricant at the airway epithelium surface and suggest that hyaluronan may play a therapeutic role in a variety of respiratory diseases.
Assuntos
Tosse/fisiopatologia , Citoproteção , Ácido Hialurônico/fisiologia , Muco/metabolismo , Transporte Biológico , Western Blotting , Morte Celular , Linhagem Celular , Cílios/metabolismo , Cílios/fisiologia , Tosse/tratamento farmacológico , Fermentação , Imunofluorescência , Junções Comunicantes/metabolismo , Junções Comunicantes/fisiologia , Humanos , Ácido Hialurônico/farmacologia , Proteínas de Membrana/metabolismo , Peso Molecular , Fosfoproteínas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Mucosa Respiratória/fisiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Streptococcus equi/química , Propriedades de Superfície , Junções Íntimas/metabolismo , Junções Íntimas/fisiologia , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment. METHODS: To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. RESULTS: We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected beta1 integrin expression nor the state of phosphorylation of FAK and RhoA. CONCLUSION: This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.
RESUMO
BACKGROUND: Staphylococcus aureus releases virulence factors (VF) that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting beta2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal) combined with a corticosteroid (fluticasone propionate, FP) was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. METHODS: A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. RESULTS: When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S) and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFalpha. CONCLUSIONS: Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting beta2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of beta2 adrenergic receptor agonist and glucocorticoid.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2 , Albuterol/análogos & derivados , Androstadienos/administração & dosagem , Mucosa Respiratória/microbiologia , Mucosa Respiratória/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Albuterol/administração & dosagem , Broncodilatadores/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Combinação de Medicamentos , Combinação Fluticasona-Salmeterol , Humanos , Mucosa Respiratória/efeitos dos fármacosRESUMO
Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the alpha7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca(2+), is involved in lung morphogenesis. Here, we have investigated the potential role of the alpha7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, alpha7 nAChR expression coincides with epithelium differentiation. Inactivating alpha7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in alpha7(-/-) mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old alpha7(-/-) mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only alpha7 nAChR, as opposed to heteropentameric alpha(x)beta(y) nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that alpha7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Células Epiteliais/fisiologia , Receptores Nicotínicos/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Humanos , Queratinas/metabolismo , Pneumopatias/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Fosfoproteínas/metabolismo , Receptores Nicotínicos/genética , Regeneração/fisiologia , Mucosa Respiratória/patologia , Proteína da Zônula de Oclusão-1 , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Live-cell imaging has become a powerful analytical tool in most cell biology laboratories. The scope of this paper is to give an overview of the environmental considerations for maintaining living cells on the microscope stage and the technical advances permitting multi-parameter imaging. The paper will then focus on two-dimensional and three-dimensional analysis of cell dispersion and migration and finally give a brief insight on computational modeling of the cell behavior.
Assuntos
Movimento Celular/fisiologia , Células/ultraestrutura , Imageamento Tridimensional , Microscopia de Vídeo , Animais , Linhagem Celular , HumanosRESUMO
The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF.
Assuntos
Albuterol/análogos & derivados , Fibrose Cística/metabolismo , Vesículas Secretórias/metabolismo , Membrana Serosa/metabolismo , Membrana Serosa/patologia , Traqueia/metabolismo , Traqueia/patologia , Albuterol/farmacologia , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Cloretos/metabolismo , Colforsina/farmacologia , AMP Cíclico/biossíntese , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Exocitose/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/ultraestrutura , Íons/metabolismo , Muramidase/metabolismo , Xinafoato de Salmeterol , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/enzimologia , Vesículas Secretórias/ultraestrutura , Traqueia/enzimologiaRESUMO
In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Doxorrubicina/farmacologia , Fibronectinas/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , HumanosRESUMO
AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.
Assuntos
Matriz Extracelular/fisiologia , Neutrófilos/fisiologia , Receptores Imunológicos/fisiologia , Adulto , Animais , Anticorpos/farmacologia , Adesão Celular , Movimento Celular , Citosol/fisiologia , Glicosilação , Humanos , Ativação do Canal Iônico/fisiologia , Peptídeos/farmacologia , Coelhos , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/efeitos dos fármacos , Valores de Referência , TendõesRESUMO
This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE(2) that was reduced by the bacterial treatment with MAFP, a cPLA(2) inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE(2). Also, in PA103-infected cells, PGE(2) was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE(2) and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Corpos de Inclusão/metabolismo , Mediadores da Inflamação/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/farmacologia , Proteínas de Bactérias/genética , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/microbiologia , Linhagem Celular , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Humanos , Metabolismo dos Lipídeos , Organofosfonatos/farmacologia , Prostaglandinas E/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
BACKGROUND: Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior. METHODS: The effect of EGCG (at concentrations lower than 10 microg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG. RESULTS: We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin. CONCLUSION: Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Brônquicas/tratamento farmacológico , Catequina/análogos & derivados , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Brônquicas/enzimologia , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/patologia , Catequina/farmacologia , Catequina/uso terapêutico , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imageamento Tridimensional , Metaloproteinase 2 da Matriz , Inibidores de Metaloproteinases de Matriz , Microscopia de Vídeo , Invasividade Neoplásica , Inibidores de Proteases/uso terapêutico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Vimentina/metabolismoRESUMO
BACKGROUND: For a better understanding of the early stages of cystic fibrosis (CF), it is of major interest to study respiratory epithelial cells obtained as early as possible. Although bronchoalveolar lavage has been proposed for this purpose, nasal brushing, which is a much less invasive technique, has seldom been used in CF infants. The aim of the present study was to examine in a few infants the feasibility of a nasal brushing technique for studies of airway epithelial functions in very young CF infants. METHODS: In 5 CF (median age 12, range 1-18 months) and 10 control infants (median age 5, range 1-17 months), a nasal brushing was performed by means of a soft sterile cytology brush, after premedication with oral paracetamol (15 mg/kg body weight) and rectal midazolam (0.2 mg/kg body weight). Samples were used for microbiological, cytological and functional studies. RESULTS: The procedure was well tolerated. Number of cells collected was similar in CF and non-CF patients (CF: median 230x10(3), range 42x10(3)-900x10(3); non-CF: median 340x10(3), range 140x10(3)-900x10(3)). Median number of viable cells was 67% (range 31-84%). Freshly obtained samples were successfully used for studies of ciliary beating frequency and cAMP-dependent chloride efflux. In 7 out of 17 cell cultures, confluence was obtained (CF: 2 out of 7; non-CF: 5 out of 10). The feasibility of studying protein release and mRNA expression of IL-8, IL-6 and TNF-alpha, under basal conditions and after stimulation by Pseudomonas aeruginosa, was demonstrated. CONCLUSIONS: By means of a simple nasal brushing technique easily performed and well tolerated, it is feasible, in infants, to harvest respiratory cells in sufficient amounts to study the airway epithelium using a broad range of techniques including cell culture.
Assuntos
Biópsia/métodos , Fibrose Cística/fisiopatologia , Mucosa Nasal/fisiopatologia , Técnicas de Cultura de Células , Cílios/fisiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mucosa Nasal/fisiologiaRESUMO
Abnormal epithelial repair to damage participates in airway remodeling in asthma by the paracrine regulation of mesenchymal cell functions. Retinoids control epithelial functions through nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR) activation, yet their expression and contribution to epithelial repair and to airway remodeling in asthma are unknown. We determined the plasma levels of retinol and the immunohistochemical expression of retinoid receptors in damaged and repaired bronchial epithelium from 9 control subjects, 10 subjects with intermittent asthma, 8 subjects with mild-to-moderate asthma, and 8 subjects with severe asthma. In addition, the effect of the retinoid receptor ligands, all-trans-retinoic acid, and 9-cis retinoic acid, on the synthesis of 38 factors potentially involved in epithelial repair and in airway remodeling was determined in human cultured airway epithelial cells and correlated with cell migration and proliferation. Circulating retinol was similar in the three patient groups. In contrast, the epithelial expression of RARgamma, RXRalpha, and RXRgamma was greater in subjects with severe asthma, as compared with patients with milder disease and to control subjects. Retinoid receptor expression correlated positively with the proportion of morphologically intact epithelium. In vitro, retinoids up-regulated the expression of the transcripts encoding transforming growth factor (TGF)-beta1, metalloproteinase-9, beta1-integrin, and hepatocyte growth factor receptor, and promoted wound repair and chemokinesis of human airway epithelial cells without altering proliferation. Cell treatment with an anti-TGF-beta1 monoclonal antibody partially reduced retinoid-induced effects. Persistent interaction between retinoids and some of their receptors, which are overexpressed by the bronchial epithelium of individuals with severe asthma, may contribute to an abnormal repair and to airway remodeling, partly through TGF-beta1 production.
Assuntos
Asma/patologia , Asma/fisiopatologia , Células Epiteliais/metabolismo , Mucosa Nasal/metabolismo , Receptores do Ácido Retinoico/metabolismo , Brônquios/metabolismo , Brônquios/cirurgia , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Integrina beta1/metabolismo , Ligantes , Metaloproteinase 9 da Matriz/metabolismo , Mucosa Nasal/citologia , Mucosa Nasal/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/metabolismo , Análise de Regressão , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Vitamina A/sangue , Cicatrização/efeitos dos fármacosRESUMO
We propose two methods for characterizing the spatio-temporal behavior of cell populations in culture. The first method, image auto-correlation microscopy (IACM), allows us to characterize the variation in the number of objects as a function of time, thus enabling the quantification of the clustering properties of cell populations to be performed. The second method, image cross-correlation microscopy (ICCM), allows us to characterize the migration properties of cell populations. The latter method does not require estimation or measurement of the trajectories of individual cells, which is very demanding when populations of >100 cells are examined. The capabilities of the two methods are demonstrated with simulated cell populations, and their usefulness is illustrated with experiments involving invasive and noninvasive tumor cell populations.
Assuntos
Células/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Animais , Catequina/análogos & derivados , Catequina/farmacologia , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Humanos , Camundongos , Neoplasias/patologia , Fatores de TempoRESUMO
In chronic obstructive pulmonary disease (COPD), exacerbations are generally associated with several causes, including pollutants, viruses, bacteria that are responsible for an excess of inflammatory mediators, and proinflammatory cytokines released by activated epithelial and inflammatory cells. The normal response of the airway surface epithelium to injury includes a succession of cellular events, varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even complete denudation of the basement membrane. The epithelium then has to repair and regenerate to restore its functions, through several mechanisms, including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. In COPD, the remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia that occur during injury, may considerably disturb the innate immune functions of the airway epithelium. In vitro and in vivo models of airway epithelial wound repair and regeneration allow the study of the spatiotemporal modulation of cellular and molecular interaction factors-namely, the proinflammatory cytokines, the matrix metalloproteinases and their inhibitors, and the intercellular adhesion molecules. These factors may be markedly altered during exacerbation periods of COPD and their dysregulation may induce remodeling of the airway mucosa and a leakiness of the airway surface epithelium. More knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to cytoprotective and regenerative therapeutics, allowing the reconstitution of a functional, well-differentiated airway epithelium in COPD.
Assuntos
Diferenciação Celular/fisiologia , Inflamação/imunologia , Pulmão/fisiologia , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Regeneração/fisiologia , Mucosa Respiratória/citologia , Animais , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Pneumonia/microbiologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/terapia , Regeneração/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologiaRESUMO
Although Staphylococcus aureus is a major cause of pulmonary infection, the role played by this bacterium in the induction of inflammation of human airway epithelial cells (HAEC) is poorly understood. In this study, we investigated the inflammatory response of HAEC to S. aureus soluble virulence factors and demonstrate that the combination of a long-acting beta2-adrenergic receptor agonist (salmeterol) with a glucocorticoid (fluticasone propionate) has an anti-inflammatory effect on HAEC. First, we demonstrate increased expression at both the mRNA and protein levels of interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF)-alpha following incubation of HAEC in the presence of S. aureus soluble virulence factors and the increase of 1) the free nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activities and 2) the phosphorylated (P-) extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), the P-c-Jun NH2-terminal kinase (JNK), and the P-isoform-alpha of the NF-kappaB inhibitor (IkappaB alpha). Next, when HAEC were preincubated with the combination of salmeterol and fluticasone propionate, the inflammatory response of HAEC was markedly attenuated in that levels of IL-8, IL-6, TNF-alpha, NF-kappaB, AP-1, P-ERK1/ERK2, P-JNK, and P-IkappaB alpha decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a beta2-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation.
Assuntos
Albuterol/análogos & derivados , Androstadienos/farmacologia , Mediadores da Inflamação/imunologia , Pneumonia/tratamento farmacológico , Mucosa Respiratória/imunologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Linhagem Celular Transformada , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Combinação de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Combinação Fluticasona-Salmeterol , Expressão Gênica/imunologia , Glucocorticoides/farmacologia , Humanos , Quinase I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Pneumonia/imunologia , Pneumonia/microbiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Solubilidade , Traqueia/citologia , Fator de Transcrição AP-1/metabolismo , VirulênciaRESUMO
Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, alpha3alpha5beta2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas alpha7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of alpha3beta2 neuronal nAChR antagonists, mecamylamine, alpha-conotoxin MII, and kappa-bungarotoxin; alpha-bungarotoxin, an antagonist of alpha7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and kappa-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary alpha-bungarotoxin had no significant effect on migrating cells. These results suggest that alpha3alpha5beta2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells.
Assuntos
Cálcio/metabolismo , Movimento Celular/fisiologia , Receptores Nicotínicos/metabolismo , Mucosa Respiratória/metabolismo , Cicatrização/fisiologia , Idoso , Idoso de 80 Anos ou mais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Immunoblotting , Imuno-Histoquímica , Líquido Intracelular/química , Pessoa de Meia-Idade , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização/efeitos dos fármacosRESUMO
To date, most of the studies in the field of cell migration have been applied to two-dimensional (2D) models. To mimic the three-dimensional (3D) conditions similar to those observed in vivo during tumor invasion, we developed a 3D model of cell migration in which cells were embedded in a collagen I matrix placed in a double-compartment chamber. Using time-lapse videomicroscopy and interactive cell tracking in a four-dimensional data set, we determined the cell trajectories and their migration kinetics. We compared the 2D and 3D migratory behavior of a noninvasive cell line (16HBE) with the migratory behavior of an invasive cell line (BZR). Our results show that the 3D migration kinetics of the noninvasive cell line were lower than the migration kinetics of the invasive cell line. In contrast, in 2D models, no significant difference was observed between the two cell lines. To validate our 3D model, we further investigated the effect of epidermal growth factor (EGF), a promoter of tumor cell motility and invasion on the noninvasive cell line (16HBE). EGF increased significantly the migration kinetics of the noninvasive cell line. Our results show that the 3D model of cell migration allowed us to differentiate the migratory behavior of invasive and noninvasive cells and that such a model can help in the development of molecular targeted therapy as it approaches the in vivo conditions.