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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 [[beta].sub.2]-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 tree nuclear factor-[kappa]B (NF-[kappa]B) 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 [NH.sub.2]-terminal kinase (JNK), and the P-isoform-[alpha] of the NF-[kappa]B inhibitor (I[kappa]B[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-[kappa]B, AP-1, P-ERK1/ERK2, P-JNK, and P-I[kappa]B[alpha] decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a [[beta].sub.2]-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation. airway inflammation; proinflammatory cytokines; nuclear fator-[kappa]B and activator protein-1 activation; glucocorticoid; bacterial virulence factors

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 matr[micro] 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. tumor invasion; metastasis; image analysis; kinetic migration; epidermal growth factor

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent ([10.sup.4], [10.sup.6], and [10.sup.8] CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the [alpha]-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus ( videomicroscopy; secretory leukoproteinase inhibitor; [alpha]-toxin; airway cell damage; bacterial adherence and internalization

The interaction between impaired mucociliary clearance and airway inflammation due to cystic fibrosis was analyzed on CF mice with mutant cystic fibrosis transmembrane conductance regulator (CFTR) genes. CF mice with targeted insertional mutagenesis in the exon 10 of the murine CFTR gene of embryonal stem cells exhibited impaired mucociliary clearance in the absence of bacterial or viral infections. Effects of CFTR gene mutations in CF mice were mediated by alterations in CFTR channel activity which impaired mucociliary transport.

The role of fibronectin and its alpha5beta1-ligand receptor on airway epithelial wound repair was investigated by the immunofluorescence localization technique. Wounded cultures displayed immunoreactivity to fibronectin at the basal cell layer of repairing cells. Wounded cells that were incubated with fibronectin showed an increase in wound-repair index compared with control and wound cells not incubated with fibronectin. Cells also showed an increase in the rate of cell migration during the wound-repair process. It was therefore concluded that fibronectin and it receptor are involved in wound repair.

Abstract 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 β1 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.

Abstract 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 β2 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 TNFα. Conclusions Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting β2 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 β2 adrenergic receptor agonist and glucocorticoid.

Abstract 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 μg/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.

The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in [Cftr.sup.tm1Hgu]/[Cftr.sup.tm1Hgu] mice compared with that in control littermates. ASL ion content from eight [Cftr.sup.tm1Hgu]/[Cftr.sup.tm1Hgu] mice and eight control littermates did not appear significantly different. The mean ([+ or -] SE) concentrations were 2,352 [+ or -] 367 and 2,058 [+ or -] 401 mmol/kg dry weight for Na, 1,659 [+ or -] 272 and 1,448 [+ or -] 281 mmol/kg dry weight for Cl, 357 [+ or -] 57 and 337 [+ or -] 38 mmol/kg dry weight for S, 1,066 [+ or -] 220 and 787 [+ or -] 182 mmol/kg dry weight for K, 400 [+ or -] 82 and 301 [+ or -] 58 mmol/kg dry weight for Ca, 105 [+ or -] 31 and 105 [+ or -] 20 mmol/kg dry weight for Mg, 33 [+ or -] 15 and 29 [+ or -] 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues. cryoprobe; ion concentration

Coraux, Christelle, Jean-Marie Zahm, Edith Puchelle, and Dominique Galliard. [[Beta].sub.1]-Integrins are involved in migration of human fetal tracheal epithelial cells and tubular morphogenesis. Am J Physiol Lung Cell Mol Physiol 279: L224-L234, 2000.--Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of [[Beta].sub.1]-integrins during development and tubular morphogenesis are still unknown. To analyze [[Beta].sub.1]-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-[[Beta].sub.1]-integrin subunit antibodies. On a collagen matrix, [[Beta].sub.1]-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, [[Beta].sub.1]-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-[[Beta].sub.1]-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating [[Beta].sub.1]-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration. cell-matrix interaction; collagen sandwich

We investigated whether cluster formation by noninvasive cells can be explained by a global attractive potential. Indices quantifying the persistence of migration in experimental conditions were compared to the same indexes computed from simulations with a density-based cellular automaton. The results indicate that the attractive potential hypothesis must be rejected. Index Terms--Cell biology, cellular automata, cluster formation, migration.