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Biomedical subjects

E Puchelle

Publications and source records attributed to E Puchelle.

At least 19 recordsLinked to original sources
PubMed2003

Fluticasone reduces IL-6 and IL-8 production of cystic fibrosis bronchial epithelial cells via IKK-beta kinase pathway.

Inhaled fluticasone propionate (FP) is widely used to reduce pulmonary inflammation in chronic obstructive pulmonary disease, but the potential effects of FP on airway epithelial cells from patients with cystic fibrosis (CF) are unknown. In CF disease, a nonregulated inflammatory lung response occurs through exaggerated nuclear factor (NF)-kappaB activation and elevated pro-inflammatory cytokines production by airway epithelial cells. To determine whether FP reduces cytokine production in bronchial epithelial cells via NF-kappaB, the authors investigated the nonstimulated and the Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulated production of NF-kappaB-dependent interleukin (IL)-6, IL-8 and RANTES (regulated on activation, T-cell expressed and secreted) along with the activation of NF-kappaB in non-CF and CF human bronchial gland epithelial cells. It was demonstrated that a relevant concentration of FP (10(-8) M) inhibited constitutive and P. aeruginosa LPS-induced IL-6 and IL-8 production of non-CF and CF bronchial epithelial cells. Interestingly, the expression of two IkappaB kinases (IKK)-alpha/beta, the degradation of cytosolic IkappaB-beta inhibitor and the NF-kappaB deoxyribonucleic acid binding activity were markedly reduced after FP treatment in both CF and non-CF bronchial epithelial cells. It was shown by the authors that fluticasone propionate exerts an anti-inflammatory effect by blocking a signal transduction leading to a reduced level of IkappaB-alpha/beta kinases in bronchial epithelial cells. In particular the strong effect on the IkappaB-beta kinase, which is known to be elevated in bronchial epithelial cells in cystic fibrosis patients, was observed.

Analysis of Variance↗
PubMed2001

Relationship between IkappaBalpha deficiency, NFkappaB activity and interleukin-8 production in CF human airway epithelial cells.

Several recent reports have suggested that airway inflammation may precede infection and relate to an endogenous dysregulation of pro-inflammatory cytokines in cystic fibrosis (CF) airways. Evidence suggests that activation of the nuclear factor kappa B (NFkappaB), which regulates the inflammatory gene transcription, depends on the degradation of the inhibitory factor IkappaBalpha. We show that, in in situ human DeltaF508 CF bronchial tissues, inhibitor factor IkappaBalpha is not present in gland cells, although endogenous levels of chemokine IL-8 are high. These data are confirmed by studying cultured CF human bronchial gland cells, in which a lack of cytosolic IkappaBalpha and high levels of activated NFkappaB, concomitant with IL-8 overproduction (a 13-fold increase) are found when compared to non-CF bronchial gland cells. Interestingly, treatment of CF gland cells with the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, results in a significant decrease ( P < 0.001) in IL-8 production down to levels released by non-CF gland cells. The addition of genistein also reverses the effects of lipopolysaccharide (LPS) Pseudomonas-aeruginosa-induced nuclear translocation of NFkappaB by increasing IkappaBalpha protein level (65%) in CF gland cells. Our data indicate that the induction of IkappaBalpha protein in CF airway glandular epithelial cells may be a novel mechanism by which IL-8-mediated lung inflammatory events are markedly reduced in CF patients, at least at the airway glandular level.

Bronchi↗
PubMed2001

Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa.

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous DeltaF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0.001). The number of T lymphocytes was higher at the CF distal level (P = 0.035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0.033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.

Adult↗
PubMed2001

Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis.

BACKGROUND: Airway inflammation and infection are early events in cystic fibrosis (CF) pathogenesis. The existence of an imbalance in the immune cell population of the CF fetal airway before infection remains completely unknown. OBJECTIVE: The aim of this study was to determine whether early signs of inflammation are observed in CF airways during human fetal development. METHODS: Tracheas and lungs were collected from 21 CF and 16 non-CF fetuses. In tissue sections, the numbers of neutrophils, mast cells, macrophages, and B and T lymphocytes were quantitatively analyzed by means of image cytometry. The presence of IL-4, IL-6, IL-8, IL-10, RANTES, IFN-gamma, TNF-alpha, and NF kappa B and its inhibitor I kappa B-alpha was qualitatively evaluated by immunofluorescent staining. RESULTS: During fetal airway development, epithelial and glandular differentiation, as well as the distribution of inflammatory markers, was similar in CF and non-CF tissues. Significant differences between CF and non-CF fetal airways were observed only in the numbers of mast cells and macrophages. In the CF trachea, the mast cell number increased slowly but continuously, whereas in the non-CF trachea this number rapidly reached a plateau. In the CF lung, the macrophage number increased with time, whereas in the non-CF lung it decreased. CONCLUSION: Although no intrinsic inflammation was demonstrated, we observed a distinct appearance of mast cells and macrophages in CF airways in comparison with non-CF airways during fetal development. These 2 cell populations were greater in CF airways at a late stage of fetal development, suggesting their possible involvement in the early onset of inflammation in CF infants.

Antigens, CD↗
PubMed2001

Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro.

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.

Bacterial Adhesion↗
PubMed2001

X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice.

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(tm1Hgu)/Cftr(tm1Hgu) mice compared with that in control littermates. ASL ion content from eight Cftr(tm1Hgu)/Cftr(tm1Hgu) mice and eight control littermates did not appear significantly different. The mean (+/-SE) concentrations were 2,352 +/- 367 and 2,058 +/- 401 mmol/kg dry weight for Na, 1,659 +/- 272 and 1,448 +/- 281 mmol/kg dry weight for Cl, 357 +/- 57 and 337 +/- 38 mmol/kg dry weight for S, 1,066 +/- 220 and 787 +/- 182 mmol/kg dry weight for K, 400 +/- 82 and 301 +/- 58 mmol/kg dry weight for Ca, 105 +/- 31 and 105 +/- 20 mmol/kg dry weight for Mg, 33 +/- 15 and 29 +/- 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.

Animals↗
PubMed2001

Improved activity of an actin-resistant DNase I variant on the cystic fibrosis airway secretions.

In cystic fibrosis (CF), actin and DNA originating from inflammatory cells contribute to the thickness of airway secretions. Actin can bind to DNA-rich fibers and potently inhibit the enzymatic activity of rhDNase. The in vitro effects of the actin-resistant rhDNase variant (A114R) were analyzed and compared with those of the wild-type rhDNase. Frozen and thawed CF airway secretions were incubated for 30 min with different concentrations (0.1, 0.5, 1, 5, or 10 microg/ml) of either actin-resistant rhDNase or wild-type rhDNase. We observed that both the wild-type and the actin-resistant rhDNase significantly decreased (p < 0.05 and p < 0.001, respectively) the airway secretion viscosity. The decrease in airway secretion viscosity was significant even at low concentrations (0.1 microg/ml) of the actin-resistant variant. Incubation with the actin-resistant variant resulted in a significant decrease (p < 0.02) of the airway secretion contact angle and cough transport. A significantly higher (p < 0.01) increase in contact angle and cough transport of airway secretions was observed at 10 microg/ml with the actin-resistant variant as compared with the wild-type rhDNase. The present study had demonstrated that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical properties and cough transport of airway secretions from patients with cystic fibrosis.

Actins↗
PubMed2001

Physiomer reduces the chemokine interleukin-8 production by activated human respiratory epithelial cells.

The authors have recently shown that the transcription factor nuclear factor-kappaB (NF-kappaB) is a central mediator in the NaCl-mediated interleukin (IL)-8 production by human airway epithelial cells. In this study, it was investigated whether Physiomer, an isotonic sea water-derived solution commercialized for cleaning the nasal mucosa, impaired the chemokine IL-8 expression and secretion by human respiratory epithelial cells compared with that obtained with an isotonic 9% NaCl solution. Primary human bronchial gland (HBG) epithelial cells were incubated either in Physiomer or in a NaCl 9% solution and activated either with 20 ng x mL(-1) tumour necrosis factor-alpha, or IL-1beta, respectively. Physiomer significantly reduced the IL-8 protein release in basal and activated HBG cells in comparison with that obtained with the 9% NaCl solution. In contrast to the effects of Physiomer observed on resting HBG cells, Physiomer did not significantly reduce the level of phosphorylation of the NF-kappaB inhibitor protein IkappaBalpha or the steady-state IL-8 messenger ribonucleic acid levels in activated HBG cells, suggesting that Physiomer would have a post-transcriptional effect on IL-8 expression in activated HBG cells. The authors conclude that Physiomer is potentially useful in the reduction of airway mucosal inflammation.

Adult↗
PubMed2000

Human airway xenograft models of epithelial cell regeneration.

Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

Animals↗
PubMed2000

High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the I kappa B kinase alpha pathway in response to extracellular NaCl content.

Increasing evidence suggests that in airways from cystic fibrosis (CF) patients, inflammation may precede bacterial infection and be related to an endogenous dysregulation of proinflammatory cytokines in airway epithelial cells. Several investigators have reported that, in CF airway fluids, elevated NaCl concentrations may also contribute to the diseased state by inhibiting the bactericidal properties of airway fluid. Because many proinflammatory cytokines are transcriptionally regulated by the NF-kappa B, we investigated whether an elevated extracellular NaCl content in airway fluids significantly impaired the regulation of the NF-kappa B/I kappa B alpha complex and the chemokine IL-8 production in primary non-CF and CF human bronchial gland epithelial cells. Exposure of non-CF gland cells to hypotonic (85 mM) NaCl solution, compared with isotonic (115 mM) NaCl and hypertonic (170 mM) NaCl solutions, resulted in a significant decrease in IL-8 production that was paralleled by a strong inhibition of activated NF-kappa B associated with an increased cytosolic expression of I kappa B alpha and a decrease in the I kappa B kinase alpha protein level. In CF gland cells, we demonstrated that, compared with the high IL-8 in an hypertonic solution, the release of IL-8 was significantly reduced 2-fold in an isotonic solution and 5-fold in a hypotonic solution. Strikingly, exposure of CF bronchial gland cells to either hypotonic or isotonic milieu did not result in a marked inhibition of the activated NF-kappa B/I kappa B alpha system. This is the first demonstration that primary human CF bronchial gland cells exhibit abnormally high IL-8 production through constitutively activated NF-kappa B and high I kappa B kinase alpha level, whatever the hypo-, iso-, and hypertonic NaCl milieu.

Adolescent↗
PubMed2000

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium.

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.

Cell Movement↗
PubMed2000

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2.

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.

Actins↗
PubMed2000

Differentiated and functional human airway epithelium regeneration in tracheal xenografts.

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.

Animals↗
PubMed2000

Early expression of beta- and gamma-subunits of epithelial sodium channel during human airway development.

The amiloride-sensitive epithelial Na(+) channel (ENaC) is an apical membrane protein complex involved in active Na(+) absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming alpha-, beta-, and gamma-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against beta- and gamma-ENaC, immunohistochemical localization of the channel was performed in fetal (10-35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of beta- and gamma-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, beta- and gamma-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of beta- and gamma-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na(+) absorption might begin significantly before birth, even if secretion is still dominant.

Adult↗
PubMed2000

Beta(1)-integrins are involved in migration of human fetal tracheal epithelial cells and tubular morphogenesis.

Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of beta(1)-integrins during development and tubular morphogenesis are still unknown. To analyze beta(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(1)-integrin subunit antibodies. On a collagen matrix, beta(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(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(1)-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating beta(1)-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration.

Antigens, Differentiation↗