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J Jacquot

Publications and source records attributed to J Jacquot.

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

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

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

Inflammation and infection in naive human cystic fibrosis airway grafts.

Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease. Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial. We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice. This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways. Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001). CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria. In non-CF grafts, these three sets of events were not observed before 6 h. Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state. After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.

Animals↗
PubMed1999

Genistein inhibits constitutive and inducible NFkappaB activation and decreases IL-8 production by human cystic fibrosis bronchial gland cells.

The inflammatory pathogenesis in airways of patients with cystic fibrosis (CF) is still unresolved. We demonstrate here that in in situ human DeltaF508 homozygous CF bronchial tissues, submucosal gland cells exhibit an absence of inhibitor factor kappaBalpha (IkappaBalpha) and high levels of chemokine interleukin-8 (IL-8) expression. These results were confirmed by cultured human CF bronchial gland cells in which a lack of cytosolic IkappaBalpha and high levels of constitutively activated nuclear factor kappaB (NFkappaB) associated with an up-regulation of IL-8 production (13-fold increase) were found when compared to non-CF (control) disease bronchial gland cells. We also demonstrated that the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, significantly reduces the endogenous and Pseudomonas aeruginosa lipopolysaccharide-induced IL-8 production in cultured CF bronchial gland cells by increasing cytosolic IkappaBalpha protein levels. Overall, results show that genistein is a potent inhibitor of the activated NFkappaB identified in CF gland cells. This strong inhibition of constitutively activated NFkappaB and the resulting down-regulation of IL-8 production by genistein in the CF gland cells highlights the key role played by cytosolic IkappaBalpha in the regulation of inflammatory processes in CF human airway cells.

Adolescent↗
PubMed1998

Extracellular Mg2+ inhibits both histamine-stimulated Ca(2+)-signaling and exocytosis in human tracheal secretory gland cells.

The effects of extracellular Mg2+ concentration have been investigated on the histamine-stimulated exocytotic process of human tracheal secretory gland (HTG) cells. The exocytosis of secretory granules (SG) was observed concomitantly with dynamic changes of intracellular Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i). The rate of SG exocytosis was appraised by the decrease of quinacrine fluorescence emission. Dynamic changes of [Mg2+]i and [Ca2+]i in HTG cells were determined by the combined use of UV-microspectrofluorometry with Mag-Indo-1 and Indo-1 probes, respectively. High Mg2+ medium significantly inhibited the histamine-stimulated secretion. The influence of the extracellular and intracellular Mg2+ concentrations on [Ca2+]i was analyzed. Basal [Mg2+]i increased from 0.8 mM in a Mg(2+)-free medium to 1.7 mM in 10 mM Mg2+ medium. Histamine induced a [Mg2+]i increase which is dependent on extracellular Mg2+ concentration. The histamine stimulated [Ca2+]i rise was reduced in the presence of elevated Mg2+ extracellular medium and inhibitory effects of extracellular Mg2+ were concomitant with changes in [Mg2+]i. Our data suggest that the inhibition by extracellular Mg2+ of stimulated exocytosis is dependent on both the increase of [Mg2+]i and the inhibition of cytosolic Ca2+ influx.

Calcium↗
PubMed1998

Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro.

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Adolescent↗
PubMed1998

Neutrophil elastase promotes rapid exocytosis in human airway gland cells by producing cytosolic Ca2+ oscillations.

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Adenosine Triphosphatases↗
PubMed1996

Spatial and temporal Mg2+ signaling in single human tracheal gland cells.

The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2+ variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2+ concentrations (up to 10 mM) induced increases in intracellular Mg2+ concentration [Mg2+]i. From a cytosolic basal concentration of 0.8 +/- 0.3 mM in a medium free of Mg2+, an increase in extracellular Mg2+ concentration from 1 to 10 mM, increased cytosolic [Mg2+]i from 1.4 +/- 0.6 to 1.8 +/- 0.8 mM after 10 min (p < 0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2+]i is distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2+ stores involved. The influx pathway for Mg2+ in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2+]i.

Bombesin↗
PubMed1996

CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be a cAMP-regulator of plasma membrane recycling in epithelial cells overexpressing CFTR. To assess its role in the different endocytic processes in human respiratory epithelial cells, the rates of internalization of membrane, fluid-phase and receptor-mediator tracers were compared, under control conditions and after treatment with the cAMP agonist forskolin in normal and cystic fibrosis (CF) cells. In both control and treated-cells, CFTR was only present in the plasma membrane of normal but not in CF cells. Similarly, activation of Cl- efflux only occurred in normal and not in CF-treated cells. The rate of membrane endocytosis was significantly decreased by 35% in normal treated-cells, whereas it was not significantly decreased (12%) in CF-treated cells. Upon forskolin treatment, the decrease of the rate of both fluid-phase and receptor-mediated endocytosis was not significantly different between normal and CF cells. These results demonstrate that CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cell Line↗
PubMed1996

Intracellular free Ca2+ dynamic changes to histamine are reduced in cystic fibrosis human tracheal gland cells.

This study documents a difference between cystic fibrosis human (CF-HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca2+]i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca2+]i peak rise in response to histamine is smaller in CF-HTG cells than in HTG cells, and the percentage of CF-HTG cells that increase [Ca2+]i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF-HTG and HTG cells generated [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF-HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

Calcimycin↗
PubMed1995

Asynchronous dynamic changes of intracellular free Ca2+ and possible exocytosis in human tracheal gland cells induced by neutrophil elastase.

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident: cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85 +/- 15 nM, HNE (1 microM) produces a [Ca2+]i increase of 91 +/- 66 nM in about 50% of responding cells. At lower concentrations of HNE (0.1 microM, 0.01 microM), the [Ca2+]i rise remains similar, but only 30-40% of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

Calcium↗
PubMed1994

Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensional collagen gel matrix.

The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three-dimensional (3-D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland-like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well-developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland-like tubules. A cell-associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland-like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3-D collagen matrix form functional tracheal gland-like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling.

Cell Differentiation↗
PubMed1993

Studies on human porin. IX. Immunolocalization of porin and CFTR channels in human surface respiratory epithelium.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

Antibodies↗
PubMed1993

Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR). From human normal tracheal submucosal gland cells in culture, we identified endogenous CFTR as a 170 kDa protein, consistent with that of fully glycosylated, mature CFTR molecule. This observation led to the hypothesis that airway secretory glands could be an important site for the CFTR expression. Using anti-human CFTR polyclonal and monoclonal antibodies, we examined the cellular and subcellular localization of the CFTR protein in airway submucosal glands from human and bovine tracheal tissues as well as in tracheal gland cell cultures. In human tracheal tissue, CFTR immunolabelling was present along both the apical and basolateral plasma membranes of glandular mucous cells. In contrast, CFTR was associated with the secretory granules of glandular serous cells. Using immunogold electron microscopy, we demonstrated that CFTR protein was more specifically associated with the membrane of serous cell secretory granules. In bovine tracheal tissue CFTR labelling was also identified in the secretory granules of glandular serous cells. In contrast, when bovine and human tracheal gland cells were cultured, no mature secretory granules were present, but a predominantly intracytoplasmic distribution of CFTR was observed. Our data thus suggest that in airway tissues, CFTR could be involved in intracellular processes of the mucus exocytosis in submucosal secretory glands.

Animals↗