Diesel exhaust particles induce an inflammatory response in airway epithelial cells: involvement of reactive oxygen species.
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Bronchi↗
Biomedical subjects
F Marano
Publications and source records attributed to F Marano.
Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles.
Epidemiological studies have shown that particulate air pollution is linked to the increase of morbidity and mortality due to respiratory diseases. Diesel exhaust particles (DEPs), which are the most important part of PM2.5 in Western European and Japanese urban areas, have been suspected. The mechanisms of proinflammatory response induced by DEPS were elucidated using a human epithelial cell line (16-HBE). It has been shown that DEPs can be phagocytosed by HBE cells, inducing the release of cytokines. MAP kinase pathways (i.e., ERK1/2 and P38) were triggered as well as the activation of the nuclear factor NF-kappaB. Reactive oxygen species (ROS) were strongly incriminated in this response because DEPs induce the increase of intracellular hydroperoxides and antioxidants inhibit the release of DEP-induced cytokines, the activation of MAP kinases and NF-kappaB. Organic compounds adsorbed on DEPs seemed to be involved in the response and the production of ROS. Moreover, we have demonstrated that DEPs can activate CYP1A1 in HBE cells. These experimental results give biological plausibility to the epidemiological findings.
Bronchi↗
Biocompatibility and biodegradation of intravitreal hyaluronan implants in rabbits.
To study the biocompatibility and the biodegradation rate in vivo of new intravitreal implants made with three different hyaluronic acid esters: Hyaff7, Hyaff11 and Hyaff11p75 (100% ethyl ester, 100 and 75% benzyl esters, respectively), the plugs were implanted through a sclerotomy at 3.5 mm from the limbus of rabbit eyes. In order to evaluate the in vivo biodegradation the shaft diameter of the plugs was measured by ultrasound biomicroscopy. Slit lamp microscopy, ophthalmoscopy and ERG were performed periodically. The effects of the implants on ocular tissues were also evaluated histologically. All the plugs showed a good biocompatibilitv. Plugs of both the total esters, Hyaff7 and Hyaff11, were found to undergo a slow dissolution process for 60 and 150 days, respectively. The partial benzyl ester, Hyaff11p75, was completely reabsorbed after 15 days. Analysis of variance showed a high correlation between biodegradation rate and the time of resorption (F = 90.5; p < 0.001). The biodegradation rate of each implant is related to the chemical structure of the three types of Hyaff (F = 4.51; p = 0.005). The present data suggest that intravitreal implants based on hyaluronic acid esters represent useful biocompatible and biodegradable devices for a potential drug delivery system in the treatment of posterior segment ocular diseases.
Animals↗
Multifocal intra-ocular lenses.
Multifocal intra-ocular lenses (IOLs) are designed to overcome the lack of accomodation to provide useful distance and near vision. Reduced contrast sensitivity has been reported. Refractive IOLs produce two or more focal points depending on the refractive power of the portion of the lens crossed by the light. Diffractive IOLs are strictly bifocal; each point of the lens can create both a distance and near focus. Several clinical investigations show that multifocal IOLs can be used as an alternative to monofocal lenses. However, patient selection, realistic expectations, and adequate control of surgical procedures are necessary.
Cataract Extraction↗
Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression.
Diesel exhaust particles (DEP) are known to enhance inflammatory responses in human volunteers. In cultured human bronchial epithelial (16HBE) cells, they induce the release of proinflammatory cytokines after triggering transduction pathways, including nuclear factor (NF)-kappaB activation and mitogen-activated protein kinase (MAPK) phosphorylation. This study compares the effects of native DEP (nDEP), organic extracts of DEP (OE-DEP), and carbonaceous particles, represented by stripped DEP (sDEP) and carbon black particles (CB), in order to clarify their respective roles. OE-DEP and nDEP induce granulocyte macrophage colony-stimulating factor (GM-CSF) release, NF-kappaB activation, and MAPK phosphorylation. The carbonaceous core generally induces less intense effects. Reactive oxygen species are produced in 16HBE cells and are involved in GM-CSF release and in the stimulation of NF-kappaB DNA binding by nDEP and OE-DEP. We demonstrate, for the first time, in airway epithelial cells in vitro that nDEP induce the expression of the CYP1A1, a cytochrome P450 specifically involved in polycyclic aromatic hydrocarbons metabolism, thereby demonstrating the critical role of organic compounds in the DEP-induced proinflammatory response. Understanding the respective contributions of DEP components in these effects is important for vehicle manufacturers in order to improve their exhaust gas post-treatment technologies. In conclusion, the DEP-induced inflammatory response in airway epithelial cells mainly involves organic compounds such as PAH, which induce CYP1A1 gene expression.
Cells, Cultured↗
Effects of retinoic acid receptor-selective agonists on human nasal epithelial cell differentiation.
Retinoids play a critical role in the maintenance of the mucociliary phenotype of epithelial cells in the upper respiratory tract. To determine the role of retinoic acid receptors (RARs) in the regulation of epithelial differentiation, we tested the effect of the synthetic retinoids CD336, CD2019, and CD666, selective agonists for RARalpha, RARbeta, and RARgamma, respectively, during differentiation of human nasal epithelial (HNE) cells in vitro. Using glutamylated tubulin and transglutaminase I (Tg I) as markers of ciliated cell and squamous cell differentiation, respectively, we showed that retinoic acid (RA) stimulated mucociliary differentiation and, in parallel, inhibited squamous cell differentiation. The agonists of the three RARs independently induced ciliogenesis and inhibited squamous cell differentiation by downregulating Tg I expression in a dose- and time-dependent manner. Antagonists specific for the three RARs abolished the effects of the corresponding agonists, demonstrating an RAR-specific mediated effect. Moreover, treatment of retinoid-deficient cultures with RAR agonists induced conversion of the squamous-like phenotype into a ciliated phenotype. In conclusion, all three RARs are potentially involved in the differentiating effects of RA in respiratory epithelial cells.
Benzoates↗
IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.
In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.
Asthma↗
[Physiopathology of particle attack: animal and in vitro models].
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Air Pollutants↗
Hereditary retinal dystrophies and choroidal neovascularization.
BACKGROUND: Choroidal neovascularization infrequently occurs in patients affected by hereditary retinal dystrophies. METHODS: We studied eight patients suffering from different hereditary retinal dystrophies (Best's disease, reticular dystrophy, butterfly-shaped dystrophy, gyrate atrophy, and retinitis pigmentosa) who developed choroidal neovascularization. All patients underwent complete ophthalmic evaluation, electrophysiology, colour vision testing, and fluorescein angiography. In some patients, ICG video-angiography was also performed. Laser treatment was carried out in only one patient. RESULTS: The mean duration of follow-up was 41.7 months (range 6-148 months). At CNV diagnosis, the mean VA was 0.23 (range 0.02-0.6). At the last follow-up, mean VA was 0.34 (range HM to 0.9). At the last follow-up, fluorescein angiography showed a focal, atrophic scar in seven eyes, a fibrotic membrane in two eyes and a still active membrane in two cases. CONCLUSION: We emphasize the relatively favourable visual prognosis in patients suffering from inherited retinal dystrophies complicated with choroidal neovascularization. Therapeutic approaches other than laser treatment could be attempted in these patients.
Adult↗
Effects of hydroxyl radicals on outwardly rectifying chloride channels in a cultured human bronchial cell line (16HBE14o-).
Respiratory pathologies can result from the exposure of airway epithelial cells to oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on an outwardly rectifying chloride channel (ORCC). In the human bronchial cell line 16HBE14o-, the cytoplasmic side of ORCC in inside-out excised membrane patches was exposed to *OH created by simultaneously superfusing Fe2+ and H2O2 in front of the patch-pipette. ORCC was activated by depolarizing voltage steps. Its open probability (Po) increased with bath [Ca2+] above 1 microM. Upon brief exposure to *OH, ORCC first closed and then alternated between periods of closure and normal activity. The duration of closure increased with the duration of *OH exposure but voltage steps could reopen the channel. After 10 min exposure to *OH, however, the channel closed irreversibly, regardless of the number of subsequent voltage steps or the duration of washing. Low [Ca2+] in the bath accelerated the irreversible closure of the channel in the presence of *OH. Intracellular application of *OH progressively inhibited ORCC activity by inducing long closure periods that increased with time. This might have important pathophysiological implications in the process of inflammation.
Bronchi↗
Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells.
The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.
Animals↗
Responses of the rabbit tracheal epithelium in vitro to H(2)O(2)-induced oxidative stress.
A model of rabbit tracheal epithelial (RTE) cells in primary culture was used to characterize specific and repair responses of airway epithelial cells to oxidative stress. Two well-known reactive oxygen species (ROS) generating systems were used: H(2)O(2) alone or in combination with Fe(2+) to produce the hydroxyl radical. RTE cells exhibited lipid peroxidation when exposed to H(2)O(2) + Fe(2+). Moreover, catalase (CAT) activity decreased after a 1-hour treatment in 3-day-old cultures but increased in 7-day-old cultures which have higher antioxidant enzyme activities. Superoxide dismutase (SOD) activity was never affected. In addition, RTE cells displayed a repair response leading to squamous metaplasia. H(2)O(2) + Fe(2+) treatment resulted in a time-dependent increase in the steady-state level of c-myc mRNA while c-jun and c-fos were not activated. Moreover, a chronic exposure induced the expression of the squamous phenotype characterized by the expression of the cytokeratin 13 confirmed both at the message and protein levels. RTE cells in primary culture react early to H(2)O(2) + Fe(2+) exposure by an increase in c-myc expression and by modifications in CAT activity. Further, a lipid peroxidation occurs and the tracheal epithelium evolves to squamous metaplasia.
Animals↗
Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro.
The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H)-phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants.
Adenosine Triphosphate↗
Efficient protection of human bronchial epithelial cells against sulfur and nitrogen mustard cytotoxicity using drug combinations.
The aim of this study was to test the efficacy of several candidate molecules against sulfur mustard (SM) and nitrogen mustard (HN2) using a human bronchial-epithelial cell line (16HBE14o-). Candidate molecules were chosen on the basis of the known cytotoxicity mechanisms of mustards or their efficacy previously observed on other cellular models. It included the sulfhydryl-containing molecules N-acetyl-cysteine (NAC) and WR-1065, the nucleophile hexamethylenetetramine (HMT), the energy-level stabilizer niacinamide (NC), the antioxidant dimethylthiourea (DMTU), L-arginine analogues such as L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME), and the anti-gelatinase doxycycline (DOX). Their efficacy was determined using 2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2Htetrazolium (WST-1) reduction by viable cells 24 h after initial exposure to 100 microM HN2 or SM. On individual immediate cotreatment, some molecules exhibited selective protection against only one mustard, such as DMTU and WR-1065 against HN2 and DOX against SM, whereas NAC and L-TC were effective against both SM and HN2 cytotoxicity. However, as the level of protection against SM was always weak compared to HN2, several combinations were investigated against SM to improve the protection. The effective combinations (L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX) combined agents, reducing the bioavailability of the mustard with compounds possibly acting on the consequences of alkylation. One of these combinations, NAC + DOX, appeared to be the most interesting, as these agents are already used in human therapy. It exhibited good efficacy in delayed cotreatment (up to 90 min) against SM.
Acetylcysteine↗
Mechanisms of GM-CSF increase by diesel exhaust particles in human airway epithelial cells.
We have previously shown that exposure to diesel exhaust particles (DEPs) stimulates human airway epithelial cells to secrete the inflammatory cytokines interleukin-8, interleukin-1beta, and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in allergic diseases. In the present paper, we studied the mechanisms underlying the increase in GM-CSF release elicited by DEPs using the human bronchial epithelial cell line 16HBE14o-. RT-PCR analysis has shown an increase in GM-CSF mRNA levels after DEP treatments. Comparison of the effects of DEPs, extracted DEPs, or extracts of DEPs has shown that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and not to the metals present on the DEP surface because the metal chelator desferrioxamine had no inhibitory effect. Furthermore, radical scavengers inhibited the DEP-induced GM-CSF release, showing involvement of reactive oxygen species in this response. Moreover genistein, a tyrosine kinase inhibitor, abrogated the effects of DEPs on GM-CSF release, whereas protein kinase (PK) C, PKA, cyclooxygenase, or lipoxygenase inhibitors had no effect. PD-98059, an inhibitor of mitogen-activated protein kinase, diminished the effects of DEPs, whereas SB-203580, an inhibitor of p38 mitogen-activated protein kinase, had a lower effect, and DEPs did actually increase the active, phosphorylated form of the extracellular signal-regulated kinase as shown by Western blotting. In addition, cytochalasin D, which inhibits the phagocytosis of DEPs, reduced the increase in GM-CSF release after DEP treatment. Together, these data suggest that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and that the effect of native DEPs requires endocytosis of the particles. Reactive oxygen species and tyrosine kinase(s) may be involved in the DEP-triggered signaling of the GM-CSF response.
Bronchi↗
Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro.
Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.
Calcium-Binding Proteins↗
Long-term visual results of bifocal intraocular lens implantation.
PURPOSE: To evaluate the long-term visual results and variability in contrast sensitivity in patients with heparin-surface-modified (HSM) diffractive bifocal intraocular lenses (IOLs). SETTING: Institute of Ophthalmology, University of Catania, Italy. METHODS: In this prospective study, visual acuity (distance and near) and contrast sensitivity were measured in 35 patients who had phacoemulsification with bifocal diffractive IOL (model 811E, Pharmacia) implantation. Patient satisfaction was also evaluated using a questionnaire. Mean follow-up was 20 months (range 18 to 24 months). RESULTS: At the last examination, mean distance visual acuity was 0.79 +/- 0.2 (SD) without correction and 1.0 +/- 0.15 with best correction. Mean uncorrected near visual acuity was J1.6 +/- J0.77 and with best distance correction, J1.19 +/- J0.49. No statistically significant changes in visual acuity were evident at the last follow-up (Student t test). No changes were found in contrast sensitivity reduction over time, nor were late postoperative complications noted. Overall patient satisfaction was rated as good by 94.3% of patients with best distance correction. CONCLUSIONS: The diffractive bifocal HSM IOL provided good visual performance for distance and near over time. In relation to the low rate of postoperative complications, the slight contrast sensitivity reduction was stable during follow-up.
Adult↗
Airborne particles evoke an inflammatory response in human airway epithelium. Activation of transcription factors.
PM10, the commonly used indicator of respirable environmental suspended particulate matter with a mean aerodynamic diameter of less than 10 microm, is composed of organic or elemental carbon aggregates containing various metals, acid salts, organic pollutants (polyaromatic hydrocarbons, quinones, nitroaromatic hydrocarbons, etc.), and biological contaminants. In urban and industrial areas, fossil fuel combustion products (e.g., diesel exhaust particles and residual oil fly ash) are the main contributors to PM10. Epidemiological data show that air pollution particulates cause adverse pulmonary health effects, especially in individuals with preexisting lung diseases. A critical cell type that encounters particles after inhalation and that is affected in a number of respiratory diseases is the epithelial cell of the airway and alveoli. In vitro studies have shown that PM10 is responsible for the production and the release of inflammatory cytokines by the respiratory tract epithelium as well as for the activation of the transcription factor NFkappaB. As many of the adsorbed materials on the particle surface are direct oxidants (metals, quinones) and indirectly produce reactive oxygen species, it is hypothesized that oxidative stress may be a component of the mechanisms by which particles activate cytokine production and NFkappaB in epithelial cells.
Air Pollutants↗