Francelyne Marano

Biological effects of atmospheric particles on human bronchial epithelial cells. Comparison with diesel exhaust particles

Epidemiological studies have associated the increase of respiratory disorders with high levels of ambient particulate matter (PM) levels although the underlying biological mechanisms are unclear. PM are a complex mixture of particles with different origins but in urban areas, they mainly contain soots from transport like Diesel exhaust particles (DEP). In order to determine whether PM biological effects can be explained by the presence of DEP, the effects of urban PM, DEP and carbon black particles (CB) were compared on a human bronchial epithelial cell line (16-HBE14o-). Two types of PM were used : reference material (RPM) and PM with an aerodynamic diameter < or =2.5 microm collected in Paris with a high volume sampler (VPM). From 10 to 30 microg/cm2, cell viability was never modified whatever the particles. However, DEP and to a lower extent PM inhibited cell proliferation, induced the release of a pro-inflammatory cytokine, GM-CSF, and generated a pro-oxidant state as shown by the increased intracellular peroxides production. By contrast, CB never induced such effects. Nevertheless CB are more endocytosed than DEP whereas PM are the less endocytosed particles. In conclusion, PM induced to a lower extent the same biological effects than DEP in 16-HBE cells suggesting that particle characteristics should be thoroughly considered in order to clearly correlate adverse effects of PM to their composition and to clarify the role of DEP in PM effects.

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 μm, 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 NFκB. 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 NFκB in epithelial cells.

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-κB. 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-κB. 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.

Inhibition of caspase-dependent mitochondrial permeability transition protects airway epithelial cells against mustard-induced apoptosis

In the present study, the toxicity of yperite, SM, and its structural analogue mechlorethamine, HN2, was investigated in a human bronchial epithelial cell line 16HBE. Cell detachment was initiated by caspase-2 activation, down-regulation of Bcl-2 and loss of mitochondrial membrane potential. Only in detached cells, mustards induced apoptosis associated with increase in p53 expression, Bax activation, decrease in Bcl-2 expression, opening of the mitochondrial permeability transition pore, release of cytochrome c, caspase-2, -3, -8, -9 and -13 activation and DNA fragmentation. Apoptosis, occurring only in detached cells, could be recognized as anoikis and the mitochondrion, involved both in cell detachment and subsequent cell death, appears to be a crucial checkpoint. Based on our understanding of the apoptotic pathway triggered by mustards, we demonstrated that inhibition of the mitochondrial pathway by ebselen, melatonin and cyclosporine A markedly prevented mustard-induced anoikis, pointing to these drugs as interesting candidates for the treatment of mustard-induced airway epithelial lesions.

Progress in outgrowth culture from rabbit tracheal explants : balance between proliferation and maintenance of differentiated state in epithelial cells

SummaryPrimary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively.

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

SummaryThe differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis.The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea

Summary— We have developed an automated image analysis method to study the ciliary beat frequency of ciliated cells of the primary culture from rabbit trachea. The ciliated outgrowth image is digitized and the variation in optical density is automatically calculated for each selected area of interest. 32 measurements of ciliary beat frequency are, in this way, calculated simultaneously in 6 min. With this reliable device, some studies on baseline frequency of control culture have been carried out. There was no variation in the mean frequencies of ciliated cells of the primary culture of different tracheas in our culture conditions. Moreover, the values of ciliary beat frequency at the starting point of the outgrowth were similar to those at the periphery of the outgrowth. There is nevertheless a slow decrease in frequencies versus the duration of culture. We have also established that the frequency of ciliary beat of some cells fluctuates in a periodic pattern whereas the majority of the ciliated population beat in a stable way. The image analysis process allows us to perform a cartography of frequencies on the video display. It also allows us to have access to the frequency of one cilium. Our method therefore seems to be reliable and furthermore simple in the evaluation of the potential effect of inhaled toxic compounds on ciliated cells of mammalian respiratory tract.

Activation of Transcription Factors by Diesel Exhaust Particles in Human Bronchial Epithelial Cells in Vitro.

Diesel exhaust particles (DEP) are suspected to be involved in the aggravation of inflammatory respiratory diseases. We have shown previously, in human bronchial epithelial cell line 16HBE 14o-, that DEP induced the release of the proinflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8) after 24 h of exposure. Gene expression of these cytokines is regulated by transcription factors including NF-κB and AP-1, which are known to be sensitive to oxidative stress. Their activation by DEP was investigated in comparison with a pure oxidant, H2O2 A 4-h exposure to DEP (10 μg/cm(2)) or to H2O2 (100 µM) increased NF-κB DNA binding in 16HBE cells as assessed by electrophoretic mobility shift assay. AP-1 was only activated by H202 in the same conditions. Organic extracts of DEP increased NF-κB DNA binding as did native DEP, suggesting the role of the polycyclic aromatic hydrocarbons (PAH) in this NF-κB increased DNA binding. Dimethylthiourea (DMTU), an antioxidant, inhibited the NF-κB DNA binding induced by DEP, suggesting an involvement of reactive oxygen species (ROS) in the transduction pathways leading to NF-κB activation. Moreover, the MEK pathway inhibitor PD98059 inhibited DEP-induced NF-κB DNA binding. The role of Erk 1/2 was likely implicated, since DEP induced an increase of Erk phosphorylation.

The flagellar apparatus ofDunaliella: Isolation of basal body-flagellar root complex

SummaryInDunaliella bioculata, a biflagellate wall-less unicellular alga, the cytoskeleton is organized around the two basal bodies. Each basal body is associated with two dissymetric flagellar roots and numerous micro tubules which constitue a regular frame around the cell. We isolated the basal body-flagellar-root apparatus and studied its ultrastructure after negative staining. The two different flagellar roots are formed of microtubules and bundles of twisted filaments 3,5–4 nm in diameter. The proximal end of each root fans out and envelopes the basal body. We have shown preliminary results on the protein composition of basal body-flagellar roots fraction.

Diesel Exhaust Particles Increase NF-κB DNA Binding Activity and c-FOS Proto-oncogene Expression in Human Bronchial Epithelial Cells

There is increasing evidence that diesel exhaust particles (DEP) could be incriminated in respiratory diseases. They have been shown to induce an inflammatory response in the lung and are suspected to be carcinogenic because of the presence of polyaromatic hydrocarbons (PAH) on their surface. DEP were tested on a human bronchial epithelial cell line (16HBE) in comparison with carbon black particles (CB) devoid of PAH. DEP and CB at 10mug/cm(2) induced the release of the lactate dehydrogenase (LDH) by 16HBE cells from 48hr of exposure. DEP at 5mug/cm(2) but not CB activated the binding of the nuclear factor kappaB (NF-kappaB) to DNA from 2hr of exposure up to 15hr. NF-kappaB is a transcription factor involved in the expression of some cytokines such as IL-8 and GM-CSF which have been shown to be released by 16HBE cells after DEP exposure. In addition, DEP as well as CB induced the expression of the c-fos proto-oncogene. Taken together, these new data suggest that the activation of NF-kappaB and the expression of c-fos could contribute to the proliferation and chronic inflammation processes induced in lungs after DEP exposure.