Diesel particles increase phosphatidylcholine release through a NO pathway in alveolar type II cells

Philippe Juvin1,*, Thierry Fournier2,*, Martine Grandsaigne1, Jean-Marie Desmonts1, and Michel Aubier1

1 Unité 408, Institut National de la Santé et de la Recherche Médicale, 75870 Paris Cedex 18; and 2 Unité 427, Institut National de la Santé et de la Recherche Médicale, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes Paris 5, 75006 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Diesel exhaust particles (DEPs) have been shown in vivo as well as in vitro to affect the respiratory function and in particular the immune response to infection and allergens. In the current study, we investigated the effect of DEPs on the production of phosphatidylcholine (PC), a major constituent of surfactant, by rat alveolar type II (ATII) primary cells in vitro. Our results demonstrate that incubation of ATII cells with DEPs lead to a time- and dose-dependent increase in labeled PC release. This effect was mimicked by nitric oxide (NO) donors and cGMP and was abolished by inhibitors of NO synthase (NOS). In addition, a NOS inhibitor inhibits by itself the basal secretion of PC. We next examined the effects of DEPs on NOS gene expression and showed that DEPs increase NO production and upregulate both protein content and mRNA levels of the inducible NOS (NOS II). Together our data demonstrate that DEPs alter the production of surfactant by ATII cells through a NO-dependent signaling pathway.

surfactant; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAJOR AIR POLLUTANTS include nitrogen dioxide, ozone sulfur dioxide, and breathable particulate matter of diameter inferior to 10 µm (PM10) (12, 27). Several studies have particularly implicated diesel exhaust particles (DEPs), a major component of PM10, as contributing to the incidence and severity of respiratory diseases during air pollution episodes. Epidemiological studies have reported that high levels of DEPs are associated with worsening peak flow, inhaler usage, respiratory symptoms, and emergency room visits in asthmatic children and adults (34, 38). In a cohort of over half a million adults residing in 151 metropolitan areas in the United States between 1982 and 1989, fine particulate pollution was associated with cardiopulmonary and lung cancer mortality (33). Similarly, a recent work has demonstrated an increased mortality and morbidity rate after DEPs exposure (26).

Airway epithelial cells are primary targets for air pollutants. Several studies have investigated the effect of DEPs on nasal, bronchial, and alveolar epithelial cells. In nasal cells, DEPs enhance cytokine expression synergistically with allergens and increase local eosinophil adhesion (15, 16, 44). In bronchial epithelial cells, DEPs are internalized, inducing airway inflammation (4, 8, 41) and hyperresponsiveness (43).

DEPs also interact with alveolar type II (ATII) cells since their small size (0.1-1 µm) allows them to reach the alveolar space (32, 46, 48). ATII cells regulate the intra-alveolar homeostasis through four main functions. They secrete a large panel of cytokines (5, 13, 31, 39), serve as progenitors to the alveolar type I cells (18), actively transport ions (23), and synthesize and secrete surfactant. We previously demonstrated that one of these functions (i.e., cytokine secretion) was altered by DEPs (24). Synthesis and secretion of surfactant are other very important functions of ATII cells. Surfactant prevents alveolar collapse at end expiration by lowering surface tension at the air/extracellular lining interface in the alveoli and distal airways. Surfactant also maintains alveolar fluid balance and possibly exhibits host defense properties (28, 37). It has previously been shown in vivo that exposure to air pollutants including DEPs may alter the composition of the surfactant (17). However, the direct consequences of DEPs exposure on the secretion of surfactant by ATII cells remain unknown, as do the mechanisms mediating these effects.

As demonstrated in bronchial cells (29, 42), various pollutants like silica and ozone may act on respiratory epithelial cells through the L-arginine-nitric oxide (NO) pathway. This pathway has also been involved in alveolar pollution-related consequences. In vivo exposure of rats to silica significantly increases NO production in bronchoalveolar lavage cells (22). Rat ATII cells produce NO after in vivo exposure to irritant-inducing doses of ozone (35). Last, NO by itself has been suggested to cause damage to surfactant homeostasis (36). However, the role of the L-arginine-NO pathway in the DEPs-related alteration of the surfactant metabolism remains unknown.

The aim of this study was to investigate 1) whether DEPs alter the secretion of surfactant by ATII cells and 2) whether the L-arginine-NO pathway is involved in this alteration.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Tissue culture media and fetal bovine serum were obtained from GIBCO BRL Life Technologies (Cergy Pontoise, France). Tissue culture plastic ware was from Costar (Cambridge, MA). Chemicals were from Sigma (La Verpillière, France) unless specified. The diesel particulate matter SRM 1650 was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). Carbon black (FR103, 95-nm diameter) was obtained from Degussa (Frankfurt, Germany).

Rat ATII cell isolation and culture. ATII cells were isolated from adult pathogen-free male Sprague-Dawley rats, 200-220 g body wt (Charles River Breeders, St.-Aubin-les-Elbeuf, France), by enzymatic dissociation and purified by differential adherence to plastic as described previously (14). ATII cells, 106/well, were plated in 12-well cell culture plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin), fungizone (1 µg/ml), and 10% fetal bovine serum (complete medium). The cultures were incubated in humidified 95% air with 5% CO2 at 37°C. After 24 h, nonadherent cells were removed by washing with DMEM, and adherent cells were used for surfactant phospholipid production or NO synthase (NOS) II gene expression experiments.

Chemical treatment. DEPs treatment was performed as described previously (8, 9, 24). Stock solutions of particles were prepared by suspension in complete culture medium and sonicated three times for 60 s each at maximal power (8 kilocycles). Concentrations were expressed in micrograms per square centimeter, because the particles rapidly sediment onto the cell cultures. Inhibitors such as NG-monomethyl-L-arginine (LNMMA) were added 30 min before DEPs treatment.

Surfactant phospholipid production by purified ATII cells. The ability of isolated ATII cells to produce surfactant was studied after incorporation of a radioactive precursor, [3H-methyl]choline, in PC as previously described (3, 30). Twenty-four hours after isolation, we washed the cell monolayers three times with DMEM before adding fresh complete medium containing 2 µCi/ml [3H-methyl]choline chloride (Amersham, Les Ulis, France) for another 24-h period. The radioactive medium was removed, and the cells were washed twice with DMEM containing 40 mM HEPES and 3 mg/ml bovine serum albumin (to remove unincorporated [3H-methyl]choline) and once with DMEM. Labeled ATII cells were cultured for various periods of time in the presence of particles or other stimuli. Supernatants from each well were collected and centrifuged to pellet loose cells, and the cells were lysed in methanol. Then a trace amount of [14C]dipalmitoylphosphatidylcholine (113 mCi/mmol; Amersham) was added to each fraction (supernatants and cell lysates) for determination of lipid extraction recovery. Lipids were extracted by the method of Bligh and Dyer (6). As previously reported, >90% of the lipid-associated label was in PC (10), thus radioactivity of the total lipid fraction was quantified. PC release was expressed as a percentage, calculated as disintegrations/min (dpm) in supernatant normalized to total dpm [dpm supernatant/(dpm supernatant + dpm cell lysate)] × 100.

Nitrite quantification in cell supernatants. Nitrite, a stable breakdown product of NO in physiological systems, was assayed using the Griess reaction. As previously described (21), the amount of nitrite produced by ATII cells was assayed in the cell supernatants after reduction of nitrate to nitrite using Escherichia coli nitrate reductase and determined by comparison with a nitrite standard curve. Values were expressed as nanomoles of nitrite per 106 ATII cells.

Detection of inducible NOS (NOS II) mRNA by RT-PCR. The oligonucleotides used for PCR were chosen to encompass at least one intron to detect amplification of contaminating genomic DNA. For NOS II, the primers were sense, 5'-TGC TTT GTG CGG AGT GTC AGT-3', and antisense, 5'-CGG ACC ATC TCC TGC ATT TCT-3' (size of the amplified products was 227 bp). For the ribosomal protein S14, used as a standard, the primers were sense, 5'-ATC AAA CTC CGG GCC ACA GGA-3', and antisense, 5'-GTG CTG TCA GAG GGG ATG GGG-3' (137 bp). Two days after isolation, ATII cells were stimulated for 6 or 24 h, and total cellular RNA was isolated from subconfluent ATII cell monolayer cultures with TRIzol reagent (GIBCO BRL) according to Chomczynski and Sacchi (11). Purified RNA was dissolved in RNase-free water and quantified by measurement of absorbance at 260 nm. All samples were demonstrated to contain undegraded RNA as assessed by electrophoresis of an aliquot of each preparation on 1% agarose gels and visualization of 18S and 28S ribosomal RNA after ethidium bromide staining. One microgram of total RNA was first incubated for 5 min at 70°C with 1 µg of oligo(dT) in a volume of 7.5 µl and then quick chilled on ice before RT reaction buffer was added. Single-strand cDNA synthesis was carried out in 12.5 µl containing 50 mM Tris · HCl (pH 8.5), 30 mM KCl, 8 mM MgCl2, 1 mM dithiothreitol, 1 mM deoxynucleotide triphosphates, 25 units of RNase inhibitor, and 200 units of Moloney murine leukemia virus RT (Amersham Pharmacia Biotech). Reaction mixture was incubated for 60 min at 37°C and then for 5 min at 99°C. cDNAs were amplified in 25 µl of reaction buffer containing 50 mM KCl, 20 mM Tris (pH 8.4), 2 mM (for S14) or 1.25 mM (for NOS II) MgCl2, 0.2 mM deoxynucleotide triphosphates, and 0.40 µM of each primer. Samples were heated to 80°C before 2.5 units of Taq DNA polymerase (Life Technologies) were added to reduce formation of nonspecific amplification products. After denaturation of DNA at 94°C for 5 min, cycling parameters were as follows: denaturation, 94°C for 30 s; annealing, 59°C (for S14) or 62°C (for NOS II) for 30 s; and extension, 72°C for 30 s. After 31 cycles, the final extension was carried out for 7 min at 72°C. The amplification products were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.

SDS-polyacrylamide gel electrophoresis/immunoblot analysis. ATII cells were cultured for 2 days as described for surfactant study. After an additional 24-h incubation period with DEPs or endotoxin from E. coli [lipopolysaccharide (LPS)], cells were scraped in buffer containing 50 mM Tris · HCl, pH 7.4, 0.1 mM EDTA, 1 µM leupeptin, 1 µM phenylmethylsulfonyl fluoride, and 1 µM aprotinin and stored at -80°C until use. Western blot analysis was performed as previously described (7). Briefly, 50 µg of total protein determined using a bicinchoninic acid protein assay were separated in SDS-7.5% polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were then blocked for 1 h at room temperature with 10% nonfat dry milk in Tris-buffered saline (25 mM Tris · HCl, pH 7.5, and 0.150 mM NaCl) containing 0.05% (vol/vol) Tween 20 (TTBS), washed in TTBS, and incubated for 1 h with a rabbit anti-murine NOS II polyclonal antibody in TTBS containing 1% BSA. Horseradish peroxidase-conjugated goat anti-rabbit IgG in TTBS containing 1% BSA was used for detection of immunoreactive proteins. After stripping, the same membrane was incubated with murine anti-human beta -actin antibody and revealed with horseradish peroxidase-conjugated sheep anti-murine IgG as previously described. Bands (130 kDa for NOS II and 42 kDa for beta -actin) were detected with chemiluminescence reagents and film per the manufacturer's instructions (NEN, Les Ulis, France).

Cytotoxicity of particles. Trypan blue uptake was performed to determine the effects of DEPs and carbon black on cell viability. Lactate dehydrogenase (LDH) release into the cell culture medium was also measured to assess cell integrity. Briefly, the medium was collected from each well and centrifuged (200 g, 10 min, 4°C) to pellet loose cells, and supernatant was used for extracellular LDH assay. The same cells were lysed with 1 ml 1% Triton in PBS for determination of intracellular LDH. LDH activity was assayed spectrophotometrically by monitoring the NADH-dependent conversion of pyruvate to lactate at 340 nm. LDH release was expressed as a percentage of total cellular LDH activity (i.e., extracellular LDH divided by extracellular plus intracellular LDH).

Statistical analysis. Values are means ± SD of at least three separate cultures performed in triplicate. Comparisons between groups were made by analysis of variance for repeated measures. Student's t-test with the Bonferroni correction for multiple comparisons was used to compare groups. The Wilcoxon test was used in carbon black particle experiments (Table 1). A P value < 0.05 was considered significant.

                              
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Table 1.   Carbon black particles do not significantly alter PC release by ATII cells


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant is a complex mixture consisting of ~90% lipids and 10% protein. Most of the lipids is phospholipid (about 90%), and PC, which is regarded as a major component for reduction in the surface tension, makes up about 80%. In the present study we show that rat ATII cells cultured in the presence of DEPs produced a time- and dose-dependent increase in PC release (Fig. 1, A and B), which was already significant from 2 µg/cm2 DEPs after 24 h of exposure. When we increased the concentration of DEPs by ten (20 µg/cm2), a maximal 50% increase was reached at 24 h without inducing any cell toxicity. However, in all the following experiments, the concentration 2 µg/cm2 was used since it induced a significant 40% increase in PC release. Interestingly, incubation of cells for 24 h with the same concentrations of carbon black particles (2 and 20 µg/cm2) did not affect PC release (Table 1).


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Fig. 1.   Concentration- and time-dependent effect of diesel exhaust particles (DEPs) on phosphatidylcholine (PC) release by rat alveolar type II (ATII) cells. A: 24 h after isolation, ATII cell primary cultures were labeled for 24 h with [3H]choline and then incubated with increasing concentration of DEPs for 24 h. Forskolin (10 µM) was used as a positive control for PC secretion. [3H]PC secretions were quantified in each well using a trace amount of [14C]dipalmitoyl PC for determination of lipid extraction recovery and normalized to total [3H]PC (intra- plus extracellular contents). B: cells were treated as previously described but incubated with 2 µg DEPs/cm2 for different times. Values represent means ± SD of at least 3 independent cultures in triplicate and are expressed relative to control values. * P < 0.05 corrected for multiple comparisons, compared with control at the same time point.

To investigate whether DEPs-induced PC release was mediated by the NO signaling pathway, we incubated ATII cells with 2 µg/cm2 DEPs for 24 h in the presence or absence of the NO synthase inhibitor LNMMA (Fig. 2). LNMMA (100 µM) totally prevented the effect of DEPs, since PC release in cells treated concomitantly with LNMMA and DEPs remained at basal levels. It is noteworthy that LNMMA by itself decreased PC release by 40% compared with untreated cells, suggesting that the NO pathway is involved in the constitutive release of PC.


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Fig. 2.   Nitric oxide synthase (NOS) inhibitors abolished the DEPs-induced increase in PC secretion. Rat ATII cells were incubated for 24 h with medium alone or 2 µg/cm2 DEPs in the presence or absence of 100 µM NG-monomethyl-L-arginine (LNMMA). Values represent means ± SD of at least 3 independent cultures in triplicate and are expressed relative to control values. * P < 0.05 compared with control. §P < 0.05 DEPs+LNMMA vs. DEPs.

We next examined whether a NO donor or NO derivative might affect the release of PC by ATII cells (Fig. 3). Incubation of cells with 50 µM s-nitroso-N-acetyl-D penicillamine (SNAP) or 100 µM 3-morpholinosydnonimine (SIN) led to a significant increase in PC release (25 and 12%, respectively). Before investigating the effects of NO donors on PC release, we provided evidence that these compounds elevated the production of nitrites as measured by Griess reaction in ATII cell supernatants (data not shown). Because the NO signaling pathway involved elevated intracellular cGMP, we next investigated the effect of the membrane-permeable dibutyryl cGMP on the release of PC. We demonstrated that, like NO donors, cGMP induced a significant 15% increase in PC release (Fig. 3).


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Fig. 3.   NO donors and cGMP increase PC secretion in rat ATII cells. Rat ATII cells were incubated for 24 h with 2 µg/cm2 DEPs, or with either 50 µM SNAP or 100 µM 3-morpholinosydnonimine (SIN) or with 20 µM dibutyryl cGMP. Values represent means ± SD of at least 3 independent cultures in triplicate and are expressed relative to control values. * P < 0.05 corrected for multiple comparisons, compared with control.

In the present study, we have demonstrated that the NO signaling pathway was involved in the DEPs-induced increase in PC release. We next examined the effect of DEPs on NO production and NOS gene expression (Fig. 4). As determined by Griess reaction, we first showed that DEPs induced a dose-dependent increase in nitrite production at 24 h (Fig. 4A). Immunoblot experiments demonstrated that DEPs induced expression of NOS II at 24 h (Fig. 4B), whereas NOS II expression remained undetectable in controls. Finally, as shown in Fig. 4C, we studied the expression of the NOS II gene using RT-PCR. The presence of NOS II transcripts remained undetectable in controls. This was not due to a lack or degradation of total RNA, as demonstrated by the equal presence of ribosomal protein S14 transcripts (Fig. 4C, top) and by the absence of 18 and 28S ribosomal RNA degradation (Fig. 4C, bottom). As shown in Fig. 4C, NOS II gene expression was induced as soon as 6 h and maintained at 24 h. Although NOS II gene expression was induced with 2 µg/cm2 DEPs, the strongest increase was obtained with 20 µg/cm2.


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Fig. 4.   Incubation of rat ATII cells with DEPs increases NO production and upregulates inducible NOS (iNOS) gene expression. A: ATII cells were incubated for 24 h with increasing concentrations of DEPs, and nitrite production was quantified in cell supernatants using Griess reaction. Values represent means ± SD of 3 independent cultures and are expressed as nmol nitrite/106 cells. * P < 0.05. B: ATII whole cell lysates obtained after a 24-h stimulation period with 2 µg/cm2 DEPs or 10 µg/ml LPS were probed with antibodies for iNOS and beta -actin. Comparison to a protein ladder and to the migration of lipopolysaccharide (LPS)-stimulated rat alveolar macrophage (AM)-cell lysates identifies iNOS. beta -Actin protein contents demonstrate equal loading of total protein in lanes. C: ATII cells were incubated with 2 or 20 µg/cm2 DEPs for 6 or 24 h before total cellular RNA was extracted. Bottom: 18 S and 28 S ribosomal RNA (10 µg) were separated on 1% agarose gels and stained with ethidium bromide to demonstrate the absence of RNA degradation. Top: iNOS mRNA levels were analyzed by RT-PCR as described in MATERIALS AND METHODS, and ribosomal protein S14 mRNA was used as an internal standard.

The cell viability from DEPs or carbon black-treated cells for 48 h was higher than 95%, as assessed by the trypan blue test, and was not different from control cells. LDH release from DEPs or carbon black-treated cells was never higher than from control cells over the 48-h incubation time.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated here that DEPs increased PC secretion by ATII cells in a time- and concentration-dependent manner. This effect was mimicked by NO donors and abolished by inhibitors of NOS. We also provided evidence that DEPs upregulated NOS II gene expression and induced increased NO production.

Our first result is that DEPs increase the secretion of surfactant by ATII cells. This result is in agreement with in vivo studies performed with DEPs (17) and another pollutant (silica) (25). Rats chronically exposed to DEPs or given a single intratracheal dose of DEPs showed increased levels of PC (i.e., surfactant) content in the lungs and in pulmonary lavage fluid (17). Silica instillation also induced an alveolar phospholipidosis in rats (25). In this study, the amount of phospholipids increased, and, interestingly, phospholipid composition of lavage material from silica-treated animals was altered by a reduction in the percentage of phosphatidylglycerol and an increase in phosphatidylinositol. This dissociation between amount and composition of surfactant suggests that other pollutants such as DEPs may also induce an inappropriate overproduction of phospholipids, which could finally lead to alterations in tensioactive properties of surfactant, which in turn could be responsible for gas exchange impairment and possibly for pulmonary infections (28).

Our second result is that the DEPs-induced increase in surfactant secretion was mediated by NO. We first demonstrated that DEPs increased both the level of NOS II mRNA and protein as well as the secretion of NO by rat ATII cells. Previous studies have demonstrated that respiratory epithelial cells express NOS II in response to proinflammatory stimuli and are thus capable of producing locally large amounts of NO (1, 19). These proinflammatory stimuli include outdoor pollutants (22, 29, 35, 40, 42). For example, in humans, the level of endogenous NO in exhaled air has indeed been found to increase on days with high outdoor air pollution (40). Similar results have been obtained in animals, with silica (22), ozone (35), or DEPs (29, 42). In vivo exposure of rats to silica significantly increased NO production by alveolar macrophages. In this work, interaction of macrophages with pneumocytes was necessary for the induction of NO synthesis, suggesting an important role for ATII cells in the pollution-related NO increase (22). This role of ATII cells in NO production was highlighted in another study performed in rats in which ATII cells themselves produced more NO after exposure to irritant-inducing doses of ozone (35). As demonstrated here with DEPs, this increase in NO synthesis after ozone was due to an overexpression of cellular NOS II mRNA and enzyme. Concerning the effects of DEPs on NO production, the only published reports have been performed with macrophages or bronchial cells (29). The effects of DEPs on these cells were very similar to those observed here in ATII cells. For instance, exposure of mice to DEPs induced a doubling both in the level of NO in the exhaled air and in NO secretion by bronchial cells. In addition, the increase of NO production by DEPs was the result of an increased synthesis of NOS II in macrophages and of constitutive NOS (NOS III) in bronchial epithelial cells (29). These results are partially in agreement with ours, since we have demonstrated that DEPs induced NOS II but not NOS III synthesis in ATII cells. Similarly, it has been shown that the DEPs-induced airway inflammation is inhibited by aminoguanidine, a relatively selective inhibitor of inducible NOS (42). Together, these results and ours strongly suggest that DEPs induce NO production by increasing expression of NOS II mRNA and protein in various cells of the airway including ATII cells.

Last, the role of NO in the DEPs-mediated effects was confirmed, since the effects of DEPs were mimicked by SNAP, a NO donor, and abolished by inhibitors of NOS. Exposure of rat ATII cells to a NO donor increases the amount of surfactant secreted by these cells. The effects of NO on the surfactant pulmonary system have been poorly studied so far. A previous study has demonstrated apparently opposite results of ours, i.e., a decrease in the rate of surfactant synthesis by ATII cells in response to NO exposure. However, this effect, not reproduced in vivo, was associated with a decrease in cell ATP levels, suggesting an alteration of the cell viability (21). A second study has shown that in vitro NO decreases surfactant protein A gene expression in a human lung tumor cell line representative of distal respiratory epithelium. However, this effect was of relatively small magnitude (2).

There are probably several mechanisms underlying the alteration of the NO-induced surfactant secretion (45). The partial effects of cGMP and of SIN-1, a peroxynitrite donor, which is the result of the reaction of NO with superoxide anion, suggest the existence of at least two mechanisms, including an oxidative pathway. This observation is in agreement with previous studies that have shown that peroxynitrite may interfere with surfactant activity (20). Furthermore, we have previously shown that DEPs-induced effects in human airway epithelial cells are partly mediated through the production of reactive oxygen species (9). We hypothesized in the present study that DEPs-induced increase in PC might be mediated by reactive oxygen species of the NO pathway such as peroxynitrite.

DEPs are composed of polyaromatic hydrocarbons (PAHs), which are adsorbed on carbon particles. No effect of carbon black particles on surfactant secretion or on NO synthesis was noted in the present study. This suggests that PAHs, which are adsorbed on DEPs but not on carbon black particles, are responsible for the effects of DEPs. This observation is in agreement with previous reports, which have emphasized the specific PAHs' role in the DEPs activity in bronchial cells (9), macrophages (47), and ATII cells (24). This role of PAHs is corroborated by the observation that the catalysts that reduce the adsorbed organic compounds also reduce the DEPs-induced increase in cytokine secretion by bronchial cells (8).

Together, our results demonstrate that DEPs induce the secretion of surfactant in ATII cells through a NO-dependent pathway. Because previous studies have suggested that NO (36) can damage pulmonary surfactant as well as DEPs can (17), our results suggest that DEPs may induce an inappropriate oversecretion of altered surfactant, which may contribute to the pathophysiology of the pollution-induced lung injuries. Further studies are needed to verify this hypothesis in vivo.


    FOOTNOTES

* P. Juvin and T. Fournier contributed equally to this work.

Address for reprint requests and other correspondence: P. Juvin, Service d'Anesthésie et de Réanimation, Centre Hospitalier Bichat Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France (E-mail: pjuvin{at}free.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00213.2001

Received 8 June 2001; accepted in final form 12 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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