Effect of Ozone on Platelet-activating Factor Production in Phorbol-differentiated HL60 Cells, a Human Bronchial Epithelial Cell Line (BEAS S6), and Primary Human Bronchial Epithelial Cells James M. Samet, Terry L. Noah, Robert B. Devlin, James R. Yankaskas, Karen McKinnon, Lisa A. Dailey, and Mitchell Friedman Center for Environmental Medicine and Lung Biology, Curriculum in Toxicology, Department of Medicine, and Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill; Health Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park; and Alliance Technologies, Inc., Chapel Hill, North Carolina
Platelet-activating factor (PAF) is a phospholipid with a wide spectrum of pro-inflammatory properties. In the lung, PAF induces airway hyperresponsiveness, neutrophil sequestration, and increased vascular permeability. The alveolar macrophage and the bronchial epithelium are tissues that are exposed to inhaled ozone (0 3) , We studied the effect of an in vitro 0 3 exposure on PAF production in a macrophage-like HL60 human cell line (dHL60), a human bronchial epithelial cell line (BEAS S6), and also in primary human bronchial epithelial cells. PAF was quantified by thin-layer chromatographic separation of lipid extracts from cells radio labeled with FH]lysoPAF and by radioimmunoassay. In vitro exposure of dHL60 cells to 0.05 to 1.0 ppm 0 3 for 15 to 120 min was found to significantly increase PAF levels above air control values at all exposure levels and time points (average increase of 92 %). Similarly, BEAS S6 cells grown on collagen-coated filter supports and exposed to 0.05 to 1.0 ppm 0 3 for 60 min released an average increase in PAF of 626 % above control values. Primary human bronchial epithelial cells also demonstrated significant increases in FH]PAF release (average increase of 289% after exposure to 1.0 ppm 0 3 for 60 min) compared with paired air controls. These findings suggest that some of the effects of 0 3 inhalation may be mediated by PAP.
Ozone (03) is an oxidant pollutant gas that is a major constituent of photochemical smog. In the United States, it is estimated that 150 million people reside in areas that exceed the current National Ambient Air Quality Standard for 0 3 of 0.12 ppm (1). 0 3 inhalation has been demonstrated to result in changes in lung function and evidence of pulmonary inflammation in human subjects, including airway hyperreactivity (2, 3), increased epithelial and vascular perme-
(Received in original form November 22, 1991 and in final form June 23, 1992)
Mitchell Friedman, M.D., Pulmonary Diseases/Critical Care Medicine Section, Department ofMedicine SL9, Tulane University Medical Center, 1430 Tulane Ave., New Orleans, LA701122699. Abbreviations: bovine serum albumin, BSA; [N-methyl-14C]1-0-alkyl-2acetyl-sn-3-glycerophosphocholine, [14C]PAF; Dulbecco's modified Eagle's mediumIHam's Fl2 medium, DMEM/Fl2; fetal bovine serum, FBS; Hanks' balanced salt solution, HBSS; 1-0-eHI-octadecyl-2-sn-glycero-3phosphocholine, fHllysoPAF; high performance liquid chromatography, HPLC; keritanocyte basal medium, KBM; Joklik's minimal essential medium, MEM; ozone, 0 3 ; platelet-activating factor, PAF; phosphate-bufered saline, PBS; phorbol 12-myristate 13-acetate, PMA; radioimmunoassay, RIA; thin-layer chromatography, TLC. Address correspondence to:
Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 514-522, 1992
ability to macromolecules (4-6), and neutrophil infiltration into the airways (5-7). Several in vivo and in vitro exposure studies have suggested that some of the pathophysiologic responses of the lung to 0 3 may involve arachidonic acid-derived inflammatory mediators produced by lung cells interacting with inhaled 0 3 , i.e., alveolar macrophages and airway epithelial cells. For example, increased levels of prostaglandins E, and F2a have been found in the bronchoalveolar lavage fluid of human subjects exposed to 0.4 ppm 0 3 for 2 h (5, 6). Primary cultures of bovine airway epithelial cells exposed in vitro to 0.1 ppm 0 3 for 2 h have been shown to release arachidonic acid and increased amounts of prostaglandin F 2a (8). Similarly, cultured rat alveolar macrophages release increased amounts of arachidonic acid, thromboxane B2, and leukotrienes B4, C4, and D4 during exposure in vitro to 1.0 ppm 0 3 for 2 h (9, 10). In addition to providing the substrate for lipoxygenasemediated and cyclooxygenase-mediated eicosanoid synthesis, the release of arachidonic acid from certain membrane phospholipids is linked to the synthesis of another important lipid mediator, platelet-activating factor (PAF). PAF is derived from 1-0-alkylphosphatidylcholine, a membrane phospholipid that is enriched in arachidonic acid at the sn-2 posi-
Samet, Noah, Devlin et al.: Effect of Ozone on PAF Production in Cultured Lung Cells
tion of the molecule (11). The enzyme phospholipase A2 catalyzes the first step in the synthesis of PAF by removing the sn-2 fatty acid from the phospholipid to produce lysoPAF. Thus, the release of arachidonic acid and the formation of lysoPAF can be simultaneous events (12). LysoPAF can then be acetylated by a specific acetyltransferase to form PAF (13, 14). PAF is a potent mediator with a wide spectrum of pro-inflammatory activities in a variety of tissues (15, 16). In the lung, PAF has been shown to induce inflammatory reactions including airway hyperreactivity, vascular permeability, and neutrophil infiltration in the airways (17). These inflammatory events are similar to those observed after 0 3 inhalation (2-7). Based on the possibility that PAF may be an important pro-inflammatory molecule involved in the response of the lung to 0 3 , we examined the effect of in vitro 0 3 exposure on the production of PAF in two relevant human cell lines, the cell line HL60 differentiated into macrophage-like cells and the bronchial epithelial cell line BEAS S6. We also exposed primary human bronchial epithelial cells in culture to 0 3 , We now report that exposure of these various human cells to 0 3 (in concentrations as low as 0.05 ppm for 15 min) resulted in significant increases in PAF levels in phorbol-differentiated HL60 cells and significant release of PAF by BEAS S6 cells and primary human bronchial epithelial cells.
Materials and Methods Materials Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/FI2), gentainicin sulfate, L-glutamine, and phosphate-buffered saline (PBS) were obtained from the UNC Lineberger Cancer Center Tissue Culture Facility (Chapel Hill, NC). Growth hormones used for the culture of primary cells were obtained from Collaborative Research (Waltham, MA). Nu Serum IV was obtained from GIBCO (Grand Island, NY). Keratinocyte basal medium (KBM) and supplements were obtained from Clonetics (San Diego, CA). Tissue culture dishes and flasks were obtained from Falcon (Oxnard, CA) or Costar (Cambridge, MA). Phorbol 12-myristate 13-acetate (PMA) , unlabeled PAF, cholera toxin, phospholipase A2 , calcium ionophore A23187, and acid phosphatase kits were obtained from Sigma Chemical Co. (St. Louis, MO). Silica thin-layer chromatography (TLC) plates were obtained from Merck (VWR; Durham, NC), RP-8 TLC plates were from Analtech (Newark, DE). 1-0-PH]-octadecy1-2-sn-glycero- 3-phosphocholine (PH]lysoPAF; specific activity, 80 to 180 Ci/ mmol) and [Nmethy1-14C]1-0-alky1-2-acety l-sn- 3-g1ycerophosphocholine ([14C]PAF; specific activity, 50 to 60 mCi/mmol) were obtained from Amersham (Arlington Heights, IL). PAFspecific radioimmunoassay kits were obtained from New England Nuclear (Boston, MA). 51Cr was obtained from NEN Dupont (Wilmington, DE). High performance liquid chromatography (HPLC) solvents and common laboratory reagents were obtained from Fisher (Raleigh, NC). Cell Culture Undifferentiated myeloid leukemic HL60 cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM/FI2 (1:1) supplemented with 10% Nu Serum IV,
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50 mg/liter gentamicin sulfate, and 2 mM L-glutamine(complete DMEM/F12) at a density of 0.5 to 2.0 x 106 cells/rnl. Cells were plated at a density of 2.0 to 4.0 X 106 cells/dish in 60-mm polystyrene tissue culture dishes and induced to differentiate toward monocytic development with 30 nM PMA (18) in complete DMEM/FI2 for 72 h. Differentiation was assessed by morphologic and enzymatic criteria, as previously described (18). HL60 cells treated with PMA (dHL60) become growth-arrested, strongly adherent, flattened, and elongated in appearance with long pseudopodlike processes. Acid phosphatase activity in the dHL60 cells at day 3 of differentiation was 96.7 ± 6.3 nmol/min/mg protein, compared with 20.0 ± 2.7 nmol/min/mg protein for the undifferentiated cells (n = 3), as measured with a standard assay kit (Sigma). At day 3 of differentiation, the dHL60 cells contained 129.6 ± 12.3 p,g of cellular protein/dish (n = 10). There were 2 X 106 cells/dish, but this value is an approximation due to the difficulty found in not being able to completely remove all intact dHL60 cells from the culture dishes. Passages 10 through 30 of the human bronchial epithelium cell line BEAS S6 (generous gift of Drs. C. Harris and 1. Lechner, National Institutes of Health, Bethesda, MD) were maintained in KBM supplemented with 10 ng/ml epidermal growth factor (human recombinant), 5 mg/rnl bovine insulin, 0.5 mg/rnl hydrocortisone, 0.15 mM calcium and bovine pituitary extract (supplements provided by Clonetics for use with KBM), on tissue culture petri dishes. In order to expose these cells and study the vectorial release of PAF, 5 x lOS BEAS S6 cells were seeded on 25-mm, collagen-coated (3-p,m pore size) polycarbonate filter supports (Transwell-COL; Costar), with 1 rnl of supplemented KBM added to the apical (top) compartment and 2.5 rnl to the basolateral (bottom) compartment. Cells became confluent (approximately 2 x 106 cells/filter) and growth-arrested after 2 days of the collagen filters. BEAS S6 cells were cultured for an additional 2 days in order to promote differentiation (19). Primary human bronchial epithelial cells were obtained from lung lobes excised from patients at the University of North Carolina Hospitals for standard clinical conditions (lung mass or localized carcinoma) within 30 min of excision. Third to sixth generation bronchi, which were not involved with the primary disease process, were isolated by sharp and blunt dissection while the lobe was bathed in 4°C lactated Ringer's solution. The tissues were rinsed in Joklik's minimal essential medium (MEM) containing penicillin (50 U/rnl), streptomycin (50 p,g/rnl), and gentamicin (40 mg/ ml). Epithelial cells were dissociated by protease treatment (Sigma type 14; 0.1%, 16 h at 4°C) as described (20). The protease was neutralized by addition of 10% fetal bovine serum (FBS), and the cells were dislodged by gentle agitation. The cell suspension was pelleted (800 X g for 5 min), washed in l(j% FBS in MEM, and plated on collagen filters in serum-free F12 medium that was supplemented with six growth factors (insulin, endothelial cell growth supplement, triiodothyronine, hydrocortisone, epidermal growth factor, and cholera toxin), as previously described (20). These primary cultures were incubated in a humidified atmosphere of 5 % CO 2 at 37°C. Media was changed 3 times/wk until the cells reached confluency.
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Before 0 3 exposure, dHL60, BEAS S6, or primary human bronchial epithelial cell cultures were radio labeled with 1 to 2 11Ci/rnl PH]lysoPAF in growth media containing 0.25% bovine serum albumin (BSA) for 0.5 to 5 h. Under these conditions, BEAS S6 cells labeled with 2 11Ci/filter incorporated an average of 25 % of the label and dHL60 cells labeled with 1.5 11Ci/cultureincorporated an average of 67 % of the label over the duration of the labeling period. The duration of the labeling period did not influence the extent of incorporation of the [3H]lysoPAF into the cells (data not shown). After labeling, the cells were washed extensively with PBS in order to remove unincorporated label before exposure. dHL60 cells were then exposed to 0 3 (see next section) in 1.5 rnl of PBS (with 1 mM calcium and magnesium) supplemented with 1 g/liter glucose, while rocking. In additional experiments, dHL60 cultures were incubated with the calcium ionophore A23l87 (at a final concentration of 10 11M in 0.01% dimethyl sulfoxide). Primary epithelial and BEAS S6 cells were washed thoroughly and then exposed on Transwell-COL filters with no media in the apical compartment and 1.6 rnl of complete Hanks' balanced salt solution (HBSS) in the basolateral compartment. Cells exposed in this manner are hydrated by the HBSS in the basolateral compartment.
In Vitro 0 3 Exposure System Two separate in vitro 0 3 exposure systems, similar in design to the previously described exposure system from our laboratory (9), were used for these experiments. The results were identical in both systems and, therefore, the data were pooled. The first system consisted of two plexiglass and steel chambers (each 15 liters in capacity) mounted on rocking platforms (Bellco Glass, Vineland, NJ) through which filtered room air at 37°C, with or without 0 3 , flowed at a rate of 7.5 liters/min. Separate input lines carried humidified air containing CO 2 into the chambers in order to maintain a 5 % CO2 atmosphere, as maintained by two CO 2 analyzers (Forma Scientific, Marietta, OH). Chambers and platforms were enclosed in 37°C incubators (Forma Scientific). Ozone was generated by passing a stream of air over a partially occluded UV pen lamp. Ozone concentrations in the chamber were monitored with a 1003-AH 0 3 Analyzer (Dasibi, Glendale, CA). The second system consisted of two glass chambers approximately 2.5 liters in volume through which humidified Zero Grade Air (containing < 1 ppm organic contaminants), with or without OJ, flowed at a rate of 1.2 liters/min. Ozone was generated with the use of a partially occluded pen lamp. Dehumidified air was analyzed in a Model 8002 Ozone Analyzer (Bendix, Lewisburg, WV). The lack of CO2 during exposures in the second system had no effect on any of the endpoints measured, including the pH of the exposure media. In each system, 0 3 concentration was maintained within ±1O% of the desired concentration. Extraction and Analysis of PAF After exposure of the dHL60 cells, the cells were scraped in the exposure media. The cell and media lipids were extracted with chlorofurm:methanol:aqueous(2:1:0.8) and spiked with approximately 50 11g of unlabeled PAF per sample to serve as a carrier (21). In some experiments, a few hundred dpm of p4C]PAF were included in the solvent mixture in
order to correct for recovery losses. The chloroform-rich phase was removed, a second extraction with chloroform was performed, and the chloroform phases were pooled and dried under nitrogen. After exposure of the BEAS S6 cells and the primary human bronchial epithelial cells using the same systems as the ones used for exposure of dHL60 cells, 1 rnl of HBSS with 0.25% BSA was added to the apical compartment and the cultures were incubated for an additional 5 min in room air. Apical and basolateral media were then removed and the lipids were extracted separately as described for the dHL60 cells. It was not possible to examine intracellular levels of [3H]PAF in the BEAS S6 cells or primary human bronchial epithelial cells because the collagen-coated membrane filter used to grow and expose these cells dissolves in organic solvents, thereby interfering with the extraction of cell-associated [3H]PAF. Therefore, only the amount of [3H]PAF released apically or basolaterally is reported for the BEAS S6 and primary human bronchial epithelial cell cultures. Lipids were resuspended in 40 111 of chloroform and chromatographed on heat-activated silica gel 60 plates, using a solvent mixture consisting of chloroform:methanol:glacial acetic acid:water (100:50:16:8) in an equilibrated tank (22). Lipids were visualized with iodine vapor and the PAF band was scraped, 0.5 rnl of methanol: water (1:1) and 4 rnl of scintillation cocktail were added to the scraped PAF band, and the sample was counted in a liquid scintillation counter. This method was used to quantitate [3H]PAF accumulation in dHL60 cells and [3H]pAF release in BEAS S6 and primary human bronchial epithelial cells. The identity of cellular PAF was verified by several independent methods: (1) dHL60 PAF was isolated by HPLC in an 8 x 10 radial compression module fitted with a silica cartridge (Waters, Milford, MA) using an isocratic solvent system consisting of acetonitrile:methanol:85% phosphoric acid (520:20:6) (23) and tested for biologic activity toward washed rabbit platelets according to the procedure described by Wykle and colleagues (24); (2) HPLC-purified dHL60 PAF was also tested for polarizing activity toward human neutrophils as described by Donabedian and co-workers (25); (3) PAF from dHL60 and BEAS S6 cells was found to co-migrate with authentic PAF on silica (22) and reversephase TLC (see below); (4) PAF from dHL60 cells was sensitive to hydrolysis by 100 U of bee venom phospholipase A2 carried out in 100 mM Tris (pH 8.5) and 2 mM calcium chloride; and (5) PAF from dHL60 and BEAS S6 cells purified by reverse-phase TLC was detected by radioimmunoassay (RIA) (see below). RIA PAF from dHL60 and BEAS S6 cells was isolated by a TLC system for subsequent analysis by RIA using a kit (New England Nuclear). This TLC system was developed in order to reduce the variability in yield that is reportedly associated with recovery of choline-containing phospholipids from silica TLC plates (23). For the dHL60 cells, cellular lipids were extracted as described previously and PAF was purified by chromatography on RP-8 TLC plates in a tank equilibrated with acetonitrile:methanol:water:acetic acid (50:50: 2:0.5). The PAF/lysoPAF band was located by exposing outside lanes containing authentic PAF to iodine vapor. The
Samet, Noah, Devlin et al.: Effect of Ozone on PAF Production in Cultured Lung Cells
PAF/lyso PAF band was then scraped into 16 x 100 mm glass tubes and 2 ml of methanol was added, the tube was sonicated and centrifuged briefly at 200 x g, and the methanol supernatant was decanted into another tube. This procedure was repeated using 1 ml of methanol, and 2 vol of chloroform and 1 vol of water were added. The chloroform phase was removed with a siliconized Pasteur pipette and evaporated under nitrogen to dryness in a glass tube. Each sample tube was then rinsed with 2 ml of methanol and dried once more in order to concentrate the lipid at the bottom of the tube. The sample was then resuspended in ix assay buffer as per kit instructions. PAF recoveries of 90% are possible using this procedure. For the BEAS S6 cells, after exposure the cells were incubated for 5 min with 1 ml of HBSS with 0.25 % BSA. The apical and basolateral media were then pooled and extracted together with chloroform and methanol as described for the dHL60 cells, but omitting the PAF carrier. In order to obtain enough material for analysis by RIA, it was necessary to pool apical and basolateral media from three BEAS S6 Transwell cultures. Extracted lipids were then dried under N2 and resuspended in assay buffer for RIA as described for the dHL60 cells. SICr Release In order to examine the possible cytotoxic effect of an in vitro exposure to 0" the amount of "Cr released from the BEAS S6 and the dHL60 cells during exposure to 0.05 to 1.0 ppm 0, was measured and compared with release by control cells exposed to air alone for the same time period using a previously reported method (26). For the dHL60 cells, cells were radiolabeled with 50 p.Ci of "Cr/dish for 90 min, washed, and then exposed to 0, or air, as described previously. Specific SICr release was computed as the fraction of "Cr released into the media (during 15 or 120 min of exposure) relative to the total radioactivity recovered (i.e., the radioactivity found in the media, the adherent cells, and cells detached during exposure) (26). For the BEAS S6 cells, the cells were radiolabeled with 50 p.Ci of "Cr/well overnight, washed, and exposed to 0, or air for 60 min, as previously described. Specific "Cr release was computed as the amount of radioactivity released into the apical and basolateral compartments relative to the total recovered radioactivity. Statistics Statistical analyses between Oj-exposed and air-exposed groups were carried out using paired or unpaired t tests.
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Multiple comparisons were tested using a one-way ANOVA, followed by a Bonferroni correction for post-hoc analyses (27). Data are expressed as the mean ± SEM. P values < 0.05 were considered significant.
Results dHL60 Cells In vitro exposure of control dHL60 cell cultures to air alone produced no significant changes in [3H]PAF levels over the 120-min period of exposure. Average dpm for [3H]PAF in the dHL60 cells were 2,105 ± 156 (n = 10), 2,266 ± 543 (n = 10), 1,809 ± 163 (n = 9), and 2,182 ± 144 (n = 10) at the 0-, 15-, 60-, and 120-min air exposure time points, respectively (P = NS). The average coefficient of variation for the control cultures over the 15- to 120-min period of air exposure was 15.8%, and the average percent difference between the 15- to 60- and 60- to 120-min control values was 10.3 ± 4.6%. Exposure of dHL60 cultures to 1.0 ppm 0, for 5 min did not result in significant elevations in [3H]PAF compared with air controls (Table 1). Based on these results, no 5-min exposure studies were conducted at lower 0, concentrations. In contrast to the 5-min exposure values, dHL60 cell cultures exposed to 0.05 ppm 0, for 15 min demonstrated a significant 1.33-fold increase in ['H]PAF accumulation compared with paired air control cultures exposed simultaneously (Table 1; P < 0.05, n = 5). Significant increases in ['H]PAF accumulation in the dHL60 cells at the 0.05 ppm exposure level were observed for the longer exposure periods (2.32-fold increase at 60 min [n = 5] and 1.72-fold increase at 120 min [n = 5]). There were no significant differences between the increased [3H]PAF levels at these various time points. Similarly, significant increases in [3H]PAF were also found after exposure to 0.1 and 0.3 ppm 0, for all time points (average increase of 1.68-fold and 1.67-fold for 0.1 and 0.3 ppm exposure levels, respectively). Exposure of the dHL60 cultures to 1.0 ppm 0, for 15 min also resulted in a significant 2.0-fold increase in [3H]PAF levels. After 60 min of exposure to 0" there was a peak 3.18-fold increase in [3H]PAF levels that decreased after 120 min of exposure to a 2A8-fold increase in [3H]PAF over control levels. The increased PAF levels at the 60-min exposure period were significantly different from the 15- and 120-min exposure period values at the 1.0 ppm 0, level. Approximately 70% of the [3H]PAF produced by
TABLE 1
Effect of ozone on PAF levels in dHL60 cells* Ozone Concentration (ppm)
Exposure Duration (min)
0.05
0.1
0.3
1.33 ± 0.24 (5)t 2.32 ± 0.37 (5)t 1.72 ± 0.29 (5)t
1.58 ± 0.32 (6)t 1.78 ± 0.59 (6)t 1.68 ± 0.33 (6)t
1.32 ± 0.11 (4)t 2.04 ± 0.40 (4)t 1.64 ± 0.18 (4)t
5 15 60 120
1.0
1.21 2.00 3.18 2.48
± ± ± ±
0.12 0.26 0.43 0.30
(4) (7)t (7)H (7)t
* Data are shown as mean ± SEM (n) and expressed as the fold increase in ['H]PAF dpm in Os-exposed dHL60 cells relative to paired, air-exposed control dHL60 cells. dHL60 cells exposed to air alone produced 1,642 ± 144 dpm ['H]PAF/culture (n = 32). t Significant difference from air control values, P < 0.05. t Significant difference from other time points at the same ozone concentration, P < 0.05.
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tures exposed to 0.3 or 1.0 ppm 0 3 for 15 min compared with air controls (average specific release of 18.0%, P = NS). In contrast, significantly increased specific 51Cr release values were found in dHL60 cells exposed to 0.3 or 1.0 ppm 0 3 for 120 min compared with air controls (average specific release of 29.8%, P < 0.05).
the dHL60 cells was found to be cell associated, with the remainder recovered in the conditioned media. Similar significant increases in [3H]PAF levels in dHL60 cells exposed to 1.0 ppm 0 3 were also found by RIA. Chromatographically separated lipid extracts from dHL60 cultures exposed to either 1.0 ppm 0 3 (or air alone as controls) for 60 min were analyzed by a PAF-specific RIA (see MATERIALS AND METHODS). dHL60 cultures exposed to air contained 4.2 ± 1.9 ng of PAF/dish (n = 5) by RIA. After exposure to 1.0 ppm 0 3 for 60 min, there was a significant 3.9 ± 1.5-fold increase in PAF levels in the Orexposed cultures (16.4 ± 6.3 ng of PAF/dish) compared with airexposed control cultures (n = 5, P < 0.05). This 3.9-fold increase in PAF levels was not statistically different from the 3.2-fold increase in [3H]PAF found using TLC methodology after exposure to the same conditions, i.e., 1.0 ppm 0 3 for 60 min (Table 1). To compare the Oi-induced effects on dHL60 PAF accumulation to a known stimulus of PAF synthesis in macrophages, we also examined the magnitude and time course of changes in [3H]PAF levels by TLC in dHL60 cultures after incubation with the calcium ionophore A23187 (Figure 1). dHL60 cultures incubated with A23187 (10 ILM) demonstrated significantly increased [3H]PAF levels compared with vehicle alone by 2 min of incubation (1.94-fold), with a maximal response (2.03-fold increase above control) observed by 5 min of incubation. [3H]PAF values returned to baseline control levels by 120 min (Figure 1). In regard to cell viability during 0 3 exposure, as assessed by specific 51Cr release, in vitro exposure of dHL60 cells to air alone resulted in baseline-specific 51Cr release values of 13.8 ± 2.9% and 18.2 ± 2.1% at 15 and 120 min of exposure, respectively (Figure 2; P = NS). Exposure of dHL60 cells to 0.05 and 0.1 ppm 0 3 for 15 and 120 min, an exposure level that did significantly increase [3H]PAF accumulation (Table 1), did not result in any significant increases in specific 51Cr release values above baseline values (average specific release of 12.5 %, P = NS). No significant increases in specific 51Cr release were found in dHL60 cul-
BEAS S6 Cells In vitro exposure of BEAS S6 cells to 0 3 for 50 min also resulted in significant increases in [3H]PAF production. BEAS S6 cells, cultured on Transwell filters and exposed to 0.05 to 1.0 ppm 0 3 for 60 min released significantly increased amounts of [3H]PAF into both the apical and basolateral compartments, as compared with paired airexposed controls. There were 2.9-, 5.6-, 13.5-, and 7.9-fold increases in total [3H]PAF release (apical plus basolateral compartments) after exposure to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P < 0.05 for all concentrations). There were no significant differences in [3H]PAF increases between the various concentrations of 0 3 used. The effects of 0 3 exposure on vectorial release of [3H]PAF are shown in Table 2. There were 1.65-, 4.77-, 10.19-, and 7.53-fold increases in [3H]PAF release into the apical compartment after exposure to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P< 0.05). In the basolateral compartment, there were 1.25-, 8.02-, 16.09-, and 8.59-fold increases in [3H]PAF release by BEAS S6 cells exposed to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P < 0.05). No statistically significant vectorial release differences were found in [3H]PAF release by BEAS S6 cells exposed to 0 3, We believe the data demonstrating significant [3H]PAF release into the apical as well as the basolateral compartments during 0 3 exposure represent true vectorial release of [3H]PAF by BEAS S6 cells. This conclusion is based on data demonstrating that when [l4C]PAF was added to either the apical or basolateral compartment of BEAS S6 cell cultures grown on collagen filters and exposed to air for 60 min, an average of only 2.9% of the [l4C]PAF was found in the opposite compartment 60 min after addition to the radiola-
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Platelet-activating factor (PAF) is a phospholipid with a wide spectrum of pro-inflammatory properties. In the lung, PAF induces airway hyperresponsiveness, neutrophil sequestration, and increased vascular permeability. The alveolar macrophage and the bronchial epithelium are tissues that are exposed to inhaled ozone (0 3) , We studied the effect of an in vitro 0 3 exposure on PAF production in a macrophage-like HL60 human cell line (dHL60), a human bronchial epithelial cell line (BEAS S6), and also in primary human bronchial epithelial cells. PAF was quantified by thin-layer chromatographic separation of lipid extracts from cells radio labeled with FH]lysoPAF and by radioimmunoassay. In vitro exposure of dHL60 cells to 0.05 to 1.0 ppm 0 3 for 15 to 120 min was found to significantly increase PAF levels above air control values at all exposure levels and time points (average increase of 92 %). Similarly, BEAS S6 cells grown on collagen-coated filter supports and exposed to 0.05 to 1.0 ppm 0 3 for 60 min released an average increase in PAF of 626 % above control values. Primary human bronchial epithelial cells also demonstrated significant increases in FH]PAF release (average increase of 289% after exposure to 1.0 ppm 0 3 for 60 min) compared with paired air controls. These findings suggest that some of the effects of 0 3 inhalation may be mediated by PAP.
Ozone (03) is an oxidant pollutant gas that is a major constituent of photochemical smog. In the United States, it is estimated that 150 million people reside in areas that exceed the current National Ambient Air Quality Standard for 0 3 of 0.12 ppm (1). 0 3 inhalation has been demonstrated to result in changes in lung function and evidence of pulmonary inflammation in human subjects, including airway hyperreactivity (2, 3), increased epithelial and vascular perme-
(Received in original form November 22, 1991 and in final form June 23, 1992)
Mitchell Friedman, M.D., Pulmonary Diseases/Critical Care Medicine Section, Department ofMedicine SL9, Tulane University Medical Center, 1430 Tulane Ave., New Orleans, LA701122699. Abbreviations: bovine serum albumin, BSA; [N-methyl-14C]1-0-alkyl-2acetyl-sn-3-glycerophosphocholine, [14C]PAF; Dulbecco's modified Eagle's mediumIHam's Fl2 medium, DMEM/Fl2; fetal bovine serum, FBS; Hanks' balanced salt solution, HBSS; 1-0-eHI-octadecyl-2-sn-glycero-3phosphocholine, fHllysoPAF; high performance liquid chromatography, HPLC; keritanocyte basal medium, KBM; Joklik's minimal essential medium, MEM; ozone, 0 3 ; platelet-activating factor, PAF; phosphate-bufered saline, PBS; phorbol 12-myristate 13-acetate, PMA; radioimmunoassay, RIA; thin-layer chromatography, TLC. Address correspondence to:
Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 514-522, 1992
ability to macromolecules (4-6), and neutrophil infiltration into the airways (5-7). Several in vivo and in vitro exposure studies have suggested that some of the pathophysiologic responses of the lung to 0 3 may involve arachidonic acid-derived inflammatory mediators produced by lung cells interacting with inhaled 0 3 , i.e., alveolar macrophages and airway epithelial cells. For example, increased levels of prostaglandins E, and F2a have been found in the bronchoalveolar lavage fluid of human subjects exposed to 0.4 ppm 0 3 for 2 h (5, 6). Primary cultures of bovine airway epithelial cells exposed in vitro to 0.1 ppm 0 3 for 2 h have been shown to release arachidonic acid and increased amounts of prostaglandin F 2a (8). Similarly, cultured rat alveolar macrophages release increased amounts of arachidonic acid, thromboxane B2, and leukotrienes B4, C4, and D4 during exposure in vitro to 1.0 ppm 0 3 for 2 h (9, 10). In addition to providing the substrate for lipoxygenasemediated and cyclooxygenase-mediated eicosanoid synthesis, the release of arachidonic acid from certain membrane phospholipids is linked to the synthesis of another important lipid mediator, platelet-activating factor (PAF). PAF is derived from 1-0-alkylphosphatidylcholine, a membrane phospholipid that is enriched in arachidonic acid at the sn-2 posi-
Samet, Noah, Devlin et al.: Effect of Ozone on PAF Production in Cultured Lung Cells
tion of the molecule (11). The enzyme phospholipase A2 catalyzes the first step in the synthesis of PAF by removing the sn-2 fatty acid from the phospholipid to produce lysoPAF. Thus, the release of arachidonic acid and the formation of lysoPAF can be simultaneous events (12). LysoPAF can then be acetylated by a specific acetyltransferase to form PAF (13, 14). PAF is a potent mediator with a wide spectrum of pro-inflammatory activities in a variety of tissues (15, 16). In the lung, PAF has been shown to induce inflammatory reactions including airway hyperreactivity, vascular permeability, and neutrophil infiltration in the airways (17). These inflammatory events are similar to those observed after 0 3 inhalation (2-7). Based on the possibility that PAF may be an important pro-inflammatory molecule involved in the response of the lung to 0 3 , we examined the effect of in vitro 0 3 exposure on the production of PAF in two relevant human cell lines, the cell line HL60 differentiated into macrophage-like cells and the bronchial epithelial cell line BEAS S6. We also exposed primary human bronchial epithelial cells in culture to 0 3 , We now report that exposure of these various human cells to 0 3 (in concentrations as low as 0.05 ppm for 15 min) resulted in significant increases in PAF levels in phorbol-differentiated HL60 cells and significant release of PAF by BEAS S6 cells and primary human bronchial epithelial cells.
Materials and Methods Materials Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/FI2), gentainicin sulfate, L-glutamine, and phosphate-buffered saline (PBS) were obtained from the UNC Lineberger Cancer Center Tissue Culture Facility (Chapel Hill, NC). Growth hormones used for the culture of primary cells were obtained from Collaborative Research (Waltham, MA). Nu Serum IV was obtained from GIBCO (Grand Island, NY). Keratinocyte basal medium (KBM) and supplements were obtained from Clonetics (San Diego, CA). Tissue culture dishes and flasks were obtained from Falcon (Oxnard, CA) or Costar (Cambridge, MA). Phorbol 12-myristate 13-acetate (PMA) , unlabeled PAF, cholera toxin, phospholipase A2 , calcium ionophore A23187, and acid phosphatase kits were obtained from Sigma Chemical Co. (St. Louis, MO). Silica thin-layer chromatography (TLC) plates were obtained from Merck (VWR; Durham, NC), RP-8 TLC plates were from Analtech (Newark, DE). 1-0-PH]-octadecy1-2-sn-glycero- 3-phosphocholine (PH]lysoPAF; specific activity, 80 to 180 Ci/ mmol) and [Nmethy1-14C]1-0-alky1-2-acety l-sn- 3-g1ycerophosphocholine ([14C]PAF; specific activity, 50 to 60 mCi/mmol) were obtained from Amersham (Arlington Heights, IL). PAFspecific radioimmunoassay kits were obtained from New England Nuclear (Boston, MA). 51Cr was obtained from NEN Dupont (Wilmington, DE). High performance liquid chromatography (HPLC) solvents and common laboratory reagents were obtained from Fisher (Raleigh, NC). Cell Culture Undifferentiated myeloid leukemic HL60 cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM/FI2 (1:1) supplemented with 10% Nu Serum IV,
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50 mg/liter gentamicin sulfate, and 2 mM L-glutamine(complete DMEM/F12) at a density of 0.5 to 2.0 x 106 cells/rnl. Cells were plated at a density of 2.0 to 4.0 X 106 cells/dish in 60-mm polystyrene tissue culture dishes and induced to differentiate toward monocytic development with 30 nM PMA (18) in complete DMEM/FI2 for 72 h. Differentiation was assessed by morphologic and enzymatic criteria, as previously described (18). HL60 cells treated with PMA (dHL60) become growth-arrested, strongly adherent, flattened, and elongated in appearance with long pseudopodlike processes. Acid phosphatase activity in the dHL60 cells at day 3 of differentiation was 96.7 ± 6.3 nmol/min/mg protein, compared with 20.0 ± 2.7 nmol/min/mg protein for the undifferentiated cells (n = 3), as measured with a standard assay kit (Sigma). At day 3 of differentiation, the dHL60 cells contained 129.6 ± 12.3 p,g of cellular protein/dish (n = 10). There were 2 X 106 cells/dish, but this value is an approximation due to the difficulty found in not being able to completely remove all intact dHL60 cells from the culture dishes. Passages 10 through 30 of the human bronchial epithelium cell line BEAS S6 (generous gift of Drs. C. Harris and 1. Lechner, National Institutes of Health, Bethesda, MD) were maintained in KBM supplemented with 10 ng/ml epidermal growth factor (human recombinant), 5 mg/rnl bovine insulin, 0.5 mg/rnl hydrocortisone, 0.15 mM calcium and bovine pituitary extract (supplements provided by Clonetics for use with KBM), on tissue culture petri dishes. In order to expose these cells and study the vectorial release of PAF, 5 x lOS BEAS S6 cells were seeded on 25-mm, collagen-coated (3-p,m pore size) polycarbonate filter supports (Transwell-COL; Costar), with 1 rnl of supplemented KBM added to the apical (top) compartment and 2.5 rnl to the basolateral (bottom) compartment. Cells became confluent (approximately 2 x 106 cells/filter) and growth-arrested after 2 days of the collagen filters. BEAS S6 cells were cultured for an additional 2 days in order to promote differentiation (19). Primary human bronchial epithelial cells were obtained from lung lobes excised from patients at the University of North Carolina Hospitals for standard clinical conditions (lung mass or localized carcinoma) within 30 min of excision. Third to sixth generation bronchi, which were not involved with the primary disease process, were isolated by sharp and blunt dissection while the lobe was bathed in 4°C lactated Ringer's solution. The tissues were rinsed in Joklik's minimal essential medium (MEM) containing penicillin (50 U/rnl), streptomycin (50 p,g/rnl), and gentamicin (40 mg/ ml). Epithelial cells were dissociated by protease treatment (Sigma type 14; 0.1%, 16 h at 4°C) as described (20). The protease was neutralized by addition of 10% fetal bovine serum (FBS), and the cells were dislodged by gentle agitation. The cell suspension was pelleted (800 X g for 5 min), washed in l(j% FBS in MEM, and plated on collagen filters in serum-free F12 medium that was supplemented with six growth factors (insulin, endothelial cell growth supplement, triiodothyronine, hydrocortisone, epidermal growth factor, and cholera toxin), as previously described (20). These primary cultures were incubated in a humidified atmosphere of 5 % CO 2 at 37°C. Media was changed 3 times/wk until the cells reached confluency.
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Before 0 3 exposure, dHL60, BEAS S6, or primary human bronchial epithelial cell cultures were radio labeled with 1 to 2 11Ci/rnl PH]lysoPAF in growth media containing 0.25% bovine serum albumin (BSA) for 0.5 to 5 h. Under these conditions, BEAS S6 cells labeled with 2 11Ci/filter incorporated an average of 25 % of the label and dHL60 cells labeled with 1.5 11Ci/cultureincorporated an average of 67 % of the label over the duration of the labeling period. The duration of the labeling period did not influence the extent of incorporation of the [3H]lysoPAF into the cells (data not shown). After labeling, the cells were washed extensively with PBS in order to remove unincorporated label before exposure. dHL60 cells were then exposed to 0 3 (see next section) in 1.5 rnl of PBS (with 1 mM calcium and magnesium) supplemented with 1 g/liter glucose, while rocking. In additional experiments, dHL60 cultures were incubated with the calcium ionophore A23l87 (at a final concentration of 10 11M in 0.01% dimethyl sulfoxide). Primary epithelial and BEAS S6 cells were washed thoroughly and then exposed on Transwell-COL filters with no media in the apical compartment and 1.6 rnl of complete Hanks' balanced salt solution (HBSS) in the basolateral compartment. Cells exposed in this manner are hydrated by the HBSS in the basolateral compartment.
In Vitro 0 3 Exposure System Two separate in vitro 0 3 exposure systems, similar in design to the previously described exposure system from our laboratory (9), were used for these experiments. The results were identical in both systems and, therefore, the data were pooled. The first system consisted of two plexiglass and steel chambers (each 15 liters in capacity) mounted on rocking platforms (Bellco Glass, Vineland, NJ) through which filtered room air at 37°C, with or without 0 3 , flowed at a rate of 7.5 liters/min. Separate input lines carried humidified air containing CO 2 into the chambers in order to maintain a 5 % CO2 atmosphere, as maintained by two CO 2 analyzers (Forma Scientific, Marietta, OH). Chambers and platforms were enclosed in 37°C incubators (Forma Scientific). Ozone was generated by passing a stream of air over a partially occluded UV pen lamp. Ozone concentrations in the chamber were monitored with a 1003-AH 0 3 Analyzer (Dasibi, Glendale, CA). The second system consisted of two glass chambers approximately 2.5 liters in volume through which humidified Zero Grade Air (containing < 1 ppm organic contaminants), with or without OJ, flowed at a rate of 1.2 liters/min. Ozone was generated with the use of a partially occluded pen lamp. Dehumidified air was analyzed in a Model 8002 Ozone Analyzer (Bendix, Lewisburg, WV). The lack of CO2 during exposures in the second system had no effect on any of the endpoints measured, including the pH of the exposure media. In each system, 0 3 concentration was maintained within ±1O% of the desired concentration. Extraction and Analysis of PAF After exposure of the dHL60 cells, the cells were scraped in the exposure media. The cell and media lipids were extracted with chlorofurm:methanol:aqueous(2:1:0.8) and spiked with approximately 50 11g of unlabeled PAF per sample to serve as a carrier (21). In some experiments, a few hundred dpm of p4C]PAF were included in the solvent mixture in
order to correct for recovery losses. The chloroform-rich phase was removed, a second extraction with chloroform was performed, and the chloroform phases were pooled and dried under nitrogen. After exposure of the BEAS S6 cells and the primary human bronchial epithelial cells using the same systems as the ones used for exposure of dHL60 cells, 1 rnl of HBSS with 0.25% BSA was added to the apical compartment and the cultures were incubated for an additional 5 min in room air. Apical and basolateral media were then removed and the lipids were extracted separately as described for the dHL60 cells. It was not possible to examine intracellular levels of [3H]PAF in the BEAS S6 cells or primary human bronchial epithelial cells because the collagen-coated membrane filter used to grow and expose these cells dissolves in organic solvents, thereby interfering with the extraction of cell-associated [3H]PAF. Therefore, only the amount of [3H]PAF released apically or basolaterally is reported for the BEAS S6 and primary human bronchial epithelial cell cultures. Lipids were resuspended in 40 111 of chloroform and chromatographed on heat-activated silica gel 60 plates, using a solvent mixture consisting of chloroform:methanol:glacial acetic acid:water (100:50:16:8) in an equilibrated tank (22). Lipids were visualized with iodine vapor and the PAF band was scraped, 0.5 rnl of methanol: water (1:1) and 4 rnl of scintillation cocktail were added to the scraped PAF band, and the sample was counted in a liquid scintillation counter. This method was used to quantitate [3H]PAF accumulation in dHL60 cells and [3H]pAF release in BEAS S6 and primary human bronchial epithelial cells. The identity of cellular PAF was verified by several independent methods: (1) dHL60 PAF was isolated by HPLC in an 8 x 10 radial compression module fitted with a silica cartridge (Waters, Milford, MA) using an isocratic solvent system consisting of acetonitrile:methanol:85% phosphoric acid (520:20:6) (23) and tested for biologic activity toward washed rabbit platelets according to the procedure described by Wykle and colleagues (24); (2) HPLC-purified dHL60 PAF was also tested for polarizing activity toward human neutrophils as described by Donabedian and co-workers (25); (3) PAF from dHL60 and BEAS S6 cells was found to co-migrate with authentic PAF on silica (22) and reversephase TLC (see below); (4) PAF from dHL60 cells was sensitive to hydrolysis by 100 U of bee venom phospholipase A2 carried out in 100 mM Tris (pH 8.5) and 2 mM calcium chloride; and (5) PAF from dHL60 and BEAS S6 cells purified by reverse-phase TLC was detected by radioimmunoassay (RIA) (see below). RIA PAF from dHL60 and BEAS S6 cells was isolated by a TLC system for subsequent analysis by RIA using a kit (New England Nuclear). This TLC system was developed in order to reduce the variability in yield that is reportedly associated with recovery of choline-containing phospholipids from silica TLC plates (23). For the dHL60 cells, cellular lipids were extracted as described previously and PAF was purified by chromatography on RP-8 TLC plates in a tank equilibrated with acetonitrile:methanol:water:acetic acid (50:50: 2:0.5). The PAF/lysoPAF band was located by exposing outside lanes containing authentic PAF to iodine vapor. The
Samet, Noah, Devlin et al.: Effect of Ozone on PAF Production in Cultured Lung Cells
PAF/lyso PAF band was then scraped into 16 x 100 mm glass tubes and 2 ml of methanol was added, the tube was sonicated and centrifuged briefly at 200 x g, and the methanol supernatant was decanted into another tube. This procedure was repeated using 1 ml of methanol, and 2 vol of chloroform and 1 vol of water were added. The chloroform phase was removed with a siliconized Pasteur pipette and evaporated under nitrogen to dryness in a glass tube. Each sample tube was then rinsed with 2 ml of methanol and dried once more in order to concentrate the lipid at the bottom of the tube. The sample was then resuspended in ix assay buffer as per kit instructions. PAF recoveries of 90% are possible using this procedure. For the BEAS S6 cells, after exposure the cells were incubated for 5 min with 1 ml of HBSS with 0.25 % BSA. The apical and basolateral media were then pooled and extracted together with chloroform and methanol as described for the dHL60 cells, but omitting the PAF carrier. In order to obtain enough material for analysis by RIA, it was necessary to pool apical and basolateral media from three BEAS S6 Transwell cultures. Extracted lipids were then dried under N2 and resuspended in assay buffer for RIA as described for the dHL60 cells. SICr Release In order to examine the possible cytotoxic effect of an in vitro exposure to 0" the amount of "Cr released from the BEAS S6 and the dHL60 cells during exposure to 0.05 to 1.0 ppm 0, was measured and compared with release by control cells exposed to air alone for the same time period using a previously reported method (26). For the dHL60 cells, cells were radiolabeled with 50 p.Ci of "Cr/dish for 90 min, washed, and then exposed to 0, or air, as described previously. Specific SICr release was computed as the fraction of "Cr released into the media (during 15 or 120 min of exposure) relative to the total radioactivity recovered (i.e., the radioactivity found in the media, the adherent cells, and cells detached during exposure) (26). For the BEAS S6 cells, the cells were radiolabeled with 50 p.Ci of "Cr/well overnight, washed, and exposed to 0, or air for 60 min, as previously described. Specific "Cr release was computed as the amount of radioactivity released into the apical and basolateral compartments relative to the total recovered radioactivity. Statistics Statistical analyses between Oj-exposed and air-exposed groups were carried out using paired or unpaired t tests.
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Multiple comparisons were tested using a one-way ANOVA, followed by a Bonferroni correction for post-hoc analyses (27). Data are expressed as the mean ± SEM. P values < 0.05 were considered significant.
Results dHL60 Cells In vitro exposure of control dHL60 cell cultures to air alone produced no significant changes in [3H]PAF levels over the 120-min period of exposure. Average dpm for [3H]PAF in the dHL60 cells were 2,105 ± 156 (n = 10), 2,266 ± 543 (n = 10), 1,809 ± 163 (n = 9), and 2,182 ± 144 (n = 10) at the 0-, 15-, 60-, and 120-min air exposure time points, respectively (P = NS). The average coefficient of variation for the control cultures over the 15- to 120-min period of air exposure was 15.8%, and the average percent difference between the 15- to 60- and 60- to 120-min control values was 10.3 ± 4.6%. Exposure of dHL60 cultures to 1.0 ppm 0, for 5 min did not result in significant elevations in [3H]PAF compared with air controls (Table 1). Based on these results, no 5-min exposure studies were conducted at lower 0, concentrations. In contrast to the 5-min exposure values, dHL60 cell cultures exposed to 0.05 ppm 0, for 15 min demonstrated a significant 1.33-fold increase in ['H]PAF accumulation compared with paired air control cultures exposed simultaneously (Table 1; P < 0.05, n = 5). Significant increases in ['H]PAF accumulation in the dHL60 cells at the 0.05 ppm exposure level were observed for the longer exposure periods (2.32-fold increase at 60 min [n = 5] and 1.72-fold increase at 120 min [n = 5]). There were no significant differences between the increased [3H]PAF levels at these various time points. Similarly, significant increases in [3H]PAF were also found after exposure to 0.1 and 0.3 ppm 0, for all time points (average increase of 1.68-fold and 1.67-fold for 0.1 and 0.3 ppm exposure levels, respectively). Exposure of the dHL60 cultures to 1.0 ppm 0, for 15 min also resulted in a significant 2.0-fold increase in [3H]PAF levels. After 60 min of exposure to 0" there was a peak 3.18-fold increase in [3H]PAF levels that decreased after 120 min of exposure to a 2A8-fold increase in [3H]PAF over control levels. The increased PAF levels at the 60-min exposure period were significantly different from the 15- and 120-min exposure period values at the 1.0 ppm 0, level. Approximately 70% of the [3H]PAF produced by
TABLE 1
Effect of ozone on PAF levels in dHL60 cells* Ozone Concentration (ppm)
Exposure Duration (min)
0.05
0.1
0.3
1.33 ± 0.24 (5)t 2.32 ± 0.37 (5)t 1.72 ± 0.29 (5)t
1.58 ± 0.32 (6)t 1.78 ± 0.59 (6)t 1.68 ± 0.33 (6)t
1.32 ± 0.11 (4)t 2.04 ± 0.40 (4)t 1.64 ± 0.18 (4)t
5 15 60 120
1.0
1.21 2.00 3.18 2.48
± ± ± ±
0.12 0.26 0.43 0.30
(4) (7)t (7)H (7)t
* Data are shown as mean ± SEM (n) and expressed as the fold increase in ['H]PAF dpm in Os-exposed dHL60 cells relative to paired, air-exposed control dHL60 cells. dHL60 cells exposed to air alone produced 1,642 ± 144 dpm ['H]PAF/culture (n = 32). t Significant difference from air control values, P < 0.05. t Significant difference from other time points at the same ozone concentration, P < 0.05.
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tures exposed to 0.3 or 1.0 ppm 0 3 for 15 min compared with air controls (average specific release of 18.0%, P = NS). In contrast, significantly increased specific 51Cr release values were found in dHL60 cells exposed to 0.3 or 1.0 ppm 0 3 for 120 min compared with air controls (average specific release of 29.8%, P < 0.05).
the dHL60 cells was found to be cell associated, with the remainder recovered in the conditioned media. Similar significant increases in [3H]PAF levels in dHL60 cells exposed to 1.0 ppm 0 3 were also found by RIA. Chromatographically separated lipid extracts from dHL60 cultures exposed to either 1.0 ppm 0 3 (or air alone as controls) for 60 min were analyzed by a PAF-specific RIA (see MATERIALS AND METHODS). dHL60 cultures exposed to air contained 4.2 ± 1.9 ng of PAF/dish (n = 5) by RIA. After exposure to 1.0 ppm 0 3 for 60 min, there was a significant 3.9 ± 1.5-fold increase in PAF levels in the Orexposed cultures (16.4 ± 6.3 ng of PAF/dish) compared with airexposed control cultures (n = 5, P < 0.05). This 3.9-fold increase in PAF levels was not statistically different from the 3.2-fold increase in [3H]PAF found using TLC methodology after exposure to the same conditions, i.e., 1.0 ppm 0 3 for 60 min (Table 1). To compare the Oi-induced effects on dHL60 PAF accumulation to a known stimulus of PAF synthesis in macrophages, we also examined the magnitude and time course of changes in [3H]PAF levels by TLC in dHL60 cultures after incubation with the calcium ionophore A23187 (Figure 1). dHL60 cultures incubated with A23187 (10 ILM) demonstrated significantly increased [3H]PAF levels compared with vehicle alone by 2 min of incubation (1.94-fold), with a maximal response (2.03-fold increase above control) observed by 5 min of incubation. [3H]PAF values returned to baseline control levels by 120 min (Figure 1). In regard to cell viability during 0 3 exposure, as assessed by specific 51Cr release, in vitro exposure of dHL60 cells to air alone resulted in baseline-specific 51Cr release values of 13.8 ± 2.9% and 18.2 ± 2.1% at 15 and 120 min of exposure, respectively (Figure 2; P = NS). Exposure of dHL60 cells to 0.05 and 0.1 ppm 0 3 for 15 and 120 min, an exposure level that did significantly increase [3H]PAF accumulation (Table 1), did not result in any significant increases in specific 51Cr release values above baseline values (average specific release of 12.5 %, P = NS). No significant increases in specific 51Cr release were found in dHL60 cul-
BEAS S6 Cells In vitro exposure of BEAS S6 cells to 0 3 for 50 min also resulted in significant increases in [3H]PAF production. BEAS S6 cells, cultured on Transwell filters and exposed to 0.05 to 1.0 ppm 0 3 for 60 min released significantly increased amounts of [3H]PAF into both the apical and basolateral compartments, as compared with paired airexposed controls. There were 2.9-, 5.6-, 13.5-, and 7.9-fold increases in total [3H]PAF release (apical plus basolateral compartments) after exposure to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P < 0.05 for all concentrations). There were no significant differences in [3H]PAF increases between the various concentrations of 0 3 used. The effects of 0 3 exposure on vectorial release of [3H]PAF are shown in Table 2. There were 1.65-, 4.77-, 10.19-, and 7.53-fold increases in [3H]PAF release into the apical compartment after exposure to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P< 0.05). In the basolateral compartment, there were 1.25-, 8.02-, 16.09-, and 8.59-fold increases in [3H]PAF release by BEAS S6 cells exposed to 0.05, 0.1, 0.3, and 1.0 ppm 0 3, respectively (P < 0.05). No statistically significant vectorial release differences were found in [3H]PAF release by BEAS S6 cells exposed to 0 3, We believe the data demonstrating significant [3H]PAF release into the apical as well as the basolateral compartments during 0 3 exposure represent true vectorial release of [3H]PAF by BEAS S6 cells. This conclusion is based on data demonstrating that when [l4C]PAF was added to either the apical or basolateral compartment of BEAS S6 cell cultures grown on collagen filters and exposed to air for 60 min, an average of only 2.9% of the [l4C]PAF was found in the opposite compartment 60 min after addition to the radiola-
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