Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, 841125820
Received April 1, 2004; accepted July 15, 2004
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ABSTRACT |
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Key Words: geological dust; physical treatment; cell culture; interleukin-6; IL-6; interleukin-8; IL-8; endotoxin; vanilloid receptor.
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INTRODUCTION |
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In vitro toxicology studies, motivated by specific biochemical signaling hypotheses, have attempted to link specific particle sources with cellular responses. Typically, lung cells are exposed to 10100 µg/cm2 of ambient particles or laboratory surrogates for specific components of air pollution. Cell culture models include immortalized lung cell lines such as BEAS-2B (Frampton et al., 1999; Ghio et al., 1999
; Steerenberg et al., 1998
) and A549 (Seagrave and Nikula, 2000
; Smith et al., 2000
), normal human bronchial epithelial cells (Carter et al., 1997
), macrophages, and cocultures of macrophages with epithelial cells (Tao and Kobzik, 2002
).
Mills et al. (1999) and Driscoll (1999)
reviewed cytokine responses in airway epithelial cells. The release of both IL-6 and IL-8 by lung cells is transcriptionally controlled by NF-
B (Fan et al., 2001
) as the result of an inflammation-regulating signal cascade involving TNF-
and other cytokines (Nelson and Martin, 2000
). Induction of IL-6 has been observed in BEAS-2B cells treated with diesel exhaust particles (Steerenberg et al., 1998
), residual oil fly ash (ROFA) (Veronesi et al., 1999
), a range of ambient and combustion particles (Veronesi et al., 2002b
), negatively charged particles (Agopyan et al., 2003
; Veronesi et al., 2002a
), and capsaicin-related compounds (Reilly et al., 2003
). Titanium dioxide particles larger than 1 µm are inert and serve as a PM negative control (Steerenberg et al., 1998
; van Maanen et al., 1999
), but mineral dust from stone quarries induces cytotoxicity and IL-6 release in A549 cells (Hetland et al., 2000
) and in rat lung type 2 alveolar cells (Becher et al., 2001
).
Identifying the most toxic particle types requires understanding the effects of the low solubility particle core compared to the soluble and adsorbed species that are rapidly released from particles under physiological conditions. Studies that associated effects with the insoluble particle fraction include work with reactive oxygen species (ROS) generation by polymorphonuclear leukocytes treated with a range of mineral dusts and ambient particles (Prahalad et al., 1999) and work with alveolar macrophages treated with concentrated ambient particles (Imrich et al., 2000
). Evidence for effects from soluble metals comes from studies of BEAS-2B cells treated with aqueous extracts of Utah Valley filters (Frampton et al., 1999
), A549 cells treated with mineral particles (Hetland et al., 2001
), rat tracheal epithelial cells treated with residual oil fly ash (Dye et al., 1999
), and urban particles instilled into mouse lung (Adamson et al., 1999
). Ghio et al. (1999)
reported oxidative stress and IL-8 induction by BEAS-2B cells in response to both the soluble and insoluble fractions from filter samples. Physical treatments to modify particle toxicological properties include leaching particles with the metal chelator desferrioxamine (Smith et al., 2000
), and applying surface-modifying coatings (Schins, 2002
).
Biogenic components, including viable organisms, endotoxin, fungal toxins, and proteins in ambient particles directly affect lung cells (Monn and Koren, 1999). Lipopolysaccharide (LPS) pretreatment is used to model inflammation in animal inhalation experiments (Elder et al., 2000
), and LPS induces IL-6 in BEAS-2B and A549 cells (Schulz et al., 2002
). Procedures to remove endotoxin from medical devices typically involve repeated washing or heating (Williams, 2001
). Heat or chemical sterilization eliminates viable organisms but may change other particle characteristics.
Veronesi et al. (1999) used the receptor antagonists capsazepine (CPZ) and amiloride in BEAS-2B cell culture studies and concluded that the acidic, soluble components of ROFA cause immediate increases in [Ca + 2] influx, followed by IL-6 and IL-8 release through activation of both TRPV1 receptors and acid sensitive ion channels (ASICs). CPZ suppressed IL-6 release in response to St Louis and Ottawa urban particles, wood stove deposits, and coal fly ash (Veronesi et al., 2002b
). Experiments with a BEAS-2B-derived cell line that overexpressed TRPV1 showed that overexpression resulted in a 100-fold decrease in the concentration of capsaicinoids needed to cause cell death and IL-6 release (Reilly et al., 2003
).
Soil dusts are sometimes assumed to be benign compared to anthropogenic emissions, but preliminary experiments in our laboratory suggested that soil dust induced cytokine release at concentrations comparable to that induced by coal fly ash, a widely studied combustion particle source. The objective of this study was to test the hypothesis that specific soil dusts could induce proinflammatory cytokine signaling in an immortalized lung cell line that is widely used to study the mechanisms by which specific types of atmospheric particulate matter induce proinflammatory signaling in airway tissues. Soil dusts, representative of fugitive dust emissions from unpaved roads, from a range of sites in the western United States were tested for IL-6 release response. A subset of four soils was selected for additional experiments that used particle-modifying treatments to gain insight into the chemical component classes (semivolatile, water soluble, etc.) in the soil that affect the observed cell responses.
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MATERIALS AND METHODS |
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Surrogate treatments were prepared from 1.0 2.0 µm APS TiO2 (Alfa Aesar, stock #43047) and Pseudomonas aeruginosa lipopolysaccharide (Sigma, catalog # L9143). Koyama et al. (2000) tested several commercial LPS materials, and P. aeruginosa LPS induced the highest levels of IL-8 in A549 cells. The particles identified as "TiLPS" were prepared by mixing TiO2 and LPS in water, drying the suspension with constant stirring overnight in a vacuum oven at 60°C, and recovering the particles by scraping.
Preparation of cell treatments. Particle samples were weighed, sterilized with 70% ethanol (approximately 50 µl alcohol for 110 mg of particles), vacuum dried, and resuspended in cell culture media, typically at 200 µg/ml. The treatment concentrations were prepared by serial dilution. We routinely used a combination of 5 min of sonication in an ultrasonic cleaner and vortexing immediately before each transfer to insure complete dispersion of the particles. Scanning electron microscopy verified that this procedure dispersed clusters but preserved the primary particles.
We physically modified samples of DD, WM, and R4 dusts. The thermally treated particles were prepared by heating in loosely covered borosilicate glass tubes in air in a muffle furnace at 150, 300, or 550°C for 1 h. Leaching treatments involved suspending the particles in 5 ml of liquid and rotating the tubes overnight at 6 rpm. The samples were centrifuged at 750 x g for 10 min and decanted, and the cycle was repeated three times. The leaching liquids were LHC-9 cell culture media, 1 mM desferrioxamine (Sigma #D9533) in phosphate-buffered saline (PBS), (Biofluids, Camarillo CA), unbuffered demineralized water, and 2:1 (v/v) mixture of chloroform and methanol. The pH values of the particle suspensions were 7.6, 7.0, and 6.0 for the LHC-9, desferrioxamine in PBS, and water, respectively. The supernatant from the first 24 h leaching with LHC-9 was recovered and used to measure the effects of the soluble fraction of the original particles. After leaching, residual solvent was removed from the chloroform-methanol-treated samples by vacuum evaporation. Desferrioxamine was removed by washing the particles with LHC-9.
Some weight change during physical treatment was expected due to evaporation of volatiles, oxidation, and dissolution, so we calculated cell exposures in terms of the original sample mass. The actual amount of particles applied to the cell culture was estimated by light absorbance using a plate reader.
Particle characterization. Endotoxin was measured using the chromogenic Limulus Amebocyte Lysate assay (LAL) kit (QCL-1000, Cambrex BioProducts, Walkersville, MD). A dilution series was prepared for each particle type to find a concentration within the range of the assay. Values are reported as endotoxin units (EU) per mg of dry particle sample or EU/ml for liquid suspensions of purchased LPS. The Veterinary Pathology Laboratory at Utah State University performed elemental analysis on samples using nitric acid digestion and ion-coupled plasma mass spectrometry.
Cell culture. BEAS-2B human bronchial epithelial cells (Reddel et al., 1989) (American Type Culture Collection, Rockville, MD) were used at passage numbers 6080. The cells were cultured in Lechner and LaVeck media (LHC-9) containing retinoic acid (33 nM) and epinephrine (2.75 µM). Culture flasks and multiwell plates were coated with LHC-basal media containing BSA (100 µg/ml), collagen (30 µg/ml), and fibronectin (10 µg/ml) for at least 4 h at 37°C. The cells were maintained in 75 cm2 flasks at 37°C and 6% CO2. Media was replaced every second day, and cells were passaged when >85% confluent by washing with Ca- and Mg-free PBS and dislodging with 0.05% trypsin.
Cell treatment experiments used 48-well polystyrene plates (Costar, Fisher Scientific) containing 0.3 ml of media per well, with cells seeded at an initial density of 20,000/cm2. After one day we applied new media containing the treatments. On the third day we harvested the media for cytokine assays and measured viable cell count. Experiments used triplicate wells for each treatment level and allocated six or nine wells per culture plate as controls. Positive controls were included to monitor changes in the BEAS-2B cell response. All experiments were replicated with at least two independent cell passages.
The role of the TRPV1 receptor was tested by pretreating the cells with the receptor antagonist capsazepine (CPZ) at 1, 2, and 4 µM for 30 min followed by cotreatment with particles and the same concentration of CPZ. The role of reactive oxygen species was tested by adding 2 mM dimethyl-thiourea to the particle-containing culture media.
Cytotoxicity assay. Cell viability was assessed using the cell counting kit (CCK-8, Dojindo Laboratories, Gaithersberg MD). We incubated the cells for 2 h in culture media containing 4% of the reagent, then transferred media containing the reacted dye to a 96-well plate. Viable cell count relative to control was calculated by absorbance at 450 nm minus the absorbance at 630 nm, corrected for the slight absorbance (approximately 0.050.07) of cell-free reagent in LHC-9. Preliminary experiments showed that the particles did not interfere with this assay.
ELISA assays. The concentrations of IL-6, IL-8, and TNF- in the cell culture media were determined using sandwich ELISA assays. For IL-6, we used both a commercial kit (R&D Systems, Minneapolis MN), and plates prepared with anti-human IL-6, biotin-conjugated anti-human IL-6, and avidin-horseradish peroxidase from eBioscience (San Diego CA). All IL-6 values were quantified using an R&D Systems recombinant human IL-6 standard. The IL-8 ELISA used the R&D Systems DuoSet IL-8 development kit antibodies and standard. TNF-
was determined using a kit from R&D Systems.
Statistics. Student's t-test and the Dunnett's test option in the JMP statistical package (SAS Institute) were used to determine the statistical significance (p < 0.05) of differences between treatment conditions.
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RESULTS |
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The IL-6 response to the LPS-containing mixtures was primarily due to the LPS, and the response to plausible amounts of LPS was much less than the response to the environmental dust particles. Figure 2 shows that the TiLPS produced less than a two-fold increase in IL-6 over control compared to over seven-fold increase observed with the soil dust positive control. There was no significant difference between treatment with TiO2 and LPS added separately and treatment with LPS alone. The treatments containing commercial LPS were less cytotoxic than DD, and the viable cell count was greater than 70% of control at all treatment levels. The TiLPS and the 2000 EU/ml soluble LPS treatment both induced IL-8 release that was significantly above control but much less than the response to the DD particles.
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In order to check for material loss from physical treatment of the particles, the absorbance versus mass concentration of treated particles was obtained by a standard curve using a dilution series of each of the untreated suspensions (Fig. 5a). The concentrations of the washed particles appeared to be within ±25% of the unmodified particle treatment with the exception of two leaching treatments for DD and R4. This suggested that the effect of desferrioxamine and water leaching on the DD and R4 soil dusts was due to material loss.
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DISCUSSION |
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The endpoints commonly measured with in vitro studies of ambient and mineral particles include the cytokines IL-6 (Becher et al., 2001; Carter et al., 1997
; Hetland et al., 2000
), IL-8 (Carter et al., 1997
; Hetland et al., 2000
; Smith et al., 2000
) and TNF-
(Carter et al., 1997
; Imrich et al., 2000
), second messenger signals such as Ca influx (Veronesi et al., 2002a
), and oxidative damage (Schins, 2002
). The focus of this study was on cell death and IL-6 release. Release of IL-8 in response to the soil dust and LPS treatments was qualitatively similar to the IL-6 responses, suggesting a common or closely related mechanism. The lack of detectable TNF-
is somewhat surprising since this cytokine has been reported to be involved in the regulation of both IL-6 and IL-8 (Nelson and Martin, 2000
). The suite of PM2.5-enriched soil dusts in the as-collected and surface-modified condition provide model particles that can be used in future mechanistic in vitro studies to elucidate the pathways by which particles interact with lung cells.
We previously reported necrotic cell death in normal BEAS-2B cells treated with capsaicin at concentrations associated with the maximal IL-6 induction (Reilly et al., 2003). The Vybrant Apoptosis Detection Kit #3 (Molecular Probes), which uses propidium iodide (PI) and annexin-FITC, was used in an attempt to assess the cell death mechanism in response to particle treatments. Control cells, cell-free particles, and particle-treated cells were examined using a fluorescence activated cell sorter (FACScan, Becton-Dickinson). However, the soil dusts contain particles of fluorescent minerals that are detected in the PI and FITC channels. Also, particles associated with cells increase the side scatter signal. Attempts to eliminate the particle signals by gating appeared to also eliminate dead cells, presumably because the dead cells were the cells associated with surface-bound or internalized particles. These particle effects seriously confounded the cell death assay data, and no conclusions could be drawn.
The physical treatment experiments supported our assumption that handling procedures such as collection of particles from filters by washing, and the subsequent drying and sterilizing of the sample, do not confound experiments with soil and mineral dusts.
New insights have been gained from the physical treatment experiments, but the specific components or characteristics of the soil-derived particles that cause the IL-6 and IL-8 release remain elusive. Many papers have been published reporting in vitro cytokine induction in response to particles. The novel contributions of this paper are that a significant range of responses, from benign to highly inflammatory and cytotoxic, is caused by soil particles that are representative of fugitive dust from unpaved roads, and that physical treatments can be used to modify soil-derived PM2.5 particles as a means of testing toxicology hypotheses related to potential active components or particle characteristics.
Hypotheses for particle-induced toxicity and cytokine release that are in the current literature include metal-dependent ROS generation, vanilloid receptor activation, and LPS-mediated events. We have investigated whether these mechanisms are involved in the responses to soil dust. The studies using soluble LPS, particle-associated LPS, heat-treated particles, and solvent-washed particles all implied that LPS was not the dominant component inducing the response. Treatments with the metal chelator desferrioxamine, the ROS scavenger dimethyl-thiourea, and the TRPV1 antagonist CPZ gave mixed results. The evidence from this study implies that the soil-dust-induced IL-6 release and cytotoxicity result from interactions between solid-phase particle components and the cells. Potential alternative hypotheses include activation of receptors other than TRPV1 by chemicals or sites on the particle surface and processes involving particle uptake by the cells.
It was unfortunate that the TRPV1 nonspecific antagonist, CPZ, was highly cytotoxic under our experimental conditions, because amelioration of IL-6 release has been reported in particle studies by others (Veronesi, 1999). Current studies with more selective TRPV1 antagonists, which are not cytotoxic at low concentrations, may provide more compelling experimental evidence for vanilloid receptor participation in the inflammatory process in response to mineral dust particles.
The physical treatment results also put constraints on the nature of the active components of the particle mixture. For example, most semivolatile and biogenic organic compounds would be removed or inactivated by heating or by washing with solvents. Speculation concerning other components of the particles that could be responsible for release of IL-6 and IL-8 by these immortalized lung cells could focus on organic components (redox-active quinones for example) or redox-active metals. However, these components would have to be very tightly associated with the particles, not affected by dimethyl-thiourea, and stable enough to survive heating to 150°C. We are not aware of any immediately compelling examples of such components.
Endotoxin is known to cause release of IL-6 and IL-8 and is associated with lung inflammation, but this study demonstrated that the response to these soil dusts was not due to particle-associated endotoxin. The evidence includes the limited response to surrogate particles containing LPS (Fig. 2), the lack of correlation between measured endotoxin and IL-6 release (Fig. 3), and the lack of an effect from thermal treatment (Fig. 4) at temperatures higher than those that are used to remove LPS from manufactured medical products.
The concentrations of particles and LPS used in this study are comparable to those used by other investigators. Table 3 indicates typical particle and LPS treatment concentrations used for IL-6 and IL-8 response experiments with BEAS-2B cells grown submerged in media. Particle concentrations of 10 to 80 µg/cm2 convert to 25200 µg/ml for the cell culture wells used in this study. The LPS concentration of 2000 EU/ml converts to 7 x 105 pg/ml. These concentrations are high compared to plausible lung deposition doses. A calculation (Lighty et al., 2000) based on typical values for particle size distribution, ventilation rate, and deposition fraction shows that a 10 µg/m3 increase in ambient PM2.5 concentration results in an increment of 0.020.05 mg of particles deposited in the lung per day (approximately 70 m2). However, particles in vitro are diluted by 35 mm of cell culture media compared to a typical lung surfactant liquid layer of 0.050.2 µm (Miller et al., 1985
). Immortalized cells in submerged culture also have reduced response to particles compared to other, presumably more realistic, systems such as cells grown on an airliquid interface (Seagrave et al., 2004
) or macrophageepithelial cell coculture (Tao and Kobzik, 2002
). Cell culture experiments using high treatment concentrations are most appropriate for mechanistic toxicology experiments where the goal is to induce and modify specific signaling responses in a simplified biological model. Cell culture is also proposed as an expedient method of screening large numbers of environmental samples to select materials for animal toxicology studies.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, 30 South 2000 East, 112 Skaggs Hall, Salt Lake City, UT 841125820. Fax: (801) 585-3945. E-mail: John.Veranth{at}utah.edu.
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