The protocol for the acquisition of NHBE cells was approved by the University of North Carolina School of Medicine Committee on Protection of the Rights of Human Subjects and by the U.S. EPA. Subjects were informed of the procedures and potential risks and each signed an informed consent. NHBE cells were obtained from healthy individuals through bronchoscopy with bronchial brushings. Cells were expanded to passage-3 in bronchial epithelial growth medium (BEGM; Clonetics, San Diego, CA), plated on collagen-coated filters with a 0.4-micron pore size (Trans-CLR, Costar, Cambridge, MA) at a density of 1 × 105 cells/filter and inserted into 12 well culture plates
[6, 23]. Cells were maintained in a 1:1 mixture of BEBM and Dulbecco’s Modified Eagles Medium (DMEM) with high glucose, growth supplements, bovine pituitary extracts, bovine serum albumin, and nystatin. Fresh medium (0.5 mL in the apical chamber and 1.0 mL in the basal chamber) was provided every 48 hours. In those cells grown at ALI, the apical medium was removed (day 0). Submerged cells were continued with 0.5 mL in the apical chamber. With confluence of the cells, retinoic acid was added to the media to promote differentiation in cells grown both at ALI; submerged cells also received this supplement. The cells were maintained for 21 days allowing those grown at ALI to differentiate into ciliated, mucus-producing cells (occurring at approximately day 10 and later). Fresh medium was provided every 48 hours (1.0 mL in the basal chamber for those grown at ALI and 0.5 mL and 1.0 mL in the apical and basal chambers for those grown submerged).
BEAS-2B cells were also used in in vitro studies. This is an immortalized line of human bronchial epithelial cells derived by transfection of primary cells with SV40 early-region genes. These cells have not been reported to undergo differentiation and cells similarly transformed with SV40 oncogene have not demonstrated such a capacity
. Comparable to the NHBE cells, BEAS-2B cells were plated on the same collagen-coated filters and inserted into 12 well culture plates. The cells were maintained in keratinocyte growth medium (KGM; Clonetics) which is essentially MCDB 153 medium with supplemented human epidermal growth factor, insulin, hydrocortisone, calcium, bovine pituitary extract, ethanolamine and phosphoethanolamine. Fresh medium (0.5 mL in the apical chamber and 1.0 mL in the basal chamber) was provided every 48 hours. Following confluence of the cells, ALI was created on day 0 by removing the apical medium. The cells were maintained for 21 days after this. Fresh medium (1.0 mL in the basal chamber) was provided every 48 hours.
NHBE cells were exposed to coarse, fine, and ultrafine fractions of ambient air pollution particle. Particles were collected outside the U.S. Environmental Protection Agency (EPA) Human Studies Facility in Chapel Hill, North Carolina using a ChemVol model 2400 high volume cascade impactor (Rupprecht & Patashnick Co., Albany, NY)
. Coarse PM (PM2.5–10 μm) and fine PM (PM0.1–2.5 μm) were collected onto polyurethane foam (McMaster-Carr, Atlanta, GA), which was previously cleaned with methanol and water and dried under sterile conditions. Ultrafine particles (PM < 0.1 μm) were collected onto G5300 filters (Monandock Non-Wovens LLC, Mt. Pocono, PA). The foam or filter was pre-wetted with a small amount of 70% ethanol, and endotoxin-free water was added to a total volume of 40 mL. The particles were removed from the foam or filter by sonication for 1 hour in a water bath (FS220; Fisher Scientific, Pittsburgh, PA). The foam was removed and particles were then lyophilized.
Cultured BEAS-2B cells were exposed to NIST 1648 (National Institute of Standards and Technology; Gaithersburg, MD). This was an ambient air pollution particle collected in St. Louis, Missouri and has been previously characterized
Immediately prior to exposure to particles, the apical chamber of the cultured cells was washed with 500 μL PBS and the buffer immediately removed. Particle in 25 μL PBS was placed on the cells in the apical chamber of the transwells and agitated on a rocking shaker (Reliable Scientific, Nesbit, MS) at an intermediate speed for a minimum of 2 minutes. Dispersion was documented by visual inspection of the cells using an inverted microscope at a magnification of 100×. Particle exposure continued until collection of the specific endpoint. For RT-PCR, this was 4 hours while for cytokines this was 24 hours. Cytotoxicity, assessed using release of LDH after exposure to particles, was demonstrated to be insignificant. The response of submerged cells of a specific age exposed to particle was compared to submerged cells of that same age without exposure and the response of cells grown at ALI to a specific age exposed to particle was compared to these cells grown at ALI of that same age without exposure.
Relative gene expression in NHBE and BEAS-2B cells was quantified using real-time quantitative PCR. RNA was reverse transcribed to generate cDNA. Primer/probe sets were obtained as Taqman pre-developed assay reagents (concentrated and pre-optimized mix of primers and FAM-labeled Taqman probe) from Applied Biosystems (University Park, IL). Quantitative fluorogenic amplification of cDNA was performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystems) primer/probe sets of interest, and TaqMan Universal PCR Master Mix (Applied Biosystems). The relative abundance of mRNA levels was determined from standard curves generated from a serially diluted standard pool of cDNA prepared from cultured respiratory epithelial cells. The relative abundance of GAPDH mRNA was used to normalize levels of the mRNAs of interest. Specific proteins for which RNA was measured included two involved in differentiation (alpha tubulin and muc5B), two involved in inflammation (interleukin-8 and interleukin-6; IL-8 and IL-6 respectively), and two involved in oxidative stress (heme oxygenase 1 and cyclooxygenase 2; HOX1 and COX2 respectively).
Cell release of interleukin (IL)-8 and IL-6
Cells were exposed to particle for 24 hr. IL-8 and IL-6 concentrations in the cell media were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN).
Cell air and hypoxia exposures
Two modular chambers (Billups-Rothenberg, Modular Incubator Chamber, Del Mar, CA) were placed in an incubator, set at 37°C, and each was ventilated with either 95% air-5% CO2 (normoxia) or 95% N2-5% CO2 (hypoxia). Humidity was supported with the inclusion of a petri dish of deionized water included in the chambers. The flow to each chamber (approximately 2 L/min) was adjusted so that there was constant exchange of the gas mixtures. The percentage oxygen in the two chambers was monitored using Handi + analyzers (Maxtec, Salt Lake City, UT).
NHBE cells were employed during investigation of the effect of hypoxia on the response to particle. Cells were grown in 12 well culture plates, maintained in KGM (0.5 mL in the apical chamber and 1.0 mL in the basal chamber), and allowed to grow to confluence. The apical medium was removed and the cells immediately placed in either normoxia or hypoxia for 24 hr. At 24 hrs, either 25 μL PBS or NIST 1648 particle in 25 μL PBS was placed on the apical aspect of the cells.
The minimum number of replicates for all measurements was nine; with NHBE cells, this included three different volunteers’ cells. Data are expressed as mean value ± standard error. Differences between multiple groups were compared using one-way analysis of variance. The post-hoc test employed was Scheffe’s test. Differences between two groups were compared using T tests of independent means. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.