Inflammatory cytokines and cell death in BEAS-2B lung cells treated with soil dust, lipopolysaccharide, and surface-modified particles

Abstract

Cultured human lung epithelial cells (BEAS-2B) were treated in vitro with PM(2.5)-enriched particles of soil-derived mineral dust from nine sites in the western United States. The particle samples simulate windblown dust and vehicle-generated emissions from unpaved roads. Five of the sites yielded relatively benign dust. Particles from three sites caused IL-6 release when cells were treated for 24 h at doses from 20 to 80 microg/cm(2), and particles from one site were highly cytotoxic. The particle components or characteristics that caused the IL-6 release were stable at temperatures below 150 degrees C, but were inactivated by treatment at 300-550 degrees C. The active factors were also associated predominantly with the insoluble fraction, and were partially attenuated by leaching with aqueous and organic solvents. The IL-6 release caused by the particles was much greater than the cytokine response to either lipopolysaccharide (LPS) or to surrogate particles of titanium dioxide mixed with LPS, suggesting that endotoxin was not a major factor in the inflammatory response. The release of IL-8 in response to particle treatment was qualitatively similar to the IL-6 response, but release of TNF-alpha was not detected at the 24-h time point. The combined results support the hypothesis that some ambient dusts from geological sources can cause cell death and cytokine release in a lung cell line that is widely used as an in vitro model to study mechanisms of environmental respiratory injury.

FIG. 1

Geological dusts are cytotoxic to BEAS-2B cells and induce IL-6 and IL-8 release at particle concentrations from 10 to 160 μg/cm². x¯±SD, n = 6. # designates not statistically different from control; > and <, respectively, designate statistically greater than and statistically less than the response for the same concentration of DD, p < 0.05.

FIG. 2

BEAS-2B cells treated with surrogates containing P. aeruginosa LPS showed no cytotoxicity and a minimal increase in IL-6 and IL-8 release compared to the particle positive control. TiLPS, micron-size TiO₂ spiked with LPS and dried, doses in μg of dry mixture/cm² of culture well; Ti + LPS, 80 μg/cm² of TiO₂ and 2000 EU/ml of LPS added separately to the culture media; LPS, 2000 EU/ml; DD80, positive control, 80 μg/cm² of soil dust. x¯±SD, n = 6. * designates statistically different from control, p < 0.05.

FIG. 3

IL-6 fold increase over control (left axis) and endotoxin concentration in the soil dusts and surrogate materials (right axis) have an inverse correlation, demonstrating that the response is not due to endotoxin. IL-6 x¯±SD, n = 6. Endotoxin n = 2.

FIG. 4

Cytotoxicity and IL-6 response of cells exposed to untreated and physically modified particles of soil dust. All exposures were 200 μg of the original particles/ml of media. Identification: WT, wild-type unmodified particles, 150, 300, 550, oxidizing thermal treatment at indicated temperature for one hour; LHC-9 S, supernatant from initial leaching in cell culture media; LHC-9 P, DF, DI, ChMe, particles recovered after three cycles of leaching in LHC-9, 1 mM desferrioxamine, ion-exchange treated water, and chloroform-methanol, respectively. x¯±SD, n = 6. * designates statistically different from untreated particles, # designates not statistically different from media-only control, p < 0.05.

FIG. 5

Light absorbance indicates that physically modified exposure concentrations were generally within ± 25% of the standard containing 200 μg/ml, but some material loss occurred during leaching. Top (5a): standard curves obtained with a dilution series for each particle type. Bottom (5b): Absorbance of the treated particle suspensions compared to unmodified particles; n = 2.