Cytokine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides compared to soil dusts

Abstract

Background: The induction of cytokines by airway cells in vitro has been widely used to assess the effects of ambient and occupational particles. This study measured cytotoxicity and the release of the proinflammatory cytokines IL-6 and IL-8 by human bronchial epithelial cells treated with manufactured nano- and micron-sized particles of Al2O3, CeO2, Fe2O3, NiO, SiO2, and TiO2, with soil-derived particles from fugitive dust sources, and with the positive controls LPS, TNF-alpha, and VOSO4.

Results: The nano-sized particles were not consistently more potent than an equal mass of micron-sized particles of the same nominal composition for the induction of IL-6 and IL-8 secretion in the in vitro models used in this study. The manufactured pure oxides were much less potent than natural PM2.5 particles derived from soil dust, and the cells were highly responsive to the positive controls. The nano-sized particles in the media caused artifacts in the measurement of IL-6 by ELISA due to adsorption of the cytokine on the high-surface-area particles. The potency for inducing IL-6 secretion by BEAS-2B cells did not correlate with the generation of reactive oxygen species in cell-free media.

Conclusion: Direct comparisons of manufactured metal oxide nanoparticles and previously studied types of particles and surrogate proinflammatory agonists showed that the metal oxide particles have low potency to induce IL-6 secretion in BEAS-2B cells. Particle artifacts from non-biological effects need to be considered in experiments of this type, and the limitations inherent in cell culture studies must be considered when interpreting in vitro results. This study suggests that manufactured metal oxide nanoparticles are not highly toxic to lung cells compared to environmental particles.

Figure 1

Treatments at the maximum particle concentrations used in this study were not highly toxic to the cells as indicated by a mitochondrial reductase assay. Data are mean ± s.d., normalized by the control, N = 9, and are typical of multiple cell passages. * designates statistically different than control. Sample ID codes are in Table 1.

Figure 2

IL-6 response of in vitro cell cultures treated with equal mass concentrations (53 μg/cm²) of nano- and micron-sized particles of the same nominal substance. A and B represent single experiments, N = 9. C and D are merged results from two independent experiments, N = 9 and N = 4 and have a broken scale to show the positive controls. A, C, : BEAS-2B cells in KGM media with 0.1% BSA; B, D. BEAS-2B cells in LHC-9 media with 0.1% BSA, IL-6 concentrations in pg/mL, mean ± s.d. *denotes statistically greater than control. Treatment codes are in Table 1.

Figure 3

Comparison of manufactured Fe₂O₃ and SiO₂ nanoparticles to soil-derived dusts (DD, JE, MC) at 53 μg/cm² and to positive controls at concentrations listed in Table 1. IL-6 concentration in pg/mL, mean ± standard error of the mean (95% confidence interval) based on 5 independent experiments. Note the discontinuity in the Y-axis scale. A. KGM media with BSA 0.1% BSA. B. LHC-9 with 0.1% BSA.

Figure 4

IL-8 response of in vitro cell cultures treated with equal mass concentrations (53 μg/cm²) of nano- and micron-sized particles of the same nominal substance. A. BEAS-2B cells in LHC-9 media with 0.1% BSA, B. NHBE cells in BEGM media with 0.1% BSA. IL-8 in pg/mL, mean ± s.d., N = 9, * denotes statistically greater than control. Treatment codes are in Table 1.

Figure 5

IL-6 response for a typical experiment with NHBE cells in BEGM media with 0.1% BSA treated at the same particle concentration as previous figures. IL-6 concentrations in pg/mL, mean ± s.d., N = 9, *denotes statistically greater than control. Treatment codes are in Table 1.

Figure 6

High surface area particles can interfere with measurement of cytokines in cell-free media containing a known aliquot of recombinant IL-6. A. Addition of 200 μg/mL of nano-SiO₂, nano-TiO₂, or kaolin to KGM media with the indicated concentration of BSA added. B. Addition of 200 μg/mL of nano-SiO₂, nano-TiO₂, or kaolin to KGM media with the indicated concentration of bovine serum added. C. The effect of adding increasing amounts of the indicated particles to KGM media without supplemental protein. Measured IL-6 divided by standard, * indicates significantly less than standard prepared in assay diluent, # indicates significantly greater than the IL-6 in the same particle suspension in media without exogenous protein addition, (two-tailed, p < 0.05). N = 3.

Figure 7

Response of BEAS-2B cells to SiO₂ particles when treated in LHC-9 media with varying concentration of BSA. Data are merged from two independent experiments, mean ± s.d., N = 12, * indicates statistically significant difference between particle sizes.

Figure 8

IL-6 response of BEAS-2B cells treated with the indicated concentrations of commercial lipopolysaccharide from three bacterial strains. A. in KGM media. B. in LHC-9 media. Mean ± standard error of the mean, N = 6–12 determinations.

Figure 9

Reactive oxygen species produced by nano- and micron-sized metal oxide particles as measured by relative fluorescence in the cell-free DCFH assay. A. comparison of nano- and micron-sized particles of the indicated oxides. * indicates statistically significant difference between particle sizes for the pair. B. comparison of two nanoparticles to soil-derived dusts. Data are mean ± s.d., N = 3.