Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells

Abstract

Background: Exposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression.

Results: Microarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 106μm2/cm2) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 106μm2/cm2) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (FOS, ATF3, IL6 and IL8) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells.

Conclusions: Cristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses in vitro are suggested as predictive in determining the pathogenicity of potentially harmful particulates.

Figure 1

Assessment of BEAS 2B cell viability after exposure to silica particles. Cell viability assessed by the Trypan blue exclusion assay of cells exposed to cristobalite (A) and amorphous silica particles (B) for 24 h. Results are expressed as the mean percent viable cells ± SEM compared to unexposed controls and are representative of 3 independent experiments (N = 3 for each group in each experiment). ^a denotes the surface area concentrations expressed as (x10⁶μm²/cm²) and ^b denotes the respective mass concentrations of particles used (μg/cm²). Concentrations presented are represented by surface area for all other figures and tables.

Figure 2

Interaction and uptake of silica particles by BEAS 2B cells. Scanning Electron Micrographs of BEAS 2B cells. Unexposed controls (A, B), exposed to cristobalite (75 × 10⁶μm²/cm²) for 2 h (C, D), 24 h (E, F) and amorphous silica (75 × 10⁶μm²/cm²) for 2 h (G, H) and 24 h (I, J). Panels on the left are at low magnification (500×) scale bar = 50 μm and on the right at high magnification (2500×) scale bar = 10 μm. White arrows indicate silica particles.

Figure 3

Microarray analysis of BEAS 2B cells in response to silica particle exposure (24 h). (A) Total number of significant gene changes (p < 0.05) with a cut-off of ≥ 2.0-fold change in mRNA compared to unexposed controls in BEAS 2B exposed for 24 h (total number of gene changes). Gene ontology analysis of BEAS 2B exposed to (B) Cristobalite at 75 × 10⁶μm²/cm², (C) 150 × 10⁶μm²/cm² and (D) amorphous silica at 150 × 10⁶μm²/cm². Positive values represent number of genes up-regulated (Black bars) and negative values represent number of genes down-regulated (white bars) (N = 3).

Figure 4

BEAS 2B steady state mRNA levels over time with exposure to silica particles of stress-related and immune response genes. Time-course analysis of mRNA levels by QRT-PCR of BEAS 2B cells exposed to 75 × 10⁶μm²/cm² cristobalite (black bars) and amorphous silica (gray bars) and unexposed controls (white bars) for 2, 4, 8 and 24 h. Fold change in mRNA of (A) FOS, (B) ATF3, (C) IL6 and (D) IL8. *P < 0.05 compared to unexposed controls. Bars denote mean ± SEM. Results from two independent experiments were pooled (N = 6/group/time-point). Values of cells exposed to amorphous silica at 2 and 4 hours (N = 5/group/time-point).

Figure 5

Bio-Plex analysis of secreted cytokines and chemokines in medium of BEAS 2B cells exposed to silica particles 24 h. Cell-free conditioned medium of BEAS 2B exposed to silica particles for 24 h was assayed for 27 cytokines and chemokines. Presented in this panel are the 11 which yielded significant differences from untreated controls in levels of secreted proteins: Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-12 (p70) (IL-12 (p70)), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Monocyte chemotactic protein-1 (MCP-1 or MCAF), Regulated upon activation, normal T-cell expressed and secreted (RANTES), Granulocyte-colony stimulating factor (G-CSF), Basic fibroblast growth factor (bFGF), Platelet derived growth factor-BB (PDGF-BB) and Vascular endothelial growth factor (VEGF). White bars (unexposed controls), black bars (cristobalite silica) and gray bars (amorphous silica), N = 3. * (P < 0.05) vs. unexposed controls, †P < 0.05 vs. amorphous silica.

Figure 6

Assessment of cell viability and microarray analysis of NHBE cells in response to silica particle exposure. Effects of silica particles on NHBE cells after 24 h exposures. (A) Cell viability assessed by the Trypan blue exclusion assay (N = 3 in 2 independent experiments) *P < 0.05 compared to controls. NS = Not Significant for comparison of all exposure groups. (B) Total number of gene changes ≥ 2.0-fold vs. controls induced by silica particle exposure for 24 h. (C-E) Gene ontology analysis of NHBE exposed to (B) Cristobalite at 15 × 10⁶μm²/cm², (C) 75 × 10⁶μm²/cm² and (D) amorphous silica at 75 × 10⁶μm²/cm². Positive values represent number of genes up-regulated (Black bars) and negative values represent number of gene down-regulated (white bars) (N = 3).