Lung Epithelial Cell (A549) Interaction with Unopsonized Environmental Particulates: Quantitation of Particle-Specific Binding and IL-8 Production

Abstract

The A549 cell line was used to model in vitro the interaction of alveolar epithelium with environmental particulates. Confocal and electron microscopy demonstrated A549 binding and internalization of titanium dioxide (TiO₂), iron oxide (Fe₂O₃), concentrated ambient air particulates (CAPs), and the fibrogenic particle α-quartz. Flow cytometry allowed quantitation of particle binding by measuring increased right angle light scatter (RAS) (TiO₂ [40 μg/mL], Fe₂O₃ [100 μg/mL], a-quartz [200 μg/mL], or CAPs [40 μg/mL] fold increase RAS: 8.1 ± 0.9, 4.3 ± 0.4, 2 ± 0.1, 1.6 ± 0.1, respectively). With this quantitative assay, binding of particles was found to be calcium-dependent for TiO₂ and Fe₂O₃ (% inhibition, 61.0 ± 1.9, 40.0 ± 5.6, respectively), while α-quartz binding was calcium-independent. A panel of polyanionic ligands known to inhibit scavenger-type receptors was used to identify binding mechanisms for environmental particulates. Both heparin and polyinosinic acid (polyl), but not the control polyanion chondroitin sulfate, caused marked inhibition of particulate binding by A549 cells (e. g., TiO₂ [40 μg/mL] binding; polyl, heparin, and chondroitin sulfate: 73.8 ± 3.5, 75.5 ± 6.0, 7.5 ± 6.7% inhibition, respectively; mean ± SE, n ± 4), indicating that scavenger receptor(s), albeit those distinct from the heparin-insensitive acetylated-LDL receptor, mediate particulate binding. The particulates ability to stimulate interleukin (IL-8) production in A549 cells was also tested, a-quartz, but not TiO₂ or CAPs, caused a dose-dependent production of IL-8 (range 1–6 ng/mL), demonstrating a particle-specific spectrum of epithelial cell cytokine (IL-8) response. The results suggest that lung epithelial cell interaction with environmental particles is mediated by distinct receptors and can lead to particle-dependent cytokine responses.

Key Words:

• A549 cells
• environmental particulates
• flow cytometry
• interleukin-8
• scavenger receptors