Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose

Abstract

Background: Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 microm and 2.5 microm) and ultrafine ( <or= 0.1 microm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number.

Methods: Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (slashed circle fine - 1.062 microm, ultrafine - 0.041 microm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy.

Results: Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations.

Conclusion: This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM.

Figure 1

Particle size characteristics. Particles were visualized by transmission electron microscopy verifying that no large agglomerates were present (A: 1 μm particles; B: 0.05 μm particles). Ultrafine particle size in RPMI medium was further analyzed by dynamic light scattering. The size distributions from three individual measurements show that the majority of ultrafine particles in RPMI medium are present as single particles or small agglomerates of two to three particles.

Figure 2

Visualization of fine and ultrafine particles by confocal laser scanning microscopy. Figure A illustrates the appearance of 1 μm fluorescent polystyrene particles (green) inside A549 cells. Figure B illustrates the appearance of 0.05 μm fluorescent particles (green) inside A549 cells after application of a deconvolution algorithm. For visualization of the cells, the actin cytoskeleton was stained with phalloidine-rhodamine (red). The panels on the right and at the bottom of each figure show the corresponding y/z and x/z projection, respectively.

Figure 3

Surface area of the apical plasma membrane of A549 cells at different particle concentrations. * = p < 0.01 vs. negative control (NC). The mass of 3 × 10⁷ 1 μm particles and the surface area of 6 × 10⁸ 1 μm particles are approximately equal to the mass and surface area of 4.5 × 10¹¹ 0.05 μm particles, respectively. Increases in the surface area of the APM were observed at the same particle surface area concentration exposed to the cells. n = 5.

Figure 4

Electron micrographs of the apical plasma membrane of A549 cells. A: Control experiments without particle exposure. B: Exposure to 6 × 10⁸ 1 μm particles. Note the changes in APM in comparison with A. Numerous particles (P) taken up by the epithelial cells are found inside the cells. N = Nucleus. Scale bar = 5 μm.

Figure 5

Cellular LDH release and IL-8 protein after particle exposure. A: LDH release after 24 h incubation with different concentrations of 1 μm and 0.05 μm particles. An exposure concentration of 6 × 10⁹ particles per cell culture well significantly increases the LDH release vs. negative control (NC) (* = p < 0.01). B: IL-8 protein after 24 h particle exposure. The concentration of 6 × 10⁹ 1 μm particles induces a significant IL-8 secretion compared to the negative control (NC) (* = p < 0.01). A positive control (PC) was generated with TNFα stimulation. At no other of the tested exposure concentrations was a significant LDH release or IL-8 secretion observed.

Figure 6

Electron micrographs of the interaction between particles and the apical plasma membrane of A549 cells. A: Exposure to 1 μm particles (P). Three particles in the process of cellular uptake, probably via macropinocytosis or phagocytosis. Scale bar = 1 μm. B: Exposure to 0.05 μm particles (P). One particle in the process of cellular uptake, probably via clathrin- or caveolae-mediated endocytosis. Scale bar = 500 nm.

Figure 7

Intracellular particle number and surface area upon exposure to the same particle number. Cells were exposed to the same number concentration of particles (3 × 10⁷ particles per well) and the number of intracellular particles was counted by LSM. From the number of intracellular particles, the total particle surface area taken up by the cells was calculated. There was a greater number (A) and surface (B) of 1 μm particles inside the cells than of 0.05 μm particles. Due to the small sample size (n = 3) and the use of the Mann Whitney u-test, these obvious differences failed to reach statistical significance (p = 0.1). Note the logarithmic scale on the y-axis in B.

Figure 8

Intracellular particle number and surface area upon exposure to the same particle surface area. Cells were exposed to the same total surface area concentration of particles (3.5 × 10⁷ μm² per well) and the number of intracellular particles was counted by LSM. From the number of intracellular particles, the total particle surface area taken up by the cells was calculated. At the same surface area concentration, the number of 0.05 μm particles exceeded the number of 1 μm particles taken up by the cells (A), however, the fine particles accounted for a greater intracellular particle surface area (B). Due to the small sample size (n = 3) and the use of the Mann Whitney u-test, these obvious differences failed to reach statistical significance (p = 0.1). Note the logarithmic scale on the y-axis in B.

Figure 9

Dose dependent particle entering. At rising exposure concentrations, the number of intracellular particles increased for both particle sizes. The increase in the number of 1 μm was steeper than that of 0.05 μm particles. Note the logarithmic scale of the x and y axis. Black circle = 1 μm particles. Black triangle = 0.05 μm particles. Black horizontal line = mean values of three experiments at each concentration. The trend lines are based on the means.