Figure 1.
Both crystalline and amorphous silica induce high rates of cell death in macrophages.
MH-S cells treated with no particles, 3 µm latex beads, crystalline silica [50 µg/cm2] or 3 µm spherical, amorphous silica [50 µg/cm2]. (A) Cell death measured by propidium iodide (PI) [0.1 µg/ml] nuclear staining at 4, 8, 12 and 24 hours. Statistically significant differences compared using ANOVA, (* = P<0.001, ** = P<0.01, as compared to untreated control, # = P<0.001, ## = P<0.01, ### = P<0.05, as compared to latex beads). (B) Crystalline or amorphous silica [50 µg/cm2] was placed in a Transwell (0.45 µm diameter pore size) chamber and placed above cells attached to the bottom of a 24 well culture dish. Cell death was measured with PI (n = 150–600 cells).
Figure 2.
Actin localization at the site of amorphous silica particle phagocytosis.
(A) MH-S mCherry-actin transfected cells were exposed to 3 µm spherical, amorphous silica and images were captured every 30 seconds. The accumulation of fluorescence, indicating the polymerization of F-actin filaments, can be seen at the point of cell-particle contact. The actin ring extends around the particle and the filaments depolymerizes as the particle is internalized. The arrow in the 370 second panel shows the location of the particle which can be seen as an unlabeled black hole in the G-actin background fluorescence of the cytoplasm. (B–D) MH-S mCherry-actin transfected cells were treated with ovalbumin-coated 3 µm spherical, amorphous silica then fixed and stained with rabbit anti-ovalbumin and then goat anti-rabbit FITC-conjugated antibody to determine if particles were inside or outside the cell membrane. (B) DIC image (C) mCherry actin image (D) FITC-antibody labeled external particles. Arrows indicate the location of internal particles. Arrowheads indicate the location of external particles. Scale bar = 5 µm. (E) The rate of uptake as measured by examining the appearance of actin rings and subsequent appearance of a particle sized black hole in the cytoplasm.
Figure 3.
Temperature affects opsonized and non-opsonized particle phagocytosis differently.
MH-S macrophages were exposed to ovalbumin coated (Ova) or ovalbumin plus anti-ovalbumin antibody coated (Ab) 3 µm spherical, amorphous silica. The cells were either maintained at 37°C throughout the assay or incubated at room temperature (RT, about 22°C,) during particle exposure. Ova-RT: closed squares; Ova−37°C: open squares; Ab-RT: closed triangles; Ab−37°C: open triangles. Cells were fixed at the indicated times and particle internalization was measured by staining with anti-ovalbumin antibody.
Figure 4.
Activation of Caspase-3 in amorphous silica treated cells.
Western Blot of MH-S cells treated with latex beads, crystalline silica [50 µg/cm2], or 3 µm amorphous silica [50 µg/cm2] for 4 hours. Blots were probed with anti-Caspase-3 antibodies and visualized colorimetrically. Cleavage of the 35 kD Caspase-3 to the 19 kD active form could be detected in both amorphous and crystalline silica treated cells but not cells treated with latex beads. As a positive control, NIH-3T3 cells were treated with staurosporine for 1 hour.
Table 1.
Quantification of Caspase-3 cleavage.
Figure 5.
Amorphous silica induces endo-lysosomal leakage in MH-S cells.
MH-S cells are pre-loaded with 20 kD FITC-dextran [1 mg/ml] for × hours. Fluorescence images of cells untreated cells (A) or treated with 3 µm spherical, amorphous silica particles [25 µg/cm2] for 1.5 hours (B). The punctate endosomal distribution of FITC-dextran is converted to a diffuse cytoplasmic localization after treatment of cells with amorphous silica.
Figure 6.
FITC-dextran particle labeling can be used to quantify the rate of particle internalization.
MH-S cells are pre-loaded with 20 kDa FITC-dextran [1 mg/ml]. (A) Paired DIC and fluorescent images showing FITC-dextran particle labeling during uptake. Arrows indicate internalized particles, scale bar = 5 µm. (B) The rate of 3 µm uncoated amorphous silica [16.5 µg/cm2] internalization using this assay (n = 26 cells).
Figure 7.
The number of internalized particles affects the timing of cell death.
MH-S mCherry-actin cells were treated with amorphous silica [16.5 µg/cm2-25 µg/cm2] and the time and number of particles internalized was determined for each cell. Time lapse imaging was continued in the presence of PI to monitor if and when cell death occurred. (A) The number of particles (per cell) that induces cell death as measure by the percent of PI positive cell nuclei, (n = 38 cells at 16 h). (B) Average length of time before a cell with a specific number of particles dies, (n = 37 cells).
Table 2.
Many cell lines take up silica particles, but only macrophages show extreme sensitivity.