The Phagocytosis and Toxicity of Amorphous Silica

BACKGROUND: Inhalation of crystalline silica is known to cause an inflammatory reaction and chronic exposure leads to lung fibrosis and can progress into the disease, silicosis. Cultured macrophages bind crystalline silica particles, phagocytose them, and rapidly undergo apoptotic and necrotic death. The mechanism by which particles are bound and internalized and the reason particles are toxic is unclear. Amorphous silica has been considered to be a less toxic form, but this view is controversial. We compared the uptake and toxicity of amorphous silica to crystalline silica. METHODOLOGY/PRINCIPAL FINDINGS: Amorphous silica particles are phagocytosed by macrophage cells and a single internalized particle is capable of killing a cell. Fluorescent dextran is released from endo-lysosomes within two hours after silica treatment and Caspase-3 activation occurs within 4 hours. Interestingly, toxicity is specific to macrophage cell lines. Other cell types are resistant to silica particle toxicity even though they internalize the particles. The large and uniform size of the spherical, amorphous silica particles allowed us to monitor them during the uptake process. In mCherry-actin transfected macrophages, actin rings began to form 1-3 minutes after silica binding and the actin coat disassembled rapidly following particle internalization. Pre-loading cells with fluorescent dextran allowed us to visualize the fusion of phagosomes with endosomes during internalization. These markers provided two new ways to visualize and quantify particle internalization. At 37 °C the rate of amorphous silica internalization was very rapid regardless of particle coating. However, at room temperature, opsonized silica is internalized much faster than non-opsonized silica. CONCLUSIONS/SIGNIFICANCE: Our results indicate that amorphous and crystalline silica are both phagocytosed and both toxic to mouse alveolar macrophage (MH-S) cells. The pathway leading to apoptosis appears to be similar in both cases. However, the result suggests a mechanistic difference between FcγRIIA receptor-mediated and non-opsonized silica particle phagocytosis

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65

LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

Background: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMPactivated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeleta

The Phagocytosis of Crystalline Silica Particles by Macrophages

Silicosis is a chronic lung disease induced by the inhalation of crystalline silica. Exposure of cultured macrophages to crystalline silica leads to cell death; however, the mechanism of cell–particle interaction, the fate of particles, and the cause of death are unknown. Time-lapse imaging shows that mouse macrophages avidly bind particles that settle onto the cell surface and that cells also extend protrusions to capture distant particles. Using confocal optical sectioning, silica particles were shown to be present within the cytoplasmic volume of live cells. In addition, electron microscopy and elemental analysis showed silica in internal cellular sections. To further examine the phagocytosis process, the kinetics of particle uptake was quantified using an assay in which cells were exposed to ovalbumin (OVA)-coated particles, and an anti-OVA antibody was used to distinguish surface-bound from internalized particles. Fc receptor–mediated uptake of antibody-coated silica particles was nearly complete within 5 minutes. In contrast, no OVA-coated particles were internalized at this time. After 30 minutes, 30% of bound silica was internalized and uptake continued slowly thereafter. OVA-coated latex beads, regardless of surface charge, were internalized at a similarly slow rate. These results demonstrate that macrophages internalize silica and that nonopsonized phagocytosis occurs by a temporally, and possibly mechanistically, distinct pathway from Fc receptor–mediated phagocytosis. Eighty percent of macrophages die within 12 hours of silica exposure. Neither OVA coating nor tetramethylrhodamine isothiocyanate labeling has any effect on cell death. Interestingly, antibody coating dramatically reduces silica toxicity. We hypothesize that the route of particle entry and subsequent phagosome trafficking affects the toxicity of internalized particles

Overexpression of LKB1 suppresses microtubule assembly, and LKB1 RNAi increases it.

<p>(A) C2C12 cells were infected with a control adenovirus (control AV) or an adenovirus that drives expression of wild type human LKB1 (LKB1 AV) for 24 hours. Cells were treated with Nocodazole for 1 hour followed by a 7 minute washout, fixation, and microtubule immunofluorescence. Representative microtubule asters are shown. (B) Quantification of aster diameter for A, expressed as mean diameter+/−s.e.m. (C) C2C12 were cells transfected with a control siRNA or an siRNA that targets expression of LKB1 and treated with Nocodazole for 1 hour followed by a 2 minute washout, fixation, and microtubule immunofluorescence. Representative asters are shown. (D) Quantification of aster diameter for C. At least two experiments were done for each condition. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031583#pone-0031583-g004" target="_blank">Fig. 4</a> for LKB1 levels.</p

LKB1 overexpression accelerates differentiation.

<p>(A) Phase contrast pictures of C2C12 cells uninfected (no virus), infected with an adenovirus overexpressing CRE recombinase and GFP (control AV), or infected with an adenovirus expressing human LKB1, and grown in differentiation media for the indicated number of days. Cells with LKB1 overexpression showed enhanced differentiation. (B) Western blotting shows increased myosin upon LKB1 overexpression. Samples from the indicated days were probed for LKB1, myosin heavy chain (myosin), and phosphorylated AMPK; tubulin, actin, and GAPDH were probed as loading controls.</p

LKB1 levels and substrate activation increases, and LKB1 redistributes to the cytoplasm during differentiation.

<p>(A) Western blotting was done on samples from the indicated days of differentiation. Myosin heavy chain (myosin) expression increases progressively. LKB1 levels increase by day 1 and peak at day 2 of differentiation. Levels of the phosphorylated forms of LKB1 substrates AMPK and MARK increase by day 1. Tubulin is shown as a loading control. (B, C) Cells were transfected with GFP-LKB1 as described in text. Representative images from undifferentiated cells (B) and cells cultured in differentiation media for three days (C) are shown. The mean ratio of nuclear to cytoplasmic fluorescence was 3.0 in undifferentiated cells and 1.2 in differentiated cells. Bars, 50 µm.</p

Myoblasts show a transient reduction in stable microtubules prior to an ultimate increase in microtubule stabilization.

<p>Cells were differentiated by serum switch for the indicated times, fixed, and immunofluorescence for detyrosinated (glu-) tubulin, a marker of stabilized microtubules, and tyrosinated microtubules, a marker of more dynamic microtubules, was performed. Upper panel shows detyrosinated tubulin, which was reduced at the 24 hour time point and then progressively increased over the next two days. Short linear structures visible with this antibody at 24 hours are primary cilia. Bottom panel shows glu- and tyrosinated tubulin immunofluorescence merged with DNA staining. Bar, 10 µm.</p