Title

The Molecular Pathway of Non-opsonized Silica Particle Phagocytosis and the Relationship to Silica-induced Cytotoxicity

Date of Completion

January 2011

Keywords

Biology, Cell|Health Sciences, Toxicology

Degree

Ph.D.

Abstract

Silica inhalation leads to the development of silicosis. Macrophages are directly affected when recruited to the site of silica-induced inflammation, however, the mechanism of particle-cell interaction, phagocytosis, and what induces cytotoxicity at the cellular and molecular level is unclear. ^ MH-S mouse alveolar macrophages were exposed to either crystalline silica or 3 μm amorphous silica for cytotoxicity studies. Non-opsonized (ovalbumin-coated, uncoated) silica is toxic to macrophages, but opsonizing silica with antibody (Ab-opsonized) significantly suppresses cell death, however it is presumed that silica particles are internalized by macrophages. For the first time, the kinetics of non-opsonized and Ab-opsonized particle uptake was established. Non-opsonized particles are internalized significantly slower than Ab-opsonized particles when cells are chilled prior to exposure, regardless of particle charge. Internalization does not seem to occur via FcR- or SR-A-mediated signaling. When this assay was performed at room temperature (21°C) or 37°C, both particles are rapidly internalized at 37°C, but non-opsonized particles are slowly internalized at 21°C. These data suggest that non-opsonized particle uptake is rate limiting below 37°C due to a change in membrane fluidity.^ FcR-mediated signaling was used as a paradigm to establish the molecular uptake pathway. Non-opsonized particle uptake is similar to FcR-mediated uptake of Ab-opsonized particles (Rac GTPase activation; accumulation of PI(3,4,5)P3 and F-actin at phagosomes; endo-lysosomal vesicle-phagosome fusion) and CR3-mediated uptake of complement-opsonized particles (RhoA GTPase activation; microtubule polymerization-dependent).These data suggest that non-opsonized particles are recognized non-specifically since the three uptake routes overlap.^ Activation of both GTPases is unique to non-opsonized particle uptake and microtubule-dependent uptake is the only dramatic difference from Ab-opsonized particle uptake at 37°C. Unlike CR3-mediated uptake, cells are not PMA-primed to internalize non-opsonized particles. Microtubule polymerization is necessary for non-opsonized particle-induced GTPase and PI3 K-I activation, and actin polymerization, which suggests that microtubules may be important in an initial uptake event.^ Silica-induced cell death is significantly suppressed unless inhibitor is absent. From these data, it can be hypothesized that uptake is important to elicit silica-induced cytotoxicity. Determining the fundamental processes by which silica induces silicosis is crucial for the development of an effective therapeutic treatment. ^