Date of Completion

2011

Embargo Period

10-2-2026

Major Advisor

David A. Knecht

Associate Advisor

Andrea K. Hubbard

Associate Advisor

Michael A. Lynes

Field of Study

Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

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 (Abopsonized) 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-Amediated signaling. When this assay was performed at room temperature (21 °C) or 3 7°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 3 7°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 Abopsonized particles (Rae 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 polymerizationdependent). These data suggest that non-opsonized particles are recognized nonspecifically 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 PB 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.

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