Influence of macrophages and neutrophilic granulocyte-like cells on crystalline silica-induced toxicity in human lung epithelial cells

Introduction:<br/>
In many industrial activities, workers are exposed to particles that are either manufactured and used for their physicochemical properties or generated unintentionally during industrial processes. Since particles may get aerosolized, the main exposure route for employees is inhalation. To predict the human health hazard of these materials, toxicological studies performed by inhalation or intratracheal instillation on laboratory animals represent the most relevant approach. However, the commitment of the international scientific community to reduce the use of laboratory animals has led us to develop an alternative predictive model for studying the pulmonary toxicity of particles. We therefore propose to develop a co-culture model (macrophages, granulocytes, and alveolar epithelial cells) designed to be more representative of the inflammatory pulmonary response occurring in vivo. <br/>
Material and Methods:<br/>
Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells were used as a surrogate for macrophages, All-trans retinoic acid (ATRA)-differentiated HL60 were used as a surrogate for granulocytes and A549 were used as epithelial alveolar type II cells. A crystalline silica sample DQ12 was used as a prototypical particle for its capabilities to induce DNA damage, inflammatory response, and oxidative stress in epithelial cells; its polyvinylpyridine-N-oxide (PVNO)-surface modified counterpart was also used as a negative particulate control. Cells in mono-, bi- or tri-culture were exposed to DQ12 or DQ12-PVNO for 24 h. Following exposure, the culture supernatants were collected for the measurement of pro-inflammatory cytokines and A549 cells were harvested for the analysis of DNA damage (Comet assay), gene expression and oxidative stress.<br/>
Results:<br/>
DQ12 but not DQ12-PVNO induced a significant increase in DNA damage in A549 cells. The presence of differentiated THP-1 reduced the genotoxic effects of this crystalline silica sample. The exposure of A549 to DQ12 but not DQ12-PVNO induced a significant change in interleukin-8 (IL-8) protein levels which was exacerbated when differentiated THP-1, and to a lesser extent, HL-60, were added. In addition, while no production of TNF was detected in the A549 monoculture, elevated levels of this cytokine were observed in the co-culture systems. These genotoxic and pro-inflammatory effects were associated with changes in the production of intracellular reactive oxygen species in A549 cells.<br/>
Conclusion:<br/>
This work shows that a cell culture model that takes into consideration the complexity of the pulmonary inflammatory response might be more dependable to study the toxicological properties of particles than simple monoculture models.