Investigating the immunotoxicology of silver nanoparticles in vitro

This study investigated the immunotoxicology of silver nanoparticles using in vitro human cell cultures. THP-1 human monocytes, primary human monocytes isolated from peripheral blood, and PMA-stimulated macrophages derived from these monocytes, as well as HaCaT human keratinocytes, were exposed to 4 types of nanosilver or ionic silver for up to 24 hours.<br><br>The nanosilver was characterised in pristine and agglomerated conditions using TEM, DLS, DCS and cryo-TEM. Cytotoxicity, cell death pathways, reactive oxygen species production, and oxidative stress of cell cultures exposed to nanosilver were measured using a variety of techniques. Immunomodulatory effects of nanosilver were assessed by measuring cytokine release, and uptake and intracellular localisation of nanosilver were also investigated.<br><br>Nanosilver was cytotoxic to all cell types investigated. On a mass dose basis, the smaller sized nanosilver materials were more potent than larger sized material, and ionic silver was the most cytotoxic.<br><br>Cytotoxic doses of nanosilver caused cell death almost exclusively by necrosis in HaCaT keratinocytes, and THP-1 monocytes also showed primarily necrotic cell death. In contrast, THP-1 PMA-stimulated macrophages primarily exhibited apoptotic cell death. This may have been due to the greater ability of macrophages to withstand oxidative stress, due to their role in eliminating invading pathogens in vivo.<br><br>Immune cells showed increased superoxide production with nanosilver exposure. Correspondingly, cellular glutathione was depleted drastically in THP-1 monocytes and macrophages, and was proportional to the decrease in cell viability. Some types of nanosilver also caused significant lipid peroxidation in these cells.<br><br>Nanosilver exposure did not cause an increase in Th1 or Th2 specific cytokine expression in THP-1 or primary isolated human monocytes and PMA-stimulated macrophages in vitro. However, a significant increase in pro-inflammatory cytokines was observed for THP-1 monocytes and PMA-stimulated macrophages.<br><br>Flow cytometric analysis of THP-1 immune cells indicated a dose dependent uptake of significant quantities of nanosilver within the first 4 hours of exposure. Synchrotron X-ray fluorescence microscopy showed a colocalisation of nanosilver agglomerates with the analysed THP-1 macrophages. A novel application of surface enhanced Raman scattering mapping was used to identify the cellular uptake of nanosilver agglomerates by immune cells grown on an aluminium foil substrate support. Ultra-thin section TEM of THP-1 monocytes and PMA-stimulated macrophages showed agglomerates of nanosilver present in cellular vesicles and cytosol. <br><br>Many common in vitro toxicology techniques were shown to be appropriate for investigating the immunotoxicology of nanosilver. Exposure to nanosilver at high concentrations in vitro caused cell death and increased oxidative stress in human immune and skin cells. It is unclear whether ROS production is responsible for the cytotoxicity of nanosilver, but it is likely to be just one mechanism amongst a number of synergistic processes involving disrupted cellular thiol homeostasis, mitochondrial dysfunction, oxidative stress and membrane damage. This simple exposure system did not indicate a specific Th1 or Th2 immune response to nanosilver exposure, but rather a strong pro-inflammatory response in THP-1 cells. More complex immune system models, such as co-culture systems and animal models, are required to further investigate the immunostimulatory potential of nanosilver.<br>