LA-ICP-MS and Immunohistochemical Staining with Lanthanide-Labeled Antibodies to Study the Uptake of CeO2 Nanoparticles by Macrophages in Tissue Sections

Abstract

Due to the increasing use and production of CeO2 nanoparticles (NPs), the likelihood of exposure especially via the air rapidly grows. However, the uptake of CeO2 NPs via the lung and the resulting distribution into various cell types of remote organs are not well understood because classical analytical methods provide limited spatial information. In this study, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was combined with immunohistochemical (IHC) staining with lanthanide-labeled antibodies to investigate the distribution of intratracheally instilled CeO2 NPs from the rat lung to lymph nodes, spleen, and liver after 3 h, 3 days, and 21 days. We selected regions of interest after fast imaging using LA-ICP-MS in low-resolution mode and conducted high-resolution LA-ICP-MS in combination with IHC for cellular localization. The lanthanide labeling, which was largely congruent with conventional fluorescent labeling, allowed us to calculate the association rates of Ce to specific cell types. Major portions of Ce were found to be associated with phagocytic cells in the lung, lymph nodes, spleen, and liver. In the lung, almost 94% of the Ce was co-localized with CD68-positive alveolar macrophages after 21 days. Ce was also detected in the lymph nodes outside macrophages 3 h post instillation but shifted to macrophage-associated locations. In the liver, Ce accumulations associated with Kupffer cells (CD163-positive) were found. Ce-containing populations of metallophilic and marginal zone macrophages (both CD169-positive) as well as red pulp macrophages (CD68-positive) were identified as major targets in the spleen. Overall, high-resolution LA-ICP-MS analysis in combination with IHC staining with lanthanide-labeled antibodies is a suitable tool to quantify and localize Ce associated with specific cell types and to estimate their particle burden under in vivo conditions.