Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis

Abstract

PurposeProgress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.MethodsHuman (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation.ResultsCell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.ConclusionsA high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs

“…Macrophages in this study differed in size and, with the exception of DMBM-2 cells, human macrophages were larger than murine ones, which is consistent with findings by Hoffman et al, who indicated the cell area of human (U937) macrophages with 123-391 µm 2 , and that of rat primary and NR8383 macrophages as 165-265 µm 2 and 113-210 µm 2 , respectively [11].…”

“…In a study using HCS LipidTOX Green phospholipidosis detection reagent for the identification of changes in rat and human macrophages after exposure to 0.1, 1, and 10 µM amiodarone, the following results were obtained after 24 h of exposure [11]. Rat primary alveolar macrophages reacted differently from NR8383 and human U937 macrophages and, overall, only a subpopulation of the cells showed changes to amiodarone.…”

“…Both stainings (HCS LipidTOX Green phospholipidosis detection reagent and LysoID), on the other hand, led to the same conclusion that murine macrophages reacted more sensitively to the tested CADs than human macrophages. Both dyes have been used already for the screening of PLD [11,24]. Increased staining with HCS LipidTOX green phospholipidosis detection reagent after exposure to amiodarone was not seen in DMBM-2 cells, making this assay less sensitive than the LysoID assay.…”

“…Despite the common use of murine macrophages for the study of human macrophage reactions, the species-specific sensitivity to PLD in vivo and in vitro suggests that human alveolar macrophages should be the most predictive screening models for PLD in human lung [2,11]. Monocyte-derived macrophages (MDM) obtained from human peripheral blood monocytic cells would be the ideal model because these cells reacted similarly to alveolar macrophages from the same donor [12].…”

“…It acts through a mechanism that is distinct from commercially available fluorescently labeled phospholipid analogs (e.g., N-(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) and has already been used to assess the effect of CADs in osteosarcoma cells [24]. HCS LipidTOX dyes were developed to indicate the accumulation of neutral or polar lipids and have been used for the analysis of PLD in macrophages [11].…”