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

Abstract

Purpose: Progress 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.

Methods: Human (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.

Results: Cell 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.

Conclusions: A 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.

Keywords: NR8383; U937; foamy macrophage; toxicology; vacuolation.

Fig. 1

Representation of data analysis approach. Single cell data was summarised as a frequency histogram to assess the reproducibility of untreated cells, control treatments and sample treatments (i) Dying cells (defined as the population with a mitochondrial activity < mean value minus 2nd SD) were excluded from the analysis (a), and the remainder of the cell population were included (b). Initial data analysis compared the median response values (d, e, f). Elevated cell responses (c and g) defined as the population of cells with cell characteristic > mean plus the 2nd SD from the untreated cell population were also calculated and data summarised as elevated response profiles (ii).

Fig. 2

Median morphology parameters of untreated and amiodarone-treated rat and human macrophage cell models. Median cell area, vacuole number per cell and percentage of cell area occupied by vacuoles for untreated cells (a, c, e) and cells exposed to 10 μM amiodarone (b, d, f). NR8383 rat macrophage cells (purple triangle), U937 human monocyte-derived macrophage cells (green circles), primary rat macrophages derived from BAL in in vitro culture for 24 h or 48 h (orange diamonds) and primary rat macrophages derived from BAL tested immediately after harvesting from naïve rats after 1 and 7 day timepoints (blue squares). Each data point represents the median value of n = 6 wells per plate. * indicates p < 0.05.

Fig. 3

Median lipid parameters of untreated vs amiodarone-treated rat and human macrophage cell models. Median phospholipid content and neutral lipid content for untreated cells (a, c) and cells exposed to 10 μM amiodarone (b, d). NR8383 rat macrophage cells (purple triangle), U937 human monocyte-derived macrophage cells (green circles), primary rat macrophages derived from BAL in in vitro culture for 24 h or 48 h (orange diamonds) and primary rat macrophages derived from BAL tested immediately after harvesting from naïve rats after 1 and 7 day timepoints (blue squares). Each data point represents the median value of n = 6 wells per plate. * indicates p < 0.05.

Fig. 4

Cell health, morphology and lipid multi-parameter baseline profiles. Baseline multi-parameter charts profiling cell health, morphology and lipid changes to the NR8383 rat alveolar macrophage cell line (a), U937 human monocyte-derived macrophage cell line (b) and primary rat macrophages obtained from BAL (c) after 24 h (black line) and 48 h (orange line) timepoints. Data is represented as mean of 3 experiments where n = 6 for each investigation.

Fig. 5

Cell health, morphology and lipid multi-parameter profiles after amiodarone exposure. Multi-parameter charts profiling cell health, morphology and lipid changes to the NR8383 rat alveolar macrophage cell line (a, b), U937 human monocyte-derived macrophage cell line (c, d) and primary rat macrophages obtained from BAL (e, f) after 24 h (a, c, e) and 48 h (b, d, f) exposure to 0–10 μM amiodarone. Data is represented as mean of 3 experiments where n = 6 for each investigation.

Fig. 6

Heat map indicating experimental variability. Variability of profile parameters for cells exposed to 0–10 μM amiodarone for 24 and 48 h for the NR8383 rat alveolar macrophage cell line (a), U937 human monocyte-derived macrophage cell line (b) and primary rat macrophages obtained from BAL (c). Each square represents one experiment. The colour gradient sets the lowest value for each given parameter in the heat map (bright green), highest value (bright red) and mid-range values (black) with a corresponding gradient between these extremes.