An in vitro multi-paramater assay development strategy towards differentiating macrophage responses to inhaled medicines

PURPOSE: Although "foamy macrophages" (FMΦ) are commonly observed during non-clinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies.

METHODS: The purpose of this study was to develop a multi-parameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine and polyvinylacetate nanoparticles were used to induce distinct FMΦ phenotypes in J774A.1 cells and compared with negative controls. Treated macrophages were evaluated for morphometry, lipid accumulation, gene expression, apoptosis, cell activation and phagocytosis.

RESULTS: Analysis of vacuolization (number/area vacuoles per cell) and phospholipid content revealed inducer-dependent distinctive patterns, which were confirmed by electron microscopy. In contrast to the other inducers, amiodarone increased vacuole size rather than number and resulted in phospholipid accumulation. No pronounced dysregulation of transcriptional activity or apoptosis was observed in response to sub-lethal concentrations of all inducers. Functionally, FMΦ induction did not affect macrophage activation by lipopolysaccharide, but reduced phagocytic capacity with different patterns of induction, severity and resolution observed with the different inducers.

CONCLUSIONS: An in vitro multi-parameter assay strategy is reported which successfully differentiates and characterizes mechanisms leading to FMΦ induction by different types of agents.