Novel method to assess resident alveolar macrophage efferocytosis of apoptotic neutrophils by flow cytometry

Macrophage efferocytosis of apoptotic neutrophils (PMNs) plays a key role in the resolution of inflammation. In these studies, we describe a novel flow cytometric method to assess efferocytosis of apoptotic PMNs. Resident alveolar macrophages and PMNs were collected from lungs of mice exposed to inhaled ozone (0.8 ppm, 3 h) followed by lipopolysaccharide (3 mg/kg, i.v.) to induce acute lung injury. PMNs were labeled with PKH26 or DilC18(5)-DS (D12730) cell membrane dye and then incubated with resident alveolar macrophages at a ratio of 5:1. After 90 min, macrophage efferocytosis was analyzed by flow cytometry and confirmed by confocal mi-croscopy. Whereas alveolar macrophages incubated with D12730-labeled PMNs could readily be identified as efferocytotic or non-efferocytotic, this was not possible with PKH26 labeled PMNs due to confounding macro-phage autofluorescence. Using D12730 labeled PMNs, subsets of resident alveolar macrophages were identified with varying capacities to perform efferocytosis, which may be linked to the activation state of these cells. Future applications of this method will be useful in assessing the role of efferocytosis in the resolution of inflammation in response to toxicant exposure.

Automated summary

This study describes a novel flow cytometric method to assess the ability of macrophages to engulf apoptotic neutrophils (PMNs). It was found that resident alveolar macrophages can be more easily identified as efferocytotic or non-efferocytotic when incubated with D12730-labeled PMNs, compared to PKH26-labeled PMNs. This method will be useful in studying the role of efferocytosis in the resolution of inflammation in response to toxicant exposure.