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

TY - JOUR

T1 - Novel method to assess resident alveolar macrophage efferocytosis of apoptotic neutrophils by flow cytometry

AU - Radbel, Jared

AU - Meshanni, Jaclynn A.

AU - Gardner, Carol R.

AU - Le-Hoang, Oanh

AU - Cervelli, Jessica

AU - Laskin, Jeffrey D.

AU - Gow, Andrew J.

AU - Laskin, Debra L.

N1 - Funding Information: This work was supported by National Institutes of Health [grant numbers ES031678 , ES004738 , ES033698 , AR055073 , ES005022 , and S10OD026876 ]. Publisher Copyright: © 2022 Elsevier Inc.

PY - 2023/2/1

Y1 - 2023/2/1

N2 - 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 microscopy. 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 macrophage 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.

AB - 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 microscopy. 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 macrophage 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.

KW - Acute lung injury

KW - Alveolar macrophages

KW - Efferocytosis

KW - Flow cytometry

KW - Neutrophils

KW - Ozone

UR - http://www.scopus.com/inward/record.url?scp=85145684132&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85145684132&partnerID=8YFLogxK

U2 - https://doi.org/10.1016/j.taap.2022.116359

DO - https://doi.org/10.1016/j.taap.2022.116359

M3 - Article

C2 - 36565939

SN - 0041-008X

VL - 460

JO - Toxicology and Applied Pharmacology

JF - Toxicology and Applied Pharmacology

M1 - 116359

ER -