In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Summary

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Abstract

Ozone (O₃) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O₃ exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O₃-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O₃ exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O₃ inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O₃ or other inhaled insults.

Introduction

The lung is constantly exposed to environmental insults, including air particulates, viruses, bacteria, and oxidant gases that trigger pulmonary inflammation^1,2,3. These insults can compromise gas exchange and induce irreversible tissue injury^4,5. Alveolar macrophages, which constitute approximately 95% of the immune cells found in murine and human lungs at homeostasis, are critical regulators of pulmonary inflammation after environmental insults^1,2,

Protocol

All methods have been approved by the Institutional Animal Care and Use Committee (IACUC) of East Carolina University.

1. Ozone (O₃) and filtered air exposures (Day 1)

1. Place a maximum of 12 female C57BL/6J mice, 8-12 weeks old, in a steel cage (with 12 separate compartments) with wire mesh lids into an O₃ exposure chamber.
2. Place the thermometer in the exposure chamber with the cage to accurately record the temperature and humidity.
3. Turn on .......

Discussion

Efferocytosis is an anti-inflammatory process in which macrophages clear apoptotic cells and debris as well as produce multiple anti-inflammatory mediators^{9,10,11,12,16,18}. Multiple models of efferocytosis have provided insight into how the macrophage is a critical cell in the resolution of inflammation⁶

Materials

Name	Company	Catalog Number	Comments
Annexin V-FITC Kit	Trevigen	4830-250-K	The TACS Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.
BCL2 Jurkat T Cells	ATCC	ATCC CRL-2899	The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.
Countess II Automated Cell Counter	Thermofisher	AMQAX1000	It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.
Cytospin 4 Cytocentrifuge	Thermofisher	A78300003	Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.
Fetal Bovine Serum, qualified, heat inactivated	Thermofisher	16140071	Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.
Kwik-Diff  Reagent 2, Eosin	Thermofisher	9990706	Eosin staining that stains cytoplasm.
Kwik-Diff Reagent 1, Fixative	Thermofisher	9990705	Fixes cells to be stained by H&E.
Kwik-Diff Reagent 3, Methylene Blue	Thermofisher	9990707	Methylene Blue staining that stains the nucleus.
Penicillin-Streptomycin	Sigma/Aldrich	P0781-100ML	Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.
RPMI 1640 Medium, GlutaMAX Supplement	Thermofisher	61870036	RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.
Stratagene UV Stratalinker 1800 UV Crosslinker	Cambridge Scientific	16659	The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.
Teledyne T400 ultraviolet light photometer	Teledyne API	T400	The Model T400 UV Absorption analyzer uses a system based on the Beer-Lambert law for measuring low ranges of ozone in ambient air.
Teledyne T703 Ozone calibrator	Teledyne API	T703	Provides feedback control of the UV lamp intensity, assuring stable ozone output.