Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Summary

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Abstract

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Introduction

The lung microenvironment is a uniquely complex ecosystem with an elaborate air conduit and vasculature. The inhaled air travels through the trachea and through numerous branches of bronchi and bronchioles before reaching the alveoli, where the blood-air gas exchange occurs. Due to direct interaction with the atmosphere, the respiratory surface requires protection from the potentially harmful effects of airborne particles and pollutants. A number of physical, chemical, and immunologic barriers protect the lungs. Notably, deployment of phagocytes at the respiratory surface serves an important first-line defense system. Alveolar macrophages (AMs) are one type of lung-resident phagocytes, and they make up the vast majority of the lung macrophage pool. As their name suggests, AMs are primarily localized to the alveolar lumen and occur as sessile cells that constantly sample the ambient atmosphere and communicate with the alveolar epithelium¹. In steady state lungs, more than 95% of the phagocytes in the alveolar space are AMs², whose composition may alter due to inflammation, infection, or chronic exposure to pollutants.

AMs participate in a wide range of functions that may be local to the lungs and/or of systemic importance. For example, AMs are essential in the development and the optimal functioning of lungs; immune surveillance; and clearance of cellular debris, invading pathogens, and inhaled particles^3,4,5,6,7. Targeted depletion of AMs is known to impair clearance of respiratory viruses and bacteria^4,8. Besides their role as phagocytes and a first-line defenders of pulmonary homeostasis, AMs are known to function as antigen-presenting cells in eliciting T cell immunity⁹, potentiating the efficacy of intranasal vaccine¹⁰ and influencing lung-restricted autoimmunity after lung transplantation^11,12. Deficiency in AM function has been linked to pulmonary alveolar proteinosis (PAP), a condition resulting from a genetic mutation, malignancy or infection that impairs clearance of pulmonary surfactants^13,14. Transplantation of AMs is now being explored as a therapeutic approach for treatment of PAP ^15,16.

AMs are known to originate during embryogenesis and to persist in the lungs throughout life without being replaced by circulating leukocytes^2,17. Although, AM turnover is undetectable in homeostatic lungs, varying levels of AM turnover have been reported in certain clinical conditions including infection by the influenza virus⁴, myeloablative irradiation¹⁸, exposure to endotoxin¹⁹, and old age²⁰. AMs are believed to self-renew via a low-grade proliferation^17,21, but some recent studies claim that monocytes can give rise to a population of intravascular lung macrophages^22,23 under experimental conditions, but functionality of these newly converted pulmonary macrophages have yet to be defined in lung diseases. Furthermore, understanding the threshold of stimulus in the context of AM activation is a potentially interesting area, as the lung attempts to preserve an equilibrium between the inflammatory signals and the immunoregulatory machinery.

The physiologic or pathologic alterations that lead to loss of immune regulation are important to evaluate in various clinical settings (e.g., respiratory infections, inflammatory lung disease and fibrotic lung disease). Nevertheless, AMs are increasingly recognized as indicators or even determinants of pulmonary health^11,24. Currently, there are no unified protocols available for harvesting, characterizing, and/or maintaining AMs from humans and preclinical murine models. Lack of a consensus on AM precursors and phenotypes, and absence of a detailed methodology had been the major roadblock in deciphering role(s) of AM in pulmonary health and disease. The following protocol offers a definitive identification, isolation, and in vitro culture strategy that will greatly advance the understanding of AM behavior and facilitate AM-targeted diagnostic and therapeutic studies.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Review Board (IRB) at St Joseph's Hospital and Medical Center.

1. Isolation of AMs from Murine Bronchoalveolar Lavage (BAL) Fluid

1. Anaesthetize an eight-week-old C57BL/6 mouse with ketamine (87.5 mg/kg body weight) and xylazine (12.5 mg/kg body weight) cocktail via an intraperitoneal injection. Proceed when mouse attains surgical anesthesia with loss of reflexes and relaxation of muscles.
2. Place the mouse on a dissection surface with the ventral side facing up. Apply ophthalmic vet ointment on eyes to prevent dehydration and wipe the entire ventral surface of mouse with sterile alcohol prep pads saturated with 70% isopropanol to disinfect.
3. Mount the mouse with all four legs restrained.
4. Gently open the abdominal cavity with micro dissection tools. Care must be taken to open as much area as possible without damaging any visceral organs or creating overhanging tissue.
5. Gently open the thoracic cavity by slowly incising the rib cage through the mediastinum. Ideally, the rib cage can be excised from side to side. Extreme care must be taken not to injure the pleura of lungs while opening the thoracic cavity.
6. Locate the inferior vena cava and make an incision to bleed the mouse. Use absorbent cotton pads to soak up blood.
7. Inject 10 mL ice-cold phosphate buffered saline (PBS) into the right ventricle (using 10 mL syringe and 25G needle) to flush circulating blood cells. Absorb the flow of blood with absorbent cotton pads. Once completely perfused, the lungs will appear blanched and are then ready for lavage.
8. Gently cut open the skin and muscle in the neck to expose the airway. Carefully excise the adjoining muscles, cartilages, and fat tissues without damaging the trachea.
9. Make a small incision (<2 mm) on the trachea posterior to the larynx. This incision should be just enough to insert a catheter into while the trachea is still attached to the larynx. Insert a 1-inch 22G catheter without a needle into the trachea towards the lungs. Secure the catheter with a silk braided suture (4-0; non-absorbable) with a square knot.
   NOTE: The catheter needs to be trimmed based on the mouse size. Extremely short or long catheters tend to leak and/or tear the airway during lavage. Catheter length should be such that when fully inserted the tip remains well within the trachea and not reach the primary bronchi.
10. Attach a 1-mL syringe filled with ice-cold BAL buffer (Ca²⁺ and Mg²⁺ free PBS + 1 mM Ethylenediaminetetraacetic acid, EDTA) to the catheter and slowly instill the buffer into the lungs. This will inflate the lungs. Keep the syringe attached to the catheter for five seconds and then aspirate the lavage fluid by gently pulling the piston. This will deflate the lungs. Collect the fluid in a 15 mL conical tube placed on ice.
11. Repeat Step 1.10 for nine more times and pool the lavage fluid. Do not exceed 1 mL buffer per flush, as that may exceed the lung capacity and lead to its rupture.
12. Euthanize the mouse with CO₂ overdose by IACUC approved protocol.
13. Centrifuge the 10 mL BAL fluid at 250 x g at 4 °C for 10 minutes. The cell pellet contains BAL cells.
    Caution: The pellet may be very small, so exercise caution when aspirating the supernatant.
14. Resuspend the BAL cells in 100 μL of flow/sorting buffer (PBS + 2% Heat inactivated fetal bovine serum (FBS) + 1 mM EDTA + 25 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)).
15. Block the nonspecific antibody binding to Fc receptor by adding mouse Fc block (clone 2.4G2, 10 μg/mL) and incubating for 20 minutes on ice.
16. Perform antibody staining along with fluorescence minus one (FMO) and single-stain controls^11,25. Evaluate the BAL cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by appropriate analysis software (see Table of Materials).
17. Isolate AMs by a fluorescence-activated cell sorter (see Table of Materials) directly into 1 mL mouse AM culture medium when AMs are intended for in vitro culture or in vivo cell transfer. Alternatively, sort AMs into 1 mL Lysis buffer (see Table of Materials) supplemented with 10 μL/mL β-Mercaptoethanol for RNA extraction.

2. Isolation of AMs from Mouse Lung, Single-cell Suspension

1. Anaesthetize an eight-week-old C57BL/6 mouse with ketamine (87.5 mg/kg body weight) and xylazine (12.5 mg/kg body weight) cocktail via an intraperitoneal injection. Proceed when mouse attains surgical anesthesia with loss of reflexes and relaxation of muscles.
2. Place the mouse on a dissection surface with the ventral side facing up. Apply ophthalmic vet ointment on eyes to prevent dehydration and wipe the entire ventral surface of mouse with sterile alcohol pads saturated with 70% isopropanol to disinfect.
3. Mount the mouse with all four legs restrained.
4. Gently open the abdominal cavity with micro dissection tools. Care must be taken to open as much area as possible without damaging any visceral organs or creating overhanging tissue.
5. Gently open the thoracic cavity by slowly incising the rib cage through the mediastinum. Ideally, the rib cage can be excised from side to side. Extreme care must be taken not to injure the pleura of lungs while opening the thoracic cavity.
6. Locate the inferior vena cava and make an incision to bleed the mouse. Use absorbent cotton pads to soak up blood.
7. Inject 10 mL ice-cold PBS into the right ventricle (using 10 mL syringe and 25G needle) to flush circulating blood cells. Once completely perfused, the lungs will appear blanched and are ready for harvest.
8. Dissect out the lungs by severing the trachea, blood vessels and ligaments into a 15 mL conical tube with 5 mL of cold Dulbecco's Modified Eagle's medium (DMEM). Maintain it on ice until the next step.
9. Euthanize the mouse with CO₂ overdose by IACUC approved protocol.
10. Transfer the perfused lungs into a sterile and pyrogen-free 60 mm culture dish with 3 mL DMEM. With help of dissecting forceps tease out the airways and other hard non-lung tissue material, if present. Mince the lung tissue with a scalpel to <1 mm size.
11. Add 300 µg/mL Liberase TL and 5 U/mL DNase I, mix by pipetting and incubate at 37° C in an incubator for 25 minutes. Gently mix once by pipetting after 10 minutes.
12. Pass the dissociated lungs through a 100 µm cell strainer installed on a 50 mL conical tube. Use the back of a plunger from 1 mL syringe to mash up cell clumps on the filter. Wash the filter with 20 mL wash buffer (PBS + 2% Heat inactivated FBS + 2 mM EDTA).
13. Centrifuge at 500 x g at 4 °C for 5 minutes. Discard the supernatant and resuspend the cell pellet in 20 mL wash buffer.
14. Repeat steps 2.12 and 2.13 twice, changing the filter and tube each time. Pre-soaking the filter in wash buffer for more than five minutes increases the AM yield. Prepare single-cell suspension by adding 100 μL flow/sorting buffer to the cell pellet.
15. Block the nonspecific antibody binding to the Fc receptor by adding mouse Fc block (clone 2.4G2, 10 μg/mL) and incubating for 20 minutes on ice.
16. Perform antibody staining along with FMO and single-stain controls^11,25. Evaluate the lung cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by analysis software.
17. Sort AMs by a cell sorter (see Table of Materials) directly into 1 mL mouse AM culture medium when AMs are intended for in vitro culture or in vivo cell transfer. Alternatively, sort AMs into 1 mL Lysis buffer supplemented with 10 μL/mL β-Mercaptoethanol for RNA extraction.

3. Isolation of AMs from Human BAL Fluid

1. Arrange transfer of the human BAL fluid from the clinic to the laboratory at 4 °C.
2. Centrifuge the BAL fluid (10 - 50 mL) at 250 x g at 4 °C for 10 minutes. The cell pellet contains BAL cells¹¹.
3. Wash the pellet with 20 mL wash buffer (PBS + 2% Heat inactivated FBS + 2 mM EDTA) and centrifuge at 250 x g at 4 °C for 10 minutes.
4. Resuspend the BAL cells are in 100 μL of flow/sorting buffer (PBS + 2% Heat inactivated FBS + 1 mM EDTA + 25 mM HEPES).
5. Block the nonspecific antibody binding to the Fc receptor by adding human Fc block (clone Fc1.3070, 25 μg/mL) and incubating for 20 minutes on ice.
6. Perform antibody staining along with FMO and single-stain controls^11,24. Evaluate the BAL cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by analysis software.
7. Sort AMs by a cell sorter (see Table of Materials) directly into 1 mL human AM culture medium or 1 mL Lysis buffer supplemented with 10 μL/mL β-Mercaptoethanol.

4. In vitro Culture of AMs

1. Following the cell sorting, harvest the cells by centrifuging at 250 x g at 4 °C for 10 min.
2. Resuspend human AMs in 1 mL human AM culture medium [Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% FBS, 20% L-929 culture supernatant (as a source of macrophage colony stimulating factor (M-CSF), 100 U/mL final concentration), 1 mM sodium pyruvate, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), and 1x penicillin/streptomycin (optional)]. Similarly, resuspend mouse AMs in 1 mL mouse AM culture medium [DMEM supplemented with 10% FBS, 20% L-929 culture supernatant (as a source of M-CSF, 100 U/mL final concentration), 1 mM Sodium Pyruvate, 10 mM HEPES, and 1x penicillin/streptomycin (optional)].
3. Enumerate the trypan blue-excluding viable cells by a cell counter and adjust the viable cell concentration at 1 x 10⁶/mL.
   NOTE: Ideally, up to 5 x 10⁵ AMs can be isolated from BAL fluid collected from an 8 - 10 week old C57BL/6 male mouse. The AM yield from the mouse lung digest is variable and may be slightly less than that of the mouse BAL fluid. AM yield from human BAL fluid depends on the volume of the BAL fluid, the total volume of saline used in the lavage, and patient's clinical condition.
4. Based on the yield and experimental necessity, seed the cells in chamber slides, culture dishes or culture flasks. Ideally, seed the AMs at 1 x 10⁵/mL with 10 mL medium in a T25 tissue culture flask.
5. Incubate the cells in a 37 °C humidified incubator with 5% CO₂ atmosphere.
   NOTE: AMs will grow as adherent cells and are extremely slow-growing (doubling time >7 days).
6. At 24 hours post-seeding, AMs should be adherent and will not be detached by a gentle change of culture medium. Remove the medium by aspiration from one end and replenish with fresh medium pre-warmed to 37 °C.
   NOTE: The cells can now be used to assess AM physiology or the effects of stimuli. AMs grown in chamber slides can be conveniently stained with appropriate antibodies and are ideal for microscopic visualization. For flow cytometric evaluation, AMs are harvested by incubation with non-enzymatic cell dissociating solution.
7. Aspirate the culture medium and add dissociating solution enough to cover the culture surface (approximately 2 mL per T25 flask) and incubate at 37 °C for 5 - 10 minutes. No scraping will be necessary, and it is not recommended to scrape the cells mechanically. Gently tap the sides, add 1 - 2 mL flow/sorting buffer and gently pipette up and down to detach the majority of adherent cells.
8. For RNA studies, lyse the cells directly on the culture dish by adding Lysis buffer.

Results

The flow cytometric approach to identify mouse AMs is shown in Figure 1. This includes analysis of a minimum set of surface markers necessary in distinguishing AMs from other lung-resident or lung-infiltrating phagocytes. Differential analysis is required to positively identify AMs from interstitial macrophages, dendritic cells, neutrophils, monocytes, and monocyte-derived pulmonary macrophages that occur in lungs. The following scheme of surface markers can ...

Discussion

AMs are long-living lung-resident macrophages that populate the lungs beginning at birth and enduring over the entire life span²⁶. Their roles in pulmonary physiology⁷ and pathology¹² and their potential to predict of pulmonary autoimmunity²⁴ have been recognized. Because AMs have a long-term presence in lungs^11,27 and because they are involved in activation and progre...

Materials

Name	Company	Catalog Number	Comments
Non-enzymatic cell dissociating solution	Millipore-Sigma	C5789
Puralube Vet Ointment	Dechra	620300
22G Catheter	Terumo Medical Products	SR-OX2225CA
4-0 Non-absorbable silk braided suture	Kent Scientific	SUT-15-2
Dulbecco’s phosphate buffered saline	Corning	21-031-CM
Mouse Fc block	BD Biosciences	553142
Lysis buffer (PureLink RNA Kit)	Thermo Fisher Scientific	12183018A
b-Mercaptoethanol	Millipore-Sigma	M6250
FACSAria II cell sorter	BD Biosciences	644832
Ketamine  (Ketathesia)	Henry Schein	56344
Xylazine  (AnaSed)	Akorn	139-236
RPMI 1640	Corning	10-040-CM
DMEM	Corning	10-017-CM
Liberase TL	Millipore-Sigma	5401020001
DNase I	Millipore-Sigma	AMPD1-1KT
100μm cell strainer	Corning	352360
Human Fc block	BD Biosciences	564220
EDTA	Corning	46-034-CI
Countess II Automated Cell Counter	Thermo Fisher Scientific	AMQAX1000
Trypan Blue Solution	Thermo Fisher Scientific	15250061
HEPES	Corning	25-060-CI
Fetal Bovine Serum	Atlanta Biologicals	S11150H
L-929 cell line	American Type Culture Collection	ATCC, CCL-1
Penicillin/Streptomycin	Corning	30-002-CI
Sodium Pyruvate	Corning	25-000-CI
T25 Tissue culture flask	Thermo Fisher Scientific	156367
60 mm culture dish	Millipore-Sigma	CLS3261
15 mL Conical tube	Corning	352097
50 mL Conical tube	Corning	352098
LSRFortessa cell analyzer	BD Biosciences	657669
FlowJo	FlowJo	v10.4	Analysis Software
Anti-CD45 (Mouse)	Biolegend	147709	Clone I3/2.3, FITC conjugated
Anti-CD11b (Mouse)	Biolegend	101228	Clone M1/70, PerCP/Cy5.5 conjugated
Anti-CD11c (Mouse)	BD Biosciences	565452	Clone N418, BV 421 conjugated
Anti-I-Ab (Mouse)	Biolegend	116420	Clone AF6-120.1, PE/Cy7 conjugated
Anti-Siglec-F (Mouse)	BD Biosciences	562757	Clone E50-2440, PE-CF594 conjugated
Anti-Siglec-H (Mouse)	Biolegend	129605	Clone 551, PE conjugated
Anti-F4/80 (Mouse)	Biolegend	123118	Clone BM8, APC/Cy7 conjugated
Anti-Ly-6C (Mouse)	Biolegend	128035	Clone HK1.4, BV605 conjugated
Anti-CD64 (Mouse)	Biolegend	139311	Clone X54-5/7.1, BV711 conjugated
Anti-CD24 (Mouse)	BD Biosciences	563115	Clone M1/69, BV510 conjugated
Anti-CD103 (Mouse)	BD Biosciences	745305	Clone OX-62, BV650 conjugated
Anti-CD317 (Mouse)	Biolegend	127015	Clone 927, APC conjugated
Anti-CXCR1 (Mouse)	Biolegend	149029	Clone SA011F11, BV785 conjugated
Anti-CD45 (Human)	Biolegend	304017	Clone HI30, AF488 conjugated
Anti-CD11b (Human)	Biolegend	101216	Clone M1/70, PE/Cy7 conjugated
Anti-HLA-DR (Human)	Biolegend	307618	Clone L243, APC/Cy7 conjugated
Anti-CD169 (Human)	Biolegend	346008	Clone 7-239, APC conjugated
Anti-CD206 (Human)	Biolegend	321106	Clone 15-2, PE conjugated
Anti-CD163 (Human)	Biolegend	333612	Clone GHI/61, BV421 conjugated