Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Summary

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Abstract

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Introduction

Distinct from classical inflammatory responses, macrophages that infiltrate tissues often display polarized activation status that plays a crucial role in regulating host tissue physiological functions^1-8. Upon stimulation, macrophage activation can be sorted into classic (M1) and alternative (M2) activation^{2, 4, 9} . M1 macrophage activation depends on Toll-like receptors (TLRs) and activation of nuclear factor kappa B (NFκB)/c-Jun N-terminal kinase 1(JNK1), leading to production of inflammatory cytokines, such as TNF-α and IL-1β and activation of iNOS that results in increased production of reactive oxygen species such as nitride oxide (NO) ^{10, 11} . In contrast, M2 macrophage activation recruits PPARγ, PPARδ, or IL-4-STAT6 pathways, leading to alternative, anti-inflammatory (M2) activation that is associated with upregulation of mannose receptor CD206, and arginase 1(Arg1) ^{6, 12-14} .

Bone marrow derived macrophages (BMDM) present an ideal in vitro model to understand the mechanisms controlling polarization of activated macrophages¹⁵. Specifically, activation of M1 macrophages can be induced by lipopolysaccharides (LPS) stimulation, while polarization of M2 macrophages can be induced by IL-4 and/or IL-13. Mature bone marrow derived macrophages and activated macrophages can be identified through flow cytometry analysis for expression of surface antigens, including CD11b, F4/80, CD11c, CD206, CD69, CD80 and CD86^{9, 16, 17}. In addition, changes in cytokine production and cell signaling pathways associated with macrophage polarization can be measured by quantitative RT-PCR and western blotting, respectively. In summary, mouse bone marrow derived macrophages can serve as a relevant model to study macrophage polarization in vitro.

Protocol

1. Isolation of Bone Marrow Cells

1. Isolate femur and tibia bones from 6-8 week old mice, rinse off hair and then cut open the bone.
2. Use a 21G needle and 10 ml syringe to flush out marrow into cold PBS+2% heat inactivated Fetal Bovine Serum (FBS) (3-5 ml/mouse).
3. Pass marrow through a 21G needle 4-6 times to dissociate the cells.
4. Pass cells through a 70 μm cell strainer to remove cell clumps, bone, hair and other cells/tissues.
5. Add 3 volume of NH₄Cl solution (0.8% NH₄Cl solution, Stemcell Technology), and incubate on ice for 10 min to remove red blood cells.
6. Spin down cells at 500 x g for 5 min at 4 °C.
7. Resuspend the cell pellet in cold PBS+2%FBS (20-50 ml, depending on the quantity of cells).

2. Induction BMDM Formation

1. Resuspend the isolated bone marrow cells in BMDM growth medium (2x10⁶ cells/ml).

BMDM growth medium:

Iscove's Modified Dulbecco's Medium (IMDM) + 10% FBS + 15% filtered (0.2 μm) L-929 cell (ATCC, CCL-1) culture supernatant (containing monocyte-colony stimulating factor, M-CSF) or 10 ng/ml M-CSF.

Note: L-929 cell supernatant contains M-CSF¹⁸. To ensure the effective activity of conditioned medium, 5 X 10⁵ L-929 cells are seeded in T75 cm² flasks for 6-7 days, conditioned medium is collected and passed through a 0.45 μM filter before use. Medium aliquots can be used immediately or stored in -80 °C for 1-2 months.

2. Seed cells in 6 or 12 well tissue culture plates (depending on the experimental design) (Corning Costar).
3. Change fresh BMDM growth medium on day 3.
4. On day 7, formation of mature BMDM is evaluated using flow cytometry analysis and fluorophore conjugated antibodies to detect cells expressing CD11b and F4/80.

3. BMDM Polarized Activation

1. On day 7, change to fresh stimulation medium: for M1 activation, use IMDM containing 10% FBS and 100 ng/ml LPS or 100 ng/ml LPS with 50 ng/ml IFNγ; for M2 activation, use IMDM containing 10% FBS with 10 ng/ml IL-4 and/or 10 ng/ml IL-13.
2. Collect stimulated BMDMs by detaching them from the dish using warm 0.05% trypsin, followed by washing the cells twice with PBS containing 10% FBS.

Note: To detach and resuspend mature macrophages after differentiation, 0.05% Trypsin solution (containing 0.48 mM EDTA, Invitrogen) or 2-5 mM EDTA in Ca- and Mg-free PBS or Hank's balanced buffer (HBSS) can be used. When using digestive medium containing trypsin, cells are treated at 37 °C for less than 10 min to avoid loss of surface proteins due to over-digestion.

3. Use antibodies to detect expression of cell surface antigens, including CD11b, F4/80, CD11c, CD206, CD69, CD80 or CD86 at various time points using standard flow cytometry staining procedures.
4. Determine expression of genes characteristic of activated M1 and M2 macrophages including IL-1β, TNF-α and IL-6 (M1 activation) or IL-10, IL-13, arginase1 and PPARγ (M2 activation) using qRT-PCR. Determine activation of cell signaling pathways involved in activation of M1 or M2 macrophages by western blotting analysis.

Results

A schematic description of the BMDM generation procedure is presented (Figure 1). High purity of mature macrophages can be observed on day 7 when they represent 95 to 99% of CD11b+F4/80+ cells (Figure 2). Polarized macrophages can be examined using antibodies against CD11b, F4/80, CD11c and CD206 followed by flow cytometry analysis. As shown in Figure 3, M1 macrophages are detected as CD11b+F4/80+CD11c+CD206- cells (Q2), whereas M2 macrophages are CD11b+F4/80+CD11c-...

Discussion

We report here a simple and readily adaptable in vitro procedure to induce activation of macrophages derived from bone marrow progenitor cells. This procedure can be used for investigation of mechanisms responsible for polarization of macrophages. The purity of mature macrophages obtained using this protocol averages 95 to 99%, and no additional purification procedures are required. To investigate the function of specific genes of interests in the context of macrophage polarization, ectopic expression or g...

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
IMDM	Thermo Scientific	SH30259.01
Fetal bovine serum	Invitrogen	10438-026
Murine GM-CSF	PeproTech	315-03
NH4Cl	StemCell Technologies	7850
L-929	ATCC	CCL-1
70 μm cell strainer	BD Biosciences	352350
10 x PBS	Thermo Scientific	AP-9009-10
Anti-mouse CD11b-APC	eBioscience	17-0112-81
Anti-mouse F4/80-FITC	eBioscience	11-4801-81
Anti-mouse CD69-PE	eBioscience	12-0691-81
Anti-mouse CD86-PE	eBioscience	12-0862-81
Propidium Iodine	Invitrogen	P3566