Introduction to Macrophages and Selection of Indicators

Macrophages (M) are specialized cells of the innate immune system with a long lifespan and phagocytic capabilities.

Macrophages participate in the recognition, phagocytosis, and degradation of cell debris and pathogens. They also play a role in presenting antigens to T cells and inducing the expression of co-stimulatory molecules on other antigen-presenting cells, thereby initiating adaptive immune responses.

In the bone marrow, macrophage/dendritic cell precursors can differentiate into two types of monocytes: Ly6C+CX3CR1loCCR2+CD62L+ and Ly6C+CX3CR1loCCR2+CD62L+. The former can enter various tissues and differentiate into various types of macrophages, as shown in the figure:

Mouse macrophage differentiation model

Chávez-Galán Leslie, Olleros Maria L, Vesin Dominique, et al. Much More than M1 and M2 Macrophages, there are also CD169(+) and TCR(+) Macrophages. [J]. Front Immunol, 2015, 6: 263.

Although five types of activated macrophage phenotypes have been discovered, including M1 macrophages, M2 macrophages, CD169+ macrophages, TCR+ macrophages, and tumor-associated macrophages (TAMs), most research in this field focuses on M1 and M2 macrophages. M1 and M2 macrophages are distinguished based on their functions and levels of inflammatory cytokine secretion.

M1 Macrophages:

· M1 macrophages, also known as classical activated macrophages, are macrophages that produce pro-inflammatory cytokines. They express high levels of major histocompatibility complex class II (MHC-II) and co-stimulatory molecules CD80 and CD86.

· Under stimulation by IFN-γ, LPS, and other factors, dormant macrophages (M0 type) can be polarized into M1 macrophages, which secrete large amounts of pro-inflammatory cytokines, including IL-1, IL-6, TNF-α, iNOS, high levels of IL-12, and low levels of IL-10. They play a major role in promoting inflammation, killing bacteria, and phagocytosis. Additionally, M1 macrophages produce chemotactic factors that attract Th1 cells, promoting a strong Th1 immune response. M1 macrophages can also enhance antigen presentation by increasing the expression of MHC-II and co-stimulatory molecules CD80 and CD86 on their cell surface.

M2 Macrophages:

· M2 macrophages, also known as alternatively activated macrophages, are primarily activated by the inflammatory cytokine IL-4. They play a core role in responding to parasites, tissue repair, angiogenesis, wound healing, and allergic diseases by secreting anti-inflammatory cytokines such as IL-10, which inhibits M1 macrophages.

· M2 macrophages release a large amount of cell factors such as VEGF, Arg-1, PDGF, TGF-β, and IL-10, and secrete chemotactic factors such as CCL17, CCL18, CCL22, and CCL24, which attract Th2 cells, eosinophils, and basophils. They produce various matrix metalloproteinases (such as MMP-2, MMP-7, MMP-9, CCL18, and CCL22), promoting cancer cell metastasis.

The following image summarizes the two classical macrophage activation phenotypes, their induction conditions, and functions:

Main macrophage polarization states in activated macrophages

Reference: Atri C, Guerfali FZ, Laouini D. Role of Human Macrophage Polarization in Inflammation during Infectious Diseases. Int J Mol Sci. 2018;19(6):1801.

Selection of Indicators for Macrophage Detection

How should we select indicators and perform gating analysis when detecting macrophages? Taking human macrophages as an example, we can refer to the following literature. After gating macrophages based on FSC/SSC and CD68, we can distinguish M1 and M2 macrophages based on CD80 and CD206, as shown in the figure: CD80+CD206- for M1 and CD80+CD206+ for M2.

Raggi F, Pelassa S, Pierobon D et al. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1. Front Immunol. 2017; 8: 1097.

The same method can be used for detecting mouse macrophages, with F4/80 and CD11b added as higher gates to gate the total macrophages.