Classifying Macrophages Beyond M1- and M2- Groups

Several rules exist in biology, including in macrophage characterization, but ongoing research is now finding increasingly more exceptions to these rules. For years, immunologists have attempted to categorize macrophages into two subtypes—classically activated M1 and alternatively activated M2 macrophages. However, there are a significant number of new activated macrophage subtypes requiring a description.

Alongside these new subtypes, there is also a shortage of recognized vocabulary. So scientists are looking for an alternative to the conventional black-and-white principle.

Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways,” cites Dr. Peter J. Murray, a cell researcher at St. Jude’s Children’s Hospital in Memphis, Tennessee, in a 2014 article on macrophage nomenclature.

In the biblical tale mentioned by Murray, differences in language stop a mission to construct a tower that reaches heaven. To prevent that negative result from occurring in macrophage research, Murray suggests adopting common terminology, and also universal experimental guidelines. Similar research outcomes and standardization are the basis for therapeutic approaches targeting macrophages in diseases like atherosclerosis, cancer, or specific autoimmune disorders.

Optimal culture conditions for macrophages: Dr Hagen Wieland from the development department at PromoCell works on standardizing solutions for cell generation and differentiation.

Optimal culture conditions for macrophages: Dr. Hagen Wieland from the development department at PromoCell works on standardizing solutions for cell generation and differentiation.

The Challenge

Plasticity and vast diversity are two advantageous traits of macrophages and they lead to a heterogeneous population of functionally distinguished cells with different origins. Macrophages residing in tissues are strategically present all over the body: in the lungs (alveolar macrophages), bones (osteoclasts), gut, brain (microglia), liver (Kupffer cells), eyes, connective tissues (histiocytes), and lymphoid organs (Murray et al., 2011). They all have important roles to play in tissue development and repair, pathogen immunity, and homeostasis. Furthermore, they can quickly change their phenotype in response to changes in their environment.

However, it is specifically their multiplicity that poses significant challenges to scientists.

We sense a general discontent in the community of myeloid cell experts and in the literature. It is therefore important to name the cells in a way that allows reproduction and standardization of experiments.

Dr Hagen Wieland, Development Department, PromoCell.

Macrophage Polarization is a Dynamic Process

Scientists have attempted to classify macrophages based on their origin, the mononuclear phagocytic system, or their functional phenotype. In the early 1990s, Michael Stein and his teammates reported the different effects of interleukin-4 (IL-4) in comparison with interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) on the activation phenotype of macrophages. The term “alternative activation” was coined to define IL-4 -stimulated macrophages, and to differentiate them from “classical activation” of IFN-γ /LPS -stimulated macrophages.

After many years, the M1 (classically activated)—M2 (alternatively activated) model was established to explain the responses of two different macrophages that reflect the T-helper cells nomenclature (Mills et al., 2000). M1 macrophages were depicted to possess inflammatory functions and were important for the resistance to pathogens and their eradication. M2 macrophages possessed anti-inflammatory functions and were vital for maintaining tissue integrity.

The limitations of the M1/M2 model for defining macrophage polarization are becoming more and more clear, as these two terms identify extremes of a continuum that do not occur in vivo,” remarked Wieland. Macrophages are generally present between M1 and M2 since the polarization process is dynamic, and cells usually exhibit characteristics of both states simultaneously.

A spectrum of macrophage activation stages: The traditional M1/M2 model is not satisfactory to reflect the heterogeneity of macrophages.

A spectrum of macrophage activation stages: The traditional M1/M2 model is not satisfactory to reflect the heterogeneity of macrophages.

A set of M2-like stages (M2a, M2b, M2c, and M2d) were incorporated (Biswas and Mantovani, 2010) based on bidirectional polarization and a band of intermediate activation states.

M1 or M2 macrophages can be activated, and this activation can influence their polarization state,” explained Wieland. “Polarization is not fixed, and there are so many intermediate stages that cannot be accurately described with the M1/M2 model.

For example, M2-polarized macrophages can be triggered to adopt an M1-like phenotype and vice versa. This process, known as re-education, is more powerful than polarization, and it is vital in “in vivoexperiments where the cells have to adapt to an ever-changing environment.

If trauma occurs—when the skin barrier is penetrated, for instance—M1 macrophages are required to start an inflammatory reaction and eradicate the invading bacteria. When the inflammation weakens, macrophages are re-educated toward an M2-like phenotype and initiate the regeneration of tissues. Otherwise, M1 macrophages would start damaging healthy tissue and therefore affect the healing process.

Recommendations for a Uniform Nomenclature of Macrophages

The range of studies exploring the plasticity of cells and categorizing macrophages into various subpopulations have led to an uncertain nomenclature and categorization. Due to this diversity of terminology, it is difficult to clearly describe scientific results, thus obstructing scientific development and translational approaches. To try and solve this issue, a research team convened at the International Congress of Immunology in Milan in August 2013. There, the researchers put together macrophage-activation nomenclature and reporting standards for in vitro experiments. The common framework proposal was released after one year (Murray et al., 2014), which included the following recommendations,

  • Reproducible in vitro experimental standards—bone marrow-derived monocytes in mice and peripheral blood monocytes in humans must be used as references for creating macrophages, which can be then activated with IL-4 or IFN-γ to result in defined subpopulations. Moreover, researchers must note if macrophages were polarized using GM-CSF or M-CSF.
  • Minimal reporting standards—exact descriptions of how macrophages are separated, polarized, activated, and examined are important and enable a direct comparison among various laboratories.
  • Definition of the activator—scientists should explain how they stimulate the macrophages, and adopt a nomenclature in relation to their activation standards, such as M(Ig), M(IL-4), M(GC), M(IL-10), M(LPS), M(IFN-γ), and prevent the complexity of M2b, M2a,  etc.
  • Avoid certain terms—terms that should not be used include “regulatory,” since all macrophages are regulatory in some capacity.
  • Markers of activation—the use of combinations of markers helps explain activation results.

In 2015, Guilliams et al. published an alternative classification system, based on the cellular origin of macrophages. This nomenclature shares three vital principles with the system recommended by Peter Murray. Both aim to remove terms that indicate functional specification, both introduce a predetermined level-one system across tissues and species, and both enable flexibility via a level-two system.

Macrophage plasticity and polarization in different types of pathologies:  Macrophages exhibit a high plasticity and can switch between polarization/activation states. This enables them to perform different functions. For example, M1-like macrophages are more predominant in the early phases of inflammatory responses, whereas M2-like macrophages are more commonly associated with chronic inflammation processes. (RA = rheumatoid arthritis) (Liu et al 2014, Murray and Wynn 2011, Sica and Mantovani 2012)Macrophage plasticity and polarization in different types of pathologies:

Macrophages exhibit high plasticity and can switch between polarization/activation states. This enables them to perform different functions. For example, M1-like macrophages are more predominant in the early phases of inflammatory responses, whereas M2-like macrophages are more commonly associated with chronic inflammation processes. (RA = rheumatoid arthritis) (Liu et al., 2014, Murray and Wynn 2011, Sica and Mantovani 2012)

There is still no unanimous acceptance of a standardized nomenclature in the scientific community. And some biologists criticize the straightforwardness of these approaches, pointing out the need for a more complex description of the macrophages’ system. However, PromoCell adheres to the nomenclature of Dr. Murray, as it precisely defines how macrophages are differentiated in vitro, and which cytokines are used for their polarization, as well as subsequent activation. This procedure allows scientists to reproduce and standardize the experimental processes. There is a noticeable increase in reviewers and scientists who have adopted Murray’s nomenclature in their works since 2014. This way, they can better present their results.

Dr. Hagen Wieland, the Development Department, PromoCell.

A Better Standardization is Crucial for New Therapeutic Approaches

The pathogenesis of many diseases requires different subsets of macrophages, where they usually play conflicting roles (Murray et al., 2011). M2-like macrophages maintain insulin sensitivity, regulate vital metabolic functions in adipose tissue, and also prevent the onset of type 2 diabetes, while disease progression is usually linked to a shift from M2 to M1 phenotype (Lumeng et al., 2007).

Tissue inflammation is a key feature of cancer biology and is usually associated with a polarization of M1 into M2 macrophages. These encourage angiogenesis, tumor growth, and metastasis (Joshi et al., 2014). Furthermore, cytokines derived from M1 macrophage are vital mediators in autoimmune diseases, atherosclerosis, and chronic inflammation.

A more comprehensive understanding of the mechanisms that control the activation of human macrophages will most likely result in the development of more effective approaches for treating a variety of significant inflammatory diseases.

The standardization and reproducibility of macrophage generation in vitro is the starting point for the development of new therapies,” concluded Wieland. “Only when different laboratories are able to generate the same cells, in the same way, will it be possible to succeed in developing robust strategies to address the multitude of macrophage-related disorders.”

References

  1. Fujiwara, Y., et al., Guanylate-binding protein 5 is a marker of interferon-gamma-induced classically activated macrophages.
    Clin Transl Immunology, 2016. 5(11): p. e111.
  2. Zizzo, G., et al., Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol, 2012. 189(7): p. 3508-20.
  3. Vogel, D.Y., et al., Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation.
    J Neuroinflammation, 2014. 11: p. 23.
  4. Iqbal, S. and A. Kumar, Characterization of In vitro Generated Human Polarized Macrophages. Journal of Clinical & Cellular Immunology, 2015. 06(06).
  5. Graff, J.W., et al., Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem, 2012. 287(26):
    p. 21816-25.
  6. Sousa, S., et al., Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res, 2015.
    17: p. 101.
  7. Murray, P.J., et al., Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 2014.
    41(1): p. 14-20.
  8. Murray, P.J. and T.A. Wynn, Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol, 2011. 11(11): p. 723-37.
  9. Wynn, T.A., A. Chawla, and J.W. Pollard, Macrophage biology in development, homeostasis and disease. Nature, 2013. 496(7446): p. 445-55.
  10. Murray, P.J. and T.A. Wynn, Obstacles and opportunities for understanding macrophage polarization. J Leukoc Biol, 2011.
    89(4): p. 557-63.
  11. Martinez, F.O. and S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment. Prime Rep, 2014. 6: p. 13.

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Last updated: May 22, 2019 at 3:30 AM

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