Inflammation is a complex and dynamic physiological process, which requires a well-balanced and controlled interaction of diverse cells. One of the cell types critically involved in initiation but also resolution of inflammation is macrophages. Across the different phases of an inflammatory response to e.g., infections or skin damage, their functions cover the production of pro-inflammatory cytokines and cytotoxic compounds, secretion of various growth factors to promote the function of other cells contributing to the resolution of inflammation, fighting invading microbes, and scavenging cell debris. In fact, these factors are neither produced simultaneously nor by the same kind of macrophages. Several recent reports have made clear that “the” macrophage does not exist, but that the term macrophage is rather used in a broader context for the description of a population of very versatile cells that share some common denominators. Due to their ability to adopt to a changing cytokine milieu and, vice versa, to influence the milieu by production of an array of soluble mediators, macrophages are nowadays considered to be very plastic and flexible cells [67
]. Acquiring diverse functional phenotypes in response to environmental cues is reflected in the classification of polarized rather than activated macrophages. Mirroring the Th1/Th2 dichotomy, polarized macrophages were sub-divided into M1 and M2. M1 polarization, also referred to as classical activation, results from stimulation with Interferon-γ (IFN-γ), alone or in concert with bacterial lipopolysaccharide (LPS) [68
]. The source of IFN-γ can be both innate and adaptive immune cells. Upon stress or first encounter to pathogens, natural killer (NK) cells, as part of the innate immunity, produce IFN-γ, thus polarizing macrophages to M1. In addition, an increased capacity of antigen presenting [69
], these M1 cells are characterized by an enhanced microbicidal and tumoricidal activity mediated by the production of increased levels of superoxide anions, and oxygen and nitrogen radicals, which in summary confers direct host resistance to infections [71
]. However, the capability of NK cells to provide sustainable amounts of IFN-γ is limited. Since M1 macrophages induce a Th1 response by secretion of high levels of pro-inflammatory cytokines, such as interleukin (IL)-12, IL-23, and tumor necrosis factor (TNF)-α, the polarization of macrophages to M1 can be amplified by a continued supply with IFN-γ by T cells. In contrast to T cells, which act in an antigen-specific manner, M1 polarized macrophages kill unspecifically, merely restricted by the distance to the target cell. While M1 macrophages represent the one extreme of polarization, the second extreme is summarized under the term alternatively activated macrophages or M2 macrophages [52
]. During the past decade, this term has been expanded to a more heterogeneous cell population [74
]. The nomenclature of alternatively activated macrophages is somewhat confusing and changes over time. Terms used are: M2, alternatively activated macrophages, type II activated macrophages, deactivated macrophages, M2a, M2b and M2c and a few more. In effort to find a more informative and clear classification of polarized macrophages Mosser and Edwards [49
] suggested the following three sub-populations based on their functions: host defense, wound healing and immune regulation. This classification is in accordance with that proposed by Mantovani et al.
], where M1 would refer to host defense, M2a to wound healing and M2b together with M2c to immune regulation. For discrimination of different macrophage phenotypes, Mantovani et al.
not only considered the function, but also the respective stimuli generating the sub-populations. Thereby, M2a is produced by IL-4 or IL-13 both of which activate STAT6 via binding to the IL-4 α-subunit, which is shared in both receptors [76
]. M2b results from a combined exposure to immune complexes with Toll-like receptor (TLR)- or IL-1 receptor (IL1R)-ligands, whereas M2c is induced by IL-10 [69
]. Supplementary to Mossers and Edwards’s classification, Mantovani et al.
discriminate between M2b macrophages, which in addition to immunoregulatory functions also induce Th2 driven inflammation, and M2c macrophages, which are thought to be predominantly responsible for negative/deactivating immunoregulation. More recently, additional sub-populations of macrophages were discovered ().
Diverse ways of polarizing macrophages are induced by distinct signals from the microenvironment. Unpolarized macrophages/monocytes (M0) acquire a M1 phenotype upon stimulation with IFN-γ and LPS. M2a macrophages, especially responsible for wound healing, result from exposure to IL-4 or IL-13. Other members of the alternatively activated macrophages include M2b (after exposure to IC + TLR-ligands or IL-1R ligands) and M2c (polarized by IL-10). M2d represents a more recently discovered macrophage subset, which results from re-polarization from M1 by adenosine signaling. Whether an initial stimulus polarizing M0 to M2d does exist is yet unknown. The apparently related subsets Mox and Mha can be generated in vitro by stimulation with Ox-PL-PPC and HH-complexes or oxRBS, respectively. M2 macrophages are a hydride-type subset with combined features of M1 and M2 macrophages. These cells were shown to be CSF/CXCL4-dependent.
Figure 2 Different polarized MΦ subsets. Abbreviations: IC, immune complex; TLR, Toll-like receptor; IL-1R, IL-1 receptor; Ox-PL-PPC, ox-PL 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphorylcholine; HH-complexes, hapto-hemoglobin; oxRBC, oxidized red blood (more ...)
In the context of atherosclerosis, a population of macrophages expressing a unique set of genes including Heme oxygenase-1 (HO-1), sufiredoxin-1, and thioredoxin-reductase in a nuclear factor, erythroid-derived 2, like 2(Nrf2) dependent manner was found within the aortas of 30 week western diet-fed Ldlr
− mice by immunohistochemistry and flow cytometry. In vitro
, this phenotype could be induced by ox-PL 1-palmitoyl- 2arachidonoyl-sn-glycero-3-phosphorylcholine resulting in the designation as Mox macrophages [81
]. Furthermore, Mox macrophages seem to be closely related to the lately proposed hemorrhage-associated macrophages (Mha) [82
]. Human monocytes can be differentiated to Mha macrophages in vitro
using hapto-hemoglobin complexes or oxidized red blood cells leading to the up-regulation of CD163, HO-1 and IL-10 in an Nrf2-dependent manner. Moreover, a CSF/CXCL4-dependent macrophage subset, discovered in 2010 by Gleissner et al.
], was termed M4. These macrophages were shown to be characterized by a mixed, but unique profile of transcripts including higher levels of CD86 and TNF-α (both M1-like), CD206, CCL18 and CCL20 (all M2-like) and lower levels of pentraxin 3 (PTX3), CD36 and IL-10 (M2-like). Although these cells have a weak phagocytic activity, the function of M4 macrophages remains poorly understood. Finally, the laboratory of Samuel Joseph Leibovich has characterized one further M2 subtype, termed M2d, which unlike previously described M2-like macrophages, are the result of a switching event from M1 macrophages as response to adenosine A2A
receptor signaling induced by TLR agonists [85
]. M2d macrophages were demonstrated to markedly decrease the expression of pro-inflammatory cytokines including IL-12 and TNF-α, while concurrently producing high levels of IL-10 and vascular endothelial growth factor (VEGF). Interestingly, the classical markers of wound healing or M2a macrophages were not up-regulated pointing to an M2c-resembling phenotype of M2d macrophages. The phenotypes of different polarized macrophages are summarized in .
Phenotypes of different polarized MΦ.
Polarized macrophages show characteristic expression profiles of surface receptors and soluble mediators. M1 macrophages are characterized by an elevated expression of the co-stimulatory receptors for T cell activation CD80 and CD86 and both MHC molecules underlining their role as potent antigen presenting cells. On the soluble level, M1 macrophages up-regulate the expression and secretion of pro-inflammatory cytokines, such as IL-12, IL-6, and TNF-α. In contrast, M2 (a-d) macrophages preferentially produce anti-inflammatory cytokines (e.g., IL-10). Another hallmark of M2 macrophages (especially M2a and M2c) is the up-regulation of the mannose receptor (CD206). Recently identified macrophage subsets (M4, Mox and Mha) have until now been poorly characterized. Abbreviations: MHCI/II, major histocompatibility complex I/II; SLAM, signaling lymphocytic activation molecule; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; iNOS, inducible nitric oxide synthase; RELM-α, resistin-like molecule α; YM1, heparin binding lectin; Arg, arginase; TGF-β, tumor growth factor β; PTX3, pentraxin 3; VEGF, vascular endothelial growth factor; CCL18/20, chemokine (C-C motif) ligand 18/20; HO-1, heme oxygenase-1 (HO-1); SD-1, sufiredoxin-1; TR-reductase, thioredoxin-reductase.
Together, this huge diversity in alternatively activated macrophages illustrates how different physiological processes might be accurately regulated by distinct macrophage subsets depending on the environmental conditions given. As already exemplified for M2d, all polarization subsets are also believed to be plastic and to undergo phenotypical and functional switches upon signals from the milieu, thus making the versatility of the macrophage system even vaster than that which emerges from the initial polarizing stimulus. However, imbalance and/or dysregulation of macrophage-controlled processes are strongly associated with a pathological outcome including several autoimmune diseases, impaired wound healing, metabolic disorders, and cancer [86
]. Mediated by an autocrine secretion, certain macrophage subsets sustainably maintain the cytokine milieu leading to the persistence of these cells for an inappropriate period of time. In addition, T cells become activated and further promote the inflammatory status. Both cell types, T cells and macrophages, are initiators of different inflammatory diseases and can be used as targets in diagnosis and therapy. Based on the recently acquired knowledge on the involvement of distinct polarized macrophages in certain diseases and the anticipated rapid increase of novel findings in this field, polarized macrophages are more and more becoming an object of interest. Selective depletion of distinct macrophage populations, without adversely affecting others would allow for selective therapy with fewer side effects. However, polarization of macrophages represents a fairly new field of research and little is presently known on the origin, function, and plasticity of different polarized macrophages. So far, no effective therapies selectively targeting one population of macrophages do exist.