MACROPHAGE POLARIZATION IN CANCER BIOLOGY : A MATTER OF GOOD OR BAD EDUCATION

Macrophages are terminally differentiated cells of the mononuclear phagocyte system that also encompasses dendritic cells, circulating blood monocytes, and committed myeloid progenitor cells in the bone marrow. Both macrophages and their monocytic precursors can change their functional state in response to microenvironmental cues exhibiting a marked heterogeneity. However, there are still uncertainties regarding distinct expression patterns of surface markers that clearly define macrophage subsets, particularly in the case of human macrophages. In addition to their tissue distribution, macrophages can be functionally polarized into M1 (proinflammatory) and M2 (alternatively activated) as well as regulatory cells in response to both exogenous infections and solid tumors as well as by systems biology approaches. Cassetta L, Cassol E, Poli G. (2012) Macrophage Polarization in Health and Disease. ScientificWorldJournal. 2011; 11: 2391–2402. Featured Photo from Wikipedia Download Formatted Pdf 1. MACROPHAGE POLARIZATION: DOGMA OR REALITY? Proinflammatory, “classical Activation” of macrophages, which was delineated in early studies from the 1960s12, depends on the secreted molecules of activated T helper 1 (Th1) CD4+ lymphocytes or natural killer (NK) cells, and in particular of interferon? (IFN-? ), and of other proinflammatory agents such as tumor necrosis factor-? (TNF-? ) and bacterial lipopolysaccharide (LPS). Classical activation results in a population of macrophages with enhanced microbicidal capacity and increased secretion of proinflammatory cytokines to further strengthen the cell-mediated adaptive immunity34. In contrast, interleukin-4 (IL-4) and IL13, signature cytokines of the CD4+ Th2 response, namely, are the major inducers of the “alternative activation” of macrophages that play an important role not only in the immune response to parasites, but also in allergy, wound healing, and tissue remodeling56. Indeed, “classically” and “alternatively” activated macrophages have been designated as “M1” and “M2” macrophages, respectively, by analogy to the Th1/Th2 division of labor of CD4 helper T cells 7. M2-polarized macrophages are further subdivided into M2a (elicited by IL-4 or IL-13), M2b (following stimulation by immune complexes in the presence of a Toll-like receptor ligand), and M2c (when exposed to anti-inflammatory stimuli such as glucocorticoid hormones, IL-10, or Transforming Growth Factor-? , TGF-? )8. Although this classification of macrophages provides a useful working scheme, it unlikely fully represents the complexity of the transitional states of macrophage activation, which is often fine tuned in response to different microenvironments. A more flexible classification has been suggested recently by mouse studies in which macrophages are considered as part of a continuum having a range of overlapping functions and in which classically activated, wound-healing, and regulatory macrophages occupy different points of the spectrum9. However, also this classification does not take into account the role of macrophages during development encompassing embryonic10 and wound-healing9 macrophages as well as irreversibly differentiated osteoclasts11. The differentiation of these different macrophages is profoundly influenced by the microenvironment, although there is considerable plasticity between distinct cell types. This concept recalls Metchnikoff’s original classification that considered macrophages as part of a continuum, keeping the self whole in development and adulthood (physiological inflammation) and differentiating it from nonself and the environment (pathological inflammation). 2. MACROPHAGE PLASTICITY: AN OBSTACLE TO STUDY MACROPHAGE POLARIZATION? Unlike lymphocytes where phenotypic changes are largely “fixed” by chromatin modifications after exposure to polarizing cytokines, macrophages have a plastic gene expression profile that is influenced by the type, concentration, and longevity of exposure to the stimulating agents, as documented extensively [9, 12–14]. For example, M2 macrophages can be readily 1 Macrophage induced to express M1-associated genes by exposure to TLR ligands or IFN-? [15–17]. Indeed, gene expression plasticity was the rule rather than the exception when experimental polarization was performed [15–17]. In this regard, gene expression plasticity represents an adaptive response to different microenvironmental stimuli, in which macrophages migrate in response to chemotactic signals or chemokines, phagocytose dead cells and debris, and functionally interact with different T cell subsets. Macrophages are renown for their apparent phenotypic heterogeneity and for the diverse activities in which they engage [18, 19]. Many of these activities appear to be opposing in nature: proversus anti-inflammatory effects, immunogenic versus tolerogenic activities, and tissuedestruction versus tissue-repair [6, 18, 19]. Indeed, significant differences between genes expressed early (up to 6h) versus late (12–24h or later) after LPS stimulation have been reported [20]. Thus, the functional pattern expressed by macrophages changes with time as the response progresses. It should also be noted that macrophages frequently respond to the early cytokines that they secrete in an autocrine/paracrine fashion. Macrophages treated overnight with IL-4 prior to stimulation with LPS display enhanced production of TNF-? and IL-12, in stark contrast to the reduced production of these cytokines observed upon stimulation with LPS in the presence of IL-4 [21]. There is a high number of factors contributing to diversity of macrophage function, including the synergistic or antagonistic effects of different cytokines and related signals on their differential expression, chemokines, hormones (including adrenergic and cholinergic agonists), TLR ligands, and other endogenous ligands (e.g., histamine, integrin ligands, peroxisome proliferatoractivated receptor ligands, apoptotic cells); this plethora of signals underlines the fact that macrophages can display a large number of distinct, functional patterns that have not yet been completely defined. Furthermore, identical macrophages placed in different microenvironments display different functions in response to a common stimulus. Stimulation of macrophages with functionally opposite cytokines, such as IFN-? and IL-4, initiates signal cascades that results in differential modulation (enhancement or inhibition) of different genes at the transcriptional or posttranscriptional level (e.g., stabilization or destabilization of mRNA). Unless the signal cascade triggered an apoptotic cascade, macrophages will eventually revert to their original, functional status after the cytokine signaling ceases. In vivo or in vitro treatment of macrophages with cytokines alters their functional response pattern to LPS. However, if the cytokines are washed away after incubation and macrophages are then maintained in the absence of cytokines for 1-2 days before LPS stimulation, the functional response pattern is usually identical to that of macrophages that had not been prestimulated with the cytokine. A similar reversion to basal macrophage phenotype is observed when IL-4 and granulocyte macrophage-colony-stimulating factor (GM-CSF) are removed from human monocyte-derived, immature dendritic cells (iDCs) and the cells are resuspended in a neutral environment [22]. Therefore, most Th1 and Th2 cytokines do not seem to induce a stable differentiation of macrophages into distinct subsets, but they rather promote a transient functional pattern of responses that return to basal levels in a few (3–7) days. 3. MARKERS OF MACROPHAGE POLARIZATION: STILL AN OPEN CHASE One of the most debated issues in the context of human macrophage polarization is the identification of unique or restricted markers to be used for research and clinical purposes. Innovative approaches, including intravital imaging and other in vivo techniques, will be of great help in the identification of “real” subsets of macrophages in addition to more static antigens expressed on their cellular surface following cell polarization. An example of this broader approach is summarized by the identification of at least 6 different subsets of mouse tumor-associated macrophages (TAMs) based on their distinct functional features (Table 1), as reviewed in [24]. According to this view, every macrophage subset not only expresses different cytokines and cytokine receptors but also plays a complete distinct role in tumor pathogenesis and evolution, from tumor initiation to tumor metastasis.