The Many Faces of Macrophage Activation.

It used to be easy. In the old days (∼8 years ago), activated macrophages were simply defined as cells that secreted inflammatory mediators and killed intracellular pathogens. Things are becoming progressively more complicated in the world of leukocyte biology. Activated macrophages may be a more heterogenous group of cells than originally appreciated, with different physiologies and performing distinct immunological functions. The first hint of this heterogeneity came with the characterization of the “alternatively activated macrophage” [1]. The exposure of macrophages to interleukin (IL)-4 or glucocorticoids induced a population of cells that up-regulated certain phagocytic receptors but failed to produce nitrogen radicals [2] and as a result, were relatively poor at killing intracellular pathogens. Recent studies have shown that these alternatively activated cells produce several components involved in the synthesis of the extracellular matrix (ECM) [3], suggesting their primary role may be involved in tissue repair rather than microbial killing. It turns out that the name alternatively activated macrophage may be unfortunate for a few reasons. First, although these cells express some markers of activation, they have not been exposed to the classical, activating stimuli, interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Second, and more importantly, the name implies that this is the only other way to activate a macrophage. Recent studies suggest that this may not be the case. Exposure of macrohages to classical activating signals in the presence of immunoglobulin G (IgG) immune complexes induced the production of a cell type that was fundamentally different from the classically activated macrophage. These cells generated large amounts of IL-10 and as a result, were potent inhibitors of acute inflammatory responses to bacterial endotoxin [4]. These activated macrophages have been called type 2-activated macrophages [5] because of their ability to induce T helper cell type 2 (Th2) responses that were predominated by IL-4 [6], leading to IgG class-switching by B cells. Thus, at this time, there appears to be at least three different populations of activated macrophages with three distinct biological functions. The first and most well described is the classically activated macrophage whose role is as an effector cell in Th1 cellular immune responses. The second type of cell, the alternatively activated macrophage, appears to be involved in immunosuppression and tissue repair. The most recent addition to this list is the type 2-activated macrophage, which is anti-inflammatory and preferentially induces Th2-type humoral-immune responses to antigen. Together, these three populations of cells may form their own regulatory network to prevent a well-intentioned immune response from progressing to immunopathology. As Yogi Berra might say, let's start from the beginning. It is important to remember that macrophages become classically activated by exposure to two signals. The first is the obligatory cytokine IFN-γ, which primes macrophages for activation but does not in itself activate macrophages [7]. The second signal is tumor necrosis factor (TNF) itself or an inducer of TNF. Exogenous TNF can act as the second signal, but the physiologically relevant second signal is generally the result of Toll-like receptor (TLR) ligation, which induces endogenous TNF production by the macrophage itself. Thus, classically activated macrophages are developed in response to IFN-γ, along with exposure to a microbe or microbial product such as LPS. In the murine system, these cells are now easily identified by virtue of their production of nitric oxide (NO) [8, 9]. Macrophages that have been primed with IFN-γ alone should not make NO, provided the system is free of LPS (a common contaminant in recombinant IFN-γ). In the human system, the activation of macrophages is somewhat more difficult to definitively determine, as monocyte-derived macrophages from peripheral blood generally do not produce NO in response to the classical activating stimuli. These cells must be identified by a variety of biochemical and functional criteria (Table 1), including the up-regulation of surface molecules such as MHC class II and B-7 (CD86) and an enhanced ability to present antigen and kill intracellular pathogens. With the application of microarrays, the analysis of classically activated macrophages has entered into a new era of complexity. Remarkably, 25% of the observed genes exhibited alterations in their expression upon macrophage activation [10]. The problem now becomes selecting which of the several hundred genes that are induced or suppressed during activation are true and reliable markers of macrophage activation. The biological functions of the activated macrophage are myriad and well documented. These cells migrate to sites of inflammation where they encounter pathogens and degrade them. Contrary to popular belief, activated macrophages are not more phagocytic than resting cells [11]. Although activated macrophages spread out more than resident cells and generally have a higher pinocytic rate, they express reduced levels of mannose receptor and Fc receptor for IgG (FcγR)II [12]. Activated macrophages do however possess a markedly enhanced ability to kill and degrade intracellular microorganisms, and for several years, this was the functional criterion used to define an activated macrophage. This killing is accomplished by an increase in the production of toxic oxygen species and an induction of the inducible NO synthase (iNOS) gene to produce NO. The restriction of various nutrients from the phagosome is an underappreciated but important aspect of microbial killing that is also used by activated macrophages. The restriction of iron [13] and tryptophan [14] from vacuolar organisms is a particularly well-established mechanism for limiting intracellular growth within activated macrophages. Tissue macrophages can be seen in organized collections of cells called granulomas, where they are frequently surrounded by T cells producing activating cytokines. These granuloma macrophages are a rich source of inflammatory cytokines, toxic radicals, and inflammatory lipid mediators. These products are capable of causing extensive tissue damage to the host. For example, the observation that tumors frequently developed proximal to old tuberculosis scars [15] illustrates the destructive and mutagenic potential of the activated macrophages that are frequently found there. The immunopathology associated with many type 1 autoimmune diseases has the activated macrophage as the main protagonist [16]. Thus, the activity of these cells must be carefully regulated to prevent uncontrolled tissue destruction. Signals to down-regulate macrophage activation may come from within the macrophage itself or from surrounding cells. The immunoregulatory cytokines transforming growth factor (TGF)-β and IL-10 play an important role in dampening macrophage activation. Knockout mice lacking either of these two cytokines have proven to be hypersusceptible to inflammatory pathologies [17, 18]. A recently described family of macrophage receptor tyrosine kinases in the stem cell-derived tyrosine kinase receptor (STK/RON) family has been implicated in the inhibition of macrophage activation [19]. The ligation of RON with macrophage stimulatory protein appears to induce arginase activity (see below) and inhibit NO production. Thus, the ligation of this family of receptors may alter the phenotype of these cells into ones that resemble the alternatively activated macrophages described below. Finally, cell death by apoptosis cannot be ignored as an important immunoregulatory mechanism. The direct removal of inflammatory cells as well as the induction of anti-inflammatory TGF-β following apoptotic cell removal [20] contribute to this effect. One of the most interesting, new areas of inquiry in this field is determining how intracellular pathogens interfere with signaling to prevent macrophage activation. The protozoan parasite Leishmania spp [21] and Mycobacterium spp [22] have provided the best examples of such interference. Infection of macrophages with either of these organisms prevents the macrophage from becoming fully activated in response to IFN-γ. The molecules and mechanisms by which these organisms circumvent cellular immunity will likely reveal important information about the process of macrophage activation itself. In the early 1990s, while examining the regulation of mannose receptor expression on elicited macrophages, Gordon and colleagues [1] noticed that a particularly efficient way to induce receptor expression was to treat these macrophages with IL-4. The authors concluded that macrophages treated with IL-4 assumed an “alternative activation phenotype.” This represents the first description of a macrophage that exhibited signs of activation but was clearly distinct from its classically activated counterpart. Subsequent studies by Goerdt and Orfanos [23] have championed this cell as a regulatory macrophage with diverse biological roles different from the classically activated cell. These cells fail to make NO by virtue of their induction of arginase [24] and consequently, are compromised in their ability to kill intracellular microbes. Although they up-regulate some MHC class II molecules, they are not efficient at antigen presentation, and in many instances, they actually inhibit T cell proliferation. The suppressive activity of these macrophages extends to mitogen-activated T cells, which show a significantly diminished proliferative and secretory response in the presence of alternatively activated macrophages [25]. The signature cytokines produced by these alternatively activated macrophages are IL-10 and IL-1 receptor antagonist. In the lung, it is thought that alternatively activated macrophages may provide negative regulatory signals to protect the host from overzealous inflammatory responses to environmental stimuli [26]. Recent studies on this cell population have begun to focus on their potential to mediate wound-healing, angiogenesis, and ECM deposition. Alternatively activated macrophages produce high levels of fibronectin and a matrix-associated protein, βIG-H3 [3], and they promote fibrogenesis from fibroblastoid cells [27]. The induction of arginase in these cells may lead to polyamine and proline biosynthesis, promoting cell growth, collagen formation, and tissue repair [28]. The in vivo schistosome infection model has proven to be an invaluable one to unravel the function of altered macrophage metabolism caused by type 2 cytokines [28]. Unlike many of the autoimmune models mentioned above, in the Schistosome model, type 2 immune responses correlate with pathology, whereas type 1 responses can ameliorate tissue destruction. This model has revealed that a competition for arginine metabolism within macrophages may be a key determinant of macrophage physiology. When macrophages are treated with type 1 cytokines, the iNOS2 enzyme produces NO from arginine. When macrophages are exposed to type 2 cytokines, Arg-1 is induced and metabolizes arginine to urea and ornithine, a precursor of polyamines and proline. Polyamines are involved in cell growth and division, whereas proline is a key component of collagen. In this elegant model system, Wynn and colleagues [28] demonstrated unequivocally that Arg-1 levels correlate directly with Schistosome egg-induced pathology, whereas iNOS levels inversely correlate with pathology. Recently, macrophages from the (Th2-prone) BALB/c mice were shown to make more arginase than macrophages from the Th1-prone C57Bl/6 mice [29]. Thus, the alternatively activated macrophage may serve more of a regulatory and recovery function than the effector killing functions that are associated with classically activated macrophages. Although the alternatively activated macrophage has been investigated for only slightly more than 5 years, there exists a solid battery of markers available to identify this cell type (Table 1). In addition to arginase induction, these cells make a signature chemokine, originally named AMAC-1 [30]. They react well with monoclonal antibodies to a specific form of CD163 and an uncharacterized MS-1 antigen. Finally, two transcription factors, FIZZ1 and Ym1, appear to be preferentially induced in IL-4-treated macrophages [31]. The type II-activated macrophage was initially identified during an examination of conditions under which IL-12 transcription was terminated. It was observed that the ligation of FcγRs on activated macrophages unexpectedly turned off IL-12 synthesis [32] and induced the secretion of large amounts of IL-10 [33]. A variety of immune complexes and a number of different activating stimuli were used, and in all cases, FcγR ligation resulted in a specific and reciprocal alteration in the production of these two cytokines [4]. Similar to the classically activated macrophages described above, the type II activation phenotype required two signals. The first signal was the ligation of FcγRs. However, receptor ligation had to be coupled with a macrophage stimulatory signal to influence cytokine production. Stimuli included those that signal through any of the TLRs, as well as through CD40 or CD44. The type II phenotypic switch following FcγR ligation worked on unprimed and IFN-γ-primed macrophages. Type II-activated macrophages are clearly distinct from alternatively activated macrophages described above, as they do not induce arginase, and with the exception of IL-12 and IL-10, the production of many of the other cytokines produced by classically activated macrophages, such as TNF, IL-1, and IL-6, remains intact [4]. The dramatic induction of IL-10 by these macrophages suggested that they would have anti-inflammatory properties. To test this, an acute model of LPS lethality was implemented. Type II-activated macrophages were generated in vitro and transferred into mice, which then received six times the lethal dose (90) of LPS. Mice that received 1 × 106 type II macrophages remained completely viable and healthy throughout the observation period, whereas mice that received control macrophages succumbed to lethal endotoxemia within 48 h [4]. To show that this effect was a result of IL-10 secretion, type II-activated macrophages from IL-10-/- mice were used in parallel. These macrophages failed to rescue mice from lethal endotoxemia. Thus, by virtue of their secretion of IL-10, the type II-activated macrophage exerts a potent anti-inflammatory effect that can be exploited to prevent acute pathologies, such as those associated with LPS endotoxemia. The name, type II-activated macrophages, was derived from their ability to preferentially induce Th2 adaptive immune responses. In the initial in vitro studies, classically or type II-activated macrophages were used as antigen presenting cells (APC). Classically activated macrophages stimulated T cells to produce primarily IFN-γ in response to antigen, whereas type II-activated macrophages induced T cells to produce high levels of IL-4 [6]. This bias in T cell cytokine production by these two populations of APC was stable and preserved when T cells were subsequently restimulated under a variety of nonbiasing conditions. T cell cytokine production correlated with APC cytokine production. IL-12 secretion by classically activated macrophages induced IFN-γ production by T cells, whereas IL-10 secretion by type II-activated macrophages resulted in IL-4 production by T cells [6]. To determine the extent to which type II-activated macrophages could influence antibody responses to antigen, mice were injected with type II- or classically activated macrophages along with ovalbumin (OVA) antigen in the absence of any other adjuvant. Mice vaccinated with OVA in the presence of classically activated macrophages made only modest IgG-antibody responses to antigen [5]. Mice vaccinated with OVA in the presence of type II-activated macrophages, however, made significantly more IgG, and the majority of the IgG was of the IgG1 isotype. This observation suggested that the presence of type II-activated macrophages alone was sufficient to induce (Th2-like) IgG class-switching. Thus, the ligation of FcγR on activated macrophages by antigen-IgG complexes induced T cells to produce IL-4, which in turn induced B cells to produce IgG1 in response to that antigen. These studies demonstrate that antigen binding to the FcγR on activated macrophages will preferentially induce a Th2-like response. Thus, they predict that IgG itself can be a strong inducer of humoral immunity and an inhibitor of cell-mediated immunity by virtue of its ability to change the phenotype of the APC. These observations may have important, practical implications, as they suggest that reagents that ligate the FcγR on activated macrophages may have anti-inflammatory properties that can be exploited to ameliorate autoimmune diseases. These studies may also have particular relevance to autoimmunity, where activated macrophages likely represent important APC's. Macrophage heterogeneity used to be a research topic on which the careers of many postdoctoral fellows were misspent. The lack of definitive markers and dubious biochemical assays prevented the unequivocal identification of specific cell subsets. There has now been significant progress in establishing the heterogeneity of activated macrophages. There are at least three distinct populations of macrophages, and each cell type appears to have different biological roles. The interplay among these populations of cells may help to shape not only the magnitude but also the character of the immune response. The manipulation of these cells may lead to new approaches to treat or prevent disease.