To assess macrophage function at distinct phases of disease and recovery, we developed a novel system for depletion of macrophages during disease. The role of the inflammatory tissue macrophage has remained a subject of debate, at least in part because macrophages are associated with both tissue injury and repair (10
). In addition, when macrophages have been depleted in a series of injury models, the results have been conflicting (38
). Macrophage have been successfully depleted during induction of several animal models of inflammatory diseases by antimacrophage serum or by administration of liposomal clodronate (1
). While both undoubtedly deplete macrophages, the former has pleiotropic effects, and the latter has been complicated by neutrophil depletion, toxicity, and ineffective depletion in some tissues (40
). To circumvent these difficulties, we developed a novel transgenic mouse in which minute injections of DT can deplete macrophages. This is possible because the mouse equivalent of the DT receptor (hbEGF) binds DT very poorly; mice are therefore insensitive to DT (27
) while tissue-specific expression of the human DT receptor confers sensitivity (25
We have now shown that the deletion of the macrophage population either during injury or during repair and resolution has dramatically different effects on the overall fibrotic response. Specifically, in progressive inflammatory injury, macrophage depletion results in amelioration of fibrosis. By contrast, depletion during recovery results in a failure of resolution with persistence of cellular and matrix components of the fibrotic response. These data represent the first definitive demonstration that macrophages play distinct roles in injury and repair and highlight the critical observation that a given cell may be both pathogenic and beneficial.
Our experiments suggest that over a period of a few days, 2 functionally distinct types of macrophages exist in the injured liver. During the injury phase (a time when chemokines, complement components, and proinflammatory cytokines are abundant) injury-associated macrophages promote myofibroblast proliferation and apoptosis. On balance, macrophages have the effect of increasing myofibroblast numbers. As a result, fibrogenesis predominates and matrix is deposited. The phenotype of macrophages associated with the injury phase is closest to that of the aa-Ms described by others (11
). However, it is present in response to injury normally associated with classically activated macrophages, as it is present at a stage when proinflammatory cytokines are released.
In contrast, during recovery from injury, a population of macrophages predominates that does not support HSC survival and promotes matrix degradation and, in that regard, resembles classically activated macrophages. This macrophage population, however, is present during resolution of injury and at a time when proinflammatory mediator levels are decreasing, an observation reinforced by our studies of TNF-α in this model. Moreover, these 2 functional phenotypes are separated chronologically in the liver by only a few days, suggesting that they may represent a single population. Therefore, we suggest that the yin and yang of the classically activated macrophage/aa-M model is inadequate to reflect the complex roles of these cells in vivo during liver injury.
In our study, the SAMs during injury are associated with high expression of TGF-β1 in the scars and high levels of liver tnfα transcript. In contrast, during recovery, SAMs are not associated with TGF-β1 generation in the scars, and the liver tnfα transcript is attenuated. We have identified 2 sources of SAMs. One is monocyte-derived, and the other is derived from a nonhematogenous source. It is likely that this other source of SAMs is the resident liver macrophages, the Kupffer cells. It is tempting to speculate as to whether one functional population of macrophages derives from monocytes and the other from Kupffer cells. Further experiments (beyond the scope of the current study) will be required to determine whether the functional distinctions are clearly related to macrophage origin.
Our study indicates that SAMs during disease are associated with high levels of proliferation and apoptosis in the scars, but that these parameters are markedly decreased by depletion of SAMs. Thus, the SAMs may induce both proliferation and apoptosis and, as such, maintain a population of HSC myofibroblasts. Macrophages are known to induce cell cycle–dependent cell death in some settings (42
), and so there is reason to consider this mechanism of cell number regulation in the injured liver. Furthermore, macrophages can liberate both potent myofibroblast growth factors, such as PDGF, and proapoptotic factors, such as TNF-α. Indeed, SAMs during disease generate TNF-α. Our data reveal that after 7 days of recovery, apoptosis and proliferation in the scars is reduced and the population of HSC myofibroblasts is much reduced. Thus, SAMs during recovery do not share these characteristics with SAMs during disease. In a previous study, we have shown that the loss of HSCs during recovery in rats is due to a wave of apoptosis occurring predominantly in the first 3 days (7
). We do not see this wave of apoptosis in the current study, in which we focused on 7 days of recovery in order to demonstrate changes in matrix components. It is likely that the wave of apoptosis occurred at an earlier time point.
Previous studies using other methods to modify or deplete macrophages have pointed to a role for macrophages in mediating the fibrotic response (4
). Those studies have been inconsistent, however, which may reflect nonspecific depletion of other cell types or the existence of distinct functional populations of macrophages in the different models of inflammation and scarring that were studied. Our new conditional ablation system is nontoxic, is cell type–specific, is effective in many tissues, and offers the possibility of time point–selective deletion of cells during a range of inflammatory processes.
We suggest that our description of the roles of macrophages in functional terms should provide the basis for a series of future functional studies in vivo to define more accurately the nature, specific attributes, and dynamics of the macrophage population(s) present in inflammation and repair. We further suggest that a description of macrophage populations based on functional studies in vivo should form the basis of a rational and clinically relevant classification of the cell.