Treatment of macrophages with cytokines such as IFN-γ or IL-4 initiates a signal cascade that results in differential modulation (enhancement or inhibition) of different genes at the transcriptional or post-transcriptional level (e.g., stabilization or destabilization of mRNA). It has been our experience that unless the signal cascade has initiated an apoptotic cascade, macrophages will eventually revert to their original, functional status after the cytokine signaling ceases. For example, in vivo or in vitro treatment of macrophages with cytokine alters their functional response pattern to LPS. However, if the macrophages are washed after cytokine treatment and held in the absence of cytokine for 1–2 days before LPS stimulation, the functional response pattern is essentially identical to that of macrophages that had not been treated with cytokine. A similar reversion to basal macrophage phenotype is observed when human monocyte-derived, immature dendritic cells (DC) are removed from IL-4 + granulocyte macrophage-colony stimulating factor (GM-CSF) and placed in a neutral environment [20
]. We have been able to corroborate this observation with immature DC (CD11c+) derived by IL-4 + GM-CSF treatment of murine BM-derived and peritoneal macrophages (unpublished data). The point is that the majority of type 1 and type 2 cytokines does not seem to induce differentiation of macrophages into stable subsets but rather induces regulatory cascades that transiently alter the functional pattern of response of macrophages.
Macrophages from the lung (interstitial and alveolar), peritoneum, liver (Kupffer cells), and brain (microglia) are usually considered to be separate lineages of macrophages with distinct and unique functions [2
]. Originally, these populations (peritoneal macrophages being the exception) were thought to be maintained during adult life by precursors that seeded the organs during development. Current evidence indicates that the slow turnover of these long-lived populations is maintained, at least in part, by immigration of blood monocytes [2
]. Establishment of these populations as distinct, differentiation lineages is based on the distinct pattern of functional responsiveness and pattern of surface molecules expressed [2
]. There does not appear to be a single membrane molecule that by itself could phenotypically distinguish these tissue macrophage populations. A comparative summary of the functional and phenotypic characteristics of these populations, adapted from Guillemin and Brew [3
], Hanisch [21
], and Laskin et al. [2
], is presented in . The majority of differences is quantitative differences in level of expression of a molecule. Thus, each population displays a unique functional and phenotypic pattern. However, the macrophages within each of these populations do not uniformly display the same functional and phenotypic pattern. Significant heterogeneity exists within the macrophage population of each tissue. For example, the functional phenotype of Kupffer cells depends on their proximity to the portal vein [2
]. Density gradient fractionation of lung interstitial and alveolar macrophage populations yields subpopulations differing in phenotype (e.g., degree of expression of FcγR and class II MHC) and phagocytic function [2
]. Each population is fully capable of changing its functional pattern, as evidenced by the response to infectious or inflammatory insult [2
]. The most dramatic are the microglia that display a ramified morphology and support neuronal survival by producing cytokines such as brain-derived neurotrophic factor and TGF-β [21
]. In vitro or during inflammatory responses in the brain, microglia lose their characteristic morphology, become migratory, and produce abundant oxidative radicals and inflammatory cytokines [3
]. How many of the distinctions between the various tissue macrophage populations are a result of reversible adaptation to the microenvironment of the tissue? If these macrophage populations are placed in identical microenvironments for several days, how many of the functional and phenotypic differences will be retained? As stated in the previous section, each of these tissues provides different environmental stimuli for macrophages. As stable acquisition of a trait is the hallmark of differentiation, we currently are pursuing this line of experimentation in an attempt to determine which of the functional traits of these tissue macrophages are a result of reversible adaptation to the host tissue’s microenvironment and which are a result of differentiation.
Phenotypic and Functional Comparison of Macrophages from Different Tissues
Osteoclasts are the clearest example of a distinct, differentiated lineage of macrophage. Trance (receptor activator of nuclear factor-κB ligand) is a potent inducer of osteoclasto-genesis [24
] and apparently, also the interesting synergistic pairing of TNF-α and TGF-β [25
]. The key genes that are expressed uniquely in osteoclasts are for the TRAP, calcitonin receptor, and cathepsin K [24
]. The end-stage cell is clearly differentiated in that it is the polykaryon product of cell fusion that has developed clear cell-body polarity. However, macrophages can be readily driven to express the osteoclast genes and display bone-resorbing activity () [25
]. Can prefusion, single-cell osteoclasts removed from bone revert to a macrophage functional phenotype given the appropriate environment? The role of prefusion osteoclasts versus macrophages with bone-resorption activity in atherogenic plaque decalcification is addressed below.