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The last decade has witnessed great advances in our knowledge and understanding of the mononuclear phagocyte system but many of the recent findings, about myeloid trafficking and fate, observed mainly in lymphoid organs,1-3 have raised more questions than they have answered about the vagaries and functional subtleties of myeloid immune cells in nonlymphoid organs such as the liver. As a consequence, there is ongoing debate about what constitutes a dendritic cell (DC) and what constitutes a macrophage, particularly in nonlymphoid organs.4-6 From these debates increasing consensus has evolved about functional definitions of these two cell types (Table 1).3, 6 Equally, there is little agreement about simple defining molecular markers that have been used historically to discriminate DCs from macrophages.4-6 In liver, defining markers for DCs and macrophages show substantial areas of overlap (Table 2). For example, it is now widely accepted that CD11b and F4/80 (classical macrophage markers) do not always define macrophages, and CD11c and MHC II (classical DC markers) do not always define DCs. There is also great debate about whether there are true lineages of distinct bone marrow (BM) precursors that give rise to functionally distinct myeloid cell subpopulations in the peripheral organs, as opposed to lineages that give rise to cells with tremendous plasticity (and therefore overlapping functions). As conventionally understood, macrophages are myeloid cells and are critical effectors and regulators of inflammation and the innate immune response, whereas DCs are myeloid or plasmacytoid cells that initiate and regulate the highly pathogen-specific immune response and are central to immunological memory and to tolerance (Table 1).3 What is emerging is that our terminologies, steeped in tradition and history, are now inadequate to define the many functions and subpopulations of the myeloid leukocyte system as we currently see it. Despite the current difficulties with definition, however, it has become clear that among the resident myeloid cells (formerly known as the reticuloendothelial system), which are present in every organ, including the liver, there is an admixture of cells that perform DC functions and cells that perform macrophage functions.

Table 1. Comparison of Recognized DC and Macrophage Functions (Adapted from Ref. 6)
  • *

    Overlapping functions. Italicized = demonstrated functions in liver.

Macrophage
Classic functions
 • phagocytosis of tissue debris
 • phagocytosis of pathogens
 • Steady state tissue homoeostasis (growth factors and cell clearance)
 • effectors of tissue injury and disease progression
 • activated phenotypes exhibit polarized functions: e.g., wound healing vs. tissue injury*
Contemporary additions
 • phagocytosis to remodel matrix
 • support for organ growth in development and cell fate decisions
 • paracrine role in angiogenesis and epithelial regeneration
 • polarized function can be fibrogenic
 • potent source of chemokines and cytokines*
 • mediate antiinflammatory/immunosuppressive effects through innate responses*
Dendritic cell
Classic functions
 • phagocytosis and endocytosis of antigen in peripheral tissues
 • migration to lymph nodes
 • antigen presentation to T lymphocytes
Contemporary additions
 • antigen presentation within the peripheral organs to re-stimulate or modify infiltrating T lymphocytes
 • mediate antiinflammatory/immunosuppressive effects through innate responses and modulation of T lymphocytes (peripheral tolerance)*
 • activated phenotypes exhibit distinct polarized functions*
 • potent source of chemokines and cytokines*
Table 2. Comparison of Mouse Hepatic Macrophage and DC Populations Defined by Molecular Markers
  • *

    NK, T cell, and B cell lineage excluded.

  • **

    Plasmacytoid DCs, not myeloid DCs.

  • ? = not tested.

Resident myeloid cells
Kupffer cells (sinusoidal macrophages) (refs. 19, 20)
 A. F4/80+, CD68+, CD11b-, CD11c+, MHCII?, CD8α?
 B. F4/80+, CD68- CD11b+/- MHCII?, CD8α?
 C. F4/80+, CD68+ CD11b+/- CD11c- MHCII?, CD8α?
 D. F4/80- CD11b+ CD11c+/- MHCII?, CD8α?
Dendritic cells (refs. 21, 22)
 A. CD11chigh*, B220-, CD11b+, CD8α+/- F4/80? CD68? MHCIIhigh
 B. CD11cint*, B220-, CD8α+/-, CD11b+ F4/80? CD68? MHCII-/high
 C. ** CD11cint*, B220+ CD11b- CD8α+/- MHCIIint F4/80? CD68?
Inflammatory myeloid cells (both DCs and macrophages) (Refs. 3, 7, 9, 12)
CD11b+ F4/80+/-, CD11c+/- Ly6C+/-, CX3CR1+, CCR2+

In 2005 a significant advance was made in understanding the role of myeloid cells in both progression and resolution of carbon tetrachloride (CCl4)-mediated liver injury with fibrosis, a rodent model for liver fibrosis/cirrhosis. The investigators used a novel transgenic mouse (Cd11b-DTR), expressing the Diphtheria toxin receptor (DTR) under the control of the CD11b promoter, to ablate CD11b+ myeloid cells simply by systemic injection of a drug (DT).7 The DT injection ablated monocytes and inflammatory monocyte-derived CD11b+, F4/80+ cells in the injured liver, which were called macrophages by the investigators. The ablation had no effect on resident (F4/80+) Kupffer cells. The DT injection also had no effect on granulocytes, including neutrophils or natural killer (NK) cells. Using this ablative strategy, the investigators found that, whereas inflammatory macrophages drive fibrosis during chronic injury under the influence of CCl4, inflammatory macrophages also orchestrate resolution of fibrosis by digesting and phagocytosing the pathological matrix, once the CCL4 toxicant was withdrawn (Fig. 1).7 The type I collagenase, matrix metalloproteinase (MMP)-13 (but not MMP-2, MMP-8, or MMP-14), was identified as a major macrophage-derived matrix degrading enzyme in this resolving process (Fig. 1).8 The investigators did not, however, investigate the expression of CD11c or MHC II in the cells they ablated.

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Figure 1. Cartoon showing recruited inflammatory myeloid cells resolving hepatic scar tissue. Current studies from Jiao et al.9 show a discrete population of CD11b+, CD11chigh inflammatory myeloid cells that are FLT3-L-sensitive, contribute to scar resolution after liver injury in mice, and generate the Collagen-IV cleaving enzyme MMP-9. These effector cells likely derive from FLT3-L-sensitive precursors in BM or spleen. On the other hand, previous studies7, 8 have identified a population of CD11b+, F480+/low inflammatory myeloid cells that traffic from the circulation and contribute to scar resolution in the liver by generating the Collagen-I cleaving enzyme MMP-13.

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In studies published in this current addition of Hepatology, Jiao et al.9 now report a critical role for DCs in resolution of fibrosis following CCl4-mediated liver fibrosis and identify MMP-9 as a key effector enzyme in DCs (Fig. 1). MMP-9 is a gelatinase that cleaves type IV collagen and elastin, also constituents of pathological matrix, and is widely reported as a major effector molecule in macrophages.10 In order to draw these conclusions the investigators used a different transgenic mouse to ablate myeloid cells, CD11c-DTR (DTR under regulation of the CD11c promoter),11 and they focused on the resolution phase after CCl4-mediated fibrosis during which accumulated scar tissue is resorbed and remodeled. To the uninitiated, these investigations might seem to have uncovered a completely novel pathway of resolution of fibrosis. However, we now appreciate that CD11c and CD11b are poor discriminators of cells with DC functions and macrophage functions (Table 2). Moreover, in peripheral organs such as the liver it seems that the vast majority of myeloid cells express both CD11b and CD11c to varying degrees.6, 9, 12 Therefore, it may simply be that the investigators have unwittingly ablated the same cells in the liver that the studies from 2005 ablated and have chosen to call them DCs, whereas the investigators in 2005 chose to call them macrophages. In keeping with that line of thought, the processes of digesting, phagocytosing, and clearance of matrix and its constituents are widely recognized as macrophage-type functions rather than DC-type functions in many organs (Table 1), and MMP-9 is widely reported in the literature as a macrophage effector, not a DC effector molecule.10 As such, therefore, the cells they have identified are not DCs, rather they are CD11c-DTR-sensitive macrophages.

However, if we step back from the controversies and problems with nomenclature of peripheral organ tissue effector cells, the investigators have identified a CD11b+, CD11chighCD11c-DTR-sensitive subpopulation of liver inflammatory myeloid cells (IMCs) that specifically are responsible for resolution of scarring, in part by producing MMP-9 (Fig. 1). They clearly identify this subpopulation in the resolving liver among other myeloid leukocytes that are presumably either not directly contributing to scar resolution or are contributing to scar resolution by other mechanisms (it is not possible to determine to what extent fibrosis-resolution is halted by ablation of this subpopulation). What is more, stimulated by their initial findings, the investigators go on to either administer Fms-like tyrosine kinase 3-ligand (FLT3-L) or transfer DCs (FLT3-L expanded, CD11c+ myeloid splenocytes) to mice with liver fibrosis to augment the resolution process. These studies have important therapeutic potential and provide new insight into our understanding of myeloid cell functions in inflammatory responses.

FLT3-ligand has been recognized as a colony-forming factor in hematopoietic stem cell and BM precursors for 20 years. It was identified as a growth factor, stimulating monocyte, macrophage, and immature DC expansion through ligation of CD135 (FLT).13-15 Myeloid cells have at least three critical growth factors: CSF-1 (M-CSF), CSF-2 (GM-CSF), FLT-3L, and possibly vascular endothelial growth factor A (VEGFA). In vitro, culture of myeloid BM precursors with CSF-1 or CSF-2 has been traditionally used to generate macrophages, whereas CSF-2 ± IL4 has been used to generate DCs. More recently, FLT3L has been used to generate DC cultures. Regardless of whether FLT3-L stimulates DC expansion or macrophage expansion, or both, the investigators clearly show that FLT3L has important therapeutic potential by expanding IMCs that effect repair of the liver after injury. This key finding should be placed in context. Investigators in Australia recently reported that administration of CSF-1 to mice with kidney injury stimulated repair and resolution processes.16 Therefore, from a purely therapeutic standpoint, administration of critical myeloid growth factors (FLT3-L or CSF-1) at the appropriate phases of disease resolution may be simple ways to stimulate the reparative forces present within the myeloid system and prevent the progression to chronic disease.

Several other important questions arise from these studies: (1) Where does the endogenous reparative subpopulation of CD11b+, CD11chigh IMCs come from? Is it from a population of resident myeloid cells in the liver, or, more likely, does it traffic to the liver as inflammatory monocytes, or does it come from a pool of cells in the spleen?17 Is it monocyte derived or derived from another more primitive BM precursor?18 (2) Does this endogenous CD11b+, CD11chigh CD11c-DTR-sensitive population of IMCs have phagocytic function, or does it simply release factors that degrade matrix? (3) Does the CD11b+, CD11chigh CD11c-DTR-sensitive population have other reparative or antiinflammatory functions in liver injury? (4) Are there distinct subpopulations of MMP-9- and MMP-13-expressing reparative IMCs that operate synergistically to mediate scar resolution? (Fig. 1) Although there is no head-to-head examination of this question, and it is quite possible that MMP-9-expressing and MMP-13-expressing reparative IMCs are the same cell subpopulation, several pointers in the new findings by Jiao et al. suggest that there may be two distinct subpopulations: (i) FLT3L-stimulated leukocytes do not up-regulate MMP-13, whereas they do up-regulate MMP-9; (ii) a large population of CD11b+CD11c+/low myeloid cells are not ablated in CD11c-DTR mice. One might expect these CD11b+CD11c+/low cells to be ablated by CD11b-DTR mice and likely harbor the IMCs that are expressing high levels of MMP-13. Further work is clearly necessary.

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