The anti-granulocyte receptor-1 (Gr-1) mAb, RB6-8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6-8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6-8C5 may deplete not only neutrophils but also other Gr-l+ (Ly6C+) cells. This study describes the use of an Ly6G-specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6-8C5 reduced blood neutrophils and Gr-1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr-1+ monocytes. Plasma TNF-alpha in endotoxemia was increased 20-fold by RB6-8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF-alpha staining in brefeldin-treated wound leukocytes was increased by pretreatment with RB6-8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6-8C5, as it preserves non-neutrophil Gr-1+ cells depleted by the anti-Gr-1 antibody. The loss of non-neutrophil Gr-1+ populations in RB6-8C5-treated animals is associated with increased TNF-alpha responses, suggesting these cells may function to suppress TNF-alpha production.

The phenotype of wound macrophages has not been studied by direct examination of these cells, yet macrophages recruited to sites of injury are described as alternatively activated macrophages, requiring IL-4 or IL-13 for phenotypic expression. This study characterized wound macrophage phenotype in the PVA sponge wound model in mice. Eighty-five percent of wound macrophages isolated 1 day after injury expressed Gr-1, but only 20% of those isolated at 7 days expressed this antigen. Macrophages from 1-, 3-, and 7-day wounds expressed markers of alternative activation,including mannose receptor, dectin-1, arginase 1,and Ym1, but did not contain iNOS. Day 1 wound macrophages produced more TNF-alpha, more IL-6, and less TGF-beta than Day 7 wound macrophages. Wound macrophages did not produce IL-10. The cytokines considered necessary for alternative activation of macrophages,IL-4 and IL-13, were not detected in the wound environment and were not produced by wound cells.Wound macrophages did not contain PStat6. Wound fluids inhibited IL-13-dependent phosphorylation of Stat6 and contained IL-13Ralpha2, a soluble decoy receptor for IL-13. The phenotype of wound macrophages was not altered in mice lacking IL-4Ralpha, which is required for Stat6-dependent signaling of IL-4 and IL-13.Wound macrophages exhibit a complex phenotype,which includes traits associated with alternative and classical activation and changes as the wound matures.The wound macrophage phenotype does not require IL-4 or IL-13.

Recent results call for the reexamination of the phenotype of wound macrophages and their role in tissue repair. These results include the characterization of distinct circulating monocyte populations with temporally restricted capacities to migrate into wounds and the observation that the phenotype of macrophages isolated from murine wounds partially reflects those of their precursor monocytes, changes with time, and does not conform to current macrophage classifications. Moreover, findings in genetically modified mice lacking macrophages have confirmed that these cells are essential to normal wound healing because their depletion results in retarded and abnormal repair. This mini-review focuses on current knowledge of the phenotype of wound macrophages, their origin and fate, and the specific macrophage functions that underlie their reparative role in injured tissues, including the regulation of the cellular infiltration of the wound and the production of transforming growth factor-␤ and vascular endothelial growth factor. The resolution of injury through replacement by scar has been preserved in evolution and applies to virtually all soft tissues in mammals. A healed wound is the result of the activities of cells that constitute what can be conceptually described as a wound organ. This transient neoorgan is assembled at the time and site of injury and disassembled once repair is complete, leaving in place a scar as evidence of the repair process. The parenchyma of the wound organ is composed of a temporally changing assembly of inflammatory cells, neovessels, fibroblasts and myofibroblasts, regenerating nerves, and other cells specific to the site of the injury (eg, keratinocytes, osteocytes, and hepatocytes).Two recent developments led to this review of wound macrophages. First, studies in both humans and rodents have demonstrated the existence of diverse subpopulations of circulating monocytes with, at least in mice, distinct and time-restricted abilities to migrate into wounds and other sites of tissue injury (Figure 1). These immediate precursors of wound macrophages are required for repair by scarring in organs as diverse as skin, heart, liver, and kidney. Second, the concept that macrophages involved in tissue repair express the alternatively activated phenotype first proposed by Gordon 1 has been retained in the literature.2 Results from studies of macrophages isolated from wounds, however, contest this paradigm by demonstrating that they are not alternatively activated. Wound macrophages exhibit phenotypes that partially reflect those of their monocyte precursors, change with time, and fail to be adequately described by macrophage classifications based on activation profiles or functional capacity (Figure 2). 3This review addresses the origin and fate of wound macrophages, their regulatory interactions with other cellular populations in the wound, and the roles of macrophagederived transforming growth factor-␤ (TGF-␤) and vascular endothelial growth factor (VEGF) in the regulation of wound fibrosis an...