Selective and specific macrophage ablation is detrimental to wound healing in mice

Abstract

Macrophages are thought to play important roles during wound healing, but definition of these roles has been hampered by our technical inability to specifically eliminate macrophages during wound repair. The purpose of this study was to test the hypothesis that specific depletion of macrophages after excisional skin wounding would detrimentally affect healing by reducing the production of growth factors important in the repair process. We used transgenic mice that express the human diphtheria toxin (DT) receptor under the control of the CD11b promoter (DTR mice) to specifically ablate macrophages during wound healing. Mice without the transgene are relatively insensitive to DT, and administration of DT to wild-type mice does not alter macrophage or other inflammatory cell accumulation after injury and does not influence wound healing. In contrast, treatment of DTR mice with DT prevented macrophage accumulation in healing wounds but did not affect the accumulation of neutrophils or monocytes. Such macrophage depletion resulted in delayed re-epithelialization, reduced collagen deposition, impaired angiogenesis, and decreased cell proliferation in the healing wounds. These adverse changes were associated with increased levels of tumor necrosis factor-alpha and reduced levels of transforming growth factor-beta1 and vascular endothelial growth factor in the wound. In summary, macrophages seem to promote both wound closure and dermal healing, in part by regulating the cytokine environment of the healing wound.

Figure 1

Macrophage ablation during wound healing. DTR transgenic mice and FVB wild-type mice were subjected to excisional wounding and either left untreated (NT) or treated with DT to induce macrophage depletion. Cryosections of wounds collected on days 1 to 10 postinjury were stained with antibodies against F4/80 (top), Ly6C (middle), and Ly6G (bottom) as markers for macrophages, monocytes, and neutrophils, respectively. The number of labeled cells was counted using a ×20 objective and normalized to volume of the wound bed. Data are presented as means ± SE; n = 4 to 6 mice/time point. *P < 0.05 when compared to nontreated controls.

Figure 2

Macrophage ablation results in delayed re-epithelialization. DTR transgenic mice and FVB wild-type mice were subjected to excisional wounding and either left untreated (NT) or treated with DT. Cryosections of wounds collected on days 1 to 10 postinjury were stained with H&E. Representative sections of wounds on day 5 postinjury showing delayed re-epithelialization in DTR-DT mice (middle) compared with DTR-NT mice (top) (images obtained with a ×5 objective). Arrows indicate ends of the migrating epithelial tongues. ep, epithelium; gt, granulation tissue; ml, subcutaneous muscle layer. Dashed line indicates the border between granulation tissue and subcutaneous muscle layer. Scale bar = 0.2 mm. Bottom: the percentage of re-epithelialization [(distance traversed by epithelium)/(distance between wound edges) × 100] was measured in each section by image analysis. Data are presented as means ± SE; n = 4 to 6 mice/time point. *P < 0.05 when compared to nontreated controls.

Figure 3

Macrophage ablation results in reduced collagen deposition. DTR transgenic mice were subjected to excisional wounding and either left untreated (NT) or treated with DT. Cryosections of wounds collected on days 3 to 10 postinjury were stained with Masson’s trichrome. Representative sections of wounds on day 7 postinjury show reduced collagen deposition, indicated by less blue staining, in DTR-DT mice (middle) compared with DTR-NT mice (top) (images obtained with a ×5 objective). Scale bar = 0.2 mm. Bottom: the area stained blue in each section was measured by image analysis and normalized to the area of the wound bed. Data are presented as means ± SE; n = 4 to 6 mice/time point. *P < 0.05 when compared to nontreated controls.

Figure 4

Macrophage ablation results in impaired angiogenesis. DTR transgenic mice were subjected to excisional wounding and either left untreated (NT) or treated with DT. Cryosections of wounds collected on days 3 to 10 postinjury were stained with an antibody against the endothelial cell marker CD31 to assess angiogenesis. Representative sections of wounds on day 5 postinjury showing impaired vessel formation in DTR-DT mice (middle) compared with DTR-NT mice (top) (images obtained with a ×5 objective). Arrows indicate the wound edges. ep, epithelium, gt, granulation tissue, ml, muscle layer. Dashed line indicates border between granulation tissue and subcutaneous muscle layer. Scale bar = 0.2 mm. Bottom: the stained area in the granulation tissue of each section was measured by image analysis and normalized to the area of the wound bed sampled. Data are presented as means ± SE; n = 4 to 6 mice/time point. *P < 0.05 when compared to nontreated controls.

Figure 5

Macrophage ablation results in reduced cell proliferation during wound healing. DTR transgenic mice were subjected to excisional wounding and either left untreated (NT) or treated with diphtheria toxin (DT). BrdU was injected into mice 1 hour before tissue collection, and cryosections of wounds collected on day 7 postinjury were stained with an antibody against BrdU. Representative sections of wounds on day 7 postinjury showing reduced proliferation in DTR-DT mice (middle) compared with DTR-NT mice (top; images obtained with a ×5 objective). Arrows indicate wound edges. Scale bar = 0.2 mm. Bottom: the number of labeled cells in each section was counted and normalized to area of the wound bed. Data are presented as means ± SE; n = 6 mice/group. *P < 0.05 when compared to nontreated controls.

Figure 6

Macrophage ablation results in altered cytokine expression. DTR transgenic mice were subjected to excisional wounding and either left untreated (NT) or treated with DT. Wounds collected on day 5 postinjury were processed for either RNA or protein analysis and levels of TNF-α, TGF-β1, and VEGF mRNA and protein were assessed by RT-PCR and enzyme-linked immunosorbent assay, respectively. Top: densitometric values for target mRNA levels were normalized to those for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and then to similarly normalized values for noninjured control samples. Bottom: protein levels expressed as picograms of protein in the total wound homogenate. Data are presented as means ± SE; n = 4 mice/group. *P < 0.05 when compared to nontreated controls.