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. 1998 Dec;153(6):1849-60.
doi: 10.1016/s0002-9440(10)65699-4.

Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing

E Engelhardt  1 , A Toksoy, M Goebeler, S Debus, E B Bröcker, R Gillitzer
Affiliations

Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing

E Engelhardt et al. Am J Pathol. 1998 Dec.

Abstract

Healing of cutaneous wounds requires a complex integrated network of repair mechanisms, including the action of newly recruited leukocytes. Using a skin repair model in adult humans, we investigated the role chemokines play in sequential infiltration of leukocyte subsets during wound healing. At day 1 after injury, the C-X-C chemokines IL-8 and growth-related oncogene alpha are maximally expressed in the superficial wound bed and are spatially and temporally associated with neutrophil infiltration. IL-8 and growth-related oncogene alpha profiles also correlate with keratinocyte migration and subsequently subside after wound closure at day 4. Macrophage infiltration reaches the highest levels at day 2 and is paralleled by monocyte chemoattractant protein-1 mRNA expression in both the basal layer of the proliferative epidermis at the wound margins and mononuclear cells in the wound area. Other monocyte-attracting chemokines such as monocyte chemoattractant protein-3, macrophage inflammatory protein-1alpha and -1beta, RANTES, and 1309 are undetectable. At day 4, perivascular focal lymphocyte accumulation correlates with strong focal expression of the C-X-C chemokines Mig and IP-10. Our results suggest that a dynamic set of chemokines contributes to the spatially and temporally different infiltration of leukocyte subsets and thus integrates the inflammatory and reparative processes during wound repair.

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Figures

Figure 1.
Figure 1.
Time course of neutrophil, macrophage, and lymphocyte infiltration during human skin wound healing. Infiltrating leukocyte subsets were identified by immunostaining with mAbs anti-CD3 (○), anti-CD68 (•), and anti-neutrophil elastase (NE) (▪), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. The results are expressed as the mean percentage of stained cells per counting field (± SEM) of 3–10 lesion sections for each time point until day 21 after wounding. In addition, the total number of cells per counting field (± SEM) between day 0 and day 21 is shown (▴).
Figure 2.
Figure 2.
Expression of IL-8 and GROα mRNA and localization of neutrophils in serial sections of a human skin wound at 1 day after wounding. In situ hybridization was carried out with 35S-UTP-labeled antisense probes of IL-8 (A, B) and GROα (C, D) and immunostaining with mAb anti-neutrophil elastase (NE) (E), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. The area of pronounced IL-8 mRNA and weak GROα mRNA expression in the superficial wound bed has a strong clustering of NE+ cells. GROα mRNA is also expressed in some single cells of the surrounding dermis (some cells are depicted by arrows in C and D), whereas IL-8 mRNA expression remains restricted to the superficial wound bed. Illumination: bright field ((A, C, E), dark field (B, D) . Bar = 50 μm.
Figure 3.
Figure 3.
Quantification of IL-8 and GROα mRNA expressing cells and neutrophil infiltration during human skin wound healing. In situ hybridization for IL-8 (filled bar) and GROα (empty bar) mRNA expression was carried out with 35S-UTP-labeled antisense RNA probes. The results are expressed as the mean percentage of specific mRNA-expressing cells per counting field (± SEM) of 4–14 lesion sections at each time point until day 21 after wounding. Neutrophils were identified by immunostaining with mAb anti-neutrophil elastase (•), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. The results are expressed as the mean percentage of stained cells per counting field (± SEM) of 3–10 lesion sections for each time point until day 21 after wounding.
Figure 4.
Figure 4.
Expression of MCP-1 mRNA and localization of CD68+ monocytes/macrophages in serial sections of human skin wounds at 1 and 10 days after wounding. In situ hybridization was carried out with 35S-UTP-labeled antisense probes of MCP-1 (A, B, D, E, F), and immunostaining with mAb anti-CD68 (C), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. A-C: serial sections of an incisional human wound at the first day after wounding. Strong MCP-1 expression (A, B) in the wound area correlates with an accumulation of CD68+ macrophages (C). D-F: serial section of an incisional human wound 10 days after wounding. At day 10, MCP-1 is strongly expressed in keratinocytes of the basal layer of the hyperproliferative epidermis surrounding the wound (E, F). The area marked by arrowheads in E is shown in higher magnification (D) to demonstrate expression of MCP-1 in both infiltrating cells in the dermis (d) and in keratinocytes (e = epidermis). Illumination: bright field (A, C, D, E); dark field (B, F). Bar = 100 μm (A, B, C, E, F); bar = 25 μm (D).
Figure 5.
Figure 5.
Quantification of MCP-1 mRNA-expressing cells and infiltration of CD68+ cells during human skin wound healing. In situ hybridization for MCP-1 (filled bar) mRNA expression was carried out with 35S-UTP-labeled antisense RNA probes. Results are expressed as the mean percentage of MCP-1 mRNA expressing cells per counting field (± SEM) of 4–9 lesion sections at each time point until day 21 after wounding. Macrophages were identified by immunostaining with mAb anti-CD68 (▪), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. The results are expressed as the mean percentage of stained cells per counting field (± SEM) of 4–9 lesion sections for each time point until day 21 after wounding.
Figure 6.
Figure 6.
Focal expression of Mig mRNA coincides with the presence of CD3+ lymphocytes during the wound healing process. In situ hybridization was carried out with 35S-UTP-labeled antisense probes of Mig (A, B) and immunostaining with mAb anti-CD3 (C), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. A-C: serial sections of an incisional human wound 7 days after wounding. Mig mRNA-specific signals (A, B) are located mainly in the dermis and are associated with infiltrates of CD3+ lymphocytes (C). Illumination: bright field (A, C), dark field (B). Bar = 50 μm.
Figure 7.
Figure 7.
Expression of MCP-1 mRNA and localization of MCP-1 immunoreactivity in serial sections of human skin wounds at day 7 after wounding. In situ hybridization was carried out with 35S-UTP-labeled antisense probes of MCP-1 (A), and immunostaining with mAb anti-MCP-1 (B), using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate. A, B: serial sections of an incisional human wound at 7 days after wounding. Strong MCP-1 mRNA expression (A) in the wound area correlates with MCP-1 immunoreactivity (B) as depicted by arrows. In addition, there is homogenous (although weaker) nonspecific staining of the epidermis. Bar = 50 μm.
Figure 8.
Figure 8.
Schematic drawing of the time course of chemokine-mediated recruitment of leukocyte subsets during normal healing of incisional human wounds.
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