Elsevier

Cytokine

Volume 71, Issue 2, February 2015, Pages 409-412
Cytokine

Short Communication
Contributions of cell subsets to cytokine production during normal and impaired wound healing

https://doi.org/10.1016/j.cyto.2014.09.005Get rights and content

Highlights

  • Cell subsets were isolated from excisional wounds of diabetic and non-diabetic mice.

  • Macrophages were the dominant producers of pro- and anti-inflammatory cytokines.

  • Macrophages were also significant producers of healing-associated factors.

  • Neutrophils and lymphocytes contributed to pro- and anti-inflammatory cytokines.

  • Non-leukocytic cells were significant producers of healing-associated factors.

Abstract

The objective of this study was to determine the relative contributions of different cell subsets to the production of cytokines and growth factors during normal and impaired wound healing. Cells were isolated from wounds of non-diabetic and diabetic mice and separated by magnetic sorting into neutrophils/T cells/B cells (NTB cell subset), monocytes/macrophages (Mo/Mp subset) and non-leukocytic cells including keratinocyte/fibroblast/endothelial cells (KFE subset). On both per cell and total contribution bases, the Mo/Mp subset was the dominant producer of pro-inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 in both non-diabetic and diabetic mice and was a significant producer of vascular endothelial cell growth factor (VEGF)-A, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1. The NTB subset was also a significant producer of TNF-α and IL-10 whereas the KFE subset contributed significant amounts of VEGF, IGF-1 and TGF-β1. Sustained production of pro-inflammatory cytokines and impaired production of healing-associated factors were evident in each subset in diabetic mice. These data will be useful for further experimental and modeling studies on the role of cell subsets in wound healing as well as for designing therapeutic strategies for improving healing.

Introduction

Skin wound healing involves a series of overlapping events involving hemostasis, inflammation, new tissue formation and remodeling. A number of cell subsets contribute to healing, including keratinocytes, fibroblasts, endothelial cells and inflammatory cells [1], [2], [3], [4]. The activity of these cells is regulated by cytokines acting in both autocrine and paracrine fashion to bring about efficient healing. In the setting of diabetes, impaired healing is associated with persistent inflammation, reduced angiogenesis and granulation tissue formation, and impaired closure [5], [6], [7], [8], [9], [10]. These defects are associated with persistent production of pro-inflammatory cytokines and reduced release of pro-angiogenic and pro-healing factors.

A number of studies have reported that multiple cellular sources may contribute to cytokine production in wounds. The majority of these studies involve either immunohistochemical assessment of tissue sections or cell culture studies using primary cells or cell lines [1], [3], [4], [11]. However, these methods are not optimal for determining the relative contribution of cell subsets to wound cytokine levels. The objective of this study was to isolate cells directly from wounds and measure cytokine release from these cells ex vivo to establish the relative contributions of different cell subsets to the production of cytokines and growth factors during normal and impaired wound healing.

Section snippets

Animals

Non-diabetic db/+ and diabetic db/db mice on a C57Bl/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). Experiments were performed on 12–16 week-old mice. All experimental procedures were approved by the Animal Care Committee at the University of Illinois at Chicago.

Excisional wounding

Each mouse was anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and its dorsum was shaved and cleaned with betadine and then alcohol swab. Four 8 mm excisional wounds

Results and discussion

Wound cells were analyzed on days 5 and 10 following excisional wounding, which correspond to the inflammatory and proliferative phases, respectively, in non-diabetic mice. In non-diabetic mice, Mo/Mp were present in the largest numbers on day 5, whereas KFE cells predominated on day 10 (Fig. 1a). In diabetic mice, the NTB and Mo/Mp cell subsets were present at similar levels which were higher than that of the KFE subset on both days 5 and 10, demonstrating the inability to progress through the

Acknowledgments

This study was supported by the National Institutes of Health (R01GM092850). The authors thank Dr. Luisa DiPietro, University of Illinois at Chicago, for critical comments on a previous draft of this manuscript.

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