Introduction

Long-lasting, nonhealing wounds of different etiologies present a major problem in different disciplines of medicine, and are a major source of disabilities, morbidity, and mortality. Causes of poor wound healing and factors leading to enhanced wound healing are of major interest but are poorly understood.

Wound healing is a complex but well-orchestrated process involving cellular and extracellular components leading to at least partial reconstitution of the wounded tissue (Singer and Clark, 1999). The initiation of wound healing involves sequential completion of coagulation, inflammation, cellular proliferation and migration, angiogenesis, matrix synthesis, remodeling, and wound contraction (Martin, 1997; Stadelmann et al., 1998; Baker and Leaper, 2000). Cytokines such as platelet-derived growth factor, epidermal growth factor, keratinocyte growth factor, transforming growth factor beta, fibroblast growth factor, ILs, chemokines, and their respective receptors coordinate different steps of the wound healing. In the past 10 years only few of these growth factors, for example, platelet-derived growth factor have been approved by the Food and Drug Administration for treatment of patients (Rees et al., 1999; Fu et al., 2005).

Although the mechanisms by which granulocyte–macrophage colony-stimulating factor (GM-CSF) influences wound healing have only been partly elucidated, GM-CSF has been analyzed in diverse animal and preclinical studies in this respect. GM-CSF has been employed in the treatment of poorly healing wounds of diverse etiologies with some success (El Saghir et al., 1997; Canturk et al., 1999; Jaschke et al., 1999; Stagno et al., 1999; Voskaridou et al., 1999; Siddiqui et al., 2000; Jorgensen et al., 2002; Mery et al., 2004). GM-CSF mRNA is detectable shortly after epidermal activation, for example, through wounding or application of a tumor promoter like TPA (Robertson et al., 1994; Pastore et al., 1997). It is hypothesized that GM-CSF influences the biologic activities of several hematopoietic and nonhematopoietic cells that take part in the tissue repair (Hancock et al., 1988; Bussolino et al., 1991; Shephard et al., 2004). In this respect, GM-CSF influences major aspects of the repair process, that is, inflammation, reepithelialization, and neovascularization. Impairment of any of these processes would result in chronic nonhealing wounds.

To identify the mechanisms underlying the in vivo effects of GM-CSF on wound healing, we modified the activity of GM-CSF in transgenic animals. A double mutant ligand (K14E/E21K) used to generate the K10-GM-CSF antagonist transgenics, had been shown to bind to the -chain of GM-CSF receptor but failed to stimulate the signal-transducing -chain of the receptor complex (Altmann and Kastelein, 1995). These animals exhibited high GM-CSF antagonist levels in skin extracts. It could be shown that the GM-CSF signaling is blocked in the skin of these animals as the antagonist was able to suppress GM-CSF-dependent keratinocyte proliferation and LC accumulation in vivo (Mann et al., 2001b).

In a previous study, we could show the beneficial effects of GM-CSF in wound healing in a transgenic mouse model overexpressing GM-CSF in the basal layer of the epidermis. It was proposed that the beneficial effects of GM-CSF are not only the direct effects but also indirect effects via the induction of secondary cytokines (Mann et al., 2001a). In the present study conducted in transgenic mice overexpressing an antogonist of GM-CSF in the epidermis, these mice exhibited delayed scab rejection and reepithelialization and extensive fibrosis in the newly formed matrix. These effects result from reduced keratinocyte proliferation, reduced neovascularization, and extensive deposition of collagen. Therefore, GM-CSF is not only beneficial for wound repair but it is also required for timely and high-quality wound healing.

Results

Scab rejection is delayed in GM-CSF antagonist transgenics. In total, 10 transgenic and wild-type control animals were wounded as described in Materials and Methods. Gross appearance of wounds, incidence of infection, and scab rejection were documented daily. At 3–4 days after wounding, wounds in the GM-CSF antagonist transgenics had a glistening moist appearance and exhibited only thin scabs, whereas the wounds in the wild-type animals were dry and covered with a proper scab at this point.

Also the differences in scab rejection were significant. GM-CSF antagonists exhibited a delayed onset of scab rejection as compared to the wild-type controls (Figure 1). At day 7 postwounding, 20% of the wild-type control animals had lost their scabs, whereas the first scabs were lost on day 8 (10%) in the antagonist transgenics. By day 9 after wounding only 40% of the scabs were shed in antagonist transgenics as compared to 60% wild-type controls. In GM-CSF antagonists, which had lost the scab at 8–9 days after wounding, the wound was oozing and got a dry, scaly appearance in the following couple of days. Antagonist transgenics needed as many as 14 days till all the scabs were shed, whereas all the wild-type animals had lost the scabs by day 11 after wounding. Also, major differences were observed in the gross appearance of the wounds after scab rejection. At 2 weeks after wounding, the middle of the wound had a scaly crust in the GM-CSF antagonist transgenics, whereas in the control group the wound area could only be recognized due to the lower hair density.

Figure 1.

Scab rejection postwounding. Ten transgenic animals overexpressing GM-CSF antagonist in skin and their littermate controls were wounded and observed on a daily basis for scab rejection. Note the delayed scab rejection in the transgenic animals as compared to the wild-type controls.

Full figure and legend (11K)

GM-CSF antagonist transgenics exhibit delayed reepithelialization of wounds. Reepithelialization is an important aspect of the repair process. GM-CSF overexpression has been associated with enhanced reepithelialization and tissue remodeling (Mann et al., 2001b). Sections through the middle of the wounds were cut at different time points after wounding and stained routinely with hematoxylin and eosin, and the extent of reepithelialization was documented. Significant differences were observed in the extent of reepithelialization between GM-CSF antagonist transgenics and wild-type controls. Complete reepithelialization was delayed in the antagonist transgenics in comparison to the control animals as exemplified in Figure 2a–d. Also on a quantitative basis, these animals exhibited significantly delayed reepithelialization as compared to the wild-type animals (Figure 2e).

Figure 2.

Progress in wound healing. (a–d) Wild-type control animals as well as animals overexpressing a GM-CSF antagonist were wounded. Wounds were removed and prepared for hematoxylin/eosin staining. Five animals per genotype and time point were taken for the analysis. (a and c) Stained sections from control animals and (b and d) represent sections from the transgenic animals at 3 days (a and b) and 10 days (c and d) postwounding. Note the differences in scab formation and scab rejection, granulation tissue formation, and reepithelialization between the transgenic and the control animals. Bars: 200 m. (e) Histograph showing % reepithelialization. HE stained sections were evaluated in a double-blinded manner for the extent of reepithelialization by morphometry and % reepithelialization was quantified. Note the longer time needed by the antagonists for the complete closure of wound.

Full figure and legend (143K)

Furthermore, differences in the granulation tissue formation were observed in these groups. Even though abundant granulation tissue formation in the antagonist transgenics was observed at earlier time points after wounding, a delay in tissue remodeling was observed at later time points. This was exhibited by the presence of scab, high dermal cellularity, and absence of hair follicles indicative of reduced wound contraction, in the newly formed tissue (Figure 2d). Also, pronounced inflammation and monocytic lymphocytes infiltrate in the wound area in the antagonist transgenics at 3 days postwounding were observed (Figure 2b). A closer analysis of the infiltrating cells exhibited twice as many neutrophils in the granulation tissue in the antagonist transgenic animals as compared to the wild-type controls at day 7, postwounding, whereas the numbers of mast cells were reduced in the antagonist transgenics at this time point. No significant differences in the numbers of T cells and macrophages could be detected (data not shown).

GM-CSF antagonists exhibit diminished mitotic indices. Under normal conditions, mitotic indices in the basal layer of the interfollicular epidermis in the GM-CSF antagonists are comparable to those in the wild-type controls (Mann et al., 2001b). The GM-CSF antagonists behaved comparable to the wild-type controls at earlier time points after wounding so that a maximum on proliferation rate was achieved on day 3 after wounding (Figure 3a, b, and e). At 10 days after wounding, proliferation is still higher in these animals, albeit significantly lower as compared to the wild-type controls (Figure 3c–e).

Figure 3.

Keratinocyte proliferation rates during wound healing. (a–d) Mice were injected intraperitonially with BrdUrd and killed after a labeling period of 2 hours. Sections were stained using an anti-BrdUrd antibody as described in the Materials and Methods. Five animals per genotype and time point were analyzed. Shown are (a and c) control animals and, (b and d) transgenic animals at (a and b) 3 and (c and d) 10 days postwounding, respectively. Both controls and transgenic animals exhibit a proliferative burst at 3 days postwounding; however, 10 days postwounding the transgenics exhibited significantly less numbers of proliferating kerationcytes. Bars: 200 m. (e) Histograph showing the numbers of keratinocyte S-phase nuclei at the wound margin. For this purpose BrdUrd-labeled nuclei were counted at the wound margin and related to 100 total basal cells. Enhanced but comparable rate of proliferation can be observed at 3 days postwounding in both transgenics and the control animals. At 10 days postwounding, the antagonist transgenics exhibit significantly lower proliferation rates (^*P<0.05).

Full figure and legend (122K)

GM-CSF antagonists exhibit reduced neovascularization. In order to determine the extent of microvessel formation, sections through the middle of the wounds were stained with an antibody directed towards CD31 (Figure 4). CD31 stained vessels were counted and expressed as numbers per field. Whereas the numbers of microvessels in the antagonist transgenics before wounding are comparable to those in the wild-type animals, the numbers at 7 and 10 days after wounding are significantly reduced (Figure 4) pointing towards a defect in neovascularization.

Figure 4.

Extent of neovascularization during the wound healing. Sections through the middle of the wounds were stained with a CD31-specific antibody for the detection of newly formed blood vesicles. The samples were photographed and the numbers of microvessels per field were counted. Five animals per genotype and time point were analyzed. Note the significantly lower numbers of microvessels in the antagonist transgenic at all the observed time points after wounding.

Full figure and legend (23K)

Wound healing in GM-CSF antagonists is accompanied by extensive fibrosis. Since the wounds in antagonist transgenics exhibited an obvious scar and extensive fibrosis in H&E stained sections 14 days after wounding, the sections were stained with Masson's trichrom stain for collagen deposition. As shown in the Figure 5, GM-CSF antagonist transgenics exhibit late granulation tissue with extensive extracellular matrix deposition (b) whereas the wild-type controls exhibited only marginal extracellular matrix deposition at the same time point (a). This exhibits that absence of GM-CSF is detrimental during wound healing.

Figure 5.

Detection of fibrosis in the repair process. Section through the middle of wounds were cut and stained with Masson's Trichrome stain. Note the extensive collagen deposition (Blue staining) in the (b) antagonist transgenics at 14 days postwounding as compared to the (a) control animals. Bars: 200 m.

Full figure and legend (227K)

Discussion

The process of wound healing is a precisely regulated sequence of cellular and biochemical events leading to at least partial restoration of tissue integrity after injury. In many cases, however, the proliferative response produces a fibrotic scar so that the injured organ is patched rather than restored to its original state (Singer and Clark, 1999). GM-CSF exerts its effects on wound healing through multiple mechanisms, affecting nearly every aspect of the repair process. It is synthesized by many cellular components of the repair process like keratinocytes, fibroblasts, endothelial cells, macrophages, and dendritic cells (Bussolino et al., 1991; Caux et al., 1992; Braunstein et al., 1994; Breuhahn et al., 2000). In this respect, it is a possible candidate and has already been used in various preclinical studies aimed to heal chronic ulcers of different origins (El Saghir et al., 1997; Canturk et al., 1999; Stagno et al., 1999; Voskaridou et al., 1999; Mery et al., 2004).

To identify the mechanisms underlying the in vivo effects of GM-CSF on wound healing, we modified the expression of GM-CSF in transgenic animals. In a previous study we could show that overexpression of GM-CSF in the epidermis of transgenic mice lead to an early healing of full thickness cutaneous wounds (Mann et al., 2001a). In the present study, transgenic mice overexpressing a GM-CSF antagonist were used and the process of wound healing was investigated. Here, we confirm not only the important role of GM-CSF in the wound healing but also that the absence of this growth factor is disadvantageous for the repair process.

GM-CSF has been shown to enhance keratinocyte proliferation, migration, and survival in vivo and in vitro (Hancock et al., 1988; Braunstein et al., 1994; Breuhahn et al., 2000). Under normal circumstances, GM-CSF antagonist transgenics did not show any alterations in the mitotic indices in the epidermis as compared to the wild-type mice, but in double transgenic mice overexpressing both GM-CSF and GM-CSF antagonist, GM-CSF antagonist was able to suppress the GM-CSF-dependent keratinocyte hyperproliferation and LC accumulation of the keratinocytes (Mann et al., 2001b). Therefore, lower keratinocyte proliferation rate in the antagonist transgenics at day 10, postwounding in comparision to the wild-type controls may be attributed to the suppression of GM-CSF-dependent keratinocyte proliferation by the antagonist. This is even true despite the fact that more wounds were reepithelialized in the wild-type animals at this time point as compared to the transgenic animals.

Neovascularization of the wound is of utmost importance for the supply of vital components necessary for the repair process. In our previous study, increased neovascularization in mice overexpressing GM-CSF could be correlated to enhanced wound healing (Mann et al., 2001a). On the opposite mice overexpressing an antagonist of GM-CSF exhibit reduced microvessel formation and delayed healing, suggesting a direct relationship between GM-CSF and neovascularization. GM-CSF has been shown to be an important factor for endothelial cell proliferation and survival (Bussolino et al., 1991). GM-CSF-dependent activation of IB kinase activity leading to subsequent activation of NFB has been shown to be an important factor for endothelial cell proliferation and survival in vitro (Ebner et al., 2003). Plenz et al. (2003) could show that GM-CSF deficiency leads to an altered composition of the vascular collagenous matrix suggesting an importance of GM-CSF for the maintenance of vessel wall integrity and resilience.

Fibroblasts that invade the granulation tissue as the repair progresses differentiate into myofibroblsts by acquiring smooth muscle cell characteristics (Tomasek et al., 2002). These express -smooth muscle actin, which confers contractile capacity to the wound tissue. Also, myofibroblasts are the principle source of extracellular matrix components in the granulation tissue (Oda et al., 1990). An important observation in the present study was the presence of extensive fibrosis in the newly formed tissue. Relation of GM-CSF and fibrosis has been discussed controversially in literature. On the one hand, intradermal transgenic expression of GM-CSF in rats by using a replication-deficient adenoviral vector lead to upper dermal fibrosis besides inducing other dermal and epidermal pathologies (Xing et al., 1997a). Also, overexpression of GM-CSF in lungs lead to an irreversible fibrotic response in rats (Xing et al., 1997b). On the other hand, mice deficient in GM-CSF developed pulmonary fibrosis besides developing pulmonary alveolar proteinosis (Dranoff et al., 1994; Stanley et al., 1994). In another study, a direct relation was observed in diminished levels of GM-CSF and bleomycin-induced pulmonary fibrosis suggesting a protective role for GM-CSF in bleomycin-induced pulmonary fibrosis (Christensen et al., 2000). We suggest that GM-CSF plays a complex role in fibrosis depending upon the circumstances and exact mechanism needs to be investigated further. Many cytokines, for example, platelet-derived growth factor, epidermal growth factor, keratinocyte growth factor, transforming growth factor beta, fibroblast growth factor and their respective receptors have also been shown to influence wound healing positively by coordinating different steps of the repair process (Rees et al., 1999; Fu et al., 2005). However, retarded wound healing in the GM-CSF antagonist transgenics demonstrates that there is no redundancy for GM-CSF mediated neovascularization, keratinocyte proliferation, and tissue remodeling.

Materials And Methods

Transgenic mouse line Tg2-Ant, overexpressing an antagonist of GM-CSF (K14E/E21K) under the control of bovine keratin 10 promoter, has been described previously (Mann et al., 2001b). These animals were maintained as hemizygotes on an FVB/N background. Animals were housed and bred in the animal facility of the Johannes Gutenberg University, Mainz, Germany. All experimental procedures were in accordance with the governmental and institutional guidelines.

Wounding protocol and analysis

Age and sex matched transgenic animals and their littermate controls, aged 8–10 weeks were anesthetized by avertin. The backs were shaved using electrical clippers and swabbed with 70% ethanol. A single 5 mm diameter full-thickness wound was inflicted on the mid-dorsum using a biopsy punch. The wound was left without sutures or dressing. Subsequently, the animals were housed individually. Four to five animals were analyzed per time point and experiments were repeated.

The wounds were observed once per day for infection, formation, and rejection of scab. At days 1, 3, 7, 10, and 14 postwounding, the animals were euthanized and wound areas cleaned carefully. The wound including 3–4 mm marginal nonwounded skin was carefully excised. The wound was divided into two halves and processed for cryosections and paraffin embedding.

Histology and immunohistology

Sections were cut at 5 m thickness, mounted on slides, and stained routinely with hematoxylin and eosin. Evaluation of the wounds was carried out by two investigators including a senior pathologist (P.S.). Extent of reepithelialization was quantified by morphometry and expressed as "% reepithelialization". Granulation tissue formation, cellularity, architecture, and inflammation were also evaluated. An antibody directed to CD31 (PECAM-1, Pharmingen, Germany) was used to detect microvessels in the wound bed. Sections were also stained routinely for fibrosis with Masson's Trichrome stain (Sigma). May–Grünwald–Giemsa staining was performed to evaluate the granulation tissue and naphthol AS-D chloroacetate estrase staining was performed to detect neutrophilic granulocytes and their precursors. The sections were also stained for T-cell subsets (CD4 and CD8) and for macrophages (CD11b) using specific antibodies (Pharmingen, Germany).

Determination of mitotic indices

Proliferation indices were conveyed by BrdUrd labeling using In Situ Cell Proliferation Kit, FLUOS (Roche, Mannheim, Germany). Mice were injected intraperitonially with 30 g BrdUrd per gram body weight and killed after a labeling period of 2 hours. Fixation and processing of the samples was carried out according to the manufacturer's instructions. The sections were further treated with anti-FITC-AP antibody (DakoCytomation, Denmark) and signal detection was carried out using Fast Red (Roche, Mannheim, Germany) as substrate. The sections were counterstained with Mayer's hemalum (Merck, Darmstadt, Germany). The sections were subsequently photographed and number of labeled nuclei in the basal layer of the epidermis at the wound margin was related to total 100 basal cells. Values were obtained from at least four animals per group and time point.

Statistics

Data are shown as meanSD. Student's t-test was used to analyze the statistical significance in the differences between two groups. Values of P<0.05 were considered significant.

Conflict Of Interest

The authors state no conflict of interest.

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Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft Grant BL 300/2-1.