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Diabetes 52:2821-2832, 2003
© 2003 by the American Diabetes Association, Inc.

Leptin and Wound Inflammation in Diabetic ob/ob Mice

Differential Regulation of Neutrophil and Macrophage Influx and a Potential Role for the Scab as a Sink for Inflammatory Cells and Mediators

Itamar Goren1, Heiko Kämpfer1, Maurizio Podda2, Josef Pfeilschifter1, and Stefan Frank1

1 Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
2 Zentrum der Dermatologie und Venerologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the role of leptin for the inflammatory response in diabetes-impaired skin repair. We demonstrated, that systemic treatment of diabetic ob/ob mice with leptin blunted polymorphonuclear neutrophil (PMN), but not macrophage influx into the wound site. Closed wounds of leptin-administered mice were characterized by tremendous numbers of macrophage within the granulation tissue. In line, leptin supplementation potently attenuated epithelium-derived CXC- but not CC-chemokine expression. PMNs were preferentially located in the scab, but macrophages predominantly resided within the wound stroma of the animals. The scabs of nonhealing wounds were most likely to serve as sinks for bioactive inflammatory mediators, which were still capable to drive gene expression in keratinocytes in vitro. Differential effects of leptin on PMN and macrophage axes of inflammation must be indirect, as topical administration of leptin onto wounds of ob/ob mice did not reduce PMN influx into the wounded areas. Moreover, caloric-restricted, pair-fed ob/ob mice were characterized by impaired healing conditions that were associated with persisting PMNs. Interestingly, we documented the absence of leptin receptor expression in human diabetic foot ulcers. Thus, we show that leptin might function as a regulatory link between the endocrine and the immune system in the context of skin repair.

Skin repair represents a highly dynamic process involving fibroplasia, angiogenesis, and reepithelialization. It is now well established that wound inflammation is central to these processes and pivotal for tissue regeneration (1,2). The first line of defense is represented by the polymorphonuclear subset of immune cell, also known as polymorphonuclear neutrophils (PMNs), which contribute to defense against a variety of infectious agents and production of inflammatory cytokines (3). A second phase of wound inflammation is characterized by a subsequent infiltration of macrophages into the wound, and depletion of wounds from macrophages resulted in a delayed healing (4,5). However, the importance of wound inflammation for skin repair is double-faced. Thus, chronic wound situations in mice, as well as humans, are associated with conditions of prolonged and dysregulated inflammation. The numbers of macrophages are markedly elevated in human chronic leg ulcers (6,7), although these immune cells appeared to not be activated (8). Accordingly, impaired healing in the genetically diabetic db/db mouse model was associated with a sustained presence of chemokines and a prolonged persistence of PMNs and macrophages at the wound site (9).

The diabetic db/db mouse and its functional counterpart, the obese (ob/ob) mouse, were characterized initially as diabetes-obesity syndromes (10). The ob gene encodes a 16-kDa protein named leptin (11), whereas the db gene encodes the leptin receptor (ObR), which turned out to be functionally inactive in db/db mice, thus mediating a leptin resistance to these animals (12). Both mouse mutants not only are obese, but also develop a complex metabolic syndrome. The animals exhibit a severe dysregulation of reproductive and hormonal traits (13) as well as disturbances of hematopoietic and immune functions (14). Thus, the severe wound healing disorders in ob/ob mice have long been regarded as a direct consequence of their disturbed metabolic phenotype. In contrast to the disturbed metabolic state of the animals, recent work performed in our laboratory strongly implicated a direct effect of leptin in skin repair (15).

We have reported a persisting inflammation during impaired healing conditions in skin tissue in leptin-resistant db/db mice (9). Nevertheless, a functional clue between impaired healing and sustained wound inflammation could not be stated. Here, we focused on inflammatory conditions during wound repair in ob/ob mice, as these animals are not leptin resistant, but respond to systemically and also topically administered leptin (15,16). We found a differential regulation of PMN and macrophage axes of wound inflammation after leptin-supplementation in ob/ob mice, which turned out to result from a heretofore unknown leptin-mediated coupling of endocrine and immune functions in these animals.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Female C57BL/6J (wild-type) or C57BL/6J-ob/ob mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and maintained under a 12-h light-dark cycle at 22°C until they were 8 weeks of age. At this time they were caged individually, monitored for body weight, and wounded as described below.

Treatment of mice.
Murine recombinant leptin (Calbiochem, Bad Soden, Germany) was injected intraperitoneally once a day at 8 A.M. (2 µg/g body wt) in 0.5-ml PBS per injection for 13 days. For local treatment, wounds of mice were covered with 1 µg leptin in 20 µl PBS twice a day (8 A.M. and 8 P.M.). Control mice were treated with PBS alone.

Wounding of mice.
Mice were wounded as described previously (17,18). For each experimental time point, tissue from four wounds each from four animals (n = 16 wounds, RNA analysis) and from two wounds each from four animals (n = 8 wounds, protein analysis) were combined and used for RNA and protein preparation. Nonwounded back skin from four animals served as a control. All animal experiments were carried out according to the guidelines and with the permission from the local government of Hessen, Germany.

RNA isolation and RNase protection analysis.
RNA isolation and RNase protection assays were carried out as described previously (18,19). The murine cDNA probes were cloned using RT-PCR. The probes corresponded to nt 816–1,481 (for lipocalin, accession no. X81627), nt 425 (exon 1) to 170 (exon 2) (for lysozyme M, M21047), nt 481–739 (for interleukin [IL]-1ß, NM008361), nt 541–814 (for tumor necrosis factor [TNF]-{alpha}, NM013693), nt 163–317 (for GAPDH, NM002046), nt 181–451 (for macrophage inflammatory protein [MIP]-2, NM009140), nt 50–290 (for keratinocyte-derived chemokine [KC], NM008176), nt 63–323 (for macrophage chemoattractant protein [MCP]-1, NM011333), nt 139–585 (for vascular endothelial growth factor [VEGF], S38083), or nt 40–298 (for RANTES, NM013653) of the published sequences.

Enzyme-linked immunosorbent assay.
Wound lysates were prepared as described previously (17,20). Total protein (10–50 µg diluted in lysis buffer [20] to a final volume of 50 µl) from skin lysates was subsequently analyzed for the presence of immunoreactive MIP-2, MCP-1, IL-1ß, and TNF-{alpha} by enzyme-linked immunosorbent assay (ELISA) using the Quantikine murine ELISA kits (R&D systems, Wiesbaden, Germany). Fifty microliters of supernatants from control and cytokine (2 nmol/l IL-1ß, 2 nmol/l TNF-{alpha})-stimulated murine PAM 212 keratinocytes were analyzed for MIP-2 by ELISA (R&D systems, Wiesbaden, Germany).

Human wound biopsies.
Wound biopsies were selected from surgery in normal individuals (normal wound), from chronic venous leg ulcers in patients without diabetes (ulcus cruris), and from diabetic foot ulcers in patients with diabetes (diabetic foot ulcers). Histological sections were analyzed from three patients in each group. All wounds were not debrided for at least 2 weeks before biopsy. All nondiabetic individuals had no medication, besides compression in the case of venous leg ulcers, for 2 weeks for biopsy, The diabetic patients were under oral antidiabetic therapy, and the diabetic foot ulcers were not of the typical neuropathic type.

Immunohistochemistry.
Complete murine wounds were isolated from the back, bisected, and frozen in tissue-freezing medium. Six-micrometer frozen sections were subsequently analyzed using immunohistochemistry as described (17). Antisera against murine F4/80 antigen (Serotec, Eching, Germany), murine Gr-1 (Ly-6G) (Pharmingen, Hamburg, Germany), and murine cyclooxygenase (COX)-2 (Santa Cruz, Heidelberg, Germany) were used for immunodetection. Human paraffin skin sections were analyzed for the leptin receptor ObR (Santa Cruz, Heidelberg, Germany), or macrophage (Dako, Hamburg, Germany). Formal consent was obtained from the subjects after the nature of the procedure was explained.

In situ hybridization.
Thirteen days after injury, wounds were excised and fixed in 4% paraformaldehyde (PFA)/PBS solution. Six-micrometer serial sections were subsequently analyzed for MIP-2 mRNA or MCP-1 mRNA expression using the HybriProbe in situ hybridization assay (Biognostik, Göttingen, Germany). MIP-2- or MCP-1-specific oligonucleotides were derived from the published sequences (see above).

Leptin, glucose, insulin, cortisol, and corticosterone levels.
Blood glucose levels were determined using the Accutrend sensor (Roche Biochemicals, Mannheim, Germany), serum leptin and insulin by ELISA (Crystal Chemicals, Chicago), and serum cortisol and corticosterone by radioimmunoassay (RIA) (ICN, Enschede, Germany) as described by the manufacturer.

Cell culture.
Quiescent murine PAM 212 keratinocytes were stimulated with the cytokines IL-1ß (1 ng/ml), TNF-{alpha} (5 ng/ml), or interferon (IFN)-{gamma} (100 units/ml) as indicated. After 24 h of stimulation, conditioned cell culture supernatants were analyzed by ELISA. Cytokines were from Roche (Mannheim, Germany). Scab lysates were prepared in lysis buffer (20) and given to quiescent keratinocytes in a final concentration of 750 µg/ml.

Western blot analysis.
Wound and cell culture lysates were prepared as described previously (17,20). Fifty micrograms of total protein from skin or cellular lysates was separated using SDS-gel electrophoresis. COX-2 and P-Tyr705-STAT3 protein were detected using polyclonal antibodies (anti-COX-2 sc-1746; Santa Cruz, Heidelberg, Germany; and anti-P-Tyr705-STAT3; New England Biolabs, Bad Schwalbach, Germany).

Statistical analysis.
Data are shown as means ± SD. Data analysis was carried out using the unpaired Student’s t test with raw data and the Sigma Plot statistics computer program (Jandel Scientific, Erkrath, Germany).


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Systemic leptin supplementation reverted the disturbed metabolic phenotype in ob/ob mice.
First, we determined the potency of leptin to improve the metabolic syndrome as well as the impaired wound healing conditions observed in ob/ob mice. ob/ob mice were injected intraperitoneally with recombinant leptin (2 µg/g body wt, once a day) for 13 days. Three hours after injection, we determined serum leptin levels (320 ± 164 ng/ml). Additionally, to circumvent systemic effects of leptin, wounds from a second group of mice were directly covered with leptin (1 µg/20 µl PBS, twice a day). Following systemic leptin treatment, blood glucose levels readjusted to normal levels (100 mg/dl) (Fig. 1A). Moreover, a 13-day systemic leptin regimen resulted in a marked loss of body weight in wounded ob/ob mice, which could not be observed after topical treatment (Fig. 1B). In line with recently published data from our laboratory, we found a complete reepithelialization of the injured areas in both the systemically and the topically treated mice. This observation could be well documented by the loss of the scab from reepithelialized wounds (Fig. 1C and D). Additionally, we assessed wound diameters in PBS- and leptin-treated ob/ob mice. We determined an average wound size of 5.5 ± 0.8 mm (for PBS-treated mice) compared with 2.8 ± 0.7 mm (for leptin-treated mice) (n = 24 wounds, P < 0.01). A detailed wound size kinetics (day 1–8 postwounding) for leptin-treated ob/ob mice versus control animals has been published recently (16).



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  		FIG. 1. Administered leptin is biologically active. A: Regulation of blood glucose levels 3 h after systemic application of recombinant leptin. **P < 0.01 vs. PBS-treated animals. Bars indicate the mean ± SD obtained from nine individual animals (n = 9). B: ob/ob mice were treated as indicated. After 13 days, the body weight of the animals was monitored. **P < 0.01; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from nine individual animals (n = 9). C: Presence of scab-covered wounds after 13 days of treatment with PBS or leptin as indicated. **P < 0.01 vs. PBS-treated animals. Bars indicate the mean ± SD of wounds (n = 54) obtained from nine individual animals. D: Status of wounds in PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice after 13 days.

 
Improved skin repair after systemic leptin treatment was characterized by normalization of PMN, but not macrophage influx at the wound site.
In this study, we focused on the role of inflammation under conditions of leptin-driven skin repair. First, we assessed PMN and macrophage populations at the wound site by determining the constitutively expressed molecular markers lipocalin (for PMNs) and lysozyme M (for macrophages). Impaired healing conditions in PBS-treated and untreated ob/ob mice revealed an increase in PMNs (Fig. 2A and B, left panels) and macrophages (Fig. 2A and B, right panels) during late repair (days 7 and 13 postwounding). This observation was confirmed by immunohistochemistry staining specific protein markers for PMN (GR-1) or macrophage (F4/80) (Fig. 2C, as indicated). Systemic administration of leptin, surprisingly, led to a strong decline only in PMN (Fig. 2A and B, left panels), but not in macrophage (Fig. 2A and B, right panels), cellular numbers at late time points of repair. Granulation tissues of wounds from leptin-treated mice were depleted from PMNs (Fig. 2C, upper right panel), but had tremendous numbers of macrophages (Fig. 2C, lower right panel). However, these cells were most likely to reflect an inactivated state. As shown in Fig. 2D, macrophages represented the COX-2-expressing cell type in the developing stroma of wounds (left panel). As we observed equal numbers of macrophages in the wound stroma for PBS- and leptin-injected mice, the massive expression of COX-2 mRNA in PBS-treated ob/ob mice probably reflected an activated state of the macrophage, which was markedly attenuated by leptin-treatment of the animals. Moreover, subsequent analysis of both the tissue and scab wound compartments revealed the predominant presence of PMNs within the wound scab, whereas macrophages tended to accumulate in the underlying wound tissue (Fig. 2E).



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  		FIG. 2. PMN and macrophage (M{phi}) influx into the wound is differentially regulated by leptin. A: RNase protection assay demonstrating the infiltration kinetics of PMNs and macrophages into wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification of lipocalin (marker for PMNs) and lysozyme M (marker for macrophages) mRNA (x-fold induction as compared with control skin) is shown in B. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 48) isolated from animals (n = 12) from three independent animal experiments. C: In situ determination of PMNs (upper panels, GR-1) and macrophage (lower panels, F4/80) in 13-day wounds of PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice as indicated. D: Macrophage-derived expression of COX-2 within the wound granulation tissue (left panel). RNase protection assay for COX-2 mRNA expression in PBS-treated (i.p.) and leptin-treated (i.p.) treated ob/ob mice. Two independent experimental series are shown (nos. 1 and 2). Note that every single experimental point represents 16 wounds (n = 16) isolated from four individual mice. E: RNase protection assay demonstrating the distribution of PMNs and macrophages in the wound tissue and wound scab compartments of PBS-injected (i.p.) mice.

 
Leptin supplementation strongly attenuated epithelium-derived expression of PMN- but not macrophage-specific chemokines.
As a next step, we hypothesized that differences in PMN-specific CXC- and macrophage-specific CC-chemokine expression might contribute to the differential infiltration behavior of PMNs or macrophages into wounded sites of leptin-treated ob/ob mice. Evidently, systemic administration of leptin strongly attenuated mRNA expression for the PMN attractants MIP-2 (Fig. 3A, left panels, and B) and keratinocyte-derived chemokine (KC) (Fig. 4A, right panels). In situ hyridization (ISH) in 13-day wounds revealed the developing neoepithelium as the MIP-2-expressing wound compartment (Fig. 3C, lower right panels). Whereas leptin completely shut off the MIP-2-specific mRNA signals in keratinocytes, the epithelia from PBS-injected control mice were still markedly immunopositive for MIP-2 mRNA (Fig. 3C). Note the well-developed granulation tissue and multilayered neoepithelium that was associated with an improved skin repair in leptin-administered mice (Fig. 3C, lower left panel). The overall decline in MIP-2 mRNA (Fig. 3B) was paralleled by a reduction of MIP-2 protein in total wound tissue of leptin-treated animals (Fig. 3D, left panel). To further analyze a compartmentalization of MIP-2 expression, we subsequently separated the scab from the underlying wound tissue before ELISA (Fig. 3D, middle and right panels). Interestingly, the scab isolated from delayed-healing wounds was characterized by high concentrations of this chemokine (Fig. 4D, right panel). Moreover, we also assessed MIP-2 protein levels from the few scabs that remained on wounds obtained from the leptin-treated ob/ob mice (see Fig. 1C). As these few scab-covered wounds of leptin-administered mice resembled the status of wounds from untreated control animals, we found, not unexpectedly, comparable amounts of MIP-2 for both healing conditions (Fig. 3D, right panel). These data suggest that the scab might function as a sink or reservoir for epithelial-derived MIP-2. Moreover, topical treatment of wounds with leptin clearly failed to attenuate PMN influx directly (Fig. 3E, upper panel), although we observed an activation of the signal transducer and activator of transcription (STAT)-3 transcription factor at the wound site (Fig. 3E, lower panel). Cytokine-stimulated cultured murine PAM 212 keratinocytes did not respond to leptin exposure with an attenuated MIP-2 expression, although PAM keratinocytes nicely responded to a leptin stimulus with activation of the janus kinase (JAK)/STAT signaling cascade (Fig. 3F).



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  		FIG. 3. Regulation of PMN-specific chemokine expression by leptin. A: RNase protection assay showing the expression of MIP-2 (left panels) and KC (right panels) in wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification of MIP-2 mRNA expression (x-fold induction as compared with control skin) is shown in B. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 48) isolated from animals (n = 12) from three independent animal experiments. C: MIP-2-specific in situ hybridization of 13-day wounds isolated from PBS-treated (i.p) and leptin-treated (i.p.) ob/ob mice as indicated (lower panels). A control hybridization using a nonspecific oligonucleotide is shown in the upper panels. D: MIP-2-specific ELISA analyses from lysates of total wounds (wound tissue and remaining scabs), wound tissue (without scabs), and scabs isolated from PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice are shown as indicated. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 18) isolated from nine individual animals (n = 9). E: Topical treatment of wounds with leptin did not influence PMN influx as shown by RNase protection analysis for PMN-specific lipocalin mRNA (upper panel). STAT3 activation in 5-day wounds of ob/ob mice that were treated as indicated (lower panel). Every data point depicted represents eight wounds from four individual animals, which have been pooled before analysis. F: Quiescent PAM 212 keratinocytes were stimulated as indicated. MIP-2 protein from the cell culture supernatants was determined by ELISA. *P < 0.05 vs. nonstimulated control cells. n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from three (n = 3) independent experiments (left panel). PAM 212 keratinocytes are responsive to a leptin stimulus, as leptin mediates an activation of STAT3 in the cells (right panel).

 


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  		FIG. 4. Regulation of macrophage (M{phi})-specific chemokine expression by leptin. A: RNase protection assay showing the expression of MCP-1 (left panels) and Rantes (right panels) in wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification of MCP-1 mRNA expression (x-fold induction as compared with control skin) is shown in B. n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 48) isolated from animals (n = 12) from three independent animal experiments. C: MCP-1-specific in situ hybridization of 13-day wounds isolated from PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice as indicated. A control hybridization using a nonspecific oligonucleotide is shown in the left panel. D: MCP-1-specific ELISA analyses from lysates of total wounds (wound tissue and remaining scabs), wound tissue (without scabs), and scabs isolated from PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice are shown as indicated. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 18) isolated from nine individual animals (n = 9).

 
Unlike the situation reported here for CXC chemokines, we observed no changes in mRNA expression for MCP-1 (Fig. 4A, left panels, and B) and Rantes (Fig. 5A, right panels) in PBS- and leptin-treated as well as in untreated ob/ob mice. Keratinocytes of the neoepithelium did not respond to a leptin-stimulus with reduction of MCP-1 mRNA expression (Fig. 4C, middle panel). In contrast to MIP-2 regulation, we found a persisting MCP-1 protein expression in wound tissue (Fig. 4D, middle panel). High levels of MCP-1 in isolated scabs again indicated that the wound crust might function as a source or sink for protein mediators (Fig. 4D, right panel). Obviously, this phenomenon may present an explanation of reduced MIP-2 (Fig. 4) and MCP-1 (Fig. 5) protein levels in total wound tissue. Leptin-treatment accelerated wound repair and treated animals lost their wound scabs earlier in time than PBS-injected control mice (Fig. 1C).



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  		FIG. 5. Presence of proinflammatory at the wound site in PBS- and leptin-treated mice. RNase protection assay showing the expression of TNF-{alpha} (A) and IL-1ß (C) in wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification of TNF-{alpha} and IL-1ß mRNA expression (given as arbitrary PhosphoImager PSL counts) is shown in the right panels. **P < 0.01; *P < 0.05 as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 18) isolated from nine animals (n = 9). TNF-{alpha}- (C) and IL-1ß-specific (D) ELISA analyses from lysates of total wounds (wound tissue and remaining scabs), wound tissue (without scabs), and scabs isolated from PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice are shown as indicated. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 18) isolated from nine individual animals (n = 9).

 
The scab as a sink for proinflammatory cytokines.
As a next step, we determined macrophage-derived proinflammatory mediators in the PBS- and leptin-treated experimental setups. We found a massive decrease in TNF-{alpha} and IL-1ß mRNA and protein levels for both healing conditions (Fig. 5A–D, left panels). The overall reduction of both cytokines was probably due to the loss of scabs from improved healing conditions (Fig. 1C). Nevertheless, TNF-{alpha} and IL-1ß proteins were much more enriched in isolated scabs than in the underlying wound tissue (Fig. 5B and D).

The scab is a source of bioactive mediators.
To investigate the possibility that the scab might function as a source for bioactive molecules during repair, we isolated fresh scab tissue from control mice on day 5 after wounding. As we had determined concentrations of ~200 pg for IL-1ß and TNF-{alpha} in 50 µg of isolated scab protein (Fig. 5B and D, right panels), we now added a total of 750 µg of scab protein to stimulate PAM 212 keratinocytes with a similar amount of scab-derived cytokines as compared with recombinant cytokine control conditions (f.c. 1–3 ng/ml) (Fig. 6). Interestingly, we were able to observe an induction of VEGF mRNA expression in cultured keratinocytes (18) in the presence of scab proteins, clearly indicating that the scab indeed included bioactive protein mediators that were capable to drive gene expression in keratinocytes (Fig. 7).



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  		FIG. 6. The scab represents a source of bioactive mediators. Quiescent murine PAM 212 keratinocytes were incubated with recombinant cytokines (1 ng/ml IL-1ß, 5 ng/ml TNF-{alpha}, and 100 units/ml IFN-{gamma}) or homogenized scab protein (f.c. 750 µg homogenate/ml) as indicated and analyzed for VEGF expression by RNase protection assay. **P < 0.01; *P < 0.05; n.s., not significant compared with control; #P < 0.05 as indicated by the brackets. Bars indicate the mean ± SD obtained from three (n = 3) independent cell culture experiments.

 


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  		FIG. 7. Reduced food intake does not improve wound repair. A: Regulation of body weight, wound closure, blood glucose, serum insulin, cortisol, and corticosterone (as indicated) in PBS- or leptin-treated and by pair-fed (caloric restricted) ob/ob mice. **P < 0.01; *P < 0.05; n.s., not significant as indicated by brackets. Bars indicate the mean ± SD obtained from four individual animals (n = 4). B: RNase protection assay showing the expression of TNF-{alpha}, IL-1ß, MIP-2, MCP-1, lipocalin (PMN), and lysozyme M (macrophage) in wounds of PBS- or leptin-treated and pair-fed (calorie-restricted) ob/ob mice as indicated. A quantification of mRNA expression by PhosphoImager (Fuji) analysis is shown. **P < 0.01; *P < 0.05; n.s., not significant as indicated by the brackets. Bars indicate the mean ± SD obtained from wounds (n = 16) isolated from four animals (n = 4).

 
Leptin, not reduced food intake, is responsible for improved healing.
Here, we determined to what extent changes in immune cell influx, cytokines, and skin repair might also be influenced by leptin-mediated reduction of food intake. To this end, we performed a wounding study in PBS- or leptin-treated and in a control group of pair-fed (caloric-restricted) ob/ob mice, which received equal amounts of nutriment as leptin-treated animals. Pair-feeding indeed resulted in a reduction of body weight comparable to the leptin-treated group of animals. However, overall wound repair was not improved (Fig. 7A, upper panels). Food restriction significantly reduced serum insulin, but not glucose, cortisol, or corticosterone levels (Fig. 7A). Analysis of cytokines (TNF-{alpha} and IL-1ß), chemokines (MIP-2 and MCP-1), and immune cells (PMNs, macrophages) revealed that caloric restriction did not contribute to beneficial effects of leptin treatment (Fig. 7B). Moreover, it became clear that systemic levels of immune-modulatory glucocorticoids were not significantly changed by those amounts of leptin that were able to regulate immune function differentially and improve skin repair (Fig. 7A, lower panels).

Absence of the leptin receptor ObR in diabetic foot ulcers.
Finally, we intended to make a case for leptin-mediated movements in chronic healing conditions in humans as well. Immunohistochemistry revealed that the leptin receptor was located in keratinocytes of the wound margins and within the dermis in undisturbed human wounds (Fig. 8A and B). Interestingly, and in clear contrast to chronic wound conditions in ob/ob mice (15), we could not detect ObR protein in wound margin epithelia of venous as well as diabetic ulcers. More importantly, whereas immune cells within the granulation tissues of venous ulcers expressed the ObR (Fig. 8C and D), we could not find any staining within diabetic foot ulcer tissue (Fig. 8E and F), although both conditions were characterized by large numbers of infiltrating macrophages and also PMNs (data not shown). Additionally, ObR-responsive cells within the scabs of diabetic ulcers did not represent macrophages, which failed to accumulate in large numbers within the scab (Fig. 8G and H).



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  		FIG. 8. Expression of the leptin receptor in normal wounds and ulcers in humans. Immunohistochemical staining of leptin receptor (ObR) expression (A–G) and macrophages (H) in undisturbed human wound (A and B), ulcus cruris (C and D), and diabetic foot ulcer (E–H) biopsies. Particularly strong immunopositive signals are indicated by arrows. d, dermis; gt, granulation tissue; seb, sebaceous gland; wme, wound margin epithelium.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chronic wound-healing disorders represent a serious problem of growing clinical importance. There are a number of known pathological conditions that severely interfere with a coordinated and successful wound closure. Three prototypic chronic wounds are of clinical importance: decubitus or pressure, venous, and the diabetic ulcers (21). Moreover, disturbances in the inflammatory and proliferation phases also delay acute-wound repair from progressing to later stages of healing (22,23). A naturally occurring mouse mutant, the db/db mouse, which suffers from a diabetes-obesity syndrome (10), has long been used as an animal model system to investigate wound healing deficiencies during acute repair. The most important function of leptin is its inhibitory ability to suppress appetite. Thus, leptin reverses the diabetes-obesity syndrome in leptin-deficient ob/ob mice and also attenuates food intake and increases activity in healthy mice (24–26). Additionally, the ob/ob and db/db mice develop syndromes such as inhibition of reproductive functions (14), hormonal disturbances related to the thyroid, hypothalamic-pituitary-adrenal, and growth hormone axes (13), as well as alterations in the hematopoietic and immune systems (14). It is reasonable to suggest that the complexity of the diabetes-obesity syndrome in these animals is most likely to interfere with normal skin repair at various levels of regulation and must not be functionally connected to the hyperglycemia of the animals.

Leptin administration led to serum leptin levels that were about fourfold higher than those described for the db/db mice model (27). However, data from this study demonstrate that changes in serum glucocorticoid and insulin levels did not contribute to leptin-mediated improvement of repair. However, our leptin/pair-fed experiments strongly suggested that attenuation of blood glucose might play a major role in acceleration of healing. Accordingly, some of the proposed mechanisms that drive hyperglycemia-induced pathological changes, such as glycation end products, hyperosmolarity, abnormal myoinositol metabolism, oxidant formation, or protein kinase C activation (28–32), might be improved by leptin treatment. However, leptin-driven skin-tissue responses in ob/ob mice could be transferred, at least partially, to normal wound-healing conditions, as reepithelialization of wounded sites also could be further accelerated by leptin in healthy mice (15). We found the leptin receptor ObR to be expressed in wound-margin keratinocytes of undisturbed human wounds. Nevertheless, a potential role for leptin in acute skin repair in humans has to be determined. However, circulating leptin levels have been described to be present in humans and even to rise in direct correlation to the body’s white fat tissue (33,34). Thus, diabetes-impaired healing disorders in humans develop in the presence of leptin, suggesting a leptin resistance or insensitivity in peripheral tissues. In line, we observed the absence of leptin receptor expression in diabetic foot ulcers. It appears that deficiencies in leptin responses in ob/ob mice as a model system and humans might be determined by different mechanisms, thus highlighting a potential shortcoming of the diabetes-impaired ob/ob mouse model.

This study provides evidence that endocrine mediator molecules may strongly influence tissue movements in injured areas of the body. A leptin-driven improvement of inflammation during repair was not altogether unexpected, as we had hypothesized a normalization of inflammatory conditions that might contribute to leptin-accelerated repair. Again, we found a severe dysregulation of the macrophage subset of immune cells in not only delayed-healing wounds, but, surprisingly, in also well-developed wounds of leptin-treated animals. Leptin here mediates improved tissue movements, as shown convincingly for epithelial sites (15), in the presence of tremendous numbers of persisting macrophages (this study). Evidently, the number of circulating macrophages was increased about fourfold in ob/ob mice (35). This over-representation might contribute to the large numbers of macrophages observed in wounds of these animals. It is noteworthy that the MIP-2 and MCP-1 chemokines and the inflammatory mediators TNF-{alpha} and IL-1ß were predominantly enriched in the wound scab. Early studies with wound dressings suggested that the formation of a scab in air-exposed wounds was superior in supporting granulation tissue formation when compared with wound dressings (36,37). Accordingly, the release of bioactive components from the scab might provide one explanation of the beneficial effects of the scab for repair.

Evidently, a systemic effect of leptin is supported by the observation that topical treatment of skin wounds did not result in an attenuation of PMN infiltration behavior. The observation that endocrine signals interfere with the immune cell population of skin wounds might be of importance for chronic wound conditions in humans. Histological analyses of chronic leg ulcers in diabetic humans indicated increased numbers of granulocytes and macrophages which were located in the center and at the edges of the wound (6,7). However, the observed macrophages in chronic diabetic wounds were probably in an inactive state (8). This observation is consistent with the absence of leptin receptor (ObR) expression in diabetic foot ulcers. In fact, debridement of diabetic ulcers impacts inflammatory cells, but does not always solve the problem of impaired healing conditions. It is reasonable to suggest that inactivation of macrophages might be due to a downregulation of several receptors for extracellular signals that control macrophage activation during repair. Data from this study clearly indicate that cells (keratinocytes, immune cells) must have lost their potency to respond to a leptin stimulus. Moreover, loss of ObR expression might be confined to diabetic conditions, as venous ulcers were characterized by ObR-expressing cells within the granulation tissue.

However, macrophages isolated from ob/ob mice express phenotypic abnormalities such as increased superoxide and hydrogen peroxide production as well as elevated IL-6 and COX-2 levels in vitro (38). Our in vivo data support this in vitro situation. Leptin-treatment triggered a downregulation of COX-2 expression at the wound site, as COX-2 expression completely disappeared in the presence of unaltered numbers of resident macrophages. Thus, the observed macrophage population of nontreated and treated ob/ob mice (which did not differ in total numbers) might be distinguished markedly by their inflammatory potencies. The role of leptin as a pivotal mediator of inflammatory processes is strengthened by a recent study of intestinal inflammation in ob/ob mice (39). However, the outcome of leptin-controlled intestinal inflammation in ob/ob mice completely contrasted the situation in skin repair in this animal model, as leptin replacement in ob/ob mice increased the severity of intestinal inflammation by augmentation of PMN influx and cytokine production. It is surprising that the same individual leptin-replaced animal would, however, attenuate MIP-2 and PMN influx at sites of inflammation in the context of skin repair (this study). It is reasonable to suggest leptin as an important endocrine signaling molecule that is tightly connected to the immune system, but the final outcome of leptin’s regulatory potency on different axes of immune responses might be fundamentally different for different organ systems.


   ACKNOWLEDGMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 553, grant FR 1540/1–1).

We thank M. Kock for help with the animal experiments, L. Raspe for critically reading the manuscript, and N. Kämpfer-Kolb and K. Weinelt for excellent technical assistance.

Address correspondence and reprint requests to Dr. Stefan Frank, Pharmazentrum Frankfurt, Institut für Allgemeine Pharmakologie und Toxikologie Klinikum der JW Goethe-Universität Frankfurt/M., Theodor-Stern-Kai 7, D-60590 Frankfurt/M., Germany. E-mail: s.frank{at}em.uni-frankfurt.de

Received for publication December 6, 2002 and accepted in revised form August 12, 2003

Abbreviations: COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; JAK, janus kinase; KC, keratinocyte-derived chemokine; MCP, macrophage chemoattractant protein; MIP, macrophage inflammatory protein; PFA, paraformaldehyde; PMN, polymorphonuclear neutrophil; RIA, radioimmunoassay; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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