Differential Regulation of Neutrophil and Macrophage Inﬂux...

Leptin and Wound Inflammation in Diabetic ob/ob MiceDifferential Regulation of Neutrophil and MacrophageInflux and a Potential Role for the Scab as a Sink forInflammatory Cells and MediatorsItamar Goren,

1Heiko Kampfer,

1Maurizio Podda,

2Josef Pfeilschifter,

1and Stefan Frank

1

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 neu-

trophil (PMN), but not macrophage influx into the

wound site. Closed wounds of leptin-administered mice

were characterized by tremendous numbers of macro-

phage within the granulation tissue. In line, leptin

supplementation potently attenuated epithelium-de-

rived CXC- but not CC-chemokine expression. PMNs

were preferentially located in the scab, but macro-

phages 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 ex-

pression 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 ul-

cers. Thus, we show that leptin might function as a

regulatory link between the endocrine and the immune

system in the context of skin repair. Diabetes 52:

2821–2832, 2003

Skin repair represents a highly dynamic processinvolving fibroplasia, angiogenesis, and reepithe-lialization. It is now well established that woundinflammation is central to these processes and

pivotal for tissue regeneration (1,2). The first line ofdefense is represented by the polymorphonuclear subsetof immune cell, also known as polymorphonuclear neutro-phils (PMNs), which contribute to defense against a vari-ety of infectious agents and production of inflammatorycytokines (3). A second phase of wound inflammation ischaracterized by a subsequent infiltration of macrophagesinto the wound, and depletion of wounds from macro-phages resulted in a delayed healing (4,5). However, theimportance of wound inflammation for skin repair isdouble-faced. Thus, chronic wound situations in mice, aswell as humans, are associated with conditions of pro-longed and dysregulated inflammation. The numbers ofmacrophages are markedly elevated in human chronic legulcers (6,7), although these immune cells appeared to notbe activated (8). Accordingly, impaired healing in thegenetically diabetic db/db mouse model was associatedwith a sustained presence of chemokines and a prolongedpersistence of PMNs and macrophages at the wound site(9).

The diabetic db/db mouse and its functional counter-part, the obese (ob/ob) mouse, were characterized initiallyas diabetes-obesity syndromes (10). The ob gene encodesa 16-kDa protein named leptin (11), whereas the db geneencodes the leptin receptor (ObR), which turned out to befunctionally inactive in db/db mice, thus mediating a leptinresistance to these animals (12). Both mouse mutants notonly are obese, but also develop a complex metabolicsyndrome. The animals exhibit a severe dysregulation ofreproductive and hormonal traits (13) as well as distur-bances of hematopoietic and immune functions (14). Thus,the severe wound healing disorders in ob/ob mice havelong been regarded as a direct consequence of theirdisturbed metabolic phenotype. In contrast to the dis-turbed metabolic state of the animals, recent work per-formed in our laboratory strongly implicated a direct effectof leptin in skin repair (15).

We have reported a persisting inflammation during im-paired healing conditions in skin tissue in leptin-resistantdb/db mice (9). Nevertheless, a functional clue between

From 1Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt am Main, Germany; and 2Zentrum der Dermatologieund Venerologie, Klinikum der Johann Wolfgang Goethe-Universitat, Frank-furt am Main, Germany.

Address correspondence and reprint requests to Dr. Stefan Frank, Pharma-zentrum Frankfurt, Institut fur Allgemeine Pharmakologie und ToxikologieKlinikum der JW Goethe-Universitat Frankfurt/M., Theodor-Stern-Kai 7,D-60590 Frankfurt/M., Germany. E-mail: [email protected].

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

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

© 2003 by the American Diabetes Association.

DIABETES, VOL. 52, NOVEMBER 2003 2821

impaired healing and sustained wound inflammation couldnot be stated. Here, we focused on inflammatory condi-tions during wound repair in ob/ob mice, as these animalsare not leptin resistant, but respond to systemically andalso topically administered leptin (15,16). We found adifferential regulation of PMN and macrophage axes ofwound inflammation after leptin-supplementation in ob/ob

mice, which turned out to result from a heretofore un-known leptin-mediated coupling of endocrine and immunefunctions in these animals.

RESEARCH DESIGN AND METHODS

Animals. Female C57BL/6J (wild-type) or C57BL/6J-ob/ob mice were obtainedfrom The Jackson Laboratories (Bar Harbor, ME) and maintained under a 12-hlight-dark cycle at 22°C until they were 8 weeks of age. At this time they werecaged individually, monitored for body weight, and wounded as describedbelow.Treatment of mice. Murine recombinant leptin (Calbiochem, Bad Soden,Germany) was injected intraperitoneally once a day at 8 A.M. (2 �g/g body wt)in 0.5-ml PBS per injection for 13 days. For local treatment, wounds of micewere covered with 1 �g leptin in 20 �l 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). Foreach 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 andprotein preparation. Nonwounded back skin from four animals served as acontrol. All animal experiments were carried out according to the guidelinesand with the permission from the local government of Hessen, Germany.RNA isolation and RNase protection analysis. RNA isolation and RNaseprotection assays were carried out as described previously (18,19). Themurine cDNA probes were cloned using RT-PCR. The probes corresponded tont 816–1,481 (for lipocalin, accession no. X81627), nt 425 (exon 1) to 170 (exon2) (for lysozyme M, M21047), nt 481–739 (for interleukin [IL]-1�, NM008361),nt 541–814 (for tumor necrosis factor [TNF]-�, NM013693), nt 163–317 (forGAPDH, 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 asdescribed previously (17,20). Total protein (10–50 �g diluted in lysis buffer[20] to a final volume of 50 �l) from skin lysates was subsequently analyzed forthe presence of immunoreactive MIP-2, MCP-1, IL-1�, and TNF-� by enzyme-linked immunosorbent assay (ELISA) using the Quantikine murine ELISA kits

(R&D systems, Wiesbaden, Germany). Fifty microliters of supernatants fromcontrol and cytokine (2 nmol/l IL-1�, 2 nmol/l TNF-�)-stimulated murine PAM212 keratinocytes were analyzed for MIP-2 by ELISA (R&D systems, Wiesba-den, Germany).Human wound biopsies. Wound biopsies were selected from surgery innormal individuals (normal wound), from chronic venous leg ulcers inpatients without diabetes (ulcus cruris), and from diabetic foot ulcers inpatients with diabetes (diabetic foot ulcers). Histological sections wereanalyzed from three patients in each group. All wounds were not debrided forat 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 diabeticfoot ulcers were not of the typical neuropathic type.Immunohistochemistry. Complete murine wounds were isolated from theback, bisected, and frozen in tissue-freezing medium. Six-micrometer frozensections were subsequently analyzed using immunohistochemistry as de-scribed (17). Antisera against murine F4/80 antigen (Serotec, Eching, Germa-ny), murine Gr-1 (Ly-6G) (Pharmingen, Hamburg, Germany), and murinecyclooxygenase (COX)-2 (Santa Cruz, Heidelberg, Germany) were used forimmunodetection. Human paraffin skin sections were analyzed for the leptinreceptor ObR (Santa Cruz, Heidelberg, Germany), or macrophage (Dako,Hamburg, Germany). Formal consent was obtained from the subjects after thenature of the procedure was explained.In situ hybridization. Thirteen days after injury, wounds were excised andfixed in 4% paraformaldehyde (PFA)/PBS solution. Six-micrometer serialsections were subsequently analyzed for MIP-2 mRNA or MCP-1 mRNAexpression using the HybriProbe in situ hybridization assay (Biognostik,Gottingen, Germany). MIP-2– or MCP-1–specific oligonucleotides were de-rived from the published sequences (see above).Leptin, glucose, insulin, cortisol, and corticosterone levels. Bloodglucose levels were determined using the Accutrend sensor (Roche Biochemi-cals, Mannheim, Germany), serum leptin and insulin by ELISA (CrystalChemicals, Chicago), and serum cortisol and corticosterone by radioimmu-noassay (RIA) (ICN, Enschede, Germany) as described by the manufacturer.Cell culture. Quiescent murine PAM 212 keratinocytes were stimulated withthe cytokines IL-1� (1 ng/ml), TNF-� (5 ng/ml), or interferon (IFN)-� (100units/ml) as indicated. After 24 h of stimulation, conditioned cell culturesupernatants were analyzed by ELISA. Cytokines were from Roche (Mann-heim, Germany). Scab lysates were prepared in lysis buffer (20) and given toquiescent keratinocytes in a final concentration of 750 �g/ml.Western blot analysis. Wound and cell culture lysates were prepared asdescribed previously (17,20). Fifty micrograms of total protein from skin orcellular lysates was separated using SDS-gel electrophoresis. COX-2 andP-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).

FIG. 1. Administered leptin is biologicallyactive. A: Regulation of blood glucoselevels 3 h after systemic application ofrecombinant 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 asindicated. After 13 days, the body weightof the animals was monitored. **P < 0.01;n.s., not significant as indicated by thebrackets. Bars indicate the mean � SDobtained from nine individual animals(n � 9). C: Presence of scab-coveredwounds after 13 days of treatment withPBS or leptin as indicated. **P < 0.01 vs.PBS-treated animals. Bars indicate themean � SD of wounds (n � 54) obtainedfrom nine individual animals. D: Status ofwounds in PBS-treated (i.p.) and leptin-treated (i.p.) ob/ob mice after 13 days.

LEPTIN AND WOUND INFLAMMATION

2822 DIABETES, VOL. 52, NOVEMBER 2003

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

RESULTS

Systemic leptin supplementation reverted the dis-

turbed metabolic phenotype in ob/ob mice. First, wedetermined the potency of leptin to improve the metabolicsyndrome as well as the impaired wound healing condi-tions observed in ob/ob mice. ob/ob mice were injectedintraperitoneally with recombinant leptin (2 �g/g body wt,once a day) for 13 days. Three hours after injection, wedetermined serum leptin levels (320 � 164 ng/ml). Addi-tionally, to circumvent systemic effects of leptin, woundsfrom a second group of mice were directly covered withleptin (1 �g/20 �l PBS, twice a day). Following systemicleptin treatment, blood glucose levels readjusted to nor-mal levels (100 mg/dl) (Fig. 1A). Moreover, a 13-daysystemic leptin regimen resulted in a marked loss of bodyweight in wounded ob/ob mice, which could not be ob-served after topical treatment (Fig. 1B). In line withrecently published data from our laboratory, we found acomplete reepithelialization of the injured areas in boththe systemically and the topically treated mice. Thisobservation could be well documented by the loss of thescab from reepithelialized wounds (Fig. 1C and D). Addi-tionally, we assessed wound diameters in PBS- and leptin-treated ob/ob mice. We determined an average wound sizeof 5.5 � 0.8 mm (for PBS-treated mice) compared with2.8 � 0.7 mm (for leptin-treated mice) (n � 24 wounds,P � 0.01). A detailed wound size kinetics (day 1–8postwounding) for leptin-treated ob/ob mice versus controlanimals has been published recently (16).Improved skin repair after systemic leptin treatment

was characterized by normalization of PMN, but not

macrophage influx at the wound site. In this study, wefocused on the role of inflammation under conditions ofleptin-driven skin repair. First, we assessed PMN andmacrophage populations at the wound site by determiningthe constitutively expressed molecular markers lipocalin(for PMNs) and lysozyme M (for macrophages). Impairedhealing 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). Thisobservation was confirmed by immunohistochemistrystaining specific protein markers for PMN (GR-1) or mac-rophage (F4/80) (Fig. 2C, as indicated). Systemic adminis-tration of leptin, surprisingly, led to a strong decline onlyin PMN (Fig. 2A and B, left panels), but not in macrophage

(Fig. 2A and B, right panels), cellular numbers at late timepoints of repair. Granulation tissues of wounds fromleptin-treated mice were depleted from PMNs (Fig. 2C,upper right panel), but had tremendous numbers ofmacrophages (Fig. 2C, lower right panel). However, thesecells were most likely to reflect an inactivated state. Asshown 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 macro-phages in the wound stroma for PBS- and leptin-injectedmice, the massive expression of COX-2 mRNA in PBS-treated ob/ob mice probably reflected an activated state ofthe macrophage, which was markedly attenuated by lep-tin-treatment of the animals. Moreover, subsequent analy-sis of both the tissue and scab wound compartmentsrevealed the predominant presence of PMNs within thewound scab, whereas macrophages tended to accumulatein the underlying wound tissue (Fig. 2E).Leptin supplementation strongly attenuated epithe-

lium-derived expression of PMN- but not macro-

phage-specific chemokines. As a next step, wehypothesized that differences in PMN-specific CXC- andmacrophage-specific CC-chemokine expression might con-tribute to the differential infiltration behavior of PMNs ormacrophages into wounded sites of leptin-treated ob/ob

mice. Evidently, systemic administration of leptin stronglyattenuated mRNA expression for the PMN attractantsMIP-2 (Fig. 3A, left panels, and B) and keratinocyte-derived chemokine (KC) (Fig. 4A, right panels). In situhyridization (ISH) in 13-day wounds revealed the develop-ing neoepithelium as the MIP-2–expressing wound com-partment (Fig. 3C, lower right panels). Whereas leptincompletely shut off the MIP-2–specific mRNA signals inkeratinocytes, the epithelia from PBS-injected controlmice were still markedly immunopositive for MIP-2 mRNA(Fig. 3C). Note the well-developed granulation tissue andmultilayered neoepithelium that was associated with animproved 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 totalwound tissue of leptin-treated animals (Fig. 3D, left panel).To further analyze a compartmentalization of MIP-2 ex-pression, we subsequently separated the scab from theunderlying wound tissue before ELISA (Fig. 3D, middle

and right panels). Interestingly, the scab isolated fromdelayed-healing wounds was characterized by high con-centrations of this chemokine (Fig. 4D, right panel).Moreover, we also assessed MIP-2 protein levels from thefew scabs that remained on wounds obtained from the

FIG. 3. Regulation of PMN-specific chemokine expression by leptin. A: RNase protection assay showing the expression of MIP-2 (left panels) andKC (right panels) in wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification of MIP-2 mRNAexpression (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 (lowerpanels). A control hybridization using a nonspecific oligonucleotide is shown in the upper panels. D: MIP-2–specific ELISA analyses from lysatesof 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 � SDobtained from wounds (n � 18) isolated from nine individual animals (n � 9). E: Topical treatment of wounds with leptin did not influence PMNinflux as shown by RNase protection analysis for PMN-specific lipocalin mRNA (upper panel). STAT3 activation in 5-day wounds of ob/ob micethat were treated as indicated (lower panel). Every data point depicted represents eight wounds from four individual animals, which have beenpooled before analysis. F: Quiescent PAM 212 keratinocytes were stimulated as indicated. MIP-2 protein from the cell culture supernatants wasdetermined by ELISA. *P < 0.05 vs. nonstimulated control cells. n.s., not significant as indicated by the brackets. Bars indicate the mean � SDobtained from three (n � 3) independent experiments (left panel). PAM 212 keratinocytes are responsive to a leptin stimulus, as leptin mediatesan activation of STAT3 in the cells (right panel).



leptin-treated ob/ob mice (see Fig. 1C). As these fewscab-covered wounds of leptin-administered mice resem-bled the status of wounds from untreated control animals,we found, not unexpectedly, comparable amounts ofMIP-2 for both healing conditions (Fig. 3D, right panel).These data suggest that the scab might function as a sinkor reservoir for epithelial-derived MIP-2. Moreover, topicaltreatment of wounds with leptin clearly failed to attenuatePMN influx directly (Fig. 3E, upper panel), although we

observed an activation of the signal transducer and acti-vator of transcription (STAT)-3 transcription factor at thewound site (Fig. 3E, lower panel). Cytokine-stimulatedcultured murine PAM 212 keratinocytes did not respond toleptin exposure with an attenuated MIP-2 expression,although PAM keratinocytes nicely responded to a leptinstimulus with activation of the janus kinase (JAK)/STATsignaling cascade (Fig. 3F).

Unlike the situation reported here for CXC chemokines,

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FIG. 4. Regulation of macrophage (M�)-specific chemokine expression by leptin. A: RNase protection assay showing the expression of MCP-1 (leftpanels) and Rantes (right panels) in wounds of PBS-, leptin-, and nontreated ob/ob or C57BL/6J control mice as indicated. A quantification ofMCP-1 mRNA expression (x-fold induction as compared with control skin) is shown in B. n.s., not significant as indicated by the brackets. Barsindicate 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 controlhybridization 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 miceare 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).



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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 untreatedob/ob mice. Keratinocytes of the neoepithelium did notrespond to a leptin-stimulus with reduction of MCP-1mRNA expression (Fig. 4C, middle panel). In contrast toMIP-2 regulation, we found a persisting MCP-1 proteinexpression in wound tissue (Fig. 4D, middle panel). Highlevels of MCP-1 in isolated scabs again indicated that thewound crust might function as a source or sink for proteinmediators (Fig. 4D, right panel). Obviously, this phenom-enon 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 treatedanimals lost their wound scabs earlier in time than PBS-injected control mice (Fig. 1C).The scab as a sink for proinflammatory cytokines. Asa next step, we determined macrophage-derived proin-flammatory mediators in the PBS- and leptin-treated ex-perimental setups. We found a massive decrease in TNF-�and IL-1� mRNA and protein levels for both healingconditions (Fig. 5A–D, left panels). The overall reductionof both cytokines was probably due to the loss of scabsfrom improved healing conditions (Fig. 1C). Nevertheless,TNF-� and IL-1� 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. Toinvestigate the possibility that the scab might function as asource for bioactive molecules during repair, we isolatedfresh scab tissue from control mice on day 5 after wound-ing. As we had determined concentrations of �200 pg forIL-1� and TNF-� in 50 �g of isolated scab protein (Fig. 5B

and D, right panels), we now added a total of 750 �g ofscab protein to stimulate PAM 212 keratinocytes with asimilar amount of scab-derived cytokines as comparedwith recombinant cytokine control conditions (f.c. 1–3ng/ml) (Fig. 6). Interestingly, we were able to observe aninduction of VEGF mRNA expression in cultured keratin-ocytes (18) in the presence of scab proteins, clearlyindicating that the scab indeed included bioactive proteinmediators that were capable to drive gene expression inkeratinocytes (Fig. 7).Leptin, not reduced food intake, is responsible for

improved healing. Here, we determined to what extentchanges in immune cell influx, cytokines, and skin repairmight also be influenced by leptin-mediated reduction offood intake. To this end, we performed a wounding studyin PBS- or leptin-treated and in a control group of pair-fed(caloric-restricted) ob/ob mice, which received equalamounts of nutriment as leptin-treated animals. Pair-feeding indeed resulted in a reduction of body weightcomparable to the leptin-treated group of animals. How-ever, overall wound repair was not improved (Fig. 7A,upper panels). Food restriction significantly reduced se-rum insulin, but not glucose, cortisol, or corticosteronelevels (Fig. 7A). Analysis of cytokines (TNF-� and IL-1�),chemokines (MIP-2 and MCP-1), and immune cells (PMNs,macrophages) revealed that caloric restriction did notcontribute to beneficial effects of leptin treatment (Fig.7B). Moreover, it became clear that systemic levels ofimmune-modulatory glucocorticoids were not significantlychanged by those amounts of leptin that were able toregulate immune function differentially and improve skinrepair (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 inhumans as well. Immunohistochemistry revealed that theleptin receptor was located in keratinocytes of the woundmargins and within the dermis in undisturbed humanwounds (Fig. 8A and B). Interestingly, and in clear con-trast to chronic wound conditions in ob/ob mice (15), wecould not detect ObR protein in wound margin epithelia ofvenous as well as diabetic ulcers. More importantly,whereas immune cells within the granulation tissues ofvenous ulcers expressed the ObR (Fig. 8C and D), wecould not find any staining within diabetic foot ulcer tissue(Fig. 8E and F), although both conditions were character-ized by large numbers of infiltrating macrophages and alsoPMNs (data not shown). Additionally, ObR-responsivecells within the scabs of diabetic ulcers did not representmacrophages, which failed to accumulate in large num-bers within the scab (Fig. 8G and H).

DISCUSSION

Chronic wound-healing disorders represent a seriousproblem of growing clinical importance. There are a

FIG. 6. The scab represents a source of bioactive mediators. Quiescentmurine PAM 212 keratinocytes were incubated with recombinantcytokines (1 ng/ml IL-1�, 5 ng/ml TNF-�, and 100 units/ml IFN-�) orhomogenized scab protein (f.c. 750 �g homogenate/ml) as indicatedand analyzed for VEGF expression by RNase protection assay. **P <0.01; *P < 0.05; n.s., not significant compared with control; #P < 0.05as indicated by the brackets. Bars indicate the mean � SD obtainedfrom three (n � 3) independent cell culture experiments.



number of known pathological conditions that severelyinterfere with a coordinated and successful wound clo-sure. Three prototypic chronic wounds are of clinicalimportance: decubitus or pressure, venous, and the dia-betic ulcers (21). Moreover, disturbances in the inflamma-tory and proliferation phases also delay acute-woundrepair from progressing to later stages of healing (22,23). Anaturally occurring mouse mutant, the db/db mouse, whichsuffers from a diabetes-obesity syndrome (10), has longbeen used as an animal model system to investigate woundhealing deficiencies during acute repair. The most impor-

tant function of leptin is its inhibitory ability to suppressappetite. Thus, leptin reverses the diabetes-obesity syn-drome in leptin-deficient ob/ob mice and also attenuatesfood intake and increases activity in healthy mice (24–26).Additionally, the ob/ob and db/db mice develop syndromessuch as inhibition of reproductive functions (14), hor-monal disturbances related to the thyroid, hypothalamic-pituitary-adrenal, and growth hormone axes (13), as wellas alterations in the hematopoietic and immune systems(14). It is reasonable to suggest that the complexity of thediabetes-obesity syndrome in these animals is most likely

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., notsignificant as indicated by brackets. Bars indicate the mean � SD obtained from four individual animals (n � 4). B: RNase protection assayshowing the expression of TNF-�, IL-1�, MIP-2, MCP-1, lipocalin (PMN), and lysozyme M (macrophage) in wounds of PBS- or leptin-treated andpair-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 fouranimals (n � 4).



to interfere with normal skin repair at various levels ofregulation and must not be functionally connected to thehyperglycemia of the animals.

Leptin administration led to serum leptin levels thatwere about fourfold higher than those described for thedb/db mice model (27). However, data from this study

demonstrate that changes in serum glucocorticoid andinsulin levels did not contribute to leptin-mediated im-provement of repair. However, our leptin/pair-fed experi-ments strongly suggested that attenuation of bloodglucose might play a major role in acceleration of healing.Accordingly, some of the proposed mechanisms that drive

HG

FE

DC

BA

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 marginepithelium.



hyperglycemia-induced pathological changes, such as gly-cation end products, hyperosmolarity, abnormal myoinosi-tol metabolism, oxidant formation, or protein kinase Cactivation (28–32), might be improved by leptin treatment.However, leptin-driven skin-tissue responses in ob/ob micecould be transferred, at least partially, to normal wound-healing conditions, as reepithelialization of wounded sitesalso could be further accelerated by leptin in healthy mice(15). We found the leptin receptor ObR to be expressed inwound-margin keratinocytes of undisturbed humanwounds. Nevertheless, a potential role for leptin in acuteskin repair in humans has to be determined. However,circulating leptin levels have been described to be presentin humans and even to rise in direct correlation to thebody’s white fat tissue (33,34). Thus, diabetes-impairedhealing disorders in humans develop in the presence ofleptin, suggesting a leptin resistance or insensitivity inperipheral tissues. In line, we observed the absence ofleptin receptor expression in diabetic foot ulcers. It ap-pears that deficiencies in leptin responses in ob/ob mice asa model system and humans might be determined bydifferent mechanisms, thus highlighting a potential short-coming of the diabetes-impaired ob/ob mouse model.

This study provides evidence that endocrine mediatormolecules may strongly influence tissue movements ininjured areas of the body. A leptin-driven improvement ofinflammation during repair was not altogether unex-pected, as we had hypothesized a normalization of inflam-matory conditions that might contribute to leptin-accelerated repair. Again, we found a severe dysregulationof the macrophage subset of immune cells in not onlydelayed-healing wounds, but, surprisingly, in also well-developed wounds of leptin-treated animals. Leptin heremediates improved tissue movements, as shown convinc-ingly for epithelial sites (15), in the presence of tremen-dous numbers of persisting macrophages (this study).Evidently, the number of circulating macrophages wasincreased about fourfold in ob/ob mice (35). This over-representation might contribute to the large numbers ofmacrophages observed in wounds of these animals. It isnoteworthy that the MIP-2 and MCP-1 chemokines and theinflammatory mediators TNF-� and IL-1� were predomi-nantly enriched in the wound scab. Early studies withwound dressings suggested that the formation of a scab inair-exposed wounds was superior in supporting granula-tion tissue formation when compared with wound dress-ings (36,37). Accordingly, the release of bioactivecomponents from the scab might provide one explanationof the beneficial effects of the scab for repair.

Evidently, a systemic effect of leptin is supported by theobservation that topical treatment of skin wounds did notresult in an attenuation of PMN infiltration behavior. Theobservation that endocrine signals interfere with the im-mune cell population of skin wounds might be of impor-tance for chronic wound conditions in humans.Histological analyses of chronic leg ulcers in diabetichumans indicated increased numbers of granulocytes andmacrophages which were located in the center and at theedges of the wound (6,7). However, the observed macro-phages in chronic diabetic wounds were probably in aninactive state (8). This observation is consistent with theabsence of leptin receptor (ObR) expression in diabetic

foot ulcers. In fact, debridement of diabetic ulcers impactsinflammatory cells, but does not always solve the problemof impaired healing conditions. It is reasonable to suggestthat inactivation of macrophages might be due to a down-regulation of several receptors for extracellular signalsthat control macrophage activation during repair. Datafrom this study clearly indicate that cells (keratinocytes,immune cells) must have lost their potency to respond toa leptin stimulus. Moreover, loss of ObR expression mightbe confined to diabetic conditions, as venous ulcers werecharacterized by ObR-expressing cells within the granula-tion tissue.

However, macrophages isolated from ob/ob mice ex-press phenotypic abnormalities such as increased super-oxide and hydrogen peroxide production as well aselevated IL-6 and COX-2 levels in vitro (38). Our in vivodata support this in vitro situation. Leptin-treatment trig-gered a downregulation of COX-2 expression at the woundsite, as COX-2 expression completely disappeared in thepresence of unaltered numbers of resident macrophages.Thus, the observed macrophage population of nontreatedand treated ob/ob mice (which did not differ in totalnumbers) might be distinguished markedly by their inflam-matory potencies. The role of leptin as a pivotal mediatorof inflammatory processes is strengthened by a recentstudy of intestinal inflammation in ob/ob mice (39). How-ever, the outcome of leptin-controlled intestinal inflamma-tion in ob/ob mice completely contrasted the situation inskin repair in this animal model, as leptin replacement inob/ob mice increased the severity of intestinal inflamma-tion by augmentation of PMN influx and cytokine produc-tion. It is surprising that the same individual leptin-replaced animal would, however, attenuate MIP-2 andPMN influx at sites of inflammation in the context of skinrepair (this study). It is reasonable to suggest leptin as animportant endocrine signaling molecule that is tightlyconnected to the immune system, but the final outcome ofleptin’s regulatory potency on different axes of immuneresponses might be fundamentally different for differentorgan systems.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsge-meinschaft (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.Kampfer-Kolb and K. Weinelt for excellent technical assis-tance.

REFERENCES

1. Martin P: Wound healing: aiming for perfect skin regeneration. Science

276:75–81, 19972. Singer AJ, Clark RA: Cutaneous wound healing. N Engl J Med 341:738–746,

19993. Hubner G, Brauchle M, Smola H, Madlener M, Fassler R, Werner S:

Differential regulation of pro-inflammatory cytokines during wound heal-ing in normal and glucocorticoid-treated mice. Cytokine 8:548–556, 1996

4. Leibovich SJ, Ross R: The role of the macrophage in wound repair: a studywith hydrocortisone and antimacrophage serum. Am J Pathol 78:71–100,1975

5. DiPietro LA: Wound healing: the role of the macrophage and other immunecells. Shock 4:233–240, 1995

6. Rosner K, Ross C, Karlsmark T, Petersen AA, Gottrup F, Vejlsgaard GL:Immunohistochemical characterization of the cutaneous cellular infiltratein different areas of chronic leg ulcers. APMIS 103:293–299, 1995



7. Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD, Middelkoop E:Differences in cellular infiltrate and extracellular matrix of chronic dia-betic and venous ulcers versus acute wounds. J Invest Dermatol 111:850–857, 1998

8. Moore K, Ruge F, Harding KG: T lymphocytes and the lack of activatedmacrophages in wound margin biopsies from chronic leg ulcers. Br J

Dermatol 137:188–194, 19979. Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S: Large and

sustained induction of chemokines during impaired wound healing in thegenetically diabetic mouse: prolonged persistence of neutrophils andmacrophages during the late phase of repair. J Invest Dermatol 115:245–253, 2000

10. Coleman DL: Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14:141–148, 1978

11. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM:Positional cloning of the mouse obese gene and its human homologue.Nature 372:425–432, 1994

12. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND,Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, MorgensternJP: Evidence that the diabetes gene encodes the leptin receptor: identifi-cation of a mutation in the leptin receptor gene in db/db mice. Cell

84:491–495, 199613. Ahima RS, Saper CB, Flier JS, Elmquist JK: Leptin regulation of neuroen-

docrine systems. Frontiers in Neuroendocrinology 21:263–307, 200014. Fantuzzi G, Faggioni R: Leptin in the regulation of immunity, inflammation,

and hematopoiesis. J Leukoc Biol 68:437–446, 200015. Frank S, Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J: Leptin enhances

wound re-epithelialization and constitutes a direct function of leptin inskin repair. J Clin Invest 106:501–509, 2000

16. Ring BD, Scully S, Davis CR, Baker MB, Cullen MJ, Pelleymounter MA,Danilenko DM: Systemically and topically administered leptin both accel-erate wound healing in diabetic ob/ob mice. Endocrinology 141:446–449,2000

17. Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J, Frank S: The function ofnitric oxide in wound repair: inhibition of inducible nitric oxide-synthaseseverely impairs wound reepithelialization. J Invest Dermatol 113:1090–1098, 1999

18. Frank S, Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J: Nitric oxidetriggers enhanced induction of vascular endothelial growth factor expres-sion in cultured keratinocytes (HaCaT) and during cutaneous woundrepair. FASEB J 13:2002–2014, 1999

19. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem

162:156–159, 198720. Kampfer H, Kalina U, Muhl H, Pfeilschifter J, Frank S: Counterregulation of

interleukin-18 mRNA and protein expression during cutaneous woundrepair in mice. J Invest Dermatol 113:369–374, 1999

21. Falanga V: Chronic wounds: pathophysiologic and experimental consider-ations. J Invest Dermatol 100:721–725, 1993

22. Morain WD, Colen LB: Wound healing in diabetes mellitus. Clin Plast Surg

17:493–501, 1990

23. Pearl SH, Kanaz IO: Diabetes and healing: a review of the literature. J Foot

Surg 27:268–270, 198824. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI,

Friedman JM: Abnormal splicing of the leptin receptor in diabetic mice.Nature 379:632–635, 1996

25. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D,Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasmaprotein encoded by the obese gene. Science 269:543–546, 1995

26. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T,Collins F: Effects of the obese gene product on body weight regulation inob/ob mice. Science 269:540–543, 1995

27. Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR,Grunfeld C: Leptin deficiency enhances sensitivity to endotoxin-inducedlethality. Am J Physiol 276:R136–R142, 1999

28. Friedman EA: Advanced glycosylated end products and hyperglycemia inthe pathogenesis of diabetic complications. Diabetes Care 22 (Suppl.2):B65–B71, 1999

29. Wautier JL, Guillausseau PJ: Diabetes, advanced glycation endproductsand vascular disease. Vasc Med 3:131–137, 1998

30. Yki-Jarvinen H: Toxicity of hyperglycaemia in type 2 diabetes. Diabete

Metab Rev 14 (Suppl. 1):S45–S50, 199831. Koya D, King GL: Protein kinase C activation and the development of

diabetic complications. Diabetes 47:859–866, 199832. Porte DJ, Schwartz MW: Diabetes complications: why is glucose poten-

tially toxic? Science 272:699–700, 199633. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce

MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF: Serumimmunoreactive-leptin concentrations in normal-weight and obese hu-mans. N Engl J Med 334:292–295

34. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, KimS, Lallone R, Ranganathan S, et al: Leptin levels in human and rodent:measurement of plasma leptin and ob RNA in obese and weight-reducedsubjects. Nat Med 1:1155–1161, 1995

35. Faggioni R, Jones-Carson J, Reed DA, Dinarello CA, Feingold KR, GrunfeldC, Fantuzzi G: Leptin-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: role of tumor necrosis factor alpha and IL-18.Proc Natl Acad Sci U S A 97:2367–2372, 2000

36. Jonkman MF, Bruin P, Hoeksma EA, Nieuwenhuis P, Klasen HJ, PenningsAJ, Molenaar I: A clot-inducing wound covering with high vapor perme-ability: enhancing effects on epidermal wound healing in partial-thicknesswounds in guinea pigs. Surgery 104:537–545, 1988

37. Ksander GA, Pratt BM, Desilets-Avis P, Gerhardt CO, McPherson JM:Inhibition of connective tissue formation in dermal wounds covered withsynthetic, moisture vapor-permeable dressings and its reversal by trans-forming growth factor-beta. J Invest Dermatol 95:195–201, 1990

38. Lee FY, Li Y, Yang EK, Yang SQ, Lin HZ, Trush MA, Dannenberg AJ, DiehlAM: Phenotypic abnormalities in macrophages from leptin-deficient, obesemice. Am J Physiol 276:C386–394, 1999

39. Siegmund B, Lehr HA, Fantuzzi G: Leptin: a pivotal mediator of intestinalinflammation in mice. Gastroenterology 122:2011–2025, 2002



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