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Expression and Activity of Arginase Isoenzymes During Normal and Diabetes-Impaired Skin Repair

2003; Elsevier BV; Volume: 121; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2003.12610.x

1523-1747

Heiko Kämpfer, Josef Pfeilschifter, Stefan L. Frank,

Silk-based biomaterials and applications

Within the past years, an important role for nitric oxide (NO) in skin repair has been well defined. As NO is synthesized from L-arginine by NO synthases (NOS), the availability of L-arginine might be one rate-limiting factor of NO production at the wound site. Upon injury, arginase-1 and -2 mRNA, protein, and activity were strongly induced reaching a maximum between day 3 and day 7 postwounding. Immunohistochemistry colocalized both arginases and the inducible NOS (iNOS) at epithelial sites at the margins of the wound. Notably, diabetes-impaired skin repair in leptin-deficient mice (diabetes/diabetes, db/db; and obese/obese, ob/ob) was characterized by an abnormally elevated arginase activity in wound tissue in the absence of an expression of iNOS. Expression analyses demonstrated that arginase-1 contributed to increased arginase activities in impaired repair. Interestingly, an improved healing of chronic wound situations in leptin-supplemented ob/ob mice was strongly associated with an adjustment of the dysregulated expression of L-arginine-converting enzymes: an attenuated iNOS expression was upregulated early in repair and an augmented arginase-1 expression and activity was downregulated in the presence of markedly elevated numbers of macrophages during late repair. These data suggest a coordinated consumption of L-arginine by the NOS and arginase enzymatic pathways at the wound site as a prerequisite for a balanced NO (via iNOS) and polyamine (via arginases) synthesis that drives a normal skin repair. Within the past years, an important role for nitric oxide (NO) in skin repair has been well defined. As NO is synthesized from L-arginine by NO synthases (NOS), the availability of L-arginine might be one rate-limiting factor of NO production at the wound site. Upon injury, arginase-1 and -2 mRNA, protein, and activity were strongly induced reaching a maximum between day 3 and day 7 postwounding. Immunohistochemistry colocalized both arginases and the inducible NOS (iNOS) at epithelial sites at the margins of the wound. Notably, diabetes-impaired skin repair in leptin-deficient mice (diabetes/diabetes, db/db; and obese/obese, ob/ob) was characterized by an abnormally elevated arginase activity in wound tissue in the absence of an expression of iNOS. Expression analyses demonstrated that arginase-1 contributed to increased arginase activities in impaired repair. Interestingly, an improved healing of chronic wound situations in leptin-supplemented ob/ob mice was strongly associated with an adjustment of the dysregulated expression of L-arginine-converting enzymes: an attenuated iNOS expression was upregulated early in repair and an augmented arginase-1 expression and activity was downregulated in the presence of markedly elevated numbers of macrophages during late repair. These data suggest a coordinated consumption of L-arginine by the NOS and arginase enzymatic pathways at the wound site as a prerequisite for a balanced NO (via iNOS) and polyamine (via arginases) synthesis that drives a normal skin repair. N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate inducible nitric oxide synthase nitric oxide macrophage phosphate-buffered saline To overcome tissue damage, the process of cutaneous wound repair represents a highly ordered process that is characterized by temporally and spatially overlapping phases of tissue movements comprising hemorrhage, inflammation, reepithelialization, granulation tissue formation, and the late remodeling phase of repair. As a result, the integrity of the body's protective layer is maintained, although repair fails to perfectly replace the original skin tissue. Loss of a functional healing process might lead to severe disabilities. Accordingly, chronic, nonhealing wound conditions represent a situation of major clinical importance. A series of pathologic changes accompanied with several diseases finally leads to severely disturbed wound healing conditions. Among those, the most prominent chronic wound situations are known as decubitus or pressure ulcers, venous ulcers, and diabetic ulcers (Falanga, 1993Falanga V. Chronic wounds. pathophysiologic and experimental considerations.J Invest Dermatol. 1993; 100: 721-725Abstract Full Text PDF PubMed Google Scholar). The factors mediating the intercellular communication during wound repair are known in part, but their number is still increasing. Proinflammatory cytokines and various peptide growth factors are known to be key players in this process (Martin, 1997Martin P. Wound healing—aiming for perfect skin regeneration.Science. 1997; 276: 75-82Crossref PubMed Scopus (3430) Google Scholar;Singer and Clark, 1999Singer A.K. Clark R.A.F. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar). Nevertheless, these protein-type factors are not unique in regulating cellular behavior in skin biology, and nitric oxide (NO), a small diffusible radical gas, has been demonstrated an important role in wound repair (Frank et al., 2002Frank S. Kämpfer H. Wetzler C. Pfeilschifter J. Nitric oxide drives skin repair. Novel functions of an established mediator.Kidney Int. 2002; 61: 882-888Crossref PubMed Scopus (136) Google Scholar). Especially, the inducible NO synthase (iNOS) isoenzyme is strongly upregulated upon skin injury (Frank et al., 1998Frank S. Madlener M. Pfeilschifter J. Werner S. Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064Crossref PubMed Scopus (96) Google Scholar). Inhibition of iNOS enzymatic activity during repair in wild-type mice using N6-(iminoethyl)-L-lysine resulted in a similar delay in complete wound closure (Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (190) Google Scholar) compared to injured iNOS knockout mice (Yamasaki et al., 1998Yamasaki K. Edington H.D. McClosky C. et al.Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer.J Clin Invest. 1998; 101: 967-971Crossref PubMed Scopus (359) Google Scholar). Inhibition of iNOS enzymatic activity during skin repair was paralleled by a severely impaired reepithelialization process, because the epithelial sites at the margins of the wound were characterized by an atrophied morphology and strongly reduced numbers of proliferating keratinocytes (Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (190) Google Scholar). These observations provide a strong explanation for the well-known fact that L-arginine is implicated in wound healing, because the amino acid L-arginine represents the only substrate for NOS enzymatic activity. An early study from nearly 25 years ago demonstrated the influence of L-arginine supplementation to improve wound healing in a rat model of incisional repair (Seifter et al., 1978Seifter E. Rettura G. Barbul A. Levenson S.M. Arginine: An essential amino acid for injured rats.Surgery. 1978; 84: 224-230PubMed Google Scholar). Consistently, the beneficial effect of L-arginine observed in animal models turned out to be true also in humans. Clinical studies demonstrated an enhanced wound healing in L-arginine-supplemented patients, which was characterized by a markedly increased wound collagen deposition (Barbul et al., 1990Barbul A. Lazarou S.A. Efron D.T. Wasserkrug H.L. Efron G. Arginine enhances wound healing and lymphocyte immune responses in humans.Surgery. 1990; 108: 331-336PubMed Google Scholar;Kirk et al., 1993Kirk S.J. Hurson M. Regan M.C. Holt D.R. Wasserkrug H.L. Barbul A. Arginine stimulates wound healing and immune function in elderly human beings.Surgery. 1993; 114: 155-159PubMed Google Scholar). Nevertheless, iNOS activities most likely face an antagonistic player that might strongly compete for the L-arginine substratum at the wound site. It is tempting to speculate that arginases, which turn over L-arginine into ornithine and urea, might influence wound NO production by limiting iNOS substrate. In line, L-arginine levels in wound fluids remain low during healing, because the amino acid is subject to the catalytic activity of arginase in late repair, releasing measurable amounts of ornithine into the wound fluid (Albina et al., 1990Albina J.E. Mills C.D. Henry Jr., W.L. Caldwell M.D. Temporal expression of different pathways of 1-arginine metabolism in healing wounds.J Immunol. 1990; 144: 3877-3880PubMed Google Scholar). It is important to recognize that two distinct arginase isoenzymes exist in mammals that are encoded by separate genes. Although enzymatic properties were nearly similar, both enzymes differ in subcellular localization, tissue expression, and expressional regulation (Grody et al., 1987Grody W.W. Dizikes G.J. Cederbaum S.D. Human arginase isozymes.Isozymes Curr Top Biol Med Res. 1987; 13: 181-214PubMed Google Scholar;Jenkinson et al., 1996Jenkinson C.P. Grody W.W. Cederbaum S.D. Comparative properties of arginases.Comp Biochem Physiol B Biochem Mol Biol. 1996; 114: 107-132Crossref PubMed Scopus (483) Google Scholar). As part of the urea cycle, arginase-1 is predominantly expressed in liver, whereas the mitochondrial arginase-2 isoenzyme seems to be more ubiquitously expressed. Nevertheless, although much work has been focused on the NOS axis of L-arginine turnover in the context of wound healing, only limited data are available for the arginases. To this end, we determined arginase expression and activity in animal models of normal and impaired repair. Our data suggest that expression and activity of both arginase isoenzymes were induced upon skin injury, but, interestingly, only a dysregulated arginase-1 activity was associated with impaired wound healing conditions. Female BALB/c or C57BLKS mice were obtained from Charles River (Sulzfeld, Germany). Female C57BL/6 J-ob/ob and C57BLKS/J-m+/+Leprdb (db/db) mice were obtained from The Jackson Laboratories (Bar Harbor, ME). The homozygous animals develop characteristics similar to those of human adult onset diabetes (Coleman, 1982Coleman D.L. Diabetes–obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar). Mice were maintained under a 12-h light/12-h dark cycle at 22°C until they were 8 wk of age. At this time they were caged individually, monitored for body weight and wounded as described below. Murine recombinant leptin (Calbiochem, Bad Soden, Germany) was injected intraperitoneally once a day at 8 a.m. (2 μg/g body weight) in 0.5 mL of phosphate-buffered saline (PBS) per injection for 13 d. Wounding of mice was performed as described previously (Frank et al., 1999Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (204) Google Scholar;Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (190) Google Scholar). Briefly, mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg body weight)/xylazine (10 mg/kg body weight). The hair on the back of each mouse was cut, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (5 mm in diameter, 3–4 mm apart) were made on the back of each mouse by excising the skin and the underlying panniculus carnosus. The wounds were allowed to form a scab. Skin biopsy specimens were obtained from the animals 1, 3, 5, 7, 10, and 13 d after injury. At each time point, an area that included the scab and the complete epithelial margin was excised from each individual wound. As a control, a similar amount of skin was taken from the backs of nonwounded mice. 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 assays were carried out as described previously (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar;Frank et al., 1999Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (204) Google Scholar). Briefly, 20 μg of total RNA from wounded or nonwounded skin was used for RNase protection assay. DNA probes were cloned into the transcription vector pBluescript II KS(+) (Stratagene, Heidelberg, Germany) and linearized. An antisense transcript was synthesized in vitro using T3 or T7 RNA polymerase and [α-32P]UTP (800 Ci/mmol). RNA samples were hybri-dized at 42°C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested with RNases A and T1 for 1 h at 30°C. Under these conditions, every single mismatch is recognized by the RNases. Protected fragments were separated on 5% acrylamide/8 M urea gels and analyzed using a PhosphoImager (Fuji, Straubenhardt, Germany). RNases A and T1 were from Roche Biochemicals (Mannheim, Germany). The murine cDNA probes were cloned using RT-PCR. The probes corresponded to nucleotides 362–660 (for arginase-1, Accession No. U51805), nucleotides 360–619 (for arginase-2, Accession No. U90886), nucleotides 3285–3574 (for iNOS, Accession No. NM010927), nucleotides 425–446 (exon 1) and 150–170 (exon 2) (for lysozyme M, Accession Nos. M21047, M21048), or nucleotides 163–317 (for GAPDH, Accession No. NM002046) of the published sequences. Skin and cell culture samples were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 15 μg/mL leupeptin). The extracts were cleared by centrifugation (Kämpfer et al., 1999Kämpfer H. Kalina U. Mühl H. Pfeilschifter J. Frank S. Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.J Invest Dermatol. 1999; 113: 369-374Crossref PubMed Scopus (65) Google Scholar;Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (190) Google Scholar). Fifty micrograms of total protein from skin or cellular lysates was separated using SDS-gel electrophoresis. After transfer to a polyvinylidene difluoride membrane, arginase-1 and -2 protein was detected using polyclonal antibodies against rat arginase-1 and -2. The arginase antibodies had been generated in rabbits using specific peptide sequences (arginase-1, H2N-CEGNHKPETDYLKPPK-COOH; arginase-2, H2N-CHLPTPSSPHESEKEE-COOH). The rabbit polyclonal antibody against murine iNOS was purchased from Alexis Corporation (Grünberg, Germany). A secondary antibody coupled to horseradish peroxidase and the ECL detection system were used to visualize the Arg-1 and -2 proteins, respectively. Phenylmethylsulfonyl fluoride, aprotinin, and leupeptin were from Sigma (Deisenhofen, Germany) or Roche Biochemicals. The ECL detection system was obtained from Amersham (Freiburg, Germany). Mice were wounded as described above. Animals were euthanized at day 5 after injury. Complete wounds were isolated from the back, bisected, and frozen in tissue freezing medium. Six-micrometer frozen sections were fixed with acetone and treated for 10 min at room temperature with 1% H2O2 in PBS to inactivate endogenous peroxidases. They were subsequently incubated overnight at 4°C with rabbit polyclonal antisera against rat arginase-1, arginase-2, murine iNOS, or murine macrophage (mφ)-specific F4/80 antigen (1:50 diluted in PBS, 0.1% bovine serum albumin), respectively (Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (190) Google Scholar). The antiserum against murine mφ-specific F4/80 antigen was from SeroTec (Hamburg, Germany). Arginase activity was determined as described (Corraliza et al., 1994Corraliza I.M. Campo M.L. Soler G. Modolell M. Determination of arginase activity in macrophages: A micromethod.J Immunol Meth. 1994; 174: 231-235Crossref PubMed Scopus (405) Google Scholar). Briefly, isolated wound tissue was homogenized in 25 mM Tris-HCl, pH 7.5/5 mM MnCl2 in the presence of protease inhibitors (10 μg/mL pepstatin A, 10 μg/mL leupeptin, 100 μg/mL phenylmethylsulfonyl fluoride). Homogenates were cleared by centrifugation (20 min, 5000×g). Equal amounts of total wound protein were subsequently activated for arginase activity at 55°C for 10 min. L-Arginine was added, and activated homogenates were incubated at 37°C for 1 h. The arginase reaction was terminated by addition of an eightfold excess (vol/vol) of H2SO4, H3PO4, and H2O (1:3:7). 1-Phenyl-1,2-propanedione-2-oxime was added for 30 min at 100°C. The amount of total urea formed was subsequently determined spectrophotometrically at 540 nm. Results were expressed as μg of urea per mg of protein per h. Quiescent murine PAM 212 epidermal keratinocytes were stimulated with a combination of cytokines (2 nM interleukin-1β, 2 nM tumor necrosis factor-α, 100 U per mL interferon-γ) or 1 mM N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (Bt2-cAMP). Bt2-cAMP was purchased from Sigma, and cytokines were from Roche Biochemicals (Mannheim, Germany). Data are shown as means±SD. Data analysis was carried out using the unpaired Student's t test with raw data. Statistical comparison between more than two groups was carried out by ANOVA (Dunnett's method). It is now well established that skin injury is accompanied by the induction of iNOS during the inflammatory phase of repair (Frank et al., 1998Frank S. Madlener M. Pfeilschifter J. Werner S. Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064Crossref PubMed Scopus (96) Google Scholar, Frank et al., 2002Frank S. Kämpfer H. Wetzler C. Pfeilschifter J. Nitric oxide drives skin repair. Novel functions of an established mediator.Kidney Int. 2002; 61: 882-888Crossref PubMed Scopus (136) Google Scholar). Moreover, inhibitor studies using the partially selective iNOS inhibitor N6-(iminoethyl)-L-lysine suggested the iNOS as the major source of NO synthesis at the wound site (Frank et al., 1999Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (204) Google Scholar). Because L-arginine, additionally, represents the only substrate also for arginase isoenzymes, we now investigated the expressional regulation of arginase-1- and arginase-2 upon skin injury. Not unexpected, both arginase isoenzymes were strongly induced at the mRNA (Figure 1a,b, top panels) and the protein (Figure 1a,b, bottom panels) level after skin wounding. It is noteworthy that injury-mediated induction of the arginases was characterized by a temporal delay, as both proteins were only hardly detectable 24 h after wounding (1d wound). Because we had generated polyclonal antibodies against both arginase proteins using peptide sequences from rat arginase-1 and -2, respectively (see Materials and Methods), we tested the specificity of the obtained antisera to unequivocally confirm our immunoblot data from wound tissue. As arginase-1 is strongly and predominantly expressed in liver tissue (Wu and Morris, 1998Wu G. Morris Jr., S.M. Arginine metabolism: Nitric oxide and beyond.Biochem J. 1998; 336: 1-17Crossref PubMed Scopus (2016) Google Scholar), we used total protein isolated from murine liver as a positive control for the anti-arginase-1 antiserum (Figure 1c). Additionally, as arginase-2 has been reported to be under positive regulatory control of the cAMP second messenger pathway in the murine mφ-like cell line RAW264.7 (Gotoh et al., 1996Gotoh T. Sonoki T. Nagasaki A. Terada K. Takiguchi M. Mori M. Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line.FEBS Lett. 1996; 395: 119-122Abstract Full Text PDF PubMed Scopus (207) Google Scholar;Morris et al., 1998Morris Jr., S.M. Kepka-Lenhart D. Chen L.C. Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells.Am J Physiol. 1998; 275: E740-E747PubMed Google Scholar), we used cell lysates from Bt2-cAMP-stimulated RAW264.7 cells to prove specificity of the arginase-2 antiserum (Figure 1d). Specificity of both antibodies was further verified, because, notably, anti-arginase-1 did not detect the cAMP-inducible arginase-2 isoform in RAW264.7 lysates (Figure 1c), and anti-arginase-2 did not bind to the liver-expressed arginase-1. Obviously, both antisera specifically recognized their respective arginase isoforms. As a next step, we assessed the overall arginase activity in nonwounded skin and wound tissue. To this end, equal amounts of isolated skin or wound protein were incubated with L-arginine, and the amount of generated urea was determined. As shown in Figure 2, the observed induction of both arginase isoenzymes was consistently followed by a marked increase of total arginase activity in wound tissue, which remained even slightly elevated at day 13 after injury. Nevertheless, it is important to note that the overall activity shown in Figure 2 reflects the enzymatic activities of both arginase isoenzymes at the wound site. Interestingly, the delayed induction of arginase-1 and -2 expression early after wounding (Figure 1a,b, bottom panels, 1d wound) was clearly reflected by a slight decrease in arginase activity in 1-d wound tissue (Figure 2). Next, we determined the localization of both arginases in wound tissue. We investigated tissue sections from 5-d wounds, because this time point of healing was characterized by maximal expression both of arginase isoenzymes (Figure 1a,b) and of the presence of iNOS (Frank et al., 1998Frank S. Madlener M. Pfeilschifter J. Werner S. Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064Crossref PubMed Scopus (96) Google Scholar) (see also Figure 4e). As shown in Figure 3, both arginase-1 and arginase-2 were expressed in wound margin keratinocytes. Whereas the main body of proliferating keratinocytes that usually builds up the hyperproliferative epithelium revealed only small or even no arginase immunoreactivity, marked immunopositive signals for arginase-1 and arginase-2 protein could be detected in epidermal keratinocytes directly adjacent to the massive hyperproliferative epithelia. Moreover, it is noteworthy that, especially, keratinocytes that were in direct contact with the developing granulation tissue, showed a marked immunoreactivity against arginase-1 and arginase-2 (Figure 3a,b, insets). Interestingly, it must be recognized that iNOS was nearly coexpressed at the same cellular sources in wound tissue (Figure 3c). Because expression of both arginase isoenzymes during repair (Figure 1a,b) resembled the infiltration kinetics of mφ into the wounded site, we assessed the presence of mφ in 5-d wound tissue using the mφ-specific F4/80 antigen (Figure 3d). Notably, infiltrating immune cells, which were predominantly represented by activated mφ at day 5 postwounding (Figure 3d), were not the major cellular source of arginase as well as iNOS expression, because iNOS-, arginase-1-, and arginase-2-immunopositive signals were only sparsely located within the granulation tissue. Evidently, cultured murine PAM 212 keratinocytes were also capable of expressing both arginases and iNOS in vitro, thus suggesting the overall capacity of keratinocytes of expressing these enzymes in general (Figure 3e). Please note that Th1 cytokines moderately reduced arginase-1 expression in the cells.Figure 3Colocalization of arginase-1, arginase-2, and iNOS protein in regenerating skin. Frozen serial sections from mouse wounds (day 5 postwounding) were incubated with polyclonal antisera directed against arginase-1 (a), arginase-2 (b), iNOS (c), and mφ-specific F4/80 antigen (d), respectively. All sections were stained with the avidin–biotin–peroxidase complex system using 3,3-diaminobenzidine tetrahydrochloride as a chromogenic substrate. Nuclei were counterstained with hematoxylin. Immunopositive signals within the sections are indicated with arrows. Wound margin and inner wound area are indicated. g, granulation tissue; he, hyperproliferative epithelium; iwa, inner wound area; Mφ, macrophages; wma, wound margin. (e) Murine keratinocytes express arginase-1, arginase-2, and iNOS in vitro. Quiescent murine PAM 212 keratinocytes were stimulated with cytokines (cytmix, 2 nM interleukin-1β, 2 nM tumor necrosis factor-α, 100 μ/mL interferon-γ) or Bt2-cAMP (1 mM) for 24 h. RNase protection assay to detect arginase-1-, arginase-2-, or iNOS-specific mRNA was performed as indicated. Ctrl, nonstimulated cells. One representative experiment is shown.View Large Image Figure ViewerDownload (PPT) As a next step, we investigated the regulation of arginase expression and activity in a mouse model of diabetes-impaired wound healing. Diabetes/diabetes (db/db) mice are leptin-resistant (Chen et al., 1996Chen H. Charlat O. Tartaglia L.A. et al.Evidence that the diabetes gene encodes the leptin receptor. Identification of a mutation in the leptin receptor gene in db/db mice.Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1859) Google Scholar;Lee et al., 1996Lee G.H. Proenca R. Montez J.M. et al.Abnormal splicing of the leptin receptor in diabetic mice.Nature (London). 1996; 379: 632-635Crossref PubMed Scopus (2060) Google Scholar) and, thus, suffer from severe metabolic disorders. Moreover, these animals are characterized by severe wound healing disorders (Wetzler et al., 2000Wetzler C. Kämpfer H. Stallmeyer B. Pfeilschifter J. Frank S. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: Prolonged persistence of neutrophils and macrophages during the late phase of repair.J Invest Dermatol. 2000; 115: 245-253Crossref PubMed Scopus (391) Google Scholar). Not unexpected, arginase-1 as well as arginase-2 were induced upon injury in db/db mice (Figure 4a,c, bottom panels) and the expression kinetics were similar to healthy control mice (Figure 1), especially with respect to the observed delayed induction of both enzymes. Nevertheless, analysis of mRNA expression in db/db mice revealed that both arginases were not significantly different in nonwounded skin but became dysregulated at late time points of repair (Figure 4a,c, top panels). In contrast to healthy animals (wt), diabetes-impaired repair (db) was characterized by a strong overexpression of both arginase isoforms at the mRNA level. It is important to note that the indicated experimental time points reflect the phase when an initiated healing in these mice transfers into chronic, nonhealing wound conditions (Wetzler et al., 2000Wetzler C. Kämpfer H. Stallmeyer B. Pfeilschifter J. Frank S. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: Prolonged persistence of neutrophils and macrophages during the late phase of repair.J Invest Dermatol. 2000; 115: 245-253Crossref PubMed Scopus (391) Google Scholar). Consistent with the mRNA data, we found markedly elevated levels of arginase-1 protein in chronic wounds (days 10 and 13) compared to wounds isolated from control animals (Figure 4a,b). Nevertheless, and as a surprise, arginase-2 protein expression appeared to be counterregulated with respect to its own mRNA and, additionally, when compared to arginase-1. Although arginase-2-specific mRNA was strongly overexpressed in chronic wounds (Figure 4c, top panel), we could only barely detect arginase-2 protein in chronic wounds (Figure 4c, bottom panel; Figure 4d). Note that arginase-2 protein expression was still present at the end of acute repair in normal healing (Figure 1b, Figure 4d, top panel), a time point of repair that was