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The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium

      The human cathelicidin anti-microbial protein, hCAP18 is a component of the innate immune system and has broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs upon inflammation and infection. We demonstrate here a novel role for this peptide in re-epithelialization of skin wounds. We show that high levels of hCAP18 are produced in skin in vivo upon wounding. The highest hCAP18 levels are attained at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. In chronic ulcers, however, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. Using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, we show that hCAP18 is strongly expressed in healing skin epithelium, and that treatment with antibodies raised and affinity purified against LL-37, inhibits re-epithelialization in a concentration-dependent manner. Immunoreactivity for the proliferation marker Ki67 is absent in the epithelium of such inhibited wounds, suggesting that LL-37 may play a part in epithelial cell proliferation. Thus, we suggest that, in addition to being an anti-microbial peptide, LL-37 also plays a part in wound closure and that its reduction in chronic wounds impairs re-epithelialization and may contribute to their failure to heal.

      Keywords

      Anti-microbial peptides are effector molecules of the innate immune system that serve to protect the integrity of the host against potentially harmful microorganisms. They are conserved through evolution and are widespread in nature ranging from plants to mammals, with several hundreds having been characterized (
      • Ganz T.
      • Lehrer R.I.
      Antibiotic peptides from higher eukaryotes: biology and applications.
      ;
      • Lehrer R.I.
      • Ganz T.
      Antimicrobial peptides in mammalian and insect host defence.
      ). In humans, only a handful have been identified so far; among which the defensins and the human cationic anti-microbial peptide hCAP18 have been implicated in epithelial defense (
      • Selsted M.E.
      • Brown D.M.
      • DeLange R.J.
      • Lehrer R.I.
      Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages.
      ;
      • Lehrer R.I.
      • Lichtenstein A.K.
      • Ganz T.
      Defensins. antimicrobial and cytotoxic peptides of mammalian cells.
      ;
      • Gallo R.L.
      • Ono M.
      • Povsic T.
      • Page C.
      • Eriksson E.
      • Klagsbrun M.
      • Bernfield M.
      Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich anti-microbial peptide from wounds.
      ;
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      A peptide antibiotic from human skin [letter] [see comments].
      ;
      • Bals R.
      • Wang X.
      • Zasloff M.
      • Wilson J.M.
      The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad anti-microbial activity at the airway surface.
      ). hCAP18 belongs to the cathelicidin gene family, which is comprised of more than 20 members in different species, and is believed to be the sole cathelicidin protein in humans (
      • Gudmundsson G.H.
      • Agerberth B.
      • Odeberg J.
      • Bergman T.
      • Olsson B.
      • Salcedo R.
      The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.
      ;
      • Larrick J.W.
      • Lee J.
      • Ma S.
      • Li X.
      • Francke U.
      • Wright S.C.
      • Balint R.F.
      Structural, functional analysis and localization of the human CAP18 gene.
      ). The holoprotein consist of an N-terminal part, cathelin, which is conserved through species and a C-terminal fragment, LL-37, which confers anti-microbial activity against both gram-positive and gram-negative bacteria (
      • Agerberth B.
      • Gunne H.
      • Odeberg J.
      • Kogner P.
      • Boman H.G.
      • Gudmundsson G.H.
      FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.
      ;
      • Gudmundsson G.H.
      • Agerberth B.
      • Odeberg J.
      • Bergman T.
      • Olsson B.
      • Salcedo R.
      The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.
      ). Cathelin was originally isolated from pig leukocytes and was proposed to act as an inhibitor of cysteine proteinases (
      • Ritonja A.
      • Kopitar M.
      • Jerala R.
      • Turk V.
      Primary structure of a new cysteine proteinase inhibitor from pig leucocytes.
      ); however, so far no definitive biologic function has been proven for human cathelin. Activation of hCAP18 requires proteolytic cleavage, which releases the C-terminus, i.e., LL-37 (
      • Gudmundsson G.H.
      • Agerberth B.
      • Odeberg J.
      • Bergman T.
      • Olsson B.
      • Salcedo R.
      The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.
      ). Serine proteinase 3 was recently shown to be responsible for extracellular cleavage of hCAP18 (
      • Sørensen O.E.
      • Follin P.
      • Johnsen A.H.
      • Calafat J.
      • Tjabringa G.S.
      • Hiemstra P.S.
      • Borregaard N.
      Human cathelicidin, hCAP-18, is processed to the anti-microbial peptide LL-37 by extracellular cleavage with proteinase 3.
      ). LL-37 is thought to function extracellularly and there is no evidence for intracellular cleavage of the propeptide. hCAP18 is synthesized in the bone marrow and stored in mature neutrophils (
      • Sørensen O.
      • Arnljots K.
      • Cowland J.B.
      • Bainton D.F.
      • Borregaard N.
      The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils.
      ) and is present in subpopulations of lymphocytes and monocytes (
      • Agerberth B.
      • Charo J.
      • Werr J.
      • et al.
      The human anti-microbial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations.
      ). hCAP18 is also expressed in skin and other epithelia, where it is upregulated in association with inflammation, infection, and injury (
      • Frohm M.
      • Agerberth B.
      • Ahangari G.
      • Ståhle-Bäckdahl M.
      • Lidén S.
      • Wigzell H.
      • Gudmundsson G.H.
      The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.
      ;
      • Goldman M.J.
      • Anderson G.M.
      • Stolzenberg E.D.
      • Kari U.P.
      • Zasloff M.
      • Wilson J.M.
      Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.
      ;
      • Frohm Nilsson M.
      • Sandstedt B.
      • Sørensen O.
      • Weber G.
      • Borregaard N.
      • Ståhle-Bäckdahl M.
      The human cationic anti-microbial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6.
      ;
      • Dorschner R.A.
      • Pestonjamasp V.K.
      • Tamakuwala S.
      • et al.
      Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
      ), consistent with a role in barrier defense. Additional support for a direct role in skin anti-microbial defense was recently demonstrated by an enhanced susceptibility to necrotizing skin infections in mice deficient for the murine cathelicidin protein, CRAMP (
      • Nizet V.
      • Ohtake T.
      • Lauth X.
      • et al.
      Innate anti-microbial peptide protects the skin from invasive bacterial infection.
      ). Interestingly, these mice also exhibited insufficient and delayed wound closure compatible with the notion that cathelicidins may have multiple roles in barrier defense and repair.
      Epithelia constitute the primary barrier between host and the potentially harmful environment, and therefore the protection of this interface is vital. Epithelial injury immediately sets in motion a series of tightly orchestrated events with the purpose of promptly reinstating the integrity of this barrier. Urgent wound closure has evolved in higher organisms, diverging from the time-consuming process of complete regeneration of tissue seen in lower species. Impaired wound healing and chronic ulcers constitute a major clinical burden and, regardless of the underlying cause, such ulcers are characterized by chronic inflammation (
      • Herrick S.E.
      • Sloan P.
      • McGurk M.
      • Freak L.
      • McCollum C.N.
      • Ferguson M.W.
      Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.
      ;
      • Phillips T.J.
      • Palko M.J.
      • Bhawan J.
      Histologic evaluation of chronic human wounds treated with hydrocolloid and nonhydrocolloid dressings.
      ). This persistent inflammation creates an environment with elevated levels of cytokines and proteases. Additionally, there is an imbalance in proteolytic enzymes and their endogenous inhibitors (
      • Trengove N.J.
      • Stacey M.C.
      • MacAuley S.
      • et al.
      Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors.
      ), all of which is thought to impair wound healing. Despite considerable advances in knowledge about the biology of normal wound repair, however, our understanding of this fundamental process is still insufficient and has led to little therapeutic progress in wound care.
      Wound extracts contain anti-microbial substances, including both LL-37, and various defensins (
      • Frohm M.
      • Gunne H.
      • Bergman A.C.
      • et al.
      Biochemical and antibacterial analysis of human wound and blister fluid.
      ). Here we demonstrate that the human anti-microbial peptide hCAP18 is upregulated in skin epithelium as a normal response to injury, which is consistent with and confirm recent data from
      • Dorschner R.A.
      • Pestonjamasp V.K.
      • Tamakuwala S.
      • et al.
      Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
      . In chronic nonhealing ulcers, however, only low levels of hCAP18 are found and immunoreactive protein is lacking in wound edge epithelium. We further show that hCAP18 is induced during re-epithelialization of organ-cultured skin wounds, and that re-epithelialization is inhibited by antibodies against LL-37 in a concentration-dependent manner. These findings suggest that LL-37 plays a crucial part in wound closure.

      Materials and Methods

      Tissue samples

      The study was approved by the Regional Committee of Ethics in Stockholm and all samples were used with informed consent. Patients (n=9) with chronic (>6 mo duration) leg ulcers due to venous insufficiency were recruited at the Department of Dermatology, Karolinska Hospital, Stockholm (Table I). Individuals with a history of diabetes mellitus, arterial insufficiency or chronic inflammatory disease were excluded. Patients with signs of eczema in the ulcer margin, clinical signs of infection or undergoing systemic or topical antibiotic treatment at the time for biopsy were also excluded. Patients included were all treated with inert local dressings and standard compression bandaging. Punch biopsies (4 mm) obtained from the wound margin, including 50% of the epithelialized area, were snap frozen.
      Table IClinical data
      Sex/Age (yr)Wound area (cm2)Wound duration (mo)Bacterial culture
      Results of bacterial culture showing colonizing bacteria in leg ulcers without clinical signs of infection.
      Normal skin
       1F 52
       2
       3
      Surgical wounds in vivo
       1F 29
       2F 63
       3F 52
      Chronic ulcers
       1M 40 4 7
       2F 84 26 16Staphylococcus aureus
      Enterococcus species
      Mixed gram negative flora
       3F 52 36Staphylococcus aureus
       4F 82 10 6Staphylococcus aureus
      Betaheamolytic group A streptococci
      Xanthomonas Maltophilia
       5M 54 9 36Staphylococcus aureus
      Enterobacter cloacae
       6M 88 9Staphylococcus aureus
      Enterococcus species
      Mixed gram negative flora
       7F 96 7Staphylococcus aureus
      Mixed gram negative flora
       8M 61 30 36Staphylococcus aureus
      Enterococcus species
      Mixed gram negative flora
       9F 84 12 21Staphylococcus aureus
      Proteus mirabilis
      Experimental wounds ex vivo
       1F 31
       2F 33
       3F 21
       4F 61
      a Results of bacterial culture showing colonizing bacteria in leg ulcers without clinical signs of infection.
      For the studies of normal wound healing in vivo, five surgical wounds were made with a 3 mm biopsy punch in the abdominal region of healthy volunteers (n=3), and the wound area was covered with sterile surgical film, Tegaderm® (3M Health Care, St Paul, MN). The wounds were subsequently excised with a 6 mm biopsy punch and snap frozen. These surgical wounds represented the sequential phases of normal wound healing at 5 min, 12 h, 2 d, 7 d, and 14 d postwounding (Table I).
      For the ex vivo wound healing model (
      • Kratz G.
      • Lake M.
      • Gidlund M.
      Insulin like growth factor-1 and -2 and their role in the re-epithelialisation of wounds; interactions with insulin like growth factor binding protein type 1.
      ), human skin was obtained from routine abdominal or breast reduction surgery. Under sterile conditions, full-thickness wounds were made, on the epidermal side, with a 3 mm biopsy punch. These ex vivo wounds were excised with a 6 mm biopsy punch and subsequently transferred to 24-well plates and covered with 2 ml of medium. Such wounds reproducibly re-epithelialize within 4–7 d (
      • Kratz G.
      • Lake M.
      • Gidlund M.
      Insulin like growth factor-1 and -2 and their role in the re-epithelialisation of wounds; interactions with insulin like growth factor binding protein type 1.
      ;
      • Inoue M.
      • Kratz G.
      • Haegerstrand A.
      • Ståhle-Bäckdahl M.
      Collagenase expression is rapidly induced in wound-edge keratinocytes after acute injury in human skin, persists during healing, and stops at re-epithelialization.
      ;
      • Kratz G.
      Modeling of wound healing processes in human skin using tissue culture.
      ). Medium, Dulbecco's modified Eagle's medium (Gibco BRL, Life Technologies, Scotland) containing 10% fetal bovine serum and antibiotics (PEST=penicillin 50 U per liter and streptomycin 50 mg per ml), was changed every third day. Wounds were harvested at different time-points, by 2, 4, and 7 d postwounding and snap frozen. In total, the experiment was repeated four times, two times for in situ hybridization and immunohistochemistry and two times for treatment with LL-37 anti-serum. Four different donors were used and triplicate wounds were made for each condition in every experiment. In each experiment, only skin from a single donor was used.
      All biopsies from the ex vivo wound healing model were serially completely sectioned. Sections representing maximal re-epithelialization in the center of the wounds were selected for evaluation.

      Preparation of RNA probes

      A 435 bp hCAP18 length cDNA (
      • Cowland J.B.
      • Johnsen A.H.
      • Borregaard N.
      hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules.
      ) was subcloned in Bluescript KS 11 and was used after linearization with BamHI and EcoRI as a template for in vitro transcription to generate 35S-labeled anti-sense and sense probes. After transcription, the RNA probes were ultrafiltered (Micron 100; Amicon Inc., Beverly, MA) before hybridization. The specificity of this probe has previously been demonstrated (
      • Frohm Nilsson M.
      • Sandstedt B.
      • Sørensen O.
      • Weber G.
      • Borregaard N.
      • Ståhle-Bäckdahl M.
      The human cationic anti-microbial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6.
      ).

      In situ hybridization

      In situ hybridization was performed as described (
      • Ståhle-Bäckdahl M.
      • Sudbeck B.D.
      • Eisen A.Z.
      • Welgus H.G.
      • Parks W.C.
      Expression of 92-kDa type IV collagenase mRNA by eosinophils associated with basal cell carcinoma.
      ). Briefly, frozen 6–7 μm thick sections were hybridized overnight with 2.5×106 cpm of 35S-labeled RNA probes at 50°C. After hybridization, slides were washed under stringent conditions including incubation with 50 μg of RNase A (Sigma, St Louis, MO) per ml for 30 min at 37°C and were then processed for autoradiography for 4–5 wk.

      LL-37 antibody

      LL-37 peptide (amino acid sequence: H-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser-OH) was synthesized (EuroDiagnostica AB, Malmö, Sweden) according to Fmoc-strategy using solid phase synthesis (
      • Fields G.B.
      • Noble R.L.
      Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.
      ) and purified by high-performance liquid chromatography to a purity of 98%. Biologic activity of the peptide was confirmed in an anti-bacterial assay (
      • Frohm M.
      • Gunne H.
      • Bergman A.C.
      • et al.
      Biochemical and antibacterial analysis of human wound and blister fluid.
      ). The peptide was used for immunization of three rabbits according to a standard protocol (AgriSera, Vännäs, Sweden). Polyclonal anti-serum was affinity purified using synthetic LL-37 peptide and the purified anti-serum was assessed with enzyme-linked immunosorbent assay. IgG concentration of the immune serum was diluted to 0.5 mg per ml. Pre-immune serum was collected from each rabbit and the IgG concentration was 2 mg per ml.

      Immunohistochemistry

      All biopsies were snap frozen and handled identically. Samples from both acute and chronic wounds were included in the same experiments. In short, 6–7 μm thick cryostat sections were incubated with polyclonal antibody, raised in rabbits against recombinant hCAP18 and affinity purified with recombinant cathelin peptide at dilutions 1: 1000 and 1: 2000 (
      • Sørensen O.
      • Cowland J.B.
      • Askaa J.
      • Borregaard N.
      An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma.
      ) and with polyclonal LL-37 antibody at dilutions 1: 1000 and 1: 2000. All sections were stained in parallel with both antibodies according to the indirect peroxidase method using a Vectastain kit (Vector Laboratories, Burlingame, CA) and following the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin solution. All experiments were repeated minimum three times to ensure reproducibility. As controls, serial tissue sections were processed in parallel without adding primary antibody and using preimmune rabbit IgG (DAKO, Glostrup, Denmark) as the primary antibody. For assessment of proliferation in the organ-cultured wound model, sections representing the different culture conditions and time-points were stained with mouse monoclonal Ki67 anti-serum (DAKO) at 1: 25 dilution as described above.

      Immunoabsorption of hCAP18

      To ascertain further the specificity of the immunostaining, we performed immunoabsorption adding the anti-hCAP18 antibody, diluted 1: 1000, to the recombinant cathelin peptide at 10−6M, 10−8M, and 10−10M. The mixture was preincubated at +4°C overnight and then centrifuged 10,600g (10,000 r.p.m., 5 min) prior to immunohistochemical analyses as described above. In parallel, control sections were processed identically, except that no peptide was added to absorb the antibody.

      Protein extraction

      Frozen biopsies (22–69 mg) from healthy volunteers (n=2) and patients (n=3) were cut in 50 μm sections. Cold extraction buffer of 60% aqueous acetonitrile containing 1% trifluoroacetic acid (
      • Frohm M.
      • Gunne H.
      • Bergman A.C.
      • et al.
      Biochemical and antibacterial analysis of human wound and blister fluid.
      ) was added and samples were eluted on an Eppendorfshaker (IKA-Vibrax-VXR, Labasco, Mölndal, Sweden) and then centrifuged. The supernatants were lyophilized and then diluted in 1000 μl of double distilled H2O. Protein concentrations were measured by Protein Assay Kit (Bio-Rad Laboratories, Hercules CA) based on the Bradford method (
      • Bradford M.M.
      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
      ) and samples were diluted to a final protein concentration of 1 mg per ml.

      Immunoblotting

      Total protein extracts were separated, adding 30 μg of each sample to a discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 16.5% Tris-Tricine Ready Gels (Bio-Rad Laboratories) and then electroblotted on to nitrocellulose filters. After blocking with 5% skim milk in phosphate-buffered saline for 1 h, the filters were incubated separately overnight with either rabbit polyclonal hCAP18 antibody affinity purified against cathelin (1: 1000) or rabbit polyclonal antibody (1: 1000), raised and affinity purified against synthetic LL-37 (AgriSera). The filters were then incubated with horseradish-peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) followed by development using enhanced chemiluminescence (Amersham Pharmacia Biotech, Sunnyvale, CA) and exposed with a CCD camera (Fujifilm, Sendai, Japan).

      Enzyme-linked immunosorbent assay

      A sandwich enzyme-linked immunosorbent assay, using the antibody affinity purified with cathelin, previously described was used to quantitate hCAP18 in protein extracts from patients (n=3) and healthy volunteers (n=2) (
      • Sørensen O.
      • Cowland J.B.
      • Askaa J.
      • Borregaard N.
      An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma.
      ).

      Anti-bacterial assay

      The classical zone assay was used as described (
      • Frohm M.
      • Gunne H.
      • Bergman A.C.
      • et al.
      Biochemical and antibacterial analysis of human wound and blister fluid.
      ). In short, thin agarose plates, 1 mm, were seeded with the test bacteria strain Bacillus megaterium. The melted agarose contained Luria–Bertani broth supplemented with medium E. The test bacteria were added in the log phase just before pouring the plate. Small wells were punched in the assay plates and loaded with 3 μl test sample. After overnight incubation at 30°C, the inhibition zones were measured. The LL-37 peptide was mixed with 10-fold molar excess of LL-37 anti-serum. The mixture was preincubated for 2 h at room temperature and then centrifuged 10,600g (10,000 r.p.m., 5 min) prior to use.

      Treatment with LL-37 antibody in the ex vivo wound healing model

      Rabbit polyclonal antibody, raised and affinity purified against synthetic LL-37 (AgriSera) was used in the experimental wound healing model described above. LL-37 antibody was added in 2 ml medium per well (Dulbecco's modified Eagle's medium+10% fetal bovine serum and PEST) to a final antibody dilution of 1: 10, 1: 100, and 1: 1000. As control we used the corresponding preimmune serum at a final IgG concentration equal to the 1: 10 dilution of the LL-37 anti-serum. Each experimental condition was made in triplicates and repeated twice. The media was changed every third day and LL-37 antibody or preimmune serum was added as described above. The ex vivo wounds were harvested 2, 4, and 7 d postwounding. All specimens were snap frozen, sectioned in completion and mounted on Superfrost Plus slides prior to staining with hematoxylin–eosin.

      Results

      hCAP18 is produced in wound edge keratinocytes during re-epithelialization ex vivo and in vivo but is lacking in the epithelium of chronic ulcers

      Upon injury, there was upregulation of hCAP18 in the epidermis at the wound border (Fig 1a,b). By 2 d, hCAP18 was clearly expressed in the migrating front during re-epithelialization both at RNA and protein levels (Fig 1c and Fig 2a–c). Following wound closure, by day 7, immunoreactivity for hCAP18 declined in the epidermis (Fig 1d) and was at 14 d similar to that of intact non-inflammatory skin (Fig 1e,g). This pattern of hCAP18 expression was reproduced in the organ-cultured wounds undergoing re-epithelialization (Fig 3). Our findings indicate that regulation of hCAP18 expression in wound edge keratinocytes does not require inflammation.
      Figure thumbnail gr1
      Figure 1Expression of hCAP18 in acute surgical in vivo wounds during re-epithelialization. (a) At 5 min there was prominent immunoreactivity for hCAP18 in the basal layer of the epidermis. Arrow indicates the wound edge. (b) After 12 h, intense immunoreactivity was found throughout the wound edge and in the wound bed infiltrate and exudate. (c) By 2 d, strong immunoreactivity for hCAP18 was detected in the epithelial front migrating over the wound bed (boxed area) and in the wound bed. (d) At 7 d, when re-epithelialization was complete, weak immunoreactivity was found in the basal layer of epidermis. Focal hCAP18 immunostaining remained in scattered dermal cells. (e) At day 14 single immunoreactive dermal cells remained and in the basal epidermal layer only weak staining for hCAP18 was detected, comparable with that of normal unwounded skin. (f) Immunoabsorption using cathelin peptide at 10−6M completely abolished the hCAP18 immunoreactivity detected by 7 d. (g) Normal skin with hCAP18 immunoreactivity in the basal cell layer. Photomicrographs show results obtained with the hCAP18 antibody at 1: 2000 dilution. Scale bars: (a,b,g) 50 μm; (c–f) 100 μm.
      Figure thumbnail gr2
      Figure 2Expression of hCAP18 in migrating epithelial front. (a) High magnification of the boxed area () visualizes prominent hCAP18 immunoreactivity in the epithelial front. (b) Dark-field photomicrograph, of a serial section, demonstrates a matching in situ hybridization signal for hCAP18 mRNA (white grains). (c) Control section hybridized with the sense hCAP18 cRNA probe lacked specific signal hCAP18 mRNA. Immunophotomicrographs show results obtained with the hCAP18 antibody at 1: 2000 dilution. Scale bar: 10 μm.
      Figure thumbnail gr3
      Figure 3Expression of hCAP18 in the organ-cultured wounds during re-epithelialization. (a) Prominent immunoreactivity was detected in the epithelial tongue migrating to cover the wound bed. (b) Higher magnification demonstrating that immunoreactivity for hCAP18 was essentially confined to the basal epithelial cell layer. (c) Dark-field photomicrograph demonstrates a matching signal for hCAP18 mRNA (white grains) in the basal epithelial layer by in situ hybridization. (d) Control section hybridized with the sense hCAP18 cRNA probe lacked signal for hCAP18 mRNA. (e) Bright-field view of the boxed area shows hCAP18 mRNA signal as black dots in keratinocytes. (f) High power view demonstrates lack of hCAP18 mRNA in the control section. Immunophotomicrographs show results obtained with the hCAP18 antibody at 1: 2000 dilution. Scale bars: (a) 100 μm; (b–d) 10 μm; (e,f) 25 μm.
      In contrast to the production of hCAP18 in the epithelial front of physiologically healing wounds, the epithelium of all nine chronic ulcers lacked immunoreactivity for hCAP18 (Fig 4). By in situ hybridization there was prominent signal for hCAP18 mRNA, but no staining for the protein was detected (Fig 4c,e). Strong immunoreactivity in the wound bed and in dermal cells served as a positive internal control (Fig 4a,b). Previous studies have shown good correlation between hCAP18 mRNA and protein levels (
      • Frohm M.
      • Agerberth B.
      • Ahangari G.
      • Ståhle-Bäckdahl M.
      • Lidén S.
      • Wigzell H.
      • Gudmundsson G.H.
      The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.
      ;
      • Frohm Nilsson M.
      • Sandstedt B.
      • Sørensen O.
      • Weber G.
      • Borregaard N.
      • Ståhle-Bäckdahl M.
      The human cationic anti-microbial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6.
      ;
      • Malm J.
      • Sørensen O.
      • Persson T.
      • et al.
      The human cationic anti-microbial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa.
      ). The results presented here, however, indicate that hCAP18 protein is not properly translated, or is rapidly degraded, in the epithelium of chronic ulcers.
      Figure thumbnail gr4
      Figure 4Expression of hCAP18 in a chronic venous leg ulcer. (a) Overview of a chronic ulcer where the left half of the micrograph represents the epithelium (EP) and the right half represents the wound bed (WB). Prominent immunoreactivity for hCAP18 was detected in the wound bed and in scattered dermal cells underneath the epithelium. (b) Higher magnification of the boxed area demonstrates the dermoepidermal junction (dotted line) in detail. Basal keratinocytes essentially lacked immunoreactivity for hCAP18, whereas strong immunostaining was seen in the dermal cells. (c) High magnification of the epithelial front (boxed area). Note lack of hCAP18 immunoreactivity in keratinocytes. The dotted line represents the dermoepidermal junction. Arrow indicates the wound edge. (d) Dark-field photomicrograph of the same tissue as in (c). No signal for hCAP18 mRNA was detected by hybridization with a control sense probe. (e) Positive signal for hCAP18 mRNA was detected by hybridization with the anti-sense cRNA probe. Note that despite the clear signal for hCAP18 mRNA, there was no immunoreactivity for the protein in this area as visualized in the panel above (). Immunophotomicrographs show results obtained with the hCAP18 antibody at 1: 2000 dilution. Scale bars: (a) 100 μm; (b–e) 25 μm.
      All tissues were immunostained with both hCAP18 and LL-37 anti-sera in parallel. Both anti-sera gave identical results concerning immunoreactivity in the epithelium. Immuno-staining in the dermis was slightly more pronounced using the hCAP18 antibody but there was no qualitative difference influencing interpretation of data. For consistency, all photomicrographs show results obtained with the hCAP18 antibody.

      LL-37 antibody inhibits re-epithelialization in a concentration-dependent manner in organ-cultured human wounds

      The distinct expression of hCAP18 in keratinocytes during re-epithelialization prompted us to investigate whether the bioactive peptide LL-37 may be directly involved in this process. The organ-cultured full-thickness wounds reproducibly re-epithelialize under standard conditions and represent a useful model for noninflammatory wound closure. Adding polyclonal anti-LL-37 IgG affected re-epithelialization in a concentration-dependent manner. Normal control wounds (Fig 5a) and wounds treated with preimmune serum at corresponding IgG concentrations (Fig 5b) were completely re-epithelialized at 7 d and comprised of two to three epithelial cell layers. The highest concentration of LL-37 anti-serum (1: 10) severely impaired re-epithelialization and prevented wound closure (Fig 5c). The lower concentration of LL-37 anti-serum (1: 100) allowed for partial wound coverage by 4 d (not shown) and at 7 d the wound bed was only covered with a thin, fragile layer of keratinocytes (Fig 5d). At 1: 1000 antibody dilution re-epithelialization occurred at a level equal to that of the controls (Fig 5e). The results from two different experiments, including triplicate wounds, were consistent and indicate that LL-37 produced by keratinocytes is required for epithelial wound closure.
      Figure thumbnail gr5
      Figure 5LL-37 antibody inhibited re-epithelialization in a concentration-dependent manner in the organ cultured full-thickness ex vivo wound model. (a) Normal control wound completely re-epithelialized at 7 d. Inset shows higher magnification of the epithelium of the right wound margin comprising two to three cell layers. (b) Preimmune serum, at a final IgG concentration equal to the 1: 10 dilution of the LL-37 anti-serum, did not affect re-epithelialization or the appearance of the epithelium in control wounds at 7 d or the appearance of the epithelium. (c–e) Adding polyclonal anti-LL-37 IgG affected re-epithelialization in a concentration-dependent manner. (c) The highest concentration of LL-37 anti-serum (1: 10) severely impaired re-epithelialization and inhibited wound closure. Higher magnification demonstrates a thin, profoundly affected epithelium. Arrows indicate the epithelial edges. (d) At 1: 100 dilution of LL-37 anti-serum, the wound bed was covered with a single layer of keratinocytes with a fragile appearance at day 7. (e) At 1: 1000 antibody dilution re-epithelialization occurred at a level equal to that of the controls. Scale bars: (a–e) 50 μm; insets 10 μm.

      Organ-cultured wounds treated with high concentration of LL-37 anti-serum lack immunoreactivity for the proliferation marker Ki67 in the epithelium

      High number of immunoreactive keratinocytes were detected in normal control wounds at day 7 (Fig 6a) but not in the epithelium of the wound samples treated with LL-37 anti-serum at 1: 10 dilution inhibiting re-epithelialization (Fig 6b), indicating that proliferation of the epithelium is impaired in these wounds.
      Figure thumbnail gr6
      Figure 6Expression of the proliferation marker Ki67 in the organ-cultured wound model. (a) Normal control wound at day 7 demonstrates multiple Ki67 immunoreactive cells in the basal epithelial layer. (b) Lack of Ki67 immunoreactive cells in the epithelium of the wound sample treated with LL-37 anti-serum at high concentration, 1: 10 dilution, which inhibited re-epithelialization. Arrow indicates margin of the fragile epithelium at day 7. Serial section of the same tissue as shown in . Scale bar: (a,b) 10 μm.

      LI-37 antibody blocks the anti-bacterial activity induced by II-37 peptide

      Inhibition zone assay demonstrated that LL-37 inhibited growth of Bacillus megaterium in a concentration-dependent manner. Adding LL-37 anti-serum in a 10-fold molar excess completely abolished the inhibition of bacterial growth induced by LL-37 peptide alone (Fig 7).
      Figure thumbnail gr7
      Figure 7LL-37 antibody inhibited the antibacterial activity induced by LL-37 peptide. Inhibition zone assay demonstrating that LL-37 peptide at (a) 0.05 mg per ml and (b) 0.5 mg per ml inhibited growth of the test bacterium Bacillus megaterium in a concentration-dependent manner, whereas addition of LL-37 antibody to LL-37 peptide completely abolishes the anti-bacterial effect. (c) phosphate-buffered saline was used as a negative control.

      High levels of hCAP18 are produced during physiologic wound healing but not in chronic ulcers

      Upregulation of hCAP18 occurs as a normal response to skin injury. Maximum hCAP18 levels with evidence of release of bioactive LL-37 peptide were attained at 12–48 h postwounding and declining to basal pre-wounding levels at 14 d (Figure 8, Figure 9). hCAP18 protein levels in the chronic ulcers were only 10–30% of maximum levels found in surgical wounds (Fig 8) and LL-37 was barely detected by immunoblotting (Fig 9). In addition to hCAP18 upregulation in wound edge keratinocytes, we observed an intense immunoreactivity in the wound bed infiltrate and in the extracellular interstitium of the surgical wounds (Fig 1b,c). After re-epithelialization, by day 7 (Fig 1d), hCAP18 immunoreactivity was less pronounced in the basal epidermal layer with focal immunoreactivity remaining in scattered dermal cells. Two weeks after the initial wounding (Fig 1e), only single immunoreactive dermal cells remained. Epidermis was still hypertrophic and in basal keratinocytes hCAP18 immunoreactivity was evident and comparable with that of normal, quiescent skin (Fig 1j). Immunoabsorption using cathelin peptide at 10−10M decreased hCAP18 immunoreactivity and at 10−8M and 10−6M completely abolish it (Fig 1f).
      Figure thumbnail gr8
      Figure 8hCAP18 protein levels in surgical wounds and in chronic ulcers. Enzyme-linked immunosorbent assay was performed on protein extracts from skin biopsies of two surgical wounds (nos 1 and 2 in ) and three chronic venous leg ulcers (nos 7–9 in ). The surgical wounds were obtained at different time-points from the abdominal region of healthy volunteers. Peak levels of hCAP18 protein were detected at 2 d postwounding. hCAP18 protein levels in chronic wounds were only 20–30% of the maximum levels in surgical wounds.
      Figure thumbnail gr9
      Figure 9Immunoblotting for hCAP18 in surgical wounds and chronic ulcers. (a) Synthetic LL-37 peptide, 10 ng, and recombinant cathelin, 8.5 ng, served as controls. Antibody raised against hCAP18 holoprotein and affinity purified against the cathelin peptide was used for immunoblotting. The antibody detects high concentration of LL-37 peptide. (b) The same antibody as in panel (a) was used and demonstrated immunoreactive bands corresponding to the intact, nonprocessed 18 kDa holoprotein both in the surgical wound at 12 h and in all three chronic ulcers. A strong band for the cathelin peptide is detected only in the surgical wound. (c) Antibody raised against synthetic LL-37 peptide and affinity purified against the same peptide demonstrated a strong band for LL-37 in the 12 h surgical wound. Faint LL-37 bands were seen in two of the chronic ulcers.
      In chronic, nonhealing ulcers hCAP18 immunoreactivity was most pronounced in the wound bed and essentially lacking in wound edge epidermis and in the basal epidermal layer (Fig 4a–c). In addition and analogous to the pattern detected in the surgical wounds at 7 d, there was prominent immunoreactivity in scattered dermal cells (Fig 4b). Intact skin from these patients, obtained from the area proximal to the knee, showed immunoreactivity for hCAP18 and LL-37 in the basal epithelial layer analogous to the pattern detected in healthy control skin (Fig 1g), indicating that there is no generalized defect in hCAP18 expression in skin from patients with chronic ulcers.
      Identification of the immunoreactive dermal cells in both acute and chronic wounds indicate that a subset of these cells were macrophages, by CD68 immunoreactivity in both types of wounds (data not shown).

      Discussion

      Anti-microbial peptides such as hCAP18 are now recognized as being key components of the epithelial defense armament (
      • Huttner K.M.
      • Bevins C.L.
      Antimicrobial peptides as mediators of epithelial host defense.
      ). Only low levels of hCAP18 are present in noninflamed intact skin, but upon injury there is strong upregulation in the wounded tissue with protein levels spiking in the initial phase of wound repair and subsiding upon wound closure. Our findings confirm recent data that show induction of cathelicidin proteins in association with acute wounding in humans and mice (
      • Dorschner R.A.
      • Pestonjamasp V.K.
      • Tamakuwala S.
      • et al.
      Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
      ).
      In contrast to abundant hCAP18 protein in acute wounds, only low levels are present in chronic nonhealing ulcers with lack of immunoreactivity for hCAP18 in the epithelium. In fact, the constitutive hCAP18 expression found in basal keratinocytes in normal skin, is practically lost in chronic ulcers (Fig 4). Despite lack of immunoreactive protein, however, there is a prominent signal for hCAP18 mRNA in the epithelium of chronic ulcers (Fig 4e), which is an unexpected result. Thus, regulation of hCAP18 protein production, or post-translational processing, appears to be disturbed in chronic ulcer keratinocytes. Basal keratinocytes are critical in cell migration and re-epithelialization and interfering with their functions is detrimental to healing. In chronic ulcers, wound edge keratinocytes lose the impetus to change from a stationary to a migratory and proliferative phenotype. The signals regulating this process under normal conditions are largely unknown but it has been shown that the environment in chronic ulcers is suppressive, as chronic wound fluid inhibits cell proliferation and migration in vitro, whereas acute wound fluid has a stimulatory effect (
      • Falanga V.
      Growth factors and chronic wounds: the need to understand the microenvironment.
      ;
      • Bennett N.T.
      • Schultz G.S.
      Growth factors and wound healing: Part II. Role in normal and chronic wound healing.
      ;
      • Bucalo B.
      • Eaglestein W.H.
      • Falanga V.
      Inhibition of cell proliferation by chronic wound fluid.
      ;
      • Phillips T.J.
      • Al-Amoudi H.O.
      • Leverkus M.
      • Park H.Y.
      Effect of chronic wound fluid on fibroblasts.
      ).
      What mechanisms may contribute to low levels of hCAP18 and LL-37 in nonhealing ulcers? A fundamental difference between the microenvironments of acute and chronic ulcers is the microbial flora. Current research introduces the novel concept in the battle between microbes and host, where pathogens develop strategies to suppress the expression of anti-microbial proteins, including cathelicidins and defensins (
      • Islam D.
      • Bandholtz L.
      • Nilsson J.
      • Wigzell H.
      • Christensson B.
      • Agerberth B.
      • Gudmundsson G.
      Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator.
      ), thereby facilitating the invasion and infection of the host. Chronic ulcers are indeed characterized by colonization of a mixture of bacteria that persist in the wound habitat. Interfering with anti-microbial effector molecules may provide a clever and effective strategy for such bacteria simultaneously to evade the host immune defense, escape death, and prevent closure of their habitat. Highly relevant to this study are recent findings demonstrating that bacterial proteinases have the capacity to degrade the anti-microbial peptides α-defensin (
      • Schmidtchen A.
      • Frick I.
      • Björck L.
      Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial alpha-defensin.
      ) and LL-37 (Schmidtchen A, personal communication). Thus, bacterial spectra in chronic ulcers and their abilities to inactivate the endogenous anti-microbial defense merit further detailed analysis. In this respect, not only bacteria considered as pathogenic but also strains regarded as more harmless should be investigated.
      Covering of an exposed wound bed with new epithelium represents a critical event in wound repair. In our ex vivo wound healing model, we studied re-epithelialization in a somewhat restricted sense, namely the simple migration and subsequent proliferation of keratinocytes to form a thin layer of provisional neoepidermis that does not constitute a complete and functional barrier. Bearing in mind that this “naked” model of epidermal restoration does not fully reflect in vivo complexity, however, our model does offer the potential to dissect mechanisms involved and the advantage of intact epithelial/stromal architecture, which is fundamental in re-epithelialization. Our finding that hCAP18 is expressed at mRNA and protein levels in keratinocytes migrating and proliferating to cover the wound indicates that the machinery regulating this process is contained within the wound edge microenvironment and does not require inflammation.
      In order to re-epithelialize reproducibly, the ex vivo wounds require medium containing at least 10% fetal bovine serum (
      • Inoue M.
      • Kratz G.
      • Haegerstrand A.
      • Ståhle-Bäckdahl M.
      Collagenase expression is rapidly induced in wound-edge keratinocytes after acute injury in human skin, persists during healing, and stops at re-epithelialization.
      ;
      • Kratz G.
      Modeling of wound healing processes in human skin using tissue culture.
      ). Human serum inhibits anti-microbial and cytotoxic functions of LL-37, which occur by direct membrane effect (
      • Johansson J.
      • Gudmundsson G.H.
      • Rottenberg M.E.
      • Berndt K.D.
      • Agerberth B.
      Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37.
      ), whereas migration of leukocytes in response to LL-37, a receptor-mediated effect, occurs independently of the presence of serum (
      • De Y.
      • Chen Q.
      • Schmidt A.P.
      • et al.
      LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells [In Process Citation].
      ). Thus, albeit the use of fetal and not human serum in our model, it is not likely that the effect of endogenous LL-37 on wound closure would be impeded by the present culture conditions.
      Our present results on the role of LL-37 in re-epithelialization are consistent with anti-microbial peptides being multifunctional proteins. Thus, LL-37 can block LPS-induced inflammation (
      • Nagaoka I.
      • Hirota S.
      • Niyonsaba F.
      • Hirata M.
      • Adachi Y.
      • Tamura H.
      • Heumann D.
      Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.
      ) and is chemotactic for leukocytes (
      • De Y.
      • Chen Q.
      • Schmidt A.P.
      • et al.
      LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells [In Process Citation].
      ). Of potential relevance for this study are the recent findings that the porcine cathelicidin protein PR-39 was shown to promote angiogenesis and induce syndecans in pig wounds (
      • Gallo R.L.
      • Ono M.
      • Povsic T.
      • Page C.
      • Eriksson E.
      • Klagsbrun M.
      • Bernfield M.
      Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich anti-microbial peptide from wounds.
      ;
      • Li J.
      • Post M.
      • Volk R.
      • et al.
      PR39, a peptide regulator of angiogenesis.
      ). Other data demonstrate that syndecans are critical in wound healing and mice lacking syndecan-4 genes show delayed wound healing and impaired angiogenesis (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      Delayed wound repair and impaired angiogenesis in mice lacking syndecan- 4.
      ). Thus, cathelicidin proteins, including LL-37, are likely to possess a broad functional repertoire.
      In summary, we show that induction of hCAP18 in wound edge epithelium is a physiologic response to injury that does not require inflammation and that blocking its bioactive peptide LL-37 inhibits wound closure in a concentration-dependent manner. The mechanisms underlying this inhibition remain to be elucidated, but the lack of Ki67 immunoreactive cells in the epithelium of the suppressed wounds suggests that proliferation of epithelial cells is affected. Furthermore, we demonstrate that hCAP18/LL-37 is lacking in chronic ulcer epithelium. These results indicate that LL-37 is required for re-epithelialization and suggest that lack of LL-37 in chronic ulcer epithelium may impair epidermal restoration. Further studies are needed to understand fully the role of LL-37 in the re-epithelialization process.

      ACKNOWLEDGMENTS

      This work was supported by grants from the Karolinska Institute and the Welander-Finsen Foundation. We thank coworkers at the Dermatologic Diagnostic Center and Anna-Lena Kastman for excellent technical assistance. Dr Birgitta Agerberth is gratefully acknowledged for advice on the antibacterial assay and Dr Kevin O'Brien for linguistic revision.

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