Title: The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium
Abstract: 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. 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. 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 and Lehrer, 1999Ganz T. Lehrer R.I. Antibiotic peptides from higher eukaryotes: biology and applications.Mol Med Today. 1999; 5: 292-297Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar;Lehrer and Ganz, 1999Lehrer R.I. Ganz T. Antimicrobial peptides in mammalian and insect host defence.Curr Opin Immunol. 1999; 11: 23-27Crossref PubMed Scopus (652) Google Scholar). 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 et al., 1983Selsted 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.J Biol Chem. 1983; 258: 14485-14489Abstract Full Text PDF PubMed Google Scholar;Lehrer et al., 1993Lehrer R.I. 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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.Proc Natl Acad Sci USA. 1998; 95: 9541-9546Crossref PubMed Scopus (610) Google Scholar). 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 et al., 1996Gudmundsson 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.Eur J Biochem. 1996; 238: 325-332Crossref PubMed Scopus (453) Google Scholar;Larrick et al., 1996Larrick 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.FEBS Lett. 1996; 398: 74-80Abstract Full Text PDF PubMed Scopus (58) Google Scholar). 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 et al., 1995Agerberth 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.Proc Natl Acad Sci USA. 1995; 92: 195-199Crossref PubMed Scopus (433) Google Scholar;Gudmundsson et al., 1996Gudmundsson 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.Eur J Biochem. 1996; 238: 325-332Crossref PubMed Scopus (453) Google Scholar). Cathelin was originally isolated from pig leukocytes and was proposed to act as an inhibitor of cysteine proteinases (Ritonja et al., 1989Ritonja A. Kopitar M. Jerala R. Turk V. Primary structure of a new cysteine proteinase inhibitor from pig leucocytes.FEBS Lett. 1989; 255: 211-214Abstract Full Text PDF PubMed Scopus (129) Google Scholar); 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 et al., 1996Gudmundsson 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.Eur J Biochem. 1996; 238: 325-332Crossref PubMed Scopus (453) Google Scholar). Serine proteinase 3 was recently shown to be responsible for extracellular cleavage of hCAP18 (Sørensen et al., 2001Sørensen 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.Blood. 2001; 97: 3951-3959Crossref PubMed Scopus (712) Google Scholar). 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 et al., 1997aSørensen 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.Blood. 1997; 90: 2796-2803Crossref PubMed Google Scholar) and is present in subpopulations of lymphocytes and monocytes (Agerberth et al., 2000Agerberth 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.Blood. 2000; 96: 3086-3093Crossref PubMed Google Scholar). hCAP18 is also expressed in skin and other epithelia, where it is upregulated in association with inflammation, infection, and injury (Frohm et al., 1997Frohm 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.J Biol Chem. 1997; 272: 15258-15263Crossref PubMed Scopus (665) Google Scholar;Goldman et al., 1997Goldman 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.Cell. 1997; 88: 553-560Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar;Frohm Nilsson et al., 1999Frohm 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.Infect Immun. 1999; 67: 2561-2566Crossref PubMed Google Scholar;Dorschner et al., 2001Dorschner R.A. Pestonjamasp V.K. Tamakuwala S. et al.Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.J Invest Dermatol. 2001; 117: 91-97Crossref PubMed Scopus (474) Google Scholar), 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 et al., 2001Nizet V. Ohtake T. Lauth X. et al.Innate anti-microbial peptide protects the skin from invasive bacterial infection.Nature. 2001; 414: 454-457Crossref PubMed Scopus (1020) Google Scholar). 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 et al., 1992Herrick 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.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar;Phillips et al., 1994Phillips T.J. Palko M.J. Bhawan J. Histologic evaluation of chronic human wounds treated with hydrocolloid and nonhydrocolloid dressings.J Am Acad Dermatol. 1994; 30: 61-64Abstract Full Text PDF PubMed Scopus (17) Google Scholar). 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 et al., 1999Trengove N.J. Stacey M.C. MacAuley S. et al.Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors.Wound Repair Regen. 1999; 7: 442-452Crossref PubMed Scopus (701) Google Scholar), 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 et al., 1996Frohm M. Gunne H. Bergman A.C. et al.Biochemical and antibacterial analysis of human wound and blister fluid.Eur J Biochem. 1996; 237: 86-92Crossref PubMed Scopus (192) Google Scholar). 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 fromDorschner et al., 2001Dorschner R.A. Pestonjamasp V.K. Tamakuwala S. et al.Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.J Invest Dermatol. 2001; 117: 91-97Crossref PubMed Scopus (474) Google Scholar. 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. 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 dataSex/Age (yr)Wound area (cm2)Wound duration (mo)Bacterial cultureaResults 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 aureusEnterococcus speciesMixed gram negative flora 3F 52– 36Staphylococcus aureus 4F 82 10 6Staphylococcus aureusBetaheamolytic group A streptococciXanthomonas Maltophilia 5M 54 9 36Staphylococcus aureusEnterobacter cloacae 6M 88– 9Staphylococcus aureusEnterococcus speciesMixed gram negative flora 7F 96– 7Staphylococcus aureusMixed gram negative flora 8M 61 30 36Staphylococcus aureusEnterococcus speciesMixed gram negative flora 9F 84 12 21Staphylococcus aureusProteus mirabilisExperimental 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. Open table in a new tab 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 et al., 1994Kratz 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.Scand J Plast Reconstr Surg Hand Surg. 1994; 28: 107-112Crossref PubMed Scopus (51) Google Scholar), 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 et al., 1994Kratz 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.Scand J Plast Reconstr Surg Hand Surg. 1994; 28: 107-112Crossref PubMed Scopus (51) Google Scholar;Inoue et al., 1995Inoue 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.J Invest Dermatol. 1995; 104: 479-483Crossref PubMed Scopus (113) Google Scholar;Kratz, 1998Kratz G. Modeling of wound healing processes in human skin using tissue culture.Microsc Res Tech. 1998; 42: 345-350Crossref PubMed Scopus (61) Google Scholar). 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. A 435 bp hCAP18 length cDNA (Cowland et al., 1995Cowland J.B. Johnsen A.H. Borregaard N. hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules.FEBS Lett. 1995; 368: 173-176Abstract Full Text PDF PubMed Scopus (301) Google Scholar) 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 et al., 1999Frohm 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.Infect Immun. 1999; 67: 2561-2566Crossref PubMed Google Scholar). In situ hybridization was performed as described (Ståhle-Bäckdahl et al., 1992Ståhle-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.J Invest Dermatol. 1992; 99: 497-503Abstract Full Text PDF PubMed Google Scholar). 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 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 and Noble, 1990Fields G.B. Noble R.L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.Int J Pept Protein Res. 1990; 35: 161-214Crossref PubMed Scopus (2330) Google Scholar) 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 et al., 1996Frohm M. Gunne H. Bergman A.C. et al.Biochemical and antibacterial analysis of human wound and blister fluid.Eur J Biochem. 1996; 237: 86-92Crossref PubMed Scopus (192) Google Scholar). 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. 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 et al., 1997bSørensen O. Cowland J.B. Askaa J. Borregaard N. An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma.J Immunol Methods. 1997; 206: 53-59Crossref PubMed Scopus (170) Google Scholar) 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. 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. 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 et al., 1996Frohm M. Gunne H. Bergman A.C. et al.Biochemical and antibacterial analysis of human wound and blister fluid.Eur J Biochem. 1996; 237: 86-92Crossref PubMed Scopus (192) Google Scholar) 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, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar) and samples were diluted to a final protein concentration of 1 mg per ml. 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). 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 et al., 1997bSørensen O. Cowland J.B. Askaa J. Borregaard N. An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma.J Immunol Methods. 1997; 206: 53-59Crossref PubMed Scopus (170) Google Scholar). The classical zone assay was used as described (Frohm et al., 1996Frohm M. Gunne H. Bergman A.C. et al.Biochemical and antibacterial analysis of human wound and blister fluid.Eur J Biochem. 1996; 237: 86-92Crossref PubMed Scopus (192) Google Scholar). 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. 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. 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 2Expression of hCAP18 in migrating epithelial front. (a) High magnification of the boxed area (Fig 1c) 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.View Large Image Figure ViewerDownload (PPT)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.View Large Image Figure ViewerDownload (PPT) 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