1. The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium 
Johan D. Heilborn, Margareta Frohm Nilsson, Ole Sørensen, Mona Ståhle-Bäckdahl 
Journal of Investigative Dermatology 
Volume 120, Issue 3, Pages (March 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of hCAP18 in acute surgical in vivo wounds during re-epithelialization. (a) At 5min there was prominent immunoreactivity for hCAP18 in the basal layer of the epidermis. Arrow indicates the wound edge. (b) After 12h, 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.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
Expression 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.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Expression 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.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Expression 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 (Fig 4c). Immunophotomicrographs show results obtained with the hCAP18 antibody at 1: 2000 dilution. Scale bars: (a) 100 μm; (b–e) 25 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
LL-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.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Expression 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 Fig 5(c). Scale bar: (a,b) 10 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
LL-37 antibody inhibited the antibacterial activity induced by LL-37 peptide. Inhibition zone assay demonstrating that LL-37 peptide at (a) 0.05mg per ml and (b) 0.5mg 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.
Journal of Investigative Dermatology  , DOI: ( /j x)
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9. Figure 8
hCAP18 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 Table I) and three chronic venous leg ulcers (nos 7–9 in Table I). 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.
Journal of Investigative Dermatology  , DOI: ( /j x)
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10. Figure 9
Immunoblotting 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 18kDa holoprotein both in the surgical wound at 12h 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 12h surgical wound. Faint LL-37 bands were seen in two of the chronic ulcers.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions