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An Intrinsic Antibiotic Mechanism in Wounds and Tissue-Engineered Skin

2001; Elsevier BV; Volume: 116; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2001.01279.x

1523-1747

Peter Schmid, Colin Osborne, David A. Cox, Olivier Grenet, J. Medina, Salah-Dine Chibout,

Dermatology and Skin Diseases

To the Editor Despite the loss of epidermal barrier function and subsequent exposure to environmental microorganisms, superficial epidermal wounds in vertebrates usually heal without major complications, suggesting the existence of a functional antimicrobial mechanism during wound healing. This general observation provides the basis for testing a hypothesis for the existence of a functional antimicrobial mechanism, which is active at cutaneous surfaces. By using standard immunohistochemical techniques, we have investigated expression of human P defensin-2 (hBD-2) in normal and lesional human skin. hBD-2 is a member of the defensin family of antimicrobial peptides, and has microbicidal activity to Gram negative bacteria and Candida albicans (Schroder and Harder, 1999). Moreover, hBD-2 has been shown to provide a link between innate and adaptive immunity (Yang et al, 1999). hBD-2 was first isolated from psoriatic scales (Harder et al, 1997), and is strongly expressed by psoriatic keratinocytes at sites of epidermal inflammation (Liu et al, 1998; Fig 1a). Although hBD-2 was not detected in the intact adult human epidermis (Fig 1b) or in the neonatal cornified foreskin epidermis (Fig 1c), it was also clearly detectable in the mucosal epithelium of the neonatal foreskin (Fig 1d), a finding that may well explain why mucosal surfaces rarely develop clinically relevant infections, despite the absence of a stratum corneum. Most notably, this study shows that hBD-2 is induced in the regenerating epidermis of acute surgical wounds (Fig 1e), suggesting that keratinocyte activation in response to loss of epidermal barrier function involves induction of an intrinsic antibiotic mechanism. Of special interest is the observation that hBD-2 is expressed in the epidermis of Apligraf (Fig 1f), a tissue- engineered human skin equivalent (HSE) produced from neonatal human foreskin. This product consisting of living fibroblasts anchored in a bovine type I collagen lattice and living keratinocytes forming a stratified epidermis has been developed for the treatment of wounds (Wilkins et al, 1994) and has beneficial effects in patients with chronic venous leg ulcers (Falanga et al, 1998). Unlike the other defensin genes, the hBD-2 gene contains several binding sites for the nuclear transcription factor NF-KB (Liu et al, 1998), which mediates responses to lipopolysaccharide (LPS) and proinflammatory cytokines. Therefore, we determined ifhBD- 2 expression is upregulated in the HSE following a topical bacterial challenge or exposure to LPS. Topical treatment with 0.5 p,g per ml (5 ng per cm2) and 1 p,g perml (10 ng per cm2) purified bacterial LPS (from E. coli serotype 0127:B8; Sigma-Aldrich, Bucks, Switzerland) for 24 h caused a strong, concentration-dependent, and statistically significant (p < 0.05) upregulation of hBD-2 expression (30.9 ± 14.6 and 59.1 ± 4.5; data represent fold change versus untreated HSE ± SEM). Recent studies have suggested that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-KB (Jiang et al 2000); however, to our knowledge, neither CD14 nor TLR-4 has been found to be expressed in keratinocytes. Therefore, a CD14/ TLR-4 independent LPS response mechanism may exist in keratinocytes. Alternatively, the possibility that a contaminant in the used LPS preparation might be responsible for the strong induction of hBD-2 in Apligraf cannot be excluded. Nevertheless, the hBD-2 induction in keratinocytes appears to be the result of pro-inflammatory processes, since other known chemical mediators of skin inflammation, e.g., phorbol 12-myristate 13-acetate and sodium lauryl sulfate, also enhanced hBD-2 mRNA expression in the HSE (data not shown). To investigate the effect of bacterial challenge on hBD-2 mRNA expression in Apliraf E. coli K12 bacteria grown over night in TY medium, pelleted, washed twice, and resuspended in DMEM/HAM-F12 (1:1) cell culture medium, were applied topically onto the epidermal surface of the HSE (approximately 106 bacteria per cm2). Bacterial challenge resulted in a weak but significant (p < 0.05) upregulation of hBD-2 mRNA expression (2.0 ± 0.4 after 24 h; 3.0 ± 0.7 after 48 h; data represent fold change versus untreated HSE ± SEM) as revealed by standard Real Time RT-PCR analysis. The observation that nonvirulent bacteria are much weaker inducers of hBD-2 than virulent bacteria, such as mucoid Pseudomonas aeruginosa (Harder et al, 2000), may provide a rationale for the fact that only a weak induction ofhBD-2 was achieved, although a rather high cell number of E. coli K12 bacteria was applied topically. To investigate if hBD-2 expression in the epidermis has functional relevance, we have soaked Apligraf with E. coli growth medium and subsequently exposed its surface to E. coli followed by a 24 h incubation at 37 °C. Bacterial colonies did not grow on the epidermal surface of the intact HSE, although colonies clearly formed on the dermal surface exposed to bacteria following mechanical separation of the epidermal layer. In conclusion, the findings of this study suggest that wound healing involves induction of an intrinsic antibiotic mechanism, which is also functional in epidermis of tissue-engineered skin. The authors thank Prof. Tomas Ganz, UCLA School of Medicine, Los Angeles, for kindly providing the anti-hBD2 antibody.