Portal de Periódicos da CAPES

The role of antimicrobial peptides and proteins in keratinocyte defense

2005; Wiley; Volume: 14; Issue: 10 Linguagem: Inglês

10.1111/j.1600-0625.2005.0355e.x

1600-0625

Jens‐Michael Schröder,

Dermatology and Skin Diseases

Healthy human skin is always covered with microorganisms, but usually not infected. This, in principle, unexpected phenomenon together with the observation that microbe densities are rather constant at skin surfaces led to the hypothesis that skin apart from the physical barrier contains also a ‘chemical barrier’, which may control the growth of microorganisms at body surfaces. Recent investigations have shown that human skin contains innate defense effector molecules – such as animals and plants – which belong to different molecular families such as defensins, cathelicidins, S100 proteins, RNases, or protease inhibitors. Some of these antimicrobial peptides and proteins are constitutively produced by keratinocytes; others are only inducibly expressed in these cells and therefore characterized by its rather focal and site-dependent production and release. Antibiotic activity of these antimicrobial peptides varies from more selective to rather broad-spectrum antibiotics. Therefore, it might be that the local pattern and concentrations of particular proteinaceous antibiotics determine the spectrum of local skin microflora as well as the number of microorganisms. Although inducible antibiotic peptides are always found on inflammation, there is now upcoming evidence that antimicrobial peptides might also be induced in the absence of inflammatory mediators, either by bacterial products or by endogenous cytokines. It remains to be elucidated which compounds are responsible for the induction of ‘epithelial effector molecules’ and whether it is possible to enhance the ‘chemical barrier’ of human skin, thus preventing skin infection by stimulating this innate epithelial defense shield. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 1987: 84: 5449–5453. This is the first demonstration of antimicrobial peptides in vertebrate skin showing that frog skin is secreting antimicrobial peptides termed ‘Magainins’ and thus explaining why freshly surgically treated frogs usually do not suffer from skin infections. Frohm M, Agerberth B, Ahangari G, et al. 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: 15258–15263. This is the first demonstration that the cathelicidin C-terminal antimicrobial peptide LL-37, originally discovered in granulocytes, was found to be produced by keratinocytes. Further, it demonstrated the upregulation of this human cathelicidin gene in inflammatory skin disorders, whereas in normal skin no induction was found. Harder J, Bartels J, Christophers E, Schröder J M. A peptide antibiotic from human skin. Nature 1997: 387: 861. This study describes the discovery of human beta-defensin-2 (HBD-2) in psoriatic scale extracts using a unique strategy of bacterial affinity chromatography and gram-negative antimicrobial activity testing as read-out. HBD-2 represents the first human epithelial peptide antibiotic that is induced by proinflammatory cytokines and by contact with bacteria. Further, it is also shown that HBD-2 is active against gram-negative bacteria, but not Staphylococcus aureus, suggesting it to be a preferentially gram-negative bacteria controlling epithelial effector molecule. Harder J, Bartels J, Christophers E, Schröder J M. Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 2001: 276: 5707–5713. Here, the isolation and purification of human beta-defensin-3 (HBD-3) from psoriatic scale extracts by using S. aureus antimicrobial activity testing as read-out as well as molecular cloning of HBD-3 is described. HBD-3 represents the second human epithelial peptide antibiotic that needs to be induced in keratinocytes, i.e. by proinflammatory cytokines and by contact with bacteria, in particular a mucoid clinical isolate of Pseudomonas aeruginosa. In contrast to HBD-2, HBD-3 was found to be a broad-spectrum peptide antibiotic with activity also against antibiotic resistant bacteria. This study also contains ultrastructural analyses showing that the morphology of HBD-3-treated S. aureus resembles that of penicillin-treated S. aureus. Harder J, Schröder J M. RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin. J Biol Chem 2002: 277: 46779–46784. This is the first report about biochemical analysis of healthy person's stratum corneum extracts for S. aureus-killing peptides and the discovery of a novel antibiotic protein termed RNase 7 in healthy skin. Like HBD-2 and HBD-3, it is also inducible by proinflammatory cytokines and contact with bacteria, in particular again with a mucoid clinical isolate of P. aeruginosa. RNase 7 was found to be a broad-spectrum antibacterial protein at low micromolar concentrations, which remarkably is active at nanomolar concentrations against a Vancomycin-resistant Enterococcus faecium. Gläser R, Harder J, Lange H, Bartels J, Christophers E, Schröder J M. Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 2005: 6: 57–64. This is a landmark report showing that healthy human skin secretes a disinfectant, which the authors identified as the S100-protein psoriasin. This protein preferentially kills Escherichia coli by depriving Zinc ions and protects human skin from E. coli skin infection, thus giving an explanation of why skin infection by E. coli is so rare. It describes that the application of neutralizing anti-psoriasin antibodies to healthy skin abolishes E. coli resistance and presents for the first time evidence that human proteins have antibiotic effects in humans in vivo. The observation that psoriasin can be induced in vivo and in vitro by bacterial products might explain the site-specific local surface psoriasin release, the focal expression of psoriasin in Malpighian layer of the epidermis. Its expression in sebocytes would explain the presence of psoriasin in skin lipids. Ali R S, Falconer A, Ikram M, Bissett C E, Cerio R, Quinn A G. Expression of the peptide antibiotics human beta defensin-1 and human beta defensin-2 in normal human skin. J Invest Dermatol 2001: 117: 106–111. In this study, it is demonstrated that human beta defensin-1 mRNA is consistently expressed in skin samples from various body sites, whereas HBD-2 expression is more variable. It further shows that immunoreactive HBD-1 and HBD-2 are localized to the Malpighian layer of the epidermis and/or stratum corneum and that there are interindividual and site-specific differences in the intensity of immunostaining and the pattern of peptide localization. Oren A, Ganz T, Liu L, Meerloo T. In human epidermis, beta-defensin 2 is packaged in lamellar bodies. Exp Mol Pathol 2003: 74: 180–182. This ultrastructural study shows that HBD-2 is packaged in lamellar bodies and might be released on differentiation. Yang D, Chertov O, Bykovskaia S N, et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999: 286: 525–528. Here, it is shown that human beta-defensins are also chemotactic for immature dendritic cells and memory T cells. HBD was reported to be selectively chemotactic for cells expressing human CCR6, a chemokine receptor preferentially expressed by immature dendritic cells and memory T cells. These observations indicate that beta-defensins may promote adaptive immune responses by recruiting dendritic and T cells to the site of microbial invasion through interaction with CCR6. Liu A Y, Destoumieux D, Wong A V, et al. Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation. J Invest Dermatol 2002: 118: 275–281. In this study, it was shown that the expression of HBD-2 peptide by human keratinocytes required differentiation of the cells as well as a cytokine or bacterial stimulus. Interleukin-1alpha (IL-1α), IL-1β, or live P. aeruginosa proved to be the most effective stimuli, whereas other bacteria and cytokines had little or no ability to induce human HBD-2 synthesis. Sorensen O E, Cowland J B, Theilgaard-Monch K, Liu L, Ganz T, Borregaard N. Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J Immunol 2003: 170: 5583–5589. The authors of this study found that the growth factors important in wound healing, insulin-like growth factor I and TGF-α, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, HBD-3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. These findings offer an explanation for the expression of these peptides/polypeptides in psoriasis and in wound healing and define a host defense role for growth factors in wound healing. Sorensen O E, Thapa D R, Rosenthal A, Liu L, Roberts A A, Ganz T. Differential regulation of β-defensin expression in human skin by microbial stimuli. J Immunol 2005: 174: 4870–4879. This study analyzed the molecular links between infection and the expression of HBD-1, HBD-2, and HBD-3 in the human epidermis. Exposure of both monocytes and lymphocytes to microbe-derived molecules stimulated the epidermal β-defensin expression. Whereas the induced expression of HBD-3 was mediated by transactivation of the epidermal growth factor receptor, the mechanisms of the induction of HBD-1 and HBD-3 were reported to be distinct from each other and from the IL-1-dependent induction of HBD-2 expression, suggesting that epidermal expression of beta-defensins is mediated by at least three different mechanisms. Harder J, Meyer-Hoffert U, Teran L M, et al. Mucoid Pseudomonas aeruginosa, TNF-alpha, and IL-1beta, but not IL-6, induce human beta-defensin-2 in respiratory epithelia. Am J Respir Cell Mol Biol 2000: 22: 714–721. This report indicates that a mucoid (biofilm forming) phenotype of Pseudomonas aeruginosa (PA), but not non-mucoid PA strains, TNF-alpha, and IL-1beta, but not IL-6, induce HBD-2 at low numbers in various epithelial cell-types. The finding that only excessively high concentrations (>10 μg/ml) of PA lipopolysaccharide preparation can induce HBD-2 indicate a minor – if any direct – role of TLR-4 in β-defensin induction. This report also revealed that the HBD-2 gene promoter region contains several putative transcription factor-binding sites, including nuclear factor kappa B(NF-κB), activator protein (AP)-1, AP-2, and NF-IL-6. Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004: 21: 241–254. Here, it is shown for the first time that the Th1 cytokine IL-22, which belongs to the IL-10-family, but does not serve the communication between immune cells, directly promotes the induction of β-defensins in keratinocytes and thus promotes innate epithelial defense in barrier tissues. Harder J, Meyer-Hoffert U, Wehkamp K, Schwichtenberg L, Schröder J M. Differential gene induction of human beta-defensins (hBD-1, -2, -3, and -4) in keratinocytes is inhibited by retinoic acid. J Invest Dermatol 2004: 123: 522–529. This investigation compares the gene expression of the four beta-defensins hBD-1 to hBD-4 in primary cultured keratinocytes during Ca++-induced differentiation and demonstrates a complete abolished inducible beta-defensin expression when the keratinocytes were pretreated with all-trans-retinoic acid (RA). Similar findings were observed with proinflammatory cytokines as defensin inducer. These findings indicate that RA is a potent inhibitor of beta-defensin induction in keratinocytes and might downregulate the inducible innate chemical defense system of human skin. Nizet V, Ohtake T, Lauth X, et al. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 2001: 414: 454–457. Several antimicrobial peptides are easily inactivated and have diverse cellular effects in vitro. Due to a lack of in vivo studies for long time, it remained speculative whether antimicrobial peptides act in vivo as antimicrobial agents. In this study, the function of CRAMP, a murine equivalent of human LL-37, was investigated in a mouse model of cutaneous infection. It is shown that cathelicidins are an important native component of innate host defense in mice which provide protection against necrotic skin infection caused by Group A Streptococcus (GAS). This is the first demonstration that antimicrobial peptides have protective effects in vivo of antimicrobial peptides which is brought into question by observations that several of these peptides are easily inactivated and have diverse cellular effects that are distinct from antimicrobial activity demonstrated in vitro. Ong P Y, Ohtake T, Brandt C, et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002: 347: 1151–1160. In this study, the levels of expression of LL-37 and HBD-2 in inflamed skin from patients with atopic dermatitis (AD) and from those with psoriasis were compared. Whereas abundant LL-37 and HBD-2 immunostainings were detected in the superficial epidermis of all patients with psoriasis, significantly decreased amounts of these peptides were detected in acute and chronic lesions from patients with AD. These findings support the hypothesis that deficiency in the expression of antimicrobial peptides may account for the susceptibility of patients with AD to skin infection with S. aureus. Nomura I, Goleva E, Howell M D, et al. Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol 2003: 171: 3262–3269. Patients with AD, but not psoriasis, suffer from frequent skin infections. This study tried to analyze the molecular basis for this phenomenon using GeneChip microarrays. Here, a decreased expression of HBD-3 was demonstrated. The finding that IL-13 and IL-4 inhibited induced HBD-3 production supports the idea that a decreased expression of antimicrobial genes occurs as the result of the local upregulation of Th2 cytokines. These observations could explain the increased susceptibility of AD skin to microorganisms, and might explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases. Marchini G, Lindow S, Brismar H, et al. The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa. Br J Dermatol 2002: 147: 1127–1134. This study describes that vernix caneosa contains some antimicrobial peptides. Furthermore, also in healthy newborn infants, antimicrobial peptides were found, indicating effective innate immune protection already during fetal and neonatal life. Harder J, Schröder J M. Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins. J Leukoc Biol 2005: 77: 476–486. This report summarizes our current knowledge about antimicrobial peptides and proteins that have been isolated from psoriatic scale extracts using a biochemical approach and antimicrobial activity testing as read-out. It gives an overview about quantitative relationship of the yet identified antimicrobial peptides and proteins of keratinocyte origin.