The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human ␤-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor ␣ and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.

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Human healthy skin is continuously exposed to bacteria, but is particularly resistant to the common gut bacterium Escherichia coli. We show here that keratinocytes secrete, as the main E. coli-killing compound, the S100 protein psoriasin in vitro and in vivo in a site-dependent way. In vivo treatment of human skin with antibodies to psoriasin inhibited its E. coli-killing properties. Psoriasin was induced in keratinocytes in vitro and in vivo by E. coli, indicating that its focal expression in skin may derive from local microbial induction. Zn(2+)-saturated psoriasin showed diminished antimicrobial activity, suggesting that Zn(2+) sequestration could be a possible antimicrobial mechanism. Thus, psoriasin may be key to the resistance of skin against E. coli.

Background: Mutations in NOD2, a putative intracellular receptor for bacterial peptidoglycans, are associated with a subset of Crohn's disease but the molecular mechanism linking this protein with the disease pathogenesis remains unclear. Human a defensins (HD-5 and HD-6) are antibiotic effector molecules predominantly expressed in Paneth cells of the ileum. Paneth cells also express NOD2. To address the hypothesis that the function of NOD2 may affect expression of Paneth cell defensins, we compared their expression levels with respect to NOD2 mutations in Crohn's disease. Methods: Forty five Crohn's disease patients (24 with NOD2 mutations, 21 with wild-type NOD2) and 12 controls were studied. Real time reverse transcription-polymerase chain reaction was performed with mucosal mRNA for HD-5, HD-6, lysozyme, secretory phospholipase A 2 (sPLA 2 ), tumour necrosis factor a, interleukin 8, and human hypoxanthine phosphoribosyltransferase (housekeeping gene). Immunohistochemistry with anti-HD-5 and histological Paneth cell staining were performed in 10 patients with NOD2 mutations or wild-type genotypes. Results: Ileal expression of HD-5 and HD-6, but not sPLA 2 or lysozyme, were diminished in affected ileum, and the decrease was significantly more pronounced in patients with NOD2 mutations. In the colon, HD-5, HD-6, and sPLA 2 were increased during inflammation in wild-type but not in NOD2 mutated patients. In both the colon and ileum, proinflammatory cytokines and lysozyme were unaffected by NOD2 status. Immunohistochemistry identified Paneth cells as the sole source of HD-5. Conclusion: As alpha defensins are important in the mucosal antibacterial barrier, their diminished expression may explain, in part, the bacterial induced mucosal inflammation and ileal involvement of Crohn's disease, especially in the case of NOD2 mutations.

We analyzed healthy human skin for the presence of endogenous antimicrobial proteins that might explain the unusually high resistance of human skin against infections. A novel 14.5-kDa antimicrobial ribonuclease, termed RNase 7, was isolated from skin-derived stratum corneum. RNase 7 exhibited potent ribonuclease activity and thus may contribute to the well known ribonuclease activity of human skin. RNase 7 revealed broad spectrum antimicrobial activity against many pathogenic microorganisms and remarkably potent activity (lethal dose of 90% < 30 nM) against a vancomycin-resistant Enterococcus faecium. Molecular cloning from skinderived primary keratinocytes and purification of RNase 7 from supernatants of cultured primary keratinocytes indicate that keratinocytes represent the major cellular source in skin and that RNase 7 is secreted. RNase 7 mRNA expression was detected in various epithelial tissues including skin, respiratory tract, genitourinary tract, and at a low level, in the gut. In addition to a constitutive expression, RNase 7 mRNA was induced in cultured primary keratinocytes by interleukin-1␤, interferon-␥, and bacterial challenge. This is the first report demonstrating RNases as a novel class of epithelial inducible antimicrobial proteins, which may play an important role in the innate immune defense system of human epithelia.