Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.

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Cathelicidin antimicrobial peptides are effectors of innate immune defense in mammals. Humans and mice have only one cathelicidin gene, whereas domesticated mammals such as the pig, cow, and horse have multiple cathelicidin genes. We hypothesized that the evolution of multiple cathelicidin genes provides these animals with enhanced resistance to infection. To test this, we investigated the effects of the addition of cathelicidins by combining synthetic cathelicidin peptides in vitro, by producing human keratinocytes that overexpress cathelicidins in culture, or by producing transgenic mice that constitutively overexpress cathelicidins in vivo. The porcine cathelicidin peptide PR-39 acted additively with human cathelicidin LL-37 to kill group A Streptococcus (GAS). Lentiviral delivery of PR-39 enhanced killing of GAS by human keratinocytes. Finally, transgenic mice expressing PR-39 under the influence of a K14 promoter showed increased resistance to GAS skin infection (50% smaller necrotic ulcers and 60% fewer surviving bacteria). Similarly constructed transgenic mice designed to overexpress their native cathelicidin did not show increased resistance. These findings demonstrate that targeted gene transfer of a xenobiotic cathelicidin confers resistance against infection and suggests the benefit of duplication and divergence in the evolution of antimicrobial peptides.innate immunity ͉ keratinocyte

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/ LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk. Antimicrobial peptides are found throughout nature and are important for the immune defense of plants, insects, and animals (1). Most of these molecules have a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and viruses. In mammals, peptides from the cathelicidin family assist the antimicrobial efficacy of neutrophils, macrophages, and mast cells (2-6). In addition, antimicrobial genes of the defensin and cathelicidin families are produced by epithelia in response to injury of lung, gut, urinary bladder, oral mucosa, and skin (7-9). At the interface with the external environment, these molecules serve as a rapid first-line defense for inhibition of microbial proliferation and invasion.Human milk contains various anti-infectious materials such as immunoglobulins, cellular components, and cytokines. It is believed that these molecules contribute to decreased morbidity and mortality of infants who are fed breast milk (10,11). Recently, peptide antibiotics have been isolated from human milk, providing additional evidence that multiple molecules can be involved in the immune defense of this material (12,13). In other systems, the clinical consequences of antimicrobial peptide expression have been observed in patients who have atopic dermatitis and are susceptible to infection and also lack the ability to increase the cathelicidin LL-37 and human ␤-defensin (hBD-2) in response to inflammatory stimuli (14). Furthermore, patients with Kostmann syndrome, a rare inherited disorder characterized by frequent infections and neutrophil dysfunction, also have a deficien...