Expression and Secretion of Cathelicidin Antimicrobial Peptides in Murine Mammary Glands and Human Milk

Abstract

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...

Host-defence-related proteins in cows’ milk

“…Lysozyme, lactoferrin and lactoperoxidase each possess the ability to suppress the growth of certain bacteria and were identified as minor milk proteins in the early part of the twentieth century . More recently, it has become apparent that milk also contains a range of additional antimicrobial proteins and peptides, including members of the b-defensin, complement, cathelicidin and S100 calgranulin families, as well as several acute phase proteins (Eckersall et al, 2001;Jia et al, 2001;Rainard, 2003;Swanson et al, 2004;Murakami et al, 2005;Lutzow et al, 2008). The role that two of these effector proteins play in contributing to the host-defence function of milk is described below, drawing on previously published work as well as our own unpublished data.…”

The Human Host Defense Peptide LL-37 Induces Apoptosis in a Calpain- and Apoptosis-Inducing Factor–Dependent Manner Involving Bax Activity

“…LL-37 is a human cationic, amphipathic host defense peptide released by proteinase 3 proteolytic processing of the COOH-terminal domain of the human cathelicidin, human cationic antimicrobial protein-18 (6). LL-37 is found in the secondary granules of neutrophils (7), is endogenously expressed by a variety of cells including keratinocytes and epithelial cells, and is released in response to multiple inflammatory stimuli (8,9). On release, LL-37 acts as an effector molecule of innate immunity, displaying anti-infective and immunomodulatory activities, including the induction of neutrophil, CD4 + T-cell and monocyte chemotaxis (10), mast cell activation (11), and neutrophil survival (12,13).…”

Vitamin D3–Triggered Antimicrobial Response—Another Pleiotropic Effect beyond Mineral and Bone Metabolism

“…Production and secretion of cathelicidin is not restricted to myeloid cells, however. It occurs also in other cells exposed to microbes, such as the epithelial cells of the mouth, tongue, esophagus, intestine, cervix and vagina (13), lung (14), and salivary, sweat (15), and mammary glands (16). Liu et al (4) pursued a remarkable species difference: In mice it had been shown that the acute antimicrobial response triggered by the heterodimer TLR2/1 depends on the generation of nitric oxide (NO) (17), yet in human macrophages the antimicrobial activity of macrophages triggered by TLR 2/1 is not dependent on the generation of NO and obviously must be mediated by alternative effectors.…”