The Human Cationic Antimicrobial Protein (hCAP-18) Is Expressed in the Epithelium of Human Epididymis, Is Present in Seminal Plasma at High Concentrations, and Is Attached to Spermatozoa

Abstract

Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 ؎ 37.8 g/ml (mean ؎ standard deviation; range, 41.8 to 142.8 g/ml; n ‫؍‬ 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/10 6 spermatozoa, corresponding to 6.6 ؋ 10 6 molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.The integrity of the human reproductive system against potentially invasive pathogenic microorganisms is crucial. Readily available, preformed antimicrobial proteins of the nonadaptive immune system serve as the body's first line of defense (5), while the adaptive immune system becomes involved if pathogens start to invade. In recent years, several components of the human nonadaptive immune system have been isolated and characterized, among them the only member of the cathelicidins known to exist in humans, the human cationic antimicrobial protein (hCAP-18) (4, 10). This protein is synthesized in neutrophil progenitors in the bone marrow and stored in the specific granules of mature neutrophils (15). It is synthesized as an 18-kDa proprotein from which a 5-kDa C-terminal fragment, LL-37, bearing all of the hitherto known biological activity, is cleaved (8). LL-37 has lipopolysaccharide-binding properties and manifests antibacterial effect against a wide range of bacterial species (11,20). Recently, expression of hCAP-18 has also been demonstrated in the epithelium of several organs, including the vagina, cervix, mouth, and lung (2, 6). The cDNA encoding hCAP-18 has been detected in a library prepared from the human testis, suggesting that the gene is expressed here (1).In the present study, we investigated the presence of hCAP-18 in the male reproductive system. We found expression in the epithelium of the epididymis by in situ hybridization and by immunohistochemistry. High levels of hCAP-18 were found in seminal plasma and in association with spermatozoa, but we were unable to detect expression of the hCAP-18 gene or the presence of hC...

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken

“…Neutrophils are a particularly rich source of cathelicidins in a variety of species. In humans, cathelicidins have also been found to be expressed in several other tissues, including the testis (Agerberth et al 1995;Malm et al 2000), squamous epithelia (Frohm Nilsson et al 1999;Nizet et al 2001), airway epithelia (Bals et al 1998), sweat glands (Murakami et al 2002b), salivary glands (Murakami et al 2002a) and colon (Hase et al 2002). Cathelicidins have a wide spectrum of antimicrobial activity and have been shown to be active against Gram-negative and Grampositive bacteria (Travis et al 2000), fungi (Shin et al 2000) and enveloped viruses (Tamamura et al 1995).…”

Effects of human cathelicidin antimicrobial peptide LL‐37 on lipopolysaccharide‐induced nitric oxide release from rat aorta in vitro

“…One of them could be human cathelicidin antimicrobial peptide (hCAP-18) (6Ð8), which is produced by human neutrophils but can also be found in a variety of human tissues (9,10). hCAP-18 belongs to the protein family of cathelicidins, which have bactericidal activity by means of their highaffinity binding to the outer bacterial cell wall (8).…”

Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions

“…Total proteins from acetone-precipitated supernatants obtained as described above were probed for CRAMP by Western blot using an antibody directed against the C terminus (C-term). The CRAMP doublet of 15 and 20 kDa has been described in human neutrophils and seminal plasma (47,54). NS denotes a nonspecific band.…”