Cryptdin-6

In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays detected Paneth cell defensin mRNAs in intestinal RNA from 1-day-old (P1) mice before crypt formation and maturation of the epithelium. Analysis of these defensin-coding RT-PCR products from P1 mice showed that 69% of clones sequenced coded for cryptdin-6, suggesting that it is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. Cryptdin immunoreactivity in the P1 mouse intestine was specific for intracellular granule contents, and immunofluorescent detection of cryptdins on mucosal surfaces suggested that the peptides are released into the intestinal lumen in P1 mice Defensin secretion may contribute to innate immunity of the neonatal intestine before the presence of distinguishable Paneth cells.

The antimicrobial peptide is one of major effectors of the innate immunity, and is common in the entire multicellular organisms. In mammals, one family of antibacterial peptide named defensins plays a central role in host defense, especially in the epithelial surface such as oral cavity, skin and the intestine. Recently, the importance of the antimicrobial peptides has been widely recognized. The epithelium of the gut is a largest surface that is exposed to various pathogens in the environment. It is the Paneth cells that produce antimicrobial peptides, α-defensins in the small intestine. Paneth cells contribute to mucosal innate immunity by sensing bacteria and releasing microbicidal activities mostly from activated α-defensins. In mice, α-defensins, named cryptdins, consisted of six major isoforms (cryptdin-1 to cryptdin-6), and among those cryptdin-4 is the most microbicidal, suggesting that cryptdin-4 has a pivotal role in innate immunity. Paneth cell α-defensins have selective activities against commensal bacteria which may be associated with compositions of intestinal microbiota in vivo and homeostasis of the entire intestine. In addition, Paneth cell α-defensins appeared to be regulated topographically to control intestinal integrity.

Defensins are widely distributed and broad-spectrum antimicrobial peptides with activities against bacteria, fungi, and enveloped viruses. Defensins have been isolated from granules of neutrophils from humans, rabbits, rats, and guinea pigs. They have also been found in lung macrophages as well as in Paneth cells of the human, rabbit, and mouse small intestine. The human beta-defensin-2 was recently isolated from human skin. In this study, we detected the expression of mRNA for the defensin cryptdin in BALB/c mouse skin by means of reverse transcriptase PCR amplification. Expression was also detected in dispase-separated epidermis and cultured keratinocytes, but expression was not detected in fibroblasts. The expression of cryptdin mRNA was found to begin on embryonic day 17.5. As determined with specific primers, the cDNA sequence cloned from the skin was found to be identical to that previously reported for cryptdin-5. cDNA derived from cultured keratinocytes demonstrated the sequences of the cryptdin-6 and cryptdin-1 isoforms. In situ hybridization analysis showed that the mRNA of cryptdin was expressed in the suprabasal keratinocytes of the skin in embryonic and neonatal days and then shifted to the hair bulbs in the skin of adult mice.