Paneth cell antimicrobial peptides: Topographical distribution and quantification in human gastrointestinal tissues

Antimicrobial peptides and proteins are key effectors of innate immunity, expressed both by circulating phagocytic cells and by epithelial cells of mucosal tissues. In the human small intestine, Paneth cells are secretory epithelial cells that express the antimicrobials human α-defensin-5 (HD5), HD6, lysozyme and secretory phospholipase A₂ (sPLA₂), and recent studies have implicated reduced HD5 and HD6 expression levels in the pathogenesis of ileal Crohn's disease. However, expression levels of these molecules have not been determined routinely by techniques that readily permit quantitative comparisons of their distribution between tissues and samples. Using quantitative real-time PCR with external standards and Northern blot analysis, we compared expression levels of mRNA encoding these four Paneth cell antimicrobial peptides, as well as circulating human neutrophil defensins in several different gastrointestinal tissues and the bone marrow. HD5 and HD6 were the most abundant antimicrobials expressed in the small intestine. The concentration of HD5 mRNA is approximately 5 × 10⁵ copies per 10 ng RNA in the jejunum and ileum; HD6 mRNA levels were about six times lower than those of HD5. With the exception of low levels in the pancreas (10³ copies/10 ng RNA), the expression of HD5 and HD6 in tissues other than small intestine was at or below detectable limits. The expression of sPLA2 and lysozyme mRNA was observed in the small intestine (approximately, 3 × 10³ and 9 × 10³ copies/10 ng RNA, respectively), but also in several other tissues. Lysozyme expression was high in the duodenum (10⁵ copies/10 ng RNA), and the protein localized to both Brunner's glands in the lamina propria and Paneth cells. By comparison, the hematopoietic α-defensins HNP1-3 mRNA were detected at 6 × 10⁵ copies per 10 ng RNA in the bone marrow. These quantitative RT-PCR data from healthy tissues represents the first quantitative topographical assessment of antimicrobial expression in the gastrointestinal tract and provides a means to directly compare expression levels between healthy tissues and disease specimens for multiple antimicrobial peptides.