Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis

Abstract

The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and alpha-defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

Fig 1

Expression levels of defensin and lysozyme mRNAs as determined by qRT-PCR in c- and l/v-IECs freshly isolated from jejunum, ileum and colon of patients with no history of intestinal inflammation. Statistically significant differences are indicated. Horizontal bars indicate median values.

Fig 2

Expression levels of defensin and lysozyme mRNAs as determined by qRT-PCR in c- and l/v-IECs freshly isolated from UC-, CD- and control colon. Statistically significant differences are indicated. Horizontal bars indicate median values.

Fig 3

Expression levels of defensin and lysozyme mRNAs as determined by qRT-PCR in c-IECs and l/v-IECs freshly isolated from CD small intestine (CD-SI) and control ileum and jejunum. Horizontal bars indicate median values.

Fig 4

Stimulation of confluent cultures of HT-29 and LS174T for 1 h with 10 µg/ml of LPS, 100 ng/ml of rhIL-1β, 1 × 10⁸ E. coli D21, 1 × 10⁸ B. megaterium Bm11, 1 × 10⁸ M. luteus, or 1 × 10⁸ S. typhimurium LT2, followed by removal of bacteria and continued culturing for 5 h in fresh medium containing 50 µg/ml gentamycin to kill remaining extracellular bacteria. Total RNA was extracted and the expression levels of defensin mRNAs were determined by qRT-PCR with copy standards.

Fig 5

Immunohistochemical staining for detection of HD-5 and lysozyme expressing cells in UC and control colon. (a) Control colon stained with anti-HD-5 antiserum. No stained cells could be detected. (b) UC colon stained with anti-HD-5 antiserum. Positive cells are seen at different locations in the crypt, but most commonly towards the crypt base. (c) Control colon stained with antilysozyme MoAb. Stained goblet cells as well as lamina propria macrophages can be seen. (d) UC colon stained with antilysozyme MoAb. Positive cells are seen at different locations in the crypt, but most commonly towards the crypt base. Original magnification: 55 × (a, d), and 220 × (b,c).