Monika Artinger

Induction of apoptosis before shedding of human intestinal epithelial cells

OBJECTIVES:Human intestinal epithelial cells (IECs) derive from stem cells at the crypt base and migrate along the so-called crypt-villus axis toward the intestinal lumen. As they reach the luminal surface in the colon or the villus tip in the small intestine, IECs are shed and their life cycle is terminated. The role of apoptosis during IEC migration along the crypt-villus axis has been subject to studies with conflicting results. In this study we use a novel approach to identify the initiation of apoptosis within normal human IECs.METHODS:Normal mucosa from the large and small human intestine was analyzed employing a novel antibody directed against activated caspase-3—an early marker of apoptosis.RESULTS:IECs initiate the apoptotic cascade as they approach the area of shedding before displaying evident morphological features of apoptosis. IECs of the small bowel also show caspase-3 activation in the small intestinal crypt base, whereas IECs of the colononic crypt base rarely show evidence of ongoing apoptosis.CONCLUSIONS:These findings indicate that apoptosis is initiated in human IECs as they reach the luminal surface/villus tip and before shedding. Furthermore, they show that different sections of the intestinal tract vary significantly in the rate of IEC apoptosis, possibly reflecting their difference in susceptibility to epithelial cell neoplasia.

Prostaglandin E2 inhibits migration of colonic lamina propria fibroblasts

Background: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. Methods: Primary cultures of CLPF were isolated from healthy controls and Crohns disease patients. Migration assays were performed in the Boyden chamber and scratch assays. EP receptors, PGE2, intracellular cyclic adenosine monophosphate (cAMP), expression and distribution of F‐actin, &agr;‐smooth muscle actin (SMA), and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry, and enzyme‐linked immunosorbent assay (ELISA). Results: All four EP receptor subtypes were present on CLPF. PGE2 and agonists to the EP2 and EP4 receptor reduced the migration of CLPF. Blockade of the EP2 and the EP4 receptor inhibited the effect of PGE2 on CLPF migration. An increase in intracellular cAMP reduced CLPF migration. PGE2 increased the concentrations of cAMP in CLPF, with abrogation after addition of EP2 and EP4 receptor antagonists. PGE2 and forskolin decreased the expression of &agr;‐SMA and F‐actin and reduced cell polarization and lamellipodium formation in a scratch assay. In addition, forskolin reduced the phosphorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Conclusions: PGE2 reduced the migration of CLPF via elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize, and a decreased amount of pMLC. This might be a possible reason for the impairment of intestinal wound healing in IBD. Inflamm Bowel Dis 2010

M1821 Prostaglandin E2 Inhibits Migration of Colonic Lamina Propria Fibroblasts

Background: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. Methods: Primary cultures of CLPF were isolated from healthy controls and Crohns disease patients. Migration assays were performed in the Boyden chamber and scratch assays. EP receptors, PGE2, intracellular cyclic adenosine monophosphate (cAMP), expression and distribution of F-actin, α-smooth muscle actin (SMA), and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA). Results: All four EP receptor subtypes were present on CLPF. PGE2 and agonists to the EP2 and EP4 receptor reduced the migration of CLPF. Blockade of the EP2 and the EP4 receptor inhibited the effect of PGE2 on CLPF migration. An increase in intracellular cAMP reduced CLPF migration. PGE2 increased the concentrations of cAMP in CLPF, with abrogation after addition of EP2 and EP4 receptor antagonists. PGE2 and forskolin decreased the expression of α-SMA and F-actin and reduced cell polarization and lamellipodium formation in a scratch assay. In addition, forskolin reduced the phosphorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Conclusions: PGE2 reduced the migration of CLPF via elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize, and a decreased amount of pMLC. This might be a possible reason for the impairment of intestinal wound healing in IBD. Inflamm Bowel Dis 2010