Jan-Michel Otte

Cell differentiation dependent expressed CCR6 mediates ERK-1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells.

The expression of CCL20 (MIP‐3α), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT‐PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas‐induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF‐α, IL‐1β, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohns disease and correlated significantly with the IL‐8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK‐1/2, and SAPK/JNK MAP kinases and increased IL‐8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6‐fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas‐induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells. J. Cell. Biochem. 97: 709–723, 2006.

Effects of the cathelicidin LL-37 on intestinal epithelial barrier integrity

The human cathelicidin LL-37 is involved in innate immune responses, angiogenesis and wound healing. Functions in maintenance and re-establishment of intestinal barrier integrity have not been characterized yet. Following direct and indirect stimulation of human colonic HT-29 and Caco-2 cells with LL-37 the cellular viability, rate of apoptosis, proliferation and wound healing were determined. Expression of mucins and growth factors was quantified by real-time PCR and Western blotting. Direct application of LL-37 stimulated migration in Caco-2 cells expressing the proposed LL-37 receptor P2X7. Intestinal epithelial cell (IEC) proliferation was not altered. Indirectly, LL-37 significantly enhanced IEC migration via release of growth factors from subepithelial fibroblasts and IEC. Furthermore, LL-37 induced the expression of protective mucins in IEC and abated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in IEC. LL-37 induced signaling is mediated in part by the P2X7 receptor, the epidermal growth factor receptor and the p38 mitogen-activated protein kinase (MAPK). LL-37 contributes to maintenance and re-establishment of the intestinal barrier integrity via direct and indirect pathways. These features, in addition to its known antimicrobial properties, suggest an important role for this peptide in intestinal homeostasis.

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re‐establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human β‐defensin 2 (hBD‐2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco‐2 and HT‐29 cells were stimulated with 0.1–10 µg/ml hBD‐2 for 6–72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo‐deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot‐blot analysis. Furthermore, anti‐apoptotic capacities of hBD‐2 were studied. Over a broad range of concentrations and stimulation periods, hBD‐2 was well tolerated by IECs and did not induce apoptosis. hBD‐2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD‐2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF‐related apoptosis‐inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD‐2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD‐2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease. J. Cell. Biochem. 104: 2286–2297, 2008.

Antimicrobial peptides in innate immunity of the human intestine.

The intestinal mucosa has to withstand exposure to a variety of substances, challenges in pH, temperature, and osmolarity; and, finally, bacterial products which might induce local and systemic inflammatory responses. The mucosal integrity is conserved by a defense system which consisting of constitutive and inducible mechanisms. These include the physical barrier function; the secretion of factors into the lumen, such as mucins and antibacterial substances; the mucosal immune system; and, finally, the ability of the mucosa to reconstitute once damage has occurred. The homeostasis and integrity of the gastrointestinal mucosa ultimately depends upon the balance between defensive and aggressive factors. While the physical barrier function was formerly believed to play the major role in mucosal protection against luminal bacteria, the recent discovery of Toll-like receptors and antimicrobial peptides in the intestinal epithelium has modified the concept of intestinal defense towards a more active character, which will be discussed in this review.

Probiotics regulate the expression of COX-2 in intestinal epithelial cells.

Cyclooxygenase-2 (COX) 2 promotes intestinal wound healing but elicits also proinflammatory effects and has been implicated in colorectal carcinogenesis. Thus, a balanced expression of COX-2 is essential for intestinal homeostasis. This study was designed to evaluate the regulation of COX-2 by probiotic organisms and to characterize ligands and receptors involved. Colo320 and SW480 intestinal epithelial cells (IEC) were stimulated with gastrin or TNF-α and pre- or coincubated with commensales, bacterial supernatants, or distinct toll-like receptor (TLR) ligands. COX-2 promoter activity was determined by luciferase assays, protein expression by Western blotting, and secretion of prostaglandin E 2 (PGE 2 ) by ELISA. Commensales differentially regulated COX-2 expression in IEC. E. coli Nissle 1917, the probiotic mixture VSL#3, and media conditioned by these organisms ameliorated induced COX-2 expression and PGE 2 secretion. Heat inactivation and DNase treatment significantly decreased these regulatory capacities. Lactobacillus acidophilus, however, significantly increased COX-2 expression and PGE 2 secretion. TLR agonists differentially ameliorated basal or induced COX-2 expression. Distinct probiotics specifically and significantly decrease induced COX-2 expression in IEC, most likely mediated by released factors and in part by bacterial DNA. A significant involvement of TLRs in these regulatory processes remains to be established.

Efficacy, safety and tolerability of vidofludimus in patients with inflammatory bowel disease: the Entrance study.

BACKGROUND Vidofludimus (SC12267) is a novel oral immunomodulator inhibiting dihydroorotate dehydrogenase (DHODH) and the expression of proinflammatory cytokines including interleukin-17 (IL17A and IL17F) and interferon-gamma. The objective of the study was to explore the efficacy, safety and tolerability of vidofludimus in steroid-dependent inflammatory bowel disease (IBD). METHODS The open label uncontrolled ENTRANCE study (ClinicalTrials.gov NCT00820365) has been conducted at 13 study centers in Germany, Bulgaria and Romania. Thirty-four steroid-dependent patients with a confirmed diagnosis of Crohns disease (CD) or ulcerative colitis (UC) were treated with a once daily 35mg oral dose of vidofludimus over 12weeks. Steroids were tapered during the first 8weeks followed by a steroid-free treatment period of 4weeks. Complete response was defined as steroid-free clinical remission at week 12; partial response was defined as being in remission at steroid dose equal or lower than the individual patients threshold dose for relapse. RESULTS Of the thirty-four patients enrolled in this trial 26 were evaluable for primary efficacy assessment. After completion of the 12weeks treatment phase 8 out of 14 (57.1%) patients with CD and 6 out of 12 (50.0%) patients with UC were in steroid-free remission (complete responders). Another 4 (28.6%) patients in CD and 5 (41.7%) patients in UC were partial responders. Vidofludimus was well tolerated, no drug-related serious adverse events were observed. CONCLUSIONS This trial provides first evidence of clinical efficacy of vidofludimus in IBD. Although the safety and tolerability profile seems favorable, long-term controlled studies are needed to further investigate its potential as novel IBD therapy.

Macrophage migration inhibitory factor (MIF) −173G/C promoter polymorphism influences upper gastrointestinal tract involvement and disease activity in patients with Crohn's disease

Background: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with increased expression in inflammatory bowel disease. The aim of the study was to analyze the role of the MIF −173G/C single nucleotide polymorphism in Crohns disease (CD). Methods: Using restriction fragment length polymorphism analysis, genomic DNA of 198 patients with CD and 159 unrelated controls was analyzed for the −173G/C SNP in the MIF promoter region. Colonic MIF mRNA expression was measured by quantitative polymerase chain reaction (PCR), serum MIF levels by enzyme‐linked immunosorbent assay (ELISA). Results: Thirty‐six of the 146 G/G wildtype carriers (24.7%) but only 3 of the 45 G/C heterozygotes (6.7%) and only 1 of the C/C homozygotes (14.3%) were diagnosed with upper gastrointestinal tract involvement (P = 0.009, odds ratio [OR] = 0.22, 95% confidence interval [CI], 0.06–0.75 for wildtype versus hetero‐ and homozygous carriers). This result was confirmed in a second prospective study, in which all patients diagnosed with upper gastrointestinal involvement (n = 13) were G/G wildtype carriers (P = 0.01 versus controls). All patients (n = 12; 100%) with a Crohns disease activity index (CDAI) > 300 were G/G wildtype carriers compared to only 65.6% wildtype carriers in the group with a CDAI < 150 (P = 0.016). MIF is expressed in the colonic mucosa of CD patients and intestinal epithelial cells but its mRNA expression does not correlate with the degree of inflammation and is not upregulated by proinflammatory cytokines. In CD, MIF serum levels are not influenced by the MIF −173G/C polymorphism. Conclusions: The MIF −173G/C polymorphism appears to be a factor contributing to a particular CD phenotype characterized by protection against upper gastrointestinal tract involvement and severe disease activity.

C-met protooncogene expression and its regulation by cytokines in the regenerating pancreas and in pancreatic cancer cells.

Background: Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on cmet expression. Methods: Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1 a (IL-1a), IL-6, tumor necrosis factor-a (TNF-a) or transforming growth factorb1 (TGF-b1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry. Results:C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a twoto threefold increase of c-met expression in vitro. Conclusion: Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.BACKGROUND Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on c-met expression. METHODS Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry. RESULTS C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a two- to threefold increase of c-met expression in vitro. CONCLUSION Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.

The +1059G/C polymorphism in the C-reactive protein (CRP) gene is associated with involvement of the terminal ileum and decreased serum CRP levels in patients with Crohn’s disease

Background  Serum C‐reactive protein (CRP) levels influence the response to anti‐tumour necrosis factor (TNF) therapies.

Mechanisms of Lectin (Phytohemagglutinin)-Induced Growth in Small Intestinal Epithelial Cells

Background/Aims: The lectin phytohemagglutinin is a mitogen for intestinal epithelial cells in vivo. The mechanisms of action are unknown and were therefore analyzed in vitro. Methods: Human (Intestine-407) and rat (IEC-6; IEC-18) intestinal epithelial cell lines were stimulated with phytohemagglutinin. Proliferation was assayed by 3H-thymidine incorporation, activation of mitogen-activated protein kinase (MAPK) by Western blotting, and induction of c-fos mRNA expression by semiquantitative polymerase chain reaction. Control experiments were performed with phenyl-N-acetyl-α-D-galactosaminide or the tyrosine kinase inhibitor tyrphostin A25. Results: Phytohemagglutinin (0.1 µg/ml) significantly stimulated proliferation in all three cell lines after 48–72 h. MAPK activation was detected after 15–30 min, and an induction of c-fos mRNA expression after 15– 30 min of stimulation. Mitogenic effects were blocked by preincubation with phenyl-N-acetyl-α-D-galactosaminide or tyrphostin A25. Conclusion: Phytohemagglutinin stimulated proliferation, MAPK activation and induction of c-fos mRNA expression. The lectin may contribute to intestinal mucosal growth and regeneration thereby preventing gut atrophy.