Attenuation of colitis in the cotton-top tamarin by anti-alpha 4 integrin monoclonal antibody.

Recent studies have demonstrated the induced expression of endothelial adhesion molecules including E-selectin (also called endothelial leukocyte adhesion molecule-1), vascular cell adhesion molecule and intercellular adhesion molecule in actively involved mucosa of patients with ulcerative colitis and Crohn's disease. Similar induction has been demonstrated in the colon of the Cotton-top tamarin (CTT), a New World primate that experiences a spontaneous acute and chronic colitis resembling ulcerative colitis. To assess the potential importance of leukocyte adhesion as a necessary step in acute colitis, the effect of parenteral mAb directed against adhesion molecules on CTT colitis was evaluated in placebo-controlled blinded trials. Serial administration of either of two anti-E-selectin mAb designated BB11 and EH8 effectively coated endothelial surfaces expressing this vascular adhesion molecule. Although colitis activity was slightly diminished after the 10-d treatment period in CTT receiving either BB11 or EH8, this reduction was not significantly different than that seen in animals given a placebo control when assessed by a previously validated standardized scale of inflammatory activity: mean histologic activity grade 2.2 +/- 0.2 pretreatment vs 1.5 +/- 0.5 posttreatment in group receiving mAb and 2.1 +/- 0.1 pretreatment vs 1.3 +/- 0.5 posttreatment in the placebo group (P > 0.2). In contrast, administration of an anti-alpha 4 integrin mAb designated HP1/2 that binds VLA4 (alpha 4 beta 1) and presumably alpha 4 beta 7 integrins resulted in significant attenuation of acute colitis when compared to both pretreatment activity index (P = 0.005) and the placebo control group (P < 0.01): mean histologic activity grade 1.6 +/- 0.3 pretreatment vs 0.2 +/- 0.1 posttreatment in the group receiving HP1/2 and 1.8 +/- 0.5 pretreatment and 1.2 +/- 0.2 posttreatment in the placebo control group. These studies using a model of spontaneous colitis in the CTT demonstrate the feasibility of modulation of leukocyte-vascular adhesion and/or other integrin-mediated events possibly including T cell aggregation and T cell-stromal interactions, as well as lymphocyte homing. These results suggest both that these processes are important and possibly essential elements in sustaining acute colitis and that their disruption may result in therapeutic benefit.

Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium.

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.

Resistance to TGF beta in SV40 large T-immortalized rat intestinal epithelial cells is associated with down-regulation of TGF beta type I receptor.

A new continuous cell line designated ESKI-1 was established by transfection of rat fetal intestinal epithelial cells with ecotropic retroviruses containing SV40 large T oncogene. The ESKI-1 cell line exhibits morphologic features of an epithelial cell line and expresses the OCI-5 and cytokeratin 8 transcripts associated with epithelial cells in the small intestine. Signal transduction and proliferation responses to TGF beta has been characterized in ESKI-1 cells, in comparison with the spontaneously-immortalized IEC cell lines originating from neonatal rat duodenum and ileum. ESKI-1 express both TGF alpha and TGF beta. However, despite a marked increase in TGF beta-stimulated p78 kinase activity observed in ESKI-1 and IEC cells, TGF beta did not modulate growth, or extracellular matrix expression in ESKI-1 cells. Resistance to growth modulation was associated with downregulation of TGF beta. Type I receptor expression in the SV40 large T-immortalized cells. Thus, proliferative resistance to TGF beta inhibition can result from depletion of the TGF beta type I receptor and disruption of the TGF beta signaling pathway downstream the p78 serine/threonine kinase. These molecular defects constitute two early events during the SV40LT-mediated immortalization and neoplastic progression of the intestinal epithelia.

Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats.

BACKGROUND & AIMS: The trefoil factors, a family of proteins abundantly expressed in gastrointestinal mucous cells, protect the epithelium in vitro. This study determines the effects of exogenously administered trefoil peptides on experimental injury in rats in vivo. METHODS: Gastric injury was induced by either intragastric absolute ethanol (1.0 mL) or subcutaneous indomethacin (20 mg/kg). Recombinant human spasmolytic polypeptide (rHSP) or rat intestinal trefoil factor (ITF) were administered at different doses and time points before or after injury. Vehicle or bovine serum albumin was used as control. The pH of the stomach contents was assessed when the rats were killed. Gastric injury was blindly evaluated macroscopically and histologically. Serum levels of rHSP and ITF were determined by an enzyme-linked immunosorbent assay. RESULTS: Oral rHSP and ITF markedly protected against both ethanol- and indomethacin-induced gastric injury (P < 0.005 at doses of 1-15 mg/rat) when given up to 2 hours before injury; no protection was noted by intraperitoneal rHSP against ethanol injury. Intraperitoneal rHSP protected against indomethacin-induced injury only at the maximal dose given (15 mg). Neither rHSP nor ITF altered gastric pH. Protection was not associated with systemic absorption of trefoil peptides. CONCLUSIONS: Topical trefoil peptides protect the gastric mucosa against ethanol- and indomethacin-induced gastric injuries. These peptides contribute to surface mucosal defense.

Molecular cloning of the rat intestinal trefoil factor gene. Characterization of an intestinal goblet cell-associated promoter.

Intestinal trefoil factor (ITF) is a small peptide bearing the unique motif of intrachain disulfide bonds characteristic of the trefoil family. Previous work had localized expression of ITF primarily within goblet cells in the small and large bowel, making it a candidate gene for the study of the molecular basis of intestinal and goblet cell-specific gene expression. In order to study the regulation of ITF expression, we have cloned the rat ITF gene and sequenced 1.7 kilobases of the 5'-flanking region. RNase protection analysis demonstrated a single transcriptional start site. Various lengths of the 5'-flanking region were linked to the reporter gene luciferase and transfected into the colon cancer cell lines LS174T and Caco-2, representing, respectively, cells with and without goblet cell-like phenotype. Expression in the goblet cell-like LS174T colon cancer cell line was nearly 10-fold greater than expression in Caco-2 cells which exhibit columnar enterocyte-like phenotype. The pattern of goblet cell-associated selective transcription required only 153 base pairs of the rat ITF 5'-flanking sequence. Transfection of a construct of human growth hormone under the control of the rat ITF promoter in the N2 subclone of HT-29 cells demonstrated expression of the reporter gene only in those cells exhibiting a goblet cell phenotype as assessed by expression of immunoreactive mucin. These initial studies of the 5'-flanking region of the ITF gene demonstrate the presence of cis-regulatory elements capable of directing goblet cell specific expression.

Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.

Trefoil peptides are a recently recognized group of small peptides abundantly produced at mucosal surfaces that offer the opportunity to define mechanisms of mucosal cell-specific differentiation and to illuminate new mechanisms for the preservation of mucosal integrity. We report the cDNA cloning of a 75-amino acid human trefoil factor expressed in small and large intestinal mucosas that is highly homologous to the intestinal trefoil factor, with 70% identity at the amino acid level of the predicted mature protein. This human intestinal trefoil factor is also homologous, although to a lesser extent, to trefoil peptides expressed at other sites in the gastrointestinal tract in man, exhibiting absolute conservation of the P domain motif (CX9CX9CX4CCX9WCF) that defines this family of peptides. These findings indicate a high degree of evolutionary conservation of organ/region-specific members of this peptide family. In situ hybridization of intestinal trefoil factor demonstrates a high degree of expression in mature small intestine villus and colonic epithelial goblet cells. Immunogold staining demonstrates high concentrations of intestinal trefoil factor in the rough endoplasmic reticulum and theca of goblet cells as well as throughout the mucosal surface, consistent with vectorial secretion of this factor by goblet cells onto the intestinal luminal surface. In addition, intestinal trefoil factor was also localized within columnar epithelial cells by immunogold labeling despite the absence of mRNA. These observations suggest that peptide secreted by goblet cells might be taken up from the luminal surface and transcytosed by enterocytes. Human intestinal trefoil factor expression was also detected in the HT-29N2 and HT-29H2 subclones in conjunction with the emergence of the goblet cell phenotype, but not in the CaCO2 cell line that exhibits enterocytic phenotype. In summary, these findings confirm the existence of a highly conserved family of peptides that are abundantly expressed in distinctive regions throughout the gastrointestinal tract in a highly cell-specific pattern reflecting a goblet cell differentiation pathway. They form one of the more abundant constituents of the interface between the mucosa and "outside" environment and may provide a new paradigm of regulation of the integrity of epithelial surfaces as well as a previously unrecognized dimension of goblet cell function.