Hormonal Signalling in the Gastrointestinal Tract

a. Peptide Growth Factors and Cytokines in Intestinal Epithelial Cells

The intestinal epithelium is sustained by a highly dynamic balance of cell proliferation, differentiation and senescence. Coordination of these processes is essential to functional and anatomic integrity. The overall goal of these studies is the delineation of the role of peptide growth factors in modulating proliferation and differentiation of intestinal epithelial cells at a molecular level. A major thrust of these efforts has been the examination of the regulation of expression of transforming growth factors alpha and beta (TGF I and J) fibroblast growth factors and cytokines by intestinal epithelial cells and their role in effecting interactions between these cell populations and their extracellular matrix. These studies will serve as a paradigm to understand the role of the cell surface receptors and cell matrix components in growth modulation by these peptide growth factors.

In addition to a detailed examination of the role of previously recognized peptide growth factors, important goals are the molecular characterization of novel and biologically complementary proteins with potent growth regulating activity which are expressed by intestinal epithelial cells. In aggregate, these studies will provide insight into the spectrum of factors maintaining mucosal epithelial homeostasis and the mechanisms which play a role in the complex coordination of this network.

a-1. Regulation of Transforming Growth Factor Expression in Rat Intestinal Epithelial Cell Lines

Autocrine and paracrine modulation of transforming growth factor expression was assessed in rat intestinal epithelial cell lines designated IEC-6 and IEC-17. Addition of the transforming growth factor I (TGFI) homologue epidermal growth factor (EGF) to media of subconfluent IEC-6 cells led to autocrine stimulation of TGFI expression as well as increased expression of the transforming growth factor J1 (TGFJ1). Increased expression of TGFI was maximal between 3 and 6 h after addition of EGF and subsequently declined coincident with increasing level of expression of TGFJ1, which achieved maximal levels 6 hours after addition of EGF and was sustained for more than 12 hours. Addition of TGFJ1 also led to autocrine induction of its own expression coincident with suppression of TGFI expression. Addition of TGFJ1 was associated with increased expression of J-actin when standardized to a constitutive transcript (GAPDH). Similar responses to addition of EGF and TGFJ1, were observed in another intestinal epithelial cell line, designated IEC-17. Modulation of expression of TGFs was attenuated when cells were grown on the complex extracellular matrix produced by the Engelbreth-Holm-Swarm tumor (Matrigel), reflecting the baseline induction of TGFJ1 expression when compared to IEC-6 and IEC-17 cells maintained on plastic. These observations suggest that expression of TGFs is controlled by autocrine mechanisms in intestinal epithelial cell lines and proliferation stimulated by TGFI may be initially self-reinforcing but ultimately down regulated by induction of TGFJ1.

a-2. Functional Cytokine Receptors on Intestinal Epithelial Cells

The presence of receptors for the cytokine interleukin-2 (IL-2) was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-interleukin 2 (IL-2) was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabelled IL-2. Both high and intermediate affinity receptors with approximate Kd of 10pM and 100pM respectively were present. Kinetic analysis were consistent with the results of Western blot analysis utilizing an antisera to the 75kd IL-2 receptor J chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 hours followed by inhibition at 48 hours. The latter correlated with enhanced expression (range: 4- to 50-fold stimulation) of transforming growth factor J, (TGFJ), by IEC-6 cells following addition of IL-2. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2RJ chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes (IEL) by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa.

a-3. Identification of a Novel Variant Form of Fibroblast Growth Factor Receptor 3 (FGFR# 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 cDNA library prepared from the human colon cancer derived cell line HT-29 with a probe containing the tyrosine kinase domain of non-receptor class tyrosine kinase (CSK) 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 form 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 IIIx variant. Genomic PCR confirmed that the IIIb containing exon occupies a position 5' relative to the IIIc containing exon within the FGFR3 gene. Using a technique combining reverse transcriptase PCR with restriction endonuclease digestion it was possible to show that the FGFR3 IIIb form is more highly expressed than FGFR3 IIIc in HT-29 colon cancer cells. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4 kb transcript in two colon cancer derived cell lines as well as normal human colonic mucosa.

a-4. Fibroblast Growth Factors Modulate Intestinal Epithelial Cell Growth and Function

Over the past decade various peptide growth factors have been found to exert a variety of functional effects among epithelial cell populations within the gastrointestinal tract. The aim of the present study was to assess the potential role of members of the fibroblast growth factor family [acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and keratinocyte growth factor (KGF)] in the regulation of intestinal epithelial cell proliferation and restitution, a process that reestablishes the mucosal integrity by rapid migration of epithelial cells. Recombinant growth factors were added to subconfluent cultures of the intestinal cell line IEC-6, and the human colon cancer derived cell lines CaCo-2 and HT-29 cells with subsequent assessment of [3H] thymidine incorporation. The effects of these factors on an in vitro model of restitution was assessed by quantitation of cells migrating into standard wounds established in confluent monolayers of IEC-6 cells. TGFJ content of growth factor treated wounded monolayers was assessed by Northern Blot and bioassay. aFGF, bFGF and KGF caused a modest increase (2-3 fold) in proliferation of IEC-6, CaCo-2 and HT-29 cell lines. aFGF and bFGF promoted intestinal epithelial cell restitution in an in vitro model up to 10-fold, in conjunction with the enhanced expression of TGFJ mRNA and protein. The promotion of IEC-6 restitution by acidic FGF and basic FGF could be completely blocked by addition of immunoneutralizing anti-TGFJ antisera. These findings suggest that members of the FGF family exert effects on fibroblast cells also exert effects on intestinal epithelial cell populations and may play an important role in the promotion of epithelial cell restitution, the initial step of intestinal wound healing through a TGFJ-dependent pathway.

a-5. Human Intestinal Epithelial Cells Express Functional Cytokine Receptors Sharing the Common Kc Chain of the Interleukin-2 Receptor

IL-2 signaling requires the dimerization of the IL-2RJ and common K(Kc) chains. The Kc is also a component of the receptors for Interleukin-4 (IL-4), Interleukin-7 (IL-7), and Interleukin-9 (IL-9). In order to assess the extent and role of the receptor signal transducing system utilizing the Kc chain on human intestinal epithelial cells, the expression of Kc, IL-2RJ, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by RT-PCR on human intestinal epithelial cell lines as well as on primary isolated human intestinal epithelial cell lines as well as on primary isolated human intestinal epithelial cells. Caco-2, HT-29, and T-84 were found to express transcripts for the Kc and IL-4R chains constitutively. IL-2RJ chain expression was demonstrated in Caco-2 and HT-29, but not T-84 cells. None of the cell lines expressed mRNA for the IL-2RI chain. After stimulation with epidermal growth factor (EGF) for 24 hours Caco-2, HT-29, and T-84 expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cell lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for Kc, IL-2RJ, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.

a-6. Interleukin 2 Modulates Intestinal Epithelial Cell Function in vitro

Although interleukin 2 (IL-2) has been presumed to have a highly circumscribed range of target cells limited largely to classic immune cell populations, the presence of functional IL-2 receptors in rat epithelial cell lines has recently been demonstrated. Limited information is available about the functional effects of IL-2 on intestinal epithelial cells. The effect of recombinant IL-2 on intestinal epithelial cell migration was assessed using a previously described in vitro model of epithelial restitution by quantitation of cells migrating into standard wounds established in confluent IEC-6 cell monolayers. Transforming growth factor J content was assessed by Northern blot and bioassay. Exogenous IL-2 enhanced epithelial cell restitution in vitro on average 3.8-fold; this effect was independent of cell proliferation. Enhancement of restitution through IL-2 could be completely blocked through antibodies directed against TGF-J1 and interleukin-2 receptor indicating that stimulation of epithelial cell restitution is specifically enhanced by interleukin-2 and mediated through a TGFJ dependent pathway. In addition, increased expression of TGF-J1 mRNA and increased levels of bioactive TGFJ peptide in wounded monolayers treated with IL-2 compared to unwounded monolayers cultured in serum-deprived medium alone support the notion that enhancement of epithelial cell restitution in vitro is mediated through a TGFJ-dependent pathway. Conclusions: These studies suggest that IL-2, a potent cytokine whose biological origin and targets have been presumed to be largely limited to lymphocyte and macrophage populations, may play a role in preserving the integrity of the intestinal epithelium following various forms of injuries.

b. Molecular Mechanisms of Healing in Intestinal Mucosa

b-1. Transforming Growth Factor J Regulation of Intestinal Epithelium

Transforming growth factor J1 (TGFJ) plays an important role in the maintenance of the integrity of the intestinal epithelium despite a rapid rate of proliferation, maturation and senescence. Studies were undertaken to assess its role in repair following injury using an in vitro wounding model in monolayers of the intestinal epithelial cell line IEC-6. Although TGFJ was a potent inhibitor of proliferation as measured by uptake of BRDU, it promoted rapid "healing" of the monolayers through stimulation of migration of the epithelial cells across the wound margin. The physiologic importance of this activity was supported by the demonstration that conditioned medium from IEC-6 cells stimulated repair of the wounded monolayers, an activity that was enhanced by prior treatment with TGFJ consistent with the previous demonstration of autocrine induction of TGFJ in this cell line. In addition, expression of TGFJ1 mRNA was significantly enhanced in the wounded monolayers. Effects of the conditioned medium on cell migration could be entirely blocked by addition of immunoneutralizing anti-TGFJ antisera indicating that it is the dominant factor intrinsically contributing to migration in the wounded monolayer. Further, addition of protease inhibitors (aprotinin, EACA) which prevented the bioactivation of latent TGFJ secreted by the IEC-6 cells also ablated the effect of the conditioned medium on the wounded epithelium.

b-2. Regulation and Function of Extracellular Matrix in Intestinal Epithelial Restitution In Vitro

Repair of epithelial injury in the gastrointestinal tract is initially accomplished through restitution in which viable epithelial cells migrate from the wound edge into the denuded area. Extracellular matrix (ECM) molecules on which epithelial cells reside are potential modulators of mucosal wound healing. In order to assess the expression and function of the ECM in the restitution phase after epithelial injury, studies were undertaken using wounded rat intestinal epithelial crypt cells (IEC-6). IEC-6 cells expressed fibronectin (FN), mRNA and protein in large amounts and lesser quantities of laminin J1 and K1 (LN J and LNK1). Type IV collagen (Col IV) was weakly expressed and laminin I1 (LNI1) was not detected. After wounding, a gradual decrease in steady-state levels of FN, LNJ1, LNK1, and Col IVI1 mRNA was observed; mean content in IEC-6 cells 24 hours after wounding was only 10-25% of that found in unwounded controls. Reduced levels of FN, LN, and Col IV proteins (30-40%) were also found at 24 hours after injury. The downregulation of these ECM transcripts and proteins could be substantially prevented by addition of 5mg/ml TFGJ1, a peptide growth factor that has been shown to promote restitution. In addition to reduced overall content of ECM components, migrating cells showed predominant cytoplasmic staining for FN whereas quiescent cells in confluent areas were surrounded by a fibrillar FN network. LN was found in streaks on basal surfaces of confluent IEC-6 cells but in migrating cells, LN was primarily intracellular. RGD peptides that recognize the major cell attachment site on FN inhibited cell migration but function-blocking anti-LN antisera did not. In conclusion, although paradoxically downregulated after wounding, ECM, in particular FN, is functionally important for restitution after mucosal injury.

c. Role of Trefoil Peptides in the Gastrointestinal Tract

Goblet cells are abundant throughout the gastrointestinal tract and play an important role in sustaining the structural and functional integrity of the mucosal surface. Although it has been long recognized that a major component of the goblet cell secretory apparatus which produces a dense viscoelastic coat present in a continuous fashion through out the gastrointestinal tract are large, densely glycosylated mucin glycoproteins, the functional contribution of the goblet cell population and the molecular basis of the specificity of gene expression within these cells have not been defined. Recently the trefoil peptide family of structurally distinct small peptides specifically expressed in a regionalized fashion at all levels of the gastrointestinal tract have been identified as a previously unappreciated major product of goblet cell populations. Circumstantial evidence implicate the importance of these proteins in preservation of mucosal integrity and modulation of mucosal function; marked induction is consistently observed in association with injury throughout the gastrointestinal tract. These peptides are categorically distinct from other modulatory peptides in their targeted secretion onto the mucosal surface where they retain biological activity due to intrinsic resistance to intraluminal degradation. However, there is little insight into the functional properties of these peptides and the molecular basis of their specific expression in a highly cell and tissue specific manner. The main goal of the studies is the elucidation of the features of the genes encoding the trefoil factors and characterization of their protein products in order to both define the molecular basis of goblet cell specific gene expression delineate the role of these abundant peptides in sustaining mucosal integrity and function. Comprehensive study of the rat and human intestinal trefoil factors (RITF and HITF), identified and cloned in this laboratory which are present in high concentrations in goblet cells throughout the small and large intestinal epithelium is being undertaken. Major specific aims include: (I) Identification of genetic elements responsible for the highly specific expression of intestinal trefoil factor in intestinal and colonic goblet cells as well as genetic elements contributing to functional modulation of ITF expression. The latter will be facilitated through the combined application of transient transfection techniques and the study of ITF expression in established cell lines. (II) The functional characterization the biological activities of ITF obtained by direct purification and expression cloning are being defined using rat and human intestinal and colonic cell lines, as well as in vitro organ culture. These studies will be complemented by the assessment of the level of expression of ITF in mucosa from patients with inflammatory bowel disease and other disorders. Collectively, these studies should provide insights into the trefoil family of proteins and a newly recognized dimension of mucosal biology as well as fundamental insights into the molecular basis of intestinal and goblet cell function. These insights promise to provide new perspective on mechanisms of mucosal destruction, repair and function in inflammatory bowel disease and other forms of injury in the gastrointestinal tract.

c-1. Molecular Cloning of the Rat and Human Intestinal Trefoil Factor Genes: 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 kb of 5'-flanking region. RNase protection analysis demonstrated a single transcriptional start site. Various lengths of 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 ten-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 bp of 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.

c-2. Structure and Expression of Murine Intestinal Trefoil Factor: High Evolutionary Conservation and Postnatal Expression.

Intestinal Trefoil Factor (ITF) is a member of a family of gastrointestinal tract peptides with region-specific expression which are enhanced at sites of injury and repair. In the present study, the murine homologue gene of ITF was molecularly cloned in order to characterize the structure and expression of this peptide in mice. The murine gene exhibits extensive homology in both its genomic structure and its sequence. Murine ITF exhibited 78, 95 and 94% nucleotide homology respectively in exons I, II and III, with overall 90% predicted amino acid identify when compared to the rat ITF. Murine ITF exhibited 70% inferred amino acid identify compared with human ITF. Interestingly, extensive homology is also present among murine rat and human ITF within introus and 5 flanking sequence. Northern blot analysis of various adult mouse tissues demonstrated that ITF is expressed abundantly in the intestine and colon, and minimally in stomach, but not in brain, lung, spleen, kidney, uterus, pancreas, liver, heart or thymus tissues. Expression of ITF appeared to occur as a post-natal event: antibody specific for ITF stains intensely goblet cells in the intestine and colon of three-day old and older mice, but not in the gastrointestinal tract of younger mice or embryos.

c-3. Trefoil Peptides Promote Gastrointestinal Epithelial Restitution Through a TGFb-Independent Pathway

The trefoil peptides are a recently recognized family of protease resistant peptides which are expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent non-transformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or non-transformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6 fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a fifteen-fold enhancement in restitution. No synergistic effect was observed by the addition of non-mucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGFJ bioactivity, trefoil peptide and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGFJ and was not affected by the presence of immunoneutralizing anti-TGFJ antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote re-establishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

c-4. Trefoil Peptide Protection of Intestinal Epithelial Barrier Function: Cooperative Interaction with Mucin Glycoprotein

Goblet cells secrete a combination of trefoil peptides and mucin glycoproteins to form a continuous gel on the mucosal surface. The functional effects of these products remain uncertain. Trefoil peptides and/or mucin glycoproteins were added to transwell monolayers of the human colonic cancer derived T84 cell line. Intact monolayers permitted penetration of < 4% of the inert marker [3H]mannitol at 4 hours. Exposure to the toxic lectin phytohemagglutinin (1 mg/ml), oleic acid (8mM)/taurocholic acid (12mM) or C. difficile toxin A (0.7 mg/ml) resulted in loss of barrier function with 36%, 62% and 45% of [3H]mannitol penetration respectively. Addition of recombinant human intestinal trefoil factor (HITF) in physiological concentrations (1-5ug/Tl) resulted in attenuation of the damage to monolayer integrity by up to 52%. Protection was enhanced (up to 95%) by the co-presence of human colonic mucin glycoproteins. Similar effects were observed when rat ITF or human spasmolysin (HSP), another human trefoil peptide, were added alone or in the presence of human mucin glycoproteins. Conversely, mucin glycoproteins isolated from rat colon or rat stomach facilitated protection when added with HSP or HITF. Trefoil peptides and mucin glycoproteins protect gastrointestinal mucosa from a variety of insults.

c-5. Oral Trefoil Peptides Protect Against Ethanol and Indomethacin-Induced Gastric Injury in Rats

The trefoil factors, a family of proteins abundantly expressed in gastrointestinal mucous cells protect the epithelium in vitro. The aim of this study was to determine the effects of exogenously administered trefoil peptides on experimental injury in rats in vivo. 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 (BSA) were used as controls. pH of stomach contents was assessed at sacrifice. Gastric injury was blindly evaluated macroscopically and histologically. Serum levels of rHSP and ITF were determined by an ELISA. Oral rHSP and ITF markedly protected against both ethanol and indomethacin-induced gastric injury (p <0.005 at doses 1-15 mg/rat) when given up to 2 hrs. prior to injury; no protection was noted by i.p. rHSP against ethanol injury. Intraperitoneal (i.p.) rHSP protected against indomethacin-induced injury only at the maximal dose given (15 mg). Neither rHSP or ITF altered gastric pH. Protection was not associated with systemic absorption of trefoil peptides. Topical trefoil peptides protect gastric mucosa against ethanol and indomethacin-induced gastric injuries and appear to contribute to surface mucosal defense.

c-6. Targeted Deletion of Murine ITF

"Knockout" of the murine ITF was achieved by homologous recombination using a targeting construct in which exon II encoding the majority of the trefoil domain was replaced with the neo selective marker. Genomic transmission was confirmed and homozygous deficient mice produced through breeding of the initial chimeric mice. Although the ITF deficient mice have normal appearing colonic mucosa, preliminary characterization suggests that these mice are especially susceptible to mucosal injury and have impaired capacity for mucosal healing. These mice should be valuable "reagents" for elucidating the functional role of trefoil peptides in the GI tract.

d. Center for the Study of Inflammatory Bowel Disease

The MGH/NERPRC Center for the Study of Inflammatory Bowel Disease is a multidisciplinary program to define fundamental mechanisms underlying Crohn's disease and ulcerative colitis. While a number of provocative and useful observations offer some insights into inflammatory bowel disease (IBD), progress in understanding these common disorders has been slow and new initiatives are needed. Clinical experience and past research efforts serve to underscore the apparent intrinsic complexity of these disorders. Most importantly, progress in IBD research has been limited by both the need for advances in a number of relevant basic fields of gastrointestinal science and sufficient interest in the scientific community to explore the relevance of findings in these areas to IBD. This Center, encompassing workers at the Massachusetts General Hospital and the New England Regional Primate Research Center, includes both scientists pursuing a broad spectrum of areas of basic science relevant to IBD and investigators with established commitment to the study of IBD to promote progress in the understanding of IBD. It is anticipated that this center for research and investigator development will serve as a national resource for the advance in our understanding of IBD.

The broad goal of advancing our knowledge of IBD is promoted through the several biomedical core resources of this center. These cores function to provide both service as well as training/consultation, include a Molecular Biology Core, a Morphology core and an Immunology/Inflammatory Core. In addition, a Primate Model Core and a Clinical/Tissue Core provide relevant animal and patient tissue for study. A major goal of the Clinical/Tissue core is to coordinate the needs of the research center with the active program of patient care and clinical investigation at the MGH and to establish a reference source of well characterized tissue and serum that can serve as a center and national resource. The Primate Model Core also serves as a unique national resource in the study of the Cotton-top tamarin as a model of chronic colitis by making it available not only for the gastrointestinal research community of this center but also nationally.

In addition to advancing the understanding of IBD per se, the goals of this center include 1) the recruitment of established investigators to the study of inflammatory bowel disease and 2) the development of a resource for the training of new investigators committed to pursuing IBD research. A pilot feasibility program in support of these goals places special relevance on proposals to explore the extension of work emerging from the laboratories of allied basic scientists in a manner relevant to IBD as well as innovative projects of other members who will benefit from the core resources. It is hoped that this mechanism will contribute to the overall goal of increasing the complement of investigators focused on the pathogenesis, diagnosis and treatment of IBD.

d-1. Expression of Transforming Growth Factors I and J in Colonic Mucosa in Inflammatory Bowel Disease

The expression of transforming growth factors Iand J (TGFI and TGFJ), two structurally unrelated growth modulating peptides, which regulate epithelial proliferation and functional features of the varied cell populations important in inflammatory bowel disease, was assessed in colonic mucosa from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls by Northern blot analysis, in situ hybridization and bioassay. Human colonic mucosa was found to contain both TGFI transcript and peptide. TGFI mRNA expression localized to the surface epithelium, the villous tips of the small intestine and the surface cells of the colon. However, TGFI expression relative to a constitutive transcript was enhanced 2.3 fold (p<2.7 x 10-6) in mucosa from UC patients with inactive disease (n=12) relative to active UC, CD or normal controls. Enhanced expression correlated with duration of disease. TGFI expression remained confined to surface cells in both UC and CD.

In contrast to TGFI, TGFJ expression was increased in affected areas of mucosa from both UC and CD patients with active disease. TGFJ1 mRNA expression localized mostly to cells of the lamina propria with highest concentration in inflammatory cells of the most superficial mucosa, i.e., closest to the luminal surface. This pattern was maintained in active inflammation in UC and CD. These findings are consistent with the concept that TGFI contributes to epithelial hyperproliferation and perhaps the increased risk of eventual malignancy in long-standing UC. In contrast, TGFJ may be a key cytokine during periods of active inflammation, modulating epithelial cell restitution and functional features of immune cell and other populations present within the lamina propria.6