Asit Panja

A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.

Differential cytokine expression by human intestinal epithelial cell lines: Regulated expression of interleukin 8

BACKGROUND To characterize the role of intestinal epithelial cells in mucosal host defense, we have examined constitutive cytokine expression and regulated expression of interleukin (IL)-8 by human colonic epithelial cells. METHODS Cytokine expression by the human colonic epithelial cell lines, T84, Caco-2, SW620, and HT29 was assessed by using polymerase chain reaction amplification of reverse-transcribed RNA. Regulated IL-8 expression was analyzed by nuclear run-off assays, Northern blot analysis, and enzyme-linked immunosorbent assay. RESULTS The cell lines constitutively expressed messenger RNA (mRNA) for IL-8 and transforming growth factor beta 1. In addition, some cell lines expressed mRNA for IL-1 alpha, IL-1 beta, IL-10 and tumor necrosis factor alpha (TNF alpha). None of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, or interferon gamma. Cell lines secreted IL-8 either constitutively or after stimulation with the physiological agonists TNF alpha, IL-1 beta, or lipopolysaccharide. Increased IL-8 secretion after TNF alpha stimulation of T84 cells was accompanied by increased IL-8 mRNA levels and an increased transcription rate of the IL-8 gene. IL-8 was preferentially secreted at the basolateral surface of polarized T84 cells. In further studies, freshly isolated human colon epithelial cells also secreted IL-8. CONCLUSIONS These results support the notion of bidirectional communication between intestinal epithelial cells and mucosal immune and inflammatory cells.

Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.

Intestinal epithelial cells (IEC) have been shown to act as antigen‐presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL‐1β, IL‐6, tumour necrosis factor‐alpha (TNF‐α), transforming growth factor‐beta (TGF‐β) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon‐gamma (IFN‐γ), IL‐1β or TNF‐α. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL‐1) or bioassay (TNF and IL‐6). Neither IL‐1β nor TNF‐α were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL‐6 at levels comparable to those of macro‐phages. IEC IL‐6 mRNA also increased approximately 200‐fold during the first 24 h of culture. LPS, IFN‐γ or TNF‐α had no effect on spontaneous IL‐6 production, and neither resulted in the secretion of IL‐1β or TNF‐α. However, IL‐1β up‐regulated IL‐6 synthesis by 6–7‐fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL‐6 expression but no detectable IL‐1β, TGF‐β or TNF‐α), adding to their uniqueness as APC.

Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins.

Repetitive mechanical deformation may stimulate intestinal epithelial proliferation. Because the extracellular matrix modulates static intestinal epithelial biology, we examined whether matrix proteins influence intestinal epithelial responses to deformation. Human Caco‐2bbe cells and nontransformed human enterocytes (HIPEC) were subjected to 10% average cyclic strain at 10 cycles/min on flexible membranes precoated with matrix proteins without or with plasma fibronectin or functional anti‐integrin antibodies in the medium. Strain stimulated proliferation, focal adhesion kinase, extracellular signal‐regulated protein kinase (ERK), p38, and Jun N‐terminal kinase similarly on collagen I or IV, and more weakly on laminin, but had no effect on fibronectin. MEK blockade (PD98059) prevented strain‐stimulated proliferation on collagen but did not affect proliferation on fibronectin. Adding tissue fibronectin to a collagen substrate or plasma fibronectin to the media suppressed strains mitogenic and signal effects, but not those of epidermal growth factor. Functional antibodies to the α5 or αv integrin subunit blocked strains effects on Caco‐2 proliferation and ERK activation, although ligation of the α2 or α6 subunit did not. Repetitive strain also stimulated, and fibronectin inhibited, human intestinal primary epithelial cell proliferation. Repetitive deformation stimulates transformed and nontransformed human intestinal epithelial proliferation in a matrix‐dependent manner. Tissue or plasma fibronectin may regulate the intestinal epithelial response to strain via integrins containing α5 or av.

Expression of growth factor receptor-encoded mRNA by colonic epithelial cells is altered in inflammatory bowel diseas

A link between inflammation of the colon in inflammatory bowel disease (IBD) and the increased risk of colon cancer in ulcerative colitis (UC) may be provided by growth factor receptor genes. Their expression may be altered in response to growth factors present in the mucosa, and this, in turn, may induce further genetic changes, linked to carcinogenesis, in the cells of the colonic epithelium. To test this hypothesis, we assayed steady-state levels of eight growth factor receptor mRNAs in colonic epithelial cells of IBD patients and controls. Four of these genes (EGF-R, IGFI-R, CSF1-R, andPDGF-R-β) were expressed in epithelial cells, whereas four (erbB-2, erbB-3, NGF-R, andmet) were not. The level of the former in involved or uninvolved IBD was considerably lower than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. In contrast, expression was much higher in IBD patients with colon tumors than in active chronic IBD. The level of PDGF-R-β mRNA was two- to fourfold higher in involved than in uninvolved areas of the colons of two UC patients, but not in one Crohns disease patient. Message abundance of its ligand,PDGF-β, however, was the same in paired UC samples. The pattern of expression ofPDGF-β andcripto was identical to that ofEGF-R, whereas the level of mRNA of amphiregulin was the same in active chronic IBD and IBD patients with tumors. A fourth growth factor,Kfgf, was not expressed. Increased levels of PDGF-R-β mRNA in involved UC relative to uninvolved UC may be related to the disease process in UC. Decreased expression of growth factor- and growth factor receptor-encoded mRNA in active chronic IBD may be related to the disease process, or it may be an effect of steroid therapy undergone by these patients. Enhanced expression of these genes in IBD patients with tumors compared to those without tumors suggests that this may be a marker for development of colon cancer in IBD.

Differential expression of interleukin 1 receptor antagonist isoforms in human intestinal epithelial cells.

BACKGROUND & AIMS Regulatory cytokines mediate intestinal epithelial cell (IEC) participation in mucosal immune responses. The aim of this study was to investigate the expression of secretory and intracellular isoforms of interleukin 1 receptor antagonist (IL-1Ra) in human primary IECs and carcinoma-derived cell lines. METHODS Primary IECs were isolated from patients with Crohns disease or ulcerative colitis and from normal controls. Isoform-specific IL-1Ra messenger RNA (mRNA) and protein were assessed by reverse-transcription polymerase chain reaction and Western blot analysis. Expression during cellular differentiation was determined by in situ immunohistochemistry on sequentially released, native IECs and in vitro differentiated cell lines. Intracellular IL-1Ra I function was analyzed by permanent transfection of Caco-2 cells. RESULTS Intracellular IL-1Ra I protein accumulated in surface IECs with extension to the crypts during inflammation. Secretory IL-1Ra and intracellular IL-1Ra II mRNA, but not the corresponding protein, was detected. Transcription of intracellular IL-1Ra I mRNA was significantly up-regulated with inflammation and in vitro by phorbol myristate acetate and interleukin 1beta. In vitro differentiated cells had higher constitutive intracellular IL-1Ra I protein content. Intracellular IL-1Ra I expression in Caco-2 cells decreased IL-1beta-stimulated interleukin 8 secretion. CONCLUSIONS Native human IECs and certain cell lines constitutively express intracellular IL-1Ra type I, which is up-regulated by inflammation, inflammatory stimuli, and cellular differentiation. Constitutive expression of this anti-inflammatory cytokine may contribute to mucosal protection.

Expression of protooncogene-encoded mRNA by colonic epithelial cells in inflammatory bowel disease

Protooncogenes are cell cycle-related genes that are involved in cell growth or proliferation. Alterations in the level of expression of these genes, or expression of aberrant gene products, have been observed in tumors and precancerous conditions. To determine if expression of these genes is altered in patients with inflammatory bowel disease (IBD)-who are at risk for development of colon cancer—we assayed transcripts of 15 protooncogenes in colonic epithelial cells of IBD patients and controls. Nine, of these genes (H-ras, c-myc, c-fos, c-jun, junB, N-myc, c-abl, c-yes, andp53) were expressed in epithelial cells, whereas two (RB1 and N-ras) were not. Expression of four other genes (c-src, K-ras, c-raf and c-myb) was observed, but the intensity of these bands was too low for densitometric analysis. The steady-state levels of transcripts of H-ras and five nuclear protooncogenes (c-myc, c-fos, c-jun, junB, and N-myc) were lower in epithelial cells from involved or uninvolved IBD samples than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. The level of c-fos mRNA was two-to threefold higher in involved than in uninvolved areas of the colons of two ulcerative colitis (UC) patients, but not in one Crohns disease (CD) patient. Message abundance of c-abl transcripts was two-to threefold lower in UC epithelial cells than in either the CD or control samples. The steady-state level of c-yes-encoded mRNA was considerably higher in IBD patients resected for colon cancer than in patients resected for active chronic IBD or in controls. The level ofp53 message was constant in these samples. Increased levels of c-fos mRNA in involved UC relative to uninvolved, UC may be related to the disease process. Decreased expression of c-abl transcripts in UC may be a diagnostic marker for UC and may be related to the rate of cell turnover in these diseases. Enhanced expression of c-yes in IBD patients with tumors compared to active chronic IBD and controls suggests that expression of this gene may be a marker for development of colon cancer in IBD.

Antigen recognition in the gastrointestinal tract: death to the dogma.

that is required of GALT by virtue of its continuous coexistence with the external environment. The exposure to Ag is limited in the spleen and lymph nodes so that antigenic challenge here should result in an active directed immune response. In the gut, the lymphoid tissue is compartmentalize d to those cells residing within the epithelial layer (IELs) and those below the basement membrane in the loose connective tissue stroma (LPLs). The origin and fate of IELs remains unclear. In both mouse and man IELs are predominantly CD8 + and are difficult to activate in vitro [6, 7]. LPLs, in contrast, are functional in vitro although they tend to be constitutively activated memory cells (activated in vivo) with poor responses to recall Ags [8]. What then governs the activation and effector function of both cell populations? Our group in man and Blands group in the rat system [9, 10] have recently described a novel role for the intestinal enterocyte, that of antigen-presenting cell (APC). Normal intestinal epithelia express class II Ags at low density and are capable of processing and presenting Ag to immunocompetent T cells [9, 10]. However, in contrast to conventional APCs, epithelial cells appear to preferentially activate CD8 + T cells. In our studies, these CD8 + T cells are antigen nonspecific and, in

2 Antigen presentation in the intestine

Abstract The intestinal mucosa is the largest surface area in the body which is continually exposed to an enormous amount of food antigens, viruses