The central role of chemokines (chemotactic cytokines) in the immunopathogenesis of ulcerative colitis and Crohn's disease.

The final composition of leukocytes present in a site of inflammation in response to chemokine stimulation and activation may depend on both the nature of the secreted chemokines as well as the relative expression of the multitude of specific chemokine cell surface receptors on many different cell types. Because related receptors with different affinities and cross-reactive binding capabilities are present on each type of leukocyte, relative differences in receptor distribution and receptor affinity for specific chemokines may significantly influence which cells are ultimately attracted to and activated by each individual chemokine. Production of IL-8, MCP-1, and ENA-78 by endothelial cells, LPMNC, and epithelial cells in IBD could establish a chemotactic gradient capable of influencing the increased migration of monocytes/macrophages, granulocytes, and lymphocytes from the blood stream through the endothelium into both the mucosa and submucosa during chronic IBD. The ability of chemokines to induce chemotaxis, leukocyte activation, granule exocytosis, increased production of metalloenzymes, and up-regulation of respiratory burst activity indicates that there may be a variety of different mechanisms by which chemokines could markedly increase chronic inflammation and chronic intestinal tissue destruction in IBD.

Increased expression of interleukin-8 mRNA in ulcerative colitis and Crohn's disease mucosa and epithelial cells.

: Interleukin-8 (IL-8) is a chemotactic cytokine (chemokine), which both attracts and activates granulocytes. IL-8 could have a central function in the initiation and perpetuation of the inflammatory bowel diseases (IBD), due to its relative resistance to inactivation and long half-life in vivo. Using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay, we have observed elevated levels of IL-8 mRNA in colonic mucosal sections obtained from surgically resected specimens from ulcerative colitis (UC) and Crohn's disease (CD) patients with actively inflamed mucosa. The level of IL-8 mRNA expression in the intestinal mucosal biopsies from UC and CD patients was much greater in involved as opposed to noninvolved mucosal sections. The highest expression of IL-8 mRNA detected by RT-PCR was in UC mucosa and in isolated intestinal epithelial cells from UC patients. Increased IL-8 production by cells in IBD intestinal mucosa as well as IBD epithelial cells may be involved in the continuous attraction and activation of granulocytes in the inflamed intestine in both UC and CD patients. Chemokines, such as IL-8, are potent chemoattractant molecules and may have a central role in the augmentation and perpetuation of inflammation in IBD.

Human intestinal epithelial cells express functional cytokine receptors sharing the common gamma c chain of the interleukin 2 receptor.

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells 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 cells 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 gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.

Mucosal barrier and immune mediators.

The intestinal mucosa functions is an immunologic organ that plays a major role in the development of oral tolerance and host-defense mechanisms. Antigens must cross the intestinal epithelium in a controlled manner to interact with dendritic antigen-presenting cells, because bacteria or their products are a primary risk factor for the development of intestinal inflammation. Therefore, the regulation of the intestinal epithelial cell barrier is central to the development of intestinal immunity and inflammation, but the involved mechanisms are largely unknown. Intestinal barrier function relies on the formation of tight junctions at the apical contact areas of intestinal epithelial cells. Tight junctions have a highly dynamic structure whose permeability, assembly, or disassembly can be regulated by a variety of cellular and metabolic mediators, including cytokines, which have major functions in the immune system. Immune modulators control tight junction dependent intestinal barrier function during development, wound healing, and pathologic processes such as cancer, infection, and chronic inflammation.

Claudins regulate the intestinal barrier in response to immune mediators.

BACKGROUND & AIMS: To determine the functional role of immune mediators in the formation of the intestinal barrier, we have examined the regulation of claudin expression by interleukin (IL)-17 in human intestinal epithelial cells. METHODS: Expression of claudins, extracellular signal-related (ERK) mitogen-activated protein kinases (MAPKs), and activated ERK MAPKs was determined by immunoblotting. Claudin membrane association was assessed by immunohistochemistry and claudin messenger RNA expression by Northern blot analysis. Intestinal epithelial barrier function was characterized through transepithelial electrical resistance and mannitol tracer flux. RESULTS: IL-17 induced the development of a paracellular barrier of T84 cell monolayers. Inhibition of ERK activation with the MEK inhibitor PD98059 blocked IL-17 as well as basal development of tight junctions in T84 cells. IL-17 induced formation of tight junctions correlated with up-regulation of claudin-1 and claudin-2 gene transcription. Inhibition of MEK reduced the activated and basal expression of claudin-2 messenger RNA and protein expression. Functional MEK was required for the expression and membrane association of claudin-2 but not claudin-1 in T84 cells. CONCLUSIONS: MEK activity is required for claudin-mediated formation of tight junctions. IL-17 is able to regulate the intestinal barrier through the ERK MAPK pathway.

Intestinal epithelial cells both express and respond to interleukin 15.

BACKGROUND & AIMS: Interleukin (IL)-15 exerts functional effects on lymphocytes similar to those of IL-2. IL-15 is expressed by nonlymphoid cells and may integrate these cells into classical immune responses. The aim of this study was to characterize the expression of IL-15 by intestinal epithelial cells and determine the functional roles of IL-15 within the mucosal immune system. METHODS: Rat IL-15 was cloned from a rat jejunal library. Expression of IL-15 in rat and human intestinal epithelial cells was assessed by Northern and Western blotting. Tyrosine kinase activation in response to IL-15 in intestinal epithelial cells was determined by immunoprecipitation. RESULTS: Rat and human intestinal epithelial cells express IL-15 messenger RNA. IL-15 activates Stat3 and stimulates the proliferation of intestinal epithelial cells. The relevance of the observations for intestinal epithelial cell function in vivo was supported by the demonstration of transcripts for IL-15 in primary human intestinal epithelial cells. CONCLUSIONS: IL-15 is expressed by intestinal epithelial cells function. These experiments suggest that IL-15 is an important mediator that could integrate intestinal epithelial cell function with the intestinal immune system.

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells.

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.

Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors.

LPS elicits several immediate proinflammatoy responses in peripheral blood leukocytes via a recently described pathway including CD14, Toll-like receptors (TLR), serine-threonine kinases, and NF-kappaB transcription factor. However, the functional responses of intestinal epithelial cells (IEC) to stimulation with LPS are unknown. Expression of mRNA and protein for CD14 and TLRs were assessed by RT-PCR, immunoblotting, and immunohistochemistry in mouse and human IEC lines. LPS-induced activation of signaling pathways (p42/p44 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), p38, p65, NF-kappaB) were assessed by immunoblotting and gel shifts. CD14 mRNA and protein expression were not detectable in IEC. However, human TLR2, TLR3, and TLR4 mRNA were present in IEC. TLR4 protein was expressed in all cell lines; however, TLR2 protein was absent in HT29 cells. Immunofluorescent staining of T84 cells demonstrated the cell-surface presence of the TLRs. LPS-stimulation of IEC resulted in activation (>1.5-fold) of the three members of the MAPK family. In contrast, LPS did not significantly induce activation of JNK and p38 in CMT93 cells, p38 in T84 cells and MAPK and JNK in HT29 cells. Downstream, LPS activated NF-kappaB in IEC in a time-, dose-, and serum-dependent manner. IEC express TLRs that appear to mediate LPS stimulation of specific intracellular signal transduction pathways in IEC. Thus, IEC may play a frontline role in monitoring lumenal bacteria.

Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa.

BACKGROUND: Monocyte-chemoattractant protein 1 (MCP-1) activates macrophages and increases the migration of monocytes into tissue during inflammation. It was hypothesized that MCP-1 expression is involved in intestinal inflammation. METHODS: MCP-1 protein was detected by immunohistochemistry and immunoprecipitation. Biological activity of MCP-1 was assessed using a chemotactic assay. MCP-1 messenger RNA (mRNA) levels were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS: In normal mucosa, MCP-1 was predominantly present in surface epithelium. In contrast, inflamed mucosa from patients with ulcerative colitis or Crohn's disease contained multiple cells immunoreactive for MCP-1, including spindle cells, mononuclear cells, and endothelial cells. Furthermore, MCP-1 mRNA expression was markedly increased in inflamed intestinal biopsy specimens from patients with inflammatory bowel disease. MCP-1 was detected in isolated intestinal epithelial cells and in conditioned media from Caco-2 cells. Caco-2 cell-conditioned media stimulated monocyte chemotaxis activity that was inhibited by anti-MCP-1 antibodies. Constituitive MCP-1 mRNA levels in Caco-2 cells were up-regulated by interleukin 1 beta and down-regulated by dexamethasone. CONCLUSIONS: In addition to lamina propria macrophages, endothelial cells, and spindle cells, intestinal epithelial cells are able to produce MCP-1. MCP-1 is expressed constitutively in the intestinal colonic mucosa and is up-regulated during inflammation.

Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease.

The perpetuation of inflammation in ulcerative colitis and Crohn's disease may be regulated in part by an increased secretion of proinflammatory cytokines due to either an appropriate response to initial stimulating agents, and/or due to an impaired down-regulation of cytokine secretion. The aim of this study was to determine the secretion patterns of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 beta, from isolated lamina propria mononuclear cells (LPMNC) isolated from colonic biopsies from patients with untreated ulcerative colitis or Crohn's disease. LPMNC isolated from involved inflammatory bowel disease (IBD) mucosa spontaneously produced increased amounts of TNF-alpha, and IL-6, and IL-1 beta. The TNF-alpha secretion from IBD LPMNC could be further enhanced by pokeweed mitogen stimulation. The secretion patterns of TNF-alpha and IL-1 beta by LPMNC from patients with either ulcerative colitis or Crohn's disease demonstrated a close correlation with the degree of tissue involvement and mucosal inflammation. LPMNC from non-involved ulcerative colitis mucosa secreted markedly increased levels of IL-6 compared with non-involved Crohn's disease mucosa or control mucosa. The heightened IL-6 secretion from LPMNC from non-involved ulcerative colitis mucosa without visible or microscopic signs of inflammation indicates that the pathophysiologic mechanisms involved in the initiation of inflammation may differ between ulcerative colitis and Crohn's disease. The determination of proinflammatory cytokine secretion by isolated LPMNC from colonoscopic biopsies may be a sensitive method for monitoring the severity of mucosal inflammation in IBD patients.

Alteration of gene expression by intestinal epithelial cells precedes colitis in interleukin-2-deficient mice.

Intestinal epithelial cells may be actively involved in the immunoregulatory pathways leading to intestinal inflammation. The aim of this study was to assess expression by intestinal epithelial cells of cytokines with potential involvement in the development of intestinal inflammation in interleukin (IL)-2-deficient [(-/-)] mice. Wild-type mice, mice heterozygous for the disrupted IL-2 gene, and IL-2(-/-) mice were studied at 6, 16, and 24 wk of age. The mRNA levels of transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, IL-15, KC, JE, and CD14 in colonic and small intestinal epithelial cells were assessed by Northern blot analysis. CD14 was also measured by Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). TGF-beta 1 mRNA was constitutively expressed in both colonic and small intestinal epithelial cells with increased expression in the colonic epithelium of colitic mice. CD14 was detected only in colonic epithelial cells, and mRNA levels increased severalfold in IL-2(-/-) mice with colitis. Northern analysis demonstrated increased levels of TGF-beta 1 and CD14 mRNA in colonic epithelial cells of IL-2(-/-) mice before the development of signs of colitis. CD14 mRNA and protein expression in the epithelial cells of colitic mice were confirmed by RT-PCR and Western blot analysis of isolated cells. In addition, IL-2(-/-) mice also expressed increased levels of IL-15 mRNA in small intestinal and colonic epithelial cells compared with heterozygous control mice. TNF-alpha, IL-1 beta, IL-6, KC, and JE mRNAs were only detectable in colonic epithelial cells of mice after the onset of colitis. Enhanced expression of TGF-beta 1, IL-15, and CD14 by colonic epithelial cells may play a role in the subsequent development of colitis in IL-2(-/-) mice.

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions.

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.

Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa.

Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control of the inflammatory mediator IL-1beta. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1beta. Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells in normal small intestine and in active Crohn's disease mucosa. Furthermore, fractalkine mRNA expression was significantly up-regulated in the intestine during active Crohn's disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa.

Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine.

The chemokine receptors CCR2 and CCR5 and their respective ligands regulate leukocyte chemotaxis and activation. To determine the role of these chemokine receptors in the regulation of the intestinal immune response, we induced colitis in CCR2- and CCR5-deficient mice by continuous oral administration of dextran sodium sulfate (DSS). Both CCR2- and CCR5-deficient mice were susceptible to DSS-induced intestinal inflammation. The lack of CCR2 or CCR5 did not reduce the DSS-induced migration of macrophages into the colonic lamina propria. However, both CCR5-deficient mice and, to a lesser degree, CCR2-deficient mice were protected from DSS-induced intestinal adhesions and mucosal ulcerations. CCR5-deficient mice were characterized by a greater relative infiltration of CD4+ and NK1.1+ lymphocyte in the colonic lamina propria when compared to wild-type and CCR2-deficient mice. In CCR5-deficient mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN-gamma was decreased, corresponding to a Th2 pattern of T cell activation. In CCR2-deficient mice, the infiltration of Th2-type T cells in the lamina propria was absent, but increased levels of IL-10 and decreased levels of IFN-gamma may have down regulated mucosal inflammation. Our data indicate that CCR5 may be critical for the promotion of intestinal Th1-type immune responses in mice.