Torsten Kucharzik

Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms

It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function. Inflammation is characterized by an influx into the mucosa of immune cells that influence epithelial function by releasing proinflammatory cytokines such as IFN-γ and TNF-α. Mucosal barrier function is regulated by the epithelial apical junctional complex (AJC) consisting of the tight junction and the adherens junction. Since the AJC regulates barrier function, we analyzed the influence of IFN-γ and TNF-α on its structure/function and determined the contribution of apoptosis to this process using a model intestinal epithelial cell line, T84, and IFN-γ and TNF-α. AJC structure/function was analyzed by confocal microscopy, biochemical analysis, and physiologic measurement of epithelial gate/fence function. Apoptosis was monitored by determining cytokeratin 18 cleavage and caspase-3 activation. IFN-γ induced time-dependent disruptions in epithelial gate function that were potentiated by coincubation with TNF-α. Tight junction fence function was somewhat disrupted. Cytokine treatment was associated with internalization of AJC transmembrane proteins, junction adhesion molecule 1, occludin, and claudin-1/4 with minimal effects on the cytoplasmic plaque protein zonula occludens 1. Detergent solubility profiles of junction adhesion molecule 1 and E-cadherin and their affiliation with “raft-like” membrane microdomains were modified by these cytokines. Inhibition of cytokine-induced apoptosis did not block induced permeability defects; further emphasizing their primary influence on the epithelial AJC structure and barrier function. Our findings for the first time clearly separate the proapoptotic effects of IFN-γ and TNF-α from their abilities to disrupt barrier function.

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohns (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohns disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

Lipoxin A4 Analogs Attenuate Induction of Intestinal Epithelial Proinflammatory Gene Expression and Reduce the Severity of Dextran Sodium Sulfate-Induced Colitis

The anti-inflammatory eicosanoid lipoxin A4 (LXA4), aspirin-triggered 15-epi-LXA4, and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA4 analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA4 analog pretreatment attenuated induction of ∼50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA4 analog pretreatment is regulated by NF-κB, suggesting that LXA4 analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA4 analog reduced NF-κB-mediated transcriptional activation in a LXA4 receptor-dependent manner and inhibited induced degradation of IκBα. LXA4 analog did not affect earlier stimulus-induced signaling events that lead to IκBα degradation, such as S. typhimurium-induced epithelial Ca2+ mobilization or TNF-α-induced phosphorylation of IκBα. To establish the in vivo relevance of these findings, we examined whether LXA4 analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA4 analog (15-epi-16-para-fluoro-phenoxy-LXA4, 10 μg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA4 analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-κB pathway can be therapeutic for diseases characterized by mucosal inflammation.

CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer’s Patch CD11b+ Dendritic Cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3α). CCL20 is chemotactic to the CD11b+ dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer’s patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b+ DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer’s patches, CCL9 (MIP-1γ, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer’s patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer’s patch CD11b+ DCs and in a chemotaxis assay, CD11b+ DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b+ DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b+ DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b+ DC number in the subepithelial dome regions of Peyer’s patches of both wild type and CCR6 −/− mice. Taken together, these results demonstrate an important role of CCL9 in CD11b+ DC recruitment to the dome regions of mouse Peyer’s patches.

Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury

Aim: Neutrophil migration in the intestine depends on chemotaxis of neutrophils to CXC chemokines produced by epithelial cells. The goal of this project was to determine if acute induction of a CXC chemokine gradient originating from intestinal epithelial cells is sufficient to induce neutrophil influx into intact intestinal tissue. Methods and results: The authors developed a double transgenic mouse model with doxycycline induced human IL-8 expression restricted to intestinal epithelial cells. Doxycycline treatment of double transgenic mice for three days resulted in a 50-fold increase in the caecal IL-8 concentration and influx of neutrophils into the lamina propria. Although neutrophils entered the paracellular space between epithelial cells, complete transepithelial migration was not observed. Doxycycline treatment also increased the water content of the caecal and colonic stool, indicating dysfunctional water transport. However, the transmural electrical resistance was not decreased. Neutrophils recruited to the intestinal epithelium did not show evidence of degranulation and the epithelium remained intact as judged by histology. Conclusions: This conditional transgenic model of chemokine expression provides evidence that acute induction of IL-8 in the intestinal epithelium is sufficient to trigger neutrophil recruitment to the lamina propria, but additional activation signals are needed for full activation and degranulation of neutrophils, mucosal injury, and complete transepithelial migration.

CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock‐in mouse

The homing properties of subsets of lymphocytes and dendritic cells (DC) are regulated in part by the profile of chemokine receptors expressed. To determine how CCR6 influences cell trafficking, a mutant allele of the mouse CCR6 gene was produced that includes an enhanced green fluorescent protein (EGFP) reporter under the control of the CCR6 promoter. In mice heterozygous for the EGFP/CCR6 knock‐in, CCR6 expression was detected on all mature B cells, subpopulations of splenic CD4+ and CD8+ T cells, and on some CD11c+ DC. Most CD11b+ myeloid DC expressed CCR6, but CD8α+ lymphoid DC were negative for CCR6. Among myeloid DC, the CD4+ subset was uniformly positive for CCR6 expression and the CD4– subset was mostly CCR6 positive. Epidermal Langerhans cells (LC) also expressed CCR6, but at lower levels than splenic myeloid DC. Culture of bone marrow precursors from the knock‐in mice with GM‐CSF for 4 to 6 days led to the appearance of a subset of CD11c+ DC expressing CCR6. The differences in CCR6 expression among the major DC subsets indicate that CCR6 and its chemokine ligand MIP‐3α participate in determining the positioning of DC subsets in epithelial and lymphoid tissues.

CD98-mediated Links between Amino Acid Transport and β1 Integrin Distribution in Polarized Columnar Epithelia

In non-polarized cells, CD98 has been shown to both influence β1 integrins and heterodimerize with LAT-2, which confers amino acid transport capability on the LAT-2/CD98 heterodimer. Since LAT-2 is most heavily expressed in intestine and CD98 associates with the β1integrin splice form selectively found in such epithelia, we investigated the relationship and polarity of these proteins using the intestinal epithelial model Caco2-BBE. CD98 was found to selectively coimmunoprecipitate with both LAT-2 and β1 integrin, and, logically, all three proteins were polarized to the same (basolateral) domain. Furthermore, expression of CD98 in polarized epithelia lacking human CD98 (MDCK cells) disrupted β1 integrin surface distribution and cytoskeletal architecture, suggesting that CD98 can influence integrin function. Expression of a CD98 mutant lacking the specific residues conferring LAT-2 binding similarly affected cells, confirming that the latter effect was not due to LAT-2 sequestration. Use of CD98 truncation mutants suggest that a 10-amino acid domain located at the putative cytoplasmic tail/transmembrane domain interface was necessary and sufficient to induce the phenotype change. We conclude that the CD98/LAT-2 amino acid transporter is polarized to the same domain on which β1 integrin resides. CD98 appears to associate with β1 integrin and, in doing so, may influence its function as revealed by disruption of the outside-in signaling that confers cytoskeletal organization. Furthermore, such findings suggest a link between classic transport events and a critical element of barrier function: integrin-mediated influences on cytoskeletal organization.