Epimorphin expression in human colonic myofibroblasts

Epimorphin is a membrane-associated protein that has been postulated to regulate epithelial morphogenesis in several tissues. However, epimorphin expression in the human intestine has not been fully investigated. In this study, we investigated epimorphin expression in the inflamed mucosa of inflammatory bowel disease (IBD). Tissue samples were obtained surgically from patients with active ulcerative colitis (UC) (n=5) and active Crohn's disease (CD) (n=5). Epimorphin and alpha-smooth muscle actin (SMA) were stained immunohistochemically. Epimorphin expression in human intestinal subepithelial myofibroblasts (SEMFs) was analyzed by Western and Northern blotting. In the normal colon, epimorphin expression was detected partly in the alpha-SMA-positive cells under the epithelial cells. Epimorphin was also expressed in alpha-SMA-positive cells in the capillary wall. In the inflamed mucosa of UC and CD patients, epimorphin expression was not altered. In isolated human SEMFs, epimorphin was detected as a single band of molecular weight 34-kDa under reducing and non-reducing conditions. In intestinal SEMFs, epimorphin mRNA expression was not affected by inflammatory cytokines and growth factors. Epimorphin was constitutively expressed in the normal colonic mucosa, and this was not altered in the inflamed mucosa of IBD patients. The localization of epimorphin may indicate a potential role in maintaining normal tissue structure in normal and IBD mucosa.     

Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in the inflamed mucosa of inflammatory bowel disease (IBD). We also investigated mechanisms regulating IL-32alpha expression. Tissue samples were obtained endoscopically or surgically from patients with ulcerative colitis (UC) (n = 10), Crohn's disease (CD) (n = 10), ischaemic colitis (n = 4) and normal colorectal tissues (n = 10). IL-32alpha expression was evaluated by standard immunohistochemical procedure. IL-32 mRNA expression was analysed by Northern blot. IL-32alpha was expressed weakly by colonic epithelial cells from normal individuals and subjects with ischaemic colitis. In the inflamed mucosa of IBD patients, epithelial IL-32alpha expression was increased markedly. In UC and CD patients, IL-32alpha expression was enhanced in affected mucosa compared to non-affected mucosa. In intestinal epithelial cell lines, expression of IL-32alpha mRNA and protein was enhanced by IL-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. A combination of TNF-alpha plus IFN-gamma exerted synergistic effects. IL-32alpha induction by IL-1beta and/or TNF-alpha was mediated by NF-kappaB activation. Epithelial IL-32alpha expression was increased in IBD patients, and in CD patients in particular. IL-32alpha might be involved in the pathophysiology of IBD as a proinflammatory cytokine and a mediator of innate immune response.     

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

Epithelial expression of interleukin‐37b in inflammatory bowel disease

Interleukin (IL)‐37 is a member of the IL‐1 cytokine family. We investigated IL‐37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL‐37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL‐37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL‐37b mRNA and protein expression were determined by real time‐polymerase chain reaction (PCR) and Western blotting, respectively. IL‐37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL‐37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL‐37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL‐37b mRNA and protein was enhanced by tumour necrosis factor (TNF)‐α. This IL‐37b induction by TNF‐α was mediated by nuclear factor (NF)‐κB and activator protein (AP)‐1 activation. Furthermore, IL‐37b inhibited TNF‐α‐induced interferon‐γ‐inducible protein (IP)‐10 expression significantly in human colonic SEMFs. Epithelial IL‐37b expression was increased in IBD patients, especially UC patients. IL‐37b may be involved in the pathophysiology of IBD as an anti‐inflammatory cytokine and an inhibitor of both innate and acquired immune responses.     

Immunoglobulin production by isolated intestinal mononuclear cells from patients with ulcerative colitis and Crohn''s disease.

We studied immunoglobulin production by isolated intestinal mononuclear cells from 25 patients with active inflammatory bowel disease (IBD) and 17 controls undergoing surgical resections for intestinal tumour or other disorders. Normal ileal intestinal mononuclear cells spontaneously produced greater amounts of IgA and IgM than did normal colon cells. In cells from patients with IBD there was a significantly reduced IgA production, but production of IgG was enhanced in both colon and ileum. The alteration in IgA and IgG production in IBD was confirmed by comparing the immunoglobulin production by mononuclear cells from inflamed with that from non-inflamed areas of mucosa in six patients with distal ulcerative colitis. The proportion of IgA-containing cells in isolated intestinal mononuclear cells from IBD mucosa was less than normal. However, the proportion of IgG-containing cells from IBD mucosa was not increased in isolated intestinal mononuclear cells although they produced more IgG than normal mucosal cells. Our study showed an altered pattern of immunoglobulin production by intestinal mononuclear cells isolated from patients with inflammatory bowel disease.     

Differential mucosal expression of three superoxide dismutase isoforms in inflammatory bowel disease

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)-SOD, manganese (Mn)-SOD, and extracellular (EC)-SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme-linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn-, Mn-, and EC-SOD in paired inflamed and non-inflamed mucosal resection specimens of patients with Crohn's disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step-wise increase in Mn-SOD protein levels was observed in non-inflamed and inflamed IBD mucosae, whereas the Cu/Zn-SOD content decreased with inflammation. EC-SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn-SOD and Cu/Zn-SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn-SOD was considerably increased in IBD, whereas epithelial Cu/Zn-SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn-SOD and Mn-SOD were prominently present in neutrophils and macrophages, and EC-SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn-, Mn-, or EC-SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn-SOD and EC-SOD, but the activity of Mn-SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn-SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients.     

Intestinal oxidative damage in inflammatory bowel disease: semi-quantification,localization, and association with mucosal antioxidants

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress-related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite-mediated protein nitration (3-NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide-producing enzyme xanthine oxidase (XO). In addition, bi- and multi-variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohn's disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3-NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3-NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3-NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3-NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite-independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothionein and with Mn-superoxide dismutase activity, suggesting the involvement of hydroxyl radicals and superoxide anions. In UC, however, the amount of MDA was associated with epithelial catalase expression and neutrophilic myeloperoxidase activity, suggesting a hydrogen peroxide- and/or hypochlorous acid-mediated mechanism. The present study underlines the importance of oxidative stress in the pathogenesis of IBD and provides clues regarding the (anti)oxidants involved which indicate that this process evolves through diverging pathways in CD and UC.     

Differential distribution of B7.1 (CD80) and B7.2 (CD86) costimulatory molecules on mucosal macrophage subsets in human inflammatory bowel disease (IBD)

The molecules B7.1 and B7.2 deliver costimulatory signals of critical importance to naive T cells, and may thus be involved in abrogation of oral tolerance in IBD. Functional disparity apparently exists among antigen-presenting cells in vivo. We wanted to examine if differential B7 expression occurs on mucosal macrophage subsets. Cryosections of bowel specimens from patients with IBD and normal controls were subjected to immunofluorescence and immunoperoxidase staining. In normal mucosa, selective subepithelial accumulation of B7.2⁺ cells was found. In inflamed IBD mucosa, however, subsets appeared consisting of both B7.2^hi and B7.1^hi cells as well as CD14^hi macrophages. Notably, outside lymphoid aggregates the prominent fraction of recently recruited CD14^hi macrophages comprised most (≈ 80%) of the B7.1^hi cells, whereas most (≈ 70%) B7.2^hi cells were identified as resident mucosal macrophages (CD14^lo or CD14⁻). Differential expression of B7.1 and B7.2 on two functionally different subsets of intestinal macrophages implies separate immunoregulatory roles for the two molecules. This finding is in keeping with recent experimental data demonstrating that monocyte-derived cells are crucial for immune responses at mucosal surfaces. Preferential B7.1 up-regulation might be critical in breaking the immunological tolerance to luminal antigens in IBD, but it cannot be excluded that it is a secondary pathogenic event.     

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)‐17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL‐17 and C3 mRNA expressions in the IBD mucosa were evaluated by real‐time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme‐linked immunosorbent assay. IL‐17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL‐17 and C3 mRNA expression in the IBD mucosa. IL‐17 stimulated a dose‐ and time‐dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115‐kDa α‐chain linked to a 70‐kDa β‐chain by disulphide bonds, which was identical to serum C3. The IL‐17‐induced C3 mRNA expression was blocked by p42/44 mitogen‐activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL‐17‐induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL‐17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL‐17.     

TL1A as a Potential Local Inducer of IL17A Expression in Colon Mucosa of Inflammatory Bowel Disease Patients

Interaction between TL1A and death receptor 3 (DR3) co‐stimulates T cells, induces production of several pro‐inflammatory cytokines and has been linked to pathogenesis of inflammatory bowel disease (IBD). This study aimed to establish a link between expression of TL1A and selected TL1A‐induced pro‐inflammatory cytokines involved in IBD pathogenesis (IL‐4, IL‐13, IL‐17A and IFN‐γ) and to investigate a connection between serum concentration of TL1A in patients with IBD and activation of peripheral blood T cells. Elevated levels of IL‐4 (2.91‐fold) and IL‐13 (4.05‐fold) mRNA were detected in the inflamed colon mucosa of patients with ulcerative colitis (UC), IFN‐γ mRNA was upregulated (3.23‐fold) in the inflamed colon mucosa of patients with Crohn's disease (CD), whereas upregulation of IL‐17A and TL1A mRNA was present in the inflamed colon mucosa of patients with both CD and UC (IL‐17A: 4.48‐fold and 2.74‐fold, TL1A: 3.19‐fold and 3.22‐fold, respectively) vs. control subjects. We did not detect any changes in DR3 mRNA expression in the investigated groups of patients. TL1A mRNA level in colon mucosa of patients with IBD correlated only with the level of IL‐17A mRNA but no other investigated cytokines. In colon mucosa, expression of TL1A and DR3 was localized to enterocytes and lamina propria mononuclear cells. We did not find any correlation between serum concentrations of TL1A and IL‐17A or changes of CD4(+) or CD8(+) lymphocytes phenotype in patients with IBD. Therefore, our data indicate that TL1A may contribute to pathogenesis of IBD via local but not systemic induction of IL‐17A but not IL‐4, IL‐13 or IFN‐γ.     

The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity

This study analyzed IL-23p19 expression in inflamed mucosa of IBD and the role in the induction of IEL and NK cell activation as well as Th17 cell differentiation. Expression of IL-23p19 was performed by immunohistochemistry and quantitative real-time PCR. Expression of IL-23R was assessed by flow cytometry. Cytolytic activities of IEL and NK cells by IL-23 were determined by a standard (51)Cr-release assay. Cytokine levels were analyzed by ELISA and quantitative real-time PCR. Expression of IL-23p19 was increased significantly in inflamed mucosa of CD compared with that in UC and healthy controls. Double-staining confirmed that IL-23p19(+) cells were mainly CD68(+) macrophages/DCs. IL-23R(+) cells were increased significantly in PB- and LP-CD4(+) and -CD8(+) T and NK cells. IL-23 markedly promoted IBD IEL and NK cell activation and cytotoxicity and triggered IBD PB- and LP-T cells to secrete significantly higher levels of IFN-γ, TNF, IL-2, and IL-17A compared with controls. Importantly, IL-23 promoted IBD PB- or LP-CD4(+) T cells to differentiate into Th17 cells, characterized by increased expression of IL-17A and RORC. Anti-TNF treatment could markedly reduce IL-23 expression and Th17 cell infiltration in inflamed mucosa of CD patients. These data indicate that IL-23 is highly expressed in inflamed mucosa of IBD and plays an important role in the induction of IEL, NK, and T cell activation, proinflammatory cytokine secretion, and Th17 cell differentiation. Targeted therapy directed against IL-23p19 may have a therapeutic role in treatment of IBD.     

Investigation of the expression of IL-1beta converting enzyme and apoptosis in normal and inflammatory bowel disease (IBD) mucosal macrophages.

Activated mucosal macrophages are derived from circulating monocytes and appear to play a major role in the pathogenesis of IBD. We have recently shown that IBD, but not normal, mucosal macrophages express the active form of IL-1beta converting enzyme (ICE) and are therefore capable of releasing mature IL-1beta. ICE expression by other mucosal cell types is unknown. Active ICE expression has also been implicated in apoptosis. The aim of this study was to investigate ICE expression (using an antibody that recognizes both active and precursor forms) in normal and IBD mucosa and to determine whether ICE-expressing macrophages are undergoing apoptosis. Normal and active IBD mucosal cells, in tissue sections and after isolation, were studied by immunohistochemistry and flow cytometry. In the mucosa, macrophages were the predominant ICE-expressing cell type. In contrast to normal, most IBD mucosal macrophages expressed ICE. Of IBD colonic macrophages 11.8 +/- 3.2%, and of normal colonic macrophages 6.6 +/- 0.6% expressed Apo2.7, a marker for apoptotic cells. Similar data were obtained when annexin V was used to identify cells undergoing apoptosis. DNA fluorescence flow cytometric analysis of normal and IBD lamina propria cells showed the presence of only small hypodiploid DNA peaks. We conclude that in the human intestinal mucosa, macrophages are the predominant ICE-expressing cell type. Expression of the active form of ICE and macrophage apoptosis are not interdependent. One mechanism of loss of resident macrophages from normal mucosa and of recruited macrophages from IBD mucosa is by apoptosis.