R. Ciccocioppo

Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease

Background and aims: Epithelium derived interleukin (IL)-15 signalling via IL-15Rα is critical for the development, activation, and survival of intraepithelial lymphocytes (IEL). We aimed to better understand the IL-15 driven effects on IEL underlying mucosal damage and lymphomagenesis in coeliac disease (CD). Methods: Enterocytes, IEL, and lamina propria mononuclear cells (LPMC) were isolated from 46 patients with uncomplicated CD (25 untreated and 21 treated) and 22 controls. IL-15 and IL-15Rα expression were determined by immunoblotting. Secretion of IL-15, interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), and granzyme B into cell culture supernatants was assessed by ELISA. The ability of IL-15 to regulate IEL proliferation, perforin/granzyme dependent cytotoxicity, and apoptosis was tested by adding different combinations of IL-15, IL-15 blocking antibody, or chloroquine to IEL cultured alone or with Caco-2 cells as target. IL-15 mucosal levels were also determined by ELISA in five patients with complicated CD (two ulcerative jejunoileites, one refractory sprue, and two enteropathy associated T cell lymphomas) tested for T cell receptor γ chain clonality. Results: IL-15 was overexpressed in untreated CD enterocytes and LPMC, and in the mucosa of complicated CD patients and uncomplicated untreated CD patients, where its levels correlated with the degree of mucosal damage. Enterocytes from untreated, but not treated, CD patients and controls secreted IL-15. Untreated CD IEL, characterised by higher IL-15Rα expression, showed increased proliferation, production of IFN-γ and TNF-α, and perforin/granzyme dependent cytotoxicity, and a decreased propensity to apoptosis in response to IL-15. Conclusions: Our findings suggest that IL-15 plays a crucial role in the generation of epithelial damage in active CD. Its promotion of IEL survival in CD may predispose to the emergence of T cell clonal proliferations. Blocking IL-15, by suppressing uncontrolled IEL activation and survival, has the potential to provide new therapeutic tools to prevent tissue damage and lymphomagenesis in CD.

Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn’s disease

Background and aims: To verify whether targeting defective mucosal T cell death underlies the sustained therapeutic benefit of infliximab in Crohn’s disease, we explored its in vivo proapoptotic effect after 10 weeks of treatment, and its in vitro killing activity on lamina propria T cells (LPT) and peripheral blood T cells (PBT), both isolated from Crohn’s disease patients. Methods: Endoscopic intestinal biopsies were collected from 10 Crohn’s disease patients (six steroid refractory and four fistulising) before and after three consecutive infusions of infliximab, administered at week 0, 2, and 6 in a single intravenous dose (5 mg/kg), and from 10 subjects who proved to have functional diarrhoea. Apoptosis was determined in vivo by TUNEL assay, and in vitro by fluorescein isothiocyanate-annexin V/propidium iodide staining on LPT and PBT from Crohn’s disease patients cultured with infliximab. The effect of the broad caspase inhibitor Z-VAD-FMK and the neutralising anti-Fas antibody ZB4 was tested in vitro on LPT and PBT treated with infliximab. Caspase-3 activity was determined by immunoblotting. Results: In Crohn’s disease patients, infliximab treatment induced a sustained LPT apoptosis, still evident four weeks after the last infusion. In vitro infliximab induced death of LPT from Crohn’s disease patients occurred via apoptosis rather than necrosis. LPT showed a higher susceptibility to infliximab induced apoptosis than PBT in Crohn’s disease patients. The signalling pathway underlying the restoration of infliximab induced LPT apoptosis occurred via the caspase pathway but not Fas-Fas ligand interaction in Crohn’s disease. Conclusions: These findings demonstrate that apoptosis is the major mechanism by which infliximab exerts its killing activity on LPT in Crohn’s disease. The sustained LPT proapoptotic action of infliximab, which extends far beyond its circulating half life, may be responsible for the sustained remission induced in Crohn’s disease patients by infliximab retreatment.

Oral butyrate for mildly to moderately active Crohn's disease

Background : Butyrate exerts anti‐inflammatory effects in experimental colitis and on Crohns disease lamina propria mononuclear cells in vitro.

Increased Enterocyte Apoptosis in Inflamed Areas of Crohn’s Disease

AbstractPURPOSE: Because increased enterocyte apoptosis has been associated with the pathogenesis of several chronic inflammatory diseases, the aim of our study was to investigate epithelial cell death in Crohn’s disease and the possible role of the Fas-Fas ligand system, E-cadherin, and matrix metalloproteinase-1 in modulating enterocyte apoptosis in this condition. METHODS: Endoscopic ileal and colonic biopsy specimens were collected from macroscopically involved and uninvolved areas of 20 patients with Crohn’s disease and 20 subjects who proved to have functional diarrhea. Diagnosis was established by clinical and pathologic criteria. Biopsy specimens were processed for traditional histology and for the immunohistochemical evaluation of Fas, Fas ligand, E-cadherin, Ki67 antigen, and matrix metalloproteinase-1 expression. For the in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine triphosphate nick end labeling was used. RESULTS: The percentages of apoptotic enterocytes were higher in involved than in uninvolved areas of Crohn’s disease patients and normal intestine. No significant difference was found between Crohn’s disease uninvolved areas and normal intestine. In Crohn’s disease, both enterocyte Fas and lamina propria mononuclear cell Fas ligand expression did not differ from controls. E-cadherin was strongly expressed by epithelium in both normal and inflamed intestine, except for the regenerative epithelium over the base of the ulcers, where a reduced E-cadherin expression was observed. The number of Ki67-positive proliferating epithelial cells did not differ either in involved or uninvolved areas of Crohn’s disease patients compared with controls. A lamina propria overexpression of matrix metalloproteinase-1 was found in involved compared with uninvolved Crohn’s disease areas and normal tissue, and a significant positive correlation between matrix metalloproteinase-1 expression and enterocyte apoptosis was found in Crohn’s disease inflamed areas. CONCLUSIONS: Enterocyte apoptosis is increased in involved areas of Crohn’s disease. This increase is not mediated by a Fas-Fas ligand mechanism or by an abnormal E-cadherin distribution. Increased matrix metalloproteinase-1 release from lamina propria mononuclear cells might be one of the possible mechanisms responsible for the increased enterocyte apoptosis in Crohn’s disease.

Increased Enterocyte Apoptosis and Fas-Fas Ligand System in Celiac Disease

Our aim was to evaluate whether increased enterocyte apoptosis was responsible for mucosal flattening in celiac disease (CD), and, since the mechanisms responsible for tissue injury in this condition are unknown, we studied the possibility that the Fas-Fas ligand (FasL) system may be involved. Endoscopic duodenal biopsy specimens from 12 patients with untreated and 12 with treated CD and 12 control subjects were evaluated for enterocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling assay and for Fas and FasL expression by immunohistochemistry. A coculture of isolated enterocytes (targets) and purified lamina propria mononuclear cells (LPMCs) (effectors) was performed in the absence or presence of an antagonistic ZB4 anti-Fas antibody. We found a significant correlation between the degree of villous atrophy, morphometrically evaluated, and the level of enterocyte apoptosis, suggesting that mucosal flattening is a consequence of exaggerated epithelial cell death. Most celiac enterocytes express Fas, and LPMCs express FasL. The abolishment of enterocyte apoptosis observed in the presence of ZB4 antibody suggests that enterocytes are potential targets of lymphocyte infiltrate. These results directly demonstrate that FasL-mediated apoptosis is a major mechanism responsible for enterocyte death in CD.

Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease

BACKGROUND Lamina propria (LPLs) and intraepithelial (IELs) lymphocytes are markedly increased in coeliac mucosa, and are thought to play a crucial role in the generation of villous atrophy in coeliac disease (CD). However, the mechanisms by which they mediate the killing of enterocytes in this condition are still poorly characterised. AIM We investigated Fas mediated cytotoxicity and apoptosis of both LPLs and IELs, isolated from 10 untreated coeliac patients, 10 coeliac patients on a gluten free diet, and 10 biopsied controls. METHODS Fas and Fas ligand expression were assessed by flow cytometry and immunocytochemistry. Lymphocyte cytotoxicity against Fas expressing Jurkat cells was determined by the Jam test. The effect of the antagonist ZB4 anti-Fas antibody on apoptotic activity exerted by coeliac lymphocytes against enterocytes was analysed. Lymphocyte apoptosis was assessed by oligonucleosome ELISA. RESULTS LPLs and IELs showed increased apoptotic activity and higher levels of Fas ligand expression in untreated CD compared with treated CD patients and controls. Enterocyte apoptosis observed after coculturing coeliac lymphocytes and enterocytes in the presence of ZB4 antibody was reduced. In active CD, LPLs manifested increased apoptosis whereas IELs showed decreased apoptosis. CONCLUSIONS Our results support the involvement of the Fas/Fas ligand system in CD associated enterocyte apoptosis. Increased LPL apoptosis is likely to downregulate mucosal inflammation whereas decreased IEL apoptosis could be responsible for autoimmune and malignant complications of CD.

Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa

Tissue transglutaminase (tTG) seems to be the target self‐antigen for endomysial antibodies in coeliac disease (CD) and to catalyse the critical deamidation of gliadin which strengthens its recognition by HLA‐restricted gut‐derived T cells. To date, it has not been demonstrated whether gliadin is cross‐linked to tTG within the gut wall, a phenomenon known to occur in vitro. We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa. The immunoprecipitation and Western blotting experiments were performed on mucosal biopsies obtained from untreated, treated CD patients and biopsied controls, by using either anti‐tTG or anti‐gliadin antibodies, in both denaturating/reducing or nondenaturating/nonreducing conditions. A subset of experiments was performed by using anti‐tTG antibodies purified by affinity chromatography from sera of untreated coeliac patients. The localization of tTG and gliadin was studied by immunofluorescence at confocal laser microscopy on seriate sections of diseased and normal duodenal mucosa by using the same antibodies of the coimmunoprecipitation section. The amounts of tTG and gliadin coimmunoprecipitated with anti‐tTG monoclonal antibody in untreated CD mucosa were significantly increased compared to those of the other two groups. When performing the experiments in nondenaturating/nonreducing conditions, a high molecular weight band formed by both molecules, was evidenciated. Also the anti‐tTG antibodies purified from patients’ sera turned out to be able to coimmunoprecipitate the two molecules. The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa. Our findings firstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls, and the ability of circulating tTG‐autoantibodies to recognize and immunoprecipitate the tTG‐gliadin complexes.

Small bowel enterocyte apoptosis and proliferation are increased in the elderly.

Background: It is known that in the elderly the small bowel does not reveal structural and functional deteriorations in normal conditions, whereas the absorptive function is impaired in stress conditions. Objective: The balance between enterocyte apoptosis and proliferation being responsible for the maintenance of tissue size, mucosal morphology and function in the gastrointestinal tract, the aim of our study was to evaluate the rates of enterocyte apoptosis and proliferation in the duodenal mucosa of aged human beings in comparison to adults. Methods: For this purpose, the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end labelling (TUNEL) technique and immunohistochemistry for MIB-1 detection were applied on histological sections of endoscopic duodenal biopsy specimens obtained from 12 healthy elderly subjects (mean age 77.6 years; M/F 7/5) and 12 healthy adult subjects (mean age 37.7 years; M/F 8/4). Counts were performed, at a constant magnification (×200), by computer-aided analysis and the results expressed as median percentages of positive enterocytes. Results: The results showed a significant increase in enterocyte apoptosis in the elderly (15.3 vs. 2.1% in the adults, p < 0.001) which was positively correlated (rs = 0.65, p < 0.05) with a significant increase in enterocyte proliferation (37.7 vs. 15.8% in the adults, p < 0.0001). Conclusions: These data suggest that the maintenance of mucosal architecture throughout the process of aging is due to either a hyperproliferative state or an exaggerated apoptosis with a consequent cellular immaturity, which may impair the absorptive function observed in stress conditions.

Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD)

The effector arm of the mucosal immune system comprises lymphocytes scattered at intraepithelial and lamina propria levels. Intraepithelial lymphocytes (IEL) are a large population of oligoclonal resting cells which exhibit phenotypic and functional characteristics of cytolytic T cells when activated. Several mechanisms have been demonstrated to account for their cytotoxicity. Among them, one is mediated by perforin and granzyme molecules, another is mediated by Fas ligand (FasL) which delivers apoptotic signals through Fas receptor on target cells. There is good evidence that a flat intestinal mucosa may be produced by activated T cells. The aim of our study was to evaluate FasL and perforin expression by IEL, and its possible correlation with the increased enterocyte apoptosis in coeliac mucosa. Endoscopic duodenal biopsy specimens from 10 untreated coeliac patients, 10 treated coeliac patients, and 10 biopsied controls were evaluated for enterocyte apoptosis by terminal deoxynucleotidyl transferase‐mediated digoxigenin‐deoxyuridine triphosphate nick end label method, for perforin expression by immunohistochemistry, and for FasL expression by immunocytochemistry. In untreated CoD there was a significant increase of percentage of both FasL+ and perforin+ IEL which positively correlated with enterocyte apoptosis in comparison with controls. All these parameters were significantly lower in treated CoD, even though they did not normalize. Our study demonstrates that in untreated CoD FasL and perforin expression by IEL is increased, and significantly correlates with the level of enterocyte apoptosis.

Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease

Since in coeliac disease mucosal flattening has been suggested to result from an increased enterocyte apoptosis triggered by Fas/Fas ligand system and perforin cytolytic granules, we looked for a similar mechanism in autoimmune enteropathy. Moreover, we tried to assess whether enterocyte autoantibodies, which are the hallmark of autoimmune enteropathy, may be involved in triggering enterocyte apoptosis in this condition. Immunohistochemical staining with anti‐Fas, ‐FasL and ‐perforin MoAb, and TUNEL technique were applied on endoscopic duodenal biopsies of two autoimmune enteropathy patients, two untreated coeliac patients and two biopsied controls. Cytotoxicity assays were carried out by incubating peripheral blood mononuclear cells from a healthy subject (effectors) with enterocytes primed with patient or control sera (targets). In autoimmune enteropathy a large number of enterocytes were apoptotic, as in coeliac disease, whereas neither Fas/Fas ligand or perforin expressions were up‐regulated. On the other hand, antibody‐dependent cellular cytotoxicity assay revealed the ability of sera from patients with autoimmune enteropathy to mediate enterocyte death through apoptosis. These results point to enterocyte autoantibody‐dependent cellular cytotoxicity as the prevalent mechanism of increased enterocyte apoptosis in autoimmune enteropathy but not in coeliac disease.