Cyclooxygenase-2 overexpression abrogates the antiproliferative effects of TGF-beta.

The influence of cyclooxygenase-2 (COX-2) overexpression on the development of tumours has been well documented. The underlying mechanism however has still not been completely elucidated. An escape of proliferating cells from the regulatory influence of TGF-beta for example in the intestine has been discussed as well as a preponderance or prolongation of growth factor stimulation. The experiments presented here demonstrated that COX-2 transfection of a TGF-beta-sensitive cell line abrogates the growth inhibitory effects of TGF-beta. However, analysis of the TGF-beta/Smad-signalling pathway clearly revealed that COX-2 overexpression did not interfere with that. Neither TGF-receptor expression nor Smad phosphorylation and signal transfer into the nucleus were influenced by COX-2 overexpression. In addition, a TGF-beta reporter assay revealed no difference between controls and COX-2-transfected cells. Thus, the proliferation inhibiting effects must have been well compensated by growth-inducing stimuli. Indications for this came from experiments showing an induction of TGF-alpha expression and secretion with a higher and prolonged stimulation of the ERK 1/2 (p42/44) pathway in COX-2 transfectants. This effect could have been triggered by direct prostaglandin receptor stimulation or changes in intracellular lipid mediators. An increase in PPAR signalling as proven by a reporter assay is indication for the latter. Therefore, inhibiting both COX-2 as well as the PPAR and TGF/EGF pathway could be effective in the inhibition of adenoma or even carcinoma development in the intestine.

Role of virulence factors, cell components and adhesion in Helicobacter pylori-mediated iNOS induction in murine macrophages.

To investigate the mechanisms involved in Helicobacter pylori-mediated inducible nitric oxide synthase (iNOS) upregulation in mononuclear cells we cocultivated human THP-1 acute monocytic leukemia cells and murine J774A.1 professional macrophages with different H. pylori wild-type strains and mutants. We have shown that H. pylori-mediated iNOS induction in J774A.1 is independent of established virulence factors but dependent on direct interaction between bacteria and cells. In J774A.1, iNOS was equally upregulated by the wild-type strains J99, 26695, P12, and P1 as well as by mutants lacking the cag pathogenicity island, vacA, katA, alpAB genes and the hp0043 gene taking part in lipopolysaccharide biosynthesis when direct cell contact was allowed but not when bacteria and cells were separated by protein-permeable filter membranes. In contrast, iNOS was not induced in THP-1. This indicates that H. pylori-mediated iNOS induction in J774A.1 is independent of important virulence factors whereas cell contact is crucial which suggests a role of adhesion or phagocytosis.

Comparison of CXC chemokines ENA-78 and interleukin-8 expression in Helicobacter pylori-associated gastritis.

Colonization of the gastric mucosa with Helicobacter pylori is associated with a dense infiltration of granulocytes into the lamina propria in the active phase of gastritis. In this study, we investigated the involvement of epithelial cell-derived neutrophil-activating protein 78 (ENA-78) in development of H. pylori-associated gastritis. Antral biopsies from 27 patients with H. pylori-associated gastritis and 25 from H. pylori-negative individuals were first analyzed for ENA-78 and interleukin-8 (IL-8) mRNA by semiquantitative reverse transcription (RT)-PCR. In H. pylori-positive patients, significantly elevated levels were found for both chemokines (P<0.05). Only IL-8 mRNA levels differed significantly (P<0.05) in H. pylori-infected individuals who had serum antibodies for cytotoxin-associated protein CagA versus H. pylori-infected CagA-negative persons. Quantification of ENA-78 transcript levels by competitive RT-PCR yielded a significant 45-fold upregulation for ENA-78 transcripts in biopsies of H. pylori-positive versus H. pylori-negative patients (P<0.05). In contrast to earlier findings with IL-8, the degree of ENA-78 mRNA upregulation was independent of the grade of activity of gastritis. Immunofluorescence studies on tissues of antral biopsies localized ENA-78 protein expression mainly to the gastric epithelium of H. pylori-positive patients, while control tissues were negative. Upregulation of ENA-78 and IL-8 mRNA and protein expression was also observed in an in vitro system using a gastric adenocarcinoma cell line. Only viable H. pylori yielded a strong ENA-78 and IL-8 induction, while H. pylori outer membrane proteins or water-soluble proteins had no significant effect. These data provide evidence for the importance of both IL-8 and ENA-78 in the development and perpetuation of H. pylori-associated gastritis.

Functional role of CD95 ligand in concanavalin A-induced intestinal intraepithelial lymphocyte cytotoxicity.

Freshly isolated murine intestinal intraepithelial lymphocytes (IEL) express CD95 ligand (CD95L), as shown by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorter (FACS) analysis. Between 15 and 25% of IEL could be stained with an antibody to CD95L. Therefore it was investigated whether the CD95L/CD95 pathway was effective in IEL cytotoxicity. Stimulation of IEL in vitro with concanavalin A (Con A) induced a strong cytotoxic response, which was much higher when using CD95-expressing target cells. This effect was most evident when comparing the specific lysis of CD95-transfected target cells of the leukaemia cell line L1210 with that of the untransfected parental cell line. In addition, an antibody to CD95 was able to dramatically reduce the specific lysis of CD95-expressing target cells. After stimulation with Con A, which is able to bind to CD95L, the effects were more obvious compared with the triggering of the T-cell receptor (TCR)-alphabeta or gamma delta. On the other hand, EGTA reduced the Con A-induced cytotoxicity. Together these findings support a role of the CD95L/CD95 pathway in IEL cytotoxicity, even though the reaction was Ca2+ sensitive. As a function, CD95L-expressing IEL should be able to contribute to the elimination of CD95-expressing target cells in the intestine.

The influence of Peyer's patches on the organ-specific distribution of IgA plasma cells.

After the surgical removal of Peyer's patches (PP) in rats, the IgA-containing cells in the thoracic duct, mesenteric lymph nodes and lamina propria of the small intestine are decreased, as shown by immunohistology. The analysis of the immunoglobulin secretion in agar of single-cell suspensions confirmed these results. Using lipopolysaccharide (LPS) 055B5 as antigen, it could be demonstrated that this reduction may be the result of an inadequate presentation of antigen and/or impaired migration of locally primed antigen (AG)-specific cells. The oral application of heat-inactivated Escherichia coli 055B5 to PP-deprived rats resulted almost exclusively in anti-LPS-secreting cells in the mesenteric lymph nodes and spleen, whereas in control animals these cells were distributed along the intestine. Therefore, in rats PP have an important function in the regulation of the intestinal immune responses.

Role of Peyer's patch in the intestinal immune response to cholera toxin in enterically immunized rats.

Cholera toxin has been widely used to obtain insight into the cellular dynamics of the antigen-specific mucosal immune response. The present study was undertaken to clarify the influence of the organized intestinal lymphoid tissue (Peyer's patches [PP]) on the distribution of anti-cholera-toxin-containing cells (ACC) after intraperitoneal immunization and intraduodenal challenge with purified cholera toxin. This was done in rats which were surgically deprived of all visible PP. In comparison with sham-operated animals, each PP-deprived rat had nearly the same amount of ACC in the spleen, the mesenteric lymph nodes, and, surprisingly, the thoracic duct lymph. In contrast, the ACC in the duodenum, the jejunum, and the ileum of each PP-deprived animal were drastically reduced. Therefore the PP are suggested as an important organizing structure for the buildup of a local antigen-specific immune response.