Characterization of M cell development during indomethacin-induced ileitis in rats.

BACKGROUND: M cells play an important role in the intestinal immune system as they have a high capacity for transcytosis of a wide range of microorganisms and macromolecules. However, little is known about the role of M cells during intestinal inflammation. AIM: We studied M cell development during indomethacin-induced intestinal inflammation in rats. METHODS: Ileitis in rats was induced by two subcutaneous injections with indomethacin (7.5 mg/kg) given 24 h apart. Rats were sacrificed after 14 days and tissue was analysed by fluorescence microscopy and electron microscopy. M cells could be visualized by using the FITC-labelled mAb anti-cytokeratin (CK)-8 (clone 4.1.18), which was recently identified as specific M cell marker in rats. The number of cytokeratin-8 positive M cells was related to the surface of the follicle associated epithelium. For morphological studies, we used both transmission electron microscopy (T.E.M.) and scanning electron microscopy (S.E.M.). RESULTS: In non-inflamed ileum M cells were scarce. Only 4% of the follicle associated epithelium were M cells, whereas an increase of M cells up to 11% was found in inflamed follicle associated epithelium (P < 0.001). The rate of M cell induction depended on the macroscopic degree of inflammation. T.E.M./S.E.M. studies showed that in inflamed tissue most M cells underwent apoptosis with typical morphological signs. In contrast to apoptotic M cells, the neighbouring enterocytes usually appeared intact. The number of mononuclear cells below the follicle associated epithelium was significantly increased. S.E.M. studies revealed that during induced ileitis mononuclear cells migrated from the lamina propria into the gut lumen by passing through apoptotic M cells. CONCLUSIONS: During indomethacin-induced ileitis in rats the increase in M cell number in association with apoptosis of M cells may alter the intestinal barrier function. These observations may play a pivotal role in the pathogenesis of chronic intestinal inflammation, e.g. in inflammatory bowel disease.

Role of M cells in intestinal barrier function.

M cells are known as specialized epithelial cells of the follicle-associated epithelium of the gastrointestinal tract. As M cells have a high capacity for transcytosis of a wide range of microorganisms and macromolecules, they are believed to act as an antigen sampling system. The primary physiological role of M cells seems to be the rapid uptake and presentation of particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. In contrast to absorptive enterocytes, M cells do not exert direct defense mechanisms to antigens and pathogens in the gut lumen. Therefore, they provide functional openings of the epithelial barrier. Although M cells represent a weak point of the epithelial barrier, even under noninflamed conditions, there seems to be a balance between antigen uptake and immunological response. The low number of M cells in the gastrointestinal tract and the direct contact to immune cells in the lamina propria usually prevent the occurrence of mucosal inflammation. During chronic intestinal inflammation we observe an increase of M cell number and apoptosis selectively in M cells. M cell damage seems to be responsible for the increase of the uptake of microorganisms that is observed during intestinal inflammation. Under inflammatory conditions in the intestine, the maintenance of the epithelial barrier is broken and M cells seem to play a major role during this process.

Differences in predominant enhancement mechanisms of superparamagnetic iron oxide and ultrasmall superparamagnetic iron oxide for contrast-enhanced portal magnetic resonance angiography. Preliminary results of an animal study original investigation.

RATIONALE AND OBJECTIVES: To determine the effect of particle size of superparamagnetic iron oxide (SPIO) contrast agents on magnetic resonance angiography of the portal venous system. METHODS: We studied eight beagle dogs by a T1-weighted 3D turbo-gradient echo magnetic resonance (MR) angiography sequence (TE 4 milliseconds, TR 11 milliseconds, flip angle 25 degrees, coronal imaging plane) before and after administration of either Resovist (SHU555A), a superparamagnetic iron oxide contrast agent with a mean particle size of 60 nm and a relaxivity ratio R2/R1 of approximately 7, or a new ultrasmall superparamagnetic iron oxide (USPIO) contrast agent with a mean particle size of approximately 20 nm and a R2/R1 ratio of approximately 2. Images were acquired on a 1.5-T MR body scanner. Both agents were injected as a peripheral bolus of 40 mumol Fe/kg body weight. Repeated scans were acquired before, immediately after, and 10, 20, 30, and 40 minutes after administration of the agent. RESULTS: After administration of Resovist, portal venous signal increased to 237% of control immediately after injection, while hepatic parenchymal signal intensity decreased to 86% of control. The maximal CNR increase to 177% was achieved immediately after injection of the agent. After USPIO, portal venous signal increased to 401% of the precontrast value immediately after injection, while hepatic parenchymal signal intensity also increased to 131% of control at this time. Hepatic signal then decreased progressively to 49% of control after 40 minutes. The maximal CNR increase to 326% was achieved 10 minutes after injection of the agent. CONCLUSIONS: It is concluded that superparamagnetic iron oxide particles of different sizes have different R2/R1 ratios and, consequently, different mechanisms of contrast improvement in T1-weighted portal MR angiograms.

Towards targeting strategies for oral immunization--identification of marker antigens in rat M cells.

Specialized M cells of the follicle-associated epithelium (FAE) of Peyer's patches in the gastrointestinal and respiratory tracts play a crucial role in the immune surveillance of mucosal surfaces and are essential for the induction of mucosal immune responses. These cells transport luminal antigens to the underlying germinal centers and thereby initiate an immune response or induce tolerance. Mucosal immune responses could be significantly enhanced if oral antigens could be directly targeted to the apical surface of M cells. However, thus far M cells have not been isolated and suitable surface markers have not been described. For the characterization and identification of potential molecular markers of M cells the follicle associated epithelium of Peyer's patches from the small intestine of rats was analyzed by indirect immunofluorescence and immunogold electronmicroscopy. The expression of intermediate filament proteins in the FAE was found to be heterogeneous. Immunoreactivity of a specific monoclonal cytokeratin 8 antibody (clone 4.1.18) was selectively found in cells which were additionally characterized by the lack of staining for alkaline phosphatase in their apical membranes. In the FAE this property is an accepted criterium for the presence of M cells. The antigen recognized by the clone 4.1.18 is expressed in substantially higher amounts in M cells of Peyer's patches as compared to neighbouring epithelial cells or "normal" enterocytes and thus can be employed as an intracellular molecular marker for M cells of Peyer's patches in rats. It is expected, that this marker will be very helpful for the isolation of M cells.

Immunocytochemical characterization of the follicle-associated epithelium of Peyer's patches: anti-cytokeratin 8 antibody (clone 4.1.18) as a molecular marker for rat M cells.

For the characterization and identification of potential molecular markers of M cells, the follicle-associated epithelium (FAE) of Peyer's patches from the small intestine of rats was analyzed by indirect immunofluorescence and immunogold electron microscopy. The expression of intermediate filament proteins in the FAE was found to be heterogeneous. Immunoreactivity of a specific monoclonal cytokeratin 8 antibody (clone 4.1.18) was selectively found in cells which were additionally characterized by the lack of staining for alkaline phosphatase in their apical membranes. In the FAE, this property is an accepted criterium for the presence of M cells. Further, these cells were found to be devoid of mucin and were thus distinct from goblet cells. The antigen recognized by the clone 4.1.18 is expressed in substantially higher amounts in M cells of Peyer's patches as compared to neighboring epithelial cells or "normal" enterocytes and thus can be employed as an intracellular molecular marker for M cells of Peyer's patches in rats.