Polarized Caco-2 Cell Monolayers Research Articles

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice.

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.

Comparison of Salmonella typhi and Salmonella typhimurium invasion, intracellular growth and localization in cultured human epithelial cells

Invasion of the cultured epithelial cell lines HeLa, Henle-407, and Caco-2 (polarized and non-polarized) by Salmonella typhi and Salmonella typhimurium were compared using conventional gentamicin invasion assays. Additionally, the mechanisms of invasion and intracellular trafficking by S. typhi and S. typhimurium were compared in HeLa cells using indirect immunofluorescence microscopy. S. typhi strain Ty2 was invasive in all human cell lines tested, including apical uptake into polarized Caco-2 cell monolayers. This strain also replicated at levels similar to S. typhimurium strain SL1344 inside HeLa and Henle-407 cells. Indirect immunofluorescence microscopy confirmed that S. typhi, like S. typhimurium, induced membrane ruffles and cytoskeletal rearrangements upon contact with HeLa cell surfaces. Ruffling induced by S. typhi and S. typhimurium was accompanied by macropinocytosis of the fluid phase endocytic marker fluorescein-dextran-sulphate and by aggregation of cell surface class I MHC heavy chain. Intracellular lysosomal trafficking of S. typhi and S. typhimurium in HeLa cells was also studied. The lysosomal membrane glycoprotein marker h-lamp-2 colocalized with S. typhi-containing vacuoles, as previously shown for S. typhimurium. The soluble lysosomal enzyme marker cathepsin D also was found within S. typhi-containing vacuoles to the same extent as previously published for S. typhimurium . The results from this study suggest that S. typhi and S. typhimurium use similar mechanisms for invasion and intracellular trafficking in cultured human epithelial cells.

Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures.

The ability of Campylobacter jejuni isolates to translocate across an epithelial cell barrier was investigated by using polarized Caco-2 cell monolayers grown on microporous membrane filters. The 4 C. jejuni isolates tested all traversed the Caco-2 cell monolayers and displayed similar translocation kinetics. The number of bacteria crossing the polarized cell monolayers continued to increase with time until 4 h after inoculation, at which time a maximum rate of translocation was observed. Transmission electron microscopy revealed that C. jejuni translocated across polarized Caco-2 cell monolayers by passing both through and between cells. Chloramphenicol, an inhibitor of bacterial protein synthesis, reduced the translocation of C. jejuni. Bacterial attachment, internalization, and translocation were inhibited at low temperature. These data indicate that adherence, penetration, and translocation of C. jejuni require active bacterial and target cell processes and further suggest a role for cellular translocation in the pathogenesis of C. jejuni-mediated enteritis.