Institut für Ernährungswissenschaften

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Transepitheliale Stimulation humaner Leukozyten durch Bakterien und ihre Oberflächenbestandteile
(2007) Bäuerlein, Annette; Parlesak, Alexandr
Background: The intestinal mucosa plays an important role in the discrimination of immune response between pathogenic and non-pathogenic bacteria as well as in mediating the systemic immunity. To adress the question whether probiotic, commensal, pathogenic germs and bacteria of food origin as well as their membrane components modify the immune response of the intestinal mucosa, we co-cultivated enterocyte-like CaCo-2 cells with human blood leucocytes in transwell cultures. Of further interest was the sequence of enteroxyte-leukocyte activation. Methods: PBMC (Peripheral blood mononuclear cells) were stimulated transepithelially in CaCo-2/PBMC co-cultures and directly challenged with probiotic, commensal, enteropathogenic and food-originating bacteria as well as with membrane components of grampositive and gramnegative bacteria. The expression of inflammatory cytokines (TNF-α, IL-8, IL-6, IL-10 and IFN-γ) was studied by enzyme linked immunosorbant assay (ELISA). The ratio of IL-8/18S mRNA was detected using quantitative reverse transcription polymerase chain reaction (qRT PCR). The permeation of endotoxin was quantified via Limulus amobocyte (LAL) assay and the integrity of the CaC-2 cell monolayer was detected via fluorescein-dextran and transepithelial electric resistance (TEER). Results: Grampositive bacteria did not activate immunocompetent cells in leukocyte-enterocyte co-cultures whereas a stimulation with the gramnegative probiotic E. coli Nissle resulted in higher expressions of TNF-α, IL-8, IL-6, IL-10 and IFN-γ than stimulation with the enteropathogenic E. coli (EPEC). The feature ?probiotic? results not necessarily in an enhanced production of inflammatory cytokines. Differences in epithelial permeability were not necessarily associated with an enhanced activation of immunocompetent cells. There was no activation of immunocompetent cells after direct or transepithelial stimulation with lipoteichoic acid (LTA) of E. faecalis. In contrast, endotoxin depending on its structure was a very potent (E. coli K12, E. coli Nissle, S. Typhimurium) or a moderately potent (B. vulgatus, B. vulgatus MPK) inducer of an inflammatory cytokine response. A neutralisation of endotoxin permeating into the basolateral compartment with Polymyxin B and Colisin resulted in a nearly total inhibition of inflammatory response. Furthermore, directly stimulated PBMC with comparable amounts of the permeating endotoxin in CaCo-2/PBMC co-cultures showed the same activation status as transepithelially stimulated cells. The probiotic, nonstimulating bacteria (Lb. rhamnosus GG, B. vulgatus, B. bifidum) were not able to reduce the E. coli K12 induced TNF-α, IL-8, IL-6, IL-10 and IFN-γ production. A co-stimulation of LPS from E. coli K12 (1 µg/ml) with non-stimulating endotoxins (B. vulgatus and B. vulgatus MPK) 100 µg/ml tended to reduce cytokine expression. Conclusion: These results show that the attribute ?probiotic?does not result in an obligatory activation of immunocompetent cells. The ability to stimulate immunocompetent cells is preferentially dependent on the presence of endotoxin, regarding the structure which is responsible for the stimulating capacity and is not mediated by enterocytes in first line. Activation of the basolaterally located lymphocytes occurs via permeating endotoxin. The permeability of the intestinal epithelial layer is only relevant when permeating endotoxin is able to stimulate immunocompetent cells to due its structural features.

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