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DOES THE CYTOSKELETON OF INTESTINAL EPITHHELIAL CELLS FUNCTION AS A CELLULAR ALARM TO IDENTIFY THE E. COLI INFECTION
Author(s) -
Li Zhe,
Elliott Elizabeth,
Gunning Peter,
O'Loughlin Edward
Publication year - 2001
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1111/j.1440-1746.2001.ca01-33.x
Subject(s) - cytoskeleton , cytochalasin d , microfilament , nocodazole , actin , biology , microbiology and biotechnology , microtubule , secretion , cell , biochemistry
Intestinal epithelial cells play an important role in regulating host immunity in response to intestinal infection. Pathogenic bacteria (EPEC and EHEC) cause profound cytoskeletal rearrangement in intestinal epithelial cells during attachment or invasion. Rearrangement of cytoskeletal proteins could be a signal to up‐regulate host defence response. Aims To determine the role of actin cytoskeleton and microtubles in IL‐8 mRNA response to E. coli infection. Methods T84 cell monolayers in 6‐well plates were infected with HB101, EPEC and EHEC (105 CFU/well) and compared with uninfected control at 3, 6 and 12 h post infection. Control and infected monolayers were treated with nocodazole (Noc, microtubule disrupter, 30 m m ), taxol (Tax, microtubule stabiliser, 10 m m ), cytochalasin D (CytoD, actin depolymeriser, 100 n m ) and Jasplakinolide (Jasp, actin polymeriser, stabilise actin filaments, 1 m m ) and studied 6 h post infection. IL‐8 gene expression was measured by semiquantitative RT–PCR in control and uninfected monolayers with and without drug treatment and IL8 protein secretion by ELISA. The morphology of F‐actin and β‐tubulin was examined by FITC‐phaloidin staining (FAS), immunohistochemistry and confocal microscopy. Results IL‐8 mRNA and IL‐8 were increased by infection with all bacterial strains at 3 and 6 h but both IL‐8 mRNA and IL‐8 in EHEC and EPEC infection were decreased compared with control and HB101 at 12 h. Disruption of microfilaments by Noc increased IL‐8 (2.7 fold) while preservation of microfilaments by Tax inhibited IL8 response (0.5 fold) to HB101 infection only. CytoD decreased (0.1–0.5 fold) IL8 expression at all time points in all infections while stabilising actin by Jasp markedly increased the IL8 response (2–6 fold) in control, HB101, EHEC and EPEC at 3 and 6 h. CytoD inhibited Noc‐induced IL8 gene expression. Confocal microscopy demonstrated that CytoD and Noc caused major morphological damage to the actin and β‐tubulin by 6 h. Similar changes were also observed in EPEC and EHEC infection at 12 h but not HB101. Jasp preserved actin stress filaments in both EPEC and EHEC. Conclusions Disruption of microtubules and exogenous rearrangement of actin by pathogenic organism may be primary stimuli to up‐regulate proinflammatory cytokine gene expression. Preservation of actin filaments is required for this response and may be necessary for signal transduction to the nucleus.