Different cytokine response of primary colonic epithelial cells to commensal bacteria

Figure 1 Evaluation of the purity and response of cultured murine CEC. A: CEC from 4-6 wk old C57BL/6 mice were cultured for 72 h in medium alone (M) or in medium containing Bacteroides ovatus (Bo), E. coli (Ec) or Lactobacillus rhamnosus (Lr) after which CEC RNA was extracted, reverse transcribed and cDNA amplified by RT-PCR using primers specific for CD45 or vimentin. PCR products were separated by gel electrophoresis and EtBr-stained amplicons visualized and digitally recorded under UV illumination. The sensitivity of CD45 detection was determined by adding spleen cells to highly purified CECs so that they comprised 2% or 10% of the total cell population prior to RNA extraction and RT-PCR analysis. Control samples (Ctrl) were spleen cells (+Ctrl) and no cDNA (-Ctrl) for CD45 RT-PCR and fibroblasts for vimentin RT-PCR assay. The results are representative of more than 10 independent experiments; B: TLR2 and TLR4 expression by CEC. The dashed line on the histogram plots represents staining with control antibody, the bold line represents staining profile of anti-TLR4 and the filled in histogram plot represents anti-TLR2 antibody staining; C: Responsiveness of TLR expressed by CEC. Supernatants from 4 h cultures of CEC in medium alone (Med) or in medium containing LPS (10 μg/mL) or PGN (1 μg/mL) were assayed for the presence of IL-6 and MCP-1 by ELISA. The results shown were collated from three independent experiments. The error bars represent SEM. ^aP<0.05 LPS vs medium values, ^bP<0.01 PGN vs medium values, ^cP<0.002 LPS vs medium values.