Journal: Infection and Immunity
Article Title: Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿
Figure Lengend Snippet: IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P
Article Snippet: To inactivate heat-sensitive proteins, the collected conditioned supernatant was boiled for 20 min. To remove most of the protein factors, the conditioned supernatant was treated with protease K (100 μg/ml; Invitrogen) at 56°C overnight, followed by a 20-min incubation at 100°C to inactivate protease K. To remove bacterial DNA, the conditioned supernatant was treated with DNase I (10 U/ml; New England Biolabs, Ipswich, MA) at 37°C for 2 h. The treated supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and agarose gel electrophoresis, confirming the removal of proteins by protease K and of bacterial DNA by DNase I (data not shown).
Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay