Journal:

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 ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter inoculation increased the secretion of IL-8 and TNF-α in polarized human intestinal epithelial T84 cells. T84 cells were cultured on transwells until the transepithelial resistance reached 1,400 Ω/cm2. C. jejuni 81-176 was inoculated from the apical chamber at an MOI of ∼10 and incubated at 37°C for 0, 4, 6, or 24 h. For the 24-h time point, the cells were washed at 6 h to remove unattached bacteria and cultured in fresh medium for another 18 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation at any time point. The supernatants from the apical and basolateral chambers were collected separately; the bacteria were removed by centrifugation; and protease inhibitors were added to prevent protein degradation. The concentrations of IL-8 (A) and TNF-α (B) were determined by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. Asterisks represent significant (P < 0.05) differences from control cells that were not inoculated with the bacteria.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Cell Culture, Incubation, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal:

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 ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-stimulated IL-8 secretion is not dependent on the secretion of TNF-α. (A) Polarized T84 cells were incubated with different concentrations of TNF-α (0, 5, 10, 50, and 100 ng/ml) basolaterally at 37°C for 24 h. The basolateral media were collected, and the concentrations of IL-8 were measured using ELISA. (B) IL-8 concentrations in the basolateral medium were measured using ELISA at 24 h after T84 cells were incubated under the following conditions: TNF-α (50 ng/ml) applied apically (Apical) (bar I), TNF-α applied basolaterally (Basol) (bar II), (III) TNF-α applied basolaterally plus mouse anti-human TNF-α antibody (5 μg/ml) applied both apically and basolaterally (Both) (bar III), or live C. jejuni 81-176 applied apically plus mouse anti-human TNF-α antibody applied both apically and basolaterally (bar IV). Averages from three independent experiments are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal:

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 ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Comparison of IL-8 secretion induced by different Campylobacter strains inoculated apically or basolaterally. (A and B) Polarized T84 cells were inoculated from either the apical (A) or the basolateral (B) side with chicken meat isolates (C. coli [C] or C. jejuni [J] strains) or with C. jejuni 81-176 or its flaA mutant at an MOI of ∼10 and were incubated for 24 h. At 6 h, T84 cells were washed and placed in fresh medium. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. Uninfected T84 cells were used as controls. The apical and basolateral media were collected separately at 24 h, and the concentrations of IL-8 were measured by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. (C) Pearson's correlation coefficients between IL-8 secretion and Campylobacter adherence, invasion, and transcytosis efficiencies (expressed as percentages of the inocula) were calculated and tested for significance (α = 0.05 by a two-tailed test).

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

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 ▿

doi: 10.1128/IAI.01317-07

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 < 0.05. (F) The cytotoxicity of CDT was measured by incubating Vero cells with serially diluted C. jejuni culture supernatants that were filtrated and treated with polymyxin B for 48 h. The viability of the Vero cells was monitored using an MTT assay. The CDT titers were expressed as the reciprocal of the highest dilution that caused 50% Vero cell death compared with the level in untreated cells. Data are averages for three independent cytotoxicity assays; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay, MTT Assay

Journal:

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 ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion depends on Campylobacter-secreted CDT and the TLR signaling adaptor MyD88 in polarized intestinal epithelial cells. (A) Polarized T84 cells were pretreated with MyD88 inhibitory peptide (100 μM) (bars III, V, and VII) or its control peptide (bars II, IV, and VI) for 24 h or with chloroquine (5 μM) (bars VIII, IX, and XIII) for 30 min before being incubated with the conditioned supernatant (ConSup) from the coculture of T84 cells with wt C. jejuni 81-176 (bars II, III, and VIII), its cdtB mutant (bars IV, V, and IX), or a C. jejuni 81-176 DNA extract (25 μg/ml) (bars VI and VII) at 37°C for 24 h. MyD88 inhibitory peptide, its control peptide, or chloroquine was also included during the 24-h incubation. Polarized T84 cells incubated with TNF-α only (bar XII) or TNF-α plus chloroquine (bar XIII) were used as controls for the effect of chloroquine on IL-8 secretion. Polarized T84 cells incubated with the medium alone for 24 h (bars I) or with live C. jejuni 81-176 for 4 h (bars X), after which the conditioned supernatants were collected, or 24 h (bars XI) were used as controls. In the 24-h incubation with live bacteria, unbound bacteria were removed at 6 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. (B) Polarized T84 cells were pretreated with chloroquine (5 μM) for 30 min before incubation with C. jejuni 81-176 (MOI, ∼10) at 37°C for 4 h. The abilities of C. jejuni 81-176 to adhere to, invade, and transcytose across chloroquine-treated T84 cells were analyzed as described in the legend to Fig. ​Fig.3.3. Averages for three independent experiments with triplicate samples are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal:

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 ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion is dependent on NF-κB activation. (A) T84 cells were incubated with C. jejuni 81-176 (MOI, ∼10) in the presence of cycloheximide for varying lengths of time. T84 cells treated with TNF-α were used as a positive control. −, no treatment. The cells were washed and lysed, and the cell lysates were analyzed by SDS-PAGE and Western blotting with probing for IκB-α. The blots were stripped and reprobed for tubulin as a loading control. Representative results from three independent experiments are shown. (B) Polarized T84 cells were pretreated with the NF-κB inhibitor SN50 (18 μM), quinazoline (28 μM), or TPCK (50 μM) for 30 min and then incubated with C. jejuni 81-176 in the presence of the inhibitor at 37°C for 24 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Activation Assay, Incubation, Positive Control, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response to Salmonella typhimurium Can Be Uncoupled from Bacterial Internalization

doi:

Figure Lengend Snippet: S. typhimurium-induced IL-8 secretion but not PEEC secretion is inhibited by mg-132. Monolayers of T84 epithelial cells were treated with the proteosome inhibitor mg-132, which inhibits NF-κb nuclear translocation, or vehicle (dimethyl sulfoxide) for 1 h and then apically colonized with S. typhimurium. IL-8 secretion (left) and PMN transmigration (right) were measured. Data are the means (±SD) of a representative experiment performed in triplicate. An asterisk indicates a significant difference (P < 0.02) between the control and the mg-132-treated model epithelia.

Article Snippet: IL-8 was measured by enzyme-linked immunosorbent assay (ELISA) as previously described ( 30 ) except for the following minor modifications: 96-well plates (Linbro/Titretek; ICN Biochemicals, Aurora, Ohio) were coated overnight with goat anti-human IL-8 (R&D Systems, Minneapolis, Minn.).

Techniques: Translocation Assay, Transmigration Assay

Journal:

Article Title: Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response to Salmonella typhimurium Can Be Uncoupled from Bacterial Internalization

doi:

Figure Lengend Snippet: IL-8 secretion but not PEEC secretion is activated by PMA. Monolayers of T84 epithelial cells were stimulated with PMA (100 ng/ml), and supernatants were collected 4 h later. Basolateral supernatants were assayed for IL-8 by ELISA, and apical supernatants were partially purified and assayed for PEEC by measuring their ability to induce an increase in PMN cytosolic Ca++ as described in Materials and Methods. Data are the means (±SD) of a representative experiment performed in triplicate.

Article Snippet: IL-8 was measured by enzyme-linked immunosorbent assay (ELISA) as previously described ( 30 ) except for the following minor modifications: 96-well plates (Linbro/Titretek; ICN Biochemicals, Aurora, Ohio) were coated overnight with goat anti-human IL-8 (R&D Systems, Minneapolis, Minn.).

Techniques: Enzyme-linked Immunosorbent Assay, Purification

Journal:

Article Title: Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response to Salmonella typhimurium Can Be Uncoupled from Bacterial Internalization

doi:

Figure Lengend Snippet: IL-8 secretion can be induced by an invasion-defective mutant which was unable to elicit PEEC secretion and PMN transmigration. Monolayers of T84 epithelial cells were colonized apically (except where otherwise indicated) with wild-type or mutant S. typhimurium (as indicated) as described in Materials and Methods. Basolateral supernatants were assayed 4 h later for IL-8 by ELISA. Data are the means (±SD) of a representative experiment performed in triplicate. An asterisk indicates a significant difference (P < 0.02) from the wild type strain applied apically.

Article Snippet: IL-8 was measured by enzyme-linked immunosorbent assay (ELISA) as previously described ( 30 ) except for the following minor modifications: 96-well plates (Linbro/Titretek; ICN Biochemicals, Aurora, Ohio) were coated overnight with goat anti-human IL-8 (R&D Systems, Minneapolis, Minn.).

Techniques: Mutagenesis, Transmigration Assay, Enzyme-linked Immunosorbent Assay