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. 2001 Oct;69(10):6140-7.
doi: 10.1128/IAI.69.10.6140-6147.2001.

Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression

C M Thorpe  1 , W E Smith, B P Hurley, D W Acheson
Affiliations

Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression

VSports手机版 - C M Thorpe et al. Infect Immun. 2001 Oct.

Abstract

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins VSports手机版. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection. .

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Figures

FIG. 1
FIG. 1
Effect of Stx1 on the accumulation of various chemokine mRNAs. HCT-8 cells were treated for 4 h with either Stx1 (1 μg/ml) or heat-inactivated Stx1 (controls; 1 μg/ml). Total cellular RNA was isolated, and then radiolabeled cDNA was prepared from total cellular RNA using reverse transcriptase and cytokine-specific primers. Paired expression arrays were probed with radiolabeled cDNA from either Stx1-treated cells or control cells. Paired arrays were washed and exposed to film. Autoradiographs were scanned using an Agfa II scanner. (A) Pertinent sections from autoradiographs. Intensity of signal corrected for background was determined for each labeled chemokine gene on both Stx1-treated and control arrays using QuantityOne software (Bio-Rad). Results (B and C) are expressed as ratios between expression of the chemokine (B) or housekeeping mRNA (C) of interest in Stx1-treated and control cells. Abbreviations: β-2 micro, beta 2-microglobulin; HLA-A, major histocompatibility complex class I lymphocyte antigen (HLA-A 0201); HPRT, hypoxanthine phosphoribosyltransferase; L19, ribosomal protein L19; Tfr, transferrin receptor. Asterisk, ratio that is ≥100.
FIG. 2
FIG. 2
Stx1 induces and superinduces GRO-α and is a slow inducer of GRO-α mRNA compared to TNF-α. Shown are Northern blots from HCT-8 cells following various treatments. (A) HCT-8 cells were treated with Stx1, Stx2, heat-inactivated Stx1, TNF-α, or both Stx1 and TNF-α for 5 h. Stxs were used at 1 μg/ml; TNF-α was used at 5 ng/ml. Total RNA was isolated, and a Northern blot was made from these samples and probed for GRO-α mRNA as well as GAPDH mRNA as a housekeeping gene. (B) HCT-8 cells were treated with TNF-α at 5 ng/ml. Total RNA was harvested at various times following addition of TNF-α as indicated. A Northern blot was made from these samples and probed as for panel A. (C) HCT-8 cells were treated with Stx1 at 1 μg/ml. Total RNA was harvested at various times following addition of Stx1 as indicated. A Northern blot was made from these samples and probed as for panel A.
FIG. 3
FIG. 3
Stx1 can superinduce ENA-78 mRNA at doses lower than those required for induction. Shown is a Northern blot of HCT-8 cells following various treatments probed for ENA-78 mRNA. HCT-8 cells were exposed for 5 h to doses of Stx1 ranging from 10 ng/ml to 100 μg/ml, heat-inactivated Stx1 at 10 or 100 μg/ml, TNF-α, or TNF-α and Stx1 at 10 or 100 ng/ml. Total RNA was harvested, and a Northern blot was made from these samples and probed for ENA-78 mRNA and GAPDH mRNA as a housekeeping gene. HI, heat inactivated; Ø, no additives.
FIG. 4
FIG. 4
Stx1 induces synthesis of IL-8 and GRO-α protein in HCT-8 cells. (A) IL-8 protein was measured by ELISA in both supernatants (black bars) and lysates (white bars) of Stx1-treated HCT-8 cells. (B) GRO-α protein was measured by ELISA in both supernatants (black bars) and lysates (white bars) of Stx1-treated HCT-8 cells. Separate measurements were done in 6 to 12 wells for both IL-8 and GRO-α supernatants and lysates. The data are mean amounts of protein per 2 × 105 cells plated and standard deviations from 6 to 12 wells per condition from a single representative experiment. Overall protein synthesis as measured at the end of the Stx1 incubation is shown as a percentage of control levels, and the average of triplicate wells is inset at the base of each black bar for each dose of Stx1. Asterisk, P value <0.05 compared with control.
FIG. 5
FIG. 5
Stx1 stabilizes IL-8 mRNA, whereas IL-1β does not, and the two stabilize GRO-α mRNA similarly. After 5 h of exposure to Stx1, TNF-α, or IL-1β, total RNA was harvested. The time of this initial harvest was time zero. Actinomycin D was then added directly to culture media for a final concentration of 10 μg/ml. Total RNA was harvested at various times (hours) after addition of actinomycin D as shown above each lane. Northern blots were prepared from these samples. (A) Northern blot probed for IL-8 mRNA and GAPDH mRNA as a housekeeping gene; (B) Northern blot probed for GRO-α mRNA and GAPDH mRNA as a housekeeping gene.
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