Chemokine expression in the monocytic cell line THP-1 in response to purified shiga toxin 1 and/or lipopolysaccharides

Abstract

Infections with Shiga toxin (Stx)-producing bacteria are associated with bloody diarrhea and postdiarrheal sequelae, including hemolytic uremic syndrome and central nervous system (CNS) abnormalities. Stx-induced intestinal, renal, and CNS vascular lesions may involve a localized production of proinflammatory cytokines in target organs, as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) up-regulate Stx receptor globotriaosylceramide (Gb(3)) expression on vascular endothelial cells. However, leukocyte recruitment to injured sites may also exacerbate vascular damage. A cytokine macroarray analysis of transcripts derived from macrophage-like THP-1 cells treated with Stx1, lipopolysaccharides (LPS), or both demonstrated a consistent up-regulation of TNF-alpha, IL-1beta, and four genes encoding the chemokines interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and growth-related oncogene beta (GRO-beta). Real-time PCR analysis verified the macroarray results. Northern blot analyses after the addition of the transcriptional inhibitor actinomycin D revealed increased IL-8 mRNA stability in THP-1 cells treated with Stx1 or Stx1 plus LPS. Finally, enzyme-linked immunosorbent assay data for Stx1- plus LPS-treated cells demonstrated a poor correlation between IL-8, MIP-1alpha, MIP-1beta, and GRO-beta mRNA levels and protein production, indicating a posttranscriptional regulatory effect. Our data suggest that in response to Stx1 and LPS, macrophages may be a source of chemokines that promote tissue damage through leukocyte recruitment and activation.

FIG. 1.

Differential gene expression by THP-1 cells after treatment with Stx1, LPS, or Stx1 plus LPS, as measured by cDNA macroarrays. Differentiated THP-1 cells were left untreated, treated with 200 ng of LPS/ml, treated with 400 ng of Stx1/ml, or treated with Stx1 plus LPS for 6 h. Total RNAs were isolated and converted into ³²P-labeled cDNA probes by the use of reverse transcriptase. The probes were hybridized to membranes containing cDNAs derived from 268 cytokine and chemokine genes spotted in duplicate. Hybridization was detected by use of a phosphorimager. Paired spots within squares correspond to the GAPDH internal control (position 1d on the membranes). Pairs of spots identified by circles correspond to IL-1β (position 3c) and TNF-α (position 14i). Pairs of spots identified by rectangles correspond to IL-8 (position 3j), MIP-1α (position 6n), MIP-1β (position 6o), and GRO-β (position 7b).

FIG. 2.

Real-time PCR verification of chemokine expression in Stx1- and/or LPS-treated THP-1 cells. Differentiated THP-1 cells (5 × 10⁶ cells/ml) were incubated with medium alone (open bars), 200 ng of LPS/ml (hatched bars), or Stx1 plus LPS (closed bars) (a) or with medium alone (open bars) or 400 ng of Stx1/ml (closed bars) (b) for 2 or 6 h. Threshold cycle values were normalized for GAPDH expression, and the levels of induction of MIP-1α, MIP-1β, GRO-β, and IL-8 expression were calculated relative to the 0-h untreated control. Data are expressed as means ± standard errors of the means (error bars) for three or four independent experiments. An asterisk (*) denotes a significant difference (P ≤ 0.05) between treatments at each time point for each cytokine. A plus sign (+) denotes a significant difference (P ≤ 0.05) between the 2- and 6-h cytokine induction levels within each treatment. A number sign (#) denotes a significant difference (P ≤ 0.05) in cytokine induction between treatments within a time point.

FIG. 3.

Comparison of IL-8 mRNA expression in THP-1 cells treated with Stx1, LPS, and Stx1 plus LPS. Differentiated THP-1 cells (5 × 10⁶ cells/ml) were treated with 400 ng of Stx1/ml, 200 ng of LPS/ml, or both for 0 to 72 h. Total RNAs (5 to 15 μg) were subjected to Northern blot analysis with ³²P-labeled IL-8 and GAPDH cDNA probes. Hybridization was detected and quantitated by use of a phosphorimager and was expressed as a percentage of IL-8 mRNA above basal (unstimulated) expression. The data are expressed as means ± standard errors of the means (error bars) for at least three independent experiments with LPS- and Stx1-plus-LPS-treated THP-1 cells (a) and Stx1-treated THP-1 cells (b). An asterisk (*) denotes a significant difference (P ≤ 0.05) between the LPS and Stx1-plus-LPS treatments. A number sign (#) denotes the first point of significant induction for Stx1-induced IL-8 mRNA; data for all time points of ≥12 h were significantly different from that at the zero time point.

FIG. 4.

Effects of Stx1 and/or LPS on IL-8 mRNA stability in THP-1 cells. Differentiated THP-1 cells (5 × 10⁶ cells/ml) were treated with LPS (200 ng/ml) for 1 h (a) or with Stx1 (400 ng/ml) for 6 h or Stx1 plus LPS for 1 h (b). Actinomycin D (ActD) was then added to each plate at a final concentration of 5.0 μg/ml. Total RNAs were isolated at each of the indicated time points after ActD addition. Northern blot analysis was performed, and IL-8 mRNA was detected and quantitated by use of a phosphorimager. The data are expressed as means of the percentages of IL-8 mRNA remaining (log) ± standard errors of the means (error bars) for at least three independent experiments. Treatments with the same symbol were significantly different (P ≤ 0.05) from each other.

FIG. 5.

IL-8 protein production by THP-1 cells treated with Stx1, LPS, and Stx1 plus LPS. Cell-free supernatants from cells treated with LPS (a), Stx1 (b), or Stx1 plus LPS (c) were collected and analyzed by a human IL-8-specific ELISA. The data shown are means ± standard errors of the means (error bars) for at least three independent experiments. Asterisks (*) denote the first significant expression difference (P ≤ 0.05) between untreated and treated cells; data for all time points beyond this point were significantly different from the zero time point.

FIG. 6.

MIP-1α, MIP-1β, and GRO-β protein production by THP-1 cells treated with Stx1, LPS, or Stx1 plus LPS. Cell-free supernatants from LPS-, Stx1-, and Stx1-plus-LPS-treated cells were collected and analyzed by ELISAs specific for human MIP-1α (a), MIP-1β (b), and GRO-β (c). The data shown are means ± standard errors of the means (error bars) for at least three independent experiments. Significant differences between treatments are indicated by asterisks (*).