Article Figures & Data
Figures
- FIG. 1.
Time course of TNF-α production in TSST-1-stimulated human PBMCs in the presence or the absence of anisodamine and related compounds (A) and the effects of various concentrations of the drugs (B). (A) PBMCs (5 × 105 cells) were stimulated with TSST-1 (10 ng/ml) for the indicated periods of time. Addition of the drugs (at 20 μg/ml) was as follows: open circles, none; closed circles, anisodamine; closed triangles, atropine (a tropane alkaloid related to anisodamine); closed squares, scopolamine (a tropane alkaloid related to anisodamine); open triangles, aspirin; open squares, TNF-α levels in control samples without TSST-1 stimulation. (B) PBMCs (5 × 105 cells) were stimulated with TSST-1 (10 ng/ml) for 48 h in the presence or the absence of the drugs. Open circles and open squares, TNF-α levels in control samples with and without TSST-1 stimulation, respectively. Addition of the drugs (at the indicated concentrations) was as follows: closed circles, anisodamine; closed triangles, atropine; closed squares, scopolamine; open triangles, aspirin. The levels of immunoreactive TNF-α in the cell-free culture supernatants (A and B) were measured by ELISA.
- FIG. 2.
Time course of proinflammatory and anti-inflammatory cytokine production in TSST-1-stimulated human PBMCs and effect of anisodamine addition. PBMCs (5 × 105 cells) were stimulated with TSST-1 (10 ng/ml) in the presence or the absence of anisodamine. Cell-free culture supernatants were periodically taken, and the levels of immunoreactive cytokines in the supernatants were measured by ELISA. Concentrations of anisodamine added: open circles, none; closed circles, 12.5 μg/ml; open triangles, 25 μg/ml; closed triangles, 50 μg/ml. Open squares, cytokine levels in control samples without TSST-1 treatment. (A) TNF-α; (B) IL-2; (C) IFN-γ; (D) IL-10.
- FIG. 3.
Direct action of anisodamine on T cells or monocytes prepared from human PBMCs. (A) Human peripheral blood T cells (5 × 105 cells) were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or the absence of anisodamine for 48 h. The culture supernatants were assayed for cytokine levels by ELISA. The concentrations of anisodamine added were as follows: open bars, none; shaded bars, 25 μg/ml; solid bars, 50 μg/ml. (B) Reconstruction experiments were performed with isolated human peripheral blood T cells and monocytes. The compositions of the cells were as follows: open bars, T cells plus monocytes; shaded bars, T cells plus monocytes pretreated with anisodamine; solid bars, T cells pretreated with anisodamine plus monocytes. The mixed cells were stimulated with TSST-1 (10 ng/ml) for 48 h, and the culture supernatants were assayed for cytokines by ELISA.
- FIG. 4.
Effects of anisodamine on TSST-1-induced production of IFN-γ by human PBMCs. Human PBMCs were stimulated with TSST-1 (10 ng/ml). Anisodamine was then added at the indicated times to the stimulated cell cultures at a concentration of 50 μg/ml (open circles). Cytokine levels in the cell-free culture supernatants were measured 48 h after TSST-1 stimulation. The data on the right (open triangle) represent cytokine levels in the absence of anisodamine. The results were obtained from triplicate experiments. *, P < 0.05 compared with the results for the TSST-1-stimulated cases in the absence of anisodamine. Similar inhibitory effects were also obtained with TNF-α and IL-2.
- FIG. 5.
Suppression of TSST-1-induced cytokine mRNA expression by anisodamine. (A) Effect of anisodamine on TNF-α mRNA expression was examined by RT-PCR. Human PBMCs (2.5 × 106 cells) were stimulated with TSST-1 (10 ng/ml) for 3 h in the presence or the absence of anisodamine (50 μg/ml). After incubation, the PBMCs were procured and total cellular RNA was extracted. mRNA was reverse transcribed and amplified by PCR with cytokine-specific primers. RT-PCR products: lanes 1 to 3, the housekeeping gene GAPDH; lanes 4 to 6, the TNF-α gene; lanes 1 and 4, no TSST-1 stimulation; lanes 2 and 5, TSST-1 stimulation; lanes 3 and 6, TSST-1 stimulation in the presence of anisodamine; lanes M, molecular size marker (HaeIII-digested φX174 replicative-form DNA fragments). (B, parts a and b) Experiments similar to those for TNF-α gene expression whose results are shown in panel A were performed by a real-time PCR assay. The relative copy numbers of the gene sequences for GAPDH (a) and TNF-α (b) were estimated as described in Materials and Methods. Samples 1 to 6 correspond to those in lanes 1 to 6 in panel A. (B, part c) IL-10 gene expression examined by a real-time PCR assay, as described above. Sample 7, no TSST-1 stimulation; sample 8, TSST-1 stimulation; sample 9, TSST-1 stimulation in the presence of anisodamine. (C) On the basis of the relative copy numbers obtained in the experiment whose results are shown in panel B, the percent inhibition of TNF-α and IL-10 mRNA expression by anisodamine was calculated and is shown. Experiments similar to the experiments whose results are shown in panel B were performed with IL-2 and IFN-γ, and the percent inhibition by anisodamine was evaluated and is also shown. *, P < 0.05 compared with the data for the proinflammatory cytokines (TNF-α, IL-2, and IFN-γ).
- FIG. 6.
Inhibition of TSST-1-induced NF-κB activation in THP-1 cells by anisodamine. THP-1 cells were stimulated with TSST-1 (10 μg/ml) for 1 h in the presence or the absence of anisodamine (50 μg/ml). Nuclear extracts were prepared, and the amount of activated NF-κB (a complex with p65) was measured with an ELISA kit with anti-65 antibodies. *, P < 0.05 compared with the NF-κB level without TSST-1 stimulation; **, P < 0.05 compared with the NF-κB level with TSST-1 stimulation in the absence of anisodamine.
- FIG. 7.
Inhibition of the TSST-1-stimulated proliferative responses of human PBMCs by anisodamine. (A) PBMCs (2 × 106 cells/ml) were stimulated with TSST-1 (10 ng/ml) for 72 h in the presence or the absence of anisodamine (50 or 100 μg/ml). To allow reexpression of the T-cell receptor, the cells were further treated with rIL-2. The cells were stained with FITC-Vβ2 and PE-CD3 antibodies, and then the labeled cells were examined by flow cytometric analysis. (Part a) CD3+ cells (T cells) in PBMCs without TSST-1 treatment; (part b) CD3+ cells (T cells) in PBMCs after stimulation with TSST-1; (part c) CD3+ cells (T cells) in PBMCs after stimulation with TSST-1 in the presence of 50 μg of anisodamine per ml; (part d) CD3+ cells (T cells) in PBMCs after stimulation with TSST-1 in the presence of 100 μg of anisodamine per ml. The numbers within each panel represent the percentage of Vβ2+ T cells. (B) PBMCs were stimulated with TSST-1 (10 ng/ml) for 72 h in the presence or the absence of various concentrations of anisodamine. The cultures were then pulsed with [3H]thymidine for 16 h, and the amount of [3H]thymidine (in counts per minute) incorporated into PBMCs was measured. *, P < 0.05 compared with the levels of [3H]thymidine incorporated in the TSST-1-stimulated cells in the absence of anisodamine.
- FIG. 8.
Effects of anisodamine on proinflammatory and anti-inflammatory cytokine levels in serum after TSST-1 injection in mice. Groups of 12 mice were injected i.p. with 20 mg of d-galactosamine 1 h before an i.p. challenge with 10 μg of TSST-1. Treatment (i.p. injection) with anisodamine at 50 mg/kg of body weight was carried out 10 min after the TSST-1 injection. Sera were obtained periodically by cardiac tapping, and the cytokine levels in sera were determined by ELISA. (A) TNF-α; (B) IL-2; (C) IFN-γ; (D) IL-10. Anisodamine treatment of mice injected with TSST-1 was as follows: open circles, none; closed circles, 50 mg/kg. Open squares, no TSST-1 injection (control). *, P < 0.05.
- FIG. 9.
Protection of mice from the lethality of TSST-1 by anisodamine. Injection of TSST-1 and treatment of d-galactosamine-sensitized mice with anisodamine (50 mg/kg) were carried out as described in the legend to Fig. 8. d-Galactosamine-sensitized mice injected only with saline (with no TSST-1) were used as controls (open squares). Treatment with anisodamine of mice injected with TSST-1 was as follows: open circles, none; open triangles, 10 mg/kg of body weight; closed triangles, 50 mg/kg of body weight. *, P < 0.05 compared with the TSST-1-injected, untreated mice (open circles).
