Inhibition of allogeneic inflammatory responses by the Ribonucleotide Reductase Inhibitors, Didox and Trimidox

Abstract

Background: Graft-versus-host disease is the single most important obstacle facing successful allogeneic stem cell transplantation (SCT). Even with current immunosuppressive therapies, morbidity and mortality rates are high. Current therapies including cyclosporine A (CyA) and related compounds target IL-2 signaling. However, although these compounds offer great benefit, they are also associated with multiple toxicities. Therefore, new compounds with a greater efficacy and reduced toxicity are needed to enable us to overcome this hurdle.

Methods: The allogeneic mixed lymphocyte reaction (MLR) is a unique ex vivo method to study a drug's action on the initial events resulting in T-cell activation and proliferation, synonymous to the initial stages of tissue and organ destruction by T-cell responses in organ rejection and Graft-versus-host disease. Using this approach, we examined the effectiveness of two ribonucleotide reductase inhibitors (RRI), Didox and Trimidox, to inhibit T-cell activation and proliferation.

Results: The compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-gamma, TNF-alpha, IL-2, IL-13, IL-10 and IL-4.

Conclusions: In conclusion, these data provide critical information to justify further investigation into the potential use of these compounds post allogeneic bone marrow transplantation to alleviate graft-versus-host disease thereby achieving better outcomes.

Figure 1

Inhibition of proliferation of T-cell obtained from B10.D2 mice spleens. Briefly, 5 × 10⁴ T-cells per well were purified and seeded in 96 well sterile plates pre-coated with PBS or anti CD3ε antibodies (5 μg/ml). Either PBS or several concentrations of Didox or Trimidox (25 μM and 50 μM) was added to RPMI 1640 growth media supplemented with 10% fetal bovine serum, 50 μM 2-Mercaptoethanol and 1% penicillin streptomycin. The plates were then incubated at 37°C and 5% CO₂ for 24, 48, 72 or 96 hours, after which spectrophotometric quantification of cell growth and viability was determined. The results shown represent data obtained in triplicate from two independent experiments. (A) Treated with Didox (B) Treated with Trimidox. Values shown (mean ± SD) represent data obtained in triplicate from two independent experiments. * indicate a significant difference compared to anti CD3ε stimulated. (p < 0.05, ANOVA + the Bonferroni test).

Figure 2

Cellular toxicity of Didox and Trimidox in T-cells from C57BL6 and B10.D2 mouse strains. Briefly, 1 × 10⁵ cells per well were seeded in 96 well culture plates in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and 2-mercaptoethanol, containing PBS or concentrations of Didox or Trimidox from 25 μM- 100 μM. The cells were then incubated at 37°C and 5% CO₂ for 4 days. After this, percentage of viable cells of (A) Didox and (B) Trimidox was determined for each drug dose. * indicates a significant difference compared to PBS treated (UT). (p < 0.05, ANOVA + the Bonferroni test; n = 3).

Figure 3

Inhibition of TH1 cytokine secretion by Didox and Trimidox. Briefly, 2 × 10⁵ T-cells per well were purified and seeded in 96 well sterile plates pre-coated with PBS or anti CD3ε antibodies (5 μg/ml). Either PBS or several concentrations of Didox or Trimidox (25 μM- 100 μM) was added to RPMI 1640 growth media supplemented with 10% fetal bovine serum, 2-mercaptoethanol (50 μM) and 1% penicillin streptomycin. The plates were then incubated at 37°C and 5% CO₂ for 24 or 48 hours, after which the levels in pg/ml of IFN-γ, IL-2 and IL-6 were determined using the Searchlight multiplex assay system. (A) B10.D2 mice; (B) C57BL6 mice. NC: Normal control, AC: Activated control. The values shown (mean ± SD) represent data obtained in triplicate from two independent experiments. * p < 0.05, ** p < 0.001 indicates a significant difference compared to activated control (AC), ANOVA + the Bonferroni test; n = 3.

Figure 4

Inhibition of TH2 cytokine secretion by Didox and Trimidox. The levels in pg/ml of IL-4, IL-13 and IL-10 (*p < 0.05, ** p < 0.001) were determined using the Searchlight procedure. (A) B10.D2 mice; (B) C57BL6 mice. NC: Normal control, AC: Activated control. The values shown (mean ± SD) represent data obtained in triplicate from two independent experiments. * p < 0.05, ** p < 0.001 indicates a significant difference compared to activated control (AC), ANOVA + the Bonferroni test; n = 3.

Figure 5

Inhibition of T-cell proliferation responses in MLRs by Didox and Trimidox. (A) Major antigen mismatch: Briefly, 2 × 10⁵ T-cells were purified from spleens C57BL6 mice (responder cells) were mixed with 4 × 10⁵ irradiated non T-cells (stimulator cells) obtained from BALB/c mice (exposed to 3000 rads of γ-radiation). (B) Minor antigen mismatch: Briefly, 2 × 10⁵ T-cells were purified from spleens B10.D2 mice (responder cells) which were mixed with 4 × 10⁵ non T-cells (stimulator cells) obtained from BALB/c and pre-exposed to 3000 rads of γ-radiation. Either PBS (untreated) or several concentrations of Didox or Trimidox (25 μM- 100 μM) was added to RPMI 1640 growth media supplemented with 10% fetal bovine serum, 50 μM 2-Mercaptoethanol and 1% penicillin streptomycin. The plates were then incubated at 37°C and 5% CO₂ for 6 days, after which spectrophotometric quantification of cell growth and proliferation was determined. The results shown represent data obtained in triplicate from two independent experiments. Values shown represent the mean ± SD obtained in triplicate from two independent experiments. * indicates a significant difference compared to PBS treated (Untreated). (p < 0.05, ANOVA + the Bonferroni test).

Figure 6

Inhibition of IFN-γ, IL-2, IL-6 and TNF-α cytokine release from MLRs by Didox and Trimidox. Major antigen mismatch (filled square): Briefly, 2 × 10⁵ T-cells were purified from spleens of C57BL6 mice (responder cells) which were mixed with 4 × 10⁵ non T-cells (stimulator cells) obtained from BALB/c mice and exposed to 3000 rads of γ-radiation. Minor antigen mismatch (open square): Briefly, 2 × 10⁵ T-cells were purified from the spleens of B10.D2 mice (responder cells) which were mixed with 4 × 10⁵ non T-cells (stimulator cells) obtained from BALB/c mice and previously exposed to 3000 rads of γ-radiation. Either PBS or several concentrations of Didox or Trimidox (25 μM- 100 μM) was added to RPMI 1640 growth media supplemented with 10% fetal bovine serum, 2-mercaptoethanol (50 μM)and 1% penicillin streptomycin. The plates were then incubated at 37C and 5% CO₂ for 6 days, after which the levels of IFN-γ, IL-2 and IL-6 were determined using the Searchlight multiplex assay system. NC: Normal control, AC: Activated control. The results shown represent data obtained in triplicate from two independent experiments (n = 3). * p < 0.05, ** p < 0.001 indicates a significant difference compared to activated control (AC), ANOVA + the Bonferroni test.

Figure 7

Inhibition of IL-13, IL-10 and IL-4 cytokine release from MLRs by Didox and Trimidox. The levels of IL-13, IL-10 and IL-4 were determined using the Searchlight multiplex assay system from minor (open square) or major (filled square) mixed lymphocyte reactions. NC: Normal control, AC: Activated control. The results shown represent data obtained in triplicate from two independent experiments (n = 3-6). (*p < 0.05, ** p < 0.001 indicates a significant difference compared to activated control (AC), ANOVA + the Bonferroni test.