A role of cellular glutathione in the differential effects of iron oxide nanoparticles on antigen-specific T cell cytokine expression

Figures & data

Figure 1 No effect of iron oxide nanoparticles on viability of splenocytes. Splenocytes (5 × 10⁶ cells/mL) were treated with iron oxide nanoparticles (1–100 μg iron [Fe]/mL) and/or vehicle (VH; Roswell Park Memorial Institute medium) and then either left unstimulated or stimulated with ovalbumin (OVA; 100 μg/mL) for 44 hours. The viability of splenocytes was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.

Notes: Data are expressed as the mean ± standard error of quadruplicate cultures. Results are representative of three independent experiments.

Abbreviation: OD, optical density.

Figure 2 Differential effects of iron oxide nanoparticles on the production of antigen-specific interleukin (IL)-2, IL-4, and interferon (IFN)-γ by splenocytes. (A–C) Splenocytes (5 × 10⁶ cells/mL) were either left untreated (naïve; NA) or treated with iron oxide nanoparticles (1–100 μg iron [Fe]/mL) and/or vehicle (VH) followed by stimulation with ovalbumin (100 μg/mL) for 48 hours. (D) Splenocytes were pretreated with N-acetyl-L-cysteine (NAC; 1 mM) prior to the treatment of iron oxide nanoparticles and ovalbumin stimulation described above. The levels of (A) IL-2, (B) IL-4, and (C and D) IFN-γ in the supernatants were measured by enzyme-linked immunosorbent assay.

Notes: Data are expressed as the mean ± standard error of triplicate cultures. The level of cytokines in the VH group was designated as 100%, and the percentage of inhibition induced by iron oxide nanoparticles (10–100 μg Fe/mL) was calculated against this standard (as indicated in parentheses). *P < 0.05, comparison with VH group. Results are representative of three independent experiments.

Abbreviation: ND, no data.

Table 1 Effects of thiol and nonthiol antioxidants on iron oxide nanoparticle-mediated suppression of interferon-γ production by splenocytes

Antioxidants	Iron oxide nanoparticle-mediated inhibition of interferon-γ production (%)a
Control	85.8 ± 2.6
N-acetyl-L-cysteine (mM)
1	39.3 ± 8.9*
2	35.6 ± 6.4*
4	27.9 ± 4.8*
Glutathione (mM)
1	41.5 ± 6.2*
2	36.1 ± 8.5*
4	29.7 ± 4.1*
Pyruvate (mM)
1	82.3 ± 6.9
4	84.3 ± 3.1
Dimethylthiourea (mM)
4	89.7 ± 1.0
Tiron (mM)
0.1	86.6 ± 1.2

a Notes: Splenocytes (5 × 10⁶ cells/mL) were pretreated with iron oxide nanoparticles (50 μg iron/mL) in the absence (control) or presence of N-acetyl-L-cysteine, glutathione, pyruvate, dimethylthiourea or tiron, followed by stimulation with ovalbumin (100 μg/mL) for 48 hours. The levels of interferon-γ in the supernatants were measured by enzyme-linked immunosorbent assay. The percentage of inhibition induced by iron oxide nanoparticles was calculated as described in Materials and methods. Data are expressed as the mean ± standard error of six samples pooled from two experiments;

* P < 0.001 compared to the control.

Figure 3 Diminishment of intracellular glutathione in the presence of iron oxide nanoparticles in splenocytes. (A) Splenocytes (5 × 10⁶ cells/mL) were treated with iron oxide nanoparticles (50 μg iron [Fe]/mL) and/or vehicle (VH) followed by stimulation with ovalbumin (100 μg/mL) for 1–12 hours. (B) Splenocytes were treated with iron oxide nanoparticles (1–100 μg Fe/mL) and/or VH, followed by stimulation with ovalbumin (100 μg/mL) for 6 hours in the absence or presence of N-acetyl-L-cysteine (NAC; 1 mM). The levels of intracellular glutathione were measured as monochlorobimane (MCB) fluorescence by flow cytometry.

Figure 4 No effect of iron oxide nanoparticles on the intracellular levels of reactive oxygen species. Splenocytes (5 × 10⁶ cells/mL) preloaded with dichlorofluorescin (DCF) diacetate (20 μM) were treated with iron oxide nanoparticles (1–100 μg iron [Fe]/mL) and/or vehicle (VH), followed by stimulation with ovalbumin (100 μg/mL) for 6 hours. The levels of intracellular reactive oxygen species were measured as DCF fluorescence using a microplate reader.

Notes: Data are expressed as the mean ± standard error of triplicate cultures. Results are representative of three independent experiments.

Notes: Data are expressed as the mean ± standard error of triplicate cultures. *P < 0.05, comparison with matched control group without nanoparticle treatment. ^#P < 0.05, comparison with matched nonNAC group. Results are representative of three independent experiments.

Figure 4 No effect of iron oxide nanoparticles on the intracellular levels of reactive oxygen species. Splenocytes (5 × 10⁶ cells/mL) preloaded with dichlorofluorescin (DCF) diacetate (20 μM) were treated with iron oxide nanoparticles (1–100 μg iron [Fe]/mL) and/or vehicle (VH), followed by stimulation with ovalbumin (100 μg/mL) for 6 hours. The levels of intracellular reactive oxygen species were measured as DCF fluorescence using a microplate reader.

Notes: Data are expressed as the mean ± standard error of triplicate cultures. Results are representative of three independent experiments.