In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells

Abstract

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4(+) T lymphocytes. We found that atrazine exposure significantly inhibited CD4(+) T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3(+) CD4(+) T cells.

Keywords: CD4+ T cell; Foxp3; atrazine; cAMP; regulatory T cells.

FIG. 1.

In vitro Atrazine (ATR) exposure significantly reduces CD4⁺ T cell proliferation and accumulation, but does not increase apoptosis. A, Male AD10 spleen cells were peptide-stimulated for 4 days in the presence of 0.1% EtOH (vehicle control) or 10 µM, 30 µM, or 50 µM ATR in 0.1% EtOH. ATR exposure results in a dose-dependent reduction of CD62L down-modulation on CD4⁺ cells. The marker in the graph indicates the CD62L⁺ population and the frequency of CD62L⁺ and CD62L⁻ cells for each dose is shown in the associated table. This data is representative of 3 separate experiments. B, The percent reduction in the absolute number of CD4⁺ T cells in the 30 µM ATR-treated cultures compared with the EtOH-only control cultures is shown for 6 separate experiments. The ATR-associated mean reduction in CD4⁺ T cells for these 6 experiments was statistically significant; **P = .0058. C, T cells were stained with Annexin V to monitor apoptosis induction and with 7-AAD to monitor necrosis. Apoptotic cells are in the Annexin V⁺ 7-AAD⁻ lower right quadrant, whereas necrotic cells are found in the upper left Annexin V⁻ 7-AAD⁺ (upper left) and Annexin V⁺ 7-AAD⁺ (upper right) quadrants. Data are representative of 3 separate experiments. D, Male TCR transgenic splenic T cells were stimulated for 4 days in the presence of 30 µM ATR (center) or EtOH vehicle control (left). Proliferation (CFSE dilution) vesus size (forward scatter) of CD4⁺Vβ3⁺ gated cells is shown. An overlay of CFSE (right panel) is also shown. These results are representative of 11 separate experiments. E, T cells from AD10 male (gray line) and female (black line) mice were CFSE-labeled before being peptide-stimulated in the presence of EtOH (gray histogram) or 30 μM ATR for 4 days. CFSE levels on CD4⁺Vβ3⁺ gated T cells are shown. The data are representative of 6 separate experiments.

FIG. 2.

In vitro Atrazine exposure inhibits the CD4⁺ T cell activation and effector cytokine production. A, Male AD10 TCR transgenic splenocytes were antigen stimulated for 4 days in the presence of EtOH (center) or 30 µM Atrazine (ATR; right). Cells were stained with anti-CD25 and anti-CD69 to assess activation status. Day 0 cells are shown for comparison (left). The data are representative of 8 separate experiments. B, On day 4 the cells from cultures stimulated in the presence of EtOH, 30 µM ATR or media-only cultures were recovered and stimulated with PMA + ionomycin for 24 h. The concentration of IFNγ in culture supernatants was quantified by ELISA. Values are shown as mean ± SEM. The difference between the EtOH and Atrazine cultures is significant (P = .011). Data are representative of 3 separate experiments.

FIG. 3.

Atrazine (ATR)-mediated inhibition of CD4⁺ T cell activation and proliferation is dose dependent. Male AD10 TCR transgenic spleen cells were antigen-stimulated for 4 days in the presence of 0.1% EtOH only (filled histogram) or 3 μM (thin black line), 15 μM (gray line), or 30 µM ATR (thick black line) in 0.1% EtOH. CFSE dilution (left) and expression of CD69 (center) and CD25 (right) on CD4⁺Vβ3⁺ cells are shown. The region marker in the left panel was used to determine the undivided frequency. The frequency of dividing cells (left panel) or the mean fluorescent intensity values (center and right panels) is found in the insets for each panel. Results are representative of 4 separate experiments.

FIG. 4.

Pharmacological reagents that elevate intracellular cAMP partially mimic Atrazine (ATR) phenotype in a dose-dependent manner. Male AD10 spleen cells were peptide-stimulated for 4 days in the presence of 0.1% EtOH vehicle control or increasing concentrations of ATR (30 and 15 µM; left column), PTX (PDE inhibitor at 500 and 250 nM; center column) and dbcAMP (cAMP analog at 100 and 50 µM; right column). A, Proliferation of CD4⁺Vβ3⁺ AD10 cells, as monitored by CFSE dilution, was measured for each treatment group. The region marker in the CFSE plots indicates the position of the non-dividing cells. B, Activation of CD4⁺Vβ3⁺ T cells was monitored by examining CD25 expression and C, CD69 expression. The region markers in the CD25 plots indicate the position of the CD25⁺ population. D, A summary table of numerical data from the plots in figures, A–C is shown. E, The expression levels of CD25 and CD69 on cells from the EtOH, 30 µM ATR, 500 nM PTX, and 100 µM dbcAMP-treated cultures are shown. The table indicates the frequency of CD25⁺ and CD69⁺ cells for each treatment. Results are representative of 5 separate experiments.

FIG. 5.

Exogenous IL-2 does not reverse Atrazine (ATR)-mediated T cell suppression. Male AD10 TCR transgenic T cells were stimulated in the presence of 30 µM ATR (gray line) or EtOH (shaded histogram) for 4 days. On day 2, 50 U/ml rmIL-2 was added to some cultures (black line). A, CFSE dilution and B, CD25 expression on the CD4⁺Vβ3⁺ gated cells for each treatment condition is shown. The region marker in the CD25 plot indicates the position of the CD25⁺ population. The table indicates the CFSE and CD25 mean fluorescent intensity values as well as the frequency of dividing and non-dividing cells. These data are representative of 4 separate experiments.

FIG. 6.

Atrazine (ATR) treatment significantly increases the frequency of Foxp3⁺ regulatory T cells. A, Male AD10 TCR transgenic T cells were antigen-stimulated in the presence of 30 µM ATR (right) or EtOH vehicle control (left) and on day 4 cells were fixed and permeabilized for intracellular Foxp3 antibody staining. The frequency of CD25⁺Foxp3⁺ cells in the CD4⁺Vβ3⁺ transgenic population is shown. Data are representative of 11 separate experiments. B, Male splenocytes from Balb/c Foxp3^gfp+ mice were stimulated for 4 days by immobilized 1 mg/ml anti-CD3 and 1 mg/ml anti-CD28 in the presence of EtOH (left) or 30 µM ATR (right). The frequency of GFP⁺ Foxp3⁺ T_regs in CD4⁺CD25⁺ gated cells is shown. At the initiation of the experiment 1.52% of the CD4⁺CD25⁺ cells were Foxp3^gfp+. Data are representative of 5 separate experiments. C, On day 4, splenocytes from EtOH- or ATR-exposed AD10 TCR transgenic male mice were recovered and the frequency of CD4⁺CD25⁺ cells was determined by flow cytometry. The CD4⁺CD25⁺ putative T_regs from the EtOH or 30 µM ATR cultures were incubated with peptide-stimulated, CFSE-labeled naïve AD10 responder cells at a ratio of 2.5 T_reg: 1 responder T cell. CFSE dilution by the responder cells in the presence of the EtOH (thick black line) or 30 µM ATR (thin gray line) CD4⁺CD25⁺ cells is shown. Proliferation of the responders in the absence of T_regs (gray histogram) is shown for comparison. Data are representative of 3 separate experiments.