Lack of in vitro effect of aglepristone on IFN-γ and IL-4 production by resting and mitogen-activated T cells of luteal bitches

Abstract

Background: Aglepristone (RU534) is an antiprogestin used for pregnancy termination, parturition induction and conservative pyometra treatment in bitches. Its molecular structure is similar to mifepristone, an antiprogestin used in human medicine. Mifepristone has been shown to suppress proliferation and cytokine production by T cells, whereas the effect of aglepristone on T cell function remains elusive. The purpose of this project was to investigate the in vitro influence of RU534 on IFN-γ and IL-4 synthesis by peripheral blood T cells isolated from healthy bitches (N = 16) in luteal phase. The peripheral blood mononuclear cells (PBMCs) were incubated with three different dosages of aglepristone, or dimethyl sulfoxide (DMSO), with or without mitogen. The production of cytokines by resting or mitogen-activated T cells was determined by intercellular staining and flow cytometry analysis or ELISA assay, respectively.

Results: Our results showed no statistically significant differences in the percentage of IFN-γ and IL-4-synthesizing CD4+ or CD8+ resting T cells between untreated and aglepristone-treated cells at 24 and 48 hours post treatment. Moreover, mitogen-activated PBMCs treated with RU534 displayed similar concentration of IFN-γ and IL-4 in culture supernatants to those observed in mitogen-activated DMSO-treated PBMCs. Presented results indicate that administration of aglepristone for 48 hours has no influence on IFN-γ and IL-4 synthesis by resting and mitogen-activated T cells isolated from diestral bitches.

Conclusions: We conclude that antiprogestins may differentially affect T cell function depending on the animal species in which they are applied.

Figure 1

Representative dot plots show synthesis of IFN-γ by CD4⁺ (A) and CD8⁺ (B) T cells isolated from diestral bitches and treated for 24 and 48 h with DMSO (control) or 30, 300 and 3000 ng/ml of aglepristone. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes. MFI of IFN-γ in T cell subsets is shown within upper right quadrants.

Figure 2

The percentage of IFN-γ-synthesizing T cell subsets (A) and the MFI of IFN-γ-positive T cell subsets (B) treated for 24 and 48 h with 30, 300 and 3000 ng/ml of aglepristone. All data are presented relative to control values (line). Each point represents an individual dog (n = 7–9), with the bar indicating the mean. *P < 0.05, ** P < 0.01.

Figure 3

Representative dot plots show synthesis of IL-4 by CD4⁺ (A) and CD8⁺ (B) T cells isolated form bitches in luteal phase and treated for 24 and 48 h with DMSO (control) or 30, 300 and 3000 ng/ml of aglepristone. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes. MFI of IL-4 in T cell subsets is shown within upper right quadrants.

Figure 4

The percentage of IL-4-synthesizing T cell subsets (A) and the MFI of IL-4-positive T cell subsets (B) treated for 24 and 48 h with 30, 300 and 3000 ng/ml of aglepristone. All data are presented relative to control values (line). Each point represents an individual dog (N = 7–8), with the bar indicating the mean. *P < 0.05, ** P < 0.01.

Figure 5

The production of IFN-γ and IL-4 by ConA- or PHA-activated control PBMCs at 24 and 48 h of culture. Each point represents an individual dog (N = 7), with the bar indicating the mean. *P < 0.05, ** P < 0.01.

Figure 6

The normalized concentration of IFN-γ (A) and IL-4 (B) produced by PBMNs after 24 and 48h of treatment with 30, 300 and 3000 ng/ml of aglepristone. All data are presented relative to control values (line). Each point represents an individual dog (N = 7–8), with the bar indicating the mean. *P < 0.05, ** P < 0.01.