Inhibition of IL-4 gene transcription in CD4⁺ T cells by salicylates.

(A) Purified PBT (≥ 97% CD3⁺CD4⁺) were treated with ASA (10⁻³ M) or the corresponding amount of DMSO carrier (0.1%) before stimulation with A23187 (0.5 μg/mL) and PMA (10 ng/mL) for 6 hours. Shown are the results of RT-PCR of total RNA extracted in a typical experiment using primers specific for the indicated cytokine gene transcripts. Shown as an RNA integrity and loading control is the expression of transcripts of the GAPDH gene. Similar results were obtained in 3 identical experiments using ASA or SA. (B) Real-time RT-PCR analysis of IL-4 RNA expression in purified PBT incubated in the presence of the indicated concentrations of ASA or with 10⁻⁵ M IM before stimulation with A23187 (0.5 μg/mL) and PMA (10 ng/mL) for 6 hours. Standard curves for IL-4 and GAPDH were generated by serial dilutions of PBT total RNA in separate experiments (data not shown). The relative IL-4 RNA levels were calculated. Shown is the mean ± SEM percentage of control IL-4 expression (corresponding to 2-ΔΔC_T × 100) in 3 independent experiments done in duplicate. The asterisk indicatesP < .05 relative to DMSO-treated controls. (C) Jurkat T cells transiently transfected with the IL-4.265 or IL-2.312 CAT reporter plasmids were treated (15 minutes) with the indicated concentrations of ASA (, 10⁻⁵ M;, 10⁻⁴ M; ▪, 10⁻³ M) or corresponding amounts of DMSO (■) before 20-hour stimulation with A23187 (0.5 μg/mL) with (IL-2) or without (IL-4) PMA (10 ng/mL). Data are expressed as fold induction of intracellular CAT relative to unstimulated controls and are the mean ± SEM results from 4 independent experiments done in duplicate. The asterisk indicates P < .05 (Wilcoxon test) relative to DMSO-treated controls.