Figure 4.

Molecular mechanism underlying the effect of dopamine on Treg. a, Expression of CTLA-4. Treg were activated for 24 hr, then incubated for 2 hr with dopamine or SKF-38393 (control cells were activated but were not incubated with either dopamine or SKF-38393; note that different cell preparations were used for each treatment, and therefore the controls used for each treatment were not the same) and were stained 24 hr later for CTLA-4 on cell surfaces. CTLA-4 expression was reduced after exposure to dopamine or to SKF-38393. Representative results of one of five independent experiments with each treatment are shown. b, Production of IL-10. Treg were activated for 24 hr with anti-CD3 and IL-2 in the presence of lethally irradiated splenocytes (APCs) and then for an additional 2 hr with dopamine. Conditioned media were collected after 24, 48, or 72 hr of culture and were assayed for IL-10 using a sandwich ELISA. At any given time, significantly less IL-10 was detected in media conditioned by dopamine-treated Treg than in media conditioned by Treg not exposed to dopamine. Statistical significance was verified using Student's t test analysis (^**p < 0.01; ^*p < 0.05). The results shown are of one of three independent experiments, performed at each time point. c, Lack of IL-2 production by Treg. Treg and Teff were activated separately for 48 hr with anti-CD3 and anti-CD28 (without mrIL-2) with or without dopamine. Conditioned media were collected after 48 hr and subjected to ELISA. Treg with or without dopamine did not secrete detectable levels of IL-2. Production of IL-2 by Teff was not affected by dopamine. d, Foxp3 expression in Treg. Treg were activated for 24 hr with anti-CD3 and anti-CD28 in the presence of IL-2, then exposed to dopamine for 2 hr, washed, and analyzed 30 min later for Foxp3 expression. No changes in Foxp3 were detected after 30 min of dopamine treatment of naive Treg. DA, Dopamine.