Figure 6.

CyP interferes with LPS-induced cytokine production even when added several hours after LPS. DCs were stimulated with 0.4 μg/ml LPS, and 20 μg/ml CyP was added at various time intervals. (A) Cells were analyzed for CD80, CD86, and HLA-DR 20 h after stimulation and for CCR7 44 h after stimulation. Shadowed profiles correspond to unstimulated cells. One representative experiment out of five is shown. (B) Graded numbers of DCs stimulated as in A were cultured with allogeneic naive CD4⁺ T cells, and proliferation was measured on day 5 by [³H]thymidine incorporation and expressed as cpm. Data represent the mean ± SD of duplicates of one experiment of four performed. (C) DCs were stimulated with LPS, and CyP was subsequently added at the indicated time points. Cytokines released in the culture supernatants at 20 h were measured by ELISA and expressed as percentage of production in absence of CyP addition (−). Data represent the mean ± SD of three independent experiments. (D) DCs were stimulated with LPS in replicate cultures. The cells were left untreated (•) or CyP was added after 1 (▿), 3 (⋄), or 6 (○) h. The kinetics of cytokine-specific transcripts were determined by quantitative real-time RT-PCR. One representative experiment of three is shown. Fig. S4 reports the kinetics of CCL5 transcripts. (E) After 4 h of LPS stimulation, DCs were left untreated or CyP was added to the culture. Cell lysates were prepared 20 min, 1 h, or 2 h after the addition of CyP and analyzed by Western blot with antibodies specific for phosphorilated c-Jun, phosphorilated p38, or IκBα.