Figure 3.

DGC and TLR agonists induce TNF production and TXNIP down-regulation in T cells. (A) TNF in the supernatant of PBMC incubated for 0 to 4 hours (mean + SEM, n = 3). (B) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from PBMC incubated for 0 to 4 hours. (C) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from untreated blood and blood treated with TLR1–5 ligands (TLR1/2, 500 ng/ml PamCSK4; TLR2, 1 × 10⁸ heat killed Listeria monocytogenes/ml; TLR3 HMW, 5 µg/ml Poly(I:C) high molecular weight; TLR3 LMW, 5 µg/ml Poly(I:C) low molecular weight; TLR4, 500 ng/ml E. coli K12 LPS; TLR5, 500 ng/ml S. typhimurium Flagellin) for 4 hours (4 h blood, Procedure II, Fig. 1B) as indicated. (D) TNF in the supernatant of blood incubated with the TLR1–5 ligands as above for 4 hours (4 h blood, Procedure II, Fig. 1B) as indicated. (E) TNF in the supernatant of blood incubated for 0 to 4 hours without (white columns) or with the TLR4 ligand (50 ng/ml) (black columns). (F) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from untreated blood and blood treated with the TLR4 ligand (50 ng/ml) in the absence or presence of the TNF inhibitors etanercept (10 µg/ml) and infliximab (10 µg/ml) for 4 hours (4 h blood, Procedure II, Fig. 1B) as indicated. (G) The frequency of CD25⁺ T cells in untreated blood, blood treated with OKT3 (100 ng/ml), TNF (10 ng/ml) and the TNF inhibitors etanercept (10 µg/ml) and infliximab (10 µg/ml) as indicated for 72 hours. The data show the mean + SEM obtained from four experiments with nine different donors. (B,C,F) Each Western blot is representative for Western blots obtained from at least 3 different biological experiments and the quantification shows the mean + SEM of the band densities of TXNIP from Western blots obtained from at least 3 different biological experiments. The positions of the relevant molecular weight markers and their molecular weight in kDa are given to the right of each Western blot.