MyD88, p38 MAP kinase and NF-κB pathway activation after stimulation of eosinophils with M bovis BCG. (A) MyD88, TIRAP, and TRAF6 expression on eosinophils was analyzed with RT-PCR. (B) Total proteins were extracted from eosinophils and neutrophils and equal protein amounts were analyzed with the use of Western blot for MyD88. The gel from 1 of 2 similar experiments is shown. (C) Measurement of ROS release by eosinophils preincubated with serially diluted MyD88 inhibitor 1 μmol/L (×), 10 μmol/L (▵), or 100 μmol/L (●) or with peptide control (100 μmol/L) (○) at 37°C for 30 minutes, further activated with BCG (10:1). ROS release by BCG (1:10 ■)–activated eosinophils without MyD88 inhibitor and by unstimulated eosinophils (−) were determined. Results are expressed as ROS cps values and 1 representative of 3 experiments was shown. (D) Purified eosinophils were pretreated with inhibitors to JNK1/2 (SP 600125, 0.1 μmol/L), ERK1/2 (PD 98 059, 0.1 μmol/L), p38 MAP kinase (SB 203580, 0.1 μmol/L), or NF-κB inhibitors (BAY 11-7082, 1 μmol/L) for 30 minutes at 37°C, before the addition of BCG (10:1). ROS release by eosinophils was analyzed by chemiluminescence. Bar graph represents the percentage ROS release inhibition. Results are expressed as mean plus or minus SEM (n = 3-4). (E) Purified eosinophils were stimulated with different numbers of BCG for the times indicated, and total cell extracts were analyzed by Western blotting using Abs against phosphorylated or nonphosphorylated forms of p38 MAP kinase. The results are representative of 2 independent experiments. *P < .05.