Figure 4.

Effect of inhibition of extracellular signal–regulated kinase (ERK) and p38 phosphorylation on tumor necrosis factor (TNF)-α and IL-12p40 production in bronchoalveolar lavage (BAL) cells of patients with sarcoidosis. Cells obtained from patients during BAL were counted and placed in culture immediately after isolation. Cells were pretreated either with (A and C) SB203580 or (B and D) PD98059 30 minutes before treatment with γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) (1 μg/ml) or LPS (1 ng/ml) for 16 hours. (A and B) TNF-α and (C and D) IL-12/IL-23p40 immunoreactivity in supernatants was analyzed by ELISA. Data presented are mean ± SEM of eight patients analyzed in triplicate. Using analysis of variance Mann-Whitney U test P < 0.05 was considered significant. *P < 0.05; **P < 0.001. (E) SB203580 abolishes p38 phosphorylation but increases ERK phosphorylation in BAL cells. Cells (5 × 10⁷) obtained from patients with sarcoidosis (n = 4) were placed in culture immediately after isolation in the presence or absence of SB203580 30 minutes before treatment with iE-DAP (1 μg/ml) or LPS (1 ng/ml) for 45 minutes or left in media without any treatment (control). Whole cell lysates were subjected to Western blot analysis and immunoblotting using antibodies recognizing the active (phosphorylated) form of ERK and p38. Several treatments with other ligands as well as PD98059 were cut from the original blot and excluded in the final image for clarity of image. Equal loading of proteins was confirmed using total ERK and p38 antibodies. As shown, SB203580 inhibited phosphorylation of p38 while increasing ERK phosphorylation. The results were replicated in six different patients. (F) Densitometric values (mean ± SEM) of phosphorylated form of p38 of four different experiments. Using analysis of variance Mann-Whitney U test P < 0.05 was considered significant. **P < 0.001. Significant differences were found between the densitometric units of pp38 in presence and absence of SB203580.