Serum inhibits TNF-α–mediated maturation of MDDC: involvement of the ERK signaling pathway.

(A) Monocytes were cultured in macrophage serum-free medium containing GM-CSF and IL-4. After 5 days, cells were treated with TNF-α in the absence or presence of 10% fetal calf serum and with or without the U0126 MEK 1/2 inhibitor. Expression of CD83 was determined by flow cytometry 24 and 48 hours later. In each panel, the percentage of positive cells (bottom) and the MFI (top) are indicated. A representative experiment is shown. (B) Specificity of the PD98059, U0126, and SB203580 signaling-pathway inhibitors. Immature MDDC were treated with TNF-α in either the absence (minus sign) or presence of the indicated inhibitors (PD indicates PD98059; SB, SB203580; and U0; U0126). After 1 hour of incubation with the inhibitors, cells were stimulated with TNF-α for 10 minutes. Cell lysates (10 μg) were subjected to Western blotting using rabbit polyclonal antiserum specific for ERK 1/2 (ERK) or p38 or against their respective phosphorylated forms (pERK or pp38). The experiment was done on cells from 2 different donors and one experiment is shown.