Fig. 6. IL-10 inhibits p38-mediated signals that activate TNF mRNA translation. (A) Impaired efficacy of IL-10 to suppress TNF production in mk2-deficient macrophages. LPS-stimulated TEPM from mk2^+/+ and mk2^–/– mice were cultured with various concentrations of rmIL-10 for 12 h, and TNF protein was detected in culture supernatants by ELISA. Results are from a representative of two experiments with cells derived from five mice/group. Data are as absolute values (upper) as well as percentages (+ SD) of the corresponding LPS values (lower). (B) Defective inhibition of polysome TNF mRNA in the absence of MK2. Sucrose gradient analysis of monosomal/polysomal-associated TNF mRNA in LPS plus IL-10-stimulated mk2^+/+, mk2^–/– TEPM as well as RAW 264.7 macrophages. Results from representative experiments shown as percentages of total cytoplasmic fractions. (C) Normal IL-10 targeting of TNF production in MKK3-deficient macrophages. TEPM from mkk3^+/+ and mkk3^–/– macrophages were stimulated with LPS (1 µg/ml) in the presence of 5 ng/ml rmIL-10. Data shown as mean (± SD) of the corresponding LPS values. Results from two experiments with macrophages from three mice/group. (D) Transfection of a constitutively active form of MKK6 inhibits IL-10-mediated targeting of TNF production. RAW 264.7 cells were transfected via electroporation with control plasmid or the expression plasmid for the mutated MKK6(Glu) (MKK6 CA). Following 18 h starvation, cells were challenged with LPS supplemented with the indicated quantities of IL-10. TNF protein quantitation was performed as before. Data shown as mean (± SEM) percentages of LPS values. Results from three independent transfected cultures for each group. *p <0.01.