Fig. 7.

ERK1/2 is required for LPS-induced TNFα production since U0126 inhibits ERK1/2 phosphorylation (A) and TNFα production (D), but does not inhibit activity of p38 (B) or JNK (C) and does not induce cytotoxicity (E). RAW264.7 macrophages were stimulated with titanium particles with adherent endotoxin or soluble LPS in the presence or absence of the indicated concentrations of inhibitors or vehicle controls. All measurements were made after 90 minutes of stimulation, except for p38 activity (B), which was measured after 30 minutes. All groups received a mixture of vehicle controls, such that all contained 0.04% PBS, 0.1% DMSO and 0.2% methanol. MAPK activation (A–C) was assessed in whole cell lysates as described in Figure 1B. Western blots are from the most representative of the seven experiments pooled in (D). TNFα protein was measured by ELISA in conditioned media collected following stimulation with titanium particles with adherent endotoxin or soluble LPS in the absence or presence of inhibitors. (D) Results (mean ± SEM of seven experiments) are presented as a percentage of the amount measured following stimulation with soluble LPS in the absence of inhibitors. (E) Cytotoxicity results are presented as the mean ± SEM of six of the seven experiments pooled in (D) since LDH activity was not measured in the seventh experiment.