ERK activation by GM-CSF reduces effectiveness of p38 inhibitor on inhibiting TNFα release

Abstract

Tumor necrosis factor α (TNFα), a pro-inflammatory factor, plays an important role in many inflammatory diseases. Inhibition of p38 is being pursued as a pharmaceutical treatment to reduce TNFα release. Since a variety of cytokines and factors may exist at different amounts in patients, we explored how differences in the cytokine environment impact p38 inhibitor potency. Cytokine co-stimulation with LPS was compared against LPS stimulation alone. In both differentiated U937 cells and peripheral monocytes, GM-CSF co-stimulation with LPS increased TNFα release and led to an increased residual TNFα levels with p38 inhibitor. Adding MEK inhibitor in the presence of p38 inhibitor further reduced TNFα release suggesting that the ERK pathway plays a role in GM-CSF induced reduction of the p38 inhibitor potency. When cells were stimulated with different concentrations of LPS and GM-CSF, the minimal TNFα level obtained by MEK inhibitor was not dependent on the stimulation condition; while it was dependent on GM-CSF level for p38 inhibitor. TNFα release in the presence of combinations of p38 and MEK inhibitors under different stimulation conditions was measured. A linear model was created using the initial relative ERK and p38 phosphorylation levels and p38 and MEK inhibitor concentrations to accurately predict released TNFα level, suggesting these four parameters are sufficient to predict TNFα levels. We then used the model to show that with same TNFα levels, higher ERK pathway activity reduces p38 inhibitor potency. These results suggest that p38 inhibitor will be a more potent anti-TNFα therapy for patients with low ERK pathway activity.