Elevated GM-CSF and IL-1beta levels compromise the ability of p38 MAPK inhibitors to modulate TNFalpha levels in the human monocytic/macrophage U937 cell line

Mol Biosyst. 2010 Oct;6(10):1956-72. doi: 10.1039/c002848g. Epub 2010 Jul 9.

Abstract

Rheumatoid arthritis (RA) is a complex, multicellular disease involving a delicate balance between both pro- and anti-inflammatory cytokines which ultimately determines the disease phenotype. The simultaneous presence of multiple signaling molecules, and more specifically their relative levels, potentially influences the efficacy of directed therapies. Using the human U937 monocytic cell line, we generated a self-consistent dataset measuring 50 cytokines and 23 phosphoproteins in the presence of 6 small molecule inhibitors under 15 stimulatory conditions throughout a 24 hour time course. From this dataset, we are able to explore phosphoprotein and cytokine relationships, as well as evaluate the significance of cellular context on the ability of small molecule inhibitors to block inflammatory processes. We show that the ability of a p38 inhibitor to attenuate TNFalpha production is influenced by local levels of GM-CSF and IL-1beta, two cytokines known to be elevated in the joints of RA patients. Within the cell, compensatory mechanisms between signaling pathways are apparent, as selective p38 MAPK inhibition results in the increased phosphorylation of other MAPKs (ERK and JNK) and their downstream substrates (CREB, c-Jun, and ATF-2). Further, we demonstrate that TNFalpha-neutralizing antibodies have secondary effects on cytokine production, impacting more than just TNFalpha alone. p38 MAPK inhibition using a small molecule inhibitor also blocks production of anti-inflammatory cytokines including IL-10, IL-1ra and IL-2ra. Collectively, the impact of cell context on TNFalpha production and unintended blockade of anti-inflammatory cytokines may compromise the efficacy of p38 inhibitors in a clinical setting. The effort described in this work evaluates the effect of inhibitors on multiple endpoints (both intra- and extracellular), under a range of biologically relevant conditions, thus providing a unique means for differentiation of compounds and potential opportunity for improved pharmacological manipulation of disease endpoints in RA.

MeSH terms

  • Granulocyte-Macrophage Colony-Stimulating Factor / blood*
  • Humans
  • Interleukin-1beta / blood*
  • Macrophages / metabolism*
  • Monocytes / metabolism*
  • Tumor Necrosis Factors / metabolism*
  • U937 Cells
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors*

Substances

  • Interleukin-1beta
  • Tumor Necrosis Factors
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • p38 Mitogen-Activated Protein Kinases