Strong inhibition of TNF-alpha production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor

Abstract

In inflammatory processes, the p38 mitogen-activated protein kinase (MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-alpha, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-alpha stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-alpha and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-alpha was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1beta, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis.

Figure 1

Expression of CD14 (bold grey line) and HLA-DR (bold black line) in monocytes (left panel) and macrophages, differentiated with 50 ng/ml macrophage-colony-stimulating factor and 1% FCS after 5 days (right panel), measured by flow cytometry and expressed in mean fluorescence intensity (MFI). The isotype control is an IgG2a antibody (dashed line); the blank is in solid fill. The MFI is shown on the x-axis, while on the y-axis the number of cells measured in the flow cytometer is expressed. PE = phycoerythrin.

Figure 2

Effect of RWJ 67657 on phosphorylation of p38 MAPK and MAPKAPK-2. (a) Representative presentation of phosphorylation of p38 mitogen-activated protein kinase (MAPK) in monocyte-derived macrophages after stimulation with lipopolysaccharide (LPS) at various time points. Phosphorylation was measured by western blotting using specific antibodies to p38 MAPK and phospho-p38 MAPK. (b) Effect of RWJ 67657 on phosphorylation of the direct substrate of p38 MAPK, MAPKAPK-2 (MAPK-activating protein kinase-2), measured after 30 minutes of stimulation. DMSO = dimethyl sulfoxide.

Figure 3

Protein production of tumor necrosis factor (TNF)-α, IL-6, IL-8, and matrix metalloproteinase 9 (MMP9) by monocyte-derived macrophages from healthy controls (n = 8, open squares) and rheumatoid arthritis patients (n = 9, filled squares). Cells were stimulated with LPS for 24 hours and pretreated 1 hour beforehand with RWJ 67657 at various concentrations. Protein production was measured in supernatants by ELISA and is expressed in ng/ml (a). Inhibition was calculated against the stimulated control (b). Bars show mean and SEM. *P < 0.05; **P < 0.001, paired t-test, calculated versus the stimulated control. unst, unstimulated.

Figure 4

Time course of induction of mRNA expression of tumor necrosis factor (TNF)-α, IL-1β, IL-6, IL-8, and cyclooxygenase 2 (COX-2). Monocyte-derived macrophages from healthy controls (n = 2) were stimulated for increasing periods of time with lipopolysaccharide (50 ng/ml). mRNA expression was determined with real-time RT-PCR and results were calculated as fold induction in comparison with unstimulated cells (fold induction = 1).

Figure 5

mRNA expression of tumor necrosis factor (TNF)-α, IL-1β, cyclooxygenase 2 (COX-2), IL-6, IL-8, and matrix metalloproteinase 9 (MMP9) in monocyte-derived macrophages from healthy controls (n = 5). Cells were stimulated with lipopolysaccharide for 4 hours and pretreated with a RWJ 67657 at various concentrations. mRNA expression was determined with real-time RT-PCR and results are expressed as fold induction in comparison with unstimulated cells (fold induction = 1). Bars show means and SEM (*P < 0.05, paired t-test, calculated against the stimulated control).