Inhibition of lipopolysaccharide-stimulated chronic obstructive pulmonary disease macrophage inflammatory gene expression by dexamethasone and the p38 mitogen-activated protein kinase inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d] pyrimidin-2-yl]amino}ethyl)guanidine (SB706504)

Abstract

p38 mitogen-activated protein kinase (MAPK) signaling is known to be increased in chronic obstructive pulmonary disease (COPD) macrophages. We have studied the effects of the p38 MAPK inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]-pyrimidin-2-yl]amino}ethyl)guanidine (SB706504) and dexamethasone on COPD macrophage inflammatory gene expression and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. Lipopolysaccharide (LPS)-stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AMs) were cultured with dexamethasone and/or SB706504. MDMs were used for gene array and protein studies, whereas tumor necrosis factor (TNF) alpha protein production was measured from AMs. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared with SB706504 alone (-2.04) or dexamethasone (-3.39). Twenty-three genes were insensitive to the effects of both drugs, including interleukin (IL)-1beta, IL-18, and chemokine (CC motif) ligand (CCL) 5. In addition, the chromosome 4 chemokine cluster members, CXCL1, CXCL2, CXCL3, and CXCL8, were all glucocorticoid-resistant. SB706504 significantly inhibited LPS-stimulated TNFalpha production from COPD and smoker AMs, with near-maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS-regulated genes, including IL-1beta, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.

Fig. 1.

Heatmap comparison of inflammatory mediator mRNA levels sensitive to dexamethasone, SB706504, or cotreatment in LPS-stimulated COPD MDMs (n = 6). mRNA levels were measured by microarray. Significantly regulated (minimum of a 2-fold change, p < 0.01) inflammatory genes were loaded into Heatmap Builder Version 1.0, and the line graph shows the mean fold change for each condition.

Fig. 2.

Gene network generated through the use of Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com) and derived from dexamethasone (A)- and SB706504 (B)-insensitive inflammatory mediator genes in LPS-stimulated COPD MDMs. Each gene or complex is classified as a node; V-shaped nodes define inflammatory mediators, circular nodes define complexes or groups of molecules. Groups/complexes shown here are defined as follows; IL1 (IL18, IL1β, IL1F5/6/7/8/10, and IL1RN), p38 MAPK (p38 MAPKα/β/δ/γ), NFκB (NFκB, NFκB-RelA, Rel/RelA/RelB), NFκB-RelA (NFκB plus RelA or p50/52 plus p65), and AP-1 (Jun, Fos). Nodes shaded in purple were from the original uploaded data set, and uncolored nodes were incorporated into the network by IPA because of the high levels of connectivity between the molecules. Interacting nodes are defined by either direct, which requires direct physical contact (closed arrows), or indirect, whereby direct physical contact is not required, relationships (dashed arrows). Arrow directionality gives the direction of the interaction, i.e., A “acts on” B.

Fig. 3.

Comparison of dexamethasone, SB706504, and cotreatment effects on cytokine mRNA levels measured by TaqMan qPCR in LPS-stimulated COPD MDMs (n = 6). Data are presented as copy number medians with interquartile range and range. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 4.

Comparison of dexamethasone, SB706504, and cotreatment inhibition of cytokine levels measured by Luminex in LPS-stimulated COPD MDMs (n = 6). Data are presented as medians with interquartile range and range. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5.

Comparison of dexamethasone, SB706504, and cotreatment inhibition of IL-8 protein levels measured by ELISA in unstimulated and LPS-stimulated COPD MDMs (n = 6). Data are presented as medians with interquartile range and range. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 6.

Comparison of dexamethasone, SB706504, and cotreatment inhibition of TNFα protein measured by ELISA in LPS-stimulated COPD (clear bars, n = 9) and HS (gray bars, n = 10) (A) and UP-LPS-stimulated COPD (clear bars, n = 7) and HS (gray bars, n = 7) AMs (B). Data are presented as medians with IQR and range. *, p < 0.05; **, p < 0.01; ***, p < 0.001.