Activation of mitogen-activated protein kinases by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) plays an important role in macrophage stimulation

Abstract

The small molecule anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways play an important role in the regulation of proinflammatory cytokines, such as TNF-α, IL-1β, as well as the responses to extracellular stimuli, such as lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPK pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.

Figure 1. DMXAA activates p38 pathway in mouse macrophages via an interferon-independent mechanism

Mouse macrophages, MHS (A, B, D) and thioglycollate-elicited peritoneal macrophages (C, E), were stimulated with 20 µg/ml DMXAA in culture. At various time points, the culture supernatants and the cells were collected and assayed for IFNβ1 protein secretion (B, C) and amount of p38 and MK2 phosphorylation (A, D, E) with ELISA and immunoblots respectively. The values for IFNβ1 protein concentrations were expressed as the mean ± SEM. Both p38 and β-actin expression were used to show equal loading of lanes.

Figure 2. Blockade of p38-mediated pathway activation attenuated DMXAA-induced TNF-α and IL-6 production at the post-transcriptional level

Both MHS cells and thioglycollate-elicited macrophages were pre-treated with BIRB796 at 0.1 µM for 2h, followed by DMXAA stimulation at 20µg/ml. The cells were harvested 2hours post-DMXAA treatment to look at p38 pathway activation by western blots (A). The culture supernatant and cell lysates were collected 5 hours after DMXAA treatment to look at TNF-α and IL-6 protein level by ELISAs (B, C) or mRNA level by real-time PCR (D, E). Results represent the mean ± SEM for at least three independent experiments. * denotes p< 0.01 and ** denotes p< 0.05, determined with student t-test.

Figure 3. DMXAA also activates ERK1/2 and SAPK/JNK1,2/3 in macrophages, but only ERK1/2 signaling pathway plays a role in DMXAA-stimulated TNF-α and IL-6 production

Mouse macrophages, MHS and thioglycollate-elicited peritoneal macrophages were stimulated with 20 µg/ml DMXAA in culture for two hours. Cells were then lysed, and amount of ERK1/2 and JNKs phosphorylation were analyzed with immunoblots (A). To investigate the role of ERK1/2 and JNKs in DMXAA-induced TNF-α and IL-6 production, FR180204 (B, D) and 420135 (C, E), the pharmacologic inhibitors for ERK1/2 and JNKs, respectively, were used. MHS cells were pre-treated with the inhibitors for one hour, and DMXAA was then added to the culture and incubate for an additional 5 hours. The culture supernatants were collected and assay for secreted TNF-α and IL-6 protein level, and the RNA were extracted from the cells and analyzed with real-time PCR to look at the mRNA expression. The data are expressed as the mean ± SEM. * denotes p< 0.01.

Figure 4. DMXAA-stimulated IP-10 and MCP-1 productions are MAPKs pathway independent

MHS cells were pre-treated with different MAPK pathway inhibitors, BIRB796 (A), FR180204 (B) or 420135 (C) for one hour, prior to DMXAA stimulation. After 5 hour incubation time, culture supernatants were collected and assayed for IP-10 (left panel) and MCP-1 (right panel) with ELISA. The data are expressed as the mean ± SEM.

Figure 5. Effect of IFN-γ priming on DMXAA-stimulated MAPK pathways activation and TNF-α production

MHS cells were pre-exposed to IFN-γ, followed by DMXAA treatment at 20µg/ml. The culture supernatants were collected to measure TNF-α production by ELISA (A), or the RNA was extracted to measure the TNF-α mRNA level with real-time PCR (B). The cells were also extracted to determine the effect of IFN-γ priming on DMXAA-induced MAPK pathways activation (C). To study the role of various MAPK pathways in the IFN-γ priming effect, MHS cells were primed with IFN-γ for 2hrs with or without the inhibitors, BIRB796 (D), FR180204 (E), or 420135 (F) pretreatment for one hour, followed by treating with DMXAA for an additional 5 hours. The culture supernatants were then collected for TNF-α ELISA. The data are presented as the mean ± S.E.M., and * denotes a statistically significant reduction of TNF-α production (p< 0.01).

Figure 6. The activation of MAPK pathways by DMXAA is RIP2 independent

Thioglycollate-elicited peritoneal macrophages from the wild type and RIP2 knockout mice were treated with DMXAA at 20µg/ml or MDP at 10µg/ml for 2h, and the whole cell lysates were extracted and analyzed by immunoblot for phosphorylated p38, ERK1/2, and JNKs. Both p38 and β-actin expression were used to show equal loading of lanes.