The kinase inhibitor imatinib mesylate inhibits TNF-{alpha} production in vitro and prevents TNF-dependent acute hepatic inflammation

Abstract

Imatinib exerts potent antileukemic effects in vitro and in vivo. Despite its well known antitumor activity, the potential of imatinib for the treatment of inflammatory diseases remains elusive so far. Our current report provides strong evidence that imatinib has potent antiinflammatory effects. It potently inhibits LPS- and Con A-induced TNF-alpha production by human myeloid cells in vitro (peripheral blood mononuclear cells, CD14-selected monocytes, and monocyte-derived macrophages). Of note, the production of the antiinflammatory cytokine IL-10 was not significantly regulated by imatinib. In line with this observation, phosphorylation of IkappaB and subsequent DNA binding of NF-kappaB, which is critically involved in TNF-alpha, but not IL-10 expression, was reduced by imatinib. Using several murine models of acute hepatitis, we could corroborate our in vitro findings, as imatinib prevented macrophage- and TNF-alpha-dependent inflammatory damage of the liver induced by injection of either Con A or d-galactosamine/LPS by inhibition of hepatic TNF-alpha production. Of note, d-galactosamine/TNF-induced hepatitis was not affected, showing that imatinib does not directly inhibit TNF-alpha-induced hepatocellular cell death. These findings suggest a potent antiinflammatory role of imatinib by modulation of TNF-alpha production in monocytes/macrophages. This observation might be of therapeutic value for the treatment of TNF-mediated diseases.

Figures

Fig. 1.

Imatinib inhibits TNF-α production in human myeloid cells. Human PBMCs, monocytes, or macrophages were exposed to either saline or imatinib (1 h, 1 μM) followed by 3-h stimulation with 100 ng/ml LPS. (A) LPS-induced TNF-α levels in supernatants of all three cell types as determined by ELISA (n = 5; *, P ≤ 0.01). (B and C) Imatinib dose-dependently inhibits LPS-induced TNF-α protein (B) (n = 5; *, P ≤ 0.01) and TNF-α mRNA expression (C) (n = 3; *, P ≤ 0.01) in human PBMCs.

Fig. 2.

Imatinib reduces phosphorylation of IκBα and subsequent NF-κB activation. (A) Monocytes were treated with increasing imatinib concentrations (0.1-10 μM) for 30 min followed by LPS (100 ng/ml) for 15 min, stained for intracellular phosphotyrosine content, and analyzed by flow cytometry. A representative example from three independent experiments is shown. The percentage of phosphotyrosine-positive cells in the monocyte gate and the mean fluorescence intensity (MFI) is given in each diagram. (B) Monocytes were exposed to either saline or imatinib followed by LPS stimulation. Whole-cell lysates were prepared at the indicated time points, and the LPS-induced NF-κB p65 activation was determined by using a chemiluminescent assay (n = 3; *, P ≤ 0.05). H&E, hematoxylin/eosin. (C) LPS-induced NF-κB DNA binding was detected by EMSA. Nuclear extracts were prepared from solvent- or imatinib-treated macrophages stimulated with LPS (50 ng/ml) for 0-60 min. One of three representative experiments is shown. For supershift assays, p50 and p65 anti-NF-κB subunit antibodies were used. (D) The phosphorylation state of IκBα was determined by Western blot using antibodies specific for phosphorylated IκBα and total IκB. A representative result from three independent experiments is shown. Densitometry confirms the decrease in IκBα phosphorylation in response to imatinib (*, P ≤ 0.01). Results are expressed as percentage of control sample.

Fig. 3.

Inhibition of TNF-α- and macrophage-dependent acute liver injury in mice. Either imatinib- or saline-pretreated BALB/c mice were challenged with Con A (15 mg/kg, n = 8 per group). (A) Plasma ALT-levels were determined 8 h after Con A injection (*, P ≤ 0.0001). (B) Imatinib prevents histomorphologic liver changes [hematoxylin/eosin (H&E) staining]. A representative example from each group is shown (magnification: ×150). (C and D) Hepatic mRNA levels of TNF-α (C) and the corresponding TNF-α plasma levels (D) were determined 2 h after Con A injection by real-time PCR and ELISA, respectively (*, P <0.05).

Fig. 4.

Imatinib prevents activation of macrophages in vivo. Con A-induced induction of hepatic IL-10 mRNA (A) and circulating plasma levels of IL-10 (B) were not modulated by imatinib (P > 0.05).

Fig. 5.

Therapeutic administration of imatinib prevents acute liver failure. Mice received either imatinib or saline 0.5 h after Con A administration (n = 6 per group). (A) Plasma-ALT levels were determined after 8 h (*, P < 0.05). (B) TNF-α plasma levels were determined by ELISA 2 h after Con A injection (*, P < 0.05).

Fig. 6.

Imatinib prevents GalN/LPS-induced but not GalN/TNF-induced hepatic failure. (A and B) Plasma-ALT (A) and circulating TNF-α levels (B) from either imatinib- or saline-pretreated mice that were subsequently injected with GalN/LPS (n = 4, *, P ≤ 0.01). (C) Plasma-ALT levels from either imatinib- or saline-treated mice that were challenged with GalN/TNF-α (n = 4, P > 0.05). (D) Imatinib rescued mice from lethal doses of GalN/LPS (n = 10 in each group; *, P ≤ 0.001).