Imatinib stimulates prostaglandin E2 and attenuates cytokine release via EP4 receptor activation
Authors: Thomas Bärnthaler, Katharina Jandl, Heinz Sill, Barbara Uhl, Yannick Schreiber, Magdalena Grill, Dominique Thomas, Rudolf Schicho, Gunther Marsche, Saša Frank, Akos Heinemann, Rufina Schuligoi
Journal: Journal of Allergy and Clinical Immunology (2018)

Imatinib is a highly efficacious therapy for leukemias that are characterized by the presence of the Philadelphia chromosome, a reciprocal translocation that fuses the genes encoding breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (ABL) resulting in the expression of the constitutively active protein kinase BCR-ABL. In addition, imatinib was shown to affect immune responses; for instance, beneficial effects have been described in rheumatoid arthritis1Pereira I. Fialho S. Castro G. Zimmermann A. Imatinib mesylate induces clinical remission in rheumatoid arthritis.Joint Bone Spine. 2010; 77: 372-373Crossref PubMed Scopus (12) Google Scholar and asthma.2Cahill K.N. Katz H.R. Cui J. Lai J. Kazani S. Crosby-Thompson A. et al.KIT inhibition by imatinib in patients with severe refractory asthma.N Engl J Med. 2017; 376: 1911-1920Crossref PubMed Scopus (127) Google Scholar In human peripheral blood monocytes and in macrophages, imatinib inhibits LPS-induced production of TNF-α, IL-6, and IL-8, via inhibition of nuclear factor kappa B (NF-κB).3Rizzo A.N. Aman J. van Nieuw Amerongen G.P. Dudek S.M. Targeting Abl kinases to regulate vascular leak during sepsis and acute respiratory distress syndrome.Arterioscler Thromb Vasc Biol. 2015; 35: 1071-1079Crossref PubMed Scopus (50) Google Scholar Because imatinib shows anti-inflammatory and immunomodulatory effects, we were interested in its effect on prostanoid synthesis, especially on prostaglandin (PG)E2. Prostaglandins (PGs) and thromboxane A2 (TXA2) are formed from arachidonic acid, which is released by cytosolic phospholipase c A2 from membrane phospholipids. Arachidonic acid is metabolized by cyclooxygenases to the unstable intermediate PGH2, which is further converted by the respective terminal synthases to PGE2, PGI2, PGD2, PGF2α, and TXA2. PGE2 is one of the most abundant PGs and exerts its effects by binding to 4 different G protein–coupled receptors (EP1-EP4), which show tissue-specific distribution. PGE2 was shown to inhibit immune responses mainly via EP4 activation, by curbing cytokine and chemokine expression and promoting a shift of the TH1 to a TH2 response, although the EP2 receptor has similar and redundant roles.4Konya V. Marsche G. Schuligoi R. Heinemann A. E-type prostanoid receptor 4 (EP4) in disease and therapy.Pharmacol Ther. 2013; 138: 485-502Crossref PubMed Scopus (112) Google Scholar Thus, we hypothesized that the inhibitory effect of imatinib on proinflammatory cytokines might be mediated by PGE2 via EP4 receptor activation. The study was approved by the Ethics Committee of the Medical University of Graz to withdraw blood of healthy donors (17-291 ex 05/06) and from patients with chronic myeloid leukemia in complete remission, receiving imatinib (28-590 ex 15/16), after signing informed consent (for patients' data, see Table E1 and Fig E1 in this article's Online Repository at www.jacionline.org; also see this article's Methods section in the Online Repository at www.jacionline.org). First, we investigated the effect of imatinib in vitro. In adherent human monocytes treated with LPS (10 ng/mL) and further incubated for 20 hours, we found that imatinib caused a significant inhibition of TNF-α that was paralleled by a pronounced (5-10-fold) increase in PGE2 levels and interestingly an inhibition of TXA2, measured as its stable metabolite TXB2 (Fig 1, A-C). Pretreatment with diclofenac (1 μΜ), which totally inhibited PGE2 synthesis, and the selective EP4 receptor antagonist ONO-AE3 208 (0.3 μM) counteracted the inhibitory effect of imatinib on TNF-α release, underscoring that this effect is indeed mediated by PGE2-induced EP4 receptor activation (Fig 1, G and H), whereas antagonism of the EP2 as well as EP1 and EP3 receptors had no effect (see Fig E2 in this article's Online Repository at www.jacionline.org). This line of reasoning is further strengthened by the fact that the imatinib-induced increase in PGE2 precedes the inhibition of TNF-α formation (Fig 1, D-F). The same results were obtained in human monocyte–derived macrophages and human whole blood (see Figs E3 and E4 in this article's Online Repository at www.jacionline.org). Most importantly, we observed the same effects also after treatment with another BCR-ABL inhibitor, nilotinib (see Fig E3, Fig E4, Fig E5 in this article's Online Repository at www.jacionline.org). In addition, we could prove that the inhibitory effect of imatinib on LPS-stimulated (100 ng/mL) NF-κB activation is dependent on PGE2 and EP4 receptor activation, as shown in the human macrophage cell line U937 containing an NF-κB–GFP reporter cassette5Jorgl A. Platzer B. Taschner S. Heinz L.X. Hocher B. Reisner P.M. et al.Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB.Blood. 2007; 109: 185-193Crossref PubMed Scopus (0) Google Scholar (Fig 1, I). This observation further corroborates the hypothesis that PGE2 via EP4 receptor activation inhibits NF-κB, which leads to reduced cytokine production. Data in the Online Repository show that upstream kinases were not involved in the TNF-α inhibition by imatinib and thus further support the notion of an essential mediator role of PGE2 and EP4 (see Fig E6 in this article's Online Repository at www.jacionline.org). In washed platelet preparations and platelet microsomes we provide evidence that imatinib and nilotinib inhibit arachidonic acid– induced thromboxane synthase (or in case of microsomes PGH2); however, because accumulating PGH2 acts on thromboxane receptors, platelet aggregation is not affected (see data in this article's Online Repository at www.jacionline.org; also see Fig E7 in this article's Online Repository at www.jacionline.org). To prove that our in vitro findings can be translated to an in vivo setting, that is, to patients who receive imatinib, blood was drawn from 19 patients with chronic myeloid leukemia (8 females/11 males; median age, 59 years; interquartile range, 14) who were in complete remission under imatinib treatment (400 mg/d) and from 19 matched healthy controls (8 females/11 males; median age, 60 years; interquartile range, 10). Whole blood was treated with vehicle or stimulated with LPS (10 μg/mL) for 4 hours. Patients treated with imatinib had increased levels of PGE2 and decreased levels of TXB2 as compared with healthy controls, although total prostanoid levels did not differ. This was obvious with regard to both baseline generation of prostanoids (vehicle treatment; Fig 2, A), and the response to LPS stimulation (Fig 2, B). In addition to PGE2, we found that PGD2 and PGF2α levels were increased, supporting the hypothesis that inhibition of thromboxane synthase leads to redirection of PGH2 to other terminal PG synthases. In agreement with our in vitro data we also found that the increase in the inflammatory cytokines TNF-α, ΙL1-β, and IL-6 and the chemokine IL-8 in LPS-stimulated blood of patients was less pronounced as compared with the cytokine response to LPS in healthy controls (Fig 2, C). Furthermore, we observed that treatment of patients with imatinib, without influencing total prostanoid levels, shifted prostanoid distribution toward higher PGE2, PGD2, and PGF2α and lower TXB2 levels both under unstimulated and LPS-stimulated conditions (Fig 2, F). In addition, we found a strong inverse correlation between TXB2 and PGE2 levels (Fig 2, D and E). Our results show for the first time that imatinib causes a pronounced increase in PGE2, both in vitro and in vivo. Interestingly, imatinib was shown to ameliorate LPS-induced lung injury by attenuating endothelial barrier dysfunction, which was attributed to inhibition of Abl-related kinase, Arg.3Rizzo A.N. Aman J. van Nieuw Amerongen G.P. Dudek S.M. Targeting Abl kinases to regulate vascular leak during sepsis and acute respiratory distress syndrome.Arterioscler Thromb Vasc Biol. 2015; 35: 1071-1079Crossref PubMed Scopus (50) Google Scholar Imatinib causes a downregulation of inflammatory cytokines, inflammatory cell infiltration, edema formation, and reactive oxygen species production.3Rizzo A.N. Aman J. van Nieuw Amerongen G.P. Dudek S.M. Targeting Abl kinases to regulate vascular leak during sepsis and acute respiratory distress syndrome.Arterioscler Thromb Vasc Biol. 2015; 35: 1071-1079Crossref PubMed Scopus (50) Google Scholar Likewise, PGE2 plays a prominent protective role in the airways; for example, it inhibits LPS-induced lung injury, and enhances microvascular barrier function by activation of the EP4 receptor, thereby ameliorating LPS-induced pulmonary inflammation.6Barnthaler T. Maric J. Platzer W. Konya V. Theiler A. Hasenohrl C. et al.The role of PGE2 in alveolar epithelial and lung microvascular endothelial crosstalk.Sci Rep. 2017; 7: 7923Crossref PubMed Scopus (9) Google Scholar Imatinib also ameliorates airway hyperresponsiveness, eosinophilic inflammation, and airway remodeling in murine models of chronic asthma,7Rhee C.K. Kim J.W. Park C.K. Kim J.S. Kang J.Y. Kim S.J. et al.Effect of imatinib on airway smooth muscle thickening in a murine model of chronic asthma.Int Arch Allergy Immunol. 2011; 155: 243-251Crossref PubMed Scopus (44) Google Scholar effects that are also described for PGE2.4Konya V. Marsche G. Schuligoi R. Heinemann A. E-type prostanoid receptor 4 (EP4) in disease and therapy.Pharmacol Ther. 2013; 138: 485-502Crossref PubMed Scopus (112) Google Scholar In patients with severe asthma, imatinib, albeit showing no effect on tissue eosinophilia and tissue remodeling, improved airway hyperresponsivness, which was attributed to inhibition of KIT,2Cahill K.N. Katz H.R. Cui J. Lai J. Kazani S. Crosby-Thompson A. et al.KIT inhibition by imatinib in patients with severe refractory asthma.N Engl J Med. 2017; 376: 1911-1920Crossref PubMed Scopus (127) Google Scholar which is essential for development, survival, and degranulation of mast cells. PGE2 was also shown to counteract mast cell–mediated bronchoconstiction.8Torres R. Picado C. de Mora F. The PGE2-EP2-mast cell axis: an antiasthma mechanism.Mol Immunol. 2015; 63: 61-68Crossref PubMed Scopus (26) Google Scholar Therefore, increased levels of PGE2 may contribute to the beneficial effects of imatinib in lung conditions. In conclusion, our data provide indirect evidence that imatinib causes pronounced inhibition of thromboxane synthase, thereby redirecting PGH2 toward increased PGE2 synthesis. PGE2 in turn activates the EP4 receptor and inhibits NF-kB activation and cytokine release. Given that various chronic inflammatory conditions could possibly benefit from increased PGE2 levels, the potential of already registered drugs that are capable of enhancing PGE2 levels along with other anti-inflammatory effects is intriguing. Because chronic inflammation is also known to promote leukemogenesis,9Koschmieder S. Mughal T.I. Hasselbalch H.C. Barosi G. Valent P. Kiladjian J.J. et al.Myeloproliferative neoplasms and inflammation: whether to target the malignant clone or the inflammatory process or both.Leukemia. 2016; 30: 1018-1024Crossref PubMed Scopus (103) Google Scholar the PGE2 mediated anti-inflammatory effects of BCR-ABL inhibitors may add to their therapeutic potential in leukemia. We thank M. Ofner and I. Lanz for their excellent technical work. All laboratory reagents were from Sigma (Vienna, Austria), unless specified. The EP4 receptor antagonist ONO AE3-208, ONO 8711, imatinib mesylate, and nilotinib were from Cayman Chemicals (Ann Arbor, Mich). Imatinib mesylate and nilotinib were dissolved in dimethyl sulfoxide and further diluted in PBS to a final concentration of the solvent less than 0.1%. Blood was obtained from nonatopic volunteers, who were not taking any medication, according to a protocol approved by the Ethics Committee of the Medical University of Graz (17-291 ex 05/06) after informed consent was provided. Before blood sampling all donors signed an informed consent form. Citrated blood (40 mL) was centrifuged at 300g for 20 minutes at room temperature to remove platelet-rich plasma. Erythrocytes were removed by dextran sedimentation. PBMCs (containing monocytes and lymphocytes), were isolated by Histopaque gradient centrifugation, and washed with PBS containing 5.5 mM glucose and 2.7 mM KCl. Thereafter, cells were resuspended in RPMI 164 supplemented with penicillin (100 U/mL), streptomycin (100 U/mL), glutamine (20 mM), nonessential amino acids, HEPES 0.05 mol, and sodium pyruvate 10 mM (all obtained from Gibco, Thermo Fisher Scientific, Vienna, Austria); 0.5 × 106 mononuclear cells/0.5 mL were placed in 24-well plates and 25 μL of human AB serum was added. After 120 minutes of incubation at 37oC (5% CO2), the nonadherent cells (about 85% of the original mononuclear cells) were removed by aspiration and the plate was washed 3 times with 500 μL PBS. The adherent monocytes were cultured in 1 mL supplemented RPMI 1640 medium at 37oC (5% CO2). Monocytes were isolated from PBMCs by magnetic separation using Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated monocytes were seeded at a density of 5 × 105 monocytes/mL in 24-well culture plates in RPMI 1640 differentiation medium containing 10% FBS, penicillin (100 U/mL), streptomycin (100 U/mL), and 50 ng/mL M-CSF (Pepro Tech, Vienna, Austria). The medium was changed every third day and after 8 days the medium was replaced by RPMI 1640 with 0.5% FBS overnight before stimulation. Adherent monocytes and macrophages isolated from the blood of healthy donors (isolation and culture conditions as described above) were exposed to vehicle or endotoxin (LPS, Escherichia coli serotype 055:B5) 10 ng/mL for adherent monocytes or 1 μg/mL for monocyte-derived macrophages, and incubated at 37oC for indicated time periods. Thereafter, the supernatants were collected for radioimmunological determination of PGE2, and ELISA for TNF-α and TXB2 (the stable hydrolysis product of TXA2), and cell viability was determined. In additional experiments, PGs were detected by mass spectrometry. Imatinib and nilotinib or furegrelate were added 20 minutes before LPS, whereas pretreatment with drugs, such as the p38 MAPK inhibitor SB202190, the MEK1/2 inhibitor PD184161, as well as the JNK inhibitor SP600125, the PI3K inhibitor LY294002, the cyclooxygenase inhibitor diclofenac, or the EP4 antagonist ONO AE3-208, started 20 minutes before the addition of imatinib and nilotinib. Aliquots (0.5 mL) of blood samples containing 10 IU/mL sodium heparin were incubated in polystyrene tubes. Imatinib, nilotinib, or vehicle was added 20 minutes before addition of LPS (10 μg/mL)E1Patrignani P. Panara M.R. Sciulli M.G. Santini G. Renda G. Patrono C. Differential inhibition of human prostaglandin endoperoxide synthase-1 and -2 by nonsteroidal anti-inflammatory drugs.J Physiol Pharmacol. 1997; 48: 623-631PubMed Google Scholar and incubation at 37oC lasted for 4 hours. Concentrations of nilotinib and imatinib in this assay were chosen with regard to plasma concentrations in patients.E2Peng B. Lloyd P. Schran H. Clinical pharmacokinetics of imatinib.Clin Pharmacokinet. 2005; 44: 879-894Crossref PubMed Scopus (489) Google Scholar, E3Fava C. Kantarjian H. Cortes J. Jabbour E. Development and targeted use of nilotinib in chronic myeloid leukemia.Drug Des Devel Ther. 2009; 2: 233-243PubMed Google Scholar, E4Xia B. Heimbach T. He H. Lin T.H. Nilotinib preclinical pharmacokinetics and practical application toward clinical projections of oral absorption and systemic availability.Biopharm Drug Dispos. 2012; 33: 536-549Crossref PubMed Scopus (25) Google Scholar Thereafter, blood was centrifuged at 2000g for 10 minutes and PGE2, TXB2, and TNF-α were determined in plasma. For experiments involving whole blood from patients receiving imatinib for the treatment of chronic myelogenous leukemia, assays were performed as described above with minor modifications. Aliquots of 2.5 mL of heparinized whole blood from patients or age- and sex-matched controls were incubated with vehicle or LPS (10 μg/mL) for 4 hours at 37°C. Blood was centrifuged, plasma was frozen at −80°C, and analyzed for eicosanoids by means of mass spectrometry, and for various cytokines by multiplex ELISA (BD Biosciences). Immunoreactive PGE2 was determined as described previously.E5Amann R. Schuligoi R. Inhibition of carrageenan-induced edema by indomethacin or sodium salicylate does not prevent the increase of nerve growth factor in the rat hind paw.Neurosci Lett. 2000; 278: 173-176Crossref PubMed Scopus (23) Google Scholar TNF-α and TXB2 were determined using commercially available ELISA kits (PeproTech, Rocky Hill, NJ, and Cayman, respectively); other cytokines were determined by multiplex ELISA (Human 13-Plex Human ProcartaPlex Panel 2, Thermo Fisher Scientific) according to the instructions. Prostanoids were quantified as described previously.E6Barnthaler T. Maric J. Platzer W. Konya V. Theiler A. Hasenohrl C. et al.The role of PGE2 in alveolar epithelial and lung microvascular endothelial crosstalk.Sci Rep. 2017; 7: 7923Crossref PubMed Scopus (23) Google Scholar In brief, 200 μL supernatant or plasma was spiked with the isotopically labeled internal standards and extracted using ethyl acetate. The chromatographic separation of the analytes was carried out using a chiral column Lux Amylose-2 (150 × 2 mm inner diameter, 3 μm) coupled to a Synergi Hydro-RP column (150 × 2 mm inner diameter, 4-μm; both from Phenomenex, Aschaffenburg, Germany) under gradient conditions. Water and acetonitrile, both containing 0.1% formic acid, were used as mobile phases and sample run time was 22 minutes. The MS/MS system consisted of a hybrid triple quadrupole-ion trap mass spectrometer QTrap 5500 (Sciex, Darmstadt, Germany) equipped with a Turbo-V-source operating in the negative electrospray ionisation mode. Analysis was done in the Multiple Reaction Monitoring mode with a dwell time of 50 ms for all analytes. Data were acquired using Analyst Software V 1.6 and quantified with MultiQuant Software V 3.0 (both Sciex, Darmstadt, Germany), using the internal standard method (isotope dilution mass spectrometry). U937T cells containing NF-κB–GFP reporter cassetteE7Jorgl A. Platzer B. Taschner S. Heinz L.X. Hocher B. Reisner P.M. et al.Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB.Blood. 2007; 109: 185-193Crossref PubMed Scopus (27) Google Scholar were kindly provided by H. Strobl (Medical University of Graz). U937T cells were maintained in RPMI 1640 with stable glutamine culture medium (PAA Laboratories) containing 10% FCS and 1 × Pen/Strep (PAA Laboratories). Cells were seeded at a density of 3 × 105 cells/well in 24-well plates and treated with vehicle, imatinib, or nilotinib 20 minutes before stimulation with LPS (100 ng/mL). Twenty hours later cells were harvested and NF-κB activation was measured by flow cytometry as an increase in GFP fluorescence (FacsCalibur, BD Biosciences, Schwechat, Austria). Pretreatment with diclofenac (1 μM) or ONO-AE3 208 (300 nM) was started 20 minutes before addition of imatinib or nilotinib. Aggregation was measured as the increase in light transmission for 6 minutes starting with the addition of proaggregatory stimuli, arachidonic acid (10 μM), or PGH2 (12 μM). Furegrelate, imatinib, or nilotinib was added 10 minutes before stimulation. In additional experiments platelets were pretreated with acetylsalicylic acid (100 μM) 15 minutes before stimulation. Data were expressed as percent of maximum light transmission, with nonstimulated washed platelets being 0% and Tyrode buffer 100%. At the end of the stimulation period, the platelet suspension was rapidly transferred into Eppendorf tubes and centrifuged at 12,000g for 2 minutes. The supernatant was stored at −80 °C. Experiments were performed as previously described with some modifications.E8Wade M.L. Fitzpatrick F.A. Nitric oxide modulates the activity of the hemoproteins prostaglandin I2 synthase and thromboxane A2 synthase.Arch Biochem Biophys. 1997; 347: 174-180Crossref PubMed Scopus (37) Google Scholar Platelet-rich plasma (obtained as described above) was centrifuged at 2000g at room temperature for 15 minutes without breaks to pellet platelets. Platelets were resuspended in 50 mM Tris-HCl (pH = 7.5) and sonicated 3 times for 20 seconds on ice. Thereafter, the resulting suspension was centrifuged at 8000g for 15 minutes at 4°C and the supernatant was centrifuged at 100,000g for 60 minutes at 4°C, the pellet was resuspended in 1.5 mL of 50 mM Tris-HCl, and frozen at −80° C. For thromboxane synthase activity assays, the protein concentrations were adjusted to 150 μg protein/mL and microsomes were preincubated with indicated substances for 10 minutes and then 12 μM PGH2 was added to start the reaction. After 2 minutes at room temperature, samples were snap frozen on liquid nitrogen and stored at −80°C until determination of TXB2 concentration. Within each experiment, samples obtained from 1 individual donor were assayed in duplicates for immunoreactive PGE2, TXB2, and TNF-α. The resulting average was used to calculate means ± SEM of n independent determinations. To determine the effect of drug treatment, data are either given as absolute values or were calculated as percent of control (LPS stimulation or vehicle treatment alone) and expressed as means + SEM. Statistical analysis was calculated with GraphPad Prism 7.0a (GraphPad Software, Inc, La Jolla, Calif) using 1-way ANOVA for repeated measurements or 2-sided Student t test when appropriate; multiple comparisons were done with Tukey or Dunnett post test. For time course experiments, 2-way ANOVA for repeated measurements and Sidak multiple comparison test values was calculated. A P value of less than .05 was considered as significant. The p38 MAPK inhibitor SB202190 (2 μM), the MEK1/2 inhibitor PD184161 (1 μM), as well as the JNK inhibitor SP600125 (10 μM) and the PI3K inhibitor LY294002 (20 μM) alone all caused a pronounced inhibition of PGE2 release from LPS-stimulated (10 ng/mL) adherent monocytes as compared with vehicle treatment (Fig E6, A). The imatinib- and nilotinib-induced increase in PGE2 release was still evident; however, in the presence of MEK1/2 and PI3K inhibitors, very low amounts were released (Fig E6, A). TNF-α release was significantly reduced by treatment with SB202190, PD184161, and SP600125, but not LY294002. The inhibitory effect of imatinib and nilotinib on TNF-α release seemed to depend on the amount of PGE2 that was still released in the presence of inhibitors (Fig E6, B). In PD184161- and LY294002-treated samples, the inhibitory effect of imatinib and nilotinib on TNF-α release was abrogated (Fig E6, B); however, when exogenous PGE2 (10 ng/mL) was added, the inhibitory effect was restored (Fig E6, C).Fig E2The inhibition of LPS-induced TNF-α release in monocytes by imatinib is not affected by EP1, EP2, or EP3 receptor antagonism. Monocytes were pretreated with the EP1 antagonist ONO8711 (1 μM), the EP2 antagonist PF04418948 (1 μM), or the EP1, EP2, and EP3 antagonist AH6809 (10 μM) 20 minutes before addition of imatinib (1 μΜ) followed by addition of LPS (10 ng/mL) and incubation for another 20 hours. Thereafter, the concentrations of (A) TNF-α and (B) PGE2 were determined in the supernatants. All experiments were done in duplicate. Values were calculated as percent of LPS stimulation alone, and are shown as mean + SEM of 6 independent experiments. Statistical analysis was performed with 1-way ANOVA followed by Tukey multiple comparison test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3The inhibition of TNF-α release by imatinib and nilotinib depends on PGE2 release and EP4 receptor activation in monocyte-derived macrophages. Cells were pretreated with diclofenac (diclo, 1 μΜ) or the EP4 antagonist ONO AE3-208 (0.3 μM), 20 minutes before addition of imatinib (1 μΜ) or nilotinib (0.05 μM), followed by addition of LPS (10 ng/mL) and incubation for another 20 hours. Thereafter, the concentrations of (A) TNF-α and (B) PGE2 were determined in the supernatants. All experiments were done in duplicate. Values were calculated as percent of LPS stimulation alone, and are shown as mean + SEM of 6 independent experiments. Statistical analysis was performed with 1-way ANOVA for repeated measurements followed by Tukey multiple comparison test. *P < .05, **P < .01, and ***P < .001 as compared with LPS stitmulation. #P < .05 as compared with imatinib or nilotinib treatment alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Imatinib and nilotinib concentration-dependently increase PGE2 and inhibit TXB2 in human whole blood; the inhibition of TNF-α release depends on PGE2 release and EP4 receptor activation. Whole blood was treated with vehicle (veh) or with (A and C) imatinib or (B and D) nilotinib at the indicated concentrations 20 minutes before stimulation with LPS (10 μg/mL) for 4 hours. In E and F, whole blood was treated with diclofenac (diclo, 1 μM) or the EP4 antagonist ONO AE3-208 (0.3 μM), 20 minutes before addition of imatinib or nilotinib at the indicated concentrations, followed 20 minutes later by addition of LPS (10 μg/mL) and incubation for 4 hours. Thereafter, the concentrations of (Fig E4, A, B, and F) PGE2, (Fig E4, C, D, and E), TNF-α, and (G and H) TXB2 were determined in plasma. Values are calculated as percent of vehicle (LPS treatment) and shown as mean + SEM of 8 to 9 independent experiments. ND, Not detectable. Statistical analysis was performed with 1-way ANOVA for repeated measurements followed by Dunnett test; *P < .05, **P < .01, and ***P < .001 as compared with vehicle stimulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Nilotinib concentration- and time-dependently promotes PGE2 biosynthesis via thromboxane synthase inhibition and abrogates LPS-induced TNF-α release through PGE2 and EP4 receptor activation. A-C, Monocytes were pretreated with vehicle (veh) or nilotinib with the indicated concentrations, before cells were stimulated with LPS (10 ng/mL) for 20 hours. D and E, In timecourse experiments, monocytes were treated with vehicle or nilotinib (0.05 μM) before stimulation with LPS and incubated for the indicated time points. G and H, Monocytes were pretreated with vehicle, diclofenac (diclo, 1 μM), or the EP4 antagonist ONO AE3-208 (0.3 μM) before addition of nilotinib, followed by addition of LPS (10 ng/mL) for another 20 hours. (Fig E5, A, D, and G) TNF-α, (Fig E5, B, E, and H) PGE2, and (Fig E5, C and F) TXB2 were determined in the supernatants. N = 6 to 11; values were calculated as percent of LPS stimulation alone, and are shown as mean + SEM. A-C, G, and H, Statistical analysis was performed with 1-way ANOVA for repeated measurements followed by Dunnett test. *P < .05, **P < .01, ***P < .001. D-F, Timecourse experiments were analyzed by 2-way ANOVA for repeated measurements followed by Sidak multiple comparison test. *P < .05, **P < .01, ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Inhibition of kinases has no direct effect on imatinib- and nilotinib-induced PGE2 release and the inhibition of LPS-induced TNF-α release. A and B, Adherent monocytes were pretreated with vehicle (veh), the p38MAPK inhibitor SB202190 (p38), the MEK1/2 inhibitor PD184161 (MEK1/2), the JNK inhibitor SP600125 (JNK), and the PI3K inhibitor LY294002 (PI3K), 20 minutes before addition of imatinib (1 μM) or nilotinib (0.05 μM) followed by addition of LPS (10 ng/mL) and incubation for 20 hours. C, To test whether the abrogation of the inhibitory effect of imatinib by PI3K and MEK1/2 inhibition was due to inhibition of PGE2 release, cells were pretrated with LY294002 and PD184161 as described for (Fig E6, A and B) and PGE2 (10 ng/mL) was added 20 minutes before stimulation with LPS. Thereafter, the concentrations of (Fig E6, A) PGE2 and (Fig E6, B and C) TNF-α were determined in the supernatants. All experiments were done in duplicate. Values are calculated as ng/mL, and shown as mean + SEM of 5 to 8 independent experiments. Statistical analysis was performed with 1-way ANOVA for repeated measurements followed by Dunnett test for comparison to vehicle treatment; *P < .05, **P < .01, and ***P < .001 as compared with the respective vehicle treatment (without imatinib or nilotinib); #P < .05 and ###P < .001 as compared with vehicle without inhibitors.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because we found a decrease in TXB2 in imatinib- and nilotinib-treated adherent human monocytes (Fig 1, C; Fig E5, C), we next investigated this effect in washed human platelets. In platelets, arachidonic acid stimulation leads mainly to formation of TXA2 via thromboxane synthase, which subsequently promotes platelet activation and aggregation. Arachidonic acid (10 μM) stimulation caused platelet aggregation and TXB2 formation. Imatinib (1 μM), nilotinib (0.05 μM), and the thromboxane synthase inhibitor furegrelate (0.3-10 μM) significantly inhibited TXB2 release (Fig E7, A) without showing an effect on platelet aggregation (Fig E7, B). Inhibition of TXB2 formation by these compounds was also observed in human platelet microsomes, where the thromboxane synthase is located,E9Needleman P. Moncada S. Bunting S. Vane J.R. Hamberg M. Samuelsson B. Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides.Nature. 1976; 261: 558-560Crossref PubMed Scopus (401) Google Scholar after addition of PGH2 (12 μM; Fig E7, E). However, both the arachidonic acid–induced aggregation and TXB2 release were abolished after pretreatment of platelets with acetylsalicylic acid (100 μM, data not shown), indirectly suggesting that inhibition of thromboxane synthase leads to accumulation of PGH2. Because it is well known that PGH2 itself causes platelet aggregation by activation of the TP-receptor,E10Vezza R. Mezzasoma A.M. Venditti G. Gresele P. Prostaglandin endoperoxides and thromboxane A2 activate the same receptor isoforms in human platelets.Thromb Haemost. 2002; 87: 114-121Crossref PubMed Scopus (39) Google Scholar we stimulated aggregation of washed platelets with PGH2 (12 μM), which was accompanied by a pronounced increase in TXB2 (Fig E7, C). As expected, neither imatinib nor nilotinib or furegrelate had an effect on aggregation (Fig E7, D) while TXB2 release was abrogated.Table E1Demographic and laboratory parameters of imatinib-treated patients and controlsSubjectsFemale/maleAge (y)CR>MMR 4.0WBC (109/L)Dosing imatinib (mg/d)Patients with CML8/1159 (14)19/1918/195.1 (1.79)400 (100)Controls8/1160 (10)NANANDNAValues are given as medians (interquartile range).CML, Chronic myelogenic leukemia; CR, complete remission; MMR, major molecular response; NA, not applicable; ND, not done; WBC, white blood cell count. Open table in a new tab Values are given as medians (interquartile range). CML, Chronic myelogenic leukemia; CR, complete remission; MMR, major molecular response; NA, not applicable; ND, not done; WBC, white blood cell count.