Pivotal Role of Phospholipase D1 in Tumor Necrosis Factor-α–Mediated Inflammation and Scar Formation after Myocardial Ischemia and Reperfusion in Mice
2014; Elsevier BV; Volume: 184; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2014.06.005
ISSN1525-2191
AutoresTanja Schönberger, Tobias Jürgens, Julia Müller, Nicole Armbruster, Christina Niermann, Simone Gorreßen, Jan Sommer, Huasong Tian, Gilbert Di Paolo, Jürgen Scheller, Jens W. Fischer, Meinrad Gawaz, Margitta Elvers,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoMyocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α–mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-β secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1−/− mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1−/− mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α–mediated inflammation and transforming growth factor-β–mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion. Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α–mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-β secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1−/− mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1−/− mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α–mediated inflammation and transforming growth factor-β–mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion. Heart disease is one of the major causes of morbidity and mortality and the leading cause of death in Western countries.1Murray C.J. Lopez A.D. Global mortality, disability, and the contribution of risk factors: global Burden of Disease Study.Lancet. 1997; 349: 1436-1442Abstract Full Text Full Text PDF PubMed Scopus (3375) Google Scholar Cardiovascular disease, in particular myocardial infarction (MI), results from coronary atherosclerosis, a chronic disease with stable and unstable periods.2Thygesen K. Alpert J.S. White H.D. Jaffe A.S. Apple F.S. Galvani M. et al.Universal definition of myocardial infarction.Circulation. 2007; 116: 2634-2653Crossref PubMed Scopus (2133) Google Scholar Unstable periods are characterized by activated inflammation in the vascular wall and may lead to MI.2Thygesen K. Alpert J.S. White H.D. Jaffe A.S. Apple F.S. 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Phospholipid mediators in the vessel wall: involvement in atherosclerosis.Curr Opin Clin Nutr Metab Care. 2005; 8: 123-131Crossref PubMed Scopus (66) Google Scholar Phospholipase D (PLD) belongs to a group of phospholipases that are responsible for the degradation of phospholipids in the plasma membrane. PLD catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid (PA) and choline.6McDermott M. Wakelam M.J. Morris A.J. Phospholipase D.Biochem Cell Biol. 2004; 82: 225-253Crossref PubMed Scopus (305) Google Scholar PA, as well as its metabolites lysophosphatidic acid (LPA) and diacylglycerol, are important second messengers.7English D. Phosphatidic acid: a lipid messenger involved in intracellular and extracellular signalling.Cell Signal. 1996; 8: 341-347Crossref PubMed Scopus (178) Google Scholar PA directly stimulates the synthesis of phosphatidylinositol 4,5-bisphosphate by activating phosphatidylinositol-4-phosphate 5-kinase8Honda A. Nogami M. Yokozeki T. Yamazaki M. 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Sanematsu F. Kanai M. Hasegawa H. Tanaka Y. Shibasaki M. Kanaho Y. Sasaki T. Frohman M.A. Fukui Y. Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.Science. 2009; 324: 384-387Crossref PubMed Scopus (217) Google Scholar cell migration,10Knoepp S.M. Chahal M.S. Xie Y. Zhang Z. Brauner D.J. Hallman M.A. Robinson S.A. Han S. Imai M. Tomlinson S. Meier K.E. Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells.Mol Pharmacol. 2008; 74: 574-584Crossref PubMed Scopus (64) Google Scholar and modulates integrin-mediated cell adhesion.11Powner D.J. Pettitt T.R. Anderson R. Nash G.B. Wakelam M.J. Stable adhesion and migration of human neutrophils requires phospholipase D-mediated activation of the integrin CD11b/CD18.Mol Immunol. 2007; 44: 3211-3221Crossref PubMed Scopus (29) Google Scholar The analysis of Pld1−/− mice identified PLD1 as a critical regulator of platelet activity essential for integrin αIIbβ3 activation and shear-dependent thrombus formation. Furthermore, the lack of PLD1 leads to protection against arterial thrombosis and ischemic brain infarction.12Elvers M. Stegner D. Hagedorn I. Kleinschnitz C. Braun A. Kuijpers M.E. Boesl M. Chen Q. Heemskerk J.W. Stoll G. Frohman M.A. Nieswandt B. Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.Sci Signal. 2010; 3: ra1Crossref PubMed Scopus (156) Google Scholar Different studies have shown that oxidized low-density lipoprotein mediates the accumulation of PA and LPA mediated by the activation of PLD, thereby promoting the proliferation of smooth muscle cells and atherosclerosis.13Natarajan V. Scribner W.M. Hart C.M. Parthasarathy S. Oxidized low density l ipoprotein-mediated activation of phospholipase D in smooth muscle cells: a possible role in cell proliferation and atherogenesis.J Lipid Res. 1995; 36: 2005-2016Abstract Full Text PDF PubMed Google Scholar, 14Siess W. Tigyi G. Thrombogenic and atherogenic activities of lysophosphatidic acid.J Cell Biochem. 2004; 92: 1086-1094Crossref PubMed Scopus (94) Google Scholar, 15Xu Y.J. Aziz O.A. Bhugra P. Arneja A.S. Mendis M.R. Dhalla N.S. Potential role of lysophosphatidic acid in hypertension and atherosclerosis.Can J Cardiol. 2003; 19: 1525-1536PubMed Google Scholar By using antisense oligonucleotides, PLD1 was shown to be involved in inflammatory processes. PLD1 activity contributes to increased expression of tumor necrosis factor-α (TNF-α) and matrix metalloproteinases such as matrix metalloproteinase 9.16Gomez-Cambronero J. New concepts in phospholipase D signaling in inflammation and cancer.ScientificWorldJournal. 2010; 10: 1356-1369Crossref PubMed Scopus (47) Google Scholar, 17Issuree P.D. Pushparaj P.N. Pervaiz S. Melendez A.J. Resveratrol attenuates C5a-induced inflammatory responses in vitro and in vivo by inhibiting phospholipase D and sphingosine kinase activities.FASEB J. 2009; 23: 2412-2424Crossref PubMed Scopus (52) Google Scholar, 18Sethu S. Pushparaj P.N. Melendez A.J. Phospholipase D1 mediates TNFalpha-induced inflammation in a murine model of TNFalpha-induced peritonitis.PLoS One. 2010; 5: e10506Crossref PubMed Scopus (23) Google Scholar However, the function and regulation of PLD1 in inflammatory cardiovascular diseases, especially in processes of myocardial ischemia/reperfusion (I/R) as well as in myocardial repair, have not been explored to date. Here, we show that PLD1 is crucial for the TNF-α–induced inflammatory response that mediates NF-κB activation, the release of cytokines, cell adhesion, and migration of neutrophils and monocytes/macrophages. Specific pathogen-free C57BL/6J mice were obtained from Charles River (Sulzfeld, Germany). Pld mutant mice were described previously.19Dall'Armi C. Hurtado-Lorenzo A. Tian H. Morel E. Nezu A. Chan R.B. Yu W.H. Robinson K.S. Yeku O. Small S.A. Duff K. Frohman M.A. Wenk M.R. Yamamoto A. Di Paolo G. The phospholipase D1 pathway modulates macroautophagy.Nat Commun. 2010; 1: 142Crossref PubMed Scopus (131) Google Scholar Animal studies were performed in accordance with the guidelines for the use of living animals in scientific studies, in accordance with German law for the protection of animals, and was approved by the Regional Council Tübingen (Regierungspräsidium Tübingen). Real-time RT-PCR analysis was performed using FastStart Universal SYBR Green Master (Rox; Roche, Basel, Switzerland) as a fluorogenic probe as described previously.20Schonberger T. Ziegler M. Borst O. Konrad I. Nieswandt B. Massberg S. Ochmann C. Jurgens T. Seizer P. Langer H. Munch G. Ungerer M. Preissner K.T. Elvers M. Gawaz M. The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice.Am J Physiol Cell Physiol. 2012; 303: C757-C766Crossref PubMed Scopus (61) Google Scholar Briefly, for the assessment of endogenously expressed PLD1, TNF-α, and IL-1, respectively, total RNA from the left ventricle of the heart of healthy mice and of mice that underwent ischemia and 24 hours of reperfusion was extracted. After reverse transcription, quantitative PCR amplification was performed using the following oligonucleotide primers: forward 5′-TGCTGAGATACCGCTGCAACTTAG-3′, reverse 5′-CTTGGCACCCTTGAGGTCGATG-3′ (PLD1); forward 5′-GCCCCCACTCTGACCCCTTT-3′, reverse 5′-GGGGCTGGCTCTGTGAGGAA (TNF-α); and forward 5′-ACCTTCGGCTTCAAAATGCCAGTT-3′, reverse 5′-CACCTAGAAAACCCTGCTGCGA-3′ (IL-1α). Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA expression levels as an internal control. Ten- to 12-week-old Pld+/+ and Pld−/− mice were anesthetized by intraperitoneal injection of a solution of 5 mg midazolam, 0.5 mg medetomidine, and 0.05 mg fentanyl each per kilogram of body weight. Myocardial ischemia was induced in Pld+/+ and Pld−/− mice by ligation of the left anterior descending artery (LAD) for 30 minutes. After 1 day of reperfusion the ischemic area (area at risk) was defined by negative staining with 4% Evans Blue (after re-ligation of the LAD at the level marked by the suture left in place), and the infarcted area (infarct size) was detected by triphenyltetrazolium staining (Sigma-Aldrich, St. Louis, MO). The ratio of infarct size/area at risk is an accurate measure to analyze infarct size within ischemic myocardium and is the primary end point, which determines the effect of the treatment strategy. Areas were quantified digitally by video planimetry. After 28 days of reperfusion, the infarct size was determined by Gomori's One Step Trichrome staining (Dako, Glostrup, Denmark). Animals were sacrificed and serial sections of the hearts (10 per mouse, 250 μm apart, up to the mitral valve) were fixed in 4% formalin, embedded in paraffin, and stained with Gomori's One Step Trichome staining. Determination of infarct size was performed using Diskus software version 2005 (Hilgers, Königswinter, Germany) and was expressed as the percentage of total left ventricular (LV) volume. Twenty-eight days after MI an echocardiography was performed, as described previously.3Baumer Y. Leder C. Ziegler M. Schonberger T. Ochmann C. Perk A. Degen H. Schmid-Horch B. Elvers M. Munch G. Ungerer M. Schlosshauer B. Gawaz M. The recombinant bifunctional protein alphaCD133-GPVI promotes repair of the infarcted myocardium in mice.J Thromb Haemost. 2012; 10: 1152-1164Crossref PubMed Scopus (11) Google Scholar Analysis of fractional shortening and ejection fraction was performed with Vevo2100 software version 1.5.0 (VisualSonics, Toronto, ON, Canada). Echocardiography was performed using a VEVO 2100 ultrasound machine and a MS400 (18 to 38 MHz) linear transducer (both from VisualSonics) as described recently.21Borst O. Ochmann C. Schonberger T. Jacoby C. Stellos K. Seizer P. Flogel U. Lang F. Gawaz M. Methods employed for induction and analysis of experimental myocardial infarction in mice.Cell Physiol Biochem. 2011; 28: 1-12Crossref PubMed Scopus (35) Google Scholar, 22Thum T. Gross C. Fiedler J. Fischer T. Kissler S. Bussen M. Galuppo P. Just S. Rottbauer W. Frantz S. Castoldi M. Soutschek J. Koteliansky V. Rosenwald A. Basson M.A. Licht J.D. Pena J.T. Rouhanifard S.H. Muckenthaler M.U. Tuschl T. Martin G.R. Bauersachs J. Engelhardt S. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.Nature. 2008; 456: 980-984Crossref PubMed Scopus (1929) Google Scholar Paraffin-embedded cardiac sections taken 1 day after MI were stained with hematoxylin and eosin. Immune staining of infarcted cardiac sections was performed with a streptavidin-biotin-immunoperoxidase method (Dako). The total number of cells was referred to a certain tissue area; the area was divided into grids, and data are shown per mm2. Paraffin-embedded cardiac sections were stained with an anti-Mac3 monoclonal antibody (BD Biosciences, San Diego, CA), an antipolymorphonuclear monoclonal antibody (GeneTex, Irvine, CA), and isotype control antibodies according to standard protocols. The percentage of Mac3-positive and polymorphonuclear-positive cells was counted from six random areas in the infarcted myocardium of each heart sample (n = 4) in a blinded manner (N.A.). For the analysis of PLD1 expression in monocytes of infarct areas, paraffin-embedded cardiac sections were stained with an anti-Mac3 monoclonal antibody (BD Biosciences) and a polyclonal PLD1 antibody (Cell Signaling, Danvers, MA). PLD activity was measured in an enzymatically coupled fluorescent in vitro assay (Amplex Red Phospholipase D Assay Kit; Molecular Probes, Eugene, OR) as described previously.23Elvers M. Grenegard M. Khoshjabinzadeh H. Munzer P. Borst O. Tian H. Di Paolo G. Lang F. Gawaz M. Lindahl T.L. Falker K. A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity.Cell Signal. 2012; 24: 1743-1752Crossref PubMed Scopus (13) Google Scholar Briefly, cell lysates were mixed with 100 μL of the Amplex Red reaction buffer. The PLD activity was determined for each sample by measuring fluorescence activity after 1 hour of incubation at 37°C in the dark with the GloMax-Multi detection system (Promega, Madison, WI). A standard curve with different concentrations ranging from 0 to 250 mU/mL was performed using purified PLD from Streptomyces chromofuscus (Sigma-Aldrich). Murine monocytes were freshly isolated from whole blood of Pld+/+ and Pld−/− mice. Four hundred grams of whole blood was centrifuged for 20 minutes at room temperature. Lymphocytes were separated using Biocoll Separating solution (Biochrom, Berlin, Germany) and centrifugation for an additional 10 minutes. Lysis of erythrocytes was followed by cell labeling using 10 μL CD11b Micro Beads (Miltenyi Biotec, Bergisch Gladbach, Germany) per 107 cells. Monocytes were separated with a Vario Macs Separator (Miltenyi Biotec) and counted. For the quantification of TNF-α and IL-1α in plasma after MI and in 1 μg/mL lipopolysaccharide (LPS)-stimulated monocytes from Pld+/+ and Pld−/− mice, supernatant was taken and the release of TNF-α and IL-1α, respectively, was measured following the manufacturer's protocol (Quantikine Mouse TNF-α Immunoassay, Quantikine Mouse IL-1α Immunoassay; R&D Systems, Minneapolis, MN). Transforming growth factor (TGF)-β in plasma of Pld+/+ and Pld−/− mice was quantified 3 days after MI following the manufacturer's protocol (Quantikine Mouse TGF-β Immunoassay; R&D Systems). Freshly isolated murine monocytes from whole blood of Pld+/+ and Pld−/− mice were seeded onto uncovered wells and stimulated with LPS for 6 hours to induce an immune response by the generation of cytokines such as TNF-α. Cells were stained with an anti-p65 antibody (Santa Cruz Biotechnology, Dallas, TX) according to a standard protocol. Chemotaxis was performed using a 48-well modified Boyden chamber (Neuro Probe, Inc., Gaithersburg, MD). Monocytes were added to the upper chamber and medium containing monocyte chemotactic protein 1 or medium alone was added to the lower compartment. The cultures were incubated for 4 hours at 37°C. Finally, cells in the lower chamber were counted using CellTiter-Glo (Promega). For adhesion experiments under flow conditions, 2 × 105 cells/mL activated murine endothelial 5-T cells were allowed to adhere onto glass coverslips until they were confluent and then were activated overnight (20 hours) with 100 ng/mL TNF-α (Pepro Tech, Hamburg, Germany). Monocytes were perfused over glass coverslips using arterial shear rates (flow rate, 7.53 mL/hour; shear rate, 1000 seconds−1). After perfusion, all experiments were recorded in real time and evaluated offline. α-Smooth muscle actin (α-SMA) staining of paraffin sections was performed using anti–α-SMA antibody (1:200, ab5649; Abcam, Cambridge, UK), antirabbit horseradish peroxidase as a secondary antibody (1:200; Santa Cruz Biotechnology, Dallas, TX), and 3,3′-diaminobenzidine reagent (Zytomed, Berlin, Germany) as a chromogen. Nuclei were stained with hemalaun solution (Merck KGaA, Darmstadt, Germany). Collagen accumulation was detected by Masson's trichrome, Gomori, and picrosirius red staining, celestine blue solution was used for nuclei staining. Fibrillar collagen was analyzed by polarized light microscopy.24Freudenberger T. Oppermann M. Heim H.K. Mayer P. Kojda G. Schror K. Fischer J.W. Proatherogenic effects of estradiol in a model of accelerated atherosclerosis in ovariectomized ApoE-deficient mice.Basic Res Cardiol. 2010; 105: 479-486Crossref PubMed Scopus (22) Google Scholar Images were captured at the indicated magnifications by using an AxioImager.M2 microscope with AxioCam HRC and AxioVS40V 4.8.2.0 software (all from Zeiss, Jena, Germany). Cardiac fibroblasts were isolated from 6- to 8-week-old female Pld1−/− mice or their respective controls as described previously.25Melchior-Becker A. Dai G. Ding Z. Schafer L. Schrader J. Young M.F. Fischer J.W. Deficiency of biglycan causes cardiac fibroblasts to differentiate into a myofibroblast phenotype.J Biol Chem. 2011; 286: 17365-17375Crossref PubMed Scopus (59) Google Scholar Cells were grown in high-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 20% fetal calf serum (Invitrogen) and 8 ng/mL basic fibroblast growth factor (BPS Bioscience, San Diego, CA) and used for experiments in passage 2. Cardiac fibroblasts were stimulated with 100 nmol/L angiotensin II (Sigma-Aldrich) or 10 ng/mL TGF-β (Sigma-Aldrich) and harvested for gene expression analysis 24 hours after stimulation with peqGOLD TriFast (PEQLAB, Erlangen, Germany) according to the manufacturer's instructions. cDNA was synthesized and analyzed as described previously. For quantitative real-time RT-PCR the following oligonucleotide primers were used: forward 5′-CAGGCATGGATGGCATCAATCAC-3′, reverse 5′-ACTCTAGCTGTGAAGTCAGTGTCG-3′ (Acta2), and forward 5′-CTAATGGTGGACCGCAACA-3′, reverse 5′-ACTGCTTCCCGAATGTCTGA-3′ (Tgfb1). Glyceraldehyde-3-phosphate dehydrogenase was chosen as an endogenous control: forward 5′-TGGCAAAGTGGAGATTGTTGCC-3′, reverse 5′-AAGATGGTGATGGGCTTCCCG-3′. For TGF-β secretion, cells were stimulated with 100 nmol/L angiotensin II and secreted TGF-β was measured after 48 hours using a murine TGF-β enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer's protocol. Cell migration was analyzed using a modified Boyden chamber. After 6 hours the migration was stopped, migrated cells were stained with Coomassie, and the cells subsequently were analyzed with the Odyssey Near Infrared Imaging System (Li-Cor Bioscience, Lincoln, NE). Data are shown as means ± SD from at least three individual experiments (n represents the number of experiments). All data were tested for significance using the paired or unpaired Student's t-test and the U-test. P < 0.05 was considered statistically significant. For quantification of α-SMA and picrosirius red staining, ImageJ version 1.45s software (NIH, Bethesda, MD) was used. Statistical analysis was performed using GraphPad Prism software version 6.0 (La Jolla, CA). The comparison of two groups was accomplished with an unpaired t-test; a P value <0.05 was considered significant. Cardiac sections from C57BL/6J mice that underwent experimental I/R (ligation of the LAD artery) were immunostained with anti-PLD1 at different time points and compared with healthy (control) mice without MI (Figure 1A). PLD1 was highly expressed in the myocardium after MI. PLD1 accumulation occurs in the infarcted ischemic area and the border zone at 1 and 7 days after MI. PLD1 levels peaked 21 days after MI and were still fivefold higher than in healthy mice at day 28 (Figure 1, A and B). This was supported further by quantitative RT-PCR, in which we quantified the mRNA of PLD1 of the left cardiac ventricle after MI and compared the mRNA level with that of healthy mice (Figure 1C). PLD1 was strongly up-regulated 24 hours after I/R in mice, suggesting an important role in cardiovascular inflammatory disease and/or repair. To analyze the role of PLD1 in the processes of I/R injury, we tested Pld1−/− mice in a model of myocardial I/R (Figure 2). After ligation of the LAD for 30 minutes, myocardial damage was assessed by 2,3,5-triphenyltetrazolium chloride staining to differentiate between metabolically active and inactive tissue. Infarct areas after 24 hours of reperfusion were determined (Figure 2, A and B). No differences in infarct size between mice were observed. However, the analysis of cardiac sections from Pld1+/+ and Pld1−/− mice 24 hours after MI showed approximately 50% reduced migration of cells into the infarct border zone in Pld1−/− mice compared with Pld1+/+ mice as seen by hematoxylin and eosin staining (Figure 2C). Previous reports have indicated that PLD1 is important for the adhesion and migration of neutrophils and macrophages.11Powner D.J. Pettitt T.R. Anderson R. Nash G.B. Wakelam M.J. Stable adhesion and migration of human neutrophils requires phospholipase D-mediated activation of the integrin CD11b/CD18.Mol Immunol. 2007; 44: 3211-3221Crossref PubMed Scopus (29) Google Scholar, 26Lehman N. Di Fulvio M. McCray N. Campos I. Tabatabaian F. Gomez-Cambronero J. Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2.Blood. 2006; 108: 3564-3572Crossref PubMed Scopus (78) Google Scholar Based on our findings that PLD1 is up-regulated 24 hours after MI and a strongly reduced number of migrating cells into the infarct border zone of Pld1−/− mice was detected 24 hours after MI, we decided to study the influence of PLD1 on the migration and chemotactic activity of specific inflammatory cells. The analysis of macrophage recruitment into the infarct border zone 24 hours after MI showed a significant reduction of macrophage recruitment after 24 hours of reperfusion on PLD1 deficiency (0.9 ± 0.17 mm2 versus 0.6 ± 0.1 mm2; P < 0.01; anti–Mac-3) (Figure 2, D and E). Similarly, neutrophil recruitment also was reduced (0.9 ± 0.1 mm2 in Pld1−/− mice compared with 2.4 ± 0.1 mm2 in Pld1+/+ mice; P < 0.001; antipolymorphonuclear) (Figure 2, D and E). TNF-α is one of the crucial cytokines involved in inflammation and acute phase reaction.27Parameswaran N. Patial S. Tumor necrosis factor-alpha signaling in macrophages.Crit Rev Eukaryot Gene Expr. 2010; 20: 87-103Crossref PubMed Scopus (899) Google Scholar Its major role is the regulation of immune cells.27Parameswaran N. Patial S. Tumor necrosis factor-alpha signaling in macrophages.Crit Rev Eukaryot Gene Expr. 2010; 20: 87-103Crossref PubMed Scopus (899) Google Scholar Thus, we decided to explore the release of TNF-α on PLD1 deficiency to analyze if PLD1 is able to modulate TNF-α release. Indeed, a lack of PLD1 led to a prominent reduction of TNF-α expression in cardiac sections and in blood plasma 24 hours after MI (Figure 3, A and B). Besides reduced TNF-α levels, we found reduced TGF-β levels in plasma of Pld1−/− mice 72 hours after MI (Figure 3C). Monocytes and macrophages are the major source of TNF-α.27Parameswaran N. Patial S. Tumor necrosis factor-alpha signaling in macrophages.Crit Rev Eukaryot Gene Expr. 2010; 20: 87-103Crossref PubMed Scopus (899) Google Scholar Thus, we decided to analyze TNF-α signaling in PLD1-deficient monocytes and macrophages in further detail. First, we measured PLD activity in CD34+ cells and macrophages using a nonradioactive enzymatically coupled assay, in which PLD-mediated PA production was investigated with a fluorescent in vitro assay (Figure 3D). These results were confirmed by immunostaining for PLD1, showing that PLD1 accumulates in macrophages of the left ventricle (Figure 3E). The stimulation of monocytes isolated from whole blood of Pld1+/+ and Pld1−/− mice showed that LPS treatment led to a strong increase of TNF-α in Pld1+/+ cells that was strongly reduced in Pld1−/− cells, showing that PLD1 is crucial for the release of TNF-α (986.7 ± 72.4 pg/mL versus 144.9 ± 31.3 pg/mL; P < 0.001) (Figure 3F). In control experiments, normal Toll-like receptor 4 expression on the plasma membrane of Pld1−/− monocytes was detected by flow cytometry, showing that differences in TNF-α release is not caused by alterations in LPS-receptor expression (Supplemental Figure S1). TNF is an endogenous pyrogen and is able to induce IL-1 production on inflammation.28Taylor P.C. Feldmann M. Anti-TNF biologic agents: still the therapy of choice for rheumatoid arthritis.Nat Rev Rheumatol. 2009; 5: 578-582Crossref PubMed Scopus (295) Google Scholar Consequently, we analyzed the IL-1α release of Pld1+/+ and Pld1−/− monocytes to investigate if defects in TNF-α release influences the activation of the proinflammatory cytokine IL-1 as a result of PLD1 deficiency (Figure 4A). Indeed, the level of IL-1α released from Pld1−/− monocytes was strongly reduced after 24 hours after LPS stimulation (32.2 ± 3.0 pg/mL versus 5.0 ± 0.3 pg/mL) (Figure 4A). Furthermore, the stimulation of monocytes with TNF-α likewise showed reduced IL-1α levels in Pld1−/− monocytes compared with controls (Figure 4B). In the next step, we explored TNF-α expression after LPS tre
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