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Activatory Properties of Lysophosphatidic Acid on Human THP1 Cells

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Excessive leukocyte proliferation and proinflammatory mediators release represent common phenomena in several chronic inflammatory diseases. Multiple evidences identify lysophosphatidic acid (LPA), a small lipid endowed with pleiotropic activities,
  Activatory Properties of Lysophosphatidic Acid on HumanTHP-1 Cells F. D _ Aquilio, 1 M. Procaccini, 1 V. Izzi, 1 V. Chiurchiu _ , 1 V. Giambra, 1 F. Carotenuto, 2 P. Di Nardo, 2 and P. M. Baldini 1,3  Abstract   —Excessive leukocyte proliferation and proinflammatory mediators release represent com-mon phenomena in several chronic inflammatory diseases. Multiple evidences identify lysophospha-tidic acid (LPA), a small lipid endowed with pleiotropic activities, as an important modulator of bothproliferation and activation of different cell types involved in several inflammation-associated patho-logies. However, its possible role on monocyte proinflammatory activation is not fully understood yet.Aim of the present study was to investigate LPA effects on THP-1 cells in terms of proliferation,reactive oxygen intermediates (ROI) production and release of arachidonic acid-derived inflammatorymediators. Actually, LPA significantly increased both DNA synthesis and ROI production as well asprostaglandin E 2  release and the upregulation of LPA 3  receptor expression. These findings identifiedLPA as both a growth factor and a triggering mediator of proinflammatory response in THP-1 cells. KEY WORDS:  arachidonic acid metabolites; LPA receptors; lysophosphatidic acid; proliferation; reactiveoxygen intermediates. INTRODUCTION Lysophosphatidicacid(LPA),amultifunctionalbioactivephospholipid, influences a broad spectrum of biologicalresponses which are species, tissue and cell specific [1]. LPA exerts many of its activities binding to a family of G protein-coupled receptors (GPCRs), nowadays termedLPA 1 , LPA 2  and LPA 3 , which are able to activate mul-tiple intracellular signalling pathways [2].In the immune system, dendritic cells, mast cells andmacrophages are the principal LPA sources [3]. Recent evidences have shown that LPA is involved in the de-velopment of normal immunological reactions modulatingimmune cell activities and functions [4]. Moreover, LPAplays an important mitogenic role which couples with itsstimulatory activity on diverse cell types implicated in thepathogenesis of several human diseases characterized bychronic inflammation [5, 6]. Excessive cell proliferation and activation areimportant processes involved in vascular damage [7]. These processes were initially believed to exclusivelyoccur in smooth muscle cells; at present, it is knownthat they can also occur in inflammatory cells includingmonocytes/macrophages and T-lymphocytes [8]. In this context, the presence of locally released solublemediators, to whom cells respond synthesizing and 0360-3997/06/0400-0129/0 #  2006 Springer Science+Business Media, Inc.  Inflammation, Vol. 29, Nos. 4  Y  6, December 2005 ( #  2006) DOI: 10.1007/s10753-006-9008-9 129 1 Department of Biology, University of Rome  B Tor Vergata’’, Viadella Ricerca Scientifica, 00133 Rome, Italy. 2 Department of Internal Medicine, Laboratory of Cellular and Molec-ular Cardiology, University of Rome  B Tor Vergata’’, Rome, Italy. 3 To whom correspondence should be addressed at Department of Biology, University of Rome  B Tor Vergata’’, Via della RicercaScientifica, 00133 Rome, Italy. E-mail: baldini@uniroma2.it  Abbreviations : AA, Arachidonic Acid; DCF-DA, DichlorofluoresceinDiacetate; DPI, Diphenylene Iodinium; EIA, Enzyme Immunoassay;FBS, Foetal Bovine Serum; GAPDH, Glyceraldehyde PhosphateDehydrogenase; GPCR s , G-Protein Coupled Receptors; LPA,Lysophosphatidic Acid; LTB 4 , Leukotriene B 4 ; NADPHox, NADPHoxidase; PBMC, Peripheral Blood Mononuclear Cells; PGE 2 ,Prostaglandin E 2 ; ROI, Reactive Oxygen Intermediates; RT-PCR,Reverse Transcriptase-Polymerase Chain Reaction; SDS, SodiumDocecyl Sulphate; TCA, TriChloroAcetate.  releasing several biologically active molecules, such asarachidonic acid metabolites and reactive oxygenintermediates (ROI), may contribute to lesion initiationand progression [9, 10]. In particular, ROI produced by NADPH oxidase (NADPHox), the principal ROI sourcein phagocytic cells [11], have been demonstrated to playan important role in determining the oxidative stress atinflamed sites, thus worsening the effects of arachidonicacid metabolites release [12, 13]. Among the latter, prostaglandins and leukotrienes generated by cycloxy-genases and 5-lypoxygenase, respectively, are the mostpowerful inflammatory lipid mediators [14]. In order to shed light on its capability to activateTHP-1 cells, LPA effects on key events of inflammatoryresponse, such as proliferation, ROI production andrelease of arachidonic acid metabolites, were investigat-ed. Our results demonstrated that LPA is able to promoteTHP-1 cell activation leading to enhanced cell growthand simultaneous ROI and prostaglandin E 2  (PGE 2 )release probably by means of a mechanism involvingthe stimulation of LPA 3  receptor. MATERIALS AND METHODSCell Culture The human monocytic cell line THP-1, used toavoid inter- and intraspecific variability of peripheralblood mononuclear cells (PBMC), was obtained fromAmerican Type Culture Collection (Manassas, VA), cul-tured in Falcon flasks with RPMI-1640 medium supple-mentedwith10%foetalbovineserum(FBS),L-glutamine(2 mM), streptomycin (100 m g/ml), penicillin (100 U/ml),sodiumpyruvate(100 m g/ml),andmaintainedat37 - Cinahumidified atmosphere containing 5% CO 2 . Cells wereserum starved before each experiment to rule outpossible interferences with cell growth due to serumcomponents. In all experiments, to determine cell via-bility, monocyte cultures were stained with Trypan blueat specific timepoints after treatment with differentreagents. Stained vs living cells were counted under amicroscope using a Neubauer modified chamber. DNA Synthesis Assay THP-1 cells were cultured in Falcon flasks 75 cm 2 and seeded in 30  15 mm dishes with serum-freemedium (1  10 6 cells/well). Cells were challenged withdifferent concentrations of LPA (0.1  Y  20  m M) for 3, 6 and9 h. In a series of experiments, cells were pretreated withKi16425 (10  m M) for 30 min before LPA addition. [ 3 H]-thymidine incorporation into DNA was used to measurethe mitogenic response of THP-1 cells. Cells were pre-treated with Ki16425 for 30 min, pulsed with [ 3 H]-thymidine (1  m Ci/ml) and then challenged with LPA for the requested times. Cells were then harvested by cen-trifugation and treated with 5% TCA at 4 - C for 15 min.The TCA-insoluble fraction was resuspended in 0.1%SDS in NaOH (200 mM) and samples were counted for radioactivity, after addition of 3.5 ml Optifluor, by aliquid scintillation counter (Tricarb 2180/TR, PackardInstruments, Downers Grove, IL). Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) and LightCycler-PCR Analysis Total RNA isolation and purification were carriedout by SV Total RNA Isolation System (Promega)according to the manufacturer  _ s instructions. Briefly, totalRNA was extracted from 3  10 6 THP-1 cells treated or not with LPA (1  m M) for 6 h. Total RNA quantity andquality were assessed by UV absorbance at 260 nm andelectrophoresis on agarose/formaldehyde gel, respective-ly. For RT-PCR analysis, cDNA was retro-transcribedfrom 2 m g of total mRNA for 1 h at 37 - C in an incubationbuffer containing deoxynucleotide triphosphate (dNTP,250  m M each), random primers p(dN) 5  (Roche), RNaseinhibitor and RNA murine Moloney leukaemia virusreverse transcriptase (200 U, Promega). For classic PCRanalysis, an aliquot of each RT product (2  m l) wasamplified in a final volume of 50  m l using PCR buffer containing dNTP (160  m M), primers designed to amplifythe mRNA of human LPA receptors (15 pmol each,Invitrogen) and Taq polymerase (1 U Amersham Pharma-cia Biotech.). All samples were subjected to the followingreaction conditions: 30 cycles at 94 - C for 2 min, 94 - C for 30s,(60 - Cfor30sforLPA 2  and 58 - C for 30 s for LPA 1 ,LPA 3  and GAPDH), 72 - C for 30 s, followed by a finalextension at 72 - C for 3 min. Expression levels of recep-tor mRNAs in THP-1 cells were quantified by normal-ising their respective mRNAs levels to the housekeepinghuman GAPDH mRNA levels. The amplified fragmentswere then separated by electrophoresis on agarose gel1  Y  2% in TAE buffer and visualized by ultraviolettransillumination.For LightCycler-PCR, an aliquot of each RTreaction product (2  m l) was amplified using the Light-Cycler FastStart DNA master SYBR Green I (Roche 130  D _ Aquilio, Procaccini, Izzi, Chiurchiu _ , Giambra, Carotenuto, Di Nardo, and Baldini  Diagnostic) and primers (10 pmol each). The reactionsfor LPA receptors were undergoing to 95 - C for 10 minfollowed by 45 cycles 94 - C for 10 s, (60 - C for 5 s for LPA 2 , 58 - C for 10 s for LPA 3 , 60 - C for 20 s for LPA 1 ),72 - C for 15 s, and one melting curve analysis from95 - C, 65 - C for 15 s and to 95 - C for 10 min, using theLightCycler instrument (Roche). The GAPDH reactionwas undergoing to 95 - C for 10 min followed by 45cycles 94 - C for 10 s, 58 - C for 20 s, 72 - C for 15 s, andone melting curve analysis from 95 - C, 65 - C for 15 sand to 95 - C, using the LightCycler instrument (Roche).The expression levels of receptor mRNAs werequantified by normalizing the mRNA levels to thehousekeeping human GAPDH mRNA by an externalstandard curve (LightCycler operator  _ s Manual). Theexternal standard curve was created with dilutions of genomic DNA, amplified with LPA 3  primers. Evaluation of ROI Production by 2 0 ,7 0 -Dichlorofluorescein (DCF) Fluorescence The samples (2  10 6 cells) were loaded with thefluorescent indicator DCF-DA (10  m M) for 30 min at37 - C in the dark. DCF-DA diffuses through the cellmembrane and is hydrolyzed by intracellular esterase tononfluorescent DCF deacetylated, which is then rapidlyoxidized to highly fluorescent DCF in the presence of ROI. The DCF fluorescence intensity is proportional tothe amount of intracellular ROI formed [15]. After the incubation with the fluorescent dye, cells were collected,centrifuged for 5 min at 1,200 rpm at room temperature,resuspended in serum-free medium and challenged withdifferentLPA concentrations(0.1  Y  20 m M) for3,6and9h.When requested, THP-1 monocytes were pretreated withdiphenylene iodinium (DPI, 10 j 8 M), a specific inhibitor of NADPH oxidase, and Ki16425 (10  m M), a LPA 1  andLPA 3 receptorantagonist,for 1hand30min,respectively,before LPA addition. Fluorescence was measured under continuous magnetic stirring and controlled temperature(37 - C) in a Perkin-Elmer luminescence spectrometer (Model LS-5) equipped with a chart recorder (Model R100A),withexcitationwavelengthat485nmandemissionwavelengthat530nmusing5and10nmslitsrespectively,foreachlightpath.ResultswereexpressedasFluorescenceIntensity, reported as Fluorescence Units (F.U.), respect tocells loaded with only DCF-DA (C). Primer SequenceLPA 1  sense 5 0 -CGGCGGGTAGTGGTGGTC-3 0 LPA 1  antisense 5 0 -GTCGCGGTAGGAGTAAATGATG-3 0 LPA 2  sense 5 0 -GTCGAGCCTGCTTGTCTTC-3 0 LPA 2  antisense 5 0 -CCAGGAGCAGTACCACCTG-3 0 LPA 3  sense 5 0 -TCGCGGCAGTGATCAAAAACAGA-3 0 LPA 3  antisense 5 0 -ATGGCCCAGACAAGCAAAATGAGC-3 0 GAPDH sense 5 0 -CATGGGTGTGAACCATGAGAAG-3 0 GAPDH antisense 5 0 -GTGGCTGTTGAAGTCAGAGGAG-3 0 Fig. 1.  LPA effects on THP-1 cell growth. THP-1 cells were grown in RPMI-1640 plus 10% FBS and serum-starved before each experiment to rule out possible interferences with cell growth due to serum components. Cellswere challenged with different LPA concentrations (1  Y  5  2 M) for 3, 6 and 9 h. [ 3 H]-thymidine incorporation intoDNA was assessed as reported in the Materials and Methods section. Data are reported as mean  T  SD of 4different experiments.  *  P < 0.05, as reported from Student _ s  t   test in respect to untreated cells (C). 131 Properties of Lysophosphatidic Acid on Human THP-1 Cells  132  D _ Aquilio, Procaccini, Izzi, Chiurchiu _ , Giambra, Carotenuto, Di Nardo, and Baldini  Roi Generation as Assessed by Microscopy Analysis ROI generation was carried out on glass chamber slides. 3  10 6 cells were labelled with the peroxide-sensitive fluorescent dye 2 0 ,7 0 dichlorofluorescein diace-tate (DCF-DA, 10  m M) and incubated for 6 h in thepresence or absence of LPA plus or minus DPI (10 j 8 M)or Ki16425 (10  m M), as described above. Intracellular fluorescence was monitored using a fluorescencemicroscope (Leica DMRB; objective:   200). Signal-based averaging was used to quantitate the fluorescencesignal from five randomly selected fields (Delta SystemSoftware). [ 3 H]-Arachidonic Acid Release Assays THP-1 cells were seeded in 30  15 mm dishes withserum-free medium (1  10 6 cells/well) and labelled for 3 h with 1  m Ci [ 3 H]-arachidonic acid (AA) (spec. act.202.4 Ci/mmol) at 37 - C as previously described [16].To remove non-specific binding of [ 3 H]-AA to cellsurface prior to agonist stimulation, culture mediumwas eliminated, cells resuspended in serum-freemedium and challenged with different LPA concen-trations (1  Y  20  m M) for 6 h. When requested, cells werepretreated with Ki16425 (10  m M) for 30 min before LPAaddition. After treatment, supernatants were collected,centrifuged at 1,200 rpm for 5 min to remove suspendedcells and 100  m l aliquots were added to 3 ml Optifluor and analyzed by a liquid scintillator counter. Resultswere expressed as [ 3 H]-AA cpm/1  10 6 cells. Measurement of Leukotriene B 4  and ProstaglandinE 2  Release 3  10 6 cells/well were pre-treated or not withKi16425 (10  m M) for 30 min, challenged or not withLPA (1  m M) for 6 h and, at the end of experimentaltime, supernatants were collected and Leukotriene B 4 (LTB 4 ) and Prostaglandin E 2  (PGE 2 ) levels in themedium were determined by enzyme immunoassay(EIA) (Cayman Chemical), following manufacturer  _ sinstructions. Briefly, 50  m l LTB 4  standard or sampleswere added to the pre-coated mouse monoclonal anti-rabbit IgG macrotitre plates. Subsequently, 50  m l LTB 4 tracer and 50  m l monoclonal antiserum of LTB 4  wereadded into each well and the mixture was incubatedovernight at 4 - C. After incubation, the content in eachwell was removed and the wells were washed 5 timeswith PBS buffer containing 0.05% Tween-20. Analiquot of 200  m l Ellman _ s reagent was added intoeach well, and the mixture was incubated for 2 h atroom temperature with occasional shaking. The solutionoptical density was determined by a multi-well spectro-photometer (ELISA reader) at 405 nm. A similar proce-dure was performed for PGE 2  release determination,using pre-coated goat polyclonal anti-mouse IgG macro-titre plates. LTB 4  and PGE 2  concentration in each samplewas calculated as the concentration corresponding tosample _ s optic density (O.D.) values plotted on respec-tive standard built-in curves. Values were reported aspercentile increase in respect to control (C). Reagents RPMI 1640, glutamine, penicillin (100 U/ml), andstreptomycin (100  m g/ml) were from Eurobio Labora-toires. Foetal Bovine Serum (FBS) was from GIBCO(Grand Island NY, USA). LPA (C18:1, 1-oleoyl-sn-glycerol-3-phosphate), trypan blue, DPI, Ki16425 and2 0 ,7 0 -dichlorofluorescein diacetate (DCF-DA) were fromSigma Chemicals. [ 3 H]-thymidine (20 Ci/mmol), [ 3 H]-arachidonic acid (202.4 Ci/mmol) from Amersham Bio-sciences. Random primers, murine Molooney leukaemiavirus reverse transcriptase, LPA receptors and GAPDHprimers were purchased from Invitrogen. Taq polymerasewas from Amersham Pharmacia Biotech. SV Total RNAIsolation System was from Promega. LightCycler Fast-Start DNA master SYBR Green I kit and all other reagentfor LightCycler-PCR were from Roche Diagnostic.Leukotriene B 4  and Prostaglandin E 2  assay kits werepurchased from Cayman Chemical. Statistical Analysis Data distribution was preliminarily verified by theKolmogorov  Y  Smirnov test. Each experiment set wasindependently performed and compared with the samecontrol by Student _ s  t  -test. Quantitative data wereexpressed as the mean T SD of at least four replicatedeterminations, except where otherwise indicated. Dif- Fig. 2.  LPAeffects on ROI production inTHP-1 cells. THP-1cells werelabelledwithDCF-DA(10 2 M) asreportedintheMaterials and Methodssection and ROI production was assessed after cell exposure to differentLPA concentrations (1  Y  5  2 M) for 3, 6 and 9 h (a) or after cell exposureto LPA (1  2 M) for 6 h (b). In the experiments with DPI (10 j 8 M), cellswere pre-treated with the inhibitor for 1 h before LPA addition. Resultsare expressed as Fluorescence Intensity, reported as Fluorescence Units(F.U.), in respect to cells loaded with DCF-DA only (C). Micrographswere acquired as reported in the Materials and Methods section.Original magnification:   200. Data are reported as mean  T  SD of 4different experiments.  *  P  <0.05, as reported from Student _ s  t   test inrespect to untreated cells (C). 133 Properties of Lysophosphatidic Acid on Human THP-1 Cells
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