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Platelet-activating factor enhancement of calcium influx and interleukin-6 expression, but not production,… Sattayaprasert, Prasongchai; Choi, Hyun B; Chongthammakun, Sukumal; McLarnon, James G Apr 15, 2005

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ralssBioMed CentJournal of NeuroinflammationOpen AcceResearchPlatelet-activating factor enhancement of calcium influx and interleukin-6 expression, but not production, in human microgliaPrasongchai Sattayaprasert†1,3, Hyun B Choi†1,2, Sukumal Chongthammakun3 and James G McLarnon*1Address: 1Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada, 2Division of Neurology, Department of Medicine, University of British Columbia, Canada and 3Department of Anatomy, Mahidol University, Bangkok, ThailandEmail: Prasongchai Sattayaprasert - mannkung@hotmail.com; Hyun B Choi - chb1202@hanmail.net; Sukumal Chongthammakun - scsct@mahidol.ac.th; James G McLarnon* - mclarnon@interchange.ubc.ca* Corresponding author    †Equal contributorsMicrogliaplatelet-activating factorinterleukin-6store-operated channelsAbstractCalcium-sensitive fluorescence microscopy and molecular biology analysis have been used to studythe effects of platelet-activating factor (PAF) on intracellular calcium [Ca2+]i and IL-6 expression inhuman microglia. PAF (applied acutely at 100 nM) elicited a biphasic response in [Ca2+]i consistingof an initial rapid increase of [Ca2+]i due to release from internal stores, followed by a sustainedinflux. The latter phase of the [Ca2+]i increase was blocked by SKF96365, a non-selective store-operated channel (SOC) inhibitor. RT-PCR analysis showed PAF treatment of microglia inducedexpression of the pro-inflammatory cytokine IL-6 in a time-dependent manner which was blockedin the presence of SKF96365. However, ELISA assay showed no production of IL-6 was elicited atany time point (1–24 h) for microglial exposures to PAF. These findings suggest that PAFstimulation of human microglia induces expression, but not production, of IL-6 and that SOC-mediated [Ca2+]i influx contributes to the enhanced expression of the cytokine.BackgroundMicroglia are resident, immunocompetent cells in thebrain. They show functional plasticity and can be acti-vated by a diversity of inflammatory stimuli includingones associated with neurodegenerative diseases [9,18].The functional responses of microglia following activa-tion include proliferation, phagocytosis and secretion. Inthe latter case microglia can secrete pro- and anti-inflam-matory cytokines, chemokines, neurotrophic factors andOne important inflammatory agent is platelet-activatingfactor (PAF), an alkyl ether phospholipid compound,which both stimulates and is produced by microglia [13].PAF contributes to inflammatory responses in the brainand is reported to be upregulated in CNS pathophysiol-ogy [2,17]. Acute application of PAF to human microgliainduces a biphasic change in levels of intracellular Ca2+([Ca2+]i) with an initial rapid phase due to intracellularrelease from endoplasmic reticulum (ER) stores and a sec-Published: 15 April 2005Journal of Neuroinflammation 2005, 2:11 doi:10.1186/1742-2094-2-11Received: 19 January 2005Accepted: 15 April 2005This article is available from: http://www.jneuroinflammation.com/content/2/1/11© 2005 Sattayaprasert et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)excitotoxins such as glutamate [20]. ondary phase due to influx through store operated chan-nels (SOC) [15,31]. Importantly, SOC has been shown toJournal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11exhibit sustained activation following stimulation ofhuman [31] and rodent [29] microglia. Prolonged entryof Ca2+ through SOC in stimulated microglia could consti-tute a coupling signal between an activating stimulus andcellular functional response. Indeed, the involvement ofsustained Ca2+ responses has been reported as a factor inthe production of arachidonic acid by rat microglia [23].The pro-inflammatory cytokine IL-6 is released from acti-vated microglia and mediates inflammatory responses inbrain. Levels of IL-6 in serum and cerebrospinal fluid havebeen found to be elevated in stroke patients [8,28] and thecytokine has also been implicated in the etiopathology ofneurodegenerative disorders such as Alzheimer's disease(AD), Parkinson's disease (PD) and HIV encephalopathy[3,14,25]. Interestingly, some evidence is also availablesuggesting that under some conditions elevated levels ofIL-6 in brain may actually be beneficial [27].In this study we have examined a role for SOC mediated[Ca2+]i influx in mediating actions of the inflammatorystimulus PAF to induce IL-6 in human microglia.Materials and methodsPreparation of cellsThe procedures for the isolation of human microglia havebeen previously reported [24]. In brief, human embryonicbrain tissues were dissected into small blocks, incubatedin phosphate-buffered saline (PBS) containing 0.25%trypsin and 40 µg/ml DNase and then dissociated intosingle cells by repeated pipetting. Cells were plated in T75flasks in a medium consisting of Dulbecco's modifiedEagle's medium (DMEM) containing 5% horse serum, 5mg/ml glucose, 25 µg/ml gentamicin, and 2.5 µg/mlamphotericin B. Freely floating microglia were harvestedfrom a medium of mixed cell cultures after 7–10 days ofgrowth in culture flasks and plated on aclar coverslips foridentification, on poly-L-lysine-coated glass coverslips forcalcium spectrofluorometry and plated on six-well multi-plates for RT-PCR or ELISA. CD11b and ricinus communisagglutinin (RCA), specific markers for microglia, wereused to confirm purity of the culture which was in excessof 98% [24,30].Calcium spectrofluorometryThe procedures used for measurement of intracellularCa2+ have been reported [6,31]. Microglia were incubatedwith 1 µM fura-2/AM (acetoxymethyl ester, MolecularProbes, Eugene, OR) plus 1 µM pluronic acid in normalphysiological saline solution (PSS) for 30 min. PSS solu-tion contained (in mM): NaCl (126), KCl (5), MgCl2(1.2), HEPES (10), D-glucose (10) and CaCl2 (1); pH of7.4. All reagents were obtained from Sigma (St. Louis,Following a 20 min wash in dye-free solution, coverslipswere placed on the stage of a Zeiss Axiovert invertedmicroscope employing a ×40 quartz objective lens. Cellswere exposed to alternating wavelengths of 340/380 nmat 6 s intervals and emission light passed through a 510nm filter. An imaging system (Empix Imaging, Missis-sauga, ON) was used to record fluorescence ratios using aCCD camera (Retiga 1300i, Burnaby, BC). Fluorescenceratios were determined and converted to values of [Ca2+]iusing published procedures [11]. All experiments weredone at room temperature (20–22°C).Reverse transcription-PCR and ELISA assayIL-6 expression was detected with the reverse-transcriptasepolymerase chain reaction (RT-PCR). Isolation of RNAswas performed using TRIzol (Gibco-BRL, Gaithersburg,MD, USA) and DNA contamination was eliminated usingDNase. cDNA synthesis was done using M-MLV reversetranscriptase (Gibco-BRL). The sequences for the humanspecific primers for IL-6 as follows: sense primer: 5'-GTGTGAAAGCAGCAAAGAGGC-3'; antisense primer: 5'-CTGGAGGTACTCTAGGTATAC-3'. Human-specific IL-6signals were generated with the GeneAmp thermal cyclerand Amplitaq Gold DNA polymerase (Applied Biosys-tems, Foster City, CA). The conditions for PCR were as fol-lows: initial denaturation at 95°C for 6 min followed by28 cycles of denaturation at 95°C for 45 sec, annealing at56°C for 1 min and extension at 72°C for 1 min. A finalextension step at 72°C for 10 min was carried out. PCRproducts (159 bp) were identified using 1.5% agarose gelscontaining ethidium bromide and visualized under UVlight. GAPDH was used as a reaction standard and humanspecific primer sequences were as follows: sense primer:5'-CCATGTTCGTCATGGGTGTGAACCA-3'; antisenseprimer: 5'-GCCAGTAGAGGCAGGGATGATGTTC-3'. Theintensities of each band were measured using NIH imageJ 1.24 software (National Institutes of Health, Bethesda,MD). Relative mRNA levels for each treatment were nor-malized to GAPDH.Enzyme-linked immunosorbent assays (ELISA) were per-formed according to manufacturer instructions (R & Dsystems, Minneapolis, MN). Cells were plated on multi-well plates (≈105 cells/well) and treated with PAF (100nM) in the absence or presence of SKF96365 (20 µM for 8hr). The cell-free supernatants were used for analysis of IL-6 production (kit detects IL-6 as low as 0.7 pg/ml). Valueswere expressed as means ± SEM and statistical significance(p < 0.05) was determined using one-way ANOVA andNewman-Keuls multiple comparison post-test.Page 2 of 8(page number not for citation purposes)MO).Journal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11ResultsEffects of SKF96365 on SOC-mediated [Ca2+]i influx by PAFPAF-induced changes in [Ca2+]i from human microgliahave previously been reported [15,21,31]. Initial studyshowed a transient increase in SOC [31] but more recentwork has shown PAF application to evoke a sustainedphase of SOC following an initial component due todepletion of Ca2+ from intracellular stores [15,21]. Thedifferences in PAF responses is considered in theDiscussion.A representative response to acute application of PAF(applied at 100 nM) is presented in Fig 1A (n = 18 cells).A plateau level of [Ca2+]i was sustained for a durationexceeding 2 min after removal of PAF. Following estab-lishment of a clearly defined plateau phase, the bath solu-tion was replaced with Ca2+-free PSS. This procedurecaused an immediate decline in [Ca2+]i to baseline levels(Fig 1A). Long durations of SOC-mediated influx of Ca2+have also been documented in mouse microglial cells[29].The results of application of the SOC inhibitor SKF96365(at 20 µM) to the plateau phase of a PAF response isshown in the representative recording of Fig 1B (n = 21cells). SOC-mediated entry of Ca2+ was reduced to base-line values by SKF96365. Amplitude of Ca2+ influxthrough SOC was measured as the difference betweenbaseline and plateau levels and in five independent exper-iments (n = 107 cells) the amplitude prior to SKF96365was 140 ± 21 nM and after SKF96365 was at baseline lev-els. Previous work has shown SKF96365 pretreatment ofhuman microglia (50 µM for 5 min) abolished a transientSOC in the cells [31].Effects of SKF96365 on microglial expression of IL-6We next examined effects of PAF on expression of the pro-inflammatory cytokine IL-6 in the absence and presenceof SOC inhibition. The time-dependence of PAF stimula-tion (100 nM) of human microglia on IL-6 are presentedin Fig 2A. The representative RT-PCR showed no constitu-tive expression of IL-6 in unstimulated microglia (lane 1of Fig 2A). IL-6 was maximally expressed at 1 h of expo-sure to PAF then declined to lower levels at longer treat-ment times (longest exposure of 6 h). A similar time-dependence for IL-6 expression was exhibited in a total offour experiments.A one hour exposure of human microglia to PAF was cho-sen for subsequent RT-PCR analysis. As shown in Fig 2B,constitutive expression of IL-6 was absent (lane 1). PAFtreatment was effective in stimulating expression of thetion (Fig 2B, lane 3). No evident IL-6 expression wasobserved for PAF application in Ca2+-free PSS (Fig 2B, lane4). SKF96365, applied alone in PSS solution, did notcause any increase in IL-6 (Fig 2B, lane 5).It was of interest to compare PAF as an inducer of micro-glial IL-6 to that of LPS (lipopolysaccharide) a potentinflammatory stimulus of cells. The results of exposure ofhuman microglia to LPS (100 ng/ml for 6 h) is presentedin Fig 2B (lane 6) showing LPS stimulation caused anintense band for IL-6. Altering the number of PCR cycleshad no apparent effect on intensity (data not shown) sug-gesting IL-6 band saturation with LPS (Fig 2B, lane 6).Comparison of band intensity indicated LPS was a moreeffective inducer of IL-6 relative to PAF. Interestingly, apartial inhibition of LPS-induced IL-6 mRNA wasobserved when SKF96365 was applied with LPS (Fig 2B,lane 7).Semi-quantitative RT-PCR analysis is presented in Fig 2Cand shows PAF as an effective stimulator of IL-6 expres-sion (n = 3). However, expression of IL-6 was considera-bly lower with PAF as a stimulus compared with LPS (Fig2B,C). Inclusion of SKF96365 with PAF or application ofPAF in Ca2+-free PSS eliminated expression of IL-6 (n = 3).Although LPS was not the subject of this study, thedecrease in LPS induction of IL-6 with SKF96365 is ofinterest and is discussed below.ELISA assay for effects of PAF on microglial production of IL-6We next investigated production of IL-6 from PAF-treatedhuman microglia using an exposure time of 8 h. No pro-duction of IL-6 was evident in four experiments (data notshown); levels of IL-6 were below the detection levels forELISA assay (≤ 1 pg/ml). In order to determine if the treat-ment time was a limiting factor in IL-6 production, a seriesof experiments using different microglial times of expo-sure to PAF were undertaken (from 1–24 h). The resultsare presented in Fig 3; no significant production of IL-6 (n= 4) was found for any treatment time (PAF applied for1,2,8 or 24 h).We also examined if a ten-fold increase in PAF concentra-tion (to 1 µM) would be effective in producing IL-6. Asshown in Fig 3, this higher concentration of PAF also hadno effect to induce IL-6 production for treatment times of8 or 24 h (n = 3 independent experiments). The effects ofLPS stimulation were also determined in these experi-ments (using 100 ng/ml for 8 h). Microglia, treated withLPS, produced high concentrations of IL-6 to levelsexceeding 400 pg/ml (n = 4 independent experiments).Page 3 of 8(page number not for citation purposes)cytokine (Fig 2B, lane 2). The expression of IL-6 was abol-ished when SKF96365 was included with the PAF applica-Journal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11PAF-induced Ca2+ responsesFigure 1PAF-induced Ca2+ responses. A: Representative trace (n = 18 cells) showing change in [Ca2+]i induced by PAF (100 nM). Following a prolonged level of SOC-mediated influx of Ca2+, the perfusion of Ca2+-free PSS abolished the response. B: Results from a separate experiment showing effects of SKF96365 (20 µM) on a PAF-induced increase in [Ca2+]i (n = 21 cells). Page 4 of 8(page number not for citation purposes)SKF96365 application, during a sustained entry of Ca2+ through SOC, effectively reduced [Ca2+] to baseline levels.Journal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11Expression of IL-6 in PAF treated human microgliaFigure 2Expression of IL-6 in PAF treated human microglia. A: RT-PCR analysis for different exposure times of microglia to PAF (applied at 100 nM). B: Effects of PAF, PAF plus SKF96365, PAF plus Ca2+-free and SKF96365 applied alone (1 h treat-ments). Also shown are effects of LPS and LPS plus SKF96365 (6 hr treatments). GAPDH was used as a reaction standard. C: Semi-quantitative RT-PCR for effects of the different treatments. * P < 0.05 compared with unstimulated control; # P < 0.05 Page 5 of 8(page number not for citation purposes)compared with PAF treated microglia.Journal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11DiscussionThe results from this work indicate that PAF-mediatedchanges in [Ca2+]i are involved in the cellular expressionof the pro-inflammatory agent, IL-6 in human microglia.In essence, activation of SOC acts as a transcriptional con-trol for expression of IL-6. Our results show that inhibi-tion of SOC with SKF96365 blocked both the influx ofCa2+ and microglial expression of IL-6. However, PAF-induced expression of IL-6 (Fig 2) did not translate intoproduction of the cytokine (Fig 3). This result could sug-gest that an additional signal or factor may be required formicroglial secretion of IL-6.As found for other types of unexcitable cells, microglia donot normally express voltage-dependent Ca2+ channels[7]. The sustained entry of Ca2+ through SOC is likely animportant pathway for microglial responses to specificinflammatory stimuli [15,22,26]. Although opening ofSOC is required for re-filling of ER stores, other roles forthis influx pathway have not been well established. Acti-vation of SOC is necessary for expression of IL-6 but anof human microglia may influence the extent of Ca2+influx through SOC. Microglia showing an ameboid mor-phology are considered representative of an activated statewhereas cells with a ramified morphology are consideredquiescent. We have found sustained SOC responses fromPAF-stimulated microglia in cells demonstrating ameboidmorphology [15,21] and also in the present work. How-ever, an initial study using a mixture of ameboid and ram-ified shaped cells, showed a transient SOC response withstimulation by PAF [31]. Further work will be useful tocorrelate expression of SOC with cell activation.A recent review has provided a detailed overview of ATP asan inducer of IL-6 expression and production in MG-5microglial cell line [12]. ATP and the purinergic agonistBzATP were both effective in increasing expression of IL-6with effects involving activation of the p38 MAPK path-way. However, ATP (activator of both metabotropic P2YRand ionotropic P2XR) but not BzATP (activator of theionotropic subtype P2X7R), was found to induce produc-tion of the cytokine. The role of SOC in MG-5 cellresponses is unclear since ATP evokes a monophasicchange in [Ca2+]i due to P2YR dependent release fromintracellular stores. In human microglia we have attrib-uted the lack of a SOC phase of [Ca2+]i due to concomi-tant ATP binding to some P2XR (not P2X7R) causingcellular depolarization and block of Ca2+ influx [6].PAF induction of IL-6 was found to be time-dependent(Fig 2A) in addition to the dependence on the presence ofextracellular Ca2+ and SOC (Fig 2B). We observed no IL-6expression at one-half hour and a maximal level at onehour of microglial exposure to PAF. Little or no IL-6 wasexpressed with longer PAF treatments of microglia. Inhibi-tion of endoplasmic reticulum Ca2+ ATPase (SERCA) hasbeen reported to increase IL-6 mRNA expression in rodentmacrophages within 15 min [4,19]. Blockade of SERCA,by compounds such as thapsigargin, and subsequentdepletion of intracellular stores is a stimulatory protocolfor activation of SOC. However, SOC-mediated entry ofCa2+ was not determined in the rodent studies.Although PAF was an effective stimulator of IL-6 expres-sion in human microglia, LPS elicited a higher expressionof the cytokine. Indeed, bands for IL-6 appeared saturated(Fig 2B) and showed no change in intensity withincreased number of PCR cycles (data not shown). Satura-tion with LPS would prevent a quantitative comparisonbetween PAF and LPS as activating stimuli for microglialexpression of IL-6 (Fig 2C). An interesting observationwas that SKF96365 partially inhibited the LPS-inducedexpression of IL-6 (Fig 2C). Although LPS has beenreported to act in a Ca2+-independent manner on macro-ELISA assays for production of IL-6 in human microgliaFigure 3ELISA assays for production of IL-6 in human micro-glia. PAF (at 100 nM) induced no significant production of IL-6 from microglia following exposures from 1–24 h (n = 4 for each time points). PAF (at 1 µM) induced no significant pro-duction of IL-6 (following exposures for 8 h and 24 h; n = 3 for both time points); these values are near the lower limits for sensitivity of the ELISA kits. LPS was used as a positive control in these experiments (n = 4); note the change of scale for the ordinate (from 10 to 400 pg/ml). * P < 0.05 compared with unstimulated control.Page 6 of 8(page number not for citation purposes)additional signal is required to produce the pro-inflam-matory cytokine in human microglia. The activation statephages [19], several studies have found the bacterial com-pound evokes changes in [Ca2+]i in microglia/Journal of Neuroinflammation 2005, 2:11 http://www.jneuroinflammation.com/content/2/1/11macrophages [1,5,16,32] suggesting possible involve-ment of SOC in LPS induction of cytokines.The present results may have relevance to roles of IL-6 inaging. Several studies have provided evidence for age-dependent increases in levels of IL-6 in rodent brain[reviewed in [10]]. For example, one finding was thatbrains from older mice showed considerable elevations inexpression and production of IL-6 compared with brainsfrom younger animals [33]. This result was correlatedwith microglial production of the cytokine [33]. It will beof interest to determine if PAF-stimulated adult humanmicroglia are more potent producers of IL-6 comparedwith fetal human cells.List of abbreviationsPAF: platelet-activating factor; SOC: store-operated chan-nels; IL-6; interleukin-6; PSS: physiological saline solu-tion; PBS: phosphate-buffered saline; [Ca2+]i:intracellular calcium; DMEM: Dulbecco's modifiedEagle's mediumCompeting interestsThe author(s) declare that they have no competinginterests.Authors' contributionsPS and HBC contributed equally to calcium imaging, RT-PCR and ELISA experiments. HBC also carried outisolation of microglia. SC participated in the design ofexperiments and reviewed and edited the manuscript.JGM designed and supervised all experiments, interpretedthe data and finalized the manuscript. All authors readand approved the final manuscript.AcknowledgementsThis work was supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon and Alzheimer's Society of Canada (to JGM) and a doctoral research award from the Heart and Stroke Foundation of Canada (to HBC).References1. Bader MF, Taupenot L, Ulrich G, Aunis D, Ciesielski-Treska J: Bacte-rial endotoxin induces [Ca2+]i transients and changes theorganization of actin in microglia. Glia 1994, 11:336-344.2. Bielenberg GW, Wagener G, Beck T: Infarct reduction by theplatelet activating factor antagonist apafant in rats. Stroke1992, 23:98-103.3. 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