High-Density Lipoprotein Reduces the HumanMonocyte Inflammatory Response
Andrew J. Murphy, Kevin J. Woollard, Anh Hoang, Nigora Mukhamedova, Roslynn A. Stirzaker,Sally P.A. McCormick, Alan T. Remaley, Dmitri Sviridov, Jaye Chin-Dusting
Objective—Whereas the anti–inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are welldescribed, such effects on monocytes are less studied.
Methods and Results—Human monocytes were isolated from whole blood followed by assessment of CD11bactivation/expression and cell adhesion under shear-flow. HDL caused a dose-dependent reduction in the activation ofCD11b induced by PMA or receptor-dependent agonists. The constituent of HDL responsible for the antiinflammatoryeffects on CD11b activation was found to be apolipoprotein A-I (apoA-I). Cyclodextrin, but not cyclodextrin/cholesterolcomplex, also inhibited PMA-induced CD11b activation implicating cholesterol efflux as the main mechanism. This wasfurther confirmed with the demonstration that cholesterol content of lipid rafts diminished after treatment with thecholesterol acceptors. Blocking ABCA1 with an anti-ABCA1 antibody abolished the effect of apoA-I. Furthermore,monocytes derived from a Tangier disease patient definitively confirmed the requirement of ABCA1 in apoA-I–mediated CD11b inhibition. The antiinflammatory effects of apoA-I were also observed in functional models includingcell adhesion to an endothelial cell monolayer, monocytic spreading under shear flow, and transmigration.
Conclusions—HDL and apoA-I exhibit an antiinflammatory effect on human monocytes by inhibiting activation ofCD11b. ApoA-I acts through ABCA1, whereas HDL may act through several receptors. (Arterioscler Thromb VascBiol. 2008;28:2071-2077)
Plasma levels of high-density lipoproteins (HDL) areinversely associated with cardiovascular morbidity and
mortality.1,2 The most comprehensively studied function ofHDL is reverse cholesterol transport. Other cardioprotectivefunctions include its antioxidative properties and its ability toincrease nitric oxide (NO) bioavailability.3,4 More recently,the antiinflammatory effects of HDL, particularly in theendothelium, have been reported.5,6
See accompanying article on page 1890
A critical event in the formation of atherosclerotic plaquesis the recruitment of monocytes into the adventitia where theydifferentiate into macrophages and ingest modified low-density lipoproteins (LDL) through scavenger receptors toform foam cells.7 The recruitment of monocytes involves theexpression of both endothelial and monocytic adhesion mol-ecules. In the multi-step adhesion cascade the initialmonocyte-endothelium attachment occurs via selectins ex-pressed on endothelial cells. Firm adhesion then occursthrough vascular cell adhesion molecule (VCAM)-1 and
intracellular adhesion molecule-1 (ICAM-1) interacting withmonocyte adhesion molecules such as CD11b/CD18 (Mac-1,CR3).8–10
A reduction in tumor necrosis factor (TNF)-�–inducedexpression of VCAM-1, ICAM-1, and E-selectin in endothe-lial cells preincubated with HDL has been reported.11,12
Similarly, decreases in reactive oxygen species production,neutrophil infiltration, and monocyte chemoattractantprotein-1 (MCP-1) have also been reported.13,14 It has beendemonstrated that inhibition by HDL of E-selectin expressionon human endothelial cells is mediated by lysosphingolip-ids,11 suggesting the involvement of both the scavengerreceptor class B-1 (SR-B1) and the S1P3 receptor, activatingendothelial nitric oxide synthase (eNOS) to produce NO.3,15,16
The in vivo effects, however, are complex and can bemediated by reconstituted HDL (rHDL) or lipid free apoA-I.13,14 In contrast, at least to the in vitro findings usingendothelial cells, studies on the antiinflammatory effects ofHDL on neutrophil activation demonstrate, with one notableexception,17 that apoA-I is responsible.18–21 Interestingly,
Original received January 2, 2008; final version accepted July 1, 2008.From the Laboratories of Vascular Pharmacology (A.J.M., K.J.W., J.C.D.) and Lipoproteins and Atherosclerosis (A.H., N.M., D.S.), Baker Heart
Research Institute, Melbourne, Victoria, Australia; Molecular Medicine (R.A.S.), The Walter and Eliza Hall Institute of Medical Research, Melbourne,Victoria, Australia; the Department of Biochemistry (S.P.A.M.), University of Otago, Dunedin, Otago, New Zealand; and the Lipoprotein MetabolismSection (A.T.R.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md.
Correspondence to Jaye Chin-Dusting, Baker Heart Research Institute, PO Box 6492, St Kilda Rd Central, Victoria 8008 Australia. E-mail [email protected]
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.108.168690
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HDL also inhibits oxidized LDL (oxLDL)–induced leukocyte–endothelial interactions without the induction of endothelialadhesion molecule expression, nor was leukocyte adhesionattenuated by blocking the endothelial adhesion molecules.22
In this article, we explore the mechanism by which HDLand apoA-I prevents and reverses leukocyte activation. Ourstudy shows that HDL and apoA-I act through variousreceptors to decrease monocyte activation and that the majorcontributing pathway for apoA-I is the monocytic ATP-binding cassette transporter A1 (ABCA1).
MethodsFull details are provided in the supplemental materials (availableonline at http://atvb.ahajournals.org.
Study Subjects: Healthy and R1068HTangier FamilyThe study was approved by the Human Ethics committees of theAlfred Hospital and the University of Otago; informed consent wasobtained from all donors. Blood was anticoagulated with sodiumcitrate (19.2mMol/L) or EDTA-vacutainer tubes (Tangier study).
Monocyte IsolationResting human monocytes were isolated by density centrifugationwith Lymphoprep followed by Dynal Negative Monocyte Isolationkit as described previously.23
Cholesterol AcceptorsHDL was isolated from plasma using sequential ultracentrifugation(density 1.085 to 1.21g/mL), and protein content was measured.Reconstituted HDL (rHDL)24,25 and phosphatidylcholine liposomeswere prepared as previously described26; all HDL treatments wereperformed using 50 �g/mL unless otherwise stated. Human plasmaapoA-I was isolated as previously described27 and used at 40 �g/mL.Beta-cyclodextrin and cholesterol saturated cyclodextrin was pre-pared as previously described.28 The L37pA peptide was synthesizedas described.29
Receptor Blocking and Trapping StudiesMonocyte receptors were blocked using specific blocking antibodies for4 hours at 4°C. Antimouse IgM (Sigma) was used as a control Ab.
Flow CytometryMonocytes were stimulated and incubated with the fluoresceinisothiocyanate (FITC)-Ab to CD11b for 15 minutes at 37°C, unlessotherwise stated. Cells were fixed and CD11b expression wasmeasured by flow cytometry. Samples were controlled for by usingthe isotype matched negative control. Results were expressed aspercentage of the unstimulated control (100%) or PMA (100%,Tangiers only). For lipid raft quantification monocytes were treatedfor 15 minutes at 37°C, centrifuged, and incubated with FITC-Cholera toxin B (CT-B) for 1 hour at room temperature and raftsmeasured by flow cytometry.
Lipid Raft StainingRafts were stained using the Vybrant lipid raft labeling kit as per themanufacturer’s instructions. Monocytes were mounted in fluores-cence mounting media and viewed on the fluorescent microscope.Staining intensity was quantified using Image Pro software.
Perfusion StudiesPerfusion Studies were conducted using the parallel plate flow-chamber as previously described.30 Prestimulated monocytes wereperfused over human coronary aortic endothelial cells (HCAECs) ata shear-rate of 150s�1 (1.1dyn/cm2) for 5 minutes with a washoutperiod of 5 minutes. Monocyte adhesion was captured and analyzedoffline.
Monocyte Spreading/Adhesion Perfusion AssayPerfusion studies were conducted in platelet coated glass microcap-illary tubes at 37°C.31 Preactivated monocytes were perfused overthe platelet monolayer for 5 minutes (t�0 seconds) followed by awashout period of 5 minutes (t�300 seconds). Monocyte-plateletinteractions were visualized according to “perfusion studies.”
Static Adhesion AssayMonocyte adhesion to immobilized fibrinogen was performed for 15minutes at 37°C as previously described.31
Migration AssayMigration assays were performed using 8.0 �mol/L Transwells.32
Monocytes with treatment were seeded in the upper chamber, andallowed to migrate for 30 minutes at 37°C to the lower chambercontaining 50 ng/mL of MCP-1. Migrated monocytes were fixed andthe number of migrated cells quantified.
Filamentous Actin ContentMonocytes were stained for F-actin with alexa fluor 488-Phalloidinand quantified by flow cytometry or further stained with DAPI andinvestigated by confocal microscopy.
Statistical AnalysisValues are presented as the mean�SEM or percentage ofcontrol�SEM. All results were analyzed for statistical significanceusing 1-way ANOVA followed by Bonferroni posthoc test, exceptPerfusion studies which were analyzed using a 2-way ANOVAfollowed by Bonferroni posthoc test. Significance was accepted atP�0.05.
ResultsHDL Inhibits PMA-Induced Activation of CD11bPMA induced monocytic integrin CD11b activation whichwas dose-dependently inhibited by HDL (2 to 50 �g/mL;Figure 1A). Although the HDL concentrations used in thisstudy are below plasma levels, they are approaching saturat-ing concentrations described in cholesterol efflux experi-ments, routinely used by others.33 The decrease in activatedCD11b was accompanied by a decrease in total CD11babundance (PMA versus HDL (50 �g/mL) � PMA: 19�1.84versus 9.7�1.89 U; n�4, P�0.001).
To assess whether the response to HDL was dependent onmonocyte heterogeneity, CD16� and CD16- monocytes wereisolated and their response compared. There was no differ-ence between the two subsets in response to HDL (supple-mental Figure I).
ApoA-I Reduces CD11b ActivationReconstituted HDL and apoA-I inhibited CD11b activation toa similar extent to HDL (Figure 1B). In contrast, neither BSAnor phosphatidylcholine liposomes had any effect (Figure 1Aand 1B). HDL and apoA-I significantly reduced both lipo-polysaccharide (LPS) and fMLP-induced CD11b activation(supplemental Figure II).
HDL Prevents and Reverses Monocyte ActivationPretreatment of monocytes with HDL followed by stimula-tion with either PMA (Figure 1C) or LPS (LPS versusPrevention; 152�1.8 versus 101�4.5, n�5, P�0.01) led to asignificant reduction of CD11b expression (Figure 1C).Likewise, prestimulation of monocytes with PMA followedby a 15-minute incubation with HDL also significantly
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reduced CD11b expression (Figure 1C), also seen with LPS(LPS versus Reversal; 152�1.8 versus 92.8�5.7, n�5,P�0.01). Monocytes preincubated with HDL or apoA-I,washed, and then challenged with either PMA (or fMLP, datanot shown) also demonstrated prevention of monocytes fromactivation (Figure 1D).
Regulation of CD11b Expression andCholesterol EffluxCyclodextrin significantly attenuated PMA-induced CD11bactivation (Figure 2A). Cholesterol-saturated cyclodextrin(Ch-CD) had no effect on PMA-induced CD11b activation(Figure 2A).
Changes in Monocyte Lipid Raft AbundanceTreatment of monocytes with apoA-I, HDL, and CD, but notliposomes and BSA, significantly decreased lipid rafts in theplasma membrane (Figure 2B). Incubation with apoA-I dra-matically modified membrane raft abundance (control versusapoA-I; 66.9�11.3 relative fluorescence units [rfu] versus37.3�4.6 rfu, n�5, P�0.05; represented visually in Figure2C), indicating rapid efflux from plasma membrane rafts.
Involvement of SR-B1Blocking SR-B1 blunted the effect of HDL on CD11bactivation, albeit not significantly (P�0.18; Figure 3A).SR-B1 blockade failed to affect the inhibition of CD11bactivation induced by apoA-I (Figure 3A). An irrelevantisotype matched Ab (cAb) had no effect.
Involvement of ABCA1The antiinflammatory effects of apoA-I but not HDL (Figure3B) were abolished in the presence of the ABCA1 blockingantibody NDF4C2. Irrelevant isotype matched Ab showed noeffect. The role for ABCA1 internalization was examinedusing NDF6F1,34 which does not affect ABCA1-dependentcholesterol efflux but prevents ABCA1 internalization anddegradation. NDF6F1 had no effect on the inhibitory actionof apoA-I on PMA-induced CD11b expression (Figure 3C).
Monocyte Adhesion to Endothelial CellsUnder Shear-FlowCompared to unstimulated monocytes, PMA significantlyincreased monocyte adhesion to endothelial cells. Coincuba-tion of monocytes with PMA and HDL or apoA-I resulted ina significant reduction in monocyte adhesion (Figure 4A and4D). The importance of the interaction between apoA-I and
Figure 1. CD11b activation. Monocytes were stimulated with (A)PMA�HDL or BSA (B) PMA�rHDL, apoA-I, or liposomes. Inprevention and reversal studies (C) monocytes were preincu-bated with HDL followed by PMA or vice versa. (D) Monocyteswere stimulated after preincubation with HDL or apoA-I whichwere removed.
Figure 2. Cholesterol efflux. (A) Monocytes were incubated with PMA�cyclodextrin (CD) or cholesterol saturated cyclodextrin (CD-CH).(B) Monocytes were incubated with apoA-I, HDL, CD, and BSA for 15 minutes, lipid rafts stained with CT-B, and cells analyzed by flowcytometry. (C) Confocal microscopy image of monocytes stained with CT-D without (top panel) and with treatment with apoA-I.
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ABCA1 was also examined under shear flow. A significantapoA-I–induced reduction in adhesion was no longer evidentin the presence of the ABCA1 antibody (Figure 4E). NDF4C2alone or an isotype matched Ab had no effect on adhesion(data not shown).
Monocyte Spreading and Adhesion on PlateletsUnder Shear-FlowTo explore the effect of HDL on monocyte spreading undershear flow conditions, PMA stimulated monocytes wereperfused over a platelet monolayer for 5 minutes (time�0seconds) followed by a 5-minute washout period (time�300
seconds; with or without HDL). Before washout there was nodifference in cell spreading between the 2 groups. Washing ofstimulated monocytes with HDL-containing buffer resulted ina significant reduction in spreading compared to washoutwith buffer alone (Figure 5A). A significant reduction inmonocyte adhesion to platelets was also observed afterwashout with HDL compared to buffer alone (supplementalFigure III).
Monocyte TransmigrationMCP-1 facilitated a significant monocytic migratory re-sponse, which was markedly reduced when the monocyteswere preincubated with either HDL or apoA-I (Figure 5B).
Monocyte F-Actin ContentStimulation of monocytes with PMA resulted in increasedF-actin levels, which was significantly reduced by coincuba-tion with HDL and PMA (Figure 5C). This observation wasconfirmed by flow cytometry (P�0.01; Figure 5D).
Figure 3. Blocking studies. (A) Monocytes incubated with SR-B1 blocking or control antibody (cAb) were treated with PMA�apoA-I orHDL. (B) Monocytes incubated with NDF4C2 anti-ABCA1 Ab(4C2) or cAb were treated with PMA�apoA-I or HDL or (C) the NDF6F1anti-ABCA1 Ab (6F1).
Figure 4. Monocyte adhesion under flow. (A) Monocytes werepretreated with PMA (F), PMA�HDL (E), or PMA�apoA-I (�)before perfusion over HCAECs. Images of cell adhesion after 5minutes; with PMA (B) PMA�HDL (C) and PMA�apoA-I (D). (E)Monocytes were treated with PMA (F), PMA�apoA-I (E), andNDF4C2 anti-ABCA1 Ab with PMA�apoA-I (�).
Figure 5. Monocyte phenotype. Monocyte spreading (A) undershear-flow �HDL (E). Monocyte transmigration. (B) Monocytes(�treatment) were seeded in the upper chamber of a transwelland allowed to migrate toward MCP-1. F-actin content. (C) Con-focal images of resting monocytes �PMA �HDL stained forF-actin (green) and nuclei (blue). (D) F-actin content measuredusing phalloidin.
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L37pA Inhibits Monocyte ActivationSimilar to ApoA-IThe apoA-I mimetic peptide L37pA29 (10 and 20 �g/mL)significantly reduced PMA stimulated CD11b expression onmonocytes as well as PMA challenged monocyte adhesion tofibrinogen coated glass cover-slips under static conditions(supplemental Figure IV).
Tangier Patient Derived MonocytesFor simplification of comparison between members of theR1068H Tangier family, results are expressed as percentageof CD11b expression after activation with PMA. Monocytesisolated from an unaffected relative, treated with PMA andapoA-I or HDL, displayed a similar reduction to that ob-served in previous assays (Figure 6A). Heterozygote mono-cytes had a diminished ability to respond to apoA-I, howevera reduction in CD11b activation was still observed. Mono-cytes derived from the Tangier disease patient failed torespond to apoA-I treatment. Although there was a cleargene-dose-dependent response to apoA-I, HDL reducedCD11b activation similarly in both heterozygote and Tangierderived monocytes (Figure 6A). Similar results were obtainedwhen assessing the ability of both apoA-I and HDL to reducethe adhesion of monocytes to fibrinogen (Figure 6B).
DiscussionThe activation of monocytes is a pivotal event in vascularinflammation and atherosclerosis. In the current study wereport that HDL and apoA-I can prevent as well as reverse theactivation of human monocytes with apoA-I exerting itseffects through ABCA1.
The main finding of this article is that HDL dose-dependently decreases CD11b expression and activation onprimary human monocytes stimulated with PMA. This find-ing was also observed with the receptor-mediated activatorsLPS and fMLP.23,35,36 Both rHDL and lipid-free apoA-Iinhibited PMA-induced activation of CD11b, however phos-pholipid liposomes or albumin had no effect. This is consis-tent with previous findings where apoA-I inhibited monocytespreading over time in response to M-colony stimulatingfactor (CSF).21 Interestingly, the mechanism by which HDLand apoA-I inhibit monocyte activation appears to be differ-ent from that of the antiinflammatory actions of these
molecules on endothelial cells, thought to be mediated viaHDL-stimulating NO production.11,16,37
Cholesterol efflux appears to be a requirement for theeffects of apoA-I and HDL because cyclodextrin effectivelymimicked HDL and apoA-I in inhibiting CD11b activation onmonocytes. Although cyclodextrin removes cholesterol non-specifically,38 it removes it from the same plasma membranepools as apoA-I as evidenced by the enhanced efflux tocyclodextrin after overexpression of ABCA1.39 Loading ofcyclodextrin with cholesterol, which converts cyclodextrin toa cholesterol donor, rendered it inactive thus confirming thatcholesterol efflux is required. Interestingly cyclodextrin andcholesterol removal has been shown to inhibit monocytespreading when coincubated with M-CSF21 and cause rapidretraction of membrane protrusions of macrophages.40 Fur-ther we demonstrated that short incubations with apoA-I,HDL, and CD, but not liposomes and BSA, resulted indepletion of raft cholesterol. It may be that the latter do notincite perturbation as potently as the former cholesterolacceptors. Regardless, the above findings suggest that rapiddepletion of lipid from cell membranes appears to be a keymechanism influencing the inflammatory response of themonocyte/macrophage.
To examine the specific mechanisms connecting the effectsof HDL on monocyte activation and cholesterol efflux, weinvestigated the involvement of 2 HDL receptors, SR-B1 andABCA1. SR-B1, along with ABCG1, has been shown to beinvolved in supporting cholesterol efflux to HDL, whereaslipid-poor apoA-I removes cholesterol exclusively throughABCA1-dependent pathways.41 Blocking SR-B1 resulted in areduction, but not elimination, of the antiinflammatory effectsof HDL, however it did not affect apoA-I. Blocking ABCA1totally abolished the inhibitory effect of apoA-I but had noeffect on HDL. Thus, the apoA-1/ABCA1 interaction is likelyto be a major pathway mediating these effects, with otherpathways, such as SR-B1 and ABCG1, also contributing tothe effects of HDL.
Shear flow adhesion assays were used to examine thefunctional outcome of HDL in reducing monocyte activation.When either HDL or apoA-I was present, PMA-stimulatedmonocyte adhesion to HCAECs was significantly attenuated.Furthermore, blocking monocyte ABCA1 reverses the apoA-I–induced decrease in adhesion, consistent with the results ofthe CD11b activation assay and confirming the involvementof ABCA1.
Although it has been previously demonstrated that HDLcan inhibit monocyte spreading,21 this is the first reportdescribing this effect under physiological shear conditions.The inhibition of the cell adhesion cascade by HDL wasfurther investigated by examining the effects of HDL andapoA-I on monocyte migration to MCP-1. Both HDL andapoA-I were able to significantly inhibit monocyte migrationto MCP-1, a finding consistent with the observations ofNavab et al.42 Because changes in the cytoskeleton arerequired to induce spreading and migration of cells which hasbeen shown to be associated with an increase in F-actincontent,43 total F-actin was quantified and demonstrated todecrease in the presence of HDL. This finding is consistentwith the decrease in spreading observed by Diederich et al.21
Figure 6. Tangier monocytes. (A) Monocytes from individuals(white�unaffected, gray�hetero, black�Tangier) were stimu-lated with PMA�apoA-I or HDL and CD11b activation mea-sured. (B) Monocytes treated with PMA�apoA-I or HDL wereadded to fibrinogen where adherence was quantified.
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The inhibitory effect of HDL on monocytes was effectivein both preventative and reversal settings. The latter finding,in particular, has important implications in disease states suchas acute coronary syndrome, where the process of inflamma-tion has already occurred. Here we have clearly demonstratedthat prestimulated monocytes can be rescued from activationby HDL. This may further explain the findings of previousstudies demonstrating that HDL/apoA-I can reduce the acti-vation of the endothelium and neutrophils accumulated in theintima-media in an in vivo inflammatory model up to 9 hourspostinjury.14 Our findings are also consistent with animalstudies where the short term elevation of HDL administrationwas atheroprotective.44
Recent termination of CETP inhibition trials suggest dif-ferent strategies for raising HDL levels are required.45 Thedevelopment of apoA-I mimetics may provide an effectivealternative. L37pA has previously been shown to effectivelystimulate cholesterol efflux and stabilization of ABCA1through the same mechanisms as apoA-I.29,46,47 In our studiesL37pA was able to mimic apoA-I antiinflammatory actionson monocytes indicating a potential for peptide based thera-peutics in inflammatory diseases.
Finally, we explored the antiinflammatory role of HDL andapoA-I in a Tangier disease subject along with a heterozygotesubject and an unaffected member from the R1068H family.48
Tangier disease patients have a dysfunctional ABCA1 unableto support cholesterol efflux to apoA-I.49 The response ofmonocytes from the unaffected family member was similar tothat of healthy subjects, monocytes from the heterozygotesubject responded to both HDL and apoA-I albeit less thancompared to monocytes of unaffected family member. Incontrast, apoA-I did not decrease CD11b activation in mono-cytes of the Tangier disease patient, although HDL stillproduced a degree of inhibition. Similar effects were ob-served when examining adhesion of monocytes of Tangierdisease patient to fibrinogen. These findings are consistentwith our hypothesis that apoA-I is working via ABCA1 toinhibit monocytic activation, whereas HDL additionally en-gages via an ABCA1-independent pathway.
In summary, this study details for the first time themechanism by which HDL and apoA-I regulate monocyteadhesion, spreading, and integrin activation. The finding thatapoA-I is equally potent to HDL in inhibiting elements ofinflammation provides important insight into the develop-ment of novel strategies such as apoA-I mimetic peptides inthe treatment and control of atherosclerosis.50 The findingthat HDL can still influence the inflammatory status ofmonocytes from Tangier disease patients also indicates thattherapeutic HDL strategies can be applied in the patientpopulation which is at risk of CVD. The ability of HDL toprevent and reverse activation of monocytes may also be ofsignificant interest for management a variety of inflammatorydiseases.
AcknowledgmentsWe acknowledge Rachel Brace for her efforts in helping with theblood collection and providing medical care for the R1068H family.
Sources of FundingA.J.M. is supported by an industry scholarship form Actelion Ltd,Sydney. K.J.W. is an Australian National Heart Foundation ResearchFellow. D.S. and J.C.-D. are Senior Research Fellows of theAustralian National Health and Medical Research Council.
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Murphy et al The Antiinflammatory Effects of HDL 2077
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Stirzaker, Sally P.A. McCormick, Alan T. Remaley, Dmitri Sviridov and Jaye Chin-DustingAndrew J. Murphy, Kevin J. Woollard, Anh Hoang, Nigora Mukhamedova, Roslynn A.High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response
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(This section to be published as an on-line supplement) Study Subjects: Healthy and R1068H Tangier family
Blood was drawn from healthy individuals into syringes containing sodium citrate
(19.2mMol/L). Blood from an unaffected, a heterozygote (carrier) and a
homozygote (affected, Tangier disease) subjects, corresponding to family
member III:5, IV:3 and III:3 respectively from the R1068H pedigree 1 was drawn
into 10mL EDTA-vacutainer tubes. The study protocol was approved by the
Human Ethics committees of the Alfred Hospital and the University of Otago;
informed consent was obtained from all donors.
Monocyte Isolation
Resting human monocytes were isolated by density centrifugation with
Lymphoprep (Axis Shield) followed by Dynal Negative Monocyte Isolation kit
(Invitrogen) as described previously 2. Monocytes were resuspended in PBS and
cell number was determined on an automated hematology analyzer (Sysmex,
KX-21N, USA).
Purification of HDL and apoA-I
HDL was isolated from pooled normolipidemic plasma supplied by the Red Cross
using sequential ultracentrifugation in KBr solutions (density 1.085-1.21g/mL).
Human plasma apoA-I was isolated as previously described 3 and the purity
determined using mass spectrometry.
Synthetic cholesterol acceptors
Reconstituted HDL (rHDL) (POPC/apoA-I 100:1mol/mol) 4, 5 and
phosphatidylcholine liposomes were prepared as previously described 6. β-
cyclodextrin (Sigma) and cholesterol saturated cyclodextrin was prepared as
described by Christian et al 7. The L37pA peptide was synthesized 8 and
dissolved in 20mM NaHCO3, 150mM NaCl.
Receptor blocking and trapping studies
SR-B1 was blocked using a specific blocking antibody (Ab) (Novus, USA).
ABCA1 was blocked by the anti-ABCA1 Ab NDF4C2 or trapped on the cell
surface with NDF6F1 9. Isolated monocytes were incubated with the
corresponding Ab for 4hr at 4˚C, centrifuged to remove the unbound Ab and
resuspended in PBS. Anti-mouse IgM (Sigma) was used as a control Ab.
Flow Cytometry
Monocytes were stimulated with either 1μmol/L PMA , 0.1μmol/L fMLP or 1μg/mL
LPS (Sigma, Australia) ± HDL (50μg/mL) or apoA-I (40μg/mL) and incubated with
the FITC conjugated Ab to either the active epitope of CD11b (eBiosciences,
USA, Clone CBRM1/5) or total CD11b (Serotec, USA, Clone ICRF44) for 15min
at 37˚C, unless otherwise stated. Cells were then fixed with 4% para-
formaldehyde. Samples were controlled for by using the isotype matched
negative control (FITC-anti-mouse IgG, Serotec, USA, Clone W3/25). CD11b
expression was measured by flow cytometry using FACS Calibur (Becton
Dickinson). Analysis was conducted using the Cell Quest Pro software. Results
were expressed as percentage of the unstimulated control (100%) or PMA
(100%, Tangiers only). For lipid raft quantitative analysis, monocytes were
treated for 15mins at 37˚C and lipid rafts quantified by incubating the monocytes
with FITC-Cholera toxin B (CT-B) for 1hr at room temperature and measured by
flow cytometry.
Lipid Raft Staining
Lipid rafts were stained using the Vybrant lipid raft labeling kit (Invitrogen).
Isolated monocytes were incubated with PBS or PBS with apoA-I (40μg/mL) for
15mins at 37˚C in 8 well chamber slides (Nunc). The cells were then washed
with PBS and incubated with PBS containing 1μg/mL of cholera toxin subunit B
(CT-B) for 10mins on ice. Monocytes were then gently washed with cold PBS
and then incubated with PBS containing anti-CT-B Ab and DAPI (5μg/mL)
(Sigma) for 15mins on ice. Monocytes were then washed and mounted in
fluorescence mounting media (Dako) and viewed on the fluorescent microscope.
Staining intensity was quantified using Image Pro software.
Perfusion Studies
Perfusion Studies were conducted using the parallel plate flow-chamber
(Glycotech, USA) described by Lawrence et al 10. Human Coronary Aortic
Endothelial Cells (HCAEC) (Cell Applications, USA) were cultured in 6-well
plates to form a confluent monolayer with HCAEC Media (Cell Applications).
Prior to perfusion, HCAECs were washed twice with PBS. Isolated monocytes
(1x106/mL) in PBS were perfused over HCAECs under negative pressure (PHD
2000, Harvard Apparatus, USA), at a shear-rate of 150s-1 (1.1dyn/cm2) for 5 min
with an additional washout period of 5min with PBS. To study the inhibitory
effect of HDL on monocyte adhesion, monocytes were pre-treated with 1µmol/L
of PMA ± 50µg/mL HDL for 15min at 37˚C. Monocyte-endothelial cell
interactions were visualised using phase microscopy (X20 lens, Olympus,
Australia) captured digitally (MDC-1004, Imperx, USA) at 30 frames/sec with
XCAP™ software v2.2 (Epix, USA) and analysed off line using Image Pro-Plus
v5.1 (Media Cybernetics, USA). Time 0 was defined by the first adhering
monocyte. Fields (274µm x 275µm) were recorded for 10sec at 0.5min, 1min (1
visual field each/time point were analysed), 2.5min, 5min and 10min (five visual
fields each/time point were analysed) for off-line analysis.
Monocyte Spreading/Adhesion Perfusion Assay
Perfusion studies were conducted in platelet coated glass micro-capillary tubes
(2x0.2x100mm) at 37˚C 11. PMA activated monocytes (1x106/mL) in Tyrodes
buffer with 1mmol/L of CaCl2/MgCl2 were perfused over the platelet monolayer
for 5min (t=0secs) followed by a washout period with Tyrodes or Tyrodes +
50µg/mL HDL for a further 5min (t=300secs). Monocyte-platelet interactions
were visualised as above. Time negative 5min was defined by the first adhering
monocyte and time 0 when the washout period began. Fields were recorded for
10sec at 0min and 5min post washout (3 visual fields/time point were analysed).
Static Adhesion Assay
Monocyte adhesion to immobilized fibrinogen was performed as previously
described 11. Monocytes (1x106/mL) were allowed to adhere for 15min at 37°C
with the appropriate treatment. Monocyte adhesion was quantified by phase
microscopy (X40) counting five random fields form each slide. Treatments were
carried out in triplicate.
Migration Assay
Migration assays were performed using 8.0μM Transwells (Costar) 12.
Monocytes (1x105 cells/well) in PBS ± HDL or apoA-I were seeded in the upper
chamber, while the lower chamber contained 50ng/mL of MCP-1. Monocytes
were allowed to migrate for 30min at 37°C. Monocytes which had migrated to
the bottom surface of the membrane were fixed with 4% formaldehyde, the
membrane was mounted onto cover-slips and the number of migrated cells
quantified by phase microscopy (X40) averaging three random fields from each
transwell. Active migration was determined by subtracting migrated cells in the
PBS only control from the different treatment groups.
Filamentous Actin Content
Monocytes were stimulated with 1μmol/L PMA ± HDL (50μg/mL) and incubated
for 15min at 37˚C. Cells were then fixed with 4% para-formaldehyde for 10min.
The fixed monocytes were centrifuged and resuspended in 0.1% triton X-100 and
permeabilised for 10min with occasional mixing. Cells were labelled with Alexa
Fluor 488-Phalloidin (33nM) (Invitrogen, Australia) for 20min at 25˚C in the dark
and F-actin content measured by flow cytometry.
Fluorescence Microscopy – F-actin
Monocyte F-actin content was examined using a fluorescence laser scanning
confocal microscope (Leica). Monocytes were treated for 15min in 8 well
chamber slides, fixed for 15min with 4% para-formaldehyde and permeabilised
with 0.1% triton X-100 for 10min. These were then blocked with 10% FCS for
15min and labelled with alexa fluor 488-Phalloidin (33nM) and DAPI (5μg/mL) for
20min in the dark. Cells were then washed twice and mounted in fluorescence
mounting media and viewed on the confocal microscope.
Statistical Analysis
Values are presented as the mean ± SEM or percentage of control ± SEM. All
results were analysed for statistical significance using one-way ANOVA followed
by Bonferroni post-hoc test, except Perfusion studies which were analysed using
a two-way ANOVA followed by Bonferroni post-hoc test. Significance was
accepted at P<0.05.
References 1. Slatter TL, Williams MJ, Frikke-Schmidt R, Tybjaerg-Hansen A, Morison
IM, McCormick SP. Promoter haplotype of a new ABCA1 mutant influences expression of familial hypoalphalipoproteinemia. Atherosclerosis. 2006;187:393-400.
2. Woollard KJ, Phillips DC, Griffiths HR. Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells. Clin Exp Immunol. 2002;130:256-262.
3. Morrison JC. Diagnosis and follow-up of primary open-angle glaucoma. Curr Opin Ophthalmol. 1990;1:109-116.
4. Matz CE, Jonas A. Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions. J Biol Chem. 1982;257:4535-4540.
5. Jonas A, Kezdy KE, Wald JH. Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs. J Biol Chem. 1989;264:4818-4824.
6. Sviridov D, Fidge N. Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I. J Lipid Res. 1995;36:1887-1896.
7. Christian AE, Haynes MP, Phillips MC, Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res. 1997;38:2264-2272.
8. Remaley AT, Thomas F, Stonik JA, Demosky SJ, Bark SE, Neufeld EB, Bocharov AV, Vishnyakova TG, Patterson AP, Eggerman TL, Santamarina-Fojo S, Brewer HB. Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway. J Lipid Res. 2003;44:828-836.
9. Mukhamedova N, Fu Y, Bukrinsky M, Remaley AT, Sviridov D. The Role of Different Regions of ATP-Binding Cassette Transporter A1 in Cholesterol Efflux. Biochemistry. 2007;46:9388-9398.
10. Lawrence MB, McIntire LV, Eskin SG. Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion. Blood. 1987;70:1284-1290.
11. Woollard KJ, Kling D, Kulkarni S, Dart AM, Jackson S, Chin-Dusting J. Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion. Circ Res. 2006;98:149-156.
12. Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA. Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol. 1993;121:163-170.
Figure Legends:
Figure 1.
CD11b activation: Monocytes were stimulated with (A) PMA (1µmol/L)±HDL (2-
50µg/mL) or BSA (50µg/mL) (B) PMA±HDL (50µg/mL), rHDL (40µg/mL), apoA-I
(40µg/mL) or POPC liposomes (10-50µg/mL) (C) Monocytes were either pre-
incubated with HDL (50 µg/mL; 30mins) before the addition of PMA (1µmol/L;
15mins “prevention”) or pre-incubated with PMA (15mins) before the addition of
HDL (30mins “reversal”) and compared to control (PMA alone, final 15mins). (D)
Monocytes were pre-incubated with HDL (50µg/mL) or apoA-I (40µg/mL) for
30mins before removal by centrifugation and the monocytes washed with PBS.
The monocytes were then simulated with either PMA (15mins) and CD11b levels
assessed by flow cytometry. Results are expressed as a percentage of
activation relative to the control (unstimulated monocytes). *P<0.05, **P<0.01,
***P<0.001.
Figure 2.
Cholesterol efflux: (A) Monocytes were incubated with PMA (1µmol/L) ± either
cyclodextrin or cholesterol saturated cyclodextrin (100-200µg/mL) for 15mins and
Supplementary Methods CD16+/CD16- monocyte isolation Human mononuclear cells (MNCs) were isolated as stated in the manuscript and
then further separated into CD16+ and CD16- by use of an CD16+ monocyte
isolation kit (miltenyi Biotec). The two monocyte populations were treated and
prepared for flow cytometry as per the methods in the manuscript.
figure legends Figure I. The effect of apoA-I on CD16+ and CD16- monocytes Monocytes were isolated from PWB and further separated into 16+ and CD16-
populations. The monocytes were incubated with 1µmol/L of PMA ± apoA-I
(40µg/mL) with a FITC-CD11b antibody for 15mins at 37°C. The samples were
fixed and CD11b activation assessed by flow cytometry. *P<0.05, **P<0.01.
Figure II.
Monocytes were stimulated with (A) LPS (1µg/mL) or (B) fMLP (0.1µmol/L) ±HDL
(50µg/mL) or apoA-I (40µg/mL) for 15mins at 37°C. CD11b activation was
assessed via flow cytometry. *P<0.05, **P<0.01.
Figure III. (A-D) Representative fields of perfused monocytes on a platelet monolayer after
washout period (300secs). (A) Buffer, (B) HDL. Magnification X200, Bar=56µm.
*P<0.05. (B3, B4) Zoomed in sections of gated areas from C and D respectively. Figure IV. apoA-I mimetic peptides: (A) Monocytes were stimulated with PMA±L37pA (10-
40µg/mL) or apoA-I (40µg/mL). CD11b activation was quantitated by flow
cytometry. (B) Isolated monocytes were treated with PMA±L37pA (20µg/mL) or
apoA-I (20µg/mL). Adhered monocytes were quantified by counting five random
fields using phase microscopy (X40). *P<0.05, **P<0.01.
