Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

doi:10.1152/ajpcell.00416.2008 297:C886-C897, 2009. First published 18 March 2009;Am J Physiol Cell Physiol

Gamboni-Robertson, Samina Y. Khan and Christopher C. SillimanLynn M. Gries, Marguerite R. Kelher, Kelly M. England, Fabia Phillip C. Eckels, Anirban Banerjee, Ernest E. Moore, Nathan J. D. McLaughlin,clathrin-mediated endocytosis in human neutrophilsAmantadine inhibits platelet-activating factor induced

You might find this additional info useful...

75 articles, 44 of which can be accessed free at:This article cites http://ajpcell.physiology.org/content/297/4/C886.full.html#ref-list-1

including high resolution figures, can be found at:Updated information and services http://ajpcell.physiology.org/content/297/4/C886.full.html

can be found at:AJP - Cell Physiologyabout Additional material and information http://www.the-aps.org/publications/ajpcell

This infomation is current as of January 14, 2011.

American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2009 by the

is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 timesAJP - Cell Physiology

on January 14, 2011ajpcell.physiology.org

Dow

nloaded from

http://ajpcell.physiology.org/content/297/4/C886.full.html#ref-list-1

http://ajpcell.physiology.org/content/297/4/C886.full.html

http://ajpcell.physiology.org/

Amantadine inhibits platelet-activating factor induced clathrin-mediatedendocytosis in human neutrophils

Phillip C. Eckels,1 Anirban Banerjee,2 Ernest E. Moore,1,2 Nathan J. D. McLaughlin,3 Lynn M. Gries,2

Marguerite R. Kelher,2 Kelly M. England,3 Fabia Gamboni-Robertson,2 Samina Y. Khan,1

and Christopher C. Silliman2,3,4

1Department of Surgery, Denver Health Medical Center; Departments of 2Surgery and 3Pediatrics, School of Medicine,University of Colorado Denver; and 4Bonfils Blood Center, Denver, Colorado

Submitted 12 September 2007; accepted in final form 11 March 2009

Eckels PC, Banerjee A, Moore EE, McLaughlin NJ, Gries LM,Kelher MR, England KM, Gamboni-Robertson F, Khan SY,Silliman CC. Amantadine inhibits platelet-activating factor inducedclathrin-mediated endocytosis in human neutrophils. Am J PhysiolCell Physiol 297: C886–C897, 2009. First published March 18, 2009;doi:10.1152/ajpcell.00416.2008.—Receptor signaling is integral foradhesion, emigration, phagocytosis, and reactive oxygen species pro-duction in polymorphonuclear neutrophils (PMNs). Priming is animportant part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF)primes PMNs through activation of a specific G protein-coupledreceptor. We hypothesize that PAF priming of PMNs requires clath-rin-mediated endocytosis (CME) of the PAF receptor (PAFr), and,therefore, amantadine, known to inhibit CME, significantly antago-nizes PAF signaling. PMNs were isolated by standard techniques to�98% purity and tested for viability. Amantadine (1 mM) signifi-cantly inhibited the PAF-mediated changes in the cellular distributionof clathrin and the physical colocalization [fluorescence resonanceenergy transfer positive (FRET�)] of early endosome antigen-1 andRab5a, known components of CME and similar to hypertonic saline,a known inhibitor of CME. Furthermore, amantadine had no effect onthe PAF-induced cytosolic calcium flux; however, phosphorylation ofp38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of theNADPH oxidase and shape change with lesser inhibition of increasesin CD11b surface expression and elastase release. Furthermore,rimantadine, an amantadine analog, was a more potent inhibitor ofPAF priming of the N-formyl-methionyl-leucyl-phenylalanine-acti-vated oxidase. PAF priming of PMNs requires clathrin-mediatedendocytosis that is inhibited when PMNs are pretreated with eitheramantadine or rimantadine. Thus, amantadine and rimantadine havethe potential to ameliorate PMN-mediated tissue damage in humans.

G protein-coupled receptor; clathrin-coated vesicle; polymorphonu-clear neutrophils

POLYMORPHONUCLEAR NEUTROPHILS (PMNs) circulate for 12–18 hbefore migrating into tissue (36). Emigration of PMNs fromthe vasculature into tissue is a complex, coordinated processthat is vital for the eradication of infectious pathogens (1, 6).This innate process of tissue migration initiates PMN primingand augmentation of the microbicidal response, both oxidativeand nonoxidative, to a subsequent stimulus (1, 67). Priming isan integral part of PMN emigration and changes PMNs from anonadherent to an adherent phenotype (1, 6, 60, 66, 67). Inaddition, priming causes rapid actin redistribution, allowingfor shape change and chemotaxis, and maximizes the mi-

crobicidal response to invading microbes for effective elim-ination (24, 31, 60, 67, 74). Priming begins via vascularendothelial attraction of PMNs to their surface through therelease of chemokines followed by E- and P-selectin-mediated tethering through contact with obligate ligands(PSGL-1) on the PMN surface and firm adhesion of thePMNs through �2-integrins and ligands [e.g., intercellular adhe-sion molecule-1 (ICAM-1), vascular cellular adhesion molecule(VCAM)] on the endothelial cell (EC) surface. PMNs thendiapedese through the EC, which involves CD31 on bothPMNs and EC, chemotax to the site of infection, and phago-cytize and kill the pathogenic invaders (1, 6, 22, 24, 74).Furthermore, priming changes the PMN phenotype to “hyper-responsive,” such that the PMNs microbial arsenal can beactivated by normally innocuous stimuli (74), and in two-eventmodels of PMN-mediated lung injury, PMN priming agents areetiologic; therefore, interruption of the priming signal canameliorate or inhibit tissue damage (49, 61).

Platelet-activating factor (PAF) is an effective physiologicalpriming agent, and its receptor is a member of the G protein-coupled receptor family (GPCR) (37, 49, 58, 67). A charac-teristic of GPCRs is clathrin-mediated endocytosis (CME),which may be sensitive to weak bases such as amantadine (33).Amantadine has classically been used for the prevention andtreatment of influenza A and Parkinson’s disease (46, 54).Amantadine has been further described to inhibit neural protonchannel activity (28) and acts as a low-affinity N-methyl-D-aspartate (NMDA) receptor antagonist (39, 48); however, itsspecificity inhibiting CME of the PAF receptor is not wellunderstood although it is frequently used as a clathrin inhibitor(3, 29, 47, 50, 52, 65). Recent work has characterized the earlyevents in PAF-mediated CME (25) including internalization ofthe ligand:receptor complex and sequestration of these ligatedreceptors to clathrin-coated vesicle (CCVs) (55). In addition,the PMN is unique for it does not contain caveolin and thusserves as an excellent cellular model to study CME (56).Therefore, we hypothesize that amantadine inhibits CME,which is required for PAF signaling and for PAF-mediatedchanges in PMN physiology. We will also compare the effectsof amantadine to hypertonic saline, a well-described antagonistof CME, to support this hypothesis (9, 26, 57).

MATERIALS AND METHODS

Unless otherwise indicated, all chemicals were purchased fromSigma Chemical (St. Louis, MO). Acrylamide, N�-methylene-bis-acrylamide, and N,N,N�,N�-tetramethylethylenediamine (TEMED)were obtained from Bio-Rad (Hercules, CA). X-ray and enhancedchemiluminescence reagents were obtained from E. I. DuPont, (Wil-

Address for reprint requests and other correspondence: C. C. Silliman,Associate Medical Director, Research Dept., Bonfils Blood Center, 717 Yo-semite St., Denver, CO 80230 (e-mail: [email protected]).

Am J Physiol Cell Physiol 297: C886–C897, 2009.First published March 18, 2009; doi:10.1152/ajpcell.00416.2008.

0363-6143/09 $8.00 Copyright © 2009 the American Physiological Society http://www.ajpcell.orgC886


Dow

nloaded from


mington, DE) and Amersham (Arlington Heights, IL), respectively.Antibodies to the clathrin heavy chain, Rab5a, and early endosomeantigen 1 (EEA-1), along with conjugated secondary anti-goat (Cy3)and anti-rabbit (FITC) IgG, were purchased from Invitrogen (Carls-bad, CA), and dual phosphorylated-p38 MAPK (Thr180/Tyr182) andsecondary anti-rabbit IgG were obtained from Cell Signaling (Bev-erly, MA). Monoclonal PAF receptor antibodies for flow cytometrywere purchased from Cayman Chemical (Ann Arbor, MI). Secondaryantibodies were purchased from Santa Cruz Biotechnology (SantaCruz, CA). FITC labeled anti-CD45 was obtained from BD Pharm-ingen (San Diego, CA), Nunc 96-well plates from Life ScienceProducts (Frederick, CO), and indo-1 AM from Molecular Probes(Eugene, OR). A Leica DRM-mechanized fluorescence microscope,equipped with a movable stage with a custom Zeiss �63 water-immersion lens, was purchased from Leica Microsystems (Exton,PA). A Zeiss LSM (Oberkochen, Germany) was used for the quanti-fication and imaging of slides for fluorescence resonance energytransfer (FRET) analysis.

Neutrophil Isolation

PMNs were isolated by standard techniques (59). Heparinizedwhole blood was drawn from healthy human donors after obtaininginformed consent and employing a protocol approved by the ColoradoMultiple Institutional Review Board and Human Subjects Committeeat the University of Colorado School of Medicine. PMNs wereisolated by dextran sedimentation, ficoll-hypaque gradient centrifuga-tion, and hypotonic lysis as previously described (59). Cells wereresuspended to a concentration of 2.5 � 107 cells/ml in Krebs-Ringer-phosphate buffer with 2% dextrose (KRPD) (pH 7.35) and usedimmediately for all subsequent manipulations. In all experiments,isolated PMNs were pretreated with amantadine, 100 �M or 1 mM,rimantadine, 1 �M or 100 �M, or hypertonic saline (HTS) at 180mmol/l NaCl for 5 min at 37°C before the addition of PAF (2 �M).

Fluorescent Visualization of Clathrin

PMNs (5.0 � 105 cells) were warmed to 37°C, incubated for 5 minwith buffer (control) or inhibitors, and were then primed with eitherbuffer or PAF for 3 and 5 min. The reaction was stopped with theaddition of an equal volume of cold, fresh 8% paraformaldehyde(PFA) and incubated at 4°C for 5 min. Approximately 1.5 � 104

PMNs were smeared on microscope slides and allowed to dry over-night. Following three washes with phosphate-buffered saline (PBS),cells were permeabilized with acetone:methanol (70:30) at �20°C for10 min. The slides were air dried and blocked for 1 h at roomtemperature with 10% donkey serum and incubated overnight at 4°Cwith goat anti-clathrin primary antibody in PBS with 1% BSA. Afterwashing three times with PBS, an AlexaFluor 488 donkey anti-goatsecondary antibody (red) was added along with anti-quenching media,and the nuclear region (blue) was visualized using bis-benzimide (60).

Subcellular Localization and Western Blot Analysis of ClathrinHeavy Chain

PMNs (1 � 108/ml) were warmed to 37°C with gentle agitation,pretreated with buffer, amantadine, or HTS for 5 min and thenstimulated with buffer or PAF for 1–3 min and placed into ice-coldrelaxation buffer [10 mM PIPES (pH 7.4), 3 mM NaCl, 100 mM KCl,3.5 MgCl2, 1.2 mM EGTA, 10 �g/ml leupeptin, 40 mM sodiumorthovanadate, 1 M nitrophenylphosphate, and 50 �g/ml PMSF] andimmediately sonicated (2 � 30 s). The lysates were placed ontosucrose gradients and ultracentrifuged as documented previously (37,38, 60). Purity of fractions was determined using p47phox as a cytosolmarker and the PAF receptor as a membrane marker (data not shown).Before protein separation, the fractions were placed in Laemmlidigestion buffer containing 40 mM sodium orthovanadate, 1 Mnitrophenolphosphate, 10 �g/ml leupeptin, and 100 mM PMSF (an

inhibitor cocktail) (17). Samples were boiled for 15 min and proteinswere separated by SDS-PAGE. The proteins were transferred tonitrocellulose, blocked with 5% BSA (fraction V) overnight, andincubated with a primary antibody to clathrin heavy chain. Afterwashing in Trizma-buffered saline plus 0.1% Tween 20 (TBST), theblots were incubated with horseradish peroxidase (HRP)-linked goatanti-rabbit secondary antibody and visualized by enhanced chemilu-minescence and exposure of X-ray film (17, 60).

FRET Analysis of Rab5a and EEA-1

Isolated PMNs were incubated at 37°C with buffer, 2 �M PAF (1min), 1 mM amantadine (5 min), or amantadine followed by PAF, andthen fixed in 4% paraformaldehyde at 4°C for 20 min and smearedonto slides. FRET determinations were obtained by direct acceptorphotobleaching FRET (adFRET), as previously described (37).Within this context, the ability of the two secondary antibodies toFRET was acquired between rhodamine (Jackson ImmunoResearch;excitation, 550 nm; emission, 570 nm, acquired on the Cy3 channel)and AlexaFluor 488 (Molecular Probes; excitation 495 nm; emission519 nm, acquired on the FITC channel). In all cases, an initial imagewas acquired of the donor and acceptor channels, and followingcapture, a region of interest was defined, a mask applied, and thespecified acceptor (Cy5 or Cy3) ablated (i.e., photo bleaching, permanufacturer’s nomenclature). Ablation was accomplished using aPhotonics FRAP laser fitted with the appropriate wavelength discrim-inator (rhodamine 610-Cy5 or rhodamine 540-Cy3). FRET efficien-cies (Ei) were calculated using the following equation:

Ei �Ipost,i � Ipre,i

Ipre,i

(1)

where Ipre,i is the mean intensity of the donor pre-photo-bleach imageand Ipost,i is the mean intensity of the donor post-photo-bleach image(73). Images are displayed in pseudocolor where blue indicates littleFRET and red indicates the most FRET.

PAF Receptor Surface Expression Via Flow Cytometry

Isolated PMNs (1 � 106 cells) were preincubated with amantadine,rimantadine, or buffer before incubation with PAF at 37°C. Thereaction was stopped with the addition of an equal volume of cold,fresh 8% paraformaldehyde and incubated at 4°C for 5 min. PMNwere coincubated with a PAF receptor antibody and Fc-receptorblocker (Accurate Chemical, Westbury, NY), to reduce nonspecificbinding, for 30 min at 4°C and washed with cold KRPD. Cells wereresuspended in a 4% PFA-PBS solution and analyzed on a BeckmanFC500 flow cytometer within 24 h.

Measurement of Cytosolic Ca2�

In selected experiments, PMN cytosolic Ca2� levels were deter-mined by indo-1 AM loading of PMNs and analyzed in a Perkin-Elmer LS50B spectrofluorimeter over real time (Perkin-Elmer, Nor-walk, CT), as previously described, employing the Grynkiewiczequation (60). Briefly, following loading of indo-1 AM, PMNs wereeither incubated in amantadine (1 mM), HTS (180 mmol/l), or buffer,stimulated with PAF, and in some cases stimulated a second timeagain with PAF following a return to basal calcium levels.

Western Blot Analysis of Phosphorylated p38 MAPK

PMNs were warmed to 37°C with gentle agitation. PMNs werepretreated with buffer, HTS, or amantadine for 5 min and stimulatedwith buffer, PAF, or N-formyl-methionyl-leucyl-phenylalanine (fMLP)for 1 min and immediately lysed in Laemmli digestion buffer con-taining 40 mM sodium orthovanadate, 1 M nitrophenolphosphate, 10�g/ml leupeptin, and 100 mM PMSF (an inhibitor cocktail) (17).Samples were boiled for 60 min, and proteins were separated by

C887AMANTADINE INHIBITS PAF-INDUCED CME IN HUMAN PMNS

AJP-Cell Physiol • VOL 297 • OCTOBER 2009 • www.ajpcell.org


Dow

nloaded from


SDS-PAGE. The proteins were transferred to nitrocellulose, cross-linked, blocked with 5% BSA (fraction V) overnight, and incubatedwith a primary antibody to diphosphorylated (Thr180/Tyr182) p38MAPK (Cell Signaling). Washed in TBST, the blots were incubatedwith HRP-linked goat anti-rabbit secondary antibody and visualizedby enhanced chemiluminescence and exposure of X-ray film (17, 60).For evaluation of p38 MAPK, the blots were stripped, incubated witha primary antibody to total p38 MAPK, washed, and visualized asdetailed above (17, 60).

Immunoprecipitation of EEA-1 and Rab5a and Western BlotAnalysis of Proteins From PMNs

PMNs (5 � 106) were incubated with buffer, 2 �M PAF for 1–5min, 1 mM amantadine, 180 mM HTS, or amantadine � PAF orHTS � PAF as indicated. The reactions were stopped with theaddition of ice-cold relaxation buffer [10 mM PIPES (pH 7.4), 3 mMNaCl, 100 mM KCl, 3.5 MgCl2, 1.2 mM EGTA, 10 �g/ml leupeptin,40 mM sodium orthovanadate, 1 M nitrophenylphosphate, and 50�g/ml PMSF] and immediately sonicated (2 � 30 s). Lysates werecleared and immunoprecipitated with beads linked to specific anti-bodies against EEA-1 or Rab5a overnight at 4°C as previouslydescribed (51). For whole cell lysates, the PMNs were lysed employ-ing Laemmli digestion buffer and boiled for 60 min. In either case,proteins were separated by 7.5% or 10% SDS-PAGE, transferred tonitrocellulose membranes, which were cross-linked and then probedwith specific antibodies to EEA-1 or Rab5a.

PAF-Mediated Changes in PMN Morphology

Aliquots of freshly isolated PMNs (5.0 � 106 cells) were added tomicrocentrifuge tubes, warmed to 37°C using adjacent plate warmer,and incubated with buffer, PAF (5 min), 1 mM amantadine (5 min),100 �M rimantadine (5 min), or either amantadine or rimantadine (5min) followed by PAF (5 min). Cells were fixed in freshly prepared4% PFA/PBS (pH 7.4) for 20 min. PMN were smeared on Superfrost/Plus microscopy slides (Fisher Scientific), allowed to air dry over-night, washed three times with PBS, and mounted with BiomedaGel/Mount (Foster City, CA) and a coverslip. Cell response to PAFwas observed under bright field illumination using a Zeiss LSMmicroscope (60).

CD11b Surface Expression

PMNs (1.0 � 106) were incubated for 5 min with buffer oramantadine. Cells were stimulated with buffer or PAF for 5 min,pelleted, and the reaction was stopped by the addition of ice-coldbuffer. The PMNs were then incubated for 30 min at 4°C with 1 �g/mlof a monoclonal antibody to CD11b (Clone Bear1, Beckman Coulter)or isotype control. Cells were fixed in 4% PFA for 10 min, diluted to1% PFA with KRPD, and analyzed within 24 h via flow cytometrywith the data expressed as mean fluorescence intensity (60).

Elastase Release

PMNs (1.25 � 106) were warmed to 37°C and incubated for 5 minwith buffer (control) or amantadine. The PMNs were primed for 5 minwith buffer or PAF, and subsequently stimulated with buffer or fMLPfor 5 min. Cells were pelleted and the amount of elastase released inthe supernatant was quantified spectrophotometrically by the reduc-tion of the specific substrate methoxy-succinyl-alanyl-alanyl-prolyl-valyl p-nitroanilide (AAPVNA) at 405 nM (60). Elastase reactionswere run in duplicate, and wells containing 5 mM of the specificelastase inhibitor methoxy-succinyl-alanyl-alanyl-prolyl-valyl chlo-romethyl ketone (AAPVCK) were run in conjunction. Elastase isreported as percentage of total cellular elastase from an identicalnumber of cells treated with 0.1% Triton X-100.

PAF Priming of the NADPH Oxidase

The maximal rate (Vmax) of superoxide anion generation wasmeasured by monitoring the superoxide dismutase-inhibitable reduc-tion of cytochrome c at 550 nm in a Molecular Devices microplatereader (Menlo Park, CA) as previously described (59). PMNs werepreincubated with buffer, amantadine, or rimantadine for 5 min at37°C with gentle agitation. Following preincubation, PMNs (3.75 �105 cells) were primed for 3 min with PAF or buffer, activated withfMLP or buffer, and the maximal rate of O2

� production was mea-sured.

Statistical Analysis

Statistical differences among groups were determined by a pairedor an independent ANOVA followed by a Tukey or Bonferroni posthoc analysis for multiple comparisons based on the equality ofvariance. All data are presented as means � SE; statistical signifi-cance was determined at the P 0.05 level.

RESULTS

Clathrin Reorganization

Because PAF ligation of the platelet-activating factorreceptor (PAFr) results in CME in PMNs, we examined theeffects of amantadine on PAF-mediated clathrin reorgani-zation in isolated PMNs (33, 37) (Fig. 1). Quiescent buffer-treated PMNs exhibited an almost uniform distribution ofclathrin (Fig. 1Aa). In contrast, PAF (2 �M) priming ofPMNs caused a redistribution of clathrin at 3 min (Fig.1Ab), with further progression at 5 min as demonstrated bythe appearance of a number of punctate fluorescent struc-tures at the membrane and within the cytoplasm (Fig. 1Ac).Amantadine (1 mM) alone did not affect clathrin distribu-tion in buffer-treated PMNs (Fig. 1Ad), similar to buffer-treated controls (Fig. 1Aa). However, amantadine pretreat-ment abolished PAF-mediated clathrin reorganization atboth 3 and 5 min such that clathrin remained uniformlydistributed throughout the cytoplasm without the formationof the fluorescent punctuate structures in either the cyto-plasm or the membrane (Fig. 1A, e and f). This distributionin amantadine-pretreated PMNs primed with PAF was sim-ilar to buffer- or amantadine-treated PMNs (Fig. 1A, a andd). To confirm these data, PMNs were treated with buffer,amantadine (5 min), PAF (5 min), or amantadine (5 min)then PAF (5 min) and subcellular fractions were made, andclathrin immunoreactivity was measured in the PMN mem-brane. As compared with buffer-treated PMNs, PAF (5 min)elicited increased amounts of clathrin immunoreactivity inthe PMN membrane, which was inhibited by pretreatment ofthese PMNs by 1 mM amantadine (Fig. 1B). HTS causedsimilar inhibition of PAF-mediated changes in clathrin spa-tial organization similar to that of amantadine (Fig. 2).Compared with controls (Fig. 2Aa) PAF induced clathrinreorganization at 3 and 5 min (Fig. 2A, b and c), which wasinhibited by pretreatment with HTS (Fig. 2A, e and f).Importantly, HTS did not affect clathrin distribution ofcontrol PMNs (Fig. 2Ad). In addition, the subcellular frac-tions of PMNs, treated with buffer, HTS (5 min), PAF (3min) and HTS (5 min) then PAF (5 min), demonstrated thatthe PAF-elicited increases of clathrin in the PMN membranewere inhibited by HTS (Fig. 2B).

C888 AMANTADINE INHIBITS PAF-INDUCED CME IN HUMAN PMNS



Dow

nloaded from


FRET Analysis of EEA-1 and Rab5a in Responseto Amantadine

Because CME initiates the assembly of the endosomal com-ponents EEA-1 and Rab5a-GTPase in the formation of CCVs,we investigated if amantadine and HTS alter this physicalassociation (Fig. 3) (2, 5). In control PMNs, there was anegligible amount of FRET between the two proteins (Fig.3m). In contrast, PAF elicited a physical (FRET�) interactionbeginning at 1 min between Rab5a and the EEA-1 sorterprotein (Fig. 3n), similar to previous data (38). Amantadine (1mM) alone did not cause any significant FRET� interactionsbetween EEA-1 and Rab5a (Fig. 3o); however, amantadinepretreatment did inhibit the PAF-mediated FRET� interactionbetween EEA-1 and Rab5a (Fig. 3p).

Amantadine Inhibits EEA-1 and Rab5a Coprecipitation

Recruitment of Rab5a to CCVs has been described as apreliminary to receptor endocytosis (2), and since bothEEA-1 and Rab5a can be phosphorylated by p38 MAPK(34), and amantadine inhibits phosphorylation of p38MAPK, we hypothesized that amantadine would inhibit

PAF-mediated colocalization of EEA-1 and Rab5a. PMNswere treated with buffer, HTS (5 min), amantadine (5 min),PAF (1 or 3 min), amantadine (5 min) then PAF (1 or 3min), or HTS (5 min) followed by PAF ( 1 or 3 min), thePMNs were lysed immediately, and Rab5a or EEA-1 wasimmunoprecipitated. The proteins in the resultant immunopre-cipitates were separated by SDS-PAGE and immunoblotted forEEA-1 or Rab5a, respectively (Fig. 4). Compared with buffer-treated controls, PAF induced colocalization of EEA-1 withRab5a at 1 min, which persisted to 3 min (Fig. 4). Amantadinealone did not cause colocalization of EEA-1 with Rab5a, butamantadine pretreatment did inhibit the PAF-induced colocaliza-tion of EEA-1 with Rab5a (Fig. 4, A and B). In addition, HTSalone did not affect the EEA-1:Rab5a colocalization, but pretreat-ment did inhibit the PAF-mediated colocalization of EEA-1 withRab5a (Fig. 4C).

Amantadine and HTS Inhibit PAF-Mediated Internalizationof the PAFr on PMNs

CME causes internalization of the activated GPCR, and inthe case of PMNs, PAF causes internalization of the PAF:PAFr

Fig. 1. Platelet-activating factor (PAF)-mediated clathrin redistribution is inhibited by amantadine. Polymorphonuclear neutrophils (PMNs) were pretreated withbuffer or amantadine (1 mM) for 5 min, incubated with buffer (control) or PAF (2 �M) for 3 or 5 min at 37°C, and immediately fixed with paraformaldehydeor separated into subcellular fractions (membrane, cytosol, and nuclear) at 4°C for protein separation by SDS-PAGE and Western blot analysis. A: for themicroscopy, PMN nuclei were stained with bis-benzimide (blue), incubated with a fluorescently labeled antibody to the clathrin heavy chain (red), and examinedby digital microscopy. Stimulation of PMNs in PAF results in recruitment of clathrin to the membrane, as seen by punctate fluorescent immunoreactivity (arrows,b and c) as compared with the controls (a). Amantadine (1 mM) treatment did not affect PMN intracellular clathrin organization (d) and inhibited PAF-inducedclathrin reorganization (e and f) such that the membrane and cytoplasmic clathrin remained dispersed throughout the cell. B: immunoblotting of subcellularfractions of PMNs demonstrate PAF-mediated recruitment of clathrin to the membrane, which was inhibited by pretreatment with amantadine. This figure isrepresentative of three independent experiments. HC, heavy chain.




Dow

nloaded from


ligand:receptor pair (37). The surface expression of the PAFrwas determined in quiescent PMNs by flow cytometry, whichwas unaffected by amantadine alone (Fig. 5). PAF caused adecrease in PAFr surface expression by 35 � 5% as comparedwith controls or amantadine or rimantadine alone (P 0.05;n 4). Both amantadine and rimantadine (1 �M) (an effectivederivative of amantadine) significantly inhibited the loss ofPAFr surface expression on PMNs (P 0.05; n 4) (16, 51,53, 71).

Amantadine Does Not Affect PAF-Mediated Changes inCytosolic Ca2�

PAF elicits rapid increases in cytosolic Ca2� concentrationin human PMNs, and this Ca2� flux was used to test theintegrity of receptor ligation and subsequent heterotrimeric Gprotein signaling (60). Stimulation of PMNs with 2 �M PAFcaused a rapid increase in cytosolic Ca2� concentration ([Ca2�]),which returned to basal levels after �160 s (Fig. 6). Pretreatmentof PMNs with 1 mM amantadine or 180 mM HTS had little effecton the PAF-induced cytosolic Ca2� flux (Fig. 6), and to quantify

the lack of effect on the rise in cytosolic Ca2�, the baseline Ca2�,the maximal Ca2� concentration reached, and the time to reachmaximal Ca2� concentration were calculated and confirmed thatneither amantadine nor HTS affected PAF-mediated changes incytosolic Ca2� (Table 1).

Amantadine and HTS Inhibit PAF-Mediated Activationof p38 MAPK

PAF priming of PMNs caused activation, dual phosphor-ylation (Thr180/Tyr182), of p38 MAPK at 1 min (17).Consistent with these data, PMNs treated with PAF induceddual phosphorylation of p38 MAPK, when compared withtreatment with buffer alone. However, amantadine pretreat-ment of PMNs caused a concentration-dependant inhibitionof PAF-induced phosphorylation of p38 MAPK, and aman-tadine alone did not cause activation of p38 MAPK (Fig. 7A).Furthermore, HTS also inhibited PAF-induced activation ofp38 MAPK and alone did not elicit activation of p38 MAPK(Fig. 7B).

Fig. 2. PAF-induced clathrin redistribution is inhibited by hypertonic saline (HTS). PMNs were pretreated with buffer or HTS (180 mmol/l) for 5 min at 37°Cand stimulated with buffer or 2 �M PAF for 1 min, and PMNs were either immediately fixed with paraformaldehyde and smeared onto slides or divided intosubcellular fractions at 4°C for protein separation by SDS-PAGE and Western blot analysis. A: for microscopy, the PMN nuclei were stained with bis-benzimideand incubated with a fluorescently labeled antibody to the clathrin heavy chain. PAF elicited reorganization of clathrin at 3 and 5 min (arrows, b and c) ascompared with buffer-treated controls (a). Pretreatment with 180 mM HTS (d) did not affect the structural organization of clathrin organization in PMNs ascompared with buffer-treated controls (a); however, HTS pretreatment did inhibit the PAF-induced structural reorganization of clathrin (arrows, e and f) at 3 and5 min compared with b and c. B: PAF (5 min) caused recruitment of clathrin heavy chain immunoreactivity to the plasma membrane, as compared withbuffer-treated or HTS-treated PMNs, which was inhibited by pretreatment with HTS. This figure is representative of two individual experiments.




Dow

nloaded from


Amantadine Inhibits PAF-Induced Changes in PMNPhysiology

Changes in PMN morphology. Because amantadine ap-peared to inhibit PAF-mediated CME, we examined its effecton PAF-induced PMN physiology. Membrane ruffling andpseudopodia formation are characteristics of primed PMNs,which is analogous to a higher degree of actin rearrangement(14, 27). In contrast, quiescent PMNs are spherical in shape,displaying a smooth outer edge and minimal cytoskeletalrearrangement (Fig. 8A). As expected, PMNs challenged withPAF for 5 min demonstrated classic membrane ruffling andpseudopodia-like protrusions (Fig. 8B). However, PMNs incu-

bated with 1 mM amantadine or 100 �M rimantadine and thenstimulated with PAF for 5 min failed to exhibit these changes(Fig. 8, C and D).

Inhibition of PAF priming of NADPH oxidase with aman-tadine and rimantadine. To determine whether PAF priming ofthe respiratory burst depends on clathrin-mediated endocytosis,PMNs were pretreated with amantadine and subsequentlyprimed with PAF, activated with fMLP, and the maximal rateof the superoxide anion (O2

�) was measured (Vmax). Amanta-dine did not inhibit fMLP-activation of the oxidase. In contrast,amantadine significantly inhibited PAF priming of the fMLP-activated oxidase burst as compared with buffer-treated con-

Fig. 3. Amantadine inhibits the PAF-induced fluorescence resonance energy transfer-positive (FRET�) interaction between early endosome antigen-1 (EEA-1)and Rab5a. Immunofluorescent detection of EEA-1 (green, a–d), Rab5a (red, e–h), their respective overlays (colocalization: yellow, i–l), or corrected FRET�

interactions (FRETC) measured in arbitrary linear units of fluorescence intensity (ALUFI; blue lowest, red highest; m–p) are visualized in human PMNstreated with buffer, PAF (1 min), amantadine (5 min), or amantadine (5 min) followed by PAF priming. The FRETC interaction is the transfer of energy fromthe donor molecule to the acceptor molecule, which is then corrected for spectral bleed-through. PAF elicited a FRET� interaction between EEA-1 and Rab5aat 1 min (n) as compared with buffer-treated or amantadine treated-PMNs (m and o). Amantadine pretreatment disrupted the EEA-1:Rab5a FRET� interaction(p). Images are representative of 50 cells/treatment group repeated as three individual experiments.




Dow

nloaded from


trols (P 0.05, n 5) (Table 2). Rimantadine, an analog ofamantadine, was more effective in antagonizing PAF primingof the fMLP-activated respiratory burst. Rimantadine did notinhibit fMLP activation of the oxidase, but it did abrogate PAFpriming of the fMLP-activated respiratory burst in PMNs (P 0.05, n 6) (Table 2).

CD11b surface expression. Under chemoattractant chal-lenge, PMNs increase ß2-integrin surface expression via fusionof the secretory vesicles and secondary granules with theplasma membrane (75). Incubation of PMNs with PAF causedincreased surface expression of CD11b as compared withbuffer-treated controls (P 0.05, n 6). Amantadine atten-

uated the PAF-elicited increase in cell surface-labeled CD11bat 1 mM (P 0.05, n 6) but did not affect the CD11bsurface expression of buffer-treated controls (P 0.11, n 6)(Fig. 9A).

Amantadine inhibits PAF priming of elastase release.Elastase release was employed as a direct measurement ofazurophilic degranulation. PMNs treated with buffer andincubated with PAF or fMLP released very little elastase,however, elastase release was greatly increased when PMNswere primed with PAF and activated with fMLP (P 0.05,n 6). Amantadine pretreatment had little effect on elastaserelease when PMNs were incubated with PAF or fMLPalone. In contrast, pretreatment of PMNs with amantadinesignificantly diminished the PAF/fMLP-induced degranula-tion in PMNs (P 0.05, n 6) (Fig. 9B).

DISCUSSION

The results of the present study demonstrate that PAFpriming of PMNs elicited clathrin recruitment to the plasmamembrane, a corrected FRET� interaction (FRETC)-positive colocalization of EEA-1 and Rab5a, and activationof p38 MAPK. Amantadine 1) abolished clathrin recruit-ment to the membrane and it remained uniformly distributedthroughout the cytoplasm, 2) disrupted endosome formationby inhibiting the FRET�, colocalization of EEA-1 andRab5a, and 3) abrogated the activation of the p38 MAPK.Furthermore, amantadine abrogated PAF-mediated changesin PMN physiology including cytoskeletal rearrangement ofactin and priming of the respiratory burst. In addition,amantadine attenuated PAF-mediated increases in �2-integrin surface expression, and priming azurophilic granulerelease. Amantadine and rimantadine antagonism of thedescribed PAF-mediated changes in PMN physiology isidentical to the inhibitory effects of HTS on PAF priming aswe have published previously (9, 26, 57). Importantly,amantadine, similar to HTS, did not affect the PAF-inducedcytosolic Ca2� flux, which is presumably the direct result ofactivation of the heterotrimeric G proteins linked to thePAFr (34). Therefore, the hypothesis that amantadine inhi-bition is due to the possible disruption of ligand binding orinterruption in PAF-mediated signal transduction, as has

Fig. 4. Amantadine inhibits EEA-1 and Rab5a coprecipita-tion. PMNs (5 � 106 cells) were incubated with buffer, 1 mMamantadine (5 min), 180 mM HTS (5 min), 2 �M PAF (1min or 3 min), amantadine � PAF, or HTS � PAF. Reac-tions were stopped by lysing the PMNs, and the lysates werecleared before immunoprecipitation (IP) of EEA-1 or Rab5a.The beads were lysed, and the proteins were separated bySDS-PAGE and transferred to nitrocellulose membranes,which were cross-linked. The IPs of EEA-1 were probed forRab5a and the IPs of Rab5a were probed for EEA-1. Aman-tadine alone did not cause coprecipitation of EEA-1 withRab5a (A) and as compared with controls or amantadine-treated PMNs, PAF caused coprecipitation of EEA-1 withRab5a at 3 and 5 min regardless of which protein wasimmunoprecipitated (A and B). Both amantadine (A and B)and HTS (C) pretreatment inhibited coprecipitation of thesetwo proteins. These images are representative of three indi-vidual experiments, and although the presented images arefrom the same gel, the order has been changed for greaterclarity to keep the identical time points as denoted by thewhite breaks in the gel.

Fig. 5. PAF-mediated decrease in cell surface-labeled PAF receptor expres-sion is inhibited by amantadine. Isolated human PMNs were treated withbuffer, 1 mM amantadine (5 min), 2 �M PAF or 100 �M amantadine and 1mM amantadine � PAF or 100 �M rimantadine � PAF. Compared withbuffer-treated controls or amantadine-treated PMNs, PAF induced a 35 � 4%decrease in mean fluorescent intensity (MFI) of the extracellularly accessiblePAF receptor (*P 0.05; n 6). Amantadine did not alter baseline surfaceexpression of the PAF receptor compared with controls; however, after PAFstimulation, amantadine-treated cells at both 100 �M and 1 mM and riman-tadine at 100 �M inhibited the loss of cell surface-labeled PAF receptorexpression as quantified by flow cytometry. Moreover, like amantadine,rimantadine alone did not affect the amount of the extracellularly accessiblePAF receptor (results not shown). This figure is representative of 50,000cells/sample and six individual experiments.




Dow

nloaded from


been shown for the NMDA-type glutamate receptor, appearsmoot (20). Taken together, these data provide evidence thatamantadine does not affect the PAF:PAFr interaction be-cause of its inability to diminish the PAF-induced increasesin cytosolic Ca2�. Instead, we observed that amantadineinhibited clathrin reorganization into clathrin-coated pitswith subsequent decrease in dual phosphorylation (activa-tion) of p38 MAPK. Many of the cellular responses elicitedby PAF are directly influenced by p38 MAPK activation,priming of the respiratory burst, and actin reorganization;however, the effects on intracellular granules, whether dueto the specific granules binding to the membrane and in-creasing the surface expression of CD11b or to the release ofproteases from azurophilic granules, is not as well under-stood and have been proposed to involve multiple mecha-

nisms including MAPKs and changes in cytosolic Ca2� (18,44, 45). Importantly, amantadine does inhibit the PAF-induced activation of p38 MAP kinase but did not affect thePAF-mediated increases in cytosolic Ca2�, which may bethe reason that amantadine only attenuates the PAF-elicitedchanges in PMN physiology related to the movement orrelease of granule constituents including both the release ofelastase and the increased surface expression of CD11b (7,42, 69). Previous work has determined that chelation ofcytosolic Ca2� does significantly attenuate PAF-inducedincreases in the surface expression of CD11b (18). Lastly,physiologic concentration of HTS (180 mM) inhibits fMLP-elicited activation of PMNs; however, amantadine did not asdocumented by its inability to decrease the amount offMLP-activation of the oxidase in the priming experiments(Table 2) (9 –12). Although PAF and fMLP are both effec-tive chemoattractants, they mediate their actions throughvery diverse signaling pathways, and previous work hasdocumented that PAF-mediated CME in PMNs directlycauses actin reorganization and priming of the oxidasethrough phosphorylation and translocation of the cytosoliccomponents of the NADPH oxidase (8, 35, 37, 38, 44).Little data are available with respect to fMLP inducing CMEvia ligation of its receptor, and further work is needed.

The sorter protein EEA-1 is a component of the endosomalfusion machinery that binds to the GTP-loaded form of Rab5a,a small GTPase that controls endocytosis and early endosomedynamics (13, 21). A 30-amino acid region upstream of theFYVE domain of EEA-1 was shown to be essential for Rab5abinding in vitro, although the functional FYVE domain was alsorequired for efficient interaction (32). Because PMN migrationdemands reorganization of actin and changes in �2-integrin sur-face expression, the effects of amantadine preincubation weretested on PAF-induced changes in cell morphology and CD11bsurface expression (6, 14). Changes in the actin cytoskeleton arerequired for the observed changes in cellular morphology as wellas chemotaxis, which is directly related to increases in the surfaceexpression of the �2-integrins (14). Rab5a is vital for thesechanges in PMN morphology, and amantadine blocked recruit-ment of Rab5a to the endosome (30, 72).

The role of CME in GPCR cell signaling has become anarea of intense research (15, 19). Several different processesappear to be conserved among many GPCR and CME eventsincluding receptor ligation, which activates the associatedheterotrimeric G protein, resulting in an increase in secondmessengers (19). Arrestin binding uncouples the receptorfrom the G� and Gß subunits, and this desensitization isaccompanied by the concomitant sequestering of receptors

Table 1. Effects of amantadine and hypertonic salineon PAF-mediated changes in cytosolic Ca2� concentration

TreatmentResting

�Ca2��CYTO, nMPeak

�Ca2��CYTO, nM Time to Peak, s

PAF 106�15 941�64 22�0.8Amantadine � PAF 104�8 940�4 3 22�0.5HTS � PAF 102�7 984�73 21�0.6

Values are means � SE and represent 4 identical experiments performed onpolymorphonuclear neutrophils (PMNs) from disparate donors. PAF, platelet-activating factor; HTS, hypertonic saline; �Ca2�� CYTO, cytosolic Ca2� con-centration.

Fig. 6. Amantadine or HTS does not inhibit PAF-mediated changes in cyto-solic Ca2�. Changes in cytosolic Ca2� were monitored in indo-1 AM-loadedPMNs in a dual-wavelength spectrofluorimeter in real time. PMNs werepretreated with buffer, amantadine (1 mM), or HTS (180 mmol/l) for 5 min at37°C and subsequently stimulated with 2 �M PAF at 40 s (arrow). Amanta-dine- or HTS-pretreated cells followed by PAF stimulation displayed nosignificant difference from PAF-stimulated PMNs, and these compounds alonedid not cause changes in cytosolic Ca2� (data not shown). This figure isrepresentative of four independent experiments.




Dow

nloaded from


to clathrin-coated pits (19). This ends the initial stage ofreceptor signaling; however, sequestering receptors to clath-rin-coated pits induces recruitment of endocytotic machin-ery and signaling complexes to the membrane (4, 15, 19, 34,56, 64). Such signaling cascades post receptor-ligand bind-ing have been implicated as potential platforms for stagingthe assembly of signaling complexes that can be traffickedto various intracellular destinations (23, 62). Indeed, PAFand its obligate receptor have the ability to induce theformation of such scaffolding complexes as the ASK1/MKK3/p38 MAPK signalosome that has been shown to berequired for CME in which clathrin plays a key role (37).The diversity of clathrin-binding proteins suggests thatclathrin-coated pits can act as signaling microdomains that

regulate signaling in a temporal and spatial manner (15, 64).The majority of available data have come from studies oftransfected cell lines, such as the Chinese hamster ovary(CHO) or COS7 cell lines, used to represent hematopoieticcells, many of which are transformed to become virtuallyimmortal (2, 5, 40, 41, 63, 68). Although important data maybe gleaned from experimentation within these models, theymay not demonstrate physiologic relevance because they areterminally differentiated cells versus primary cells, e.g.,PMNs.

The goal of the present study was to investigate inhibition ofCME using PAF priming of PMNs as a model. However, theconcentrations of amantadine employed herein are not physi-ological. The average maximal concentration (Cmax) of aman-

Fig. 7. Amantadine and HTS inhibit PAF activation ofp38 MAPK. PMNs were treated with buffer, amantadine(100 �M or 1 mM) for 5 min (A) or HTS (180 mM; B)and stimulated with buffer or 2 �M PAF for 1 min.Activation of p38 MAPK was determined by SDS-PAGEelectrophoresis and immunoblotting with an antibodyagainst dual phosphorylated (activated) p38 MAPK(Thr180/Tyr182). Following these experiments, the blotswere stripped and incubated with an antibody against p38MAPK to ensure that the concentrations of this enzymewere constant per well, and the observed differences inp38 MAPK activation were not due to loading unequalamounts of this enzyme. Compared with buffer-treatedand amantadine-treated PMNs, PAF elicited activation ofp38 MAPK as demonstrated by a band immunoreactivityto the activated p38 MAPK (A and B). Amantadine (A)and HTS (B) pretreatment caused inhibition of PAF-induced p38 MAPK activation that was concentrationdependent for amantadine (A). The total amounts of p38MAPK did not differ across the wells as demonstrated bythe immunoblots of the stripped gels (A and B). Theseimages are representative of three individual experiments,and the white breaks are to demonstrate that data fromdifferent time points that are not discussed have beenremoved and that these bands are from the identical gel.

Fig. 8. Inhibition of PAF-mediated cytoskeletalchanges by amantadine. PMNs were preincubatedwith buffer (A), 2 �M PAF (5 min) (B), or prein-cubated with amantadine (1 mM) (C) or rimantadine(1 �M) (D) for 5 min at 37°C, and subsequentlystimulated with PAF for 5 min. PMNs were visual-ized using phase-contrast microscopy (Nomarski).Membrane ruffling and pseudopodia formation wereobserved in the PAF-primed cells (B) that is char-acteristic of chemotactically challenged PMNs. Incontrast, amantadine- and rimantadine-pretreatedPMNs appeared spherical in shape, displaying asmooth outer edge (C and D). These images repre-sent three individual experiments.




Dow

nloaded from


tadine in the plasma is 0.22 �g/ml (11.7 �M) 3 h after an initialdose of 100 mg. Thus, while we find amantadine to inhibitmany aspects of PAF priming, it is with concentrations 100times greater than that used clinically. In contrast, we findrimantadine to significantly inhibit PAF priming of the fMLP-activated oxidase at clinically achievable concentrations. Riman-tadine is an analog of amantadine, differing only in the replace-

ment of the amino group at position one with a methyl aminegroup; rimantadine pretreatment significantly inhibited PAFpriming of the fMLP-activated respiratory burst at concentra-tions 10-fold lower (70). The Cmax of rimantadine is 74 ng/ml(0.3 �M) 6 h after an initial dose of 100 mg, indicating thatphysiologic concentrations are close to the in vitro concentra-tions required to inhibit PAF-mediated changes in PMN phys-iology (25, 43). Thus, rimantadine may provide a better meansto curtail the effect of PMN-mediated organ injury, e.g., theacute respiratory distress syndrome and postinjury organ fail-ure, yet its effects on other clathrin-dependent and -indepen-dent endocytotic mechanisms in different tissues need to bemore clearly defined. Importantly, both amantadine and riman-tadine were effective in inhibiting PAF signaling and PAF-induced changes in PMN physiology similar to HTS. It isimportant to remember that HTS represents a clinically rele-vant strategy for attenuating the cytotoxic responses of PMNsand is known to inhibit CME (9, 26, 57). Therefore, HTS hasserved as a competent foil for amantadine and rimantadine,thus implicating their ability to inhibit CME in a similarfashion.

In conclusion, amantadine inhibited multiple PAF-mediatedchanges in PMN physiology at the level of clathrin recruitmentto the membrane such that downstream events including earlyendosomal formation are dependent on EEA-1 and Rab5a

Table 2. Amantadine and rimantadine inhibition of PAFpriming of the fMLP-activated respiratory burst

Buffer

Amantadine

10 �M 100 �M 1 mM

Buffer 0.2�0.1 0.2�0.1 0.1�0.1 0.1�0.1fMLP 0.8�0.3 0.9�0.2 0.6�0.2 0.6�0.1PAF/fMLP 2.6�0.3* 2.5�0.2* 1.8�0.3*† 1.6�0.2†

Buffer

Rimantadine

1 �M 10 �M 100 �M

Buffer 0.3�0.1 0.2�0.1 0.3�0.1 0.1�0.1fMLP 1.0�0.3 0.9�0.1 0.8�0.1 0.7�0.2PAF/fMLP 4.9�0.3* 4.2�0.6* 3.7�0.4*† 2.8�0.4†

Values (in nmol O2�/min) are means � SE. *P 0.05 compared with

buffer-primed PMNs activated with N-formyl-methionyl-leucyl-phenylalanine(fMLP). †P 0.05 compared with buffer-pretreated PAF/fMLP.

Fig. 9. A: amantadine inhibition of PAF-mediatedchanges in CD11b surface expression. PMNs were pre-incubated with buffer (black bar) or amantadine [100�M (shaded bar) or 1 mM (open bar)] for 5 min andsubsequently stimulated with 2 �M PAF. The PMNswere then incubated with phycoerythrin-labeled anti-bodies to CD11b and fixed in paraformaldehyde, andCD11b cell surface labeling was measured by flowcytometry. Amantadine alone did not affect the amountof cell surface accessible CD11b as compared withbuffer controls. In contrast, PAF increased extracellu-larly available CD11b as compared with buffer-treatedcontrol PMNs. Amantadine pretreatment inhibited thePAF-induced increase in extracellularly accessibleCD11b in a concentration-dependent fashion. Data areexpressed as mean fluorescence intensity � SE. *Sta-tistical differences between buffer-treated and PAF-primed PMNs (P 0.05, n 6); †statistical differencefrom PAF and PAF/N-formyl-methionyl-leucyl-phenylalanine (fMLP) buffer-treated groups (P 0.05,n 6). B: amantadine inhibition of PAF-mediatedelastase release. PMNs were incubated with buffer oramantadine (100 �M–1 mM) for 5 min and primed with2 �M PAF or buffer and activated with fMLP (1 �M)or buffer. Amantadine at either concentration had asignificant decrease on the PAF/fMLP elastase releasein PMNs, whereas no difference was detected witheither PAF or fMLP alone. *Statistical significance(P 0.05) compared with PAF/fMLP and controlPMNs. This figure is representative of six individualexperiments.




Dow

nloaded from


colocalization. Furthermore, rimantadine appears to be a morephysiological inhibitor than amantadine because of its ability toinhibit PAF priming at clinically relevant concentrations (25,43), and may represent an effective pharmacological approachto reduce PMN-mediated tissue damage in the injured orcritically ill patient.

NOTE ADDED IN PROOF

Figures 4 and 7 and the legends differ from the early postedonline version of this article because of questions raised by theeditors with regard to the presentation of these figures. Specif-ically, for Fig. 4 (A and C), spaces have been added to showthat the image is not one contiguous gel. As the legendindicates, these images are representative of three individualexperiments and although the presented images are from thesame gel, the order has been changed for greater clarity to keepthe identical time points in order. Figure 7 has been replacedwith that from the identical gel, and it shows results from threeindividual experiments, with white breaks to demonstrate thatdata from different time points that are not discussed in thearticle have been removed.

GRANTS

This work was funded by National Institute of General Medical SciencesProgram Project Grant P50-GM-49222 and National Heart, Lung, and BloodInstitute Grant HL-59355.

REFERENCES

1. Albelda SM, Smith CW, Ward PA. Adhesion molecules and inflamma-tory injury. FASEB J 8: 504–512, 1994.

2. Barbieri MA, Roberts RL, Gumusboga A, Highfield H, varez-Dominguez C, Wells A, Stahl PD. Epidermal growth factor and mem-brane trafficking. EGF receptor activation of endocytosis requires Rab5a.J Cell Biol 151: 539–550, 2000.

3. Bates SR, Dodia C, Tao JQ, Fisher AB. Surfactant protein-A plays animportant role in lung surfactant clearance: evidence using the surfactantprotein-A gene-targeted mouse. Am J Physiol Lung Cell Mol Physiol 294:L325–L333, 2008.

4. Brady AE, Limbird LE. G protein-coupled receptor interacting proteins:emerging roles in localization and signal transduction. Cell Signal 14:297–309, 2002.

5. Callaghan J, Nixon S, Bucci C, Toh BH, Stenmark H. Direct interactionof EEA1 with Rab5b. Eur J Biochem 265: 361–366, 1999.

6. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules.Blood 84: 2068–2101, 1994.

7. Cavalli V, Vilbois F, Corti M, Marcote MJ, Tamura K, Karin M,Arkinstall S, Gruenberg J. The stress-induced MAP kinase p38 regulatesendocytic trafficking via the GDI:Rab5 complex. Mol Cell 7: 421–432,2001.

8. Chen LW, Jan CR. Mechanisms and modulation of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced Ca2� mobilization in human neu-trophils. Int Immunopharmacol 1: 1341–1349, 2001.

9. Ciesla DJ, Moore EE, Biffl WL, Gonzalez RJ, Silliman CC. Hypertonicsaline attenuation of the neutrophil cytotoxic response is reversed uponrestoration of normotonicity and reestablished by repeated hypertonicchallenge. Surgery 129: 567–575, 2001.

10. Ciesla DJ, Moore EE, Gonzalez RJ, Biffl WL, Silliman CC. Hypertonicsaline inhibits neutrophil (PMN) priming via attenuation of p38 MAPKsignaling. Shock 14: 265–269, 2000.

11. Ciesla DJ, Moore EE, Musters RJ, Biffl WL, Silliman CA. Hypertonicsaline alteration of the PMN cytoskeleton: implications for signal trans-duction and the cytotoxic response. J Trauma 50: 206–212, 2001.

12. Ciesla DJ, Moore EE, Zallen G, Biffl WL, Silliman CC. Hypertonicsaline attenuation of polymorphonuclear neutrophil cytotoxicity: timing iseverything. J Trauma 48: 388–395, 2000.

13. Clague MJ, Urbe S. The interface of receptor trafficking and signalling.J Cell Sci 114: 3075–3081, 2001.

14. Coates TD, Watts RG, Hartman R, Howard TH. Relationship ofF-actin distribution to development of polar shape in human polymorpho-nuclear neutrophils. J Cell Biol 117: 765–774, 1992.

15. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature422: 37–44, 2003.

16. Dolin R, Reichman RC, Madore HP, Maynard R, Linton PN, Web-ber-Jones J. A controlled trial of amantadine and rimantadine in theprophylaxis of influenza A infection. N Engl J Med 307: 580–584, 1982.

17. Elzi DJ, Bjornsen AJ, MacKenzie T, Wyman TH, Silliman CC.Ionomycin causes activation of p38 and p42/44 mitogen-activated proteinkinases in human neutrophils. Am J Physiol Cell Physiol 281: C350–C360, 2001.

18. Elzi DJ, Hiester AA, Silliman CC. Receptor-mediated calcium entry isrequired for maximal effects of platelet activating factor primed responsesin human neutrophils. Biochem Biophys Res Commun 240: 763–765,1997.

19. Ferguson SS. Evolving concepts in G protein-coupled receptor endocy-tosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24, 2001.

20. Fourie J, Escobar MR, Sitar DS. NMDA receptor antagonists to char-acterize rat renal organic cation transporter function. Eur J Pharmacol452: 1–10, 2002.

21. Galperin E, Sorkin A. Visualization of Rab5 activity in living cells byFRET microscopy and influence of plasma-membrane-targeted Rab5 onclathrin-dependent endocytosis. J Cell Sci 116: 4799–4810, 2003.

22. Granger DN, Kubes P. The microcirculation and inflammation: modu-lation of leukocyte-endothelial cell adhesion. J Leukoc Biol 55: 662–675,1994.

23. Grimes ML, Zhou J, Beattie EC, Yuen EC, Hall DE, Valletta JS, ToppKS, LaVail JH, Bunnett NW, Mobley WC. Endocytosis of activatedTrkA: evidence that nerve growth factor induces formation of signalingendosomes. J Neurosci 16: 7950–7964, 1996.

24. Grundy JE, Lawson KM, MacCormac LP, Fletcher JM, Yong KL.Cytomegalovirus-infected endothelial cells recruit neutrophils by the se-cretion of C-X-C chemokines and transmit virus by direct neutrophil-endothelial cell contact and during neutrophil transendothelial migration.J Infect Dis 177: 1465–1474, 1998.

25. Hayden FG, Hoffman HE, Spyker DA. Differences in side effects ofamantadine hydrochloride and rimantadine hydrochloride relate to differ-ences in pharmacokinetics. Antimicrob Agents Chemother 23: 458–464,1983.

26. Heuser JE, Anderson RG. Hypertonic media inhibit receptor-mediatedendocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389–400, 1989.

27. Howard TH, Meyer WH. Chemotactic peptide modulation of actinassembly and locomotion in neutrophils. J Cell Biol 98: 1265–1271, 1984.

28. Hu J, Fu R, Cross TA. The chemical and dynamical influence of theanti-viral drug amantadine on the M2 proton channel transmembranedomain. Biophys J 93: 276–283, 2007.

29. Jain D, Dodia C, Bates SR, Hawgood S, Poulain FR, Fisher AB. SP-Ais necessary for increased clearance of alveolar DPPC with hyperventila-tion or secretagogues. Am J Physiol Lung Cell Mol Physiol 284: L759–L765, 2003.

30. Kelly RB. Microtubules, membrane traffic, and cell organization. Cell 61:5–7, 1990.

31. Khan SY, Kelher MR, Heal JM, Blumberg N, Boshkov LK, Phipps R,Gettings KF, McLaughlin NJ, Silliman CC. Soluble CD40 ligandaccumulates in stored blood components, primes neutrophils throughCD40, and is a potential cofactor in the development of transfusion-relatedacute lung injury. Blood 108: 2455–2462, 2006.

32. Lawe DC, Patki V, Heller-Harrison R, Lambright D, Corvera S. TheFYVE domain of early endosome antigen 1 is required for both phospha-tidylinositol 3-phosphate and Rab5 binding. Critical role of this dualinteraction for endosomal localization. J Biol Chem 275: 3699–3705,2000.

33. Le GC, Parent JL, Rola-Pleszczynski M, Stankova J. Structural andfunctional requirements for agonist-induced internalization of the humanplatelet-activating factor receptor. J Biol Chem 272: 21289–21295, 1997.

34. Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, DellaRocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG,Lefkowitz RJ. Beta-arrestin-dependent formation of beta2 adrenergicreceptor-Src protein kinase complexes. Science 283: 655–661, 1999.

35. M’Rabet L, Coffer PJ, Wolthuis RM, Zwartkruis F, Koenderman L,Bos JL. Differential fMet-Leu-Phe- and platelet-activating factor-induced




Dow

nloaded from


signaling toward Ral activation in primary human neutrophils. J BiolChem 274: 21847–21852, 1999.

36. Malech HL, Gallin Current concepts: immunology JI. Neutrophils inhuman diseases. N Engl J Med 317: 687–694, 1987.

37. McLaughlin NJ, Banerjee A, Kelher MR, Gamboni-Robertson F,Hamiel C, Sheppard FR, Moore EE, Silliman CC. Platelet-activatingfactor-induced clathrin-mediated endocytosis requires beta-arrestin-1 re-cruitment and activation of the p38 MAPK signalosome at the plasmamembrane for actin bundle formation. J Immunol 176: 7039–7050, 2006.

38. McLaughlin NJ, Banerjee A, Khan SY, Lieber JL, Kelher MR,Gamboni-Robertson F, Sheppard FR, Moore EE, Mierau GW, ElziDJ, Silliman CC. Platelet-activating factor-mediated endosome formationcauses membrane translocation of p67phox and p40phox that requiresrecruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol3-kinase in human neutrophils. J Immunol 180: 8192–8203, 2008.

39. Medvedev IO, Malyshkin AA, Belozertseva IV, Sukhotina IA, Sevos-tianova NY, Aliev K, Zvartau EE, Parsons CG, Danysz W, BespalovAY. Effects of low-affinity NMDA receptor channel blockers in two ratmodels of chronic pain. Neuropharmacology 47: 175–183, 2004.

40. Merithew E, Stone C, Eathiraj S, Lambright DG. Determinants ofRab5 interaction with the N terminus of early endosome antigen 1. J BiolChem 278: 8494–8500, 2003.

41. Mills IG, Urbe S, Clague MJ. Relationships between EEA1 bindingpartners and their role in endosome fusion. J Cell Sci 114: 1959–1965,2001.

42. Mocsai A, Jakus Z, Vantus T, Berton G, Lowell CA, Ligeti E. Kinasepathways in chemoattractant-induced degranulation of neutrophils: therole of p38 mitogen-activated protein kinase activated by Src familykinases. J Immunol 164: 4321–4331, 2000.

43. Nahata MC, Brady MT. Serum concentrations and safety of rimantadinein paediatric patients. Eur J Clin Pharmacol 30: 719–722, 1986.

44. Nick JA, Avdi NJ, Young SK, Knall C, Gerwins P, Johnson GL,Worthen GS. Common and distinct intracellular signaling pathways inhuman neutrophils utilized by platelet activating factor and FMLP. J ClinInvest 99: 975–986, 1997.

45. Nusse O, Lindau M. The dynamics of exocytosis in human neutrophils.J Cell Biol 107: 2117–2123, 1988.

46. Oxford JS, Galbraith A. Antiviral activity of amantadine: a review oflaboratory and clinical data. Pharmacol Ther 11: 181–262, 1980.

47. Perry DG, Daugherty GL, Martin WJ. Clathrin-coated pit-associatedproteins are required for alveolar macrophage phagocytosis. J Immunol162: 380–386, 1999.

48. Phonphok Y, Rosenthal KS. Stabilization of clathrin coated vesicles byamantadine, tromantadine and other hydrophobic amines. FEBS Lett 281:188–190, 1991.

49. Rabinovici R, Bugelski PJ, Esser KM, Hillegass LM, Vernick J,Feuerstein G. ARDS-like lung injury produced by endotoxin in platelet-activating factor-primed rats. J Appl Physiol 74: 1791–1802, 1993.

50. Ruckert P, Bates SR, Fisher AB. Role of clathrin- and actin-dependentendocytotic pathways in lung phospholipid uptake. Am J Physiol LungCell Mol Physiol 284: L981–L989, 2003.

51. Sanchez CM, Alonso F, Garcia GM, Gomez-Cambronero J, NietoML. Synthesis of platelet-activating factor from human polymorphonu-clear leukocytes: regulation and pharmacological approaches. Int J TissueReact 7: 345–349, 1985.

52. Schlegel R, Dickson RB, Willingham MC, Pastan IH. Amantadine anddansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. Proc Natl Acad Sci USA79: 2291–2295, 1982.

53. Schnell JR, Chou JJ. Structure and mechanism of the M2 proton channelof influenza A virus. Nature 451: 591–595, 2008.

54. Schwab RS, Poskanzer DC, England AC Jr, Young RR. Amantadine inParkinson’s disease. Review of more than two years’ experience. JAMA222: 792–795, 1972.

55. Scott MG, Benmerah A, Muntaner O, Marullo S. Recruitment ofactivated G protein-coupled receptors to pre-existing clathrin-coated pitsin living cells. J Biol Chem 277: 3552–3559, 2002.

56. Sengelov H, Voldstedlund M, Vinten J, Borregaard N. Human neutro-phils are devoid of the integral membrane protein caveolin. J Leukoc Biol63: 563–566, 1998.

57. Sheppard FR, Moore EE, McLaughlin N, Kelher M, Johnson JL,Silliman CC. Clinically relevant osmolar stress inhibits priming-inducedPMN NADPH oxidase subunit translocation. J Trauma 58: 752–757,2005.

58. Shimizu T, Honda Z, Nakamura M, Bito H, Izumi T. Platelet-activatingfactor receptor and signal transduction. Biochem Pharmacol 44: 1001–1008, 1992.

59. Silliman CC, Clay KL, Thurman GW, Johnson CA, Ambruso DR.Partial characterization of lipids that develop during the routine storage ofblood and prime the neutrophil NADPH oxidase. J Lab Clin Med 124:684–694, 1994.

60. Silliman CC, Elzi DJ, Ambruso DR, Musters RJ, Hamiel C, HarbeckRJ, Paterson AJ, Bjornsen AJ, Wyman TH, Kelher M, England KM,McLaughlin-Malaxecheberria N, Barnett CC, Aiboshi J, Bannerjee A.Lysophosphatidylcholines prime the NADPH oxidase and stimulate mul-tiple neutrophil functions through changes in cytosolic calcium. J LeukocBiol 73: 511–524, 2003.

61. Silliman CC, Voelkel NF, Allard JD, Elzi DJ, Tuder RM, Johnson JL,Ambruso DR. Plasma and lipids from stored packed red blood cells causeacute lung injury in an animal model. J Clin Invest 101: 1458–1467, 1998.

62. Sorkin A, Eriksson A, Heldin CH, Westermark B, Claesson-Welsh L.Pool of ligand-bound platelet-derived growth factor beta-receptors remainactivated and tyrosine phosphorylated after internalization. J Cell Physiol156: 373–382, 1993.

63. Su X, Lodhi IJ, Saltiel AR, Stahl PD. Insulin-stimulated interactionbetween insulin receptor substrate 1 and p85alpha and activation of proteinkinase B/Akt require Rab5. J Biol Chem 281: 27982–27990, 2006.

64. Takei K, Haucke V. Clathrin-mediated endocytosis: membrane factorspull the trigger. Trends Cell Biol 11: 385–391, 2001.

65. Van de Walle GR, Favoreel HW, Nauwynck HJ, Van OP, PensaertMB. Involvement of cellular cytoskeleton components in antibody-in-duced internalization of viral glycoproteins in pseudorabies virus-infectedmonocytes. Virology 288: 129–138, 2001.

66. Vedder NB, Harlan JM. Increased surface expression of CD11b/CD18(Mac-1) is not required for stimulated neutrophil adherence to culturedendothelium. J Clin Invest 81: 676–682, 1988.

67. Vercellotti GM, Yin HQ, Gustafson KS, Nelson RD, Jacob HS.Platelet-activating factor primes neutrophil responses to agonists: role inpromoting neutrophil-mediated endothelial damage. Blood 71: 1100–1107, 1988.

68. Vlahos CJ, Matter WF, Brown RF, Traynor-Kaplan AE, HeyworthPG, Prossnitz ER, Ye RD, Marder P, Schelm JA, Rothfuss KJ.Investigation of neutrophil signal transduction using a specific inhibitor ofphosphatidylinositol 3-kinase. J Immunol 154: 2413–2422, 1995.

69. Ward RA, Nakamura M, McLeish KR. Priming of the neutrophilrespiratory burst involves p38 mitogen-activated protein kinase-dependentexocytosis of flavocytochrome b558-containing granules. J Biol Chem275: 36713–36719, 2000.

70. Warnick JE, Maleque MA, Bakry N, Eldefrawi AT, Albuquerque EX.Structure-activity relationships of amantadine. I. Interaction of the N-alkylanalogues with the ionic channels of the nicotinic acetylcholine receptorand electrically excitable membrane. Mol Pharmacol 22: 82–93, 1982.

71. Wintermeyer SM, Nahata MC. Rimantadine: a clinical perspective. AnnPharmacother 29: 299–310, 1995.

72. Wissel H, Schulz C, Rudiger M, Krull M, Stevens PA, Wauer RR.Chlamydia pneumoniae affect surfactant trafficking and secretion due tochanges of type II cell cytoskeleton. Am J Respir Cell Mol Biol 29:303–313, 2003.

73. Wouters FS, Bastiaens PI, Wirtz KW, Jovin TM. FRET microscopydemonstrates molecular association of non-specific lipid transfer protein(nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J 17:7179–7189, 1998.

74. Wyman TH, Bjornsen AJ, Elzi DJ, Smith CW, England KM, KelherM, Silliman CC. A two-insult in vitro model of PMN-mediated pulmo-nary endothelial damage: requirements for adherence and chemokinerelease. Am J Physiol Cell Physiol 283: C1592–C1603, 2002.

75. Zen K, Utech M, Liu Y, Soto I, Nusrat A, Parkos CA. Association ofBAP31 with CD11b/CD18. Potential role in intracellular trafficking ofCD11b/CD18 in neutrophils. J Biol Chem 279: 44924–44930, 2004.




Dow

nloaded from


Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

Documents