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2005 11: 310Mult SclerMinagar

Joaquin J Jimenez, Wenche Jy, Lucia M Mauro, Lawrence L Horstman, Eugene R Ahn, Yeon S Ahn and Alirezaand transendothelial migration of monocyte-endothelial microparticle complexes

1b on release of endothelial microparticles, formationβand the inhibitory effects of interferon-monocyte complexes induced by multiple sclerosis plasma−−Elevated endothelial microparticle

  

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Elevated endothelial microparticle�/monocyte complexes induced bymultiple sclerosis plasma and the inhibitory effects of interferon-b1bon release of endothelial microparticles, formation andtransendothelial migration of monocyte�/endothelial microparticlecomplexes

Joaquin J Jimenez*,1, Wenche Jy1, Lucia M Mauro1, Lawrence L Horstman1, Eugene R Ahn1,Yeon S Ahn1 and Alireza Minagar2

1Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine,Miami, FL 33136, USA; 2Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA71130, USA

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis(MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitatetransendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction withmonocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypicprofiles of EMP elicited by MS plasma and (iv) the effects of IFN-b1b on release of EMP and on TEMIG of monocytes (mono) andmonocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-b1b on the release of EMP and the TEMIG of mono andmono:EMP was assessed by preincubating BMVEC cultures of IFN-b1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62Eand CD31 were assayed. Plasma specimens from 20 patients with relapsing�/remitting MS (11 in exacerbation, MS-E, and 9 in remission,ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E andEMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively.Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was nosignificant effect with MS-R or control plasmas. The presence of IFN-b1b inhibited TEMIG of mono and mono:EMP by 20% and 30%,respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-b1binhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-b1b in MS.Multiple Sclerosis (2005) 11, 310�/315

Key words: endothelial microparticle; exacerbation; interferon-b1b; monocyte; multiple sclerosis; remission

Multiple sclerosis (MS) is an immune-mediated neurolo-

gical disease characterized by the formation of multiple

foci of inflammatory demyelination within the white and

grey matter of the central nervous system (CNS).1 Adhe-

sion of activated leukocytes �/ chiefly myelin basic

protein-specific CD4�/ T lymphocytes and monocytes �/

to cerebral endothelial cells (ECs) and their transendothe-

lial migration (TEMIG) through a disrupted blood�/brain

barrier (BBB) into the CNS contributes to formation of

demyelinating lesions.2 �5 The sequence of events in

TEMIG are known to include activation of leukocytes

and ECs, chemoattraction, leukocyte-EC adhesion, proteo-

lysis of the basal membrane surrounding the BBB by

matrix metalloproteinases (MMP),6 and finally, extravasa-

tion of activated leukocytes.IFN-b1b has been approved by the FDA for treatment of

relapsing�/remitting MS (RRMS),7 although the mechan-

isms of its beneficial effects on leukocyte trafficking

through the BBB have only been partially explored.8,9

EC dysfunction appears to be centrally involved in the

disruption of the BBB. In this regard, several studies have

documented elevated circulating markers of EC distur-

bance in MS patients, especially during exacerbations.10

We demonstrated that MS patients in exacerbation but not

remission exhibit significantly elevated plasma endothe-

lial microparticles (EMP).11

EMP are small (�/B/1 mm) membranous vesicles bearing

many of the adhesion molecules and other antigens of the

parent EC, notably including platelet-EC adhesion mole-

cule-1 (PECAM-1/CD31).12,13 Plasma levels of CD31�/

EMP (EMPCD31) were elevated during exacerbations of

MS and were associated with disease progression as

judged by clinical activity and presence of contrast-

*Correspondence: Alireza Minagar, Department ofNeurology, Louisiana State University Health SciencesCenter, 1501 Kings Highway, Shreveport, LA 71130, USA.E-mail: aminag@lsuhsc.eduReceived 11 June 2004; revised 5 October 2004;accepted 21 December 2004

Multiple Sclerosis 2005; 11: 310�/315www.multiplesclerosisjournal.com

# 2005 Edward Arnold (Publishers) Ltd 10.1191/1352458505ms1184oa at UNIV TORONTO on August 12, 2014msj.sagepub.comDownloaded from

enhancing lesions on brain magnetic resonance imaging(MRI).11 However, while that study documented thepresence of CD31�/ and CD51�/ EMP (EMPCD51), it didnot address their possible functional role in MS. Previouswork has shown that some EMP, particularly thosecarrying CD54 (EMPCD54), preferentially interact withmonocytes to form leukocyte�/EMP complexes. Sabatieret al. have also demonstrated that EMP bind to monocytes,eliciting the expression of tissue factor.14 Jy et al. demon-strated that EMPCD54 bind to monocytes and form EMP-monocyte conjugates in MS.15

We therefore hypothesized that EMP may interact withleukocytes and influence their TEMIG through the en-dothelial barrier. Thus, the present report evaluates theeffect of MS-E plasma on the release of phenotypicallydistinct EMP as well as TEMIG of monocytes and mono:-EMP complexes. Additionally, the effect of IFN-b1b on therelease of EMP and TEMIG of monocytes and mono:EMPcomplexes was assessed.

Patients and methods

PatientsA total of 20 patients who met the criteria for MS16 werestudied and compared to 10 normal controls. The studywas approved by the Institutional Review Board and allpatients signed informed consent forms. Prior to enrol-ment, all patients underwent neurological examination.Eleven of them had exacerbation documented by theexamining neurologist within 24 hours of onset of theattack. The other nine MS patients were confirmed to be inremission. Exacerbation was defined as a worsening ofneurologic impairment or the appearance of a newsymptom or abnormality attributable to MS andpersisting�/24 hours, preceded by stability of at leastone month. No patient had received any immunosuppres-sive treatment for at least three months prior to enrolment;i.e., no corticosteroids or immunomodulators such as IFN-b1a, IFN-b1b or glatiramer acetate. None of the 20 MSpatients had any infections at the time of presentation.The control group consisted of 10 healthy volunteers.After neurologic evaluation, peripheral blood was drawnin citrate Vacutainers and plasma was separated asdetailed below.

NeuroimagingBrain and spinal cord MRI were performed in all patientson a 1.5 T machine with standard head coils. The imagingprotocol included sagittal T1-, axial T1-, T2- and FLAIRimages. All MRI scans were performed after infusion ofgadolinium diethylenetriamine pentaacetic acid (Gd).Axial T2- and T1-weighted postcontrast images wereused for assessment of MS plaques. The images wereindependently interpreted by neuroradiologists blinded tothe study, using visual inspection.

Antibodies and other reagentsThe following fluorescent-tagged anti-human monoclonalantibodies (mAbs) were obtained from commercial sup-

pliers: from Pharmingen, anti-CD31 (Cat. #555446, PElabel), anti-CD54 (Cat. #F-0549, FITC label) and anti-CD62E (Cat. #F-0674, FITC label). The lectin, Ulex euro-paeus FITC-labelled, was obtained from Sigma (Cat.#L9006).

BMVEC culturesMVEC of brain origin (BMVEC) were obtained from CellSystems (Kirkland, WA, USA; Cat. no ACBRI 128) andcultured as previously described.17 To assess the effect ofMS plasma on the release of EMP, BMVEC were plated on12-well multiwell clusters at a concentration of 5�/105/well. After 48 hours, cultures were confluent and ready foraddition of plasmas. For TEMIG studies, BMVEC wereresuspended in CS-C medium and replated in 6.5-mm poreTranswell multiwell clusters precoated with collagen.Cultures were closely monitored until confluency wasachieved, then MS-E, MS-R or control plasma was added(20% v/v) and incubated for 18 h. To assess effects of IFN-b1b on TEMIG of monocyte:EMP complexes and mono-cytes alone, BMVEC was preincubated with 1000 IU/wellof IFN-b1b for 6 hours prior to addition of plasmas. MS-E,MS-R and normal plasma specimens were added andincubated for 18 hours.

Monocyte and mono:EMP complexes for TEMIGThe monocytic cell line U937, here abbreviated to mono,was obtained from ATCC (Cat. #CRL-1593.2) and wascultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS). Mono:EMP binding was per-formed as follows. Briefly, EMP were generated by treatingBMVEC cultures for 24 hours with TNF-a (10 ng/mL), thensupernatants were collected by centrifugation for 15minutes at 42 000�/g and the resulting pellets wereconcentrated 10-fold relative to original volume by resus-pending in culture medium devoid of growth factors orserum. EMP preparations were then labelled with FITC-tagged U. europaeus for 15 minutes, washed 2�/ withmedium (RPMI 1640) and resuspended in medium with-out FBS. They were then incubated with 1�/105 mono/mLin a ratio of 1:100 mono/EMP for 30 minutes. As EMP aremuch smaller and less dense than mono, centrifugation at100�/g allowed separation of the EMP:mono complexesfrom unbound EMP. Aliquots of EMP:mono conjugateswere assayed by flow cytometry as before. More than 75%of the mono formed complexes (bound at least 1 EMP) asjudged by fluorescent intensity of FITC-Ulex label on themono.

Transendothelial migration (TEM) assayThese were done using U937 cells (mono), untreated orpretreated by exposure to EMP as above (mono:EMP).Cells (1 mL) at 1�/105 cells/mL were added to a confluentmonolayer of BMVEC and the percentage of cells passedthrough after six hours was evaluated by flow cytometry.

Assay of endothelial microparticles (EMP)

Anti-CD31-PE (4 mL) or anti-CD62E-FITC (4 mL) was addedto 50 mL of supernatant, then incubated for 20 min withgentle (100 rpm) orbital shaking. Diluting buffer (PBS)

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(1 mL) was then added for flow cytometry on a Coulter

EPICS XL flow cytometer. Detection of particles was by

triggering on the CD31 or CD62E fluorescence signal

exceeding noise, and fluorescent particles were further

separated on a histogram based on size. EMP are defined

as particles of size B/1.5 mm expressing either marker.

Statistical analysis

One-way ANOVA (analysis of variance) was used to

analyse differences among three or more groups. If there

were significant differences (P B/0.05), then Student’s t test

was used to evaluate significance between pairs of groups.

If the data failed the normality test, then the Mann�/

Whitney rank sum test was used. The Chi-square test

was used to compare frequency data between groups. Data

analyses were performed using Statmost and Sigmaplot v.

4.0.

Results

Patient demographics and MRI findingsThe mean age of the 20 MS patients was 33 years and 80%

were female. The 10 healthy controls had a mean age of 35

years and 80% were female. Eleven of the MS patients

were in exacerbation confirmed by the examining neurol-

ogist and contrast-enhancing lesions on their T1-weighetd

postcontrast MRIs of brain. Nine MS patients in exacer-

bation had one and two had two contract-enhancing

lesions on their T1-weighted postcontrast brain MRI.

The other nine MS patients were clinically in remission

and exhibited no contrast-enhancing lesions on their brain

MRI.

EMP phenotypes elicited in response to MS plasmaWe used flow cytometry to enumerate EMP by threeendothelial markers, CD54 (EMPCD54), CD62E (EMPCD62E)and CD31 (EMPCD31), in the supernatants of BMVECcultured with 20% plasma. As shown in Figure 1 (leftpanel), incubation of BMVEC with MS-E plasma yielded119/2 EMPCD54 while incubation with MS-R and controlplasma yielded 1.39/0.3 and 0.909/0.06, respectively (all�/106/mL); P B/0.0001. Similarly, MS-E plasma yielded9.09/2.0 EMPCD62E but MS-R and control plasma yieldedonly 1.59/0.3 and 0.959/0.07, respectively; P B/0.0001.Increased EMPCD31 was also observed with MS-E plasma,yielding 3.89/0.9 as compared to 1.19/0.25 and 0.759/0.09for MS-R and control plasma, respectively; P B/0.0001. Bycriteria previously established,13 this EMP phenotypicprofile is consistent with EC activation, not apoptosis, inBMVEC exposed to MS-E plasma.

Effect of IFN-b1b on EMP releaseIFN-b1b significantly inhibited production of EMP fromBMVEC induced by MS-E plasma, to less than half thelevel without IFN-b1b, shown also in Figure 1 (rightpanel). Interestingly, IFN-b1b had only a modest inhibi-tory effect on the lesser levels of EMP elicited by MS-R orcontrol plasmas.

Effect of MS plasma on transendothelial migration (TEMIG) of

monocytes and mono:EMP complexesThe central finding of these experiments was that onlyMS-E plasma, not MS-R plasma, significantly promotedTEMIG of mono through this model of the BBB relative tocontrol plasma (see Figure 2A, solid bars). Specifically,359/8.5% of the monocytes passed through the BMVECmonolayer exposed to MS-E plasma for six hours, but only89/1.2% and 5.09/0.6% of the monocytes migrated withMS-R and control plasma, respectively; P 5/0.001.

Figure 1 Effect of MS plasma on EMP release by BMVEC. BMVEC were exposed to MS-E, MS-R or normal plasma for 24 h. Supernatants

were then collected and assayed for EMP by flow cytometry. EMP counts (�/106/mL) are depicted in A. To assess the effect of IFN-b1b,cultures were preincubated with 1000 U prior to the addition of plasmas, as seen in B. All experiments were conducted in triplicate.

(9/SEM).

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Effect of bound EMP (EMP:mono) on TEMIG of monocytesThe presence of EMP bound to the monocytes signifi-cantly increased the percentage of cells migrated. Speci-fically, TEMIG results with mono:EMP and MS-E plasmawere 519/13% of cells migrated (Figure 2B, solid bars), upfrom 35% with untreated monocytes (compare Figure 2A,solid bars); P B/0.001. Results using MS-R and controlplasma with mono:EMP TEMIG were not significantlydifferent from their effect on TEMIG of untreated mono-cytes.

Effect of IFN-b1b on TEMIG of EMP:mono complexes

Preincubation of BMVEC cultures with IFN-b1b inhibitedTEMIG of monocytes alone (Figure 2A, open bars) andmono:EMP complexes (Figure 2B, open bars) in thepresence of all three plasmas (MS-E, MS-R and control).The magnitude of this effect was greatest on EMP:monocomplexes with MS-E plasma (Figure 2B).

Comment

Disruption of the BBB is a consistent initiating event information of demyelinating plaques in RRMS.18,19 Undernormal circumstances the tight junctions of cerebral ECsand the lack of expression of adhesion molecules restrictextravasation and adhesion of leukocytes from peripheralcirculation into the CNS. In the pathogenesis of MS,however, proinflammatory cytokines such as TNF-a andIFN-g released by inflammatory cells promote breach ofthe BBB by upregulating adhesion molecules, loss ofjunctional integrity20 and release of EMP.11 Measurementof plasma EMPCD31 is a sensitive indicator of inflamma-tory activity in MS, as it was previously demonstrated thatelevated EMPCD31 was associated with contrast-enhancinglesions on brain MRI.11

In the present study, we showed that EMP derived from

incubation of MS plasma with BMVEC or TNF-a-stimu-lated BMVEC binding to monocytes, enhancing their

migration through BMVEC monolayers. As we previously

demonstrated, two EMP subpopulations, EMPCD54 and

EMPCD62E, bind rather specifically to monocytes.15,21 �23

We have shown here that these complexes facilitate themigration of monocytes through a monolayer of cerebral

ECs. This novel finding sheds more light on the role of

EMP in pathogenesis of MS. Our findings suggest that

shed EMP from the activated ECs in MS patients promotesthe migration of monocytes through the cerebral ECs and

assist in the formation of demyelinating lesions.The inhibitory effect of IFN-b1b on some components

involved with TEMIG has been documented. In clinical

studies, almost 90% of MS patients treated with IFN-b1b

showed a rapid and robust decrease in the number ofgadolinium-enhancing lesions on MRI.24,25 IFN-b1b has

been shown to affect disruption of the endothelial barrier

of the BBB by inhibition of T-cell entry to the CNS,25

possibly interfering with leukocyte adhesion to endothe-lium,26 shifting the cytokine balance from Th1 predomi-

nance to Th2 predominance,26 and increasing the

expression of occludin at endothelial tight junctions.19

IFN-b1b alters expression of adhesion molecules oncerebral ECs,25,27,28 facilitates conversion of cell-anchored

VCAM-1 into soluble VCAM-1,25 lowers the expression of

several chemokines and of the CCR5 receptor by

leukocytes,29 �31 decreases the production of MMP9 inthe peripheral circulation while increasing generation of

its inhibitor TIMP32 and blocks the disintegrating effects

of IFN-b on endothelial tight junctions and barrier func-

tion.20

In the current study, we observed that IFN-b1b effec-

tively inhibited release of EMPCD31, EMPCD54 and

EMPCD62E and TEMIG of mono and mono:EMP. These invitro effects of IFN-b1b are in accord with its stabilizing

Figure 2 TEMIG of mono (A) and mono:EMP (B). Confluent BMVEC grown in filter chambers were stimulated with MS-E, MS-R orcontrol plasma. Eighteen hours later, 1�/105 mono (A) or mono:EMP (B) were added. Filtrates were assayed for the presence of mono

(A) and mono:EMP (B) 6 h later. To assess the effect of IFN-b1b, cultures were preincubated with 1000 U for 6 h prior to the addition

of plasmas, as seen in the grey bars in A and B. All experiments were conducted on triplicates. (9/SEM).

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effects20,25,28 on endothelial integrity and activity. Inaddition, Corsini et al ., using an in vitro model of humanumbilical vein endothelial cell monolayers, demonstratedthat treatment of MS patients with IFN-b1b induced areduction in TEMIG of monocytes through ECs.9 Theinvestigators attributed their finding to a decrease in theserum levels of MMP-9. It is known that under stress,expression of MMP-9 by cerebral ECs is increased33 and itis possible that in our in vitro model, the expression ofMMP-9 by BMVEC was inhibited by the IFN-b1b. Alter-natively, IFN-b1b may directly interact with monocytesand impair the formation of monocyte:EMP complexes. Ithas been shown that increased migration of monocytesacross a fibronectin matrix in response to the chemokinesRANTES, MIP-1 and MCP-1 is antagonized by IFN-b1b.34

MCP-1 treatment of monocytes elevates their mRNA leveland secretion of a matrix degrading enzyme, MMP-9,which is abrogated by IFN-b1b.

However, our findings suggest a novel protective me-chanism for IFN-b1b in patients with MS. This inhibitoryeffect of IFN-b1b on TEMIG of monocytes and monocy-te:EMP complexes may be explained by our findings. Wepreviously demonstrated that treatment of MS patientswith IFN-b1a reduces plasma concentrations ofEMPCD31.35 It is possible that as demonstrated in vitro ,IFN-b1b reduces EMP release, thereby leading to de-creased formation of EMP�/monocyte complexes and alowered rate of TEMIG of these cells in vivo .

In summary, our findings suggest a novel effect ofplasma EMP in MS patients, namely that these particlesmay enhance monocyte migration through the endothelialmonolayer in vivo . The enhancing effect is furtherinhibited by IFN-b1b; our TEMIG model demonstrates anovel mechanism for its beneficial effects in patients withRRMS. Further studies of EMP in the context of MS canhighlight more details of TEMIG of activated leukocytesthrough the cerebral EC into the CNS.

AcknowledgementsThis work was supported by the Wallace H CoulterFoundation and Funds from Mary Beth Weiss, Jane andCharles Bosco, and gifts from Frank Smather.

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