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Serum and cerebrospinal fluid levels of soluble adhesion molecules in multiple sclerosis: predominant intrathecal release of vascular cell adhesion molecule-1
Journal of Neumimmunology
EISEVIER
Serum and cerebrospinal fluid levels of soluble adhesion molecules in multiple sclerosis: predominant intrathecal release of vascular cell adhesion molecule-l
Activated ccrcbral varulsr endothdiel cells express leukrxyte. ~ascula cell. and intmellutar adhesionmokcdcs (E&win. VCAM-l and ICAM- which facilitatekukccytcadhssirmto endotheliumand migrationinto in%mnutory k&ml. Pairedsaum and cercbmspimlfluid (CSF) lcvclsaf soluble0) E-stain. sVCAM-I andslCAM-I were determinedby ELISA in patientswith clinically definiteMS in relapse.and @ens with other inflammatory(IND) and non-in”ammntcqneurologicaldisease(NIND). CSF levctsof sVCAM-I and slCAM-I were rigniticmtly increased in MS padentscomparedU, IND andNIND paticms.Elevationof CSF sVCAM-I in MS padentswas the rnc6tmarkedfinding (P-O.O@SI) and an increasedsVCAM-I index indicate3that this WSIdue to intratkcal releaseof sVCAM-I. There were no differencesin semmand CSF sE-sebctinlevelsbetweenthe studygroups.Mcawemcnt of the sVCAM-I indexmay providea markerof diseaseactivityin patienlswith clinically definiteMS.
1. Introduction An early event in multiple sclerosis(MS) lesion fomtation is increasedpermeability of the blood-brain barrier in association with invasion of the central nervous system (CNS) by T cells. Tote inflammatory responseand consequent pri-axial demyeliiation lie in close relationship to the cerebral vascula~ua,principally the veins and venules. Beyond the demyelinating plaques there is evidence of more widestwad perivasculw inflammation and collasenizaticmof bled vesselsin the CNS and meninges(Alien et al., 1981: Hauser et al., 1986). smlstinc that altered functioning of cerebral vascularend&&n-plays an important role in MS pathogen&. Factors determining Le composition and temporal evolution of the cellulw infittrate in MS brain are unknown, abhwgh regulation may be determined in part by the profile of adhesion molecule
expressionrd the site of Le lesion. Extravasationof kukccytes into the CNS parenchymais facilitated by expression of adhesion molecules on both leukocytes and cerebral vascularendothelial cells (Wang and Domvini-Zis, 1992). Leukocyte-endothelial interactionsare regulatedby a ca!+ cade of adhesion molecules including seleerins, integrins and molecules of the immunoglobutin superfamily. Activated endotbelium expressesendothelial leukwzyte, vascu1.x cell and invacellular adhesion molecules (E-srlectin, VCAM-I and ICAM-I). all involved in leukqte-endotbelial cell bindbtg. [CAM-I binds to LFA-I on lymphocytes, VCAM-I binds to VLA-4 and VLA-5 cm lymph& cytes. and E-selectin hinds to sialyl-Lewis X on new tmphils. Adhesion molecules on astmcytesand micmglia may also play a mle in leukocyte trafficking through the CNS. T cell interactions with lymphoid high endothelial venuleshave been well characterised(Springer, 1994). but the mechanisms of T cell recruitment to CNS sites of inflmnmation ate not well undentaod. Immunahistahemical studies have demonstratedthat cerebral vascular endothelium exhibits no constitutiveexpressionof E-selectin
sod VCAM-I (Washittg(on et al., 1994). aIdtough ICAM-I is oresat at low levels on astmcvtes and on some endodteliai cells &be1 et al., 1990). Expression of ICAM-I is elevated in MS plaques at all stages of disease activity, whereas VCAM-I is present at highest levels in chronic active lesions (Raine et al., 1990; Camella and Raine, 1995). E-selectin is expressed on some MS microvessels doe to waosieot upregulation during endothelial cell a&a tion (Warhinetan et al.. 1594). Downreeulation of cell surface moleeuks by intemalizttion or shedding is an essential feature of the immune response. and soluble forms of ICAM-I. VCAM-I and E.xkcdn have been detected in worn (Rotblein et al., 1991; Seth et al., 1991). Increased levels of soluble ICAM-I (JCAM-I) have been described in a variety of inflammatory diseases and in certain malignaocies with the suggestion that they are markers of endothelial cell activation @earing and Newma,,. 1993). Ekvakd levels of sICAM-I have been reported in serum and CSF of MS padents. suggesting that ~asorentent of soluble adhesion molecule levels may movide a marker of disease zxivitv in MS. This study &wed on expression ol circulating forms of ICAM-i. VCAM-I and E-sekctin. We wished to determine if there is a specifk alteration in circulating adhesion mok~ule levels in clinically active multiple sclerosis and whether or *ot the response is pmdominaody intrathecal. 1
2 Materials
and methods
Paired serum and CSF samples were obtained from 129 patknts v&b neurological disease who artended the New mlogy Dcpamnent over a 6.month paiod. The clinical diagnosie was recorded and cootirmed in all patients at kast one year after the samples were taken. Fifty-six patients (37 female) diagnosed as clinically definite multipk sclerosis, according to the criteria of Pow et al. (1983). entered the study. All patients witi MS were in relapse at dte time of sampling. A relapse was defined as the appearance of symptoms of neumlogical dysfonction witb objective confirmation lasting more tbao 24 h. All blood and CSF samples from MS patients were obtained within 3 weeks of onset of relapse. No patient had received corticostemids OT other immunosuppressive treatment in the 6 months prior to entry into the study. Control groups iocloded 30 patients with inflammatory neomlogical disease (IND) (viral encephalitis, cerebral vascolitis, acute disseminated eocephalomyelitis, transverse myelitis, stroke, sarcoidosis, Ckilliao Barr& syndrome and chronic inflammatory demyelinating polyndiculopathy) and 43 patients with non-inflammatory cxwological disease (NIND) (cervical spondylotic myelopathy, syringomyelia, psychogenic symptoms, compression neuropathy, motor new ran disease, epilepsy and vertebral disc prolapse). Written
informed consent was obtained from all palienh. The protocol was approved by the Queen’s University of Belfast Medical Research Ethical Committee.
Blood samples were collected by venepuncture sod semm SW prepared by centrifogation of the clotted blood. A paired CSF sample. obtained from each patient, was centrifwed for 10 min at 200 x I and sopematant was obmiz Serum and CSF sample; were s&d hozen at - 7vC until we. Only CSF samoks that showed no visual evidence of a bloody -& were &!lysed. L?.boratory Malysis was performed on all samples including qoantitation of albumin and immtmoglobulin G (IgO). calcolatioo of CSF IgG/albomin ratio (Tibbing et al.. 1977), CSF IgO index (Delpech and Lichtbku. 1972) and detection of oligoclonal 180 bands tGCB) (Walker et al., 1983).
Levels of slCAivl-I, #CAM-I and SE-sekctin were detemdned in mired serum and CSF samples from all patients. Com&rcklly available sandwich &ISA assays were used in accordance with the instmctions of the manufactorer (R&D Systems Europe Ltd.). To optimise assay sensitivity, IO CSF samples were firs1 assayed for sICAM-I. sVCAM-I and SE-selectlo at serial dilutions of I:,, I:5 and 1:lO. As linearity of results was obtained for all samples. with no sigtdficaot matrix effects, CSF sampier in-tie study werediluted 1: I in appropriate sample dikent instead of the recommended dilution. Seturn sampks were tested at the recommended dilution. Samples diluted with buffered protein were applied to microwell strips coated with tbe respective murine monoclonal antibodv (mAb) aeainst human ICAM-I. VCAM-I. and Es&tin. A&cr&iog washed twice wiL a buff& (polysorbate 240. the microwell strips were incubated wilh the respective horseradish pemxidase-conjugated anti-adhesion mokcuk murine mAb diluted with the assay buffer. The reaction was stopped after 30 min by the addition of 4 N sulfuric acid. The absorbance of each well was measured at 4.50 nm osine an ELISA reader. A standard cwve was used to determine the adhesion mokcole concentration in each samok. Samales were all examined mine ELISA assay kits-with the &woe batch number. The de&on limit of the assavs was: 1.0 w/ml sICAM-I: 3 w/ml sVCAM-I: 0.i ng/ml sE-s&tin. Sample. con&trations below the detection limit of the assay were recorded as zero. The mean intro-assay variability was S-7% and Le mean inter-assay variability was S-IO%. The adhesion molecule index levels (according m the IgG index) Klelpecb and Lichtblao. 1972) were calc~ktwl as follows: s adhesionmolecuka, s adhesionmoleeuk_,
albumincsp ‘albumin,,,’
were considered significant. Spearman’s rank-order comlation test was applied to identify asso&dions betwen group parametw. Two-tailed P-values lower than 0.05 were considered significant.
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3. Results Serum [CAM-I levels ranged fmm M) ng/ml to loo0 ng/ml, with a mean value in the non-inflammatory disease patient? (mean f SD., 308 f I I8 n&!/ml) similar to praviou8 reports (Jan&r et al.. 1993, Hartung et al., 199393). Serum slCAM-1 levels were lower in MS pafiats (P = 0.04) and in IND patients compared with the NIND comml group (Fig. 1). CSF concentrations of sICAM-I io Lbe patients studied ranped from undetectable levels (< 1.0 I NIND patient) to 9.9 &ml. CSF slCAM-I was sienificwdlv elevated in MS oatieow cornowed with NlND p&sots (P= 0.02). Serum ‘SWAM-I Was in the range 271% 1900 “g/ml and no significant differences were found between the patient groups. TIP mean CSF #CAM-I concentration in h4S oatients was sienificardlv elevated compared to inflammatory controls (P - 0.001) and noninflammatory controls (P = O.ouDl) (Fig. 2). Serum levels of SE-selectin were normal in all patient groups. CSF E-s&&n concentration ranged from undetectable (4 0.2 nc/mk 77/129 aatiems) to 6.6 w/ml and no sienificaot differences were found between the patient groups. Calculat~on of tbe corresponding soluble adhesion molcudc iodizes showed that the SWAM-I index was significantly higher in tbe MS Sroup compwcd with IW (P - O.OfJl)
ng/ml:
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Analysis was carried out using SPSS/PC + software (SPSS Corpaaion, 1988). The Mann Whitney U-test was used 10 compare test groups. P-values lower than 0.05
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and NlND patients (P = 0.X11) indicating that inuathecal production of VCAM-I occurs predominandy in MS (Table I). Significant positive correlation was abserved between lhe sVCAM-I index and the slCAM-I index in
patientswith MS (r = 0.40, P = 0.005) and IND (r = 0.40, P = 0.031, but not in patients with NIND (r = 0.24. P = 0.23). Interestingly, the presenceof oligoclonal bands in the CSF of MS patients was associatedwith higher CSF levels of SWAM-I (17.5 “g/ml vs. 7.3 “g/ml. P = 0.006) and sICAM-I: (2.0 “g/ml vs. 1.4 “g/ml. P = 0.005). and a higher VCAM-I index (6.1 vs. 3.1. P=O.OLlX). This associarion was not found in the inflammatory control group. In MS patientsno asscciationwith soluble adhesion molecules was found whh other indicators of intrathecal immunoglobulin production, including CSF IgG concenuation, CSF IQ ratio and CSF IgG index.
4. Discussion
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Leukocyte migration acrossthe blood-brain barrier en. dotielium involves at least three steps: slowing of leukocyte flow by transient selectin interactions, followed by triggering of integrin activation and strong adhesionmediated by mokcules of the immunoglobulin superfamily such as lCAM-1 and VCAM-I. Activation of vascular endolhelial cells resultsin rapid upregulationand shedding of E-selectin, ICAM- and VCAM-I from the cell surface. The anmunl of soluble adhesionmolecules releasedcwrelates directly with their cell surface expression(Leeuwenberg et al., 1992). In this study we demonsuareda marked increase in #CAM-l levels in CSF during MS relapse. Elevation of the VCAM-I index indicated that this was due to inirathecal expressionand releaseof VCAM-I. The
mean CSF sVCAM-I concentration was signiFica.ndy hither in oatiettts who had CSF oligoclonal bands, emphasioittg the~importattce of the itnmo~e response within the intralhecal compartmettt in MS. This finding confirms recent reports of increased CSF sVCAM-I levels during MS relawe (~ore-~eff~ et al.. 1995; Matsuda et al., 1995). Elevated CSF &CAM-l levels have also been detected during clinical remission (DoresDuffy et al., 199% and doting the chronic progressive phase of the disease @ore-DoFFy et al., 1995; Matsoda et al., 199% suggesting that episodic or persistent activatioo of the cerebral endothelium is sustained throughout the diwase cowse. mere are few repow of CSF VCAM-I levels in other diseases, although elevation of VCAM-I without an increase in E-s&& and ICAMhas been detected in CSF of attitnals with simian immunodeficiency virus-t&ted enceohalitis &sseville et al.. 1992). Serum levels of sVC.&l-l were normal in active MS. as previously reported (Dare-Duffy et al., 1595). although increased levels have been detected in sotoe patienls (Marsoda et al., 19995). We demonstrated that slCAM-I levels were increased in CSF but not in serum during MS relapse, with no elevation of the sICAM-I index. Data on slCAM-I levels in active MS have been CooFlicting, with reports of normal or elevated levels in serum (Hartung et al., 1993: Iaoder et al.. 1993: Sharief et al.. 1993) and in CSF (Rieckmann et al., 1993: Tsukada et al., 1993: Dare-Duffy et al., 1995). Tote cause of these divergent results is unknown but may be doe to differences in methodology and time of sampling in relation to disease ,activity. The cellular origin and kinetics of release of ICAM-I and VCAM-I may also differ in response to cylokine stimulation of the endothelium as the MS lesion evolves. Our observations confirm the finding of normal sentto sEselectin levels in active MS (Rieckmann et al., 1994). and demonsuate that SE-selectin levels are also normal in CSF in active MS. Higher serum sE-wlectin levels in the inflammatory control group were similar to results in other patients with acute and chronic inflammatory demyelinating polyradicolopathy (Oka et al., 1994: HttrNng et PI.. 1994). Neutmphils in tbe semm of MS patients show impaired adherence and chemotaxis (Pcdikogloo et al., 1994), and normal sE+electin levels reaflirm the low level of oeutrophil-endotheliel cell interactions in relapsing remining MS. Inlerestingly. increased seturn sE-wlectin levels have been detected in patients with primary progressive
MS Kiiovannoni et al., 1594; Doie-Duffy et al., 199% suggesting that the dynamics of the inflammatory response may differ in relapsing remitting and primary progressive forms of the disease (Revesz et al.. 1994). The site of origin of these circulating adhesion molecules in MS is not known. sVCAhl-I may arise from cerebral or meningeal endothelial cells, macrophages or reactive astrocvtes. WAM-I ma” be relcascd from maw cell WDeS i&loding endothelitd~cells, lymphocyles, tnactbphage;&d microglial cells. VCAM-I and ICAM- 8re the tttosl abuttdant activation markers present on blood vessels in MS plaques. The most active lesions appear to be HLA-DR oaitive whereas chronically active lesions also ewress high levels of VCAM-I &d ICAM-I (Traugott, i987: Hayashi et al.. 1988; Raioe et al., 1990: Washington et d 1994). Adhvsion molecule expression extends beyond the areas of celktlar infiltration, and in periplaque areas 50% of microvessels co-express VCAM-I and HLA-DR (Wasltington et al., 1994). Tote differential shedding of adhesion molecules in MS suppa the idea that endothelial cell activation may involve a sequence of steps regulated by locally released cvtokiner. Dose- and time-decendent induction of Eselec&I. ICAM-I, and VCAM-I &pression occurs on vascular endotbelium in vitro in response to a range of inflammatory cytokines, with resultant shedding of adhesion molecules into the sopematant (Leeuwenberg et al., 1992; Pigott et al., 1992: Schieffer et al., 1992). Tumor necrosis Factor (TNF), interleokin-I (IL-I). interleukin-4 (IL-4) and interferon-v (IPN-y) induce expression of E-selectin and ICAM-I. v&as optimal expre&ion of VCAM-I requires a combination of IL-I and/or TNP together with IL-4. Cytokines induce expression of ICAM-I and E-selectin oa both arteries and veins but VCAM-I levels are increased only on venous endothelial cells (Szekaoecz et al., 1994). The oredilection of MS lesions for petivcnous sites may d&ore explain the predominant l&thecal release OF sVCAM-I in MS. EN-y can induceclinical telapseOf MS (Panitch et al., 1987) and administration of anti-IFN-y antibodies to animals with experimentalautoimmuneencephalomyelitis blocks the uptegulation of VCAM-I, but not of ICAM-I, suggesting that VCAM-I may be more imponant in the exacerbation of disease activity (Barten and Roddle, 1994). The activation state OFvenoo~ endothelium is of critical importance to the recruitment of resting and effector T cells during developmentof the inflamma-
tory lesion. lntratbecal release of VCAM-I during relapse may correlate with the regulated migration of immune cells inm the brain. VCAM-I is a l&and for the integrin a4pl 6X4-4) and binds weakly m a4p7 Khan et al.. 1992). hagrins provide the main adhesive force between T cells and vascular endotheliwn. and VCAh&a4pI interactions mediate the rolling and stable arre8, of T cells on Ule endothelial surface prior to vansmigration (Luxinskas et al., 1995). A8 such. e4 integrins are crucial 10 the effective homing of activated T cells across Ule blood-brain barrier ,o shes of CNS inflammation (Yedncck et al., 1992; Baron et al.. 1993). CSFT cells in MS show increased expression of many adhesion molecules. in&ding VLA-4 and VLA-5 (Svamingsson et al., 1993). al$augh Ule distribution of a4 in,eg,ins on selective T cell subpwdadans is no, known. Soluble adhesion molecule8 may exen functional effects such as inhibition of adhesion by compedtion (Rolhlein et al.. 1991). so sVCAM-I mav act to modulate the recruitn&l of iymphocyles u, sit& of tissue desvuction. Both ICAM-I and VCAM-I, but no, E-selectin, are imponam in T cell bindine 10 activated cerebral endoLhelium in viuo (Wang et al.,-1994). However, selective elevation of the VCAM-I index highlights the decisive role of VCAMl/a4 integrin interactions in lymphocyte trafficking thrwzh areas of CNS inllammalion in MS. Serum levels of &AM-I are consistently raised in patients with systemic vasculitis and appear 10 correlate well with disease activity (Janssen et al.. 1994). Accumulating evidence far significant elevation of CSF sVCAM-I in MS implies that measuranen, of sVCAM-I may allow the detection of subclbdcal disease activity. The sVCAM-I index may therefore provide a surrogate marker of an adequate respcmse ,o ,,ea,men,. Blockade of VCAM-I on cerebral end&&an by monalonal andbalies may also have considerable lhempeutic potential as a disease modifying therspy in MS.
This study was supported by a gran, from the Multiple Sclerosis Society of Great Britain and Nanhem Ireland.
Powr, C.M.. PaIy. nW.. Scheinbrrg. L... McDcn~ld. W.I.. Davis. F.A.. Ebcrr. G.C., Jolmron. KP.. Sibley. W.A.. SBberbwg. D.H. and Tou~Il~r. W.W. (1983) New diagnostic crivria for MuIdpIo Sclw Pasir: gukkIi.c for rerearch pmtc&r. Ann. Ncurnl. 13.227~23I. Rainc. CS.. Lee. SC.. Scheinberg. L.C.. Dnijvcslcin. A.M. and Cross. A.H. (l%XI) Adhesion mo!+mlcs on w&Ihcli~l cells in !be ccnlml nervous system: an enxrging mea in neuroimmunology of mullipk sckldsI*. Clln. lmmunol. Immunof&ol. J?.l73-181. Reverr. T.. Kid& D.. liwmp.wn. AJ.. Bsmmd. R.O. and McDonald. W.I. (1994) A compuiwm ai hc pa!hology of primary and aecondm~ progressive muldplc sclerosis. Brain 117. 759-765. Riwkmarm. P.. Nunke. K.. Bwchhard~ M.. Albnchr. M.. Wdltang. 1.. LIlti. M. uld Felgenhruer. K. (19%) Soluble indrc~llular adhesion nwkcule-I in ccrebmspln# fluid: an indicrvx for tie inflmmnalory imprinnenr di ti blaad-cerebrosplnrl fluid barrier. J. Ncuroimmunal. 47. 133-149, Ritckmann, P.. Marlin, S.. Wckhxlbrsun, I.. Albrcchr. hl.. Kiue. E., Weber.T.. TUmMi. H.. Brooch. A.. Lwr, W.. Helwi% A. nnd Pow, S. (IS941 Scriai ul.%Iy?vsis di circulating tihesian molecules and TNF receptor in semen imm palients v&b multiple sclemsis: clCAM-I is an indiiamr ior s&p%. Neumlqy 44.2367-2372. R.,,h,ein. R,. Mzdnolfi. EA., Cz+ws!& M. ad Martin. S.D. tl’Z+l) A iorm of cirwlding ICAMin hvmln sxum. 1. Immunol. 147. 3788-3793. Saswillc. V.. Newman, W. md Lwkner. A. (1992) Ekvared wccular cell adhesion molecule-l in AIDS cnce&ditis induced by simian immunodeficieocy virus. Am. 1. Palhol. Ihl. IOU- 1030. _ Schictfer, S.. Kcm. P.. W&u, C. and Schcrbaum. W. (1992) Cytokinc indeed erprcrsim oi adhcrion molccukr 011 HUVEC and shedding oi rlCAM-I in vim. pd$Ier al Lhe iolemllional cmference on tic w.culw endmhelium in inflrmmuion. Schloss Elmau. Gcrmsny. octobw l I-15. 1992. Seth, IL. Raymond. F.D. and Makvbba. M.W. (1991) Circuloing ICAM-I ~solorms: diagnostic prospecs fox infkmmalory and immune disorders. LzmceI 338.83-U. Shzrief. M.K.. Nmri. MA.. Cianli. M.. Cirelli. A. and ‘fhompsan. E.I. (1993) lacrcnsed levels of circutaling ICAM-I in serum and webrospinal fluid of p&ems wilh &YF multiple s&rods. Comlalion v&h TNFa ad blood-tin bticr dmnge. 1. Ncumimmunol. 43. 15-21. sobel. R.A.. Mdchcll. M.E. and Fcndren. G. (15%) lnrrccllulw adhesion mok_cu~I GCAhl-I) in cellular immune nwlidns in d-& human ecn,mI neryo”~ sysIcm. Am. 1. PaIhol. 136. 1309-1316.
Sprinzeer. ‘LA. (1994) TmttE signlln for Iympbocyle rccirculnioo and Ie~kocyte cmigmdcn: tie mu&ep p&Il& &II 76.301-314. Svcnningsson. k. HIIISIMI.G.K.. Anderson. 0.. Anderson. R.. Wlpmyo, M. &d Stemme. S. (1993) Adhcsi~l mokcule exprrasion on cercbmspinal fluid 7 lymphwyes: evidence tar common lccnilmcnl mechanism in muldde &rosis. ascmic mrnioeilis and normal canII&. Ann. Ncurol. ;A. 155-161; M.R.. Pcnrce. W.H. and Koch. A.E. (IS%) Inurc=zllulnr ndl&ii &cu~-! IICAM-I) enprersiw and wlubla [CAM-I (XAM-I) prcdwion by cymkinccactiwed human aoltic endcibelial cells: i possible role for ICAM-I and s1CAM-I in .wlwwkmtic aardc aneurysms. Clin. Enp. Immunol. 98. 337-343. Tibbing. G.. Link, H. pnd Ohman. S. (1977) Principkr of albumin and IrG analvris in neumloeical disaders. 1. EsIablisbmenI of reiemwc ranger. seti. I. ciin. in=,. 37.385 -390. Tmunoa. U. (1987) Muld~le xlcwis: relcvanco of Class I and Class II MHC.cxprcssing celk’w Icsion dcvclotxmcnt. 1. Ncuwimmenvl. IO. 283-302 Tsukada. N.. Malsuda, M., Miyw, K. and Yansgisawa. N. (1993) Increased lcvelo of inwccllular aJhesio0 molecule-1 (ICAM and turnournecrosis faclor vzceptm in ulc cercb=ospinul tInid oi patients will? Multiple Sclerorir. NewoIogy 43. 2679-2682 Walker, R.W.H.. Weir. G.. Johnston. M.H. an4 Tlbxnpsdn. E.J. (1983) A mpid memod ior dclecling aligc&nal IgG in unconccnlmlcd CSF by agarare iyYkctric lowing. wansfcr 10 cclluI0~ nitnlc and immunoperowldsre slaining. .I. Neuroimmunol. 4. 141-148. Wshington. R.. BUMI. J.. Tcdd. R.F. Ill. Newman. W.. Dragavie. L. uld Dore.Duffy. P. (1994) Expression of immunoIoglcaIIy relevanl xtivation anhgens on imlsiled cenVaI neryws ryrrrm micrwcsxIs from palicnls wi* muhipk sclerosis. Ann. Ncuml. 35. 69-Q. Wang. D. and Domvini-Zis. K. (1992) Upregulation of iolencellular zidhesion molccule.1 (ICAM-I) expression in @ima CUI~CS oi human brain microvesrel cndolhellal cells by cyldiincs and lipopoly saccharide. 1. Neuroimmunal. 39. I l-22. Wang. D.. Pmmcyo. R. yld Dorovini-Zis. K. (19%) Monocl~lamibzdier IO endolhelial adhesion makcuIcs VCAM-1 and (CAM-I. but not E-selecda. inhibit adhesion of T-lymphayes IO cymkinc slimulaled human bmin microvesscl rndothelial cclIs in vitm. Bnin Paled. 4. 498. Ycdnock. T.A.. Cannon. C.. Fritz. L.C.. SzmcbwMadrid. E. SIeinman. L. and Cxmn. N. (1992) PfeYentiMl of experimcnlsl Buloimmune encrpb.%llamyeIilis by antibodies against 014pl intogrin. Naiurc 356. 63-66.
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EISEVIER
Serum and cerebrospinal fluid levels of soluble adhesion molecules in multiple sclerosis: predominant intrathecal release of vascular cell adhesion molecule-l
Activated ccrcbral varulsr endothdiel cells express leukrxyte. ~ascula cell. and intmellutar adhesionmokcdcs (E&win. VCAM-l and ICAM- which facilitatekukccytcadhssirmto endotheliumand migrationinto in%mnutory k&ml. Pairedsaum and cercbmspimlfluid (CSF) lcvclsaf soluble0) E-stain. sVCAM-I andslCAM-I were determinedby ELISA in patientswith clinically definiteMS in relapse.and @ens with other inflammatory(IND) and non-in”ammntcqneurologicaldisease(NIND). CSF levctsof sVCAM-I and slCAM-I were rigniticmtly increased in MS padentscomparedU, IND andNIND paticms.Elevationof CSF sVCAM-I in MS padentswas the rnc6tmarkedfinding (P-O.O@SI) and an increasedsVCAM-I index indicate3that this WSIdue to intratkcal releaseof sVCAM-I. There were no differencesin semmand CSF sE-sebctinlevelsbetweenthe studygroups.Mcawemcnt of the sVCAM-I indexmay providea markerof diseaseactivityin patienlswith clinically definiteMS.
1. Introduction An early event in multiple sclerosis(MS) lesion fomtation is increasedpermeability of the blood-brain barrier in association with invasion of the central nervous system (CNS) by T cells. Tote inflammatory responseand consequent pri-axial demyeliiation lie in close relationship to the cerebral vascula~ua,principally the veins and venules. Beyond the demyelinating plaques there is evidence of more widestwad perivasculw inflammation and collasenizaticmof bled vesselsin the CNS and meninges(Alien et al., 1981: Hauser et al., 1986). smlstinc that altered functioning of cerebral vascularend&&n-plays an important role in MS pathogen&. Factors determining Le composition and temporal evolution of the cellulw infittrate in MS brain are unknown, abhwgh regulation may be determined in part by the profile of adhesion molecule
expressionrd the site of Le lesion. Extravasationof kukccytes into the CNS parenchymais facilitated by expression of adhesion molecules on both leukocytes and cerebral vascularendothelial cells (Wang and Domvini-Zis, 1992). Leukocyte-endothelial interactionsare regulatedby a ca!+ cade of adhesion molecules including seleerins, integrins and molecules of the immunoglobutin superfamily. Activated endotbelium expressesendothelial leukwzyte, vascu1.x cell and invacellular adhesion molecules (E-srlectin, VCAM-I and ICAM-I). all involved in leukqte-endotbelial cell bindbtg. [CAM-I binds to LFA-I on lymphocytes, VCAM-I binds to VLA-4 and VLA-5 cm lymph& cytes. and E-selectin hinds to sialyl-Lewis X on new tmphils. Adhesion molecules on astmcytesand micmglia may also play a mle in leukocyte trafficking through the CNS. T cell interactions with lymphoid high endothelial venuleshave been well characterised(Springer, 1994). but the mechanisms of T cell recruitment to CNS sites of inflmnmation ate not well undentaod. Immunahistahemical studies have demonstratedthat cerebral vascular endothelium exhibits no constitutiveexpressionof E-selectin
sod VCAM-I (Washittg(on et al., 1994). aIdtough ICAM-I is oresat at low levels on astmcvtes and on some endodteliai cells &be1 et al., 1990). Expression of ICAM-I is elevated in MS plaques at all stages of disease activity, whereas VCAM-I is present at highest levels in chronic active lesions (Raine et al., 1990; Camella and Raine, 1995). E-selectin is expressed on some MS microvessels doe to waosieot upregulation during endothelial cell a&a tion (Warhinetan et al.. 1594). Downreeulation of cell surface moleeuks by intemalizttion or shedding is an essential feature of the immune response. and soluble forms of ICAM-I. VCAM-I and E.xkcdn have been detected in worn (Rotblein et al., 1991; Seth et al., 1991). Increased levels of soluble ICAM-I (JCAM-I) have been described in a variety of inflammatory diseases and in certain malignaocies with the suggestion that they are markers of endothelial cell activation @earing and Newma,,. 1993). Ekvakd levels of sICAM-I have been reported in serum and CSF of MS padents. suggesting that ~asorentent of soluble adhesion molecule levels may movide a marker of disease zxivitv in MS. This study &wed on expression ol circulating forms of ICAM-i. VCAM-I and E-sekctin. We wished to determine if there is a specifk alteration in circulating adhesion mok~ule levels in clinically active multiple sclerosis and whether or *ot the response is pmdominaody intrathecal. 1
2 Materials
and methods
Paired serum and CSF samples were obtained from 129 patknts v&b neurological disease who artended the New mlogy Dcpamnent over a 6.month paiod. The clinical diagnosie was recorded and cootirmed in all patients at kast one year after the samples were taken. Fifty-six patients (37 female) diagnosed as clinically definite multipk sclerosis, according to the criteria of Pow et al. (1983). entered the study. All patients witi MS were in relapse at dte time of sampling. A relapse was defined as the appearance of symptoms of neumlogical dysfonction witb objective confirmation lasting more tbao 24 h. All blood and CSF samples from MS patients were obtained within 3 weeks of onset of relapse. No patient had received corticostemids OT other immunosuppressive treatment in the 6 months prior to entry into the study. Control groups iocloded 30 patients with inflammatory neomlogical disease (IND) (viral encephalitis, cerebral vascolitis, acute disseminated eocephalomyelitis, transverse myelitis, stroke, sarcoidosis, Ckilliao Barr& syndrome and chronic inflammatory demyelinating polyndiculopathy) and 43 patients with non-inflammatory cxwological disease (NIND) (cervical spondylotic myelopathy, syringomyelia, psychogenic symptoms, compression neuropathy, motor new ran disease, epilepsy and vertebral disc prolapse). Written
informed consent was obtained from all palienh. The protocol was approved by the Queen’s University of Belfast Medical Research Ethical Committee.
Blood samples were collected by venepuncture sod semm SW prepared by centrifogation of the clotted blood. A paired CSF sample. obtained from each patient, was centrifwed for 10 min at 200 x I and sopematant was obmiz Serum and CSF sample; were s&d hozen at - 7vC until we. Only CSF samoks that showed no visual evidence of a bloody -& were &!lysed. L?.boratory Malysis was performed on all samples including qoantitation of albumin and immtmoglobulin G (IgO). calcolatioo of CSF IgG/albomin ratio (Tibbing et al.. 1977), CSF IgO index (Delpech and Lichtbku. 1972) and detection of oligoclonal 180 bands tGCB) (Walker et al., 1983).
Levels of slCAivl-I, #CAM-I and SE-sekctin were detemdned in mired serum and CSF samples from all patients. Com&rcklly available sandwich &ISA assays were used in accordance with the instmctions of the manufactorer (R&D Systems Europe Ltd.). To optimise assay sensitivity, IO CSF samples were firs1 assayed for sICAM-I. sVCAM-I and SE-selectlo at serial dilutions of I:,, I:5 and 1:lO. As linearity of results was obtained for all samples. with no sigtdficaot matrix effects, CSF sampier in-tie study werediluted 1: I in appropriate sample dikent instead of the recommended dilution. Seturn sampks were tested at the recommended dilution. Samples diluted with buffered protein were applied to microwell strips coated with tbe respective murine monoclonal antibodv (mAb) aeainst human ICAM-I. VCAM-I. and Es&tin. A&cr&iog washed twice wiL a buff& (polysorbate 240. the microwell strips were incubated wilh the respective horseradish pemxidase-conjugated anti-adhesion mokcuk murine mAb diluted with the assay buffer. The reaction was stopped after 30 min by the addition of 4 N sulfuric acid. The absorbance of each well was measured at 4.50 nm osine an ELISA reader. A standard cwve was used to determine the adhesion mokcole concentration in each samok. Samales were all examined mine ELISA assay kits-with the &woe batch number. The de&on limit of the assavs was: 1.0 w/ml sICAM-I: 3 w/ml sVCAM-I: 0.i ng/ml sE-s&tin. Sample. con&trations below the detection limit of the assay were recorded as zero. The mean intro-assay variability was S-7% and Le mean inter-assay variability was S-IO%. The adhesion molecule index levels (according m the IgG index) Klelpecb and Lichtblao. 1972) were calc~ktwl as follows: s adhesionmolecuka, s adhesionmoleeuk_,
albumincsp ‘albumin,,,’
were considered significant. Spearman’s rank-order comlation test was applied to identify asso&dions betwen group parametw. Two-tailed P-values lower than 0.05 were considered significant.
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3. Results Serum [CAM-I levels ranged fmm M) ng/ml to loo0 ng/ml, with a mean value in the non-inflammatory disease patient? (mean f SD., 308 f I I8 n&!/ml) similar to praviou8 reports (Jan&r et al.. 1993, Hartung et al., 199393). Serum slCAM-1 levels were lower in MS pafiats (P = 0.04) and in IND patients compared with the NIND comml group (Fig. 1). CSF concentrations of sICAM-I io Lbe patients studied ranped from undetectable levels (< 1.0 I NIND patient) to 9.9 &ml. CSF slCAM-I was sienificwdlv elevated in MS oatieow cornowed with NlND p&sots (P= 0.02). Serum ‘SWAM-I Was in the range 271% 1900 “g/ml and no significant differences were found between the patient groups. TIP mean CSF #CAM-I concentration in h4S oatients was sienificardlv elevated compared to inflammatory controls (P - 0.001) and noninflammatory controls (P = O.ouDl) (Fig. 2). Serum levels of SE-selectin were normal in all patient groups. CSF E-s&&n concentration ranged from undetectable (4 0.2 nc/mk 77/129 aatiems) to 6.6 w/ml and no sienificaot differences were found between the patient groups. Calculat~on of tbe corresponding soluble adhesion molcudc iodizes showed that the SWAM-I index was significantly higher in tbe MS Sroup compwcd with IW (P - O.OfJl)
ng/ml:
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Analysis was carried out using SPSS/PC + software (SPSS Corpaaion, 1988). The Mann Whitney U-test was used 10 compare test groups. P-values lower than 0.05
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and NlND patients (P = 0.X11) indicating that inuathecal production of VCAM-I occurs predominandy in MS (Table I). Significant positive correlation was abserved between lhe sVCAM-I index and the slCAM-I index in
patientswith MS (r = 0.40, P = 0.005) and IND (r = 0.40, P = 0.031, but not in patients with NIND (r = 0.24. P = 0.23). Interestingly, the presenceof oligoclonal bands in the CSF of MS patients was associatedwith higher CSF levels of SWAM-I (17.5 “g/ml vs. 7.3 “g/ml. P = 0.006) and sICAM-I: (2.0 “g/ml vs. 1.4 “g/ml. P = 0.005). and a higher VCAM-I index (6.1 vs. 3.1. P=O.OLlX). This associarion was not found in the inflammatory control group. In MS patientsno asscciationwith soluble adhesion molecules was found whh other indicators of intrathecal immunoglobulin production, including CSF IgG concenuation, CSF IQ ratio and CSF IgG index.
4. Discussion
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Leukocyte migration acrossthe blood-brain barrier en. dotielium involves at least three steps: slowing of leukocyte flow by transient selectin interactions, followed by triggering of integrin activation and strong adhesionmediated by mokcules of the immunoglobulin superfamily such as lCAM-1 and VCAM-I. Activation of vascular endolhelial cells resultsin rapid upregulationand shedding of E-selectin, ICAM- and VCAM-I from the cell surface. The anmunl of soluble adhesionmolecules releasedcwrelates directly with their cell surface expression(Leeuwenberg et al., 1992). In this study we demonsuareda marked increase in #CAM-l levels in CSF during MS relapse. Elevation of the VCAM-I index indicated that this was due to inirathecal expressionand releaseof VCAM-I. The
mean CSF sVCAM-I concentration was signiFica.ndy hither in oatiettts who had CSF oligoclonal bands, emphasioittg the~importattce of the itnmo~e response within the intralhecal compartmettt in MS. This finding confirms recent reports of increased CSF sVCAM-I levels during MS relawe (~ore-~eff~ et al.. 1995; Matsuda et al., 1995). Elevated CSF &CAM-l levels have also been detected during clinical remission (DoresDuffy et al., 199% and doting the chronic progressive phase of the disease @ore-DoFFy et al., 1995; Matsoda et al., 199% suggesting that episodic or persistent activatioo of the cerebral endothelium is sustained throughout the diwase cowse. mere are few repow of CSF VCAM-I levels in other diseases, although elevation of VCAM-I without an increase in E-s&& and ICAMhas been detected in CSF of attitnals with simian immunodeficiency virus-t&ted enceohalitis &sseville et al.. 1992). Serum levels of sVC.&l-l were normal in active MS. as previously reported (Dare-Duffy et al., 1595). although increased levels have been detected in sotoe patienls (Marsoda et al., 19995). We demonstrated that slCAM-I levels were increased in CSF but not in serum during MS relapse, with no elevation of the sICAM-I index. Data on slCAM-I levels in active MS have been CooFlicting, with reports of normal or elevated levels in serum (Hartung et al., 1993: Iaoder et al.. 1993: Sharief et al.. 1993) and in CSF (Rieckmann et al., 1993: Tsukada et al., 1993: Dare-Duffy et al., 1995). Tote cause of these divergent results is unknown but may be doe to differences in methodology and time of sampling in relation to disease ,activity. The cellular origin and kinetics of release of ICAM-I and VCAM-I may also differ in response to cylokine stimulation of the endothelium as the MS lesion evolves. Our observations confirm the finding of normal sentto sEselectin levels in active MS (Rieckmann et al., 1994). and demonsuate that SE-selectin levels are also normal in CSF in active MS. Higher serum sE-wlectin levels in the inflammatory control group were similar to results in other patients with acute and chronic inflammatory demyelinating polyradicolopathy (Oka et al., 1994: HttrNng et PI.. 1994). Neutmphils in tbe semm of MS patients show impaired adherence and chemotaxis (Pcdikogloo et al., 1994), and normal sE+electin levels reaflirm the low level of oeutrophil-endotheliel cell interactions in relapsing remining MS. Inlerestingly. increased seturn sE-wlectin levels have been detected in patients with primary progressive
MS Kiiovannoni et al., 1594; Doie-Duffy et al., 199% suggesting that the dynamics of the inflammatory response may differ in relapsing remitting and primary progressive forms of the disease (Revesz et al.. 1994). The site of origin of these circulating adhesion molecules in MS is not known. sVCAhl-I may arise from cerebral or meningeal endothelial cells, macrophages or reactive astrocvtes. WAM-I ma” be relcascd from maw cell WDeS i&loding endothelitd~cells, lymphocyles, tnactbphage;&d microglial cells. VCAM-I and ICAM- 8re the tttosl abuttdant activation markers present on blood vessels in MS plaques. The most active lesions appear to be HLA-DR oaitive whereas chronically active lesions also ewress high levels of VCAM-I &d ICAM-I (Traugott, i987: Hayashi et al.. 1988; Raioe et al., 1990: Washington et d 1994). Adhvsion molecule expression extends beyond the areas of celktlar infiltration, and in periplaque areas 50% of microvessels co-express VCAM-I and HLA-DR (Wasltington et al., 1994). Tote differential shedding of adhesion molecules in MS suppa the idea that endothelial cell activation may involve a sequence of steps regulated by locally released cvtokiner. Dose- and time-decendent induction of Eselec&I. ICAM-I, and VCAM-I &pression occurs on vascular endotbelium in vitro in response to a range of inflammatory cytokines, with resultant shedding of adhesion molecules into the sopematant (Leeuwenberg et al., 1992; Pigott et al., 1992: Schieffer et al., 1992). Tumor necrosis Factor (TNF), interleokin-I (IL-I). interleukin-4 (IL-4) and interferon-v (IPN-y) induce expression of E-selectin and ICAM-I. v&as optimal expre&ion of VCAM-I requires a combination of IL-I and/or TNP together with IL-4. Cytokines induce expression of ICAM-I and E-selectin oa both arteries and veins but VCAM-I levels are increased only on venous endothelial cells (Szekaoecz et al., 1994). The oredilection of MS lesions for petivcnous sites may d&ore explain the predominant l&thecal release OF sVCAM-I in MS. EN-y can induceclinical telapseOf MS (Panitch et al., 1987) and administration of anti-IFN-y antibodies to animals with experimentalautoimmuneencephalomyelitis blocks the uptegulation of VCAM-I, but not of ICAM-I, suggesting that VCAM-I may be more imponant in the exacerbation of disease activity (Barten and Roddle, 1994). The activation state OFvenoo~ endothelium is of critical importance to the recruitment of resting and effector T cells during developmentof the inflamma-
tory lesion. lntratbecal release of VCAM-I during relapse may correlate with the regulated migration of immune cells inm the brain. VCAM-I is a l&and for the integrin a4pl 6X4-4) and binds weakly m a4p7 Khan et al.. 1992). hagrins provide the main adhesive force between T cells and vascular endotheliwn. and VCAh&a4pI interactions mediate the rolling and stable arre8, of T cells on Ule endothelial surface prior to vansmigration (Luxinskas et al., 1995). A8 such. e4 integrins are crucial 10 the effective homing of activated T cells across Ule blood-brain barrier ,o shes of CNS inflammation (Yedncck et al., 1992; Baron et al.. 1993). CSFT cells in MS show increased expression of many adhesion molecules. in&ding VLA-4 and VLA-5 (Svamingsson et al., 1993). al$augh Ule distribution of a4 in,eg,ins on selective T cell subpwdadans is no, known. Soluble adhesion molecule8 may exen functional effects such as inhibition of adhesion by compedtion (Rolhlein et al.. 1991). so sVCAM-I mav act to modulate the recruitn&l of iymphocyles u, sit& of tissue desvuction. Both ICAM-I and VCAM-I, but no, E-selectin, are imponam in T cell bindine 10 activated cerebral endoLhelium in viuo (Wang et al.,-1994). However, selective elevation of the VCAM-I index highlights the decisive role of VCAMl/a4 integrin interactions in lymphocyte trafficking thrwzh areas of CNS inllammalion in MS. Serum levels of &AM-I are consistently raised in patients with systemic vasculitis and appear 10 correlate well with disease activity (Janssen et al.. 1994). Accumulating evidence far significant elevation of CSF sVCAM-I in MS implies that measuranen, of sVCAM-I may allow the detection of subclbdcal disease activity. The sVCAM-I index may therefore provide a surrogate marker of an adequate respcmse ,o ,,ea,men,. Blockade of VCAM-I on cerebral end&&an by monalonal andbalies may also have considerable lhempeutic potential as a disease modifying therspy in MS.
This study was supported by a gran, from the Multiple Sclerosis Society of Great Britain and Nanhem Ireland.
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