Ž .Journal of Neuroimmunology 93 1999 81–91

Antigen presenting capacity of brain microvasculature in altered peptideligand modulation of experimental allergic encephalomyelitis

L. Santambrogio a,b,c, M. Pakaski a,c, M.L. Wong a,b, B. Cipriani d, C.F. Brosnan d,M.B. Lees a,c, M.E. Dorf b,)

a Biomedical Sciences DiÕision, E.K. ShriÕer Center, Waltham, MA 02452 USAb Department of Pathology, HarÕard Medical School, Boston, MA 02115 USAc Department of Neurology, HarÕard Medical School, Boston, MA 02115 USA

d ( )Department of Pathology Neuropathology , Albert Einstein College of Medicine, Bronx, New York, NY 10461 USA

Received 11 May 1998; revised 10 August 1998; accepted 21 August 1998

Abstract

Ž .Co-immunization with an altered peptide ligand LR partially protects SJL mice from proteolipid protein peptide 139–151-inducedwexperimental allergic encephalomyelitis Kuchroo, V.K., Greer, J.M., Kaul, D., Ishioka, G.Y., Franco, A., Sette, A., Sobel, R.A., Lees,

M.B., 1994. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire. J.Immunol. 153, 3326-3336; Santambrogio, L., Lees, M.B., Sobel, R.A., 1998. Altered peptide ligand modulation of experimental allergic

xencephalomyelitis: immune responses within the CNS. J. Neuroimmunol. 81, 1-13 . Clinical protection was noted despite extensivecentral nervous system inflammation observed after co-immunization with native and altered peptides. To extend our previous reports onthis model, we now compare MHC class II expression and antigen presenting cell activity of cells associated with the blood–brain barrierin diseased and protected mice. Immunohistochemical studies identified MHC class II products on both the endothelial andmicroglialrmacrophage populations. Ex vivo experiments suggested a correlation between the reduced clinical disease observed in theco-immunized mice and the antigen presenting activity of cells at the blood–brain barrier. The results suggest that antigen presentingactivity is primarily mediated by macrophage-lineage cells of the central nervous system. q 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Experimental allergic encephalomyelitis; Perivascular macrophages; Brain endothelial cells; Inflammation; Proteolipid protein

1. Introduction

We have carried out extensive studies on the proteolipidŽ .protein PLP -induced experimental allergic encepha-Ž .lomyelitis EAE model in SJL mice using the major

encephalitogenic epitope constituted by PLP residues 139–Ž . Ž .151 p139 reviewed by Sobel et al., 1994; Tuohy, 1994 .

Co-immunization of SJL mice with the native peptide andŽ .an altered peptide LR in which the amino acids at the TŽ .cell binding sites tryptophan 144 and histidine 147 were

Ž . Ž .substituted with leucine L and arginine R , respectively,blocked clinical disease. Despite clinical protection in mostof the mice, meningeal and inflammatory cell infiltration

Žare observed in the protected animals Kuchroo et al.,.1994 . Further, the co-immunized mice develop high levels

) Corresponding author. Tel.: q1-617-4321978; Fax: q1-617-4322789; E-mail: dorf@warren.med.harvard.edu

of CD4rCD8 cell infiltration of the central nervous systemŽ .CNS unexpected for mice with little or no clinical signs

Ž .of disease Santambrogio et al., 1998 . Our recent im-munohistochemical and PCR studies of the cytokines pre-sent within the brain also revealed decreased levels ofTNF-a and IFN-g in mRNA from co-immunized mice. Bycontrast, TGF-b mRNA and immunoreactivity were in-creased and protection from clinical EAE was partiallyabolished by treatment with anti-TGF-b antibodyŽ .Santambrogio et al., 1998 .

This system, in which closely related peptides induceextensive inflammation but different clinical outcomes,directed our investigations to comparisons of MHC expres-sion and antigen presenting capacity of the cells whichmight be involved in disease, especially cells associated

Ž .with the blood–brain barrier BBB . Potential antigen pre-Ž .senting cells APC of the BBB include endothelial cells,

perivascular macrophages, microglia, astrocytes, and peri-

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 98 00203-3

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–9182

Ž .cytes Traugott et al., 1985; Balabanov et al., 1996 . Inrodents MHC class II expression on these cell types isinducible in the presence of proinflammatory cytokines

Žsuch as IFNg Pober et al., 1983; Suzumura et al., 1987;.Male and Pryce, 1989 .

The present study was undertaken to determine if theobserved differences in clinical course and cytokine pat-terns within the CNS were related to differences in theantigen presenting capacities of cells of the CNS microvas-culature. MHC class II expression and related function onendothelial cells and perivascular macrophages in p139immunized, LR immunized and co-immunized mice werecompared. The results identify functional differences in theantigen presenting capacity of the microvessels among thethree groups and suggest that the differences are associatedwith clinical signs. Furthermore, the data suggest thatperivascular macrophages and parenchymal macrophage-lineage cells are the primary APC of the CNS.

2. Materials and methods

2.1. Mice

Ž .Female SJLrJ mice 4 to 6 weeks of age were pur-Ž .chased from Jackson Laboratories Bar Harbor, ME and

housed in the animal quarters of the E.K. Shriver Center orat Harvard Medical School. They were maintained inaccordance with the guidelines of the Committees onAnimals of Harvard University, the Shriver Center, and theCommittee on Care and Use of Laboratory Animals of theInstitute of Laboratory Animal Resources, National Re-

Žsearch Council Department of Health and Human Ser-.vices Publication 85-23, Revised 1987 .

2.2. Antigens

ŽProteolipid protein peptide 139–151 HSLGKWL-.GHPDKF was prepared according to the published se-

quence, except that serine was substituted for cysteine atŽ .position 140 Tuohy et al., 1989 . The serine replacement

was used to prevent potential disulfide interactions of thepeptide. In the altered peptide ligand LR, amino acid

Žsubstitutions were introduced at residue 144, tryptophan. Ž .to leucine and residue 147, histidine to arginine . p139–

151 and LR were synthesized in the laboratory of Dr.ŽRichard Laursen Department of Chemistry, Boston Uni-

.versity, Boston MA . All peptides were )90% pure asdetermined by HPLC.

2.3. Induction of EAE

Mice were injected subcutaneously at the base of thetail and the nape of the neck with 50 mg of peptide

Ž .consisting of PLP residues 139–151 group 1 , 50 mg ofŽ .p139–151 mixed with 500 mg LR group 2 or 500 mg of

Ž .LR peptide alone group 3 and 400 mg of MycobacteriumŽ .tuberculosis H37Ra Difco Laboratories, Detroit, MI in an

emulsion consisting of equal parts of peptide in PBS andCFA. Each mouse was also injected i.v. on days 1 and 3

9 Žwith 10 heat killed Bordetella pertussis bacilli Mas-sachusetts Public Health Biological Laboratories, Boston,

.MA .Mice were observed daily for clinical signs of EAE.

Clinical signs were scored according to their severity asfollows: grade 0, no symptoms; grade 1, decreased tailtone or slightly clumsy gait; grade 2, limp tail and hind

Ž .limb weakness waddling gait and or poor righting ability;grade 3, severe hind limb weakness; grade 4, limb paraly-sis; and grade 5, moribund state. Animals in all experimen-tal groups were sacrificed when essentially all mice in the

Žnative peptide immunized group developed EAE usually 2.days after onset of clinical signs . Mice were sacrificed by

overdose of CO and perfused via the left ventricle with2

100 ml of isotonic saline solution prior to isolation of brainmicrovessels, resident microglia, or infiltrating lympho-cytes.

2.4. Immunohistochemistry

Seven-mm sections were prepared from snap-frozentissue, air-dried for 10 min and fixed in ice-cold acetone

Žfor 10 min at y208C. After three washes with TBS Tris.buffered saline , the sections were blocked with 10% goat

serum for 1 h at room temperature. In a pilot experiment,s Žfive different mAb reactive with IA were compared MK-

Ž .S4, American Tissue Culture Collection ATCC ,Rockville, MD; 10-2.16, ATCC; 10-3.6.2, ATCC; MRC

.OX-6, Serotec, Washington, DC; MRC OX-3, Serotec .For single color staining slides were incubated with 0.2 ml

Ž .anti-MHC class II antibody 1 mgrml; clone MRC OX-6overnight at 48C, washed three times in TBS, incubatedwith alkaline phosphatase conjugated goat anti-mouse IgG1Ž .Southern Biotechnology Associates, Birmingham, AL for2 h at room temperature, washed as before, and incubatedwith alkaline phosphatase substrate HistoMarke Red for10 min at room temperature for chromogenic detection ofalkaline phosphatase staining, and with True Blue for 2

Žmin as counterstain Kirkegaard and Perry Laboratories,.Gaithersburg, MD . Control tissues were incubated with

normal mouse serum as primary antibody and processed asabove. After the last wash with TBS, sections were dehy-

Ždrated and mounted in Permount Fisher Scientific, Pitts-.burgh, PA .

To detect the co-expression of MHC class II and CD11bŽ .Mac-1 molecules, tissues were air-dried, fixed, and

Žblocked as above. Biotinylated anti-MHC class II mAb 10.mgrml, MRC OX-6 and rat IgG2b anti-mouse CD11b,

Žclone M1r70 10 mgrml; Boehringer Mannheim, Indi-.anapolis, IN were incubated overnight at 48C. The slides

were washed three times in TBS and the TRITC conju-Ž .gated anti-rat IgG Southern Biotechnology at 18.5 mgrml

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–91 83

was added to detect Mac-1 and incubated for 45 min atroom temperature in the dark. After three washes in TBS,

Ž .Streptavidin-FITC Becton Dickinson, San Jose, CA wasadded for the detection of MHC class II staining. Theslides were incubated for 45 min at room temperature.Negative controls consisted of species or isotype-matchednormal immunoglobulins. After washing, the slides were

Žmounted in Aqua-mount Lerner Laboratories, Pittsburgh,.PA and examined by confocal fluorescence microscopy.

2.5. Isolation of brain microÕessel cells

Microvascular cells were isolated from pools of three tofour mice using a modification of the method of Diglio et

Ž .al. 1982 . In preliminary experiments, preparations fromintact brains were compared with those from brains fromwhich meninges and white matter had been removed andno significant differences in the composition or purity ofthe microvessels were found. Therefore, microvessels iso-lated from whole brains were used in the analyses.

Cerebra from each of the three groups of immunizedŽ .mice were placed in Hanks Balanced Salt Solution HBSS

Ž .Life Technologies, Grand Island, NY containing 0.4 mMEDTA and homogenized. The homogenate was filteredthrough 100 mm nylon mesh and then centrifuged at1000=g, for 10 min. The pellet was resuspended in

Ž . Ž .HBSS containing 15% wrv dextran Sigma and 5%Ž . ŽFCS Life Technologies . After centrifugation 4000=g,.20 min the microvessels formed a pellet while the floating

thick band of myelin was removed with the supernatant.The microvessel pellet was resuspended in HBSS contain-ing 10% FCS. The suspension of microvessels was passed

Ž . Ž .through a glass bead column 150–212 mm Sigma andwashed with HBSS containing 10% FCS. The microvesselsuspension was centrifuged at 1000=g, for 10 min. Thepellet was resuspended in HBSS. For T cell proliferationstudies, the preparations were treated with 0.05% collage-nase and digested for 60 min at 378C. At the end of theincubation, the cell suspension was centrifuged at 1000=gfor 10 min and the pellet resuspended in Dulbecco’s

Ž .modified Eagles Medium DMEM q10% FCS. Viabilityof the collagenase treated cells was )90% as determinedby trypan blue dye exclusion.

The following antibodies were utilized to characterizethe microvessel preparations: a 1:200 dilution of rabbit Ab

Žto human von Willebrand factor Factor VIII-related anti-. Ž .gen Sigma followed by FITC conjugated goat anti-rabbitŽ . Ž . Ž .IgG 1:200 Sigma ; 1 mg phycoerythrin PE conjugated

Ž .anti-MHC class II mAb clone MRC OX-6 ; FITC coupledŽ .anti-a-smooth muscle actin 1:500, Sigma ; antibody toŽ . Ž .glial fibrillary acidic protein GFAP Sigma at a 1:100

dilution followed by a 1:200 dilution of FITC goat anti-Ž .rabbit IgG Sigma as the secondary antibody; and rat

Ž . Žanti-mouse Mac-1 CD11brCD18 Boehringer Mann-. Ž .heim detected by FITC conjugated anti-rat Ig Sigma .

The incubation temperatures and times for staining fol-

lowed manufacturers recommendations. Negative controlsincluded species or isotype-matched control immuno-globulins. Slides were read on a Leica microscope fittedwith I3 filters.

2.6. Isolation of endothelial cells

Endothelial cells were obtained from the collagenasetreated microvessel preparations by selective lysis ofperivascular microglia and other Mac-1q cells with anti-CD11b mAb plus complement. Immediately after isolation

Ž 6.collagenase treated brain microvascular cells 1=10Žwere incubated with 1 mg anti-CD11b Boehringer

.Mannheim in 100 ml DMEM for 1 h at 378C. Afterwashing twice with DMEM, 100 ml of a 1:2 dilution of

Ž .rabbit complement Sigma was added. Lysis was per-formed at 378C in a 10% CO atmosphere. Immediately2

Ž 4 .after isolation endothelial cells 10 viable cellsrwellŽwere plated in collagen treated 96-well plates Falcon-Be-

.cton Dickinson Labware, Franklin Lakes, NJ for prolifera-tion assays. The retrieved cells were )95% viable asassessed by trypan blue dye exclusion and )95% factorVIII positive by staining. No Mac-1 bearing cells weredetectable.

2.7. Isolation of microglia and macrophage-lineage cells

Ž .Macrophage-lineage cells Mac-1q from each of thethree groups were prepared as described by Sedgwick et al.Ž .1991 . Briefly, perfused brains and spinal cords from fourto six mice were passed through a stainless steel mesh andthe cells were collected in HBSS containing 3% FCS. Thedissociated material was enzymatically digested for 60 min

Ž .at 378C with 1 ml of 0.75% wrv type II collagenaseŽ . 4 Ž .0.95 Urmg, Serva and 10 U DNase I Sigma in

Ždissociation buffer 42 mM MgCl r23 mM CaCl r502 2.mM KClr153 mM NaCl . The digested CNS tissues from

individual mice were pelleted and resuspended in 5 mlŽ .isotonic 1.088 grml Percoll Pharmacia Uppsala, Sweden .

ŽThis suspension was underlayered with 5 ml Percoll 1.122.grml and subsequently overlayered with 9 ml each of

Percoll at 1.072 grml, 1.030 grml, and finally 9 ml ofHBSS. The tubes were centrifuged at 1250=g for 45 min.Microglia were collected from the 1.072 grml and 1.088grml interfaces. After washing, viability was assessed bytrypan blue dye exclusion. Insufficient perfusion of theCNS was determined by the presence of erythrocytes ontop of the 1.122 grml interface or in the pellet. If erythro-cytes were seen, the isolated cells were discarded. Thetotal number of cells recovered from each mouse dependedon the degree of inflammation, with 1=105 "20% re-trieved from p139 and co-immunized mice and 3=104 "

20% from mice immunized with LR alone. The retrievedpopulations were phenotyped with the following mAb:

Ž .hamster anti-mouse CD3´ FITC Boehringer Mannheim ;Ž .rat anti-mouse Mac-1 CD11brCD18 ; rat anti-mouse

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–9184

Ž .B220 Boehringer Mannheim and FITC anti-rat Ig. TheŽ .retrieved cells were either Mac-1 positive 70–75% or

Ž .CD3 positive 25–30% . In one experiment a few B220Ž .bearing B cells were detected -2% .

2.8. Isolation of T cells from neural tissue

T cells from the brains of peptide-immunized mice wereseparated and Ag-specific T cell lines generated and char-

Žacterized as previously described Santambrogio et al.,.1998 . Before use in T cell proliferation assays, microves-

sel preparations and T cells were purified over Ficollgradients. Aliquots were stained with anti-B220 and anti-CD11b mAb to ensure that exogenous APC were notadded to the cultures.

2.9. Lymphocyte proliferation assays

Collagenase-treated microvessel cell preparations andCNS microgliarmacrophages obtained from each of thethree groups or naıve syngeneic spleen cells were used as¨

Ž .APC after irradiation with 1600 rad cesium irradiation .Ž 4APC were seeded in 96-well plates 1=10 viable

.cellsrwell together with 10 mgrml specific or irrelevantantigen. The plates were incubated for 2–3 h at 378C in a5% C0 atmosphere. Subsequently 1=105 T cells were2

added at a concentration of 1=106rml. After 48 h thecells were pulsed with 1 mCi 3H-thymidine per well andplates were harvested 18–24 h later using a PHD cell

Ž .harvester Cambridge Technology, Cambridge, MA . Thesamples were counted in a LS5000 scintillation counterŽ .Beckman Instruments, Columbia, MD . The data repre-sent mean cpm of incorporated thymidine in insolubleDNA from triplicate cultures.

3. Results

3.1. Clinical comparisons

In this study, 43 of 46 mice immunized with 50 mgŽp139 developed clinical signs of EAE ascending paralysis

.and cachexia , whereas only 20 of 44 mice co-immunizedwith a mixture of 50 mg p139 and 500 mg LR showed anyclinical signs. In addition to the lower incidence of diseasethe co-immunized mice had a much milder disease. Af-fected mice in the co-immunized group generally had tail

Žweakness and little involvement of the hind limbs themaximal clinical score of diseased mice was 3.5"1.1 in

.p139 primed vs. 1.1"0.2 in p139qLR primed mice . Inthose mice that showed clinical signs, the mean day ofonset for the p139 and co-immunized groups were similarŽ .12.6"1.9 vs. 12.8"2.2 days and peak disease severityoccurred 15 days after priming in both groups. None of theanimals immunized with 500 mg LR alone developedclinical signs. Light microscopic examination of CNS tis-sues showed that in p139 and co-immunized mice thedistribution and cellular profile of lesions within the brainand spinal cord were typical of EAE in the SJLrJ mouseŽ .Fig. 1 .

3.2. MHC Class II expression

Most MHC class II bearing cells were found in theperivascular areas of the brain and spinal cord of p139 andco-immunized mice. Infiltration of class II positive cellsinto the cord parenchyma appeared to be greater in tissuesfrom p139 primed animals than in p139qLR immunizedmice. In the latter group, inflammatory cells tended to be

Ž .confined to perivascular sites Fig. 1A, B . Large andsmall vessels throughout the cord parenchyma, even atsites that did not show evidence of perivascular inflamma-

Ž .tion, were immunoreactive for class II MHC Fig. 1F . Inanimals sensitized with LR alone, class II MHC immuno-reactivity was found only in association with a few vesselsŽ .Fig. 1E and on cells within the meninges. Smaller vessels

Ž .were generally nonreactive Fig. 1G .To further define the nature of the cells that were

immunoreactive for MHC class II, double immunofluo-rescent labeling was performed. In inflammatory foci in allthree groups of mice there was prominent staining for IAs

Ž . Žsee, for example, Fig. 1I and Mac-1 see, for example,

Ž .Fig. 1. see right Cross sections of the lumbar spinal cord stained for MHC class II or Mac-1. Panels A, B, and E illustrate similar regions of the anteriorŽ . Ž . Ž . Ž .columns from animals sensitized with either p139 panel A , p139qLR panel B or LR panel E stained for MHC class II OX-6 mAb using

Ž .HistoMarkRed as the chromogen and counterstained with TrueBlue, magnification 480= . In animals sensitized with p139 panel A , inflammatory cellsŽ . Ž .strongly immunoreactive for MHC class II red are detected within the CNS parenchyma. In co-immunized animals panel B , inflammatory cells were

detected principally at meningeal and perivascular sites, where they showed strong immunoreactivity for MHC class II. In animals sensitized with LRŽ .panel E , inflammatory cells were rarely noted and MHC class II reactivity was restricted to large caliber vessels near the meningeal surface. Panels F and

Ž .G are at higher magnification 1200= to show MHC class II immunoreactivity associated with smaller caliber vessels within the CNS parenchyma thatŽ . Ž .was detected in both p139 and co-immunized animals panel F whereas in animals sensitized with LR alone panel G these smaller caliber vessels were

Ž .negative for MHC class II 1200= . Panels C, D, and H are the negative controls for Panels A, B, and F, respectively. Panels I and L illustrate staining for.MHC class II; panels J and M show staining for Mac-1; and panels K and N reveal double staining, Mac-1 TRITC-red and class II FITC-green . Note that

most of the cells that are immunoreactive for MHC class II also react with the Mac-1 antibody, indicating that these cells were of the monocytermacro-Žphage lineage. The images shown in panels I–K are from co-immunized mice. Panels L–N are a penetrating vessel as determined by phase contrast

.microscopy from a LR primed mouse and are consistent with low level MHC class II immunoreactivity on LR primed perivascular macrophage-lineagecells.

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–91 85

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–9186

.Fig. 1J . Double staining showed that many cells wereco-labeled, suggesting that these were of macrophage lin-eage. However, some of the cells in these foci stained only

Ž .for class II MHC Fig. 1K . In LR primed mice, immuno-reactivity was also associated with large and small vessels

Žthat did not show perivascular inflammation, Fig. 1L, M.and N . In these preparations, however, it was difficult to

distinguish stained endothelial cells from perivascularmacrophages or microglia.

3.3. Characterization of microÕessel preparations

Fig. 2 depicts a typical microvessel preparation beforeŽ . Ž .Fig. 2A and after Fig. 2B collagenase treatment. The

morphological appearance of these preparations by lightmicroscopy was similar among p139, LR and p139qLRprimed donors. 75 to 87% of the collagenase-treated popu-lation were endothelial cells as determined by cytoplasmic

Ž .staining for factor VIII Fig. 2D .Perivascular macrophages were identified by staining

Ž .with anti-CD11 b Mac-1 antibody. Positive staining wasevident on intact microvessels and, after collagenase treat-ment, quantitation of these cells was possible. Mac-1positive cells comprised 12–20% of the cells in the mi-

Ž .crovessel preparations Fig. 2E . No staining of pericyteswas observed before collagenase treatment, but after colla-genase, 1–5% of the cells stained with anti-smooth muscle

Ž .Fig. 2. Characterization of microvessel preparations from the brains of SJLrJ mice. Phase micrographs of microvessels before panel A and afterŽ . Ž . Ž .collagenase treatment panel B . Microvessels stained with anti-Factor VIII Ab before Panel C and after collagenase treatment panel D . Collagenase-

Ž . Ž .treated microvessels stained for Mac-1 panel E or for smooth muscle actin panel F . Panels A and B from p139 primed donors, panels C, D and E fromLR primed mice, and panel F from co-immunized animals.

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–91 87

Fig. 3. Factor VIII and MHC class II expression on microvessel preparations from the brains of SJLrJ immunized mice. Microvessels from the threeŽ . Ž . Ž .groups of immunized mice: p139 primed panels A and B ; p139qLR primed panels C and D ; and LR primed panels E and F . Double-staining of

Ž .microvessels with rabbit Ab to von Willebrand factor Factor VIII-related antigen followed by an FITC conjugated anti-rabbit Ig to detect endothelial cellsŽ . s Ž . Ž .panels A, C and E and microvessels stained with PE conjugated anti-I-A Clone 0X-6 to detect MHC class II positive cells panels B, D and F .

Ž .Representative data from three independent experiments 100=magnification .

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–9188

Ž .actin Fig. 2F . GFAP positive astrocytes were not de-Ž .tected in the microvessel preparations data not shown .

We next examined MHC class II expression on mi-crovessels from the three groups of immunized mice bydouble-staining with antibodies against Factor VIII andMHC. No consistent differences were seen in the expres-

Žsion of Factor VIII among these preparations Fig. 3A, C.and E . However, analysis of class II expression on these

preparations demonstrated differences. The Factor VIII-positive cells expressed class II in the p139 and co-im-munized groups; but expression was consistently stronger

Ž .on cells from p139 immunized mice Fig. 3B than cellsŽ .from the co-immunized group Fig. 3D . Little MHC class

II reactivity was observed in the vessels derived from LRŽ .immunized mice Fig. 3F . From the staining pattern of the

microvessel fragments it was not possible to definitivelyallocate class II reactivity to particular cell types. There-fore, double staining experiments were carried out withcollagenase-treated dispersed microvessel cells. All cellpreparations contained similar percentages of endothelial

Ž . Ž .cells Factor VIIIq Table 1 . However, analyses of cellsthat were double stained for MHC class II and Factor VIIIshowed a higher percentage of double positive cells fromp139 primed mice than from co-immunized mice. Evenfewer class IIq endothelial cells were found in prepara-

Ž .tions derived from LR primed animals Table 1 . On theother hand, comparable percentages of class IIq , Mac-1qcells were found in the p139 and co-immunized groupswhile lower percentages of double stained cells were found

Ž .in LR or CFA immunized mice Table 1 . Thus, whileclass II expression was extensively upregulated on bothendothelial cells and perivascular macrophages from thep139 primed group, class II was upregulated on perivascu-lar macrophages but to a lesser percentage on endothelialcells from the co-immunized mice, and only slight in-creases of class II were detected on cells from LR immu-

Ž .nized animals Table 1 .

Table 1MHC Class II expression on microvessel-derived cells from primed micea

Peptide Cells Percent positively staining cellss sfor priming counted Factor VIII VIIIqIA Mac-1 Mac-1qIA

p139 968 84"5 43"10 15"4 10"2p139qLR 1102 80"4 18"6) 16"4 11"2LR 1115 79"3 9"3) 16"4 3"1)

None 323 85"6 0"0 15"4 1"0

a Microvessel preparations were obtained from p139, p139qLR, LR, orCFA primed SJLrJ mice. Collagenase digested microvessel preparationswere double stained as described in Section 2. The total number of cellscounted and the percentages of Factor VIIIq or Mac-1q cells areindicated. The percent Factor VIII positive cells that were double stained

Ž s.with anti-class II IA and the percent of Mac-1 positive cells that doublestained with anti-IAs are listed. The data represent pooled results from

Ž .three independent experiments. An asterisk ) indicates a significantdifference between the p139qLR or LR and p139 groups. P -0.001 bystudent’s t-test.

3.4. Comparisons of antigen presenting capacity

To compare the functional capacity of microvesselpreparations, we evaluated their ability to elicit prolifera-tive responses from T cell lines isolated from each of thethree groups. Syngeneic spleen cells were used as positive

Ž .controls for antigen presentation data not shown . Mi-crovessels from the p139 immunized mice were able toinduce antigen-specific T cell proliferation of p139, LR or

Ž .p139qLR primed T cell lines Fig. 4 . The response ofthe p139 T cell line was highest when stimulated with the

Ž .homologous antigen p139 or p139qLR . Stimulation ofp139 T cells with LR produced little or no proliferative

Ž .response Fig. 4A . In contrast, when the LR primed T cellline was stimulated with microvessels derived from miceprimed with p139 or p139qLR, the responses were essen-

Ž .tially equal Fig. 4C . These results are consistent with ourprevious observations that the p139-derived T cell linedoes not respond to LR whereas the LR-derived T cell lineis cross-reactive and responds to both p139 and LRŽ .Santambrogio et al., 1998 . The T cell line derived fromco-immunized mice also proliferated in response to each ofthe antigens, although levels of thymidine incorporationwere less.

Importantly, microvessel APC derived from the co-im-munized mice present p139 antigen to all three T cell linesbut the proliferative responses were reduced when com-pared with those induced by microvessels obtained from

Ž .p139 immunized donors Fig. 4 . The weaker p139 re-sponses using microvessel APC from co-immunized vs.p139 primed mice may in part be relevant to the decreasedclinical disease. Finally, microvessels from LR immunizedanimals displayed little APC activity with all the T cell

Ž .lines Fig. 4 .In summary, the microvessel preparations isolated from

p139 immunized mice had better APC activity than mi-crovessels from co-immunized animals. The results sug-gest that the observed differences in proliferative responseswere related to the source of APC and not the T cell linesutilized.

To provide a better definition of the APC responsiblefor T cell activation the microglialrmacrophage compo-nents were depleted from the microvessel preparations bycomplement lysis with anti-CD11b mAb. Although themAb treated endothelial cells were )90% viable andexpressed class II antigens, the cells lacked APC activity

Ž .with all three T cell lines tested Table 2 . Since we wereunable to purify sufficient Mac-1 positive cells from themicrovessel preparations, macrophage-lineage cells wereisolated from the perfused brains of p139 andror LRimmunized mice. These Mac-1q populations includedparenchymal microglia and the monocytesrmacrophagesthat infiltrated the parenchyma. Maximal antigen present-ing activity was observed with Mac-1q macrophage-lin-eage populations obtained from p139 immunized mice,whereas intermediate degrees of specific T cell prolifera-

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–91 89

Ž 5.Fig. 4. Antigen presenting activity of microvessels from primed mice. Wells containing p139, p139q LR, or LR elicited T cell lines 1=10 wereŽ . Ž . Ž .challenged with 10 mgrml p139 square with dense fine dots , p139q LR square with sparse dots , or LR square with parallel lines , respectively.

Ž 4 . Ž . Ž . Ž .Collagenase treated microvessel derived cells 1=10 rwell from p139 panel A , p139q LR panel B , or LR-primed mice panel C were used as3 Ž . 6APC. The data are presented as net H-thymidine uptake mean cpm–background cpm . The responses to 10 splenic APC were used as positive controls;

values ranged from 150–200=103 cpm. Background proliferation ranged from 11410 to 15726 cpm for spleen and 252 to 3015 cpm for themicrovessel-derived cells. Standard deviations of triplicate cultures were -20%. Representative data from three to five experiments.

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–9190

Table 2Comparison of antigen presenting activity by macrophage-lineage vs.endothelial cells from primed micea

Source of APC Priming of APC donors

p139 p139qLR LR

Macrophage-lineage cells 14.0"3.8 2.5"0.3 0.1"0.5Endothelial cells 0.4"0.2 0.3"0.3 0.1"0.1

a Ž 5.Wells containing p139 primed T cell lines 1=10 were challengedwith 10 mgrml p139 antigen. 1=104 microvessel derived endothelial

Ž .cells or macrophage-lineage cells microglia and macrophages per wellwere used as APC. The data presented are the net 3H-thymidine uptake=

y3 Ž .10 mean cpm–background cpm "S.D. Background proliferation was-1000 cpm with microgliarmacrophages and -200 cpm with endothe-lial cells.

tion were observed with cells derived from co-immunizedŽ .mice Table 2 . Little or no responses were observed using

cell populations obtained from LR primed animals.

4. Discussion

Altered peptide ligands have been shown to protectmice from clinical EAE induced by immunization with

Ž .p139 Franco et al., 1994; Nicholson et al., 1995 . How-ever, the mechanisms of protection from clinical disease inmice co-immunized with native and altered peptides arenot understood. The overall numbers of infiltrating CD4qand CD8q T cells are similar between protected, co-im-munized mice and diseased, native peptide primed ani-mals. However, the fine specificity and cytokine profiles

Ž .differ between these groups Santambrogio et al., 1998 .Greater proportions of the T cells derived from the brainsof co-immunized and LR-immunized mice showed reactiv-ity to both p139 and LR whereas T cells from the nativepeptide-immunized mice were p139 specific. In addition,co-immunized mice display higher levels of TGF-b andlower levels of TNF-a and IFN-g mRNA. The greaterTGF-b immunoreactivity in perivascular inflammatory focifrom co-immunized mice along with indications ofperivascular infiltrates suggested that events at the BBBmight be critical for disease protection. In other systems ithas also been shown that the source of APC plays a major

Žrole in the response of T cells Fabry et al., 1993; Sun et.al., 1993 . The present study was, therefore, designed to

characterize differences in antigen presenting function ofthe CNS microvasculature in p139, LR and co-immunizedmice.

A critical factor for antigen presentation is the expres-sion of class II MHC molecules. Whereas p139 and co-im-munized mice showed extensive CNS inflammation andenhanced class II expression along the microvasculature,LR-immunized mice displayed few inflammatory foci and

Ž .low levels of class II expression Fig. 1 , suggesting thatthe class II expression on microvessels may be a pre-requisite for inflammation as well as presentation. Since

several different cell types associate with the microvesselsit was not possible to definitively identify the specific classII positive cells by immunocytochemistry. Even with dou-ble labeling of tissue sections, not all class II-positive cells

Ž .were identifiable as perivascular macrophages Fig. 1 . Tolocalize MHC class II expression further, intact and colla-genase-treated microvessels were isolated from the threegroups of mice. Analysis of these and similar preparationsshowed that they were composed predominantly of en-dothelial cells with smaller amounts of Mac-1q perivas-cular macrophages and pericytes. Co-localization of FactorVIII and I-As in double staining experiments suggestedthat a majority of the class II-positive cells in the prepara-tions from p139 immunized mice were endothelial cellsŽ .Fig. 3A, B . The microvessels from co-immunized miceappeared to have lesser amounts of I-As while microves-sels from the LR-immunized mice displayed even lowerlevels of I-As. Thus, despite an inability to differentiatelevels of MHC expression within inflammatory foci be-tween p139 and co-immunized mice by immunohistochem-istry, differential expression of I-As was evident on themicrovessel components. On the other hand, Mac-1qcells from p139 and p139qLR primed mice containedcomparable percentages of I-A bearing cells, while Mac-1positive cells from LR and CFA primed control mice

Ž .expressed little class II Table 1 . These results indicatethat class II antigens are differentially induced on endothe-lial cells and perivascular macrophages following immu-nization with native or altered peptide antigens.

Microvessels of the CNS constitute a complex anatomi-cal structure composed of endothelial cells, perivascularmacrophages, pericytes, and astrocytes that together form adynamic structural and metabolic barrier to maintain nor-

Ž .mal brain function Hickey et al., 1992; Ford et al., 1995 .The ex vivo data obtained from this study indicate thatperivascular macrophages are the primary antigen present-ing cell associated with the brain microvessels, confirming

Žthe conclusions of others Ford et al., 1995; Benveniste,.1997 . The absolute numbers and percentages of class II

positive macrophage-like cells in the microvasculature ofp139 and co-immunized mice were similar. However, dif-ferences in the levels of APC activation andror expression

Ž .of class II or co-stimulatory molecules e.g., B7.1 may beresponsible for the weaker proliferative responses of APCfrom co-immunized mice.

In vitro studies have shown that IFN-g stimulated en-Ždothelial cells can also present antigen Briscoe et al.,

.1997 , but we were unable to confirm this using endothe-lial cells that were directly isolated from the brains of thethree groups of mice and which had not been stimulatedexogenously. To ensure complete removal of contaminat-ing Mac-1q cells from endothelial cell preparations, com-plement mediated lysis with anti-CD11b antibodies wascarried out. The conclusion that endothelial cells cannotpresent antigen is at variance with that of McCarron et al.Ž .1986 who reported that unfractionated microvessel prepa-

( )L. Santambrogio et al.rJournal of Neuroimmunology 93 1999 81–91 91

Žrations from inflamed brains containing 5–15% nonen-.dothelial cells presented antigen in vitro. However, the

functional cells in the population were not establishedŽ .unequivocally. Pryce et al. 1989 found that endothelial

cells in vitro had the capacity of presenting antigen butonly under limited conditions that included treatment withhigh doses of IFN-g for a prolonged time to induce class IIexpression and addition of indomethacin to down regulateprostaglandins. They concluded that endothelial cells areprobably poor APCs and that, if endothelial cells presentantigen in vivo, it is likely that the kinetics of presentationdiffer from that of conventional APC. Endothelial cellsprobably play some role in the immune response, but itmay not be as APC. Rather, they may secondarily increasethe immune response by secreting important accessorymolecules involved in promoting the inflammatory pro-cess.

The current study expands our knowledge of the pro-cesses occurring within the brain which contribute to theprotective effects of altered peptide ligands in EAE. Wenow show clear differences in MHC class II expressionand in the antigen presenting capacity of the brain mi-crovasculature between native and co-immunized mice.The data indicate that up-regulation of MHC class IIexpression is insufficient for optimal APC activity. Itremains to be shown whether the class II differences are aconsequence or cause of changes in cytokine patterns

Ž .previously described Santambrogio et al., 1998 . In sum-mary, the combined observations demonstrate a potentialrole for the BBB in modulating clinical disease in EAE.Events at the BBB may also be major determinants ofclinical disease in multiple sclerosis.

Acknowledgements

The authors thank Dr. Raymond Sobel for extensivediscussions throughout this project. This work was sup-ported with grants from the National Multiple Sclerosis

Ž .Society PP0480T , the Multiple Sclerosis Foundation, theŽNational Institutes of Health NS16945, NS31152, and

.HD04147 , and a fellowship from the Italian MultipleSclerosis Society.

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