Proc. Natl. Acad. Sci. USAVol. 86, pp. 1684-1688, March 1989Neurobiology

Immunological function of the blood-cerebrospinal fluid barrier(choroid plexus/antigen presentation/lymphocytes/epithelial cells/nervous system immunology)

JAMES A. NATHANSON AND LINDA L. Y. CHUNDepartment of Neurology and Program in Neuroscience, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114

Communicated by John E. Dowling, November 21, 1988

ABSTRACT Because the brain lacks a true lymphaticsystem, it is unclear how peripheral lymphocytes recognizeforeign antigens present in the central nervous system. Thisreport demonstrates that the choroid plexus, which constitutesthe blood-cerebrospinal fluid barrier, is able to present foreignantigen to, and stimulate the proliferation of, peripheral helperT lymphocytes through an Ia-dependent, major histocompat-ibility complex-restricted mechanism. Furthermore, in vivo,choroid plexus epithelial cells have access to, and are capableof taking up, virus-sized particles injected elsewhere into thecerebrospinal fluid. Thus these data suggest that the blood-cerebrospinal fluid barrier may play a role in immunologicalcommunication between the central nervous system and pe-riphery, a function relevant to the initiation of immunologicalresponses to central nervous system infections and autoimmuneprocesses and for the surveillance of tumor cells in thecerebrospinal fluid.

During disease states, the brain contains immunoglobulin-producing plasma cells and T lymphocytes (1, 2). These cells,which are normally present in only small numbers, appear toenter the central nervous system (CNS) from the systemiccirculation (3). Surprisingly, little is known about how thisimmune process is initiated; particularly, how, at a time whenlymphocytes are largely absent from the brain, foreignantigens in the CNS are recognized and processed forpresentation to helper T cells (a necessary step for theinitiation of most types of cellular- and humoral-mediatedimmunity). In the periphery, interaction among lymphocytes,antigen-presenting cells (e.g., macrophages), and antigen isfacilitated by a highly developed lymphatic drainage system.However, the brain parenchyma, itself, contains no compa-rable lymph system and, furthermore, is largely isolated fromthe systemic circulation by the blood-brain and blood-cerebrospinal fluid (CSF) barriers. Even with the help ofendogenous CNS antigen-presenting cells, such as microgliaor astrocytes (4-8), it is unclear how antigens present in theCNS can interact with unstimulated lymphocytes located inthe periphery.

Prior studies have suggested that foreign substances pres-ent in brain extracellular fluid can move by bulk flow into theCSF (9, 10), which itselfundergoes a slow movement throughthe lateral, third, and fourth ventricles, into the cisternamagna. It occurred to us that the choroid plexuses (whichalso contribute to CSF formation) might be well-suited todetect foreign antigens present in the CSF and to communi-cate this information to the periphery. Choroid plexus tissuenot only comes in close contact with CSF in the ventricularcavities but also is strategically present at the narrow foram-ina through which the CSF passes from lateral to thirdventricles and from fourth ventricle to cisterna magna. Inaddition, the tightly joined choroid epithelial cells (consti-tuting the blood-CSF barrier) form an interface between CSF

and periphery, since their apical side faces the CSF and theirbasal side, facing fenestrated capillaries, has potential accessto peripheral blood cells. The present report investigates thepossible immune functions of the choroid plexus, in partic-ular, its potential access to and uptake of foreign antigens,and its ability to present foreign antigens to peripherallymphocytes.

MATERIALS AND METHODSChoroid plexus tissue consists of numerous capillary andarteriolar loops containing fenestrated endothelium andsmooth muscle cells covered with a single layer of cuboidalsecretory epithelium (11). Present on the CSF-facing surfaceof the epithelium are occasional stellate-shaped epiplexuscells. For antigen presentation experiments, choroid epithe-lium was purified by modifications of prior techniques (12,13). Briefly, intact lateral, third, and fourth ventricularchoroids from 4- to 6-week-old BALB/c mice were dissectedin Dulbecco's modified Eagle's medium (DMEM) and me-ticulously cleaned of any adhering meninges or brain under ahigh-power dissecting scope. Pooled choroid plexuses werefurther washed (for three 10-min periods) in DMEM by gentlytumbling (90 rpm) in an apparatus that allowed suspendedcells to be separated and removed from intact tissue. Sub-sequently, tissue was tumbled in calcium-free artificial CSF(12) containing 0.05% trypsin (Sigma T 0511) and 0.015 mMEDTA. Every 15 min for 4 hr, choroids were gently trituratedand fractions containing suspended cells were separated fromintact tissue; in this manner, a large peak of highly enrichedepithelial cells was obtained (usually fractions 3-6) prior tothe onset of dissociation of the choroid plexus stroma(usually fractions 10-15). Cells from pooled fractions 3-6were filtered through 150-,um Nitex mesh to remove any cellaggregates and then plated in DMEM with 5% (vol/vol) fetalbovine serum in tissue culture dishes. After 4 hr, epithelialcells, which do not adhere, were removed by gentle swirling,leaving behind a small number of adherent nonepithelial cells.Using this procedure, %105 epithelial cells were obtained permouse.For measuring T-cell proliferation by thymidine incorpo-

ration in antigen-presentation experiments, it was necessaryto prevent cell division of the antigen-presenting cell. Al-though choroid epithelium from postnatal animals does notnormally proliferate in culture, isolated epithelial cells werenonetheless routinely irradiated with 2300 rads (1 rad = 0.01Gy), a dose known to prevent proliferation of other types ofantigen-presenting cells (6, 14). After irradiation, epithelialcells (usually 5 x 104 cells per microtiter well) were co-cultured in the absence or presence of antigen (chickenovalbumin) with an equal number of antigen-specific lym-phocytes. The lymphocytes were obtained from an antigen-

Abbreviations: CNS, central nervous system; MHC, major histo-compatibility complex; CSF, cerebrospinal fluid; IFN-y, y-inter-feron; DiI-Ac-LDL, fluorescent acetylated low density lipoprotein;DARPP-32, dopamine- and cAMP-regulated phosphoprotein.

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specific helper T-cell clone (03) (L3T4',Ly2-) that had beenderived from the spleen ofa BALB/c mouse immunized withchicken ovalbumin (14). At 24 hr, [3H]thymidine (1 ,uCi perwell; 1 Ci = 37 GBq) was added to each culture. Then 16-20hr later, the cells were harvested on a PHD cell harvester(Cambridge Technology, Cambridge, MA) and [3H]thy-midine incorporation was measured. Data points were car-ried out in triplicate. Antigen presentation by choroid cellswas demonstrated in eight experiments done on six occasionsover a period of 2.5 years. Morphological observations ofantigen-induced aggregation (Fig. 1) were carried out in sevenexperiments and aggregation was a consistent finding. Anti-gen dose-response experiments were replicated three timesand the data shown (Fig. 2A) are typical. Controls showingantigen presentation to 03 by BALB/c spleen cells werecarried out three times using conditions identical to those forchoroid-03 experiments. For those experiments (donetwice), which used antibody directed against Ta, antigen (100Ag/ml) and antibody were added at the time of coculture.Antibody was purified by (NH4)2SO4 precipitation fromculture supernatants prepared from hybridoma 34-5-3S (15),whereas, for antibody from hybridoma 144-4S (16), ascitesfluid (Cedarlane Laboratories, Hornby, ON) was used.For histological identification of cell type (carried out for

most experiments), parallel cultures in 15-mm wells wereplated and incubated for 48 hr. Four hours prior to fixation,cells were incubated with fluorescent acetylated low densitylipoprotein (DiI-Ac-LDL; Biomedical Technologies, Stough-ton, MA; 1:30 dilution), a marker taken up by macrophagesand endothelial cells but minimally taken up by choroid

epithelium. After fixation in cold 1% paraformaldehyde,wells were washed three times with isotonic phosphate-buffered saline (PBS) and once with 4% (vol/vol) goat serumand incubated overnight at 40C with rabbit antibody (diluted1:150) to the a form of Na,K-ATPase or with mouse mono-clonal antibody (diluted 1:300) to dopamine- and cAMP-regulated phosphoprotein (DARPP-32). (Antibodies werediluted in 0.3% Triton X-100 in PBS.) After five washes withPBS, cultures were incubated for 1 hr at 22°C with afluorescein-conjugated second antibody (Cappel Laborato-ries, 1:100 dilution), washed, and viewed by epifluorescencewith an inverted microscope. In some experiments, afterfixation, cells were harvested by scraping, transferred tocentrifuge tubes, and then immunostained in suspension, asdescribed below for particle uptake experiments.For histochemical demonstration of Ta expression in cho-

roid organ culture, minced pieces of intact BALB/c choroidplexus were cultured in DMEM containing recombinantmouse y-interferon (IFN-y) (Genentech) at 1000 units/ml, 1AM indomethacin, and Escherichia coli lipopolysaccharide(Difco 3123-25) at 10 ,ug/ml, all added initially and again at 24hr. (IFN-y or lipopolysaccharide, alone, caused similarthough less intense Ia staining. In the absence of either therewas no Ta staining.) After 42 hr of culture, unfixed tissue wastransferred to centrifuge tubes, washed, and stained over-night at 4°C with 34-5-3S (diluted 1:2), followed by fluores-cein-conjugated second antibody (Cappel Laboratories, di-luted 1:50). Subsequently, after washing and fixation in 1%paraformaldehyde, tissue was stained (to identify epithelialcells) overnight at 4°C with rabbit antibody to Na,K-ATPase(diluted 1:150), followed by rhodamine-conjugated second

FIG. 1. Co-cultures of03 lymphocytes with mouse choroid epithelial cells in the presence ofantigen (a, d, and g), lymphocytes with choroidplexus cells in the absence of antigen (b, e, and h), and lymphocytes with spleen cells in the presence of antigen (c, f, and i). (a-c) Culturesat 48 hr. (Phase-contrast; bar = 25 ,um.) Note that in the presence of the specific antigen, ovalbumin at 100 ,g/ml (a and c), cells form aggregatesassociated with lymphocyte proliferation (see Fig. 2). (d-f) Fluorescence ofDiI-Ac-LDL (a fluorescent marker avidly taken up by macrophagesand endothelial cells) present in spleen cell aggregates but absent from choroid co-cultures. (g-i) Bright immunostaining for the a form ofNa,K-ATPase of choroid epithelial cells present in cell aggregates (g) and (in the absence of antigen) in dispersed co-cultures (h); aggregatespresent in spleen cell co-cultures stain only weakly with this concentration of antibody.

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FIG. 2. (A) Stimulation, by antigen, of the proliferation oflymphocytes co-cultured with choroid plexus cells (solid circles).The specific antigen, chicken ovalbumin, failed to stimulate thymi-dine incorporation into lymphocytes cultured in the absence ofchoroid plexus cells (open circles), or into choroid plexus cellscultured alone (triangles). (B) Inhibition of antigen-dependent stim-ulation of lymphocyte proliferation by monoclonal antibody (34-5-3S) directed against the histocompatibility glycoprotein I-Ad.Values shown are the mean ± SEM for triplicate determinations.

antibody (Cappel Laboratories, diluted 1:100). After mount-ing on coated slides, multiple fields were observed byepifluorescence microscopy to locate doubly labeled cells. Inthis experiment (which was done twice), Ia staining ofepithelium was generally less intense than that seen onepiplexus cells (identified by their stellate morphology andgenerally negative ATPase staining).For studies of particle uptake by choroid plexus, 100 nM

fluorescein-conjugated, noncarboxylated polystyrene micro-spheres (Polysciences 17150) were injected (100 A.l of a 1:1dilution in artificial CSF) stereotaxically into the lateralventricle of a rabbit. Eighteen hours later the rabbit wassacrificed, the fourth ventricular choroid plexus was re-moved and extensively washed in artificial CSF, and theepithelial cells were then isolated as described above. Cellswere distributed into test tubes, where they were then fixed,washed, and immunostained for either DARPP-32 or Na,K-ATPase as described for antigen-presentation experimentsexcept that rhodamine-conjugated second antibodies (CappelLaboratories, 1:100 dilution) were used, and cells weresedimented by centrifugation after each incubation and wash.Aliquots of cells were air-dried on coated slides, mounted,and viewed by epifluorescence microscopy for doubly la-beled cells. To determine the percentage of epithelial cellstaking up beads, cells in 10 fields at a magnification of x200

were counted. Similar results were seen in two separateexperiments. On a third occasion, when the intraventricularinjection missed the lateral ventricle, labeling of fourthventricular choroid cells was not seen at 18 hr.

RESULTS AND DISCUSSIONWe first wished to determine the ability of the choroid plexusto initiate an immune response. For this purpose, a highlyenriched suspension of choroid epithelial cells was preparedfrom BALB/c mice. By a variety of criteria, these cells werefree of contaminating macrophages, endothelial cells, andastroglia (see below). The purified epithelial cells wereirradiated to prevent cell division and then co-cultured inmicrotiter wells, or in slide wells, with the antigen-specifichelper T-cell clone 03 in the absence or presence of theantigen chicken ovalbumin. The co-cultures were observedover a 48- or 72-hr period for morphological changes asso-ciated with 03 lymphocyte activation and for [3H]thymidineincorporation indicative of 03 cell proliferation.

Co-cultures of lymphocytes and choroid plexus cells incu-bated in the presence of ovalbumin at 100 gg/ml developednumerous activated centers, consisting of clusters of mixedlymphocytes and epithelial cells (Fig. la). In the absence ofantigen, cells remained largely dispersed (Fig. lb). Likewise,lymphocytes in the presence of antigen alone, or choroidplexus cells in the presence of antigen alone, failed to showthe formation of activated centers. The activated centersobserved were quite similar in appearance to those seen whenperipheral spleen cell-derived macrophages were co-culturedwith lymphocytes in the presence of antigen (Fig. ic).Although associated with lymphocyte activation, aggre-

gate formation can also involve non-antigen-specific, non-genetically restricted factors (17). Therefore, to supply directevidence for lymphocyte proliferation, [3H]thymidine incor-poration was measured. Fig. 2A demonstrates a markedincrease in isotope incorporation when choroid cells andlymphocytes were co-cultured in the presence of variousconcentrations of ovalbumin. The maximal response toantigen resulted in a >100-fold increase in incorporation, andthe dose of ovalbumin causing 50% maximal activation ofproliferation (EC50) was about 5 ug/ml. Under identicalconditions, 03 lymphocytes co-cultured with irradiatedBALB/c spleen cells showed a similar maximal increase inincorporation with an EC50 of 0.5 JUg/ml. In the absence ofeither choroid epithelial cells or T cells, addition of ovalbu-min caused no significant stimulation of thymidine incorpo-ration (Fig. 2A). Also, in an experiment in which bovineserum albumin was used as antigen instead of chickenovalbumin, stimulation of 03 proliferation was not seen.

In a choroid plexus cell dose-response experiment, inwhich T-cell number (5 x 104 cells) and antigen dose (300gg/ml) were held constant, we found that choroid plexuscells were highly efficient in presenting antigen, fewer than103 cells producing a detectable proliferative response. Thislow presenting-cell/T-cell ratio of 1:50 makes it highlyunlikely that the proliferative response observed was due toa nonspecific effect related to a high cell density created bythe choroid cells. Inability of 03 to proliferate in response tocell density alone is also supported by other observationsshowing that 03 lymphocytes, in the presence of antigen,cannot be stimulated even by large numbers of other celltypes, such as fibroblasts, that lack Ia on their surface (A.Rao, personal communication).A variety of evidence indicated that the proliferative

response observed was not due to the presence of blood- orependymal-derived macrophages or endothelial cells con-taminating the isolated choroid cells. (i) Choroid epithelialcells initially >95% pure (see below) were isolated by afractionation procedure that removed only the epithelium,

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leaving behind the connective tissue and vascular compo-nents that are the largest potential source of contaminatingmacrophages. (ii) Prior to use, the isolated cells were furtherpurified by an adherence separation in which choroid epi-thelial cells, which do not adhere to tissue culture plastic,were separated from contaminating macrophages and endo-thelial cells, which do adhere. This resulted in a histologicallyhomogenous population of cells >99.9% positive for thechoroid epithelial cell markers described below. (iii) Inhistochemical studies, <0.1% of the purified epithelial cellsstained with DiI-Ac-LDL, a marker that is avidly taken up bymacrophages and vascular endothelium (18). More impor-tantly, as shown in Fig. ld, no DiI-Ac-LDL-positive cellswere observed in the aggregates present in the choroid-03co-cultures, in marked contrast to aggregates present inspleen cell-lymphocyte co-cultures, which showed numer-ous DiI-Ac-LDL-labeled macrophages (Fig. if). (iv) Anti-body to membrane attack complex type 1 (MAC-1), a surfacemarker directed against an epitope present on murine mac-rophages, also failed to stain any choroid epithelial cells, incontrast to positive MAC-1 staining that we obtained withspleen-derived macrophages. (As with choroid plexus cells,the 03 line itself was free of contaminating macrophages, asindicated by negative MAC-1 and DiI-Ac-LDL staining.)Finally, in other immunohistochemical studies, antibody toglial fibrillary acidic protein (an astroglial marker) did notlabel any cells present in choroid-03 activated centers.

Utilizing a different approach, and to provide additionalevidence for cell purity, we employed two cell markers tospecifically label choroid epithelial cells. (i) Polyclonal anti-body specific for the a form of Na,K-ATPase, an enzymepresent in very high concentration in choroid plexus andother transporting epithelium (11, 19), brightly labeled nu-merous epithelial cells present in the choroid-03 aggregates(Fig. ig). At the antibody dilution used, all other cells in thechoroid, as well as macrophages and endothelial cells,stained only very faintly since they contained much lowerconcentrations of Na,K-ATPase. Also, the antibody onlyfaintly labeled cells present in spleen cell-03 aggregates (Fig.ii). (ii) In other experiments (Fig. 3a), epithelial cells labeledbrightly with monoclonal antibodies to DARPP-32, a proteinpresent in the choroid plexus but not detected in macro-phages or endothelial cells (13, 20). Likewise, in co-culturestudies, antibodies to DARPP-32 labeled cells in choroid-03

aggregates but did not label cells in spleen cell-03 aggregates(data not shown).

In the periphery, antigen-presenting cells, such as macro-phages, need to express class II histocompatibility glycopro-teins (Ia antigen) on their surface to present antigen to T cells.Therefore, to determine if Ia might be involved in choroid-stimulated activation of T cells, we attempted to blockchoroid-stimulated T-cell proliferation by adding a monoclo-nal antibody (34-5-3S) that recognizes mouse I-Ad (15) to theco-cultures. Fig. 2B shows that increasing the amounts ofanti-Ia resulted in a progressive loss of choroid-stimulatedT-cell activation. (In another experiment, similar amounts of34-5-3S were required to inhibit antigen presentation byspleen cells.) This experiment, indicating the presence of Ia,was consistent with an immunohistochemical study of IFN-t-stimulated choroid plexus in organ culture (Fig. 4), in whichIa antigen staining was observed on a number of (but not all)epithelial cells that had been identified by their co-labelingwith Na,K-ATPase. In the absence of IFN-y, little stainingwas seen. [Presumably, in situ, stimulation of Ia expressionon epithelium would occur through interaction with T cellspassing through the choroid or as a result of contact withvirus particles, independent of IFN-y, as has been demon-strated for astrocytes (8).]

Consistent with major histocompatibility complex (MHC)restriction requirements, monoclonal antibody (14-4-4S),which recognizes I-Ed (16), failed to block proliferation of 03cells by choroid plexus cells (Table 1). Additional evidencethat the antigen-presenting capability of choroid plexus cellsis MHC-restricted was obtained by using another T-lymphocyte clone, BC4, reactive to bovine insulin and ofMHC type H-2b (21). When choroid cells from BALB/c(H02d) were co-cultured with BC4 T cells in the presence ofantigen, no stimulation occurred, in contrast to the stimula-tion that occurred when BC4 was co-cultured with macro-phage-containing spleen cells from a B10 (H-2b) mouse (Table1). Such results, plus the above data, strongly suggest thatchoroid plexus cells are capable of specific activation ofperipheral T cells in vitro.We next wished to determine whether, in vivo, choroid

plexus cells have access to particulate antigens entering theCSF at some distance from the choroid and whether suchantigens could approach the choroid and be taken up by theepithelial cells. Accordingly, we injected, into the lateralventricle of a rabbit, virus-sized (100 nm in diameter) fluo-rescent noncarboxylated polystyrene microspheres. Eigh-teen hours later, the fourth ventricular choroid was removedand washed extensively, and the epithelium was isolated. The

a

b

FIG. 3. Ability of identified fourth ventricular choroid epithelialcells to ingest virus-sized fluorescent particles injected into the CSFof the lateral ventricle. (a) Group of epithelial cells fixed andimmunostained for the epithelial cell protein DARPP-32 (rhodamine-selective optics). (b) The same epithelial cells show bright fluores-cence due to the presence of numerous microspheres (fluorescein-selective optics). (c) Another group of epithelial cells is immuno-stained for the a isozyme of Na,K-ATPase (rhodamine-selectiveoptics). (d) Outline of nuclei by ingested microspheres indicates anintracellular localization of particles (fluorescein-selective optics).

C

FIG. 4. Ia expression on epithelial cellsof organ-cultured mouse choroid plexus.After 42 hr of culturing in the presence ofIFN-y, a small piece of intact choroid isshown by phase-contrast microscopy (a),after staining with monoclonal antibody 34-5-3S, which recognizes I-Ad (b), and afterstaining by polyclonal antisera to the a formof Na,K-ATPase (c). Note the loop of epi-thelial cells (top right of choroid) staining forboth Ia and Na,K-ATPase surrounding a(possibly vascular) cell that fails to stain.Other positive cells can also be seen.

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Table 1. MHC restriction of choroid epithelial cellantigen presentation

[3H]ThymidineExperimental components uptake, cpmCP [H-2d] + 03 [H-2d] 1,860 ± 220CP + 03 + OV 47,170 ± 320CP + 03 + OV + anti-I-Ad 5,260 ± 620CP + 03 + OV + anti-I-Ed 46,510 ± 950CP + BC4 [H-2b] 2,440 ± 200CP + BC4 + Ins 2,040 ± 210Spleen [H-2b] + BC4 + Ins 64,680 ± 1000

BALB/c choroid plexus epithelial cells (CP) were co-cultured withT-lymphocyte clone 03, reactive to chick ovalbumin (OV), or withT-lymphocyte clone BC4, reactive to insulin (Ins). Stimulation wasobserved with choroid plexus and 03 cells, which are MHC-matched, whereas no activation was seen with choroid plexus andBC4 cells, which differ in MHC. Furthermore, activation of 03 cellsby choroid plexus was blocked by anti-Ia antibody directed againstI-Ad but not I-Ed. Dilutions of antibodies used (1:12.5 and 1:50,respectively) were determined experimentally to maximally inhibitantigen presentation in their respective MHC cell types. Ovalbuminwas added at 100 ,gg/ml and insulin was at 50 ,ug/ml. Values shownare the mean ± SEM for triplicate determinations.

cells were labeled with antibody to DARPP-32 or to Na,K-ATPase (to identify the choroid epithelium) (Fig. 3 a and c)and then examined for the presence of fluorescent micro-spheres. Fig. 3b shows that many cells [47 ± 6% (mean +SEM) of all DARPP-32-positive epithelial cells] showed thepresence of microspheres. In some cells (Fig. 3d), labelingwas so intense that the outline of the cell nucleus could bevisualized, strongly suggesting an intracellular localization ofthe microspheres. These results indicate that, in vivo, thechoroid epithelium has access to particulate material intro-duced elsewhere into the CSF and, despite the large rate offluid production from its apical surface, the epithelium iscapable of taking up this material intracellularly. Choroidepithelium is also known to be capable of absorbing solubleproteins from the CSF (22), and other studies have shownthat ventricular ependymal cells, from which choroid epithe-lium is derived, have specific receptors for several viruses(23).In vivo, it is known that peripheral T cells can pe w. -rate the

fenestrated choroid capillaries, thereby gaining po-tentialaccess to the choroid epithelium (24, 25). Of inter-est. theseepithelial cells also contain large concentrations cf ?c ecep-tors on their surface (26). Given the results of our stu(ies, itis conceivable that in vivo the choroid plexus ccold sake upantigens from the CSF and present them to peripheral -i cells,thereby functioning as a type of immunological commu-nica-tions link between the CSF and blood. Such activatedlymphocytes might function peripherally or, at so.nme latertime, enter the CNS through the cerebral vasculatlre. (Suchactivation of T cells could be an important factor- gye .rningthe ability of a lymphocyte to cross the blood-borna ,arrier,thereby providing a mechanism by which the Inrain canrestrict lymphocyte traffic only to "relevant" T c+AHs.)The present results have implications for the initiation of

immunological responses in CNS infections and autoimmuneprocesses and for the surveillance of tumor cells in the CSF.Because substances present in brain parenchyma are knownto enter the CSF, the choroid has potential access to antigenspresent not only exclusively in the CSF but also arising fromelsewhere in the CNS outside the ventricular system (in-cluding, e.g., antigens coming from brain parenchymal in-fections). Our findings also have implications for the recog-nition of certain systemic viruses, parasites, and otherantigens (e.g., lymphocytic choriomeningitis virus and try-panosomes) that may enter the CSF through, or proliferatewithin, the choroid plexus (27-30). While our experiments

have focused on the involvement of choroid epithelium inantigen presentation, these studies do not rule out thepossibility that other choroid plexus cell types, such asendothelium and epiplexus cells, might also have immuno-logical functions. We have found, for example, that, inunpurified, intact choroid plexus organ culture, exogenousIFN-y can stimulate Ia expression on these latter cells as wellas on the epithelium. Epiplexus cells have also been observedto take up microspheres. Finally, our experiments do not ruleout the possibility that, in addition to the choroid, otherroutes of immunological communication between CNS andperiphery may also exist, such as the possible escape ofantigens from the CSF into the venous system by way of thearachnoid granulations, the exit of some CSF into nasallymphatics (31, 32), or communication through the endothe-lial cells of the blood-brain barrier (33, 34) or throughcircumventricular organs that lack a blood-brain barrier.

We thank A. Rao and S. R. Abromson-Leeman for the helperT-cell clones, for the monoclonal antibodies to I-Ad and I-Ed, and formany helpful discussions. We also thank K. Sweadner for theantibody to the a form of Na,K-ATPase, P. Greengard for theantibody to DARPP-32, and W. Schwartz and E. Amento for advice.This work was supported by Public Health Service Grants NS16356and EY5077 to J.A.N., NS21269 to L.L.Y.C., and the KatharineDaniels Research Fund.

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Proc. Natl. Acad. Sci. USA 86 (1989)

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