Antigen presentation by rat brain and retinal endothelial cells

Journal of Neuroimmunology Journal of Neuroimmunology

ELSEVIER

61 (1995) 23 1-239

Antigen presentation by rat brain and retinal endothelial cells Y’. Wang, V.L. Calder, S.L. Lightman, J. Greenwood Department

*

ofClinical Science, Institute ofOphthalmology, Bath Street, London, EC1 V 9EL, UK Received

17 April 1995; revised 7 June 1995; accepted 7 June 1995

Abstract Brain and retinal endothelial cells (EC) form the blood-brain and vascular blood-retinal barriers, respectively, and are believed to play a role in mediating T cell responses in the central nervous system. In this study, Lewis rat retinal and brain EC grown in vitro were capable of expressing MHC class II I-A but not I-E molecules following treatment with interferon-y. In the presence of their antigen, CD4+ antigen-specific T cells were able to mediate lysis of retinal EC monolayers to a similar extent as brain EC. T cell proliferation was poorly supported by confluent retinal or brain EC monolayers, but subconfluent EC monolayers supported proliferation in a MHC class II (I-A)-restricted manner (P < 0.001). Exposure of T cells to confluent retinal EC monolayers resulted in them becoming less responsive to subsequent antigen presentation by thymocytes. Conversely, pre-exposure with subconfluent EC had no such effect. These results suggest that a non-proliferating EC monolayer is able to downregulate T cell responsiveness which may have important implications during lymphocyte traffic across the blood-tissue barriers of the central nervous system. Keywords:

Antigen presentation;

Elk&-brain

barrier; Blood-retinal barrier; Endothelium; Lymphocytes

1. Introduction

Activation of CD4+ T cells by antigen involves the presence of a variety of molecules on the antigen-presenting cell (APC). Expression of the major histocompatibility complex (MHC) class II molecules and other co-stimulatory factors are normally restricted to professional ARC. However, under certain inflammatory conditions, MHC class II molecules can be induced on a wide variety of diverse cell types, suggesting that these cells might also be able to present antigen providing that the co-stimulatory factors are present (Nickoloff and Turka, 1994). Since vascular endothelial cells (EC) occur at the blood-tissue interface, their potential role as ARC has been widely investigated (Hughes et al., 1990). The retinal vascular EC {that form the anterior aspect of the blood-retinal barrier (BRB) are structurally and functionally identical to those of the blood-brain barrier (BBB). These cellular barriers, which restrict the transvascular movement of circulating mmolecules and cells, are situated

Corresponding author. Phone +44 (171) 608 6858; Fax +44 (171) 608 6810. l

0165-5728/95/$09.50 Q 1995 Elsevier Science B.V. All rights reserved SSDI 0165-5728(95)00096-8

at a critical interface in the communication between the CNS parenchyma and the immune system. In addition to this structural barrier, both brain and retinal endothelia have been found to be poor at binding lymphocytes (Hughes et al., 1988; Male et al., 1990; Wang et al., 1993) and under normal conditions lack MHC class II as detected immunocytochemically, which together leads to the relative isolation of the CNS from the immune system. However, lymphocyte infiltration of the brain and retina does occur in conditions such as multiple sclerosis (MS) and posterior uveitis. In the animal models of these diseases, experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveoretinitis (EAU), lymphocytic infiltration into the parenchyma (Raine et al., 1990; Greenwood et al., 1994) and enhanced class II expression have also been reported (Fontana et al., 1987; Lightman, 1987). Despite brain and retinal EC being in an ideal anatomical location, the question of whether they are capable of acting as ARC in vivo remains unclear. Antigen presentation by endothelium in vitro has been reported in several studies. It has been shown that human umbilical vein endothelial cells (HUVEC) can be induced to express MHC class II molecules by the cytokine interferon (IFN)-y and are able to present antigens to either

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of Neuroimmunology 61 (1995) 231-239

allogeneic or syngeneic T cells resulting in T cell activation and proliferation (Pober et al., 1983). Brain endothelia have also been shown to express MHC class II molecules (Male et al., 1987) support CD4’ T cell-mediated cytotoxicity (Sedgwick et al., 1990) but cause only minimal T cell proliferation (Pryce et al., 1989; Risau et al., 1990). In contrast to these results, others have shown that brain EC can present antigens to antigen-specific T cells resulting in a significant degree of T cell proliferation (McCarron et al., 1985; Wilcox et al., 1989). Explanations for such discrepancies include species differences and contamination of the culture system with APC. Despite this there still remains uncertainty over the role of brain and retinal EC in T cell stimulation during the development of MS, posterior uveitis and their experimental counterparts. In this study we have investigated the antigen-presenting capacity of EC cultures derived from two sites within the Lewis rat CNS: the cerebral cortex and retina. The expression of MHC ciass II molecules, the effect of EC number and their state of activation on T cell proliferation was determined.

2. Materials

and methods

2.1. Animals Specific pathogen-free 4-6-week-old female Lewis rats (Charles River Ltd., Kent, UK) were used throughout the experiments. 2.2. Endothelium Both retinal and brain EC were isolated and cultured according to previously described methods (Hughes and Lantos, 1986; Abbott et al., 1992; Greenwood, 1992) and which routinely produce primary cultures of > 95% purity as determined by their morphological characteristics and immunohistochemical profile. Briefly, rat retina or chopped cerebral cortex was dispersed by enzymic digestion and microvessel fragments separated from other material and single cells by density-dependent centrifugation. The microvessel fragments were washed and plated out in medium into collagen (type 1, Sigma, Dorset, UK)_coated plastic tissue culture plates and flasks. The cultures were maintained at 37°C in 5% CO, and medium replaced every 3 days until confluent monolayers had formed. By confluence, the EC had reached 2 10’ cells/well. To obtain single-cell suspensions for replating or flow endothelial monolayeri were washed in cytometry, Mg2+/Ca2’-free Hanks’ balanced salt solution (HBSS; Gibco, Paisley, UK) containing 0.02% ethylene diamine tetra-acetic acid disodium salt (EDTA; BDH, Leicestershire, UK) followed by enzymatic dissociation with collagenase/dispase (0.1%; Boehringer-Mannheim, Sussex, UK) for 1 h. The single cells prepared by this procedure exhibited > 80% viability by Trypan blue staining.

2.3. S-Ag-specific CD4 ’ T cell lines Lewis rat T cell lines specific for purified bovine retinal soluble antigen (S-Ag) were prepared as described in detail elsewhere (Sedgwick et al., 1989). Briefly, lymph nodes were collected from S-Ag-immunized rats and the lymphocytes propagated by periodically alternating antigen activation with IL-2 stimulation. The cell lines express the marker of the CD4+ T cell subset, are CD45Rc”‘” and recognize S-Ag in the context of MHC class II determinants. Prior to the antigen presentation assay, cell lines were maintained in IL-2 medium (IL-2 containing supernatant from IL-2 cDNA transfected hybridoma cells; Karasuyama and Melchers, 1988) for 10 days to obtain relatively ‘rested’ T cells. 2.4. Flow cytometry Retinal or brain EC cultures were washed three times with HBSS and treated for 18 h, 3 days and 5 days with 200 U/ml rat recombinant IFN-7 (Holland Biotechnology b.v., Leiden, Netherlands) in fresh culture medium in the absence of endothelial cell growth supplement (ECGS; Sigma, Dorset, UK). Control cells received no treatment. After washing three times, the monolayer was dissociated and the cells were resuspended in phosphate-buffered saline. 5 x lo4 cells per vial were incubated on ice for 1 h with monoclonal antibodies (mAbs) to MHC class I (OX18), MHC class II I-A (0X-6) and MHC class II I-E (0X-17). The cells were then incubated for a further 1 h with fluorescein isothiocyanate conjugated rabbit antimouse Ig F(ab’), antibody (FITC-RAMIG) in the presence of 20% normal rat serum. After washing twice, cells were resuspended in phosphate-buffered saline and used for analysis by flow cytometry (FACScan Plus, Becton-Dickinson, Oxford, UK). Unstained cells were used to set the parameters, and cells stained with FITC-RAMIG alone were used to set background control. Splenic non-adherent cells served as positive controls for OX-6 (I-A) and OX-17 (I-E) staining. These cells were prepared from splenic mononuclear cell suspensions after removal of adherent cells by 2 h incubation on plastic Petri dishes (Gibco, Paisley, UK). The fluorescence staining procedures and flow cytometric analyses were carried out as described above. The mAbs OX-6 and OX-17 were used as supernatants from cultured hybridoma cell lines which were a generous gift of Dr. M. Puklavec (MRC, Oxford, UK). The OX-18 mAb and the FITC-RAMIG, which was affinity-purified and non-crossreactive with rat, were obtained from Serotec (Oxford, UK). 2.5. Preparation of antigen-presenting cells Enzymatically dissociated retinal or brain EC (resting or pretreated for 3 days with 200 U/ml of IFN-y) were

Y. Wanget al./ Joumul of Neuroimmumlogy61 (1995) 231-239

seeded onto collagen-coated 96-well plates at concentrations of between 5 x lo3 and 1 X 10’ cells/well and incubated overnight in endothelial cell culture medium. These cells were then washed to remove non-adherent cells prior to the proliferation assay. Thymocytes were prepared from 4-6-week-old Lewis rats by passing the thymus through a fine wire mesh and resuspending the resulting single cells in RPM1 medium containing 10% FCS (both from Sigma, Dorset, UK). The cells were then finally resuspended in the same medium and exposed to 2000 Rads yradiation. Splenic adherent cells (enriched macrophages) were prepared from a splenic mononuclear cell suspension after removal of the non-adherent cells by 2 h incubation on plastic Petri dishes (Gibco, Paisley, UK). 2.6. Cytotoxicity assay The quantitative assay ‘of cytotoxicity was carried out according to a previously described method (Risau et al., 1990). Confluent retinal EC grown in 96-well plates were incubated for 3 days with 200 U/ml IFN-7 prior to the assay. To each well was adided 0.1 &i of 51Cr (Amersham International, Bucks, UK) in 50 ~1 of fresh medium (without ECGS) and incubated at 37°C for 18 h. The cells were then washed with HBSS and 5 X 10’ S-Ag-specific CD4+ T cells per well were added in 200 ~1 of medium (10% FCS in RPM compllete medium) in the presence of 10 pg/ml S-Ag or 10 pg/ml BSA. After 18 h incubation, the co-culture supematants were taken and the released 51Cr measured on a y-emission counter. Maximal release of 5’Cr was obtained by lysis of 5’Cr-labelled retinal EC with 10% sodium dodecyl suifate (SDS: Sigma, Dorset, UK). The specific release of 5’Cr was calculated by the following formula: Specific release (%) =

cpm of experiments -- cpm of spontaneous release maximal possible release x 100.

2.7. Proliferation assay The T cell proliferation assay was performed in 96-well flat-bottom plates. 2 X 10” S-Ag-specific T cell line lymphocytes in 200 ~1 were added to each well into which previously determined nurnbers of retinal or brain EC had been added. The cells were then co-cultured in serum-free (Nutridoma-SP, BCL, Sussex, UK) conditioned RPM1 medium. The wells were treated with 5-20 Fg/ml of S-Ag or the mitogens conc:anavalin A (ConA; 5 pg/ml) or phytohaemagglutinin (PHA; 10 pg/ml; both from Sigma, UK). In addition, 5 pg,/ml indomethacin (Sigma) was added to prevent eicosanoid production by endothelium

233

which is known to suppress lymphocyte division. After 3 days of co-culture in 5% CO, at 37”C, IL-2 was added and 8 h prior to the termination of the 5-day proliferation assay the wells were pulsed with 1 &i [3H]thymidine (Amersham International, Bucks., UK). The cells were then harvested onto nitrocellulose membranes with a cell harvester (Dynatech, Sussex, UK) and the radioactivity was determined by P-scintillation counting (Tricarb, Canberra-Packard, Berks., UK). The results are expressed as the means f S.E.M. of a minimum of 12 wells. To investigate the effect of pretreating the EC with IFN-y upon T cell proliferation, retinal EC were incubated with 200 U/ml IFN-y for 3 days prior to the proliferation assay. To investigate whether proliferation was MHC class II-restricted, wells containing 5 X lo4 retinal EC were treated with 10% of either anti-I-A (0X-6) or anti-I-E (0X-17) mAb. The T cells and EC were then co-cultured in the presence of 10 pg/ml S-Ag and the proliferation assay performed as described above. Stimulation of S-Ag-specific T cells by professional APC was carried out in an identical manner to that described for EC, except that 1 X lo6 thymocytes per well were co-cultured with 2 X lo4 S-Ag-specific T cell line lymphocytes in the presence of different concentrations of S-Ag (5-20 pg/ml). Thymocytes were chosen as they have been shown to be superior to rat splenocytes at supporting rat T cell proliferation (Sedgwick et al., 1989). To confirm this, splenic adherent cells (5 X lo4 cells/well) were also used in a proliferation assay. Proliferation was measured by [ 3Hlthymidine incorporation as described above. To determine whether T cells pre-incubated with EC affects their subsequent proliferative response to thymocyte antigen presentation, the following protocol was employed. Retinal EC were incubated with IFN-y (200 U/ml) for 3 days in culture medium without ECGS after which they were enzymatically dissociated with collagenase as described above. The EC were re-plated into 96-well plates at either 5 X lo4 or 1 X lo5 cells/well and left overnight to produce subconfluent and confluent monolayers, respectively. S-Ag-specific T cells were then added at 2 X lo4 cells/well to sub-confluent EC, confluent EC or empty wells in the presence of 10 pg/ml S-Ag and incubated for 3 days in serum-free media as outlined above. The T cells were then removed from the wells containing EC by mechanical disruption of the EC monolayers. The T cells were then counted and added to thymocytes in non-coated wells (in which the EC are non-viable) for the proliferation assay described above. 2.8. Statistical analysis The results are expressed as the mean It standard error of the mean (S.E.M.) or standard deviation (S.D.) as indicated. Significant differences between groups were assessed using Student’s t-test.

Y. Wang et al. / Jnurnal of Neuroimmunology 61 (1995) 231-239

234

OX-6

ox-17

C

0 days

l’s h

3 days

5 days

0 days

18 h

3 days

5 days

OX-18

OX-6

L L

1’1

1

RAMIG

1”

Y. Wang et al. / Journal of Neuroimmunology 61 (1995) 231-239

3. Results 3.1. MffC expression on retinal and brain EC Under normal conditions, the majority of retinal and brain EC were found to express MHC class I molecules (90.4 &-5.3% and 83.2 f 7.:3%, respectively; mean f SD.). Treatment of these EC with 200 U/ml IFN-y for 18 h did not significantly affect the percentage of class I-positive cells as most were already positive but by 5 days there was a marked increase in the intensity of expression (Fig. 1). In contrast, resting retinal and brain EC were found to be negative for MHC class II molecules (both I-A and I-E). A significant expression of the I-A molecule was observed in retinal (23.4 f 4.3%; P < 0.001) and brain EC (13.3 f 4.7%; P < 0.001) 3 days after treatment with IFN-y, with a further significant increase in retinal EC expression by day 5 (42.9 f 6.9%; P < 0.001). With brain EC, the further increase in expression of class II I-A molecules from days 3 to 5 was small (19.1 f 3.8%), remaining significantly lower than with retinal EC (P < 0.05). The expression of class II I-E molecules on brain and retinal EC was low (< 5%; Fig. 1) with only a small but insignificant increase in expression on brain EC following 5 days of treatment with IFN-y. No differences in the levels of MHC expression were observed between confluent and subconfluent monolayers. In parallel experiments, freshly isolated splenic non-adherent cells, which served as a positive control, were found to express both I-A (47.1 f 5.8%) and I-E molecules (24.7 f 4.3%). 3.2. T cell lysis of EC monolayers Observations made by phase-contrast light microscopy (not shown) revealed substantial damage to the EC monolayers. The quantitative cytotoxicity assay revealed that after 18 h of co-culture there was a significant degree of EC lysis as demonstrated by the release of approximately 40% 5’Cr from the labelled EC. This occurred when either S-Ag or ConA was present in the co-culture system and in each case the level of release was significantly higher than in the absence of antigen (P < 0.01). The presence of non-specific antigen (BSA) did not cause any additional cytotoxicity to retinal EC monolayers over that found in the S-Ag free controls (P > 0.05, Fig. 2.). 3.3. Endothelial cell support of T cell proliferation The ability of both brain and retinal EC to stimulate S-Ag-specific syngeneic CD4+ T cell proliferation was

235

60 1

a

0”

u

Fig. 2. Cytotoxic effects of S-Ag-specific T cells on retinal EC monolayers. “Cr release was measured following co-culture of confluent retinal EC monolayers with S-Ag-specific T cells in the absence of antigen (control), and in the presence of S-Ag (10 pg/mI), ConA (5 @g/ml) and an irrelevant antigen (BSA, 10 pg/ml).

investigated. IFN-y-activated ietinal and brain EC were able to stimulate T cell proliferation in the presence of S-Ag under conditions in which the EC were sub-confluent (Fig. 3). The m agnitude of T cell proliferation depended on the number of EC per well with low numbers or confluent monolayers being poor stimulators of T cell proliferation. Maximal T cell proliferation was observed with EC numbers of 5 X lo4 (approx. 50% of the confluent number), a T cell to EC ratio of 2:5. At this ratio, [ 3H]thymidine incorporation was significantly higher than those of the controls (P < 0.001) or with low numbers of EC (5 X 103, P < 0.001) or at confluence (> 105, P < 0.001) EC pre-treated for 3 days with IFN-y and used at the optimal ratio as described above brought about a significant proliferation of lymphocytes that was antigen-dependent (Fig. 4) with 20 pg/ml S-Ag leading to a six-fold increase in T cell proliferation. The mitogens ConA and PHA were also found to induce a comparable T cell response (Fig. 4). The ability of non-treated retinal EC to support T cell proliferation was investigated and it was found that these cells could also stimulate S-Ag-specific CD4+ T cell

Fig. 1. Flow cytometric analysis of MHC antigen expression on retinal (A) and brain (B) EC following treatment for O-5 days with 200 U/ml IFN-y. Cells were stained with mAb against MHC class I (0X-18), MHC class 11 I-A (0X-6) and class II I-E (0X-17). FITC-RAMIG controls are shown in each case (dotted line). Untreated splenic non-adherent cells (Cl served as a positive control.

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22 20

0

5

S-Ag concentration EC concentration

(x~O-~J

Fig. 3. Antigen presentation to S-Ag-specific T cells by retinal (hatched bars) and brain (solid bars) EC. Both confluent EC monolayers (Con0 and a range of EC concentrations plated onto 96-well plates were co-cultured for 5 days with 2 X lo4 S-Ag-specific T cells per well in the presence of 10 pg/ml S-Ag. Thymidine incorporation into subconfluent EC (5 x lo4 cells/well) in the absence of T cells was also determined (no T cells). T cell proliferation was measured by [ 3H]thymidine incorporation (cpm rt:S.E.M.).

I

TT

T

t

10

20

[kg/ml]

Fig. 5. Support of T cell proliferation by retinal EC (cross-hatched bars), brain EC (shaded bars), splenic adherent cells (hatched bars) and thymocytes (solid bars). T cells (2X lo4 cells/well) were co-cultured for 5 days with subconfluent brain or retinal EC (5 X lo4 cells/well), splenic adherent cells (5 X IO4 cells/well) or thymocytes (1X IO6 cells/well) in the presence of S-Ag (5-20 pg/ml). T cell proliferation was measured by [‘Hlthymidine incorporation (cpm f S.E.M.).

proliferation in the presence of either S-Ag or mitogens to the same degree as IFN-y-treated endothelia (Fig. 4). In those experiments in which an optimal number of irradiated thymocytes were used as APC, T cell proliferation was significantly higher than with retina1 or brain EC at all concentrations of S-Ag (P < O.OOl), giving a ten-fold proliferative response with 20 Fg/ml of S-Ag (Fig. 5). Thymocytes were also found to be significantly better at supporting T cell proliferation than splenic adherent cells (Fig. 5). 3.4. MHC class II restriction The antibodies to rat class II I-A (0X-6) and I-E (0X-17), which have previously been shown to inhibit functional recognition by T cells (Matsumoto et al., 19921, were added to co-cultures to determine the role of these molecules in EC-induced T cell proliferation. Treatment of the EC with OX-6 was found to significantly inhibit S-Ag-specific T cell proliferation on both non-activated or IFN-y-treated retinal EC (Fig. 6). In contrast, OX- I7 failed to block the induction of T cell proliferation.

Fig. 4. Antigen presentation to S-Ag-specific T cells by testing (hatched bars) and IFN-+reated (3 days pm.-treatment with 200 U/ml IFN-y) retinal EC (solid bars). T cells (2 X lo4 cells/well) were co-cultured with retinal EC (5 X lo4 cells/well) in the presence of either S-Ag (5-20 ~g/ml) or the mitogens PHA (10 pg/ml) and ConA (5 pg/ml). T cell proliferation was measured by 13H]thymidine incorporation (cpm f S.E.M.).

3.5. Effect of EC/T cell co-culture cyte-induced T cell proliferation

on subsequent

thymo-

S-Ag-specific T cell lines co-cultured with subconfluent retinal EC monolayers or cultured on their own for 3 days were able to respond to subsequent antigen presentation by

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4. Discussion

+ve Control

OX-6 (I-A)

I 0

8 1000

20oC1 3000

3H-thymidiine

4000

5000

incorporation

6000

7000

(cpm)

Fig. 6. The effect of anti-MHC class II mAbs on resting (solid bars) and IFN-y-activated (hatched bars) retinal EC mediated S-Ag-specific T cell proliferation measured by 13Hkhymidine incorporation (cpmf S.E.M.). The negative control was carried out in the absence and the positive control in the presence of S-Ag. * Significant inhibition from positive control (P < 0.001).

thymocytes to almost the same degree (Fig. 7). Pre-exposure of the T cells to a confluent monolayer of retinal EC, however, caused a pronounced and significant attenuation in response to a subsequent antigenic challenge in the presence of thymocytes (Fig. 7). The viability of the T cells following pre-exposure to confluent EC monolayers was checked by Trypan blue exclusion and found to be greater than 90%. The addition of supernatants (at 10% dilution) from both confluent and subconfluent retinal EC monolayers failed to elicit any effect upon thymocyte-mediated T cell proliferation (data not shown).

‘10 0 5 S-Ag concentrations

20 [ug/ml]

Fig. 7. The effect of pre-culturing S-Ag-specific T cells on theii own (shaded bars) or with confluent (hatched bars) or subconfluent (solid bars) retinal EC for 3 days in thle presence of antigen prior to culturing with professional APC (thymocytes). T cell proliferation was measured by [sH]thymidine incorporation tcpm * S.E.M.).

Previous investigations into the pathogenesis of immune-mediated diseases of the CNS have indicated that cellular interactions between immune cells and cells of the CNS are important in the induction and perpetuation of CNS inflammatory diseases (Fabry et al., 1994). Amongst these interactions antigen presentation within the CNS is thought to be crucial in the initiation of T cell-mediated inflammation (Fontana et al., 1987). Despite their ideal location at the blood-tissue interface it remains unclear whether vascular endothelial cells of the CNS can act as antigen-presenting cells. Numerous in vitro studies have failed to elicit any significant T cell response to EC-mediated antigen presentation from confluent monolayers (Pryce et al., 1989; Risau et al., 1990). In this study, we have demonstrated that both retinal and brain EC grown to confluence are unable to support T cell proliferation. Furthermore, such conditions are able to render T cells non-responsive to subsequent antigen presentation by professional APC. In contrast to this, subconfluent monolayers of CNS derived EC are capable of presenting antigen to CD4+ T cells, inducing a low but significant degree of T cell proliferation. Expression of MHC class II molecules is one of the requirements for antigen presentation to CD4+ T cells. Both retinal and brain EC were induced to express significant levels of I-A but not I-E molecules following IFN-y stimulation. The lack of I-E expression was not due to low binding affinity of the antibody, as we were able to detect I-E expression with the same antibody on rat splenic adherent cells. The levels of MHC expression are similar to those found in rat brain endothelia (Male et al., 1987). As in other models (Rao et al., 1989; Risau et al., 1990), we have shown that it is MHC class II I-A, but not I-E, that is required by the CNS EC to induce a CD4+ T cell response and antigen-specific T cell proliferation. Furthermore, these retinal EC can support effector CD4+ T cell functions, including modest cytotoxicity of EC monolayers to the same degree as shown for brain endothelia (Sedgwick et al., 1990; Risau et al., 1990). In all of these studies, a single time-point (18 h) was examined, to allow for both Th-1 and Th-Zmediated cytotoxity to occur (Tite, 1990). The results presented here demonstrate that only subconfluent retinal and brain EC can present antigen and induce proliferation to S-Ag-specific CD4+ T cells. Interestingly, using microglia as APC, it has been reported that T cell proliferation is also dependent on the ratio of T cells to microglia, with increasing numbers of microglia inhibiting proliferation (Matsumoto et al., 1992). The reason that confluent EC monolayers are poor stimulators of T cell proliferation is not yet clear and it remains to be seen whether this effect is due to the state of EC confluency or to the EC/T cell ratio. The poor support of T cell proliferation by confluent EC, however, may be due to either

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inhibitory factors produced by non-proliferating confluent cells or to the switching off of stimulatory factors. Eicosanoid production by EC is unlikely to be involved, since indomethacin did not improve T cell proliferation (data not shown). In contrast, retinal pigment epithelial (RPE) cells, which form the posterior BRB, also cause T cell immunosuppression which was attributed to PGE, since it could be overcome by indomethacin (Liversidge et al., 1993). Furthermore, this group have also implicated the production of NO by RPE as an additional immunosuppressive mechanism within the neuroretina (Liversidge et al., 1994) although its production by retinal EC has not yet been examined. Inhibitory cytokine production, such as TGF-P @hull et al., 1992), may play a downregulatory role. Interestingly, we have found that confluent retinal EC are able to produce significant levels of this cytokine (unpublished observations) and differential levels of production could explain the difference between subconfluent and confluent EC in supporting a T cell proliferative response. The induction of T cell proliferation by IFN-y-activated subconfluent retinal EC was shown to be class II I-A-restricted. However, EC that were not pre-treated with IFN-y were also able to induce a comparable level of proliferation over 5 days, which was inhibited by anti-class II I-A antibody, indicating the crucial role of class II in these co-cultures. This suggests that the low levels of IFN--y or other cytokines produced by these T cells is sufficient to upregulate class II expression on the untreated EC. In fact, these T cell lines produce increased levels of IFN-7 following activation with S-Ag or mitogens (Zhao et al., 1994). Other co-stimulatory molecules needed for activation are also likely to be induced by T cell-derived signals. For example, HUVEC have been shown to produce the co-stimulatory cytokines IL-l, IL-6 and TNF (Hughes et al., 1990) and we are currently examining the production of these factors by retinal EC. Both retinal and brain EC were not as proficient as professional APC in supporting T cell proliferation. This is likely to be due to either the presence of inhibitory cytokines or to the lack of important accessory molecules. In a recent study using cornea1 endothelial cells to induce T cell proliferation, a similar lack of induction was observed that was found to be due to a block at the level of IL-2 production. The effect was overcome by adding exogenous IL-2 (Kawashima et al., 1994). Although these results were obtained using non-vascular EC it is tempting to speculate that a similar process occurs in confluent retinal EC cultures. However, preliminary data have demonstrated that the addition of exogenous IL-2 given on day 3 of the proliferation assay (Chain et al., 1987) did not reverse the poor proliferation induced by confluent EC (unpublished observations). This suggests that in our system IL-2 responsiveness appears to be only partially involved. Recently, it has been shown that IL-12 can induce proliferation and cytokine production by activated human T cells,

61 (1995) 231-239

which is largely independent of IL-2 (Kubin et al., 1994). Thus, a lack of IL-12 or other such factors may explain the poor T cell responsiveness induced by confluent EC. By demonstrating that T cells previously exposed to confluent retinal EC were subsequently unable to respond to thymocyte stimulation suggests that the T cells might have become non-responsive. Recently, another group using an immortalized rat brain EC line (RBE4) have demonstrated a similar non-responsiveness in myelin basic protein-specific T cell lines which was reversible with IL-l/I but not IL-2, IL-4 or IL-6 (Bourdoulous et al., 1995). In preliminary investigations, we have been unable to reverse the EC-induced non-responsiveness by adding IL-2 (unpublished data). It remains to be seen, however, whether these T cells are truly anergic. To address this question, we are currently investigating the cytokine production of these T cells and whether this effect can be reversed. In contrast, the observation that subconfluent EC failed to inhibit thymocyte-induced T cell proliferation suggests that conditions for inducing non-responsiveness in the T cells can only be achieved with confluent EC cultures. In addition, it remains unclear whether this conferred ‘anergy’ requires the presence of antigen and whether it can be maintained in vivo, where the T cells are likely to be influenced by other cell types. We would suggest, therefore, that in vitro there is a line balance between the threshold number of EC needed to provide the necessary signals to induce T cell proliferation and the induction of a non-responsive state (anergy) produced by a non-dividing confluent EC monolayer. Whether these results can be extrapolated to the in vivo situation is unclear. If the vascular EC, the main component of the blood-CNS barrier, can induce a non-responsive state, this has important implications for migrating T cells in both the normal and immune-mediated diseases of the CNS. These T cells, which must be activated to cross the BBB and BRB (Hickey et al., 1991; Greenwood and Calder, 1993) may be subjected to signals from the EC, converting them to a non-responsive state as they enter the CNS parenchyma. Although this could occur at the luminal or abluminal side, the latter is more likely due to the persistence of T cells in the perivascular space following diapedesis. One may speculate, therefore, that a non-responsive state is induced at the normal barrier whereas in chronic inflammation this may be overcome by other factors. Further studies are clearly required to investigate this hypothesis in vivo.

References Abbott, N.J.. Hughes, C.C.W., Revest, P.A. and Greenwood, J. (1992) Development and chnracterisation of a rat brain capiky endothelial culture: towards an in vitro blood-brain banier. J. Cell Sci. 103, 23-31. Bourdoulous, S., Bkaud, E., Le Page, C., Zamora, A.J., Ferry, A.. Bernard, D., Strosberg, A.D. and Couraud, P.-O. (1995) Anergy

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