The potential role of dendritic cells in immune-mediated inflammatory diseases in the central nervous system

Pergamon

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Neuroscience Vol. 74, No. 2, pp. 599–608, 1996 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/96 $15.00+0.00 S0306-4522(96)00160-1

THE POTENTIAL ROLE OF DENDRITIC CELLS IN IMMUNE-MEDIATED INFLAMMATORY DISEASES IN THE CENTRAL NERVOUS SYSTEM M. K. MATYSZAK* and V. H. PERRY University Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K. Abstract––Dendritic cells of the rat were studied immunohistochemically with MRC OX62 monoclonal antibody and using electron microscopy. In normal CNS, a small number of OX62+ cells was detected in the choroid plexus and meninges. These cells were absent from other CNS and peripheral nervous system sites studied. Dendritic cells were also studied in two models of immune-mediated inflammatory conditions in the CNS. These were: acute experimental allergic encephalomyelitis and aberrant delayedtype hypersensitivity lesions induced as a response to heat-killed bacillus Calmette-Gue´rin sequestrated behind the blood–brain barrier. In addition, a group of animals with a delayed-type hypersensitivity response was treated with dexamethasone to assess the effect of steroid treatment on T-cells and OX62+ cells in CNS lesions. Dendritic cells were present in many but not all lesions in acute experimental allergic encephalomyelitis and their numbers were small. In experimental allergic encephalomyelitis lesions, dendritic cells were found predominantly in perivascular cuffs, where they constituted approximately 2% of the total number of major histocompatibility complex class II+ cells. Some of these cells were also detected in the CNS parenchyma, close to the perivascular cuff. In contrast, dendritic cells were present in all delayed-type hypersensitivity lesions studied. Their number in delayed-type hypersensitivity lesions was significantly higher than in experimental allergic encephalomyelitis lesions. Numerous OX62+ cells were found, even in three-month-old lesions. Electron microscopy studies revealed that these cells were often in close contact with lymphocytes. There was no significant change in the density of OX62+ cells, IL2R+ cells and OX19+ T-cells in delayed-type hypersensitivity lesions after seven-day treatment with dexamethasone, although there was a considerable reduction in the number of CD45RA+ T-cells. The high numbers of dendritic cells found in the delayed-type hypersensitivity lesions may be important in contributing to the chronicity of the response. They may also initiate autoimmune responses to CNS antigens uncovered during bystander tissue damage which occurs as a consequence of aberrant delayed-type hypersensitivity responses. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: dendritic cells, inflammatory diseases, CNS, experimental allergic encephalomyelitis, delayedtype hypersensitivity.

Dendritic cells (DCs) are a population of highly specialized antigen-presenting cells (APC) found not only in primary lymphoid organs but also in most non-lymphoid peripheral tissues.17,47 Dendritic cells that reside in the lymphoid organs originate from precursor cells distributed in non-lymphoid peripheral tissues39,44 and the blood.18,41,43 After acquiring an antigen, precursor DCs migrate out of the tissue and through the afferent lymphatics into the T-cell-rich regions of local lymphoid organs. In the steady state, there is a continuous low level of *To whom correspondence should be addressed. Abbreviations: APC, antigen presenting cell; BBB, blood– brain barrier; BCG, bacillus Calmette-Gue´rin; DC, dendritic cell; DTH, delayed-type hypersensitivity; EAE, experimental allergic encephalomyelitis; GM-CSF, granulocyte-macrophage colony-stimulating factor; LC, Langerhans cell; mAb, monoclonal antibody; MHC, major histocompatibility complex; PLP, paraformaldehyde–lysine–periodate fixative; PNS, peripheral nervous system; TCR, T-cell receptor.

traffic from the periphery to the primary lymphoid organs; however, this traffic is greatly increased after administration of a foreign antigen to the peripheral tissue.29 In lymphoid organs, dendritic cells play a key role in initiating T-cell responses.34,35,50 The mechanisms by which these professional APC activate T-cells have been largely unravelled in recent years. DCs express constitutively high levels of major histocompatibility complex (MHC) class I and II antigens. However, in addition to MHC antigens, they express multiple other ligands which serve to form receptor– ligand pairs with receptors present on the surface of T-cells. The binding of these ligands to their counterreceptors is an important co-stimulatory signal in T-cell activation. The most important of these interactions are the binding of accessory molecules such as intercellular adhesion molecule-1 (ICAM-1); leucocyte function associated molecules (LFA1 and LFA3) and B7 on the surface of DCs to LFA1, cluster of differentiation (CD2 and CD28) on

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T-cells.19,24 The binding of the B7 ligand to its counter-receptor CD28 has been shown to be of special importance in stimulating interleukin (IL)2 production by T-cells and their subsequent proliferation.49 The efficiency of antigen presentation by dendritic cells is now well recognized,1,47 not only during primary but also during secondary responses. Their potential importance has been reported in many inflammatory conditions ranging from viral infection3,8,9 to autoimmune disease.9,11 However, little is known as yet about dendritic cells in inflammatory diseases of the CNS. Here we investigated the presence of these cells in acute experimental allergic encephalomyelitis (EAE) lesions26 and in more chronic delayed-type hypersensitivity (DTH) lesions.31 Acute EAE is immune mediated. The disease is characterized by multiple lesions in the brain and the spinal cord. Acute EAE is a monophasic autoimmune disease which lasts for approximately three weeks and resolves spontaneously.28 In the DTH model, chronic inflammatory lesions are induced as a response to heat-killed bacillus CalmetteGue´rin (BCG) sequestrated behind the blood–brain barrier (BBB). Lesions which are composed predominantly of T-cells and macrophages were found to persist for months. EXPERIMENTAL PROCEDURES

Animals Male Lewis rats, four and eight weeks old, were obtained from Charles River (U.K.). The animals were housed under standard conditions. Induction of experimental allergic encephalomyelitis To induce EAE, eight-week-old rats were immunized with 100 ìg of MBP prepared as previously described,7 omitting cation exchange chromatography. MBP was emulsified in complete Freund’s adjuvant (containing 1 mg/ml of mycobacterium tuberculosis H37Ra; Sigma, U.K.) and given subcutaneously. Animals were killed on days 5, 7, 9, 10, 11, 12, 15 and 17. At least two animals were used for each time-point. In addition, healthy adult Lewis rats were used as normal controls. Delayed-type hypersensitivity response in the central nervous system DTH response in the CNS was induced as described earlier.31 In summary, four-week-old Lewis rats were injected with 105 organisms of heat-killed BCG into the dorsal hippocampus using a sterotaxic technique. Four weeks later animals were challenged subcutaneously with 107 organisms of heat-killed BCG incorporated in complete Freund’s adjuvant. Animals were perfused on days 8, 12, 14, and 19, and CNS lesions were assessed immunohistochemically. Electron microscopy study DTH responses in the brain were induced as described above. At different times post-subcutaneous injection, from day seven to three months, animals were deeply anaesthetized with pentobarbitone sodium and transcardially perfused with 3% paraformaldehyde in 0.1 M phosphate buffer, containing 3% glutaraldehyde. The brains were dissected and further post-fixed for 1 h. The tissue was washed in

0.1 M phosphate buffer. Vibratome sections, 100 ìm thick, were cut through the region of the lesion. The sections were further processed using the method previously described.4 Vibratome sections were washed in 0.1 M phosphate buffer and osmicated, using 1% osmium solution (Chemox, U.K.) in phosphate buffer. After osmication, the sections were dehydrated through a graded series of alcohol. During dehydration, sections were treated with 1% uranyl acetate (FSA, U.K.) in 70% ethanol for 40 min. Dehydrated sections were left in propylene oxide for 20 min and then immersed in Durcupan resin (Fluka, Switzerland), prepared as recommended by the supplier, and left overnight. Using fine brushes and forceps, sections were flattened on warm glass slides and left for 48 h at 60)C to allow the resin to polymerize. Areas containing inflammatory cells were selected under a light microscope and ultrathin sections were cut. Ultrathin sections were stained with lead citrate and studied under an electron microscope (Philips PW 6006, The Netherlands). Treatment with dexamethasone A group of five animals was injected with 105 organisms of heat-killed BCG into the dorsal hippocampus. Four weeks later animals were challenged subcutaneously with 107 BCG organisms in complete Freund’s adjuvant. From day five after subcutaneous challenge, animals were treated with dexamethasone for seven days. Each animal was injected subcutaneously with 0.1 mg/kg of dexamethasone once a day at approximately the same time each day. Lewis rats were killed at day 12 and tissues processed for immunohistochemistry. Immunohistochemistry All animals were deeply anaesthetized with pentobarbitone sodium (Sagatal, RMB Animal Health, U.K.) and transcardially perfused with heparinized, 0.9% NaCl solution, followed by 2% paraformaldehyde–lysine–periodate fixative (PLP).32 The brain, spinal cord (cervical and lumbar parts), eyes and spleen were removed from all EAE animals. In control animals the pituitary gland, sciatic and trigeminal nerves and the spinal roots were also saved. Only the brains were dissected from animals with the DTH lesions. Dissected tissues were further post-fixed in PLP for 4–6 h and cryoprotected in 30% sucrose. Blocks were embedded in Tissue Tek OCT compound (Lamb) and rapidly frozen. Blocks were cut on a cryostat and 10-ìm sections were labelled immunohistochemically. The primary antibodies used were: OX62 monoclonal antibody (mAb),6 OX6 mAb (anti-MHC class II),33 OX19 mAb (anti-CD5),12 OX22 (anti-CD45RA),46 OX39 (anti-IL2R),42 V65 mAb (anti-ãä T-cell receptor; TCR)22 and polyclonal rabbit anti-F4/80 antibody.2 MRC-OX62 is a monoclonal antibody which was raised against density gradient-enriched rat dendritic cells purified from lymph. This antibody has been shown to recognize an á-like integrin subunit present on dendritic cells. However, this antibody has also been shown to recognize a subpopulation of T-cells. Therefore, to differentiate between dendritic cells and T-cells, we used double-labelling techniques with OX6 mAb directly fluorescein conjugated and OX62 detected either by antibody conjugated with horseradish peroxidase (HRP) or by Texas Red. F4/80 antiserum was generously provided by Prof. S. Gordon (Sir William Dunn School of Pathology, Oxford, U.K.). Anti-ãä TCR Ab was obtained from Pharmingen (U.S.A.). The remaining antibodies were generously provided by the MRC Cellular Unit, Sir William Dunn School of Pathology, Oxford. The primary antibodies were detected using the avidin–biotin peroxidase method as previously described.27

DCs in inflammatory diseases in the CNS Analysis Cell counts. Activated T-cells were assessed by staining with anti-IL2R mAb (OX39). CD45RA+ T-cells were detected with OX22 mAb and dendritic cells were detected with OX62 mAb. The total number of cells was counted in a 10-ìm section traversing the middle of the DTH lesion. The area occupied by MHC class II+ inflammatory macrophages was used to calculate cell density in 0.1 mm2. The area occupied by MHC class II+ inflammatory macrophages was traced using a drawing tube attached to the microscope. The size of the area was subsequently calculated with a Sigma Scan (RPLACE) measurement system. The mean value of the respective cell numbers was calculated for each lesion. Statistical evaluation was carried out using a two-tailed Student’s t-test. RESULTS

OX62 staining in normal central and peripheral nervous systems We looked for OX62-positive cells (OX62+) in a number of different sites in the CNS and peripheral nervous system (PNS), and compared the staining of OX62 mAb with that of OX6 mAb. OX62+ cells were absent from most of the PNS and CNS sites studied, including the brain and the spinal cord parenchyma. However, there was a small number of OX62+ cells in the choroid plexus and meninges. To assess quantitatively the number of OX62+ cells in the choroid plexus, the staining of OX62 was compared to that of OX6. We counted OX62+ and OX6+ cells in adjacent sections through the choroid plexus of both the lateral and the fourth ventricle. The counts were carried out on sections obtained from three control animals, with a minimum of four sections (separated by 250-ìm intervals) per choroid plexus. There were approximately 150–250 OX6+ in the choroid plexus in any one coronal section studied. The number of OX62+ cells was small and found to constitute approximately 1% of all OX6+ cells in the choroid plexus (Fig. 1A,B). These numbers were comparable in the choroid plexus of both the lateral and fourth ventricle. An occasional OX62+ cell was seen in the meninges. However, we found it difficult to carry out a quantitative analysis of OX62+ cells at this site, because a large part of the meninges was lost during the dissection of the brain and any attempt at quantitative analysis would have been very imprecise. OX62+ cells in experimental allergic encephalomyelitis lesions OX62+ cells were studied in the CNS of EAE animals at different times after the initiation of the disease starting from day five through to day 17. The first lesions were detected on day 10. The maximum number of lesions was detected at days 12 and 15. Most lesions were in the spinal cord, brainstem and cerebellum. However, numerous lesions were also detected in the forebrain, especially in the brains of animals killed at the later times. A small number of

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OX62+ cells was found in many, but not all, lesions (Fig. 1C). They were mainly confined to perivascular cuffs, although some of these cells were also detected in the CNS parenchyma surrounding a perivascular cuff. In the cuffs, OX62+ cells constituted approximately 2% of all leucocytes. In the CNS parenchyma the density of OX62+ cells declined rapidly with distance from a perivascular cuff. As described in the Experimental Procedures, OX62 mAb recognizes not only dendritic cells but also a subpopulation of T-cells (presumed ãä T-cells). To characterize further the OX62+ cells, sections were double stained with OX6-fluorescein and OX62HRP. The majority of OX62+ cells in the CNS parenchyma were also MHC class II+. However, there was an occasional cell with OX62+/OX6" phenotype. Additional staining with anti-ãä TCR antibody revealed that EAE lesions were generally devoid of ãä T-cells. Only in a small number of lesions was an occasional ãä T-cell observed. In order to eliminate the possibility that the antigen recognized by OX62 mAb is up-regulated on activated macrophages or microglia, we carried out double staining with OX62 mAb and polyclonal F4/80 Ab. F4/80 is a 160,000 mol. wt plasma membrane glycoprotein present on murine macrophages and detectable with antiserum on rat macrophages. F4/80 is down-regulated on dendritic cells and is absent from lymphocytes.2 We found that all OX62+ cells were F4/80". Delayed-type hypersensitivity responses to bacillus Calmette-Gue´rin The cellular composition of a DTH lesion directed against heat-killed BCG sequestrated behind the BBB has been previously described in some detail.31 In summary, these lesions are composed predominantly of T-cells and macrophages, and are larger than EAE lesions. Unlike acute EAE lesions, the DTH lesions have been shown to persist for months. They also lead to an extensive breakdown of the BBB and myelin loss. OX62+ cells in delayed-type hypersensitivity lesions The presence of OX62+ cells has been studied in CNS lesions eight, 12, 14 and 19 days after subcutaneous challenge using light microscopy. OX62+ cells were detected in all DTH lesions studied including eight-day-old lesions. There was about a 10-fold increase in the number of OX62+ cells between day 8 and days 12–14. The cell count of the total number of OX62+ cells in a section passing through the centre of the lesion, eight days after subcutaneous injection of BCG, was 21&18 cells (n = 3). The number of OX62+ cells by 12 days increased to 222&78 cells (n = 6). OX62+ cells in a typical 12-day-old lesion are shown in Fig. 1D. Double staining of OX62 mAb with OX6 mAb showed that most OX62+ cells were

Fig. 1. Micrographs showing OX62+ cells in (A) the choroid plexus in the normal rat brain. Note that OX62+ cells constitute only a small proportion of MHC class II+ cells (B). (C) Typical EAE lesion 12 days after subcutaneous injection of MBP in CFA. (D) Typical DTH lesion 12 days after subcutaneous injection of BCG in CFA. Note a significant difference between the number of OX62+ cells in the DTH lesion and in the EAE lesion. Scale bar = 50 ìm.

Fig. 2. (A, B) Double labelling of a DTH lesion with OX62 mAb deteced with Texas Red (A) and fluorescein-conjugated anti-MHC class II mAb (B). Arrows show examples of cells which are double labelled. Note that all OX62+ cells in this field were also MHC class II+. (C) ãä TCR+ cells in a DTH lesion. Note that ãä T-cells have a characteristic round morphology distinct from OX62+ dendritic cells (arrowheads in Fig. 1C). Scale bar = 50 ìm.

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M. K. Matyszak and V. H. Perry Table 1. Profile of 12-day-old delayed-type hypersensitivity lesions after treatment with dexamethasone

Cell type OX62+ cells IL2R+ cells OX19+ cells CD45RA+ cells

Cell number/0.1 mm2 DexamethasoneControl group treated group (mean&S.D.) (mean&S.D.) 21& 8 21&11 55&15 38&13

20&9 22&9 39&13 18&3

P-Value 0.760 0.848 0.089 0.009

The difference between the experimental and control groups, in both T-cell numbers and OX62+ cell numbers, was assessed with a two-tailed Student’s t-test. Five animals were used for each experimental and control group.

MHC class II+ (Fig. 2A, B). These cells were classified as dendritic cells. The number of DCs in these lesions was much higher than in a typical 12–14-dayold EAE lesion (compare Fig. 1C with Fig. 1D). There was no significant change in the total number of T-cells between day 12 (426&220 cells per lesion in a 10-ìm section, n = 6) and day 19 (491&301 cells per lesion in a 10-ìm section, n = 6). However, there was approximately a 50% reduction in the number of OX62+ cells in the same lesions (124&33 cells/section, n = 5) when compared with lesions after 12 days. To substantiate further the presence of dendritic cells in DTH lesions, some lesions were studied using electron microscopy. We found numerous examples of cells with a characteristic DC morphology in these lesions, including three-month-old lesions. These cells were characterized by a pale cytoplasm with perinuclear accumulation of the organelles and organellefree rounded processes. Perinuclear organelles were rich in large mitochondria and rough endoplasmic reticulum but there were relatively few lysosomal compartments. Some dendritic cells showed elongated morphology with extensive processes stretching for some distance (Fig. 3A), whereas others had rounded morphology (Fig. 3B). Many of these cells were seen in close contact with lymphocytes (Fig. 3A). ãä T-cells ãä T-cells were not detected in CNS lesions, eight days after subcutaneous injection of BCG. In 12-dayold lesions we also detected a small population of ãä T-cells (Fig. 2C). All of these cells had a round morphology, resembling that of áâ T-cells. Cell counts in 10-ìm sections through the middle of the lesions revealed that the total number of ãä T-cells was 39&18 cells per section (3&1 cells/ 0.1 mm2). There were virtually no ãä T-cells present in CNS lesions studied 19 days after subcutaneous challenge. Therefore ãä T-cells constituted only a minor population of cells in DTH lesions.

Dexamethasone treatment of delayed-type hypersensitivity animals There was an overall reduction in the size of DTH lesions in animals treated with dexamethasone. However, in none of the animals studied was there a full inhibition of immune-mediated responses in the CNS. The changes in the number of OX62+ cells as compared with different populations of T-cells are illustrated in Table 1. CD45RA+ T-cells were most affected by the dexamethasone treatment. CD45RA+ T-cell density was reduced by about 59%. There was no significant reduction in the density of either OX62+ cells, IL2R+ cells and OX19+ T-cells (pan-T-marker) in these lesions. In three out of five animals which were treated with dexamethasone, DTH lesions were devoid of ãä T-cells. In the remaining two animals, only an occasional ãä T-cell was detected in sections cut through the middle of the lesion.

DISCUSSION

The results show that OX62+ dendritic cells are present in acute EAE lesions as well as in DTH lesions induced in response to BCG sequestrated in the CNS parenchyma. There were more OX62+ dendritic cells in the DTH lesions than in the acute EAE lesions. Earlier studies have shown that dendritic cells are absent from the normal CNS parenchyma (for review, see Ref. 47), and our present studies confirmed this. We also found that DCs are absent from other CNS and PNS compartments apart from the choroid plexus and meninges. The small number of OX62+ cells in the choroid plexus is consistent with our earlier report of rare dendritic cells (MHC class II+/F4/80" cells) within the stroma of this tissue.30 Since similar numbers of OX6+/F4/80" cells and OX62+ cells were detected in the choroid plexus and there were no ãä TCR+ cells, we suggest that the OX62-positive cells seen within the choroid plexus are dendritic cells.

DCs in inflammatory diseases in the CNS

Fig. 3. Electron micrographs showing examples of dendritic cells in a DTH lesion in the CNS parenchyma. (A) DC in direct contact with a lymphocyte. The DC shows extensive rounded processes which are organelle free (*), and an organelle-rich perinuclear area (arrow). (B) DC with more rounded morphology. Note only a small number of lysosomal compartments. Scale bars = 1 ìm.

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Most OX62+ cells in both acute EAE lesions and the DTH lesions were also OX6+ and were classified as dendritic cells. The identity of these cells in the DTH model was further confirmed by electron microscopy. In acute EAE, OX62+ dendritic cells were present in many, but not all, lesions studied. The number of these cells in EAE lesions was small. In contrast, OX62+ dendritic cells were detected in all DTH lesions studied, including early (eight-day-old) lesions. The highest number of dendritic cells was found in two-week-old lesions. Electron microscopy studies revealed that DCs are also present in threemonth-old lesions. Dendritic cells in inflammatory lesions The presence of dendritic cells in CNS inflammatory lesions has not been previously reported, although their importance in inflammatory conditions in tissues other than the CNS has been well recognized. It is now recognized that DCs play an important role in alloimmunity,47,23 although the exact mechanisms of their action are still unclear.14 Two possible hypotheses have been suggested. Firstly, progenitor donor DCs may undergo a process of maturation and subsequently migrate into local lymphoid organs where they can stimulate T-cell responses. Alternatively, alloantigens are shed from the graft and picked up by host dendritic cells. Recent studies have shown that dendritic cells are very efficient at presenting viral antigens to cytotoxic T-cells40,3 as well as bacterial and parasite antigens.16,37 For example, studies with Leishmania major have shown that Langerhans cells (LCs), which are the progenitor dendritic cells in the skin, are capable of phagocytosis of the parasites and subsequent induction of the Th1 response to Leishmania.38 Bone marrow progenitor DCs can digest BCG and subsequently present BCG antigens to naive T-cells.16 The presence of dendritic cells in inflammatory lesions in the CNS raises the question of what triggers the migration of these cells into the CNS parenchyma. In a recent review, Ibrahim and his colleagues put forward a hypothesis which considers tissue injury as a trigger that activates dendritic cells. They argue that an injured parenchyma may itself be the source of soluble cytokines involved in DCs activation. Among these, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-á and IL1 are probably the most important.15 For example, injection of GM-CSF into the dermis increases the number of LCs in the skin.20 Also, GM-CSF up-regulates B7 ligands on DCs in vitro.24 Indeed, if the tissue injury hypothesis is correct, it could explain why the number of DCs in EAE lesions differs from the number in the DTH lesions directed against BCG sequestrated in the CNS. In acute EAE, there is minimal tissue damage.25 In some cases,

perivascular demyelination has been reported,10 but the myelin loss was small. In the DTH lesions, however, there is extensive CNS tissue damage, including myelin damage.31 Once inside the lesion, DCs may contribute to the chronicity of the disease by in situ activation of T-cells. The fact that dendritic cells may be activated by tissue injury may have further implications. These include the induction of autoimmune responses. There is indirect evidence that this may indeed be the case. For example, self-limiting autoimmune responses have been observed as a consequence of ischaemia or physical injury to the heart.15 Studies are now under way in our laboratory to find out whether bystander CNS tissue damage during DTH responses may give rise to autoimmmune responses to CNS antigens. ãä T-cells and experimental allergic encephalomyelitis lesions As discussed earlier, OX62 antibody recognizes not only dendritic cells but also a subpopulation of T-cells. Studies in the skin have shown the presence of a small number of OX62+ cells which were CD3+ but áâ TCR". These cells were classified as possible ãä T-cells. It was therefore important here to find out the relative contribution of ãä T-cells to lesions in both the acute EAE and the DTH model, especially since the presence of these cells has already been reported in acute and chronic multiple sclerosis lesions.45,48 ãä T-cells are a population of cytotoxic T-cells which show a high affinity for heat-shock proteins. They recognize heat-shock proteins independent of MHC antigens. In multiple sclerosis, it has been postulated that they may contribute to the chronicity of the disease and prevent remyelination by cytotoxic activity toward oligodendrocytes.13 Our results showed that ãä T-cells are present in EAE lesions only in very small numbers. There were more of these cells in early DTH lesions (maximum numbers in 12–14-day lesions). However, after 19 days they were almost completely absent from DTH lesions. This suggests that ãä T-cells may be involved acutely but are unlikely to contribute to the long-term chronicity of these responses. Changes to the inflammatory responses in the central nervous system induced by treatment with dexamethasone Corticosteroid treatment is often used therapeutically in many autoimmune diseases. Treatment with dexamethasone inhibits the induction of acute EAE.5 Methylprednisolone is widely used to treat clinical relapse of multiple sclerosis. Steroid treatment has a beneficial short-term effect in multiple sclerosis patients, but has a relatively small effect on the long-term outcome of the disease. Magnetic resonance imaging studies revealed that a high dose of

DCs in inflammatory diseases in the CNS

methylprednisolone reduces the oedema21 and the breakdown of the BBB in multiple sclerosis.36 Our earlier study showed the same acute effects on the DTH lesions in the CNS after treatment with a high dose of dexamethasone (Matyszak and Perry, unpublished observations). Our present study was set up to investigate the changes in the cellular composition of CNS lesions in more detail. In doing so, we were looking for clues as to why the effect of corticosteroids is only transient. The present study focused on two subsets of cells, namely dendritic cells and T-cells, because of their primary role in initiating immune-mediated responses. We found that dexamethasone was effective in reducing the density of CD45RA+ T-cells in DTH lesions, but not IL2R+ cells and dendritic cells. There was also no significant reduction in the total number of T-cells, assessed with OX19 mAb. The persistence of both dendritic cells and T-cells (some of them activated) in CNS lesions may answer, at least in

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part, the question of why the effects of corticosteroids are only transient. The continued presence of both populations of cells in the CNS may mean that there will be renewed activity in the persisting lesions after treatment is discontinued. CONCLUSIONS

Dendritic cells are absent from the normal CNS parenchyma; however, they are present in both acute EAE lesions and the DTH lesions. The number of DCs in DTH lesions is significantly larger than in EAE lesions. Furthermore, in DTH lesions DCs have been found to persist for months. We also found that these cells persist in the CNS lesions after steroid treatment and therefore may be involved in renewing CNS inflammation. Acknowledgement—This work was supported by The Multiple Sclerosis Society.

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