Methylprednisolone prevents rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in adult rats

BRAIN RESEARCH ELSEVIER

Brain Research 712 (1996) 199-212

Research report

Methylprednisolone prevents rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in adult rats Wei-Ming Duan *, Patrik Brundin, Eva Maria Grasbon-Frodl, H~tkan Widner Section for Neuronal Surt, it,al, Department of Physiology and Neuroscience, Biskopsgatan 5, S-223 62 Lund, Sweden Accepted 24 October 1995

Abstract

We studied the effects of high-dose methyiprednisolone on the survival of intrastriatal neural xenografts and the host responses against them. Dissociated mesencephalic tissue from inbred mouse (CBA-strain) embryos was transplanted to the intact striatum of adult Sprague-Dawley rats. The rats received either daily injections of methylprednisolone (30 mg/kg), or cyclosporin A (10 mg/kg), or no immunosuppressive treatment. Two or six weeks after transplantation, there was good survival of xenografts in both the methylprednisolone- and cyclosporin A-treated rats. In contrast, the xenografls in untreated control rats were all rejected by six weeks. There was no marked difference in the degree of expression of MHC class I and lI antigens and the accumulation of activated astrocytes and microglial cells/macrophages between the three groups. However, both methylprednisolone and cyclosporin A reduced infiltration of T lymphocytes to the transplantation sites. The expression of pro-inflammatory cytokines (interferon-% tumour necrosis factor-a, interleukin-6) in and around the grafts was lower in the methylprednisolone- and cyclosporin A-treated groups than in untreated control rats. Although high-dose methyiprednisolone caused significant body weight loss, we conclude that this treatment can prevent rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in the adult. Keywords: Methylprednisolone; Cyclosporin A; Neural transplantation; Xenograft; Immunology; Inflammation; Cytokine; Brain: Rat

I. Introduction

Although the brain constitutes an immunologically privileged site [3,56], many studies have shown that intracerebral neural xenografts are rejected if the recipient animals are not immunosuppressed [8,9,23,24,27,28,40,44,48,58]. In a recent review of the literature, a meta-analysis of several studies revealed that immunosuppression increases the survival rate of intracerebral xenografts from 30% to 74% [45]. We have also observed that surviving and behaviorally functional intracerebral neural xenografts in immunosuppressed animals are rejected and the behavioral function is lost, if the immunosuppressive treatment is withdrawn [9]. In contrast, only a few studies have reported that some, but not all, xenografts survive transplantation for prolonged periods of time even without immunosuppression [7,13]. The rejection mechanisms of intracerebral xenografts are not well understood. In addition to the cell-mediated

* Corresponding author. Fax: (46) (46) 222-3065; E-mail: [email protected] 0006-8993/96/$15.(10 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - S q q 3 ( q 5 )01 409-8

immune responses, as normally observed when an allogeneic graft is rejected, antibody-dependent cellular cytotoxicity by preformed cross-reacting antibodies and direct complement activation are involved [2,20,39,49]. In the normal adult brain, neural tissue does not express detectable levels of major histocompatibility complex (MHC) molecules [16,17,31,33,52,57]. However, MHC expression can be induced on the neural tissue as a consequence of, e.g., the inflammatory reaction that occurs when grafting into the brain or other immunological diseases of the brain [16,17,24,32,33,37,43]. This inflammatory reaction may result in the release of several pro-inflammatory cytokines, such as interferon-y (IFN-y), tumour necrosis factor-it ( T N F - a ) and interleukin-6 (IL-6). Each of these contributes to various inflammatory events within the brain, such as I F N - y induced MHC expression on brain cells [21,30,31,35,42,51,52,55,57], or increased expression of adhesion molecules on the vascular bed [14,19,32,38,51], or a c t i v a t i o n o f glial and microglial cells [5,22,24,41,43,46,50]. Agents that reduce the acute inflammatory reaction could therefore be beneficial to the survival of grafts of histoincompatible neural tissue.

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Primarily cyclosporin A (CsA) has been employed as an immunosuppressive drug in transplantation of xenogeneic neural tissue [8,9,23,28]. CsA, a macrolide, inhibits Tlymphocyte activation by binding to the intracellular protein calcineurin, which in turn modulates intracellular calcium release [34]. In clinical practice, CsA which combined with steroids and sometimes azathioprine or various antilymphocyte antibodies, has enabled routine organ transplantation across MHC barriers. However, CsA has a variety of adverse side effects, is relatively expensive and requires chronic administration. Even when the blood concentrations are controlled for, there is an inevitable late nephrotoxicity [29]. With higher doses, there are increased risks of opportunistic infections, lymphomas and several other organ toxicities. Therefore, it is of interest to explore whether other immunosuppressive agents can support long-term survival of intracerebral neural xenografts. Methylprednisolone (MP) is a well known synthetic adrenocorticosteroid drug with anti-inflammatory and immunosuppressive properties, which specifically inhibits the production of TNF-o~ and IL-6 [6,47] and also reduces the expression of adhesion molecules under inflammatory conditions [11,53]. MP is extensively used in clinical transplantation practice as a backbone in immunosuppressive protocols [29]. In the current experiment, we have studied the effects of high-dose MP on the survival of intrastriatal mouse-to-rat xenografts of embryonic dopamine neurons. The study was also designed to monitor the effects of MP on inflammatory and immune reactions at the transplantation site using different immunocytochemical markers.

2. Materials and methods

2.1. Experimental design A total of 30 adult female Sprague-Dawley rats (B & K Universal, Sollentuna, Sweden, weighing about 225 g at the beginning of the experiment) were used as graft recipients. All the rats received intrastriatal xenografts of dissociated mesencephalic tissue from CBA mouse (B & K Universal) embryos and were divided into three groups. Ten rats were given daily injections of MP (30 m g / k g i.p.) 10 days before neural transplantation and then up to the time of sacrifice; ten rats were given daily CsA (10 m g / k g i.p.) one day before neural transplantation and then up to the time of sacrifice; and ten untreated control rats did not receive any immunosuppressive treatment. The rats were perfused at 2 or 6 weeks after neural transplantation (five rats for each group and time-point) and the brain tissue was processed for eleven different

immunocytochemical markers. The size of the grafts was assessed by counting the number of tyrosine hydroxylase (TH) immunopositive cells. The expression of MHC class I and class II antigens at the implantation site and the host cellular responses including macrophages, activated microglia and astrocytes, cytotoxic and helper T-lymphocytes were analyzed semi-quantitatively, and cytokine staining patterns were assessed.

2.2. Donor tissue, dissection, tissue preparation and transplantation surgery Xenogeneic grafts were prepared from mesencephalic tissue of embryonic day 13-14 mouse embryos (crownto-rump length, 10 mm), of an inbred CBA strain with H-2 (mouse MHC) haplotype b as described earlier [16]. The viabilities of all cell suspensions were over 90%, as determined using acridine orange and ethidium bromide dye exclusion. Two /zl of the cell suspension were stereotaxically injected into the right striatum of equithesin anesthetized rats using a 10 tzl Hamilton microsyringe (Hamilton Co., Reno, NV, USA) fitted with a steel cannula (inner diameter = 0.25 mm, outer diameter = 0.47 mm) at the following coordinates: 1.0 mm rostral to bregma; 3.0 mm lateral to the midline; 4.5 mm ventral to the dural surface, with the tooth-bar set at zero.

2.3. Immunocytochemistry Two and six weeks after transplantation, the rats were deeply anesthetized with chloral hydrate and perfused transcardially with saline and cold 4% paraformaldehyde as described previously [16]. The rat brains were immersed in 20% sucrose in 0.1 M phosphate buffer at 4°C, then frozen and sectioned coronally at 30 /zm thickness on a sliding microtome. Throughout the region of the graft, adjacent serial sections were collected in a cryoprotective solution in four glass vials, allowing for comparison of the same region stained with different antibodies. One spleen from a Sprague-Dawley rat was prepared for immunocytochemistry using the same fixation protocol as used for the brain tissue.

2.3.1. MHC, complement receptor (CR) 3, cluster of differentiation (CD) 4 and 8, IFN-T, TNF-a, IL-6 immunocytochemistry Adjacent brain sections were stained immunocytochemically using the avidin-biotin complex immunoperoxidase technique, as described in detail previously [15-17]. The following mouse monoclonal antibodies (dilutions in parentheses) (Serotec, Oxford, UK) were used: MRC OX-

Fig. 1. Arbitrary ratings of immunocytochemicalstaining for the sections. Bars indicate the median values, dots representthe individual ratings for the rats and the filled dots represent the rats from which photomicrographswere taken, shown in Fig. 3, Fig. 4 and Fig. 5. The antisera used were, in A: MHC class I; B: MHC class II; C: CR3; D: GFAP; E: CD4; and F: CD8.

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18 (1:400), reacting with rat M H C class I antigens; MRC OX-6 (1:400), reacting with rat MHC class II antigens; MRC OX-42 (1:400), reacting with rat CR3 expressed on microglia and macrophages; MRC W 3 / 2 5 (1:300), reacting with helper T-lymphocyte antigen CD4; MRC OX-8 (1:400), reacting with cytotoxic T-lymphocyte antigen CD8; MRC B-B1 (1:50), reacting with human INF-T; MRC B-C7 (1:50), reacting with human T N F - a ; and MRC B-E8 (1:50), reacting with human IL-6. The secondary antibody was biotinylated horse anti-mouse (rat-adsorbed) immunoglobulin (1:200) (Vector Laboratories, Burlingame, CA, USA). The sections were incubated in ABC solution (Vectastain ABC Elite kit, Vector Laboratories), and the immunoreactive products were visualized by 3,3'-diaminobenzidine (Sigma). Sections of spleen were used to test whether the current immunocytochemical protocols could detect cells which normally should be stained intracellularly for cytokines. To assess the degree of unspecific staining on brain sections, normal mouse serum was used as primary antibody on selected control sections. 2.3.2. TH and glial fibrillary acidic protein (GFAP) immunocytochemistry Adjacent brain sections were processed for TH and GFAP immunocytochemistry, using a protocol similar to that of the MHC immunocytochemistry [17]. The first antibody was a rabbit anti-TH polyclonal antibody (1:500) (Pel-Freez, Rogers, AR, USA) or a bovine anti-GFAP polyclonal antibody (1:500) (Dakopatts, Copenhagen, Denmark). The secondary antibody was biotinylated swine anti-rabbit immunoglobulin (1:200) (Dakopatts).

2.4. Semi-quantitatiL, e and quantitative et~aluation of brain sections

The brain sections processed for MHC-, CR3-, GFAP-, CD4- and CD8-immunostaining were semi-quantitatively evaluated microscopically under bright-field illumination on blind-coded slides, as described previously [16,17]. In short, each section was rated by three independent raters, into one of the following rated categories: (0) no specific immunostaining in the grafting site; (1) low number of positive cells, distributed as scattered single cells or clustered in a few small patches in or around the graft; (2) several positive cells distributed as single cells or clustered in multiple, prominent patches; (3) dense immunostaining of the graft area and a large number of positive cells in and around the graft; (4) very dense immunostaining of the whole graft area and a very large number of positive cells in and around the graft. The final score for each graft was determined based on the highest score observed and the median value for each group was plotted. The rating scale only took activated astrocytes and microglia and the distinctly stained round CD4- and CD8-positive cells into account (as detailed in Section 3). Cytokine stainings were evaluated based on the morphological pattern and distribution. TH-positive neurons were microscopically counted under bright-field illumination on every fourth section throughout the graft. The raw counts of TH-positive cell were multiplied with a correction factor of 2.9 in accordance with the Abercrombie formula [1]. The cell numbers were regarded to be binomially distributed and were there-

Fig. 2. Photomicrographs of TH-immunocytochemically stained sections through the grafted striatum. A depicts a section from an MP-treated rat, B a CsA-treated rat and C an untreated control rat at 6 weeks after transplantation. There were mature TH-positive cells with a rich fibrous network in both MP- and CsA-treated rats. No significant differences in the number of surviving TH-positive cells were found between these two groups. Only scar tissue remained at 6 weeks in the untreated control group. Scale bar = 100 /xm.

W.-M. Duan et al. /Brain Research 712 (1996) 199-212

fore analysed statistically using a non-parametrical analysis of variance (Kruskal-Wallis test), at the 95% probability level, with Dunn's post-hoe test to determine group difference.

3. Results

All the rats survived the experiment, and there was no infection evident. The MP-treated rats lost body weight during the experiment, whereas the CsA-treated rats and

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the untreated control rats gained body weight as expected (Table 1). The semi-quantitative ratings of immunocytochemistry are summarized in Fig. 1, with MHC class ! ratings given in (A), MHC class II (B), CR3 (C), GFAP (D), CD4 (E) and CD8 (F). The morphology of cells stained with these antibodies was similar to that described by us in detail previously [16,17].

3.1. TH immunocytochemistry There were significant differences in the mean number of TH-positive cells between the treatment groups and the

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Fig. 3. Photomicrographs from sections stained with antisera against MHC class I and I1 antigens. A shows MHC class I-positive cells in and around the graft in an MP-treated rat at 2 weeks after grafting, B the M H C class I staining from a CsA-treated rat at 6 weeks, and C MHC class 11 staining from a untreated control rat 2 weeks after grafting. D shows MHC class II staining from an MP-treated rat at 6 weeks. There were fewer MHC class ll-positive cells at 6 weeks compared to 2 weeks. Scale bar = 100 /xm.

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untreated control group, but not between the MP- and the CsA-treated groups, at both time points (Table 2). In both the MP- and the CsA-treated groups, all the rats had surviving xenografts in the right striatum at 2 and 6 weeks after transplantation (Fig. 2A and B). The morphology of the TH-positive cells in the grafts was similar to that observed in syngeneic and allogeneic grafts in previous studies [15-17]. However, one CsA-treated rat at the 6week time-point had a big cavity in the graft, suggestive of an ongoing rejection. In the untreated control group, only two rats at the 2-week time-point displayed small surviving graft tissue containing detectable TH-positive neurons.

These cells had fewer processes than those in the MP- and the CsA-treated groups. The remaining rats at 2 weeks and 6 weeks, only had scars at the transplantation sites (Fig. 2C). 3.2. M H C i m m u n o c y t o c h e m i s t r y

Generally, endothelial cells in blood vessels and ependymal cells lining the ventricles uniformly expressed M H C class I antigens. The expression of MHC class I antigens at the graft site in the MP-treated rats was moderate at both 2 and 6 weeks after transplantation (Fig. 3A).

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Fig. 4. A shows CR3-staining from an MP-treated rat 2 weeks after grafting, with a majority of the positive cells that resembled microglia, and fewer, rounded, large cells that resembled macrophages. B shows CR3-staining from a CsA-treated rat 6 weeks after grafting. There was little palisade formation around the graft, and the cells were less intensely stained. C depicts GFAP-staining from an untreated control rat 2 weeks after grafting, with only scar tissue remaining and D the GFAP-staining from an MP-treated rat 6 weeks after grafting. Scale bar = 100 /xm.

W.-M. Duan et aL / Brain Research 712 (1996) 199-212

However, there were no clear perivascular cuffs. In the CsA-treated group, the expression of MHC class I antigens tended to be lower (Fig. 3B), except for one rat with an ongoing rejection at 6 weeks, which exhibited a very high expression of MHC class I antigens in the graft area. In the untreated control group, three grafts were rejected at 2 weeks, leaving only scars. In two of these cases there were relatively few MHC class I positive cells, whereas the remaining three rats in the group exhibited a moderate

205

immunostaining. At 6 weeks after transplantation, all the grafts in the untreated control rats were rejected and there was a variable, but circumscript MHC class I immunostaining. For MHC class II antigen staining, there was a tendency for a lower number of positive cells in the MP-treated group at 6 weeks, when compared with the 2 weeks time-point (Fig. IB and Fig. 3D). There was a lower number of positive cells in the CsA-treated group corn-

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Fig. 5. A shows CD4-immunopositive T-helper lymphocytes, found around the graft in an MP-treated rat 6 weeks after grafting. B shows CD8-immunostained T-cytotoxic lymphocytes, found in and around the graft in a CsA-treated rat 6 weeks after implantation. C depicts IL-6 producing macrophages after 6 weeks in a CsA-treated rat with an ongoing rejection. The IL-6-immunopositive cells were located in the remnants of the graft tissue and in the periphery of a cavity from which tissue has probably been phagocytized. Scale bar = 50 /xm and is the same for A-C. D - F show IFN-y immunoreactivity 2 weeks following grafting in the three groups. Two cell types were found, one roundish cell, resembling lymphocytes and one smaller and with fibrous outgrowths, resembling type II astrocytes. D: the expression of IFN-y in a MP-treated rat, with few positive cell bodies and fibrous network in small clusters, mainly outside of the graft. E: 1FN-y immunoreactivity in a CsA-treated rat. There were very few positive fibers and fewer positive cell bodies, which were mainly located at the bottom of the graft. The cells resembled astroglial type II cells and few of them were lymphocytes. F: IFN-y immunoreactivity in an untreated control rat. The cells formed a palisade around the remnants of the graft. Scale bar - 100 # m and is thc same for D-F.

W.-M. Duan et aL /Brain Research 712 (1996) 199-212

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Table 1 The body weight (mean_+ S.E.M.) of rats before and after transplantation Group (n rats)

Before transpl. (g)

2 weeks after transpl. (g)

6 weeks after transpl. (g)

MP (5) CsA (5) Control (5) MP (5) CsA(5) Control (5)

214+2.3 214 _+3.0 210_+2.4 211 _+ 1.7 211-+2.3 212+2.4

1 9 2 + 8 . 8 *'* * 255 + 3.9 * 230+9.5 -

192 + 11.6 * * 262__ 4.3 * 238_+ 9.9

* P < 0.05 compared to before transplantation (paired, two-tailed Student's t-test). * * P < 0.01 compared to the other two groups at the same time-point (one-factor A N O V A with post-hoc Scheff6 F-test).

pared to the other two groups at two weeks. At 6 weeks, the degree and patterns of immunostaining were similar in the three groups. However, in the one rat with an ongoing rejection, there was a high expression of MHC class II antigens and the immunopositive cells were spread over half the surface area of the grafted striatum. The untreated control rats contained only a small area of scar tissue which was richly stained for MHC class II (Fig. 3C).

3.3. CR3 immunocytochemistry At 2 weeks there was a slight trend for an increase in the number of activated microglia and macrophages in the untreated control group compared to the two immunosuppressed groups (Fig. 4A). The CR3-positive cells infiltrated the grafts completely in the two untreated control rats which still had surviving transplants. In the remaining three rats, with completely rejected grafts, the CR3-positive cells were found along a scar. At 6 weeks, there was no apparent difference in the total number of CR3-positive cells between the three groups. However, the distribution of positive cells differed. Since the grafts in the untreated control group were all rejected, the stained cells were densely focused around a thin scar, whereas in both immunosuppressed groups there was moderate infiltration along the host-graft interface (Fig. 4B). The rat in the CsA-treated group which displayed a cavity in the graft had the most intense CR3-immunoreactivity, with activated microglia cells throughout the transplanted hemisphere.

3.4. GFAP immunocytochemistry At both time-points, the untreated controls and the CsA-treated rats exhibited approximately equal moderate

Table 2 The number ( m e a n + S.E.M.) of grafted TH-immunopositive cells in the three groups at the two time-points

2 weeks 6 weeks

MP

CsA

Control

952+313 1581 + 5 6 0

915+289 567+219

1 7 + 14 " 0+ 0 "

* P < 0.0001 compared to the other two groups at the same time-point (Kruskal-Wallis test followed by a post-hoc Dunn's test).

expression of reactive astrocytes in the grafted striatum (Fig. 1D), but in the untreated control group there was no graft remaining in most rats, and the distribution of the GFAP-positive cells (Fig. 4C) was different from that observed in the immunosuppressed groups. The MP-treated rats tended to have slightly fewer activated astrocytes than rats in the other two groups at both time-points (Fig. 4D).

3.5. CD4 and CD8 immunocytochemistry At 2 weeks after transplantation, there were very few CD4- and CD8-positive cells in the transplanted striatum in both the MP- and the CsA-treated groups (Fig. 1E). In contrast, the untreated control group had several lymphocytes in and around the transplant at this time-point (Fig. 1E). At 6 weeks after transplantation, the number of both CD4- and CD8-positive cells had increased compared with the earlier time-point, but was still low, in the MP-treated group (Fig. IF and Fig. 5A), and the number of lymphocytes was somewhat higher in the CsA-treated group (Fig. 1F and Fig. 5B). In contrast, there were fewer lymphocytes at the transplantation sites in untreated control rats than 2-week time-point.

3.6. Cytokine immunocytochemistry There was an intense and specific staining for all of the three different antibodies against IFN-y, TNF-a or IL-6 in the normal spleen. Most of the IFN-y and IL-6-immunopositive cells resembled lymphocytes, whereas the TNF-a immunoreactivity was found in macrophage-like cells, and only in a few lymphocytes. The IFN-y and TNF-a immunoreactivities were stronger than that observed for the IL-6 antibody, which may be related to the fixation protocol and the avidity of the respective antibodies. The brain sections were comparatively less intensely stained, although there was clear and specific staining for all of the three cytokines. In the non-grafted, intact hemi-

Fig. 6. A is a high-magnification photomicrograph of a coronal section through the striatum of an MP-treated rat, stained with the monoclonal antibody against CR3. The CR3-positive cells are evenly distributed across the striatum. Scale bar = 200 ,u.m. B is a camera lucida drawing of T N F - a expressing cell bodies in microglial cells at 6 weeks after grafting. There was a halo with less immunostaining of T N F - a around the graft. The inserted hatched-lined box indicates from where the photomicrograph A was taken. C shows an even immunostaining for T N F - a 2 weeks after grafting, in contrast to that found at 6 weeks in B. Scale bar = 1 mm, and is the same for B and C. D depicts a high-magnification photomicrograph demonstrating that the TNF-a-positive cells resembled activated microglial cells. Scale bar = 50 /xm.

W.-M. Duan et al. / B r a i n Research 712 (1996) 199-212

sphere of all animals, there was TNF-a immunoreactivity within apparent microglia cells, and this was regarded as a normal background level of intracellularly stored TNF-a.

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3.6.1. IFN- T immunocytochemistry

There were clear group differences for the intensity and pattern of expression of IFN-y immunoreactivity. In the

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W.-M. Duan et al. // Brain Research 712 (1996) 199-212

MP-treated rats, the IFN-y expression was less intense and less uniform compared to in the untreated control rats. Areas close to the grafts were completely devoid of staining, whereas there were clusters of immunostained cells close nearby blood vessels. At 2 weeks after grafting, the immunoreactivity was primarily located in astrocyte-like cells that formed palisades around the cannula tract. Inside the grafts there were occasional lymphocyte-like cells, and in 2 / 5 animals several round cells were clustered close to blood vessels (Fig. 5D). At 6 weeks after grafting, there was very little IFN-',/-immunopositive staining. In the CsA-treated group, there was very little IFN-yimmunopositive staining. Only occasional round, lymphocyte-like cells were found in the grafts at 2 weeks (Fig. 5E). At 6 weeks most rats contained only a thin line of cells and fibers around the transplants. In contrast, there was a high number of lymphocyte-like, intensely stained cells invading the graft in the rat with signs of an ongoing rejection at 6 weeks. In the untreated control rats, there was very intense IFN-y-immunostaining at 2 weeks after grafting. The immunoreactivity was primarily located in astrocyte-like cells arranged in palisades around the implantation site (Fig. 5F). There were also a few positive round, lymphocyte-like cells. At 6 weeks, there was still some IFN-y expression around the scars and scattered in the brain parenchyma.

tion sites. These cells resembled small round lymphocytes, slightly larger oval macrophages, and multipolar microglia. At 6 weeks there were very few TNF-a-positive cells found around the scars. They were mainly microglia-like and the level of TNF-a immunoreactivity was close to that of the cells found in the normal, contralateral hemisphere. 3.6.3. IL-6 i m m u n o c y t o c h e m i s t r y

At 2 weeks, in the MP-treated group, there were two rats with a small number of large, oval cells, macrophagelike cells within the grafts. At 6 weeks, there was a very limited expression in a few macrophage-like and microglia-like cells. In the CsA-treated group, the IL-6 expression was similar to that in the MP-treated group. At 6 weeks, in the graft undergoing rejection, there was a large number of IL-6-immunopositive cells, in and immediately surrounding the graft (Fig. 5C). There were round cells, thought to be lymphocytes, larger oval cells thought be macrophages and stellate cells thought to be microglia cells. In the other CsA-treated rats, there was no IL-6-immunopositive staining. In the untreated control animals, there were no immunopositive cells around the scars and the remnant of the grafts at 2 and 6 weeks.

3.6.2. T N F - a i m m u n o h i s t o c h e m i s t r y

The distribution of TNF-a-immunoreactive cells differed between the three groups. In the MP-treated group, there was an accumulation of positive cells within the grafts at 2 weeks (Fig. 6C). They were both round, lymphocyte-like cells and stellate, microglia-like cells (Fig. 6D). At 6 weeks there were fewer TNF-a-immunopositive cells within the grafts than at 2 weeks. There was a very distinct halo, which was devoid of TNF-a immunostaining, around each graft in 4 of 5 rats (Fig. 6B). In contrast, CR3-staining for microglia on adjacent sections (see above) revealed an even distribution of microglia throughout the striatum (Fig. 6A). Thus, there seems to be a down-regulation of the TNF-a immunoreactivity in microglial cells around the grafts. In the CsA-treated group, the pattern of staining was similar to that of the MP-treated group but there was a slightly higher number of densely stained cells. At 2 weeks, most were inside the graft area, resembled macrophages and a few appeared to be lymphocytes and microglia. At 6 weeks, generally there were very few cells stained and no signs of a staining gradient around the grafts. The few positive cells resembled lymphocytes and microglia-like cells. In the rat with an ongoing rejection, there was a very large number of TNF-a-positive lymphocytes, macrophages and microglia throughout the graft, which in some areas formed perivascular-cuffs. In the untreated control group, there was a very small number of TNF-a-immunopositive cells in the implanta-

4. Discussion The present study demonstrates that systemic administration of high-dose MP to rats can prevent rejection, as efficiently as CsA, of intrastriatal xenografts of mouse mesencephalic tissue. The survival of xenografted THpositive neurons was unimpaired in the MP-treated rats at both 2 and 6 weeks after grafting. Treatment with MP did not reduce the expression of MHC class I and II antigens, or the microglial and astrocytic reactions in the transplantation sites two weeks after surgery. After 6 weeks there was a slightly reduced expression of MHC class II antigens at the implant sites in the MP- and the CsA-treated groups, as compared to the untreated control group. Both MP and CsA also decreased the accumulation of both CD4- and CD8-positive T-lymphocytes in the transplanted sites at 2 and 6 weeks. Moreover, the IFN-3, immunoreactivity was lower in both the MP- and the CsA-treated groups than in the untreated control group. In the MPtreated group there was also reduced expression of TNF-a in a halo surrounding the grafts 6 weeks after graft implantation, indicating MP-induced suppression of the cytokine expression. The hypothesis in the present study was that reduction of inflammatory reactions in conjunction with the implantation surgery may lead to prolonged survival of intracerebral histoincompatible neural grafts. The expression of MHC antigens and cellular reactions at the grafting site are

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essential for the triggering of host immune reactions, as well as involved in responsible for graft destruction. Normally, the brain endothelium expresses low levels of adhesion molecules, and most cells in brain tissue do not express detectable MHC antigens [16,17,32,34,53,58]. In addition, the antigen presenting capacity is reported to be relatively low in the brain, with no detectable dendritic cells, which have the highest capacity to elicit immune responses in other tissues [25,26]. However, several other cell types within the brain have been reported to be able to function as antigen presenting cells [21,22,32,36] and MHC antigens are expressed in the brain under certain conditions [15-17,24,34,38]. Although MP supported prolonged xenograft survival and prevented the otherwise inevitable complete rejection of the xenografts, there was no dramatic drug effect on the expression of MHC antigens and activation of microglia and astrocytes at 2 weeks after transplantation. At 6 weeks there was only a tendency for a lower expression of MHC class II antigen in the MP-treated rats. Thus, these results provide no direct evidence that a nonspecific inflammatory reaction, manifested by MHC expression or altered numbers of activated microglial cells in conjunction with neural transplantation, is reduced by MP. On the other hand, we have observed changes in the cyt0kine pattern, indicating a functional regulation of the inflammatory reactions. Is the beneficial effect of MP due to interference with other aspects of the rejection process such as a reduction of the activation of endothelial cells and lymphocytes, or intervention with the migration of host defence cells? The control of expression of vascular adhesion molecules on endothelial cells has been linked to cytokines. It has been shown in other organs than the brain that corticosteroid treatment can reduce the cytokine-induced expression of vascular homing signals for the white blood cells [11]. Recently, a similar effect of steroid treatment has been observed on the interaction between white blood cells and the cerebral endothelium both in vivo and in vitro. In models of cerebral inflammation, such as experimental allergic encephalitis, there is an active upregulation of adhesion molecules on vasculature, promoting the infiltration of white blood cells into the brain [12,19,33]. Cytokines, such as IFN-3, and TNF-a, regulate the expression of the these adhesion molecules and it has been shown that corticosteroids and transforming growth factor (TGF)-fl can specifically inhibit their expression [14,37,39,54]. In animal models of brain or spinal cord injury, secondary pathological processes which augment tissue damage have been shown to occur already within a few hours after the trauma [4,10,18]. Bartholdi et al. [4] found that MP efficiently inhibited the infiltration of white blood cells into injured spinal cord within 24 h of hemitransection. In line with this, the current study revealed reduced numbers of T-lymphocytes at the transplantation site in MP- and CsA-treated rats 2 weeks after surgery. Previously, we and others [16,24] have found that a few

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T-lymphocytes are present at the graft-host interface 3-4 days following xenograft implantation and their number increases with time. In the present study, we detected the pro-inflammatory cytokines IL-6, TNF-a and IFN- 7 immunocytochemically in paraformaldehyde-fixed brains. There are many potential sources of errors in the detection of the these cytokines using immunocytochemical techniques, e.g. effects of the fixation protocol on antigen preservation and speciesspecificity of the antibodies employed. We have tried to control for these potential errors. Using our fixation protocol we did detect immunoreactivity in spleen cells, as well as in the brain of a rat with an ongoing rejection. However, the IFN-7 immunostaining revealed an unexpected pattern, with immunoreactivity found within astrocyte like cells, which are thought not to produce IFN-y normally. Possibly we were visualizing IFN-'y bound to the surface of these cells and the IFN-7 was produced and released by nearby lymphocytes. Indeed, often the cell bodies of the astrocytes were not stained, supporting the possibility that the detected IFN-'y was bound to the surface of the cells. We also detected a few stellate cells, of a possible neuronal cell lineage, that expressed IFN-7 immunoreactivity. Neuronal IFN-y has been described recently, although the exact nature and function of this substance remains controversial [31,43]. Among cytokines, IFN-T is kn'.own to be one of the most efficient at increasing both .'~MHC class I and II antigen expression in vitro [21~56,58] and in vivo [52,53,55]. Recently, Subramanianfand co-workers [55] found that retinal xenografts whicl~ normally survived if placed in the mesencephalon of neonatal rats, were rejected after the host rats received recombinant IFN-7 systemically over several days. The MP- and CsA-treated rats in the present study exhibited less IFN-'y immunostaining than the untreated control rats at 2 weeks after surgery. At 6 weeks when the grafts in the untreated control group had been rejected, there was no major difference in INF-7 expression between the groups. We observed an interesting pattern for the TNF-a immunostaining at both 2 and 6 weeks in the MP-treated rats. At 2 weeks after grafting, there was an even distribution of the TNF-a-immunopositive cells in the grafted striatum. This distribution was similar to that of CR3-positive microglia, although only 10-20% microglial cells expressed TNF-a. At 6 weeks the number of microglial cells expressing TNF-a was reduced in a halo around the transplants in the MP-treated rats, although the number and distribution of microglial cells immunostained with the antibody against CR3 was unchanged. This could possibly indicate an active down-regulation of TNF-a production, induced by MP or an endogenous factor such as TGF-/3 or Interleukin-10 (IL-10), as has recently been suggested to occur [5]. TGF-/3 has also been shown to regulate the expression of adhesion molecules on cerebral endothelium [14].

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The immunological reactions in discordant (denoting a large distance between the donor and recipient species) xenograft combinations are not dominated by anti-MHC reactivity. One major biological hindrance for primarily vascularized organ xenografts to survive is direct complement activation [2,40], and pre-formed cross-reactive antibodies against several non-MHC antigens [50]. The most important target for the pre-formed antibodies in solid tissue grafts is the endothelial cells. These host immune mechanisms are most probably operative against intracerebral neural tissue grafts as well, but there are several important differences that may favour neural xenografts. First of all, the graft preparation used in this experiment is secondarily vascularized, with a large proportion of the graft most probably being vascularized via host vessels. Secondly, once reformed, the blood-brain barrier impairs both the passage of complement factors as well as the pre-formed, largely IgM-type xenoreactive antibodies. Although the brain is an immunologically privileged site, it has so far been necessary to immunosuppress recipients of neural xenografts in order to obtain reliable long-term transplant survival [9,46]. CsA is commonly used as an immunosuppressive drug experimentally and clinically, but it has a several toxic side effects when given for prolonged periods of time. In addition to increasing the risk of side effects, it is also expensive. It is of considerable interest to find alternative agents for immunosuppression regimens. Long-term administration of high-dose MP may increase the risk of infections caused by agents cleared by cellular responses, such as fungal infections, viruses and certain bacteria. In addition, there are numerous negative metabolic effects of long-term steroid treatment. None of the MP-treated rats in the present study became infected or died during the experiment. However, one obvious side effect was that the rats significantly lost body weight. This was apparent already 2 weeks after transplantation and resulted in a 20% reduction in body weight by 6 weeks post-surgery. The precise reason for the body weight loss is unknown. Body weight loss was also reported in a study where high-dose MP was given in rats with an acute spinal cord injury [10]. Possibly, low-dose MP may not result in this body weight loss, however, when a lower dose of MP (15 m g / k g , i.p.) was given to rats in a pilot study, 4 of 5 xenografted rats had no surviving graft tissue at 2 weeks after transplantation and the remaining contained only a very small graft (unpublished observation). In conclusion, the experiments show that high-dose steroid treatment offers an additional mode of immunosuppressive treatment for intracerebral neural grafts, as well as offers an insight into the mechanisms of regulation of immune response within the brain. MP may exert effects through a reduction of the vascular adhesion signals. It may also change the balance between pro-inflammatory cytokines and endogenous down-regulatory cytokines to favour the latter, allowing for prolonged

immunologically incompatible graft survival within the brain.

Acknowledgements This study was supported by grants from the Medical Faculty at University of Lund, the Swedish Society for Medical Research, the Neurologiskt Handikappades Riksf/Srbund, the Swedish Medical Research Council (B9412X-10818-01A and K93o/16P 10135-02B), the Elsa Schmitz Foundation, the Ake Wiberg Foundation, the Thorsten and Elsa Segerfalk Foundation, the Kock Foundation and the Crafoord Foundation. E.G.F. was supported by the Boehringer Ingelheim Foundation for Biomedical Research.

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