Temporal pattern of host responses against intrastriatal grafts of syngeneic, allogeneic or xenogeneic embryonic neuronal tissue in rats

Exp Brain Res (1995) 104:227-242

9 Springer-Verlag 1995

Wei-Ming Duan 9HSkan Widner 9Patrik Brundin

Temporal pattern of host responses against intrastriatal grafts of syngeneic, allogeneic or xenogeneic embryonic neuronal tissue in rats

Received: 28 September 1994 / Accepted: 6 December 1994

Abstract The host response to immunologically incom-

patible intrastriatal neural grafts was studied using immunohistochemical techniques. Dissociated ventral mesencephalic tissue from embryonic donors of either syngeneic, allogeneic or xenogeneic (mouse) origin was stereotaxically implanted into adult rats. The brains were analysed 4 days, 2 weeks or 6 weeks after grafting with antibodies against the following antigenic structures: major histocompatibility complex (MHC) class I antigens; MHC class II antigens; complement receptor (CR) 3 (marker for microglia and macrophages); helper T-lymphocyte antigen-cluster of differentiation (CD) 4; cytotoxic T-lymphocyte antigen-CD8; tyrosine hydroxylase (TH) (marker for transplanted dopaminergic neurons). The number of surviving TH-positive cells was not different at the various time points in either the syngeneic or allogeneic groups, whereas the xenogeneic cells were all rejected by 6 weeks. The host reactions were similar in character in the syngeneic and allogeneic groups. At 4 days after implantation, there were increased levels of expression of MHC class I and II antigens. In and around the grafts, there were cellular infiltrates consisting of activated microglia, macrophages, CD4- and CD8-positive lymphocytes. At 6 weeks, MHC expression was reduced and the cellular infiltrates had subsided with only low numbers of activated microglia cells and CD8-positive lymphocytes remaining. In the xenogeneic group, at 4 days, some grafts contained cavities, possibly reflecting acute rejection. At later stages, the xenografts were heavily infiltrated by macrophages, activated microglial cells and T-lymphocytes, and at 6 weeks all the xenografts were rejected. Taken together, the results suggest that there is an inflammation caused by the implantation process which leads to an accumulation of host defence cells. This, in W.-M. Duan (~;) 9H. Widner 9R Brundin Section for Neuronal Survival, Department of Medical Cell Research, Biskopsgatan 5, S-223 62 Lund, Sweden Tel: 46-(0)46-107929, Fax: 46-(0)46-103065 e-mail: [email protected]

turn, leads to increased MHC expression in and around the grafts. In syngeneic grafts, these reactions are short lasting and weak; for allografts slightly more pronounced and longer lasting than syngeneic grafts, but not sufficient to cause rejection. For xenografts, the reactions are more intense and lead to transplant rejection. Thus, a strong sustained inflammatory response may be an important determinator for the failure of histoincompatible neural grafts. It can be speculated that a short-term anti-inflammatory treatment of graft recipients may be a sufficient immunosuppressive regimen to allow long-term graft survival. Key words Neural transplantation. Allogeneic 9 Xenogeneic 9Major histocompatibility complex antigens 9 Rat

Introduction Transplants of immature neural tissue can reduce neurological deficits in progressive neurodegenerative disorders (for review see Lindvall 1991). An increased understanding of the time-course of immunological reactions against intracerebral grafts may increase the utility of neural transplantation as a clinical therapy. The brain is an immunologically privileged transplantation site (Barker and Billingham 1977; Widner and Brundin 1988). The underlying reasons for this are not fully understood. Several contributing factors have been suggested to favour long-term survival of intracerebral neural grafts: the fact that there are relatively few antigen-presenting cells present in the brain parenchyma (Hart and Fabre 1981); paucity of direct drainage to the lymphatic system from the brain interstitial space (Widner and Brundin 1988); a blood-brain barrier that may hinder white blood cells from passing into the brain parenchyma (Cross et al. 1990; Wekerle et al. 1986); and a lack or paucity of transplantation antigen expression on the donor neurons (Lampson 1987). The expression of major histocompatibility complex (MHC) antigens in the grafts is of particular interest,

228

since their presence are considered a prerequisite for immune reactions to be initiated and effected. Recent studies have shown that expression of both MHC class I and class II antigens can occur in and around intracerebral grafts of immature brain tissue (Backes et al. 1990; Date et al. 1988; Duan et al. 1993, 1995; Finsen et al. 1988; Finsen etal. 1991; Isono et al. 1993; Lawrence etal. 1990; Mason et al. 1986; Nicholas et al. 1987; Pollack et al. 1990; Poltorak and Freed 1989, 1991; Rao etal. 1989; Sloan et al. 1990; Wood et al. 1992). The degree of induced MHC antigen expression seems to vary considerably depending on several factors: first, the degree of genetic disparity between the donor tissue and host, with syngeneic grafts giving rise to only short-term, lowlevel MHC expression (Mason et al. 1986); second, the transplantation technique is important, since the greater the trauma to the host brain the more extensive the initial inflammatory response becomes; third, the transplantation site may be critical, with intraventricular as opposed to intraparenchymal grafts having been suggested to be more prone to be rejected (Sloan et al. 1990; Widner and Brundin 1988); fourth, the time that elapses between the transplantation surgery and analysis of MHC antigen expression is crucial since the expression may subside over time. We have previously hypothesised that the duration of marked MHC expression may determine whether a genetically incompatible neural graft will be rejected or survive in the brain (Widner and Brundin 1988). In the existing literature, there is no uniform picture regarding the transplantation conditions under which MHC expression is marked, how long it can be expected to persist, and when it is likely to be part of a rejection process. There is a need to systematically compare the expression of MHC antigens and other markers of host responses at different times following surgery, using donor-host combinations with variable histoincompatibility. Therefore, in the present study we stereotaxically implanted dissociated mesencephalic tissue of syngeneic, allogeneic or xenogeneic origin into the striatum of adult rats. The hosts were sacrificed at 4 days, 2 weeks or 6 weeks after surgery and their brains were processed for MHC class I and II antigens, complement receptor (CR) 3 antigens on microglia and macrophages, helper T-lymphocyte antigen-cluster of differentiation (CD) 4, cytotoxic T-lymphocyte antigen-CD8 and tyrosine hydroxylase (TH) immunohistochemistry.

quently sectioned and adjacent sections through the grafts were stained using antibodies against MHC class I, MHC class II, CR3, CD4, CD8 and TH. Graft survival was assessed by counting the number of TH-immunoreactive cells. The expression of MHC class I and class II antigens and the accumulation of activated microglia, macrophages, and helper and cytotoxic T-lymphocytes in and around the grafts were rated in a semi-quantitative fashion. Graft recipients Normal female SD rats (B&K Universal AB, Sollentuna, Sweden, weighing 200-225 g at the start of the experiment), outbred from an original inbred strain that was RT-1 (rat MHC) haplotype b, were used as graft recipients. Skin grafts were performed and exhibited no signs of rejection, confirming the syngeneic status of these outbred animals (Widner and Brundin 1993).

Donor tissue Syngeneic grafts were prepared from VM tissue of embryonic (E) 14- to 15-day-old (crown to rump length, CRL, 13-14 mm) SD rat embryos (B&K Universal AB, Sollentuna, Sweden). Allogeneic grafts were prepared from VM tissue of E 14- to 15-day-old (CRL 12 mm) Lewis rat embryos (Mr Kcge, Denmark). This Lewis strain is strictly inbred, RT-1 haplotype l. Xenogeneic grafts were prepared from VM tissue of E l 3 - to 14-day-old (CRL 10 mm) mouse embryos, inbred strain C57B1/6, H-2 (mouse MHC) haplotype b (B&K Universal AB).

Dissection, nssue preparation and transplantation surgery Embryonic syngeneic, allogeneic or xenogeneic tissue was dissociated and transplanted into the striatum as described earlier (Brundin and Strecker 1991). In short, pregnant dams were anaesthetised with equithesin (3 ml/kg body weight, i.p.), and the uterine horns were removed and rinsed in Hank's balanced salt solution (HBSS). The VM was dissected under aseptic conditions in HBSS and incubated in 0.1% trypsin (crude type II, Sigma, St Louis, Mo., USA) at 37 ~ C for 20 min. The tissue was then rinsed with sterile HBSS four to five times before gentle dissociation by trituration using fire-polished Pasteur pipettes of decreasing bore diameters. The tissue was dissociated until it was made up of a mixture of single cells and small cellular aggregates. For each dissected VM, around 5.0 btl of medium was added. The viability of all cell suspensions was determined using acridine orange and ethidium bromide as a vital stain, and it was found to be over 90%. Two microlitres of the cell suspension were stereotaxically injected into the right striatum of equithesin-anaesthetised recipient rats using a 10-gl Hamilton microsyringe (Hamilton, Reno, Nev., 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. The injection was performed over 2 rain and the cannula was left in situ for an additional 2 4 min before it was withdrawn.

Materials and methods

Immunohistochemistry

Experimental design

The rats were deeply anaesthetised with chloral hydrate and perfused transcardially with 50-100 ml saline followed by approximately 250 ml cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) over 5 min. After 4 h post-fixation in the same fixative, the brains were kept overnight in 20% sucrose in 0.1 M phosphate buffer at 4 ~ C, then frozen and sectioned coronally at 30 btm 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. Alternate sections were processed for MHC, CR3, CD4, CD8, and TH immunohisto-

Forty-seven Sprague-Dawley (SD) rats were divided into three groups, denoted the syngeneic (S) group (n=17), the allogeneic (A) group (n=15), and the xenogeneic (X) group (n=15). They were implanted with syngeneic (SD rat to SD rat), allogeneic (Lewis rat to SD rat) or xenogeneic (C57/B1/6 mouse to SD rat) grafts prepared from embryonic ventral mesencephalic (VM) tissue. The transplanted rats in each group were further divided into three subgroups that were perfused at one of three different times after surgery: 4 days, 2 weeks or 6 weeks. The brains were subse-

229 chemistry utilising the avidin-biotin complex immunoperoxidase technique (Vectastain Elite ABC; Vector Laboratories, Burlingame, Calif., USA). One complete series of sections (about 12-14 sections) per rat was processed for TH immunohistochemistry. For each of the other antibodies, four to six sections per rat which contained graft tissue were selected and stained.

MHC, CR3, CD4 and CD8 immunohistochemistry. The following monoclonal antibodies from Serotec (Oxford, UK) were used: MRC OX-18, reacting with rat MHC class I antigens; MRC OX-6 reacting with rat MHC class II antigens; MRC OX-42, reacting with rat CR3 present in high amounts on microglia and macrophages; MRC W3/25, reacting with CD4, to a lesser degree with macrophages/microglia and to a low degree with subsets of neurons; and MRC OX-8, reacting mainly with CD8. Normal mouse serum was used as a negative control. Phosphate-buffered saline (PBS) instead of the primary monoclonal antibodies was also used as a negative control. Briefly, free-floating sections were rinsed and treated with 3% hydrogen peroxide (H202) and 10% methanol in PBS to quench endogenous peroxidase activity. The sections were then sequentially incubated in 10% normal horse serum (NHS) and 0.3% Triton X-100 in PBS for 1 h to block unspecific immunostaining; mouse monoclonal antibodies (OX-18, OX-6 or OX-42) diluted 1:400 for 16 h and mouse monoclonal antibodies (W3/25 or OX-8) diluted 1:400 for 1 h in PBS containing 5% NHS and 0.5% bovine albumin (Sigma), biotinylated horse antimouse (rat-adsorbed) immunoglobulins (Vector Laboratories) diluted 1:200 in PBS containing 0.5% bovine albumin for 1 h and diluted ABC solution (one drop A and one drop B solution in 5 ml PBS) prepared from Vectastain ABC kit (Vector Laboratories) for 1 h. Each incubation was performed at room temperature and the sections were washed three times in PBS following each incubation except the pre-incubation. After the final wash, the sections were incubated with 0.05% 3,3'-diaminobenzidine (Sigma) as a chromogen and 0.04% H202 in PBS to visualise the immunoreactire products. The sections were mounted on glass slides, dehydrated through graded concentrations of alcohol, defatted in xylene, and cover-slipped using DePeX mountant (Serva, FRG).

Tyrosine hydroxylase immunohistochemistry. Adjacent sections were processed for TH immunohistochemistry, using a protocol similar to that described above. The difference was that the sections were pre-incubated in 5% normal swine serum prior to incubation with a rabbit polyclonal antibody against TH (Pel-Freez, Rogers, Ark., USA). The secondary antibody was biotinylated swine anti-rabbit immunoglobulins (Dakopatts, Copenhagen, Denmark). Semi-quantitative and quantitative evaluation of brain sections The sections processed for MHC Class I and II, CR3, CD4 and CD8 immunostaining were semi-quantitatively evaluated microscopically under bright-field illumination on blind-coded slides. For each antibody, four to six sections containing graft tissue were rated. Each section was assigned by two independent raters to one of the following categories: (0) no specific immunostaining in the graft area; (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. In general, the correspondence between the two raters was very high. After all sections had been rated, the final score for each graft was determined based on the highest score given. The median value for each group was plotted. The cell morphology and the level of immunostaining of microglia located in the hemisphere contralateral to the graft were used to define resting microglial cells. These resting cells were consistently labelled by the OX-42 antibody, but were smaller and less intensely stained than the macrophages and activated micro-

glial cells found in the grafted hemisphere. Only activated microglial cells were included in the rating. The size of the CD8-positire cells was estimated by measuring the mean diameter of 40 randomly selected cells for each survival time point in each group using computer-assisted image analysis (Nakao et al. 1994). THimmunoreactive neurons were counted microscopically under bright-field illumination on blind-coded slides. The raw counts for TH-immunoreactive cells were multiplied with a correction factor of 2.7 according to the Abercrombie formula (Abercrombie 1946). Statistical analysis Statistical comparisons of TH-positive cell numbers were performed parametrically using an unpaired Student's t-test between the 4 days and the 6 weeks groups in order to detect any rejection at the latter time. P values below 0.05 were regarded as statistically significant.

Results T h e results section is d i v i d e d into three m a i n parts w h i c h d e s c r i b e the results for the s y n g e n e i c , allogeneic, and xen o g e n e i c groups, separately. In e a c h part, the results for the five different a n t i b o d i e s are d e s c r i b e d . O n e rat in the a l l o g e n e i c group d i e d during the e x p e r i m e n t . F i g u r e 1 A - E s u m m a r i s e s the s e m i - q u a n t i t a t i v e ratings o f the i m m u n o c y t o c h e m i c a l staining for M H C class I (A) and class II (B), CR3 (C), C D 4 (D) and C D 8 (E) antigens. B l a c k dots in this figure indicate the rats from w h i c h the p h o t o m i c r o g r a p h s in Figs. 3 - 6 have b e e n taken.

S y n g e n e i c grafts

TH immunohistochemistry A l l rats had surviving grafts at all three t i m e - p o i n t s . T h e r e was no significant d i f f e r e n c e in the m e a n n u m b e r o f T H - i m m u n o p o s i t i v e cells b e t w e e n the different timep o i n t s (P>0.05, Table 1). A t 4 d a y s after transplantation, the cells w e r e quite evenly d i s t r i b u t e d t h r o u g h o u t the graft tissue (Fig. 2A). A t 2 w e e k s after transplantation, the T H - i m m u n o p o s i t i v e n e u r o n s t e n d e d to be l o c a t e d a r o u n d the p e r i p h e r y o f the grafts (Fig. 2B), and at 6 w e e k s after transplantation, this was even m o r e o b v i o u s , l e a v i n g the centre o f the grafts r e l a t i v e l y d e v o i d o f T H i m m u n o r e a c t i v i t y (Fig. 2C). T h e T H - p o s i t i v e neurons p o s s e s s e d a m u l t i p o l a r cell b o d y with several c l e a r l y stained neurites.

MHC immunohistochemistry T h e i m m u n o s t a i n i n g s for M H C class I a n d II a n t i g e n s w e r e l o w at all t i m e s (Fig. 1A, B). A t 4 d a y s after t r a n s p l a n t a t i o n , t h e r e was d e t e c t a b l e s t a i n i n g for b o t h M H C class I a n d II a n t i g e n s on a l o w n u m b e r o f cells a r o u n d a n d i n s i d e the grafts. T h e r e w a s no e v i d e n c e o f p e r i v a s c u l a r cuffing. T h e c e l l s w e r e p r e d o m i n a n t l y r o u n d a n d o f two d i f f e r e n t sizes a n d r e s e m b l e d l y m p h o -

230 Fig. 1A-E Arbitrary rating of the immunohistochemical staining using different antibodies. The bar represents the median value for all rats in the group, and the circles represent the individual rats. Filled circles indicate rats in which the photomicrographs for Figs. 3-6 were taken. A MHC class I. B MHC class II. C CR3. D CD4 lymphocyte. E CD8 lymphocyte. (S Syngeneic grafts, A allogeneic grafts, X xenogeneic grafts). The following categories were used: (0) no specific immunostaining in the graft area; (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

A 4

B 4

3

2

1

', A X

S A X

S A X

s A X

S A X

S A X

4 days

2 weeks

6 weeks

4 days

2 weeks

6 weeks

C 4

D4

CR3

CD4

3

O

2

00

i .........

s A X 4 days

S A X 6 weeks

S A X 2 weeks E4

-

S A X

S A X

S A X

4 days

2 weeks

6 weeks

o

CD8

3 -

2 -

J 1

m

o

N Table 1 The mean number of TH-immunopositive neurons (mean_+SEM) in the neural grafts in each group at three timepoints (figures in parentheses represent number of rats in group)

Syngeneic Allogeneic Xenogeneic

4 days

2 weeks

6 weeks

993+232 (6) 936+184 (5) 662_+138(5)

1525_+354(6) 1506_+438(4) 650+390 (5)

870_+245(5) 1393_+213(5) 2_+2 (5)*

* P