Introduction

The brain is considered to be an immunologically privileged site [2]. Immune reactions that take place are rarer and weaker than in other organs. This privilege has been attributed to the presence of local immunosuppressive factors [32], the blood-brain barrier [20], the lack of a conventional lymphatic drainage [9], the lack of classical antigen-presenting cells (APC) [19], or to the paucity of MHC antigen expression [3]. However, in contrast to the transplant immunology of other organs such as heart, liver or kidney, only relatively few studies have addressed questions of graft immunology in the central nervous system (CNS).

Currently transplantation of fetal cells, cell lines or, probably even more importantly, stem cells is under close investigation and intense discussion as an experimental therapeutic option in neurodegenerative disorders such as Parkinson’s [14, 17, 21, 27, 34, 37] or Huntington’s disease [1, 16], or even in multiple sclerosis [39]. In clinical trials, these experimental treatments will still require allogeneic graft material and raise questions of graft rejection and immunosuppression.

So far, experimental studies that have addressed these questions have yielded to diverging results. Some authors observed a spontaneous rejection of neural grafts [6, 26, 35], while others demonstrated transplant rejection only after induction [12, 15, 40]. Furthermore, there is evidence that some neural allografts do not undergo rejection even after systemic sensitization [24, 41]. The most probable reasons for these conflicting results are differences in the experimental protocols. For instance, it has been shown that not only the degree of genetic difference between donor and host influences the outcome of neural grafts [31], but also the age of donor and host [30] as well as the severity of the transplantation trauma [4].

Dopaminergic cell suspension grafts represent the gold standard in transplantation for Parkinson’s disease. These show a better survival and integration into the host brain than do solid grafts and are considered to be less immunogeneic. Brandis et al. [5], for example, demonstrated good survival of allogeneic fetal dopaminergic cell suspensions in the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats without immunosuppression. Only in 1 of 31 animals was a vivid immune response observed at 1 week after transplantation [5]. Interestingly, in this animal, parts of the cell suspension had accidentally been placed into the ventricular system, which is supposed to be an immunologically less privileged site than the CNS parenchyma [47]. However, no immunological studies investigating intraventricular cell suspension grafts have been performed so far. This is even more surprising since the ventricular system is considered to be an alternative grafting site for fetal dopaminergic grafts [22, 49].

Therefore, the aim of the present study was to analyze the development of allogeneic fetal dopaminergic cell suspension grafts in the ventricular system of the rat without immunosuppression and to evaluate the immunological donor-host interactions with particular attention to donor- and host-specific MHC expression and cellular infiltration in a possible rejection response.

Materials and methods

Experimental design

Adult Lewis rats received allogeneic dopaminergic single cell suspension grafts from Dark Agouti fetuses into the right lateral ventricle. Rats with syngeneic grafts served as control. No immunosuppression was administered. At 4 days, and 1, 3, 6, and 12 weeks after transplantation, the animals were killed, and the brains were processed for immunohistology to stain donor- and host-specific MHC class I and II molecules, microglia, macrophages, and various lymphocyte subtypes, as well as dopaminergic neurons and astrocytes. Results were obtained qualitatively and semiquantitatively at a light microscopic level.

Animals

Adult female Lewis1.W rats (LEW1.W, Zentrales Tierlabor, Hannover Medical School, Hannover, Germany), weighing 200–250 g at the time of surgery, were used as graft recipients. LEW1.W is an inbred congenic Lewis strain with the “u” haplotype of the MHC locus (RT1u), corresponding to a high responder type [8]. Ventral mesencephalic grafts were obtained from fetuses at embryonic day (E) 14–15 (crown-rump length 11–13 mm) either from the same strain for syngeneic controls (n=12 control) or from Dark Agouti rats (DA, Zentrales Tierlabor, Hannover Medical School) for allogeneic grafts (n=28 allo). DA is an unrelated inbred rat strain with an “av1” haplotype at the MHC locus (RT1av1) and differs from LEW1.W rats with respect to MHC and non-MHC antigens [18]. All animals were housed according to standard conditions and had free access to food and water.

Surgery

All surgical procedures were performed under deep general anesthesia (0.1 ml 10% Ketamine/100 g body weight). Ventral mesencephalon of E14–15 DA or LEW1.W fetuses was dissected under a stereomicroscope and a single-cell suspension was prepared according to the modified version [36] of the standard protocol by Björklund et al. [4]. In brief, dissected ventral mesencephali were incubated in 1 ml 0.1% trypsin/0.05% DNase/DMEM at 37°C for 20 min, followed by three to four rinses with 300–400 µl each of 0.05% DNase/DMEM. Mechanical dissociation of the tissue pieces was performed in 250 µl 0.05% DNase/DMEM by repeated trituration until the suspension became milky. After centrifugation at 600 rpm for 5 min and resuspension in the predetermined final volume of 0.05% DNase/DMEM, a total cell number of about 100,000 cells/µl resulted. The viability of the cells was estimated by the Trypan blue dye exclusion method and scored 98–99%. Immediately after the preparation procedure, each animal received 7 µl of the single-cell suspension stereotactically implanted using a 10-µl Hamilton microsyringe fitted to a steel cannula with an outer diameter of 0.5 mm. Allogeneic or syngeneic grafts were slowly injected into the right lateral ventricle using a stereotactic frame with the following coordinates: AP +1.0, L 1.5, V 4.0 with the toothbar set at 0.

Histology

Animals were killed at 4 days (n=2 allo), and 1 (n=5 allo, n=2 control), 3 (n=7 allo, n=4 control), 6 (n=6 allo, n=2 control), and 12 weeks (n=8 allo, n=4 control) after transplantation. The unfixed brains were removed after exsanguinating the deeply anesthetized animals by opening the left cardiac ventricle. A coronal slice of the brain containing both lateral and the 3rd ventricle was excised, embedded in OCT medium and immediately quick frozen in precooled isopentane. Each coronal slice was completely worked up in serial cryostat sections. Every 100 µm, a section was stained with H&E, methyleneblue or thionine for orientation. Each graft was completely cut up into 5-µm serial cryostat sections, collected on poly-l-lysine-coated glass slides. Alternating series of sections were either air-dried overnight and stored at −20°C for further MHC and lymphocyte typing or immediately fixed in 1% glutaraldehyde, followed by methanol and acetone for further tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) staining.

Immunohistochemistry

All steps were performed in TRIS-buffered saline (TBS; 0.5 M TRIS-HCl pH 7.6, 0.85% NaCl). Twelve different primary antibodies were used for visualization of MHC donor and host class I and II molecules, macrophages, microglia, lymphocytes, astrocytes and dopaminergic neurons as shown in Table 1. For staining of MHC, macrophages and lymphocytes, sections were sequentially incubated with the primary antibody after fixation with isopropanol for 10 min at 4°C, diluted in TBS/1% bovine serum albumin, biotinylated rabbit anti-mouse antibody, diluted 1:50 in TBS/5% normal rat serum and in a third step with a streptavidin-biotinylated horseradish peroxidase complex. For TH staining, all steps were performed in TBS/0.1% Triton X-100. Following preincubation with 5% normal swine serum, sections were incubated with the primary antibody for 48 h at 4°C. A biotinylated swine anti-rabbit serum (1:200) was used as secondary antibody, followed by StreptABC. For GFAP staining, incubation with the primary antibody was followed by a secondary swine anti-rabbit serum, conjugated to horseradish peroxidase (1:100 in TBS/5% normal rat serum). At the end of each staining protocol, peroxidase activity was visualized by incubation for 10 min with 0.5 mg/ml 3,3’-diaminobenzidine-tetrahydrochloride in TBS containing 0.01% H2O2. After rinsing in TBS, sections were slightly counterstained with 10% hemalaun, dehydrated and coverslipped with corbit. Positive and negative controls were run with each series. Negative controls were prepared by replacing of the primary antibody with an antibody of the same IgG subclass that is non-reactive with rat tissue.

Table 1 Primary antibodies
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Morphological evaluation

Graft morphology was evaluated qualitatively with respect to the following criteria: time-dependent graft development; arrangement and morphology of TH-positive neurons; vascularization and glial cell reaction; cellular reaction at the graft-host interface and within the grafts; signs of an immune response, such as types and localization of cells expressing donor- or host-specific MHC I antigens, types and localization of cells expressing MHC II antigens, types of infiltrating cells and time course of infiltration; and signs of necrosis, death or survival of grafted cells including TH-positive neurons.

In addition, the number of TH-positive neurons at 3, 6 and 12 weeks after grafting was counted. The extent of the MHC expression and the cellular reaction (macrophages, lymphocytes) were assessed semiquantitatively according to criteria previously described [5]. Each section was rated according to one of the following categories: (0) no specific immunostaining above background staining; (1) very low number of positive cells, distributed as scattered single cells in the graft or at the graft border, no clusters of positive cells; (2) low number of positive cells, either singly or in small clusters; (3) medium number of positive cells, mostly arranged in patches, (4) large number of positive cells in clusters, which may be confluent, plus focal dense immunostaining of the graft area without discernible cell contours; and (5) very dense immunostaining of the whole graft and graft border with a very large number of positive cells. From each animal, between six and ten sections containing graft tissue were evaluated for each primary antibody and the median value per staining reaction was calculated.

Results

Graft morphology

On average three to four grafts (range two to ten) were found in each animal. Most transplants were located in the right lateral ventricle, some in the left lateral or third ventricle. All grafts lay in contact to the ventricular wall, and partly to the choroid plexus. No signs for hydrocephalus were detected.

At 4 days and 1 week after grafting, syngeneic and allogeneic grafts imposed as aggregates of closely packed immature neuroblasts with large hyperchromatic nuclei and scanty cytoplasm. Some of these cells showed weak immunoreactivity for TH. The grafts contained a few ED1- and MHC class II-positive macrophages located close to the graft-host interface. The latter were exclusively host derived, as seen by the OX3 staining in the allogeneic transplants.

At 3 and 6 weeks after transplantation, the grafts appeared larger and in close contact to the host parenchyma. In syngeneic grafts, a few macrophages were located in the grafts (Fig. 1a). In some cases, small vessels penetrating the grafts from the host parenchyma were seen. In many transplants, the ependymal cells at the graft-host interface had partly disappeared, and a new ependymal lining grew continuously over the free ventricular surface of the grafts. Polygonal neuronal cells with large pale nuclei and Nissl substance were crowded in the center of the grafts, and were embedded in a fibrillary matrix resembling neuropil of mature gray matter. A small number of these cells were dopaminergic neurons as seen by TH staining. The astrocytic response was vivid at these time points. Reactive astrocytes with broad cytoplasm and thickened processes were mainly aligned along the graft-host interface and at the free ventricular surface of the grafts. In addition, allogeneic grafts showed an intense astrocytic reaction in areas of cellular infiltration (see below).

Fig. 1 a
figure 1

OX18 staining of a syngeneic graft in the right lateral ventricle at 3 weeks after transplantation. The grafts were larger and closely attached to the ventricular wall. A few MHC class I-expressing macrophages are indicated by arrowheads. b TH staining of several neurons (arrowheads) in a well-integrated allogeneic graft in the left lateral ventricle at 12 weeks after grafting. Arrows indicate the estimated graft-host interface (TH tyrosine hydroxylase). a, b ×125

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At 12 weeks after grafting, transplants with a broad contact area to the host parenchyma were hardly discernible. The ependymal layer at the graft-host interface had often completely disappeared, and a new continuous ependymal layer covered the grafts at their ventricular surface (Fig. 1b), thus separating them from the ventricular system (53 of 62 grafts). Graft morphology corresponded to mature gray matter with TH-positive neurons (Fig. 1b). The other 9 transplants did not show such an intimate contact to the host parenchyma and presented with features similar to those observed at 3 and 6 weeks after grafting. These grafts showed a more intense astrocytic response between graft and host parenchyma, as well as in the center of the grafts and in the region of the ventricular surface.

Immune response

Animals with allogeneic grafts showed an immune response that was most intense at 3 and 6 weeks after grafting and declined in most grafts thereafter.

At 3 and 6 weeks, parenchymal and choroidal vessels in the vicinity of the grafts were dilated and showed marginated cells which, in addition, formed perivascular cuffs and diffusely infiltrated the grafts (Fig. 2a, b). Most were ED1-positive macrophages, followed by OX8-reactive cytotoxic T lymphocytes; a smaller number of cells stained for OX35 and looked like lymphocytes rather than macrophages; only few cells turned out to be His14-positive B-lymphocytes. Simultaneously to the cellular infiltration, a strong MHC expression occurred. The transplants stained intensely for donor-MHC class I (Fig. 2a) as well as common MHC class I, respectively, the perivascular cuffs and the cellular infiltrates reacted for host-MHC class II (Fig. 2b). No donor-MHC class II expression was observed.

Fig. 2 a
figure 2

Donor-MHC class I expression of the hole allogeneic graft (T) in the right lateral ventricle at 3 weeks after transplantation (MN4-91-6-staining). b Host-MHC class II expression of the infiltrating cells in the graft of a. a, b ×125

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At 12 weeks after transplantation, the cellular infiltration had remarkably decreased. Out of the 57 allogeneic transplants found at this time point, 47 did not show strong signs of an immune response, such as cellular infiltration or strong MHC class I expression except some residual macrophages within the grafts. Interestingly, these grafts displayed a continuous ependymal layer at their free ventricular surface. These grafts possessed TH-positive neurons, which appeared to be intact (Fig. 1b). In contrast, the remaining 10 transplants demonstrated more clearly remnants of an immune reaction with clusters of lymphocytes and macrophages and MHC class I expression, similar to the findings at 3 and 6 weeks after transplantation. These grafts were not well integrated into the host parenchyma and either adhered only loosely to the ventricular wall or showed an incomplete ependymal layer at their ventricular surface.

Neuronal cell count and semiquantitative evaluation

A mean of 26 TH-positive grafted neuronal cells (range 19–30) was counted per animal at 3, 6, and 12 weeks after grafting. There was no significant difference in the mean number of positive cells per animal for syngeneic and allogeneic grafts (26 cells vs 24 at 3 weeks, 24 vs 28 at 6 weeks, 27 vs 27 at 12 weeks after transplantation).

In the semiquantitative evaluation, the values for MHC class I and MHC class II expression and those for infiltrating cells corresponded to the time schedule observed. All values reached a peak at 3 weeks after grafting, declining thereafter to a variable degree. There was a significant difference in the MHC expression between allogeneic and syngeneic grafts from 3 weeks on. In detail, allogeneic as well as syngeneic grafts were assessed as grade 1 for MHC class I at 4 days and 1 week. While the syngeneic grafts stayed at this level, the allogeneic transplants showed a strong increase with a peak of grade 5 (common MHC class I) and grade 4 (only donor-MHC class I) at 3 weeks, which slowly declined thereafter to grade 3 (common MHC class I) and grade 2 (donor-MHC class I) at 12 weeks after transplantation. For donor-MHC class II, the stainings were assessed as grades 0 and 1 for syngeneic and allogeneic grafts at 4 days and 1 week. In the syngeneic grafts, the value returned to 0 from there on. In contrast, in the allogeneic grafts, the donor-MHC class II stainings reached a peak of grade 3 at 3 weeks which declined to grade 2 thereafter. The stainings for infiltrating cells in the allogeneic transplants corresponded to the pattern described with a peak infiltration at 3 weeks after grafting. The stainings for infiltrating cells in the syngeneic grafts were not assessed semiquantitatively.

Discussion

Since transplantation of allogeneic fetal dopaminergic cells or allogeneic stem cells is under close investigation as an experimental treatment option for neurodegenerative disorders such as Parkinson’s or Huntington’s disease or for multiple sclerosis, interest in neural graft immunology is increasing. The present study showed that fetal dopaminergic cell suspension grafts survive well in the ventricular system. However, in contrast to previous reports of intraparenchymal transplants [5], the study also gives evidence that a spontaneous specific immune response to allogeneic dopaminergic fetal cell suspension grafts can occur in the ventricular system. Surprisingly, the results also indicate that such a strong immune reaction can subside without rejection, leaving intact, healthy appearing grafts behind. So far this has seemed to be a contradiction in terms. Thus, the results raise the questions what the characteristics of the observed immune response were, why it subsided without complete graft rejection and finally why, with respect to previous studies [5], the ventricular system appears to be less immunologically privileged than the host striatum.

Does the observed immune response correspond to immunological graft reactions of previous reports?

MHC expression is thought to play an important role in the initiation of allogeneic transplant rejection and killing of the targeted graft cells. MHC class II expression is an important characteristic of APC which initiate the immune reaction. The antigen is presented to T helper lymphocytes, which stimulate other lymphocytes including cytotoxic T lymphocytes and infiltrate the transplants. The MHC class I antigen of the transplanted cells is recognized by the cytotoxic T lymphocytes which consecutively kill the MHC class I-positive cells [43].

In the present study, MHC class II-positive host microglia and host macrophages infiltrated the grafts at an early stage, indicating their potential role as APC for the observed immune reaction. This supports the results of earlier reports where microglia [11, 29, 41, 44] or brain macrophages [7] were considered to function as APC within the CNS. However, in contrast to other authors [13, 26, 48, 50] who suggested an important role of donor MHC class II-positive APC for the initiation of an immune response to allogeneic grafts within the CNS, a strong immune reaction to allogeneic grafts was initiated without a significant number of donor MHC class II-positive cells in the present study. Thus, most likely donor MHC class II-positive cells are not required to provoke a vivid immunological response to allogeneic grafts in the brain.

However, the graft and host MHC class I expression as well as the infiltration pattern by macrophages and lymphocytes in the present study is in concordance with the results of rapidly rejected allogeneic neural grafts [10, 12, 26, 35, 46]. This reaction characteristically ends with necrosis of the graft. To our knowledge, such a strong donor MHC class I expression and cellular infiltration have not been reported with well surviving intracerebral transplants.

It remains speculative what the reasons responsible for this subsidence were. It has been shown that the selected rat strains reject peripheral nerve grafts after transplantation to each other [25], so it appears to be unlikely that there is a basic immunological deficit for transplant rejection between them. Nevertheless, it cannot be excluded that the host immune system was insufficiently activated, for example by lacking MHC class II-positive donor-APC as previously discussed, or that the immune attack was not long enough maintained for transplant rejection. On the other hand, it has been shown that intracerebral antigen injection leads to an activation of lymphocytes in the deep cervical lymph nodes [38]. Furthermore, intraventricular antigen deposition provokes a much stronger cellular reaction by the host than antigen deposition into the host parenchyma [51]. Together with the finding in this study that the immune reaction was especially weak in transplants that were separated from the ventricular system by a new ependymal layer, this might indicate that the limitation of exchange of cell debris or substances between the graft and the ventricular system caused by a new ependymal cell layer at the free graft surface led to a decline of the immune reaction by a lack of continuous activation of the host immune system.

What are the specific characteristics of the ventricular system that could contribute to a more immunoreactive environment for allogeneic fetal dopaminergic cell suspension grafts?

The present study enables in combination with a previous report of corresponding intrastriatal grafts [5] a comparison of the immunoreactivity of the ventricular system and the striatum. In both studies almost identical experimental protocols were employed with the exception that in the earlier study a smaller volume was grafted and that the recipients possessed a 6-OHDA lesion. Nevertheless, a major influence of these parameters on the outcome of the grafts appears to be unlikely because, as mentioned earlier, also in the study of intrastriatal grafts a strong infiltration of one transplant could be observed after accidental grafting into the lateral ventricle. In contrast to the present results with intraventricular grafts, the intrastriatal transplants were not subjected to a vivid immune response and did not strongly express donor-MHC class I or II at any time point examined. Therefore, the ventricular system seems to be immunologically less privileged for allogeneic fetal dopaminergic cell suspension grafts than the striatum.

Possible causes for this different immunoreactivity of the ventricular system are several factors. The choroid plexus epithelium possesses additional macrophage populations, which histologically resemble classical MHC class II-positive APC [33] and possess a high phagocytotic activity [28]. Although under discussion as immunosuppressive functions [45], they might contribute to the different immunoreactivity of the ventricular system. Other factors may be the partial lack of the blood-brain barrier within the ventricular system [28], or the stronger antigen drainage to cervical lymph nodes after antigen deposition into the ventricular system than into the host parenchyma [51]. The findings of MHC class II-positive macrophages in the graft area adjacent to the choroid plexus and the possible influence of the ependymal layer on the subsidence of the immune reaction in the present study particularly point to a potentially role of choroid plexus macrophages and the higher antigen drainage for the stronger immunoreactivity of the ventricular system.

In all, the present study demonstrated that the ventricular system provides a suitable environment for fetal cell suspension grafts. It further showed that a strong immune response to allogeneic grafts can occur in the ventricular system. Thus, in comparison to previous reports, the ventricular system seems to provide a more immunoreactive environment than the striatum. Possible reasons for this difference are additional populations of potential APC, different pathways for drainage of interstitial fluid and the absence of the blood-brain barrier. Additionally, the present study seems to indicate that a strong specific immune reaction, which corresponded in all investigated characteristics to classic rejection reactions in the CNS, can decline and at least even partly run out, although intact healthy looking grafts are left. The reasons for this subsidence remain unknown, and further studies are needed. Based on the results of the present study, the ependymal cell layer could play an important role in preserving the immunological privilege of the CNS. Also, donor-MHC class II expression could be important for graft rejection. In conclusion for any clinical trials with allogeneic graft material in the CNS, the current study highly recommends the administration of immunosuppression until more studies further enlighten the peculiar characteristics of neural graft immunology.