Both Bone Marrow- and Non-Bone Marrow-Associated Factors Determine Susceptibility to Experimental Autoimmune Encephalomyelitis of BUF and WAG Rats

CELLULAR IMMUNOLOGY ARTICLE NO.

168, 39–48 (1996)

0047

Both Bone Marrow- and Non-Bone Marrow-Associated Factors Determine Susceptibility to Experimental Autoimmune Encephalomyelitis of BUF and WAG Rats MICHEL VAN GELDER,* ELS P. M. KINWEL-BOHRE´,* ANDRIES H. MULDER,†

AND

DICK W.

VAN

BEKKUM*

*Introgene, P.O. Box 3271, 2280 HV Rijswijk, The Netherlands; and †Pathologisch Laboratorium voor Dordrecht e.o., Jkvr. van den Santheuvelweg 2a, 3317 NL Dordrecht, The Netherlands Received September 19, 1995; accepted October 24, 1995

In the case of experimental autoimmune encephalomyelitis (EAE, an AID of the central nervous system (CNS) induced by immunization with myelin antigens (13)) in rodents, most authors agree that susceptibility and resistance of hemopoietic chimeras are determined by the BM donor (14–18). However, one group of investigators reported a reversed relationship (19, 20) and another group showed that the response of BM chimeras to induction of EAE was modified by the host (21). As these studies are limited to a few rat and mouse strains, there is no guarantee that the results with other strains will be the same. We have developed an EAE model in BUF rats for the purpose of evaluating the therapeutic effect of BMT (7, 22). Because the influence of the BM in determining susceptibility had not been established in this model, we determined the susceptibility to EAE of allogeneic BM chimeras, using BUF and WAG rats as well as their F1 hybrid. The WAG rat strain was selected for being low responsive to EAE. We grafted T cell-depleted (TCD) BM in most experiments, in order to prevent graft-versus-host disease (GVHD). The TCD also precluded the grafting of immunocompetent lymphocytes which may transfer resistance or susceptibility to EAE, thereby masking the influence of non-BM-derived factors. Our results show that the susceptibility of hemopoietic chimeras of WAG and BUF rats is not exclusively determined by the responsiveness of the BM donor; host factors, not associated with the BM, modulate the response. Thus far, only one such host-associated factor has been identified, i.e., an association between the magnitude of the stress-induced corticosterone response and susceptibility to EAE, as originally described in PVG and Lewis rats (23). Accordingly, we have investigated the role of the adrenals in our high and low responder strains, by recording the stress-induced corticosterone response and the effect of adrenalectomy. We also attempted to identify the BM-associated factor of the low responsiveness to EAE in WAG rats. To

To determine the feasibility of treatment with allogeneic bone marrow for experimental autoimmune encephalomyelitis, we investigated the susceptibility to experimental autoimmune encephalomyelitis of bone marrow chimeras using BUF, (WAG1BUF)F1 (high responder), and WAG (low responder) rats. In contrast to previous studies in which other rat strains were used, the response was largely determined by the genotype of the grafted bone marrow, but the influence of a non-bone marrow-associated factor was evident. The latter factor was identified as a low corticosterone response in BUF rats and a high corticosterone response in WAG rats. After adrenalectomy, a significant proportion of WAG rats developed clinical experimental autoimmune encephalomyelitis. The bone marrowassociated factor of resistance appeared to be the activity of cyclophosphamide-sensitive suppressor cells. The latter cells were found to interfere with the formation of inflammatory foci in the central nervous system, while corticosterone primarily prevents the clinical expression of lesions. q 1996 Academic Press, Inc.

INTRODUCTION Susceptibility to experimental autoimmune diseases (AID) can be transferred to resistant rodent strains by bone marrow transplantation (BMT) (1). Ikehara et al. (2) showed that even purified hemopoietic stem cells may transfer susceptibility to AID. This led them to postulate that AID originate from defects in hemopoietic stem cells. In various experimental studies (3–7) and a limited number of clinical cases (8–12) beneficial effects have been reported of treatment of AID with high-dose cytoreductive treatment and allogeneic BMT. If the stem cell theory were universally valid, the use of allogeneic BM would represent a sound approach, because the ‘‘susceptible’’ stem cells of the patient would be replaced by disease-resistant donor cells. 39

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0008-8749/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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date, only one such factor has been described in various rat and mouse strains, being the cyclophosphamide (CY)-sensitive suppressor cell (24–29). Therefore, the effect of pretreatment with CY on the response of WAG rats to induction of EAE was investigated. Finally, we excluded the possibility that the low response to the induction of EAE of WAGrBUF rats is caused by a relative immune deficiency, which may occur after allogeneic BMT in certain donor–recipient combinations. For this purpose, we tested the immune responsiveness of WAGrBUF BM chimeras by inducing another AID, i.e., type II collagen-induced arthritis (CIA). This disease was chosen because WAG rats are high responders and BUF rats are low responders (30).

anti-mouse IgG1 combined with streptavidin–TRITC were used (Serotec, Oxford, UK). Anti-BUF and anti-WAG erythrocyte antibodies were raised in WAG and BUF rats, respectively, by intraperitoneal immunization with an amount of washed erythrocytes equivalent to 10 ml of whole blood, in complete adjuvant. Five weeks later, rats were reimmunized with the same amount of red blood cells in incomplete adjuvant. Seven to 10 days later, the rats were bled by aortic puncture. The titer and specificity of the antisera were determined by flow cytometry of target erythrocytes, employing FITC-conjugated goat anti-rat IgG as secondary antibody (Tago, Burlingame, CA). Induction and Evaluation of EAE

MATERIALS AND METHODS Animals The inbred rat strains BUF/SimRij (RT1b, RT7.2), WAG/Rij (RT1u, RT7.1), and their F1 hybrid were bred in our own animal breeding facilities (now Harlan CPB, Zeist, The Netherlands) and kept under SPF conditions until weaning. BUF/SimRij rats were introduced in our laboratory in 1982 from Simonsen Laboratories, Inc. (Gillroy, CA). WAG/Rij rats were developed originally by Bacharach, Glaxo Lab. (UK) and introduced in our laboratory in 1953. All animals were 12 to 16 weeks old at the onset of the experiments. Antibodies For staining rat T lymphocytes, the monoclonal antibodies OX19 (anti-rat CD5, mouse IgG1) (31) and ER1 (anti-rat ‘‘W3/13’’ antigen, mouse IgG2a) (32) were used. OX34 (anti-rat CD2, mouse IgG2a) (33) and R73 (anti-rat T cell receptor ab, mouse IgG1) (34) were used for T cell depletion from BM grafts. U9F4 (anti-RT1.Au, mouse IgG2a) (35) and HIS41 (anti-RT7.2, mouse IgG2b) (36) were used to determine the origin of mononuclear leukocytes in BM chimeras by flow cytometry. For the latter purpose, rat anti-mouse IgG (H/L) FITC (Jackson, West Grove, PA) was used as secondary antibody. ED1 (mouse IgG1) was used for staining macrophages (37). IL-2 receptor-expressing (IL-2-R/) cells were determined by staining with OX39 (mouse IgG1) (38). Hybridoma cell lines OX19, OX34, and OX39 were kindly donated by D. Mason (Oxford, UK), as was the R73 hybridoma by T. Hu¨nig (Wu¨rzburg, Germany). Supernatant of HIS41 and biotinylated OX19 and R73 were generous gifts of J. Kampinga (Groningen, The Netherlands). The U9F4 and ER1 secreting hybridomas were a gift from J. Rozing (Leiden, The Netherlands), and ED1 was a gift from C. D. Dijkstra (Amsterdam, The Netherlands). For immunohistochemistry, sheep anti-mouse IgG2a FITC and biotinylated sheep

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EAE was induced by immunization with BUF rat spinal cord (RSC) homogenate and Mycobacterium tuberculosis H37RA enriched complete adjuvant. RSC homogenate was prepared as follows. The spinal cord was mixed with phosphate-buffered saline (PBS) and homogenized using an IJstral bar mixer. PBS was added to obtain a final concentration of 0.5 g RSC/ml, and this was stored at 0207C until use. The RSC homogenate was emulsified with complete adjuvant (1 mg M. tuberculosis H37 RA/ml, Difco) supplemented with 10 mg M. tuberculosis H37 RA (Difco) per milliliter. Immunization was by subcutaneous injection in the dorsum of the right hind foot with amounts of RSC and M. tuberculosis which are indicated in the text. Immunized rats were observed 6 days per week for neurological symptoms, which were graded as follows: 0, no symptoms; 1, limp tail; 2, hind leg paresis; 3, hind leg paralysis; 4, fore leg paresis; 5, fore leg paralysis; 6, death due to EAE. Paresis of the hind and fore legs was spastic in all cases. Bone Marrow Transplantation Procedures Conditioning prior to BMT was with a lethal dose of total body irradiation (TBI, males 9.8–10 Gy, females 9.5 Gy 300 kV X rays) 4 to 24 hr before BMT. The absorbed radiation was measured with a ionization chamber, which referred to the midline dose in a rat phantom. For more details of the radiation setup see Ref. (22). Bone marrow cells were harvested from the tibias, femurs, and humeri and suspended in Hanks’ balanced salt solution (HBSS) as described previously (22). Because the transplantation of 5 1 107 unmanipulated WAG BM cells, among which were approximately 2.5% T lymphocytes, resulted in lethal acute GVHD in all BUF recipients, T cell depletion of the allogeneic BM grafts was performed prior to grafting. This was done in vitro by the ‘‘panning’’ technique, as described by Wysocki and Sato (39), with minor modifications. Briefly, polystyrene petri dishes (94.0/16 mm, Greiner, Alphen a/d Rijn, The Netherlands) were coated with

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40 mg goat anti-mouse IgG (H/L) (Caltag, South San Francisco, CA) dissolved in 8 ml PBS at 47C, followed by washing with 0.2% BSA (Sigma, St. Louis, MO). Bone marrow cells, suspended in HBSS containing 1% decomplemented normal goat serum (NGS), were incubated with ascites of the mouse anti-rat T cell antibodies R73 and OX34 for 1 hr at 47C. After four washings, 3 1 107 cells in a total volume of 1.5 ml were placed on the precoated dishes and incubated for 1 hr at 47C. The free cells were then removed by careful pipeting and washed. The number of mononuclear cells was determined in a Bu¨rker-type hemocytometer using Turk’s solution, and the viability was tested by eosin exclusion. T cells were determined before and after depletion by flow cytometry with biotinylated mouse anti-rat T cell OX19 or R73. Depletion was between 5 and 111. T cell-depleted BM cells (5–10 1 107) were transplanted, and this resulted in the complete absence of symptoms of GVHD. Determination of the Origin of Peripheral Blood Mononuclear Leukocytes and Erythrocytes in BM Chimeras Four to 7 months after BMT, the origin (donor or host) of peripheral blood mononuclear cells (PB-MNC) and T lymphocytes was determined by flow cytometry. Leukocytes were purified from heparinized whole blood by ammonium chloride shock. Leukocytes (0.5 1 106) in 25-ml aliquots, suspended in FCS buffer (i.e., PBS, containing 10% FCS and 0.1% sodium azide (Merck)), were incubated with primary antibody in appropriate dilutions for 45 min at 47C. After two washings, cells were incubated for 45 min at 47C with 50 ml rat antimouse FITC (1:300 diluted in FCS buffer) and subsequently washed. For typing of T cells, leukocytes were first incubated with U9F4 (WAG-type RT1) or HIS 41 (BUF-type RT7) and rat anti-mouse IgG FITC and then with biotinylated mouse anti-rat T cell antibody (OX19 or R73); as the fourth step, streptavidin–phycoerythrin (Becton–Dickinson, San Jose, CA) was used. For typing of erythrocytes, an equivalent of 0.2 ml washed whole blood (approximately 106 erythrocytes) was incubated with optimal dilutions of the anti-rat erythrocyte antisera. In all cases, every sample had its own control, where the primary monoclonal antibody was omitted. Cells were washed as above, resuspended in 0.25 ml FCS buffer, and stored in the dark at 47C prior to analysis. Propidium iodide (PI, final concentration 5 mg/ml) was added 5 min before analysis for staining of dead cells. Fluorescent cells were analyzed on a FACScan flow cytometer operating with an argon laser (Becton–Dickinson, Mississanga, Ontario, Canada). Data analysis was performed with the FACScan research software program Consort 30 (Becton–Dickinson). For two-color procedures, compensation settings corrected for the

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overlap of the emission spectra of FITC and PE or PI. Ten thousand events were collected in the case of onecolor labeling, while at least 25,000 events were collected in two-color procedures. Debris and dead cells were excluded from data analysis by gating. The number of positively stained cells was calculated by subtracting the matching control values. The sensitivity of the detection of peripheral blood mononuclear leukocytes, which were positively stained with U9F4, HIS 41, OX19, and R73, was 0.5–1%; the sensitivity of the rat anti-BUF and rat anti-WAG erythrocyte antibodies was 5–10%. Histological Techniques For routine histological examination of the CNS, rats were sacrificed by exposure to carbon dioxide at 3 to 4 weeks after immunization; rats which displayed neurological symptoms were killed when the degree of neurological symptoms declined. The brain, spinal cord, and sciatic nerves were dissected out and fixed in 4% buffered formalin at 47C. Two horizontal sections of the brain (corresponding to 4.28 and 6.82 mm from Bregma), eight cross sections equally spaced over the whole length of the spinal cord, and a longitudinal section of each sciatic nerve were embedded in paraffin and stained with hematoxylin–azofloxin (HA). For immunohistochemistry, rats were anesthetized with ether and perfused with ice-cold PBS. The brain and spinal cord were cut in the same way as described above, snap frozen in liquid nitrogen, and stored at 0207C until further handling. Six-micrometer cryostat sections were cut and allowed to dry at room temperature for 30 min. Sections were then fixed with 100% acetone. When peroxidase-conjugated antibody was to be used, the sections were exposed to a mixture of methanol and 0.5% H2O2 for 15–20 min to inactivate endogenous peroxidase. The sections were washed once with distilled water and three times with PBS. Incubation of the sections with antibodies was in the automated Sequenza system (Shandon, Zeist, The Netherlands). The primary antibody was diluted in 0.2% bovine serum albumin in PBS and applied to the sections for 1 hr at room temperature. The sections were then washed and incubated for 30 min at room temperature with rabbit anti-mouse IgG peroxidase conjugate (DAKO-ITK diagnostics, Glostrup, Denmark), which was diluted in 1% normal rat serum in PBS. After three washings with PBS, the sections were washed with 0.2 M acetate buffer (pH 4.6), containing 133.3 g sodium acetater3H2O, 58.8 ml 99% acetic acid (Merck, Darmstadt, Germany), and 5 ml Tween 20 (Merck). Peroxidase activity was developed in a solution of 200 ml 30% H2O2 , 10 ml 1% 3-amino-9-ethylglycol (Sigma) in N,Ndimethylformamide (Merck) and 190 ml 0.2 M acetate buffer for 30 min. After one washing with the 0.2 M acetate buffer and one washing with distilled water,

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the sections were mounted in Aquatex (Merck). For immunofluorescent double staining, sections were incubated with antibodies diluted in 0.2% bovine serum albumin in PBS and washed after each staining with PBS. The sections were finally mounted in Imsol/ Mount (Klinipath, Duiven, The Netherlands), which polymerizes after 5 min incubation at 557C. The extent of CNS inflammation, i.e., the histological EAE score, was quantified either on HA-stained sections from formalin-fixed tissues or on ED1-stained sections from frozen CNS, as follows. The number of inflammatory lesions was determined blindly for the two sections of the brain separately, as well as for the eight sections of the spinal cord separately, and scored per section as follows: 0, absence of histological lesions; 1, slight mononuclear infiltrates only in submeningeal areas; 2, 1 to 4 perivascular infiltrates; 3, 5 to 10 perivascular infiltrates; 4, more than 10 perivascular infiltrates (after Mostarica-Stojkovic et al. (29)). The final histological EAE score represents the average of the mean score for the two sections of the brain and the mean score for the eight sections of the spinal cord. The origin of IL-2-R/ T cells in the CNS was determined by immunofluorescent double staining on two sections of the brain and eight different sections of each of the eight sections of the spinal cord. The sections were first stained with U9F4 (WAG-type RT1) and sheep anti-mouse IgG2a FITC conjugated, and subsequently with OX39 (IL-2-R), biotinylated sheep antimouse IgG1, and streptavidin–TRITC. Sixty to 100 IL2-R/ T cells were scored for staining with U9F4, except for one WAGrBUF chimera, in which only 17 IL-2-R/ T cells were counted. Adrenalectomy, Determination of Baseline and StressInduced Plasma Corticosterone Levels, and Pretreatment with Cyclophosphamide Bilateral adrenalectomy was performed by bilateral dorsolateral incisions through the muscles of the flank. Adrenalectomized rats were given a subcutaneous pelleted implant of 25 mg corticosterone dissolved in 75 mg cholesterol after the operation, to restore baseline serum corticosterone levels (after Mason et al. (40)). Samples for the determination of baseline plasma corticosterone levels were obtained in the morning by decapitation. To minimize the stress reaction associated with the presence of the investigator, rats were housed in four different rooms. Per room, one rat was decapitated within a few seconds after entrance. This procedure was repeated on the 2 consecutive days. Samples for stress-induced plasma corticosterone levels were obtained from six other rats of both strains, as described by Mason et al. (40). Briefly, rats were anesthetized by ether inhalation and 1 ml of blood was taken via the tail vein. Forty minutes later, rats were anesthetized again and tail vein blood samples were

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taken for corticosterone determination. Plasma levels of corticosterone were determined by radioimmunoassay (ICN, Costa Mesa, CA). Pretreatment with low-dose CY was at 2 days before immunization, by a single ip injection of 20 or 40 mg/ kg CY (Endoxan, a generous gift of Asta, Diemen, The Netherlands) dissolved in PBS. Induction and Evaluation of Type II Collagen-Induced Arthritis Immunization with type II collagen (CII), performed as described by ’t Hart et al. (30), was employed to evaluate the recovery of the immune system in WAGrBUF chimeras in terms of its capacity to mount an autoimmune reaction. Briefly, rats were injected intracutaneously at five sites at the tail base with a 1:1 emulsion of bovine–CII in Freund’s incomplete adjuvant (Difco). The severity of arthritis was expressed as an arthritic score, calculated for individual rats as the sum of the thicknesses (measured in millimeters with an industrial caliper) of the two ankles subtracted by measures taken before immunization (3). Statistical Analysis Statistical analysis was carried out using the x2 test with Yates’s correction for the comparison of incidences between two groups. The Wilcoxon rank sum test was used when results of individual rats in two different groups were compared. Differences were considered significant when P õ 0.02. RESULTS Susceptibility to EAE of BUF Rats BUF rats proved to be highly responsive to induction of EAE by immunization with syngeneic RSC and complete adjuvant (Table 1). The severity of neurological symptoms depends on the amount of RSC and M. tuberculosis in the sensitizing inoculum. Injection of 50 mg RSC and 2.24 mg M. tuberculosis results in severe disease and a high mortality. Lowering the amounts of RSC and M. tuberculosis decreases the incidence and the severity of the disease as quantified by the EAE score, time of onset, and complete and lasting recovery from the disease. The disease was self-limiting in almost all rats which had not succumbed to severe EAE. Histological examination was performed in one experiment in which BUF rats had been immunized with 6.25 mg RSC and 0.14 mg M. tuberculosis. Two or three rats were sacrificed at various intervals after immunization, i.e., at Days 12, 18, 21, 25, 28, 35, 42, 50, and 60 pi. Perivascular and infiltrating inflammatory lesions were found in the whole spinal cord, cerebellum, and brainstem, while lesions were also frequently found in the basal ganglia and periventricularly. A large num-

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TABLE 1 Susceptibility to Induction of EAE of the BUF, WAG, and Their (WAG 1 BUF)F1 Hybrid

Rat strain BUF

WAG (WAG 1 BUF)F1

BUFa WAGa

Dose of RSC (in mg)

Dose of M. tuberculosis (in mg)

Incidence of clinical EAE

Mortality (%)

50.0 12.5 6.25 6.25 3.13 3.13 3.13 50.0 6.25 50.0 25.0 12.5 6.25 6.25 50

2.24 0.28 0.28 0.14 0.28 0.14 0.07 2.24 0.14 2.24 0.56 0.28 0.14 0.14 2.24

10/10 10/10 10/10 10/10 12/12 6/10 7/10 0/20 0/10 5/5 7/7 3/6 3/12* 10/10 0/7

90 10 80 10 66 0 0 — — 80 43 0 0 10 —

Mean highest EAE score of responding rats 5.9 4.7 5.6 4.0 5.4 1.8 1.7

5.8 4.3 3.0 2.7 3.3

(0.3) (0.6) (0.8) (1.4) (0.9) (1.6) (1.2) — — (0.4) (1.5) (0) (0.9)** (1.4) —

Time of onset b 13.4 13.8 12.1 18.6 12.7 16.3 16.9

(0.7) (0.8) (0.3) (1.2) (0.7) (1.7) (1.7) — — 13.8 (1.2) 15.9 (2.3) 26.7 (9.5)** 25.3 (8.5) 13.4 (1.5) —

Time of complete recovery b 46 59 48, 60 46.0 (8.3) 51.8 (10.4) 38.7 (17.5) 33.6 (12.2) — — 56 26.5 (2.7) 41.3 (12.3) 31.3 (9.3) 33.2 (2.8) —

Note. Rats were immunized with BUF RSC and M. tuberculosis in the dorsum of one or two hind feet at Day 0; the rats marked with were immunized with WAG RSC. Rats were observed for the occurrence of clinical symptoms for 65 to 90 days. b Time is in days pi. * 0.01 ú P ú 0.001. ** P õ 0.005 compared with BUF rats immunized with the same amount of antigens.

ber of lesions were already present in the spinal cord and the brain from Day 12 pi on (i.e., before onset of paresis). From 3 to 4 weeks after immunization onward, multiple infiltrates were also found in the cerebrum. A small number of lesions was present in the CNS of one rat, which had not developed clinical symptoms till the time of sacrifice (5 weeks pi). Lesions were still present after complete recovery of EAE (Day 60 pi). The infiltrates in the CNS mainly contain macrophages and activated microglial cells (positively staining with ED1) and T cells (stained with ER1), which are present in almost equal numbers. Only 1 of the examined 20 rats had a few inflammatory lesions in the sciatic nerves. Susceptibility to EAE of WAG and (WAG1BUF)F1 Rats WAG rats were found to be low responders to EAE after immunization with either a high dose of RSC and M. tuberculosis (50 and 2.24 mg, respectively) or a lower dose (6.25 and 0.14 mg); none of the 30 immunized WAG rats showed neurological symptoms (Table 1). However, in later studies a few WAG rats (5/48) did develop neurological symptoms (Table 3), indicating that the resistance is not absolute. The F1 hybrid of the low-responder WAG and the high-responder BUF rat strains appeared to be highly responsive to EAE (Table 1). However, the extent of responsiveness appeared to be less than in BUF rats as judged by the amount of RSC and M. tuberculosis

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needed to induce similar incidence, time of onset, and severity of EAE. The encephalitogenicity of BUF or WAG RSC did not differ, because immunization of BUF rats with WAG RSC induced EAE with similar disease characteristics as with BUF RSC (Table 1). Also, WAG rats remained low responsive to EAE when immunized with a high dose of WAG RSC. Susceptibility to EAE of Hemopoietic Chimeras To determine if the response to EAE of the BUF and WAG rats and their F1 hybrid is associated with the hemopoietic stem cell or controlled by non-BM-derived factors, we prepared BM chimeras by grafting BM from the low-responsive (L) WAG donor to high-responder (H) BUF or F1 recipients and vice versa. In combinations where GVHD was expected (i.e., strain Arstrain B or parentrF1), the grafts were TCD. Four to 7 months after BMT, PB-MNC and T cells were genotyped. Between 90 and 99% of the PB-MNC were of donor type (mean 95%). In most chimeras, a small percentage (less than 8%) of residual host T lymphocytes was found. More than 90% of erythrocytes were of donor type. One month after genotyping of the blood cells, the BM chimeras were immunized with 50 mg RSC and 2.24 mg M. tuberculosis. In this experiment (Table 2), all BUF and F1 rats and all HrH chimeras developed neurological symptoms after immunization (Table 2, lines a–c, e–g). The disease characteristics (onset and severity of neurologi-

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TABLE 2 Transfer of Susceptibility and Resistance to EAE by TCD BM and Genotyping of Activated (IL-2R/) T Cells in the CNS

Strain or BM chimera Donor r recipient a b c d e f g h i k l m

BUF BUF r BUF BUF r F1 BUF r WAG F1 F1 r BUF F1 r F1 F1 r WAG WAG WAG r BUF WAG r F1 WAG r WAG

Incidence of clinical EAE

Mean time of onset in days pi (SD)

Mean highest EAE score of clinically responding rats (SD)

Incidence of histological EAEa

12/12 7/7 13/13 9/9 14/14 9/9 9/9 8/9 0/10 2/11 7/19e 0/7

9.7 (1.1) 8.4 (0.5) 8.5 (0.8) 11.0 (2.4)b 12.6 (2.1) 12.4 (1.5) 13.9 (1.7) 18.9 (2.3)c — 19, 21 18.6 (1.9) f —

4.6 (1.0) 4.9 (0.6) 4.4 (0.8) 3.9 (1.4) 3.1 (0.9) 2.1 (1.5) 2.6 (1.8) 1.6 (0.9) — 1, 1 1.0 (0) —

6/6 ND 11/11 8/8 11/11 8/8 5/5 9/9 1/9 4/10g 11/19 0/3

Histological score of individual rats

% IL-2R/ T cells in CNS with RT1.Au (WAG/F1) haplotype

1

2

3

4

No. of rats with IL-2R/ T cells in CNS/total no. of rats studied

—

—

4

2

3/3

0 (31)

— — — — — 1 1 — — —

— 1 — 1 — 3 — 3 5 —

1 2 2 — — 4 — 1 5 —

10 5 9 7 5 1d — — 1 —

3/3 2/2 5/5 4/4 ND 3/3 0/3 3/5g ND ND

10, 13, 14 1, 4 99, 100 (41) 96, 97 (21), 100 100 (31) — 82, 88, 100

Note. BM chimeras were prepared with 10 Gy TBI and BMT. Five months later genotyping of the peripheral blood mononuclear leucocytes revealed that more than 95% were of donor type. Six months after BMT BM chimeras were immunized with 50 mg RSC and 2.24 mg M. tuberculosis. a Histological examination of the CNS was performed on a proportion of the rats in the various groups. b P õ 0.005 compared with BUF r BUF and BUF r F1 (lines b, c). c P õ 0.005 compared with F1 r BUF and F1 r F1 (lines f and g). d P õ 0.025 compared with F1 r BUF and F1 r F1 (lines f and g). e Not statistically significantly different from WAG r BUF (line k). f P õ 0.005 compared with BUF r F1 and F1 r F1 (lines c and g). g Among which are the two clinically responding animals.

cal symptoms and extent of CNS inflammation) of the chimeras were similar to those of the high-responder BM donor strains. Among the low-responsive recipients of BM from high-responder donors (HrL), all but one (17/18) developed neurological symptoms (Table 2, lines d and h). However, in comparison to HrH chimeras, the onset of clinical EAE in HrL chimeras was significantly delayed. After transplantation of BM from the F1 hybrid to WAG recipients (Table 2, line h), the intensity of CNS inflammation was significantly less than in syngeneic F1 chimeras (Table 2, line g). The one F1(H)rL chimera, which did not develop clinical symptoms, had only slight mononuclear infiltrates in the meninges of the brain and no lesions in the spinal cord. Normal low-responsive rats and LrL chimeras did not develop clinical EAE or inflammatory lesions in the CNS (Table 2, lines i and m). In contrast, some LrH chimeras displayed minor neurological symptoms (Table 2, lines k and l). The percentage of LrH chimeras in which CNS inflammatory lesions occurred was increased in comparison with that in normal WAG rats. All LrH chimeras with neurological symptoms had histological lesions in the CNS. The incidence of clinical EAE and the proportion of rats with histological lesions did not differ statistically significantly between both types of LrH (WAGrBUF and WAGrF1) chimeras. The onset of clinical EAE in the responding LrH chi-

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meras was later and the severity of neurological symptoms was less than that in HrH chimeras (Table 2, lines b, c, f, g). Origin of Activated Lymphocytes in the CNS of Allogeneic BM Chimeras after Induction of EAE The occurrence of clinical EAE in 9 of the 30 LrH BM chimeras is significantly higher than in normal WAG rats (9/30 versus 5/68, P õ 0.01) and might be due to a modifying effect of the high-responsive host on the donor-derived immune system. On the other hand, it can be postulated that residual host-type (BUF or F1) lymphocytes, which were found in low numbers in almost all chimeras, caused the inflammatory lesions in the CNS and subsequently the neurological symptoms. Although two of the responding LrH chimeras had 99% PB-MNC and T cells of donor-type, the possibility remains that a very small number of hosttype lymphocytes induced the disease in these chimeras. Therefore, we typed the activated (IL-2-R/) T lymphocytes in the CNS of both responding and three asymptomatic WAGrBUF chimeras, as well as of some HrH and HrL chimeras (Table 2). For this purpose we used double-immunofluorescent staining with U9F4 (specific for WAG-type MHC class I) and anti-rat IL-2R (OX39). In the CNS of normal susceptible rats, HrH and HrL chimeras, nearly all activated T cells were of

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TABLE 3 Factors Determining Resistance to EAE in the WAG Rat Strain

Strain

Pretreatment

Incidence of clinical EAE

WAG WAG WAG BUF

— Adx CY (controls)

5/48a 7/13d 2/22 13/13

Highest EAE score of clinically responding rats 1 (21), 3 (31) 1 (21), 2, (21), 4 (31) 1, 3 3 (61), 4 (41), 5, 6 (21)

Mean time of onset in days pi { SD 15.6 16.7 15.0 10.7

{ { { {

Histological score of individual rats Incidence of histological lesions in the CNS

1

2

3

4

17/46a 8/13 19/22c 12/12

6 — 11 —

3 2 4 —

5 2 2 1

3b 4e 2 11

2.3 4.5 1.0 1.4

Note. Rats were immunized at Day 0 with 50 mg BUF RSC and 2.24 mg M. tuberculosis. Pretreatment with CY or adrenalectomy was at Day 02. a P õ 0.001 compared with BUF rats. b P õ 0.005 compared with BUF rats. c P õ 0.001 compared with non-pretreated WAG rats. d 0.01 ú P ú 0.001 compared with non-pretreated WAG rats. e P õ 0.05 compared with non-pretreated WAG rats.

the BM donor phenotype (Table 2, lines a to h). A smaller number of IL-2-R/ T cells were present in the CNS of the two paretic WAGrBUF chimeras and in an asymptomatic one. The large majority of activated T lymphocytes in the CNS of these three chimeras had the genotype of the WAG BM donor (Table 2, line k). These results indicate that in all chimeras, the development of neurological symptoms resulted from the activation of T lymphocytes derived from the BM of the donor. The increased incidence of EAE in LrH chimeras in comparison to normal WAG rats must then be due to a host-associated factor of the high-responsive host. Inversely, the low-responsive host influenced the response to induction of EAE in HrL chimeras, as judged by the delayed onset and less intense inflammation in the CNS. These results indicate that the response to EAE of BUF, F1, and WAG rats is determined by both BM-associated and non-BM-associated (host) factors. Factors Determining Low Responsiveness of the WAG Rat We attempted to identify the host factor by investigating the role of corticosterone in the susceptibility of WAG and BUF rats to EAE. A high corticosterone response is thus far the only known host factor involved in resistance to EAE. It was also investigated whether BM-derived CY-sensitive suppressor cells were operative in WAG rats. The results of several experiments are summarized in Table 3. The 20 WAG rats shown in Table 1 were not included in Table 3, because the CNS of these rats was not processed for histological examination; the 10 WAG rats of Table 2, line i, were included. First, it should be noted that WAG rats are not completely resistant to EAE. Five of 48 WAG rats developed neurological symptoms, and the strain is therefore

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designated as low responsive to EAE. The severity of EAE in the WAG rats was significantly less than that in BUF rats. At histological examination of the CNS, inflammatory lesions were found in 37% (17/46) of the WAG rats, including all five responders. The intensity of inflammation varied from mononuclear infiltrates only in submeningeal areas (score 1) to multiple perivascular inflammatory lesions in the parenchyma (score 4), but was on the average significantly less than that in immunized BUF rats. The five paretic WAG rats had histological scores of 3 and 4. Adrenalectomy of WAG rats prior to immunization resulted in an increased incidence of neurological symptoms, and the numbers of inflammatory lesions in the CNS of the responding rats were higher than those in WAG rats which were not subjected to adrenalectomy (Table 3). The percentage of adrenalectomized WAG rats with CNS inflammatory lesions was slightly (not significantly) higher than that of normal WAG rats. Only one of the six asymptomatic adrenalectomized rats showed histologically perivascular inflammatory lesions, while the other five asymptomatic rats had no lesions. The increased incidence of clinical EAE following adrenalectomy in WAG rats indicates that the adrenal glands are involved in the low responsiveness of the WAG rat strain. In analogy to the difference found by Mason et al. (40) in the stress-induced corticosterone response of susceptible Lewis and resistant PVG rats, we determined baseline and stress-induced corticosterone plasma levels in normal WAG and BUF rats (Table 4). Baseline corticosterone plasma levels varied in individuals from both strains, but the levels tended to be higher in WAG rats than in BUF rats. After exposure to ether and venepuncture, the plasma corticosterone levels in WAG rats rose significantly higher than those in BUF rats (P õ 0.005). These results indicate

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Rat strain

Baseline

Stress-induced

susceptibility to CIA is also associated with the genetic constitution of the hemopoietic stem cell. One of the five BUFrBUF rats developed CIA. This is not exceptional, because in larger experiments with BUF rats an occasional BUF rat has been seen to develop arthritis (30); the BUF rat strain was therefore designated as low responsive to CIA.

WAG BUF

130 { 49 78 { 23

1778 { 52* 1356 { 72

DISCUSSION

TABLE 4 Baseline and Stress-Induced Plasma Corticosterone Levels in WAG and BUF Rats Plasma corticosterone levels (in nM) { SD

Note. Baseline levels of corticosterone were determined in blood samples obtained by rapid decapitation of six rats of either strain in the early morning. Stress induction was performed in six other rats of both strains by ether inhalation and venepuncture, which was repeated after 40 min; the latter samples were used for the determination of corticosterone. * P õ 0.005 compared with stress-induced levels in BUF rats.

that the low responsiveness to clinical EAE of the WAG rat strain is—at least in part—determined by its corticosterone response. We also investigated if a CY-sensitive suppressor cell may represent the BM-associated factor which determines the low responsiveness to EAE of WAG rats. For this purpose, WAG rats were treated with low-dose CY (20 or 40 mg/kg) 2 days before immunization. The treatment did not influence the incidence of clinical EAE in comparison with normal WAG rats, but there is no doubt that the incidence of subclinical EAE was significantly increased in comparison with normal WAG rats: 86% of the CY-pretreated rats (19/22) had inflammatory lesions in the CNS (P õ 0.001). In this respect and with regard to the incidence of clinical symptoms, the effect of pretreatment with CY differs from adrenalectomy. Immune Reconstitution in Long-Term WAGrBUF BM Chimeras It could be argued that the incidence of EAE in LrH BM chimeras was low as a result of post-BMT immunodeficiency instead of a result of the genetic constitution of the donor. In an attempt to exclude this possibility, we investigated the responsiveness of WAGrBUF BM chimeras to CII 3 months after BMT. Immunization with CII induces arthritis (CIA) in a high proportion of WAG rats, while BUF rats are low responders (30). In this way, the ability of the grafted immune system to mount an autoimmune reaction could be established. As is shown in Table 5, all but one of the BUF recipients of WAG BM developed arthritis. The severity of arthritis of the WAGrBUF BM chimeras was similar to that of WAGrWAG and WAG controls. These results indicate that the WAGrBUF BM chimeras are immunocompetent. The lower incidence of EAE in such chimeras must therefore be attributed to properties of the grafted BM. In addition, our data demonstrate that

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In this study it is shown that BUF rats are highly susceptible to induction of EAE. The main site of involvement was the CNS, as most lesions were found in the spinal cord, white matter of the cerebellum, and brainstem. The fact that in the large majority of BUF rats paresis was spastic supports the dominant role of CNS lesions in accounting for the neurological symptoms. The inflammatory lesions consisted of T cells and ED1/ macrophages and activated microglial cells, which is similar to EAE in the Lewis rat (41). In view of the option of treating MS patients with allogeneic BM from healthy donors, we were interested in whether the EAE low-responsive WAG rat would be a suitable BM donor for treatment of EAE in the highresponsive BUF rat. Our results show that after immunization with 50 mg RSC and 2.24 mg M. tuberculosis 9 of 30 high-responsive recipients (BUF and F1) of WAG BM develop EAE, which is significantly lower than the incidence of EAE in normal high responders (Tables 1 and 2: 22/22 (BUF) and 19/19 (F1), P õ 0.001), although higher than in normal WAG rats (Tables 1 and 3: 5/68, P õ 0.01). In the WAGrBUF chimeras the majority of activated (IL-2R/) T cells in the CNS were of WAG type, which indicates that the development of EAE did not result from residual T cells from the highresponsive recipient but from donor (WAG) cells. Evidence that these IL-2R/ T cells are indeed causing the inflammatory reaction in the CNS is from literature

TABLE 5 Type II Collagen-Induced Arthritis in Allogeneic Bone Marrow Chimeras Rat strain or BM chimera (donor r recipient) WAG (12 weeks old) WAG r WAG (40 weeks pBMT) WAG r BUF (11–14 weeks pBMT) BUF r BUF (11 weeks pBMT)

Incidence

Mean swelling of the ankles of arthritic rats in mm ({ SD)

6/6

3.9 { 1.3

6/6

2.7 { 0.9

9/10

3.4 { 1.0

1/5*

4.0

Note. Rats were immunized with CII as described under Materials and Methods. * 0.05 ú P ú 0.02 compared with WAG r BUF.

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EAE SUSCEPTIBILITY AS PARTLY BONE MARROW ASSOCIATED

data: despite the fact that most T cells present in the CNS during EAE are presumed to be nonmyelin specific (42), only myelin-reactive activated T cells accumulate in the CNS during EAE (43, 44). These activated T cells are in blastic phase (45) and express the IL-2R (46–48). It can therefore be concluded that the high responsiveness of BUF and F1 rats to EAE and the low responsiveness of the WAG rat to EAE are largely determined by properties of the hemopoietic stem cell, but that nonBM-derived host factors modify the response. These results are in contrast to the results in guinea pigs (strains 2 and 13) (14), Lewis and Le-R rats (18), and SJL/J and B10.S mice (15), where host factors did not influence the response to induction of EAE in BM chimeras. Only Singer et al. showed that the incidence of EAE in resistant BN recipients of BM from the susceptible Lewis rat strain was significantly lower than that in LewisrLewis chimeras (21). A likely candidate for the host factor involved in the responsiveness of BUF and WAG rats to EAE is the activity of the adrenal cortex, as both adrenalectomy and low or high levels of stress-induced corticosterone levels in the blood have been demonstrated to be associated with susceptibility of Lewis and PVG rats to EAE, respectively (40). We found that the incidence of EAE in the WAG rat was increased from 7% (5/68) to 54% (7/13) after adrenalectomy. This is less than reported for PVG rats, all of which developed clinical EAE after adrenalectomy (40). The stress-induced corticosterone levels were higher in low-responsive WAG rats (1778 nM) than in high-responsive BUF rats (1356 nM). Although the levels in our rats were lower than those in the PVG (2344 nM) and Lewis rats (1656 nM), respectively, the trend is similar. The delayed onset of EAE in the HrL chimeras compared to normal high responders (BUF, F1) may be explained by the higher release of corticosterone by the adrenals of the WAG host. Along the same line of reasoning, the lower release of corticosterone in BUF rats may be responsible for the higher incidence of EAE in the LrH chimeras in comparison with WAG rats. However, only a minority of the LrH chimeras developed EAE. We excluded the possibility that this low incidence of EAE was due to immunosuppression related to the BMT, by demonstrating that all but one WAGrBUF chimeras were fully competent to develop CIA. Thus, it seems that the genotype of the BM is the overriding factor. Our data indicate that the BM-associated factor is related to the presence of CY-sensitive suppressor cells. These suppressor cells seem to interfere at some level upon the process from sensitization of myelin-reactive lymphocytes to the formation of inflammatory lesions in the CNS of normal WAG rats, as the proportion of WAG rats with CNS inflammatory lesions was significantly higher after pretreatment with CY than in nor-

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47

mal WAG rats (Table 3: 86% versus 37%, respectively, P õ 0.01). It can be concluded from our results that the low responsiveness of the WAG rat to clinical EAE is determined by at least two factors, which act at different levels of the disease process: (i) CY-sensitive suppressor cells, which prevent lesion formation in the CNS; and (ii) a high corticosterone response, which prevents lesion formation to some extent and the clinical expression of CNS inflammatory lesions to a great extent. This lends support to a study of Gasser et al. (49), who showed that the genetic control of the inflammatory reaction as observed histologically in the CNS is not strictly linked to the genetic control of the clinical expression of EAE. Our results indicate that the hypothesis proposed by Ikehara et al. (2), that susceptibility and resistance to experimental AID reflect a property of the hemopoietic stem cell, does not apply unrestrictedly to EAE. This conclusion has practical implications, as we (1) and others (50) are interested in the application of BMT for the treatment of human AID. Because it is unknown whether the genetic predisposition to MS is only partially or completely determined by hemopoietic stem cell-derived factors, which might also differ between individuals, it seems justified to consider the use of allogeneic BMT as a therapeutic option for MS at best with some caution. Nevertheless, allogeneic BMT might be beneficial for life-threatening severe progressive or malignant MS. In support of this view are the excellent therapeutic results obtained with WAG BMT in paretic BUF rats (7). Only 8% (5/61) of the treated rats developed a spontaneous relapse, and the induced relapse rate was 14% (8/59), which was considerably lower than that in untreated rats or that in rats treated with syngeneic BMT. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical support of Ms. N. Stouten and Mr. A. Timmermans (Department of Pathology, Erasmus University of Rotterdam) for the histology and immunohistofluorescence. We thank Professor F. Tilders (Department of Pharmacology, Free University, Amsterdam) for determining the plasma corticosterone levels. Dr. S. Knaa¨n-Shanzer and Dr. L. A. ’t Hart are hereby indebted for their critical discussions and Dr. L. A. ’t Hart also for his assistance with the induction of CIA. This work was financially supported by the Dutch ‘‘Stichting Vrienden MS Research.’’

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