immunodeficiency mice by mononuclear cells from cerebrospinal fluid ...

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6157-6161, July 1992 Medical Sciences

Transfer of multiple sclerosis into severe combined immunodeficiency mice by mononuclear cells from cerebrospinal fluid of the patients (autoimmune disease/demyelination/local engraftment)

YUKIHIKO SAEKI*, TORU MIMA*, SABURO SAKODAt, HARUTOSHI FUJIMURAt, NORIo ARITAt, TATSUJI NOMURA§, AND TADAMITSU KISHIMOTO* Departments of *Medicine III, tNeurology, and tNeurosurgery, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan; and §Central Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216, Japan

Contributed by Tadamitsu Kishimoto, April 2, 1992

protein- or proteolipid protein-reactive T lymphocytes were cloned from CSF of MS patients (10, 11). Although the initial insult leading to MS is unknown, autoreactive T lymphocytes and the infiltrating cells can be assumed to be closely involved in pathogenesis of the disease. However, the evidence to support this possibility is fragmentary and indirect. Mosier et al. (12) and McCune et al. (13) demonstrated that the human immune system could be reconstituted in severe combined immunodeficiency (SCID) mice by the transfer of either human peripheral blood mononuclear cells (PBMC) or lymphoid tissues. Recent reports have shown that PBMC from the patients with several autoimmune conditions-such as primary biliary cirrhosis, systemic lupus erythematosus, and rheumatoid arthritis-could transfer some characteristics of these diseases into SCID mice, such as autoantibody production, but the typical diseases could not be reproduced (14-16). In this study, we report the development of MS-like pathology in SCID mice by intracisternal injection of mononuclear cells from CSF of MS patients undergoing the exacerbation stage of the disease.

To investigate the mode of the pathogenesis of ABSTRACT multiple sclerosis (MS), we transferred cerebrospinal fluid (CSF) cells, predominantly mononuclear cells, from MS patients at both exacerbation and remission stages of the disease into severe combined immunodeficiency mice by intracisternal Injection. As controls, (a) CSF cells from patients with cervical spondylosis and (ii) peripheral blood mononuclear cells from normal individuals were transferred. Four to 6 weeks after transfer, most mice transferred with CSF cells from MS patients at the exacerbation stage of the disease developed paralysis and ataxia. The histopathological examination on the sacrificed mice revealed multiple scattered, discrete lesions localized in the white matter of the brainstems and spinal cords. These lesions were characterized by various degrees of tissue necrosis, involving inflammatory-cell infiltration. Most infitrating cells were macrophages, although a smaller number of granulocytes appeared in several foci. Reactive astrocytic gliosis was also seen around the necrotic foci. Furthermore, these lesions exhibited demyelination. These histopathological changes are similar to those seen in MS. In contrast, none of the severe combined immunodeficiency mice transferred with CSF cells from MS patients at the remission stage of the disease, or with CSF cells from the patients with cervical spondylosis, or with peripheral blood mononuclear cells from normal individuals showed any such histopathological changes. These observations provide convincing direct evidence of encephalitogenicity of mononuclear cells in CSF from MS patients at the exacerbation stage of the disease.

MATERIALS AND METHODS Patients and Cell Sources. CSF samples were obtained by lumbar puncture from four female MS patients being followed at the hospitals described below (patients MS-H.M. and MS-S.Y., Department of Neurology, Osaka University Medical School; patient MS-T.O., Kinki Central Hospital of the Mutual Aid Association of Public School Teachers; patient MS-S.T., Center for Adult Diseases, Osaka). All the MS patients were diagnosed according to the standard criteria of Schumacher et al. (17). CSF samples from MS-H.M. were obtained at both exacerbation and remission stages of the disease. The CSF sample from MS-T.O. was obtained at the exacerbation stage of the disease. CSF samples from MS-

Multiple sclerosis (MS) is one of the commonest demyelinating diseases of the central nervous system, the etiology of which and the mode of its pathogenesis still remain largely unknown. Experimental allergic encephalomyelitis, a prototypic animal model, which has many histopathological similarities with MS, suggests that the autoimmune process may be involved in the pathogenesis of MS (1, 2). Cell-transfer experiments in experimental allergic encephalomyelitis indicate that T lymphocytes sensitized to the specific autoantigens, such as myelin basic protein and proteolipid protein, are responsible for induction of the disease (3). This result suggests the possible involvement of autoreactive T lymphocytes in the development of MS. Indeed, it is evident that most infiltrating cells at the active demyelinating foci in MS patients are T lymphocytes and macrophages, and the cerebrospinal fluid (CSF) cells of MS patients are predominantly T lymphocytes. Furthermore, recent investigations have demonstrated the oligoclonality of these T lymphocytes by analysis of T-cell-receptor gene use (4-9), and myelin basic

S.Y. and MS-S.T. were obtained at the remission stage of the disease. Control CSF samples were obtained from two patients with cervical spondylosis being followed at the Department of Neurology, Osaka University Medical School. PBMC from three normal individuals were isolated by Ficoll/ Hypaque (Pharmacia) separation. Mice. The SCID mice used in this experiment were 6- to 8-week-old female CB-17 scid/scid mice obtained from the Central Institute for Experimental Animals. The SCID mice were maintained under specific-pathogen-free conditions. Transfer of CSF Cells into SCIDI Mice. CSF cells were transferred into SCID mice by intracisternal injection. CSF Abbreviations: MS, multiple sclerosis; CSF, cerebrospinal fluid; SCID, severe combined immunodeficiency; PBMC, peripheral blood mononuclear cells.

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cells (3 x 103) suspended in 50 A.l of Hanks' solution were injected directly into the cisterna magna of an anesthetized SCID mouse by microsyringe. Assessment of Clinical Symptoms. Mice were observed daily for clinical symptoms by two independent observers. The incidence of hypomotility was assessed by comparing the motility between a "transferred"-SCID mouse and an untransferred-SCID mouse in the same cage. The incidence of motor paralysis or ataxia was assessed by the presence of limb paralysis or gait disturbance. Histopathological Examination of the Transferred-SCID Mice. Four to six weeks after cell transfer, the SCID mice were sacrificed. Under deep anesthesia with pentobarbital, mice were perfused intracardially with saline followed by 10%o buffered formalin. The brains and spines were removed and fixed in the same fixative for 3 days. Representative coronal sections of cerebrum and horizontal sections of brainstem, cerebellum, and spinal cord were embedded in paraffin and cut at 4-,um thickness. Sections were stained with hematoxylin/eosin, luxol fast blue, or luxol fast blue/ Bodians silver impregnation.

RESULTS AND DISCUSSION In a representative experiment, we transferred 3 x 103 CSF cells, composed mainly of mononuclear cells (>95%, based on morphological definition) and derived from a MS patient (MS-H.M.) undergoing the exacerbation stage of the disease, into six individual SCID mice by intracisternal injection. Fig. 1 shows that all injected mice (SCID-hu-MS-H.M.) gradually showed hypomotility within 4 weeks. Moreover, 4 weeks after the transfer four out of six SCID-hu-MS-H.M. mice developed either motor paralysis or ataxia similar to the clinical manifestations of the patient. All SCID-hu-MS-H.M. mice showing obvious clinical symptoms except one, which had died just before histopathological examination, were sacrificed as described, and brains and spines were examined for histopathology. Encephalomyelitis with demyelination was observed in all of five SCID-hu-MS-H.M. mice. Multiple scattered, discrete lesions were seen mainly in the white matter of the brainstems and spinal cords. These lesions were characterized by various degrees of tissue necrosis with the infiltration of inflammatory cells, of which most were macrophages, although a smaller number of granulocytes appeared in several foci. The number of astrocytes was increased around the necrotic foci, indicative of a reactive gliosis (Fig. 2A). Furthermore, these lesions were

also showing demyelination with relative preservation of axons by double staining with luxol fast blue and Bodians silver impregnations (Fig. 2 B and C). On the other hand, the SCID mice injected with cell-free supernatant of CSF prepared from the same patient (MS-H.M.) did not show either such clinical symptoms or histopathological changes (Fig. 1). These results suggest that induction of the disease in SCIDhu-MS-H.M. mice was mediated by the cellular, but not the humoral, components of the CSF. Next, to confirm reproducibility of the observation, CSF cells from another MS patient (MS-T.O.) at the exacerbation stage of the disease were transferred into four SCID mice. At this time we injected variable numbers of CSF cells from the patient into each pair of SCID mice. Table 1 shows that the two SCID mice received the same number (3 x 103 cells per mouse) of CSF cells as in the previous experiment and that they developed exactly the same clinical symptoms and histopathological changes. The SCID mice that received 5 x 102 CSF cells from the patient showed similar histopathological changes, although the lesions were relatively milder than those seen in the first group of SCID mice. However, the SCID mice that received CSF cells from two patients with cervical spondylosis did not show any significant changes (Table 1). Interestingly, the SCID mice reconstituted with CSF cells from the MS patients at the remission stage of the disease (MS-S.Y. and MS-S.T.) did not develop the disease, although we transferred the same number (3 x 103 cells per mouse) of CSF cells (Table 1). Even when the CSF cells were derived from the same patient (MS-H.M.), the disease did not develop; the SCID mice developed the disease only with CSF cells obtained at the exacerbation stage ofthe disease and not with CSF cells obtained at the remission stage of the disease (Table 1). At the remission stage, the number of mononuclear cells in the CSF was small (Table 1), and these cells might be at the nonactivating stage. Involvement of graft-versus-host reactions in development of the disease in SCID mice caused by the transfer of lymphoid cells from the patients with primary biliary cirrhosis into SCID mice was suggested (14). To examine the possible involvement of graft-versus-host reactions, we transferred PBMC from three normal individuals into SCID mice via the same route. As shown in Fig. 1 and Table 1, the SCID mice transferred with PBMC from the normal individuals did not show any clinical symptoms or histopathological changes, indicating that the observations from the SCID-hu-

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FIG. 1. Incidence and time course of clinical symptoms on the SCID mice transferred with CSF cells from a MS patient (MS-H.M.) at the exacerbation stage of the disease. CSF cells (3 x 103) obtained from a MS patient (MS-H.M.) were transferred into six individual SCID mice by intracisternal injection. Clinical symptoms were assessed as described in text. Every transferred mouse (o) showed hypomotility 4 weeks after transfer, and four out of six mice developed motor paralysis or ataxia similar to the clinical manifestations of the patient. In contrast, the SCID mice transferred with PBMC from normal individuals (m) and the SCID mice injected with supernatant of CSF from MS-H.M. (0) did not develop any such symptoms.


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FIG. 2. Histopathological examinations on the SCID-hu-MS-H.M. mice. Four weeks after the transfer of CSF cells, five SCID-hu-MS-H.M. mice were sacrificed under anesthesia and subjected to histopathological examination. (A) Section of the mid-pons of SCID-hu-MSH.M. mice with hematoxylin/eosin staining. (B) Section of the lumbar spinal cord of the same mouse with luxol fast blue staining, showing demyelination. (C) Section of the mid-pons of the same mouse stained with luxol fast blue and Bodians silver impregnation, showing relative preservation of axons. Multiple scattered necrotic foci with infiltrations of macrophages and reactive astrocytic gliosis appear in A and B; the lesions were mainly localized in the white matter.

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Table 1. Summary of incidence of encephalitis in SCID-hu-MS and control mice Encephalitis, Transferred SCD Mononuclear CSF cells, Clinical incidence at 4-6 weeks substance/mouse recipients cells, % no./Al stage Donor 5/5* 3.0 x 103 CFS cells 6 >95 31/3 Exacerbation MS-H.M. 2/2 2 3.0 x 103 CSF cells >95 20/3 Exacerbation MS-T.O. 1/1* 2 5.0 x 102 CSF cells >95 20/3 Exacerbation MS-T.O. 0/1 1 4.0 x 103 CSF cells >95 4/3 Remission MS-H.M. 0/1 1 5.0 x 103 CSF cells >95 5/3 Remission MS-S.Y. 0/3 3 3.0 x 103 CSF cells >95 11/3 Remission MS-S.T. 0/2 3.5 x 103 CSF cells 2 ND 8/3 Cervical spondylosis-1 0/1 3.0 x 103 CSF cells 1 ND 4/3 Cervical spondylosis-2 0/3 CSF alone 3 MS-H.M. 0/3 CSF alone 3 MS-T.O. 0/2 5.0 x 103 PBMC 2 Normal-1 0/2 2 5.0 x 103 PBMC Normal-2 0/5 3.0 x 103 PBMC 5 Normal-3 0/4 4 Hanks' solution ND, not done; PBMC, peripheral blood mononulear cells. *One mouse of each group was not examined histopathologically because they died just before scheduled sacrifice.

MS-H.M. mice are unlikely to have resulted from graftversus-host reactions. In the present experiments, we transferred CSF cells directly into the central nervous system of the SCID mice by intracisternal injection. Mosier et al. (12) reported that the peritoneal cavity injection of human lymphoid cells, but not i.v. injection, caused the production of human immunoglobulins in the serum, showing successful human lymphoid-cell engraftment. However, even for peritoneal-cavity injection, there was no evidence of cells moving from the peritoneal cavity into other tissues/organs-such as the spleen, lymph nodes, or peripheral blood. Recent reports have shown that PBMC from the patients with autoimmune conditions, such as primary biliary cirrhosis (14), systemic erythematosus (15), and rheumatoid arthritis (16), could reconstitute some characteristics of the disease in SCID mice by peritonealcavity injection. However, these studies reported the observations, such as production of autoantibodies or minimal histopathological changes in the absence of any other typical clinical symptoms and signs. In the cell-transfer experiment of experimental allergic encephalomyelitis, migration of T lymphocytes through blood-brain barrier was shown. However, the efficiency of the migration may be low, particularly for the transfer of a small number of cells. Therefore, in this study we attempted intracisternal injection for the local engraftment of the effector cells. The histopathological changes observed in the SCID mice transferred with CSF cells from MS patients at the exacerbation stage of the disease closely resembled those in MS patients: (i) the lesions were multiple scattered, discrete, and localized in the white matter of the brainstem and the spinal cords; (ii) the lesions showed demyelination with relative preservation of axons; (iii) the number of astrocytes increased around the foci, indicative of a reactive gliosis; and (iv) the lesions were characterized by various degrees of tissue necrosis with infiltration of inflammatory cells, of which most were host macrophages. However, lymphocytes were hardly seen around the lesions, which contrasted with the histopathological change seen in MS patients. It may be reasonable to assume that the autoantigens involved in the disease are common beyond species. The patients' antigenpresenting cells, such as macrophages and B lymphocytes, which had been transferred, processed, and presented the antigenic peptides together with autologous major histocompatibility complex molecules to the transferred T lymphocytes of the patient. Then, the activated T lymphocytes released certain lymphokines, which can recruit and activate inflammatory cells including host macrophages. Because of a

small number of transferred cells, host-derived inflammatory cells could be mainly detected in the lesions. The observations reported here provide direct evidence that CSF cells from MS patients at the exacerbation stage of the disease hold encephalitogenicity. However, the possibility of the transfer of infectious agents, such as a virus, could not be excluded, even though the transfer of CSF alone did not initiate either the clinical symptoms or the histopathological changes during the test period. Details of the mechanisms still remain to be clarified. Although CSF cells from MS patients at the exacerbation stage were mainly T lymphocytes, we did not show any direct evidence for involvement of T lymphocytes in disease transfer into SCID mice because of the limited number of patients in the exacerbation stage and the small number of CSF cells. Treatment of CSF cells with anti-CD3 antibody or various anti-variable-region antibody before transfer should provide information on the involvement of T lymphocytes and their specificity. A study on expression of the cytokine genes in the lesions may contribute to the identification of cytokines responsible for the inflammatory process in the lesions. This information could result in therapeutic advances in the treatment of MS patients. We thank Drs. Toyoki Maeda, Yusuke Oji, Tsuyoshi Azuma, Naoyoshi Kohjiro, and Hisaomi Kawai for referring patients for this study; Dr. Masaji Okada for useful information on the handling of SCID mice; Prof. Takehiko Yanagihara and Dr. Takeshi Tabira for critical comments on the histopathological examination; Drs. Hitoshi Kikutani and Edward Barsumian for helpful discussions and revisions of the manuscript; and Miss Ayumi Nobuhara for excellent assistance in preparing the manuscript. 1. McFarlin, D. E. & Mcfarland, H. F. (1982) N. Engl. J. Med. 307, 1183-1188. 2. Hafler, D. A. & Weiner, H. L. (1989) Immunol. Today 10,

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