Immune response to isolated oligodendrocytes

Journal of the Neurological Sciences, 1979, 43 : 157-167 © Elsevier/North-Holland Biomedical Press

157

I M M U N E RESPONSE TO ISOLATED O L I G O D E N D R O C Y T E S

ODED ABRAMSKY*, ROBERT P. LISAK, DONALD H. SILBERBERG, TALMA BRENNER* and DAVID PLEASURE Department of Neurology, University of Pennsylvania School of Medicine, and the Multiple Sclerosis Research Center of the University of Pennsylvania and the Wistar Institute, Philadelphia, PA (U.S.A.)

(Received 15 March, 1979) (Accepted 4 April, 1979)

SUMMARY Oligodendrocytes were isolated from bovine white matter and were injected with complete Freund's adjuvant (CFA) into experimental animals. Indirect immunofluorescence studies using fluoresceinated goat anti-rabbit or anti-guinea pig immunoglobulin (GARIg; GAGPIg) showed that rabbit and guinea pig anti-oligodendrocyte (RAO, GPAO) sera reacted specifically with the surface of isolated oligodendrocytes in suspension, as well as with oligodendroglia in bovine and human brain sections, and in mouse cerebellum cultures. This activity of RAO was blocked by non-fluoresceinated GARIg and by GPAO, and absorbed by oligodendrocyte preparation (OP) or whole white matter, but not by purified myelin, neuroblastoma or non-brain tissue. Low levels of anti-basic protein antibodies were found in many RAO (but not GPAO) sera by radioimmunoassay, and a few showed significant anti-galactocerebroside antibody by agglutination and radioimmunoprecipation techniques. Guinea pigs sensitized with isolated oligodendrocytes in CFA showed cell-mediated immunity (CMI) to OP as manifested by delayed type skirt test and induced in vitro lymphocyte transformation. CMI to purified myelin basic protein was not detected. The demonstration of humoral and CMI to the cell responsible for the production of CNS myelin may be related to some aspects of the immunopathogenesis of demyelinating disorders.

This study was supported by grants from NMSS (NMS 894-B-2)and USPSHS (1P01-HD-08536, NS-11037, NS-08075, 5 K07-NSll061), and fellowships (O.A.) from the Fogarty center of the NIH and the National Multiple Sclerosis Society. Presented in part at the 29th Annual Meeting of the American Academy of Neurology, Atlanta, April 1977. Reprint requests should be addressed to: Dr. Lisak, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. * Present address: Department of Neurology, Hebrew University Hadassah Medical School, Jerusalem (Israel).

158 INTRODUCTION In recent years a number of procedures have been developed for the isolation of oligodendrocytes from mammalian brain (Poduslo and Norton 1972; Fewster et al. 1973; Chao and Rumsby 1977; Iqbal et al. 1977). Some chemical and morphological properties of these cells have been examined (Poduslo and Norton 1972; Fewster et al. 1973, 1974; Norton et al. 1975; Pleasure et al. 1977; Poduslo and McKhann 1977) and the cells have been maintained in short term culture (Fewster and Blackstone 1975; Pleasure et al. 1977; Poduslo and McKhann 1977). Recently, Poduslo et al. (1977) and also ourselves in a preliminary report Abramsky et al. (1978), have demonstrated by indirect immunofluorescence technique the presence of antioligodendroglia antibody in rabbits immunized with oligodendrocyte preparation (OP). The relationship of the experimentally induced antioligodendroglia antibody to demyelinating disease stems from the possibility that demyelination might be the consequence of an immune response involving the CNS myelin and/or the oligodendroglia, the cells responsible for the production of CNS myelin (Peters 1964; Bunge and Glass 1965; Hirano 1968). Our demonstration of similar antioligodendroglia antibody in patients with multiple sclerosis (Abramsky et al. 1977a, b) supports this possibility. We present here our study of some aspects of the humoral and cellular immune response to oligodendroglia in experimental animals. This study supports the usefulness of OP for further studies of oligodendrocyte surface components and of their possible relationship to the immunopathogenesis of demyelinating disorders. MATERIALSAND METHODS

Oligodendroglial preparation Oligodendrocytes were isolated by sterile technique from fresh calf white matter according to the method of Poduslo and Norton (1972) with some modifications. The tissue was dissected, minced (5 ~ glucose, 5 ~ fructose, 1 ~ albumin, antibiotics and KH~PO4-NaOH buffer, pH 6.0) and incubated with 0.25 ~ trypsin at 37 °C for 75 min. The trypinized tissue was cooled, treated with cold buffered calf serum to inactive the trypsin, and then washed several times with medium. The white matter pieces were then suspended in 0.9 M sucrose solution and put through a nylon monofilament and a stainless steel screen in order to obtain a cell suspension. The suspension was layered onto discontinuous gradients consisting of 0.9 M, 1.4 M and 1.55 M sucrose, and centrifuged at 3200 rpm (Sorvall RC-2B centrifuge with HS-4 rotor) for 15 min. The main oligodendroglial fraction on the 1.55 M sucrose layer was collected, pooled, diluted with minimal essential medium (MEM) and concentrated by centrifuging at 1000 rpm for 10 min. Using this procedure 4.5-7.5 × 107 cells were isolated from 150-200 g wet white matter obtained from 4-5 calf brains. Phase and Nomarski interference contrast light microscopy showed 90-98 ~o homogeneous cell population. The cells were characterized morphologically by scanning and transmission electron microscopy, and biochemically by the specific activity of several enzymes, as described by us previously (Pleasure et al. 1977; Silberberg et al. 1977).

159

Immunization of animals Cells for immunization were lyophilized immediately after isolation and stored at --45 °C. 3-6 x 107 lyophilized oligodendrocytes emulsified in complete Freund's adjuvant (CFA) were injected subcutaneously once a month for 3 months, into 7 New Zealand albino rabbits. Serum samples were collected for determination of antibody response. 1-3 x 107 lyophilized oligodendrocytes were injected 1-3 times, subcutaneously in CFA, into Hartley albino guinea pigs, in order to determine antibody and the cellular immune response. All rabbits and guinea pigs were examined daily from the 7th day post first sensitization. Three rabbits and all guinea pigs (n=16) were examined histologically.

Indirect immunofluorescence assay (1F) Aliquots of sera obtained 3 weeks after each injection from rabbits and guinea pigs injected with isolated oligodendrocytes were incubated (20 ~ v/v) for 2 hr at 37 °C and overnight at 4 °C, with bovine liver, whole bovine white matter, bovine myelin purified according to Norton and Poduslo (1973), lyophilized bovine oligodendrocytes, or mouse neuroblastoma. The absorbed sera were then diluted 1:2, 1:10, 1:40 and 1:100 with phosphate buffered saline (PBS, pH 7.8), and incubated for 30 min at 37 °C with: (1) 4 × 106 isolated oligodendrocytes, maintained for 2 hr in MEM suspension culture containing 2.5 ~o calf serum; or (2) acetone-fixed coverslips of bovine or human white matter, and gray matter, or either cerebral or cerebellar origin; or (3) 3-weeks-old mouse cerebellum cultures (MCC). The preparations were washed in PBS and fluorescein isothiocyanate-conjugated goat anti-rabbit or anti-guinea pig immunoglobulin (Baltimore Biological Laboratories), which had been previously incubated with whole bovine white matter, was applied, respectively. Thirty minutes later the preparations were washed again, mounted in solution of 50~o glycerol:PBS and examined under a Zeiss fluorescence microscope. For blocking experiments, the isolated oligodendrocytes which had been incubated with the previously absorbed sera, were incubated with non-fluoresceinated goat anti-rabbit or anti-guinea pig immunoglobulin, before the final addition of the fluoresceinated antisera. Control sera were provided by incubation with pre-immunization normal rabbit or guinea pig sera (NRS, NGPS) and sera from such animals injected with either CFA alone or with incomplete Freunds' adjuvant and whole brain homogenates. Control tissue included both acetone-fixed cell suspensions of minced bovine liver cells, A9 line of mouse fibroblasts and both differentiated and undifferentiated mouse neuroblastoma cells (cholinergic clone NS 20, adrenergic clone NIE 115, inactive clone N 18).

Determination of anti-galactocerebroside (GC) antibody Anti-galactocerebroside antibody titers in rabbits were determined using both an agglutination test and a radioimmunoprecipitation (RIP) test as previously described by us (Fry et al. 1976). In the RIP assay, the precipitation of liposomes containing galactocerebroside and [tritium-ZH]labeled cholesterol is employed as a marker of antibody directed against galactocerebroside.

160

Radioimmunoassay for determination of anti-basic protein antibody Bovine basic protein (B-BP) and guinea pig basic protein (GP-BP) were isolated according to the methods of Hirshfeld et al. (1970) and Diebler et al. (1972), respectively. The BP was labeled with 125I using chloramine-T (Hunter 1973). Excess iodide was removed by incubation of [lzSI]BP with NRS (5:1, v/v) at room temperature for 1 hr and at 4 °C for another hour, and then with goat anti-rabbit or anti-guinea pig serum (GARS; GAGPS) at 37 °C for 1 hr and at 4 °C overnight, followed by centrifugation and washing in PBS. The iodinated antigen (10-20 × l0 s cpm in each tube) was incubated at 37 °C for 1 hr with 2-10 #1 of rabbit or guinea pig test serum, and then with an optimal amount of goat anti-rabbit or guinea pig Ig as second antibody at 37 °C for 1 hr and at 4 °C overnight (Lisak et al. 1970). After centrifugation at 3500 rpm for 20 min the pellet was resuspended and counted. Results were expressed as the percentage of counts in the pellet compared to the total counts at the beginning of the reaction.

Skin test Guinea pigs were skin-tested 10-14 days after first or second sensitization with OP, and and the tests read 24 hr later. Intradermal injections of 0.1 ml containing 5 × 106 lyophylized oligodendrocytes, 50 #g purified bovine myelin, 15 #g GP-BP, 15 #g calf thymic histone, 100 #g old tuberculin, and PBS, were the skin test antigens. A diameter of < 5 mm induration was considered positive.

Lymphocyte transformation Peritoneal exudate cells (PEC) were obtained by peritoneal lavage 3 days after the i.p. injection of sterile mineral oil, from guinea pigs that had been immunized 10-20 days before, with either OP in CFA or CFA alone. The cells were washed twice with Hank's solution and then passed over a column of coil cotton in order to enrich the lymphocyte population (Lisak and Zweiman 1973, 1974). The cells eluted from this column consisted of 60-80 ~ lymphocytes, with the remaining cells being macrophages by the criterion of the incorporation of latex particles. The cells were cultured at a final lymphocyte concentration of 1 x 106 ml per vial in MEM with added glutamine (2 ~), antibiotics and 10 ~ fetal calf serum. The following antigens were added to the cultures: 5 × 105, 1 × 106 and 5 × 106 lyophilized oligodendrocytes; 25 #g and 50 #g purified bovine myelin; 5 #g, 25 #g and 75 GP-BP; 15 #g calf thymic histone; 500 #g old tuberculin (OT, Jensen-Salisbury Co.); 0.75 mg phytohemagglutinin (PHA, Difco). After 4 days 1 #Ci of triatated thymidine (New England-Nuclear Corp.) was added. Eighteen hours later the cells were processed for measurement of the incorporation of this isotope into TCA-precipitated material by scintillation counting. The proliferative responses were defined by the stimulation index (SI) defined as: mean cpm of replicate cultured with antigen (or mitogen) SI=

mean cpm of replicate cultured without antigen (or mitogen)

161 RESULTS S e r u m f r o m 7 r a b b i t s a n d five g u i n e a pigs i n j e c t e d 2 o r 3 t i m e s w i t h o l i g o d e n drocyte preparation

(OP) and

a b s o r b e d w i t h b o v i n e liver to r e m o v e n o n s p e c i f i c

b i n d i n g , r e a c t e d specifically as s h o w n b y t h e i n d i r e c t i m m u n o f l u o r e s c e n c e t e c h n i q u e (IF), w i t h t h e s u r f a c e o f i s o l a t e d o l i g o d e n d r o c y t e s in s u s p e n s i o n ( T a b l e 1). T h e p o s i t i v e r i n g l i k e f l u o r e s c e n c e a r o u n d t h e cell s u r f a c e (Fig. 1A) was d e m o n s t r a t e d in a d i l u t i o n o f _> 1:100. T h e r a b b i t a n d g u i n e a p i g a n t i - o l i g o d e n d r o c y t e ( R A O ; G P A O ) sera d i d n o t b i n d to n e u r o b l a s t o m a , f i b r o b l a s t s o r liver cells. B i n d i n g to o l i g o d e n d r o c y t e s in s u s p e n s i o n was also d e m o n s t r a t e d b y a n t i - w h o l e b r a i n s e r u m , b u t n o t b y NRS, NGPS or serum from such animals injected with CFA. RAO and GPAO

sera also r e a c t e d specifically w i t h t h e s u r f a c e o f o l i g o d e n -

d r o g l i a in b o v i n e a n d h u m a n b r a i n sections (Fig. 1B). I n c e r e b r a l a n d c e r e b e l l a r g r a y m a t t e r o n l y f l u o r e s c e n t s t a i n i n g o f t h e s m a l l per±neural o l i g o d e n d r o g l i a c o u l d be seen. I n t h e w h i t e m a t t e r s o m e satellite a n d m a n y i n t e r f a s c i c u l a r o l i g o d e n d r o g l i a , e i t h e r s e p a r a t e l y o r in g r o u p s a n d rows, w e r e d e m o n s t r a t e d . O t h e r c o m p o n e n t s s u c h as TABLE 1 H U M O R A L ANTIBODIES IN RABBITS INJECTED WITH O L I G O D E N D R O C Y T E PREPARATION Rabbit Injected Injection IF No. with No. oligo 1 : 100 1

2

OP

OP

3

OP

4

OP

5

OP

6

OP

7 8 9 10 11

12 13

OP CFA (NRS) GP-BP B-BP GC BRAIN

IF Anti-B-BP Anti-GP-BP myelin RIA ( ~ cpm RIA 1 : 10 bound 4. SD)

Anti-GC Anti-GC agglutination RIP

--

1:32 1:8 1:8 1:8 1:4 1:8 1:16

2

+

3

4. -+ + + 4. 4. + + + 4. 4" 4" -+ ---

4± -------------

-4-

+

1 2 3 4 2 3 2 3 2 3 2 3 0 2 2 1 1 2 1

4-

10.7 4- 0.93

14.7 10.0 12.1 10.9

± 1.16 4- 0.83 4- 0.39 4- 0.39

8.7 4- 0.50 9.8 4- 0.13 7.8 3.8 6.0 3.5 4.8 15.2 15.9

4. i 4± 44, 4,

0.39 0.16 0.25 0.08 0.39 0.78 0.45

9.3 + 0.82

8.0 9.2 7.0 7.6 6.0 4.4 5.5 5.5

4, 0.75 4- 0.97 4- 0.52 -4- 0.97 4- 0.32 4- 0.40 4- 0.54 4, 0.64

6.4 5.4 5.7 4.0 5.0 15.0 12.7

± 0.56 4- 0.68 :]- 0.12 4- 0.43 4- 0.40 4- 1.15 4- 0.15

0 1:16 0 1:128

1:2 1:2 1:32

1:174 1:64 1:64 1:174 1:128

1:64 1:64 1:1659

1:64

Abbreviations: IF = immunofluorescence; OLIGO = oligodendrocytes in suspension; OP = oligodendrocyte preparation; B-BP = bovine basic protein; GP-BP = guinea pig basic protein; RIA = radioimmunoassay; GC = galactocerebroside; RIP = radioimmunoprecipitation expressed as titer; CFA = complete Freund's adjuvant; NRS = normal rabbit serum.

162

L

Iqiii

.........

Fig. 1. Immunofluorescence demonstration of rabbit anti-oligodendrocyte serum binding to: A: isolated oligodendrocytes in suspension, × 400; B: interfascicular oligodendroglia in bovine white matter, x 240; C: oligodendroglia in mouse cerebellum culture, x 400.

163 myelin, blood vessels, neurons and other glial cells were stained by antibrain serum, but not by RAO or GPAO serum. The identification of oligodendroglia was confirmed in these experiments by Giesma staining of brain slices parallel to the sections used for the IF and by phase microscopy. Similarly, the RAO serum reacted specifically with the surface of oligodendroglia in the outgrowth areas of mouse cerebellum cultures (MCC) (Fig. 1C). The RAO and GPAO sera reacted with isolated oligodendrocytes in suspension and with oligodendroglia in brain sections even after preincubation with purified myelin or neuroblastoma preparations, but not after absorption with oligodendrocyte preparation of whole white matter (Table 2). The demonstration of binding of RAO serum to oligodendroglia by IF was blocked by their preincubation with GPAO serum or by incubation with non-fluorescein labeled goat anti-rabbit serum. Many rabbits injected with OP developed low or moderate levels of anti-basic protein (BP) antibodies (Table 1). None of the guinea pigs developed anti-BP antibody. The degree of the response with the bovine BP tended to be higher with RIA than the degree of the response with the guinea pig BP. There was no difference in binding of bovine BP and GP-BP by normal rabbit sera. The levels of anti-BP antibodies were usually similar in samples taken after the second or third OP injections. Only two rabbits developed significant titers of anti-GC antibodies, as determined by agglutination test ( > 1:32) (Table 1). Only one animal had a striking elevation of anti-GC antibody by RIP test (Rabbit No. 6). No correlation was demonstrated between anti-BP antibody, and anti-oligodendroglia activity in individual RAO sera. Guinea pigs injected with OP in CFA demonstrated cell-mediated immune response to OP, as determined by both the in vivo delayed type skin test and the in vitro lymphocyte transformation (Table 3). No significant response in either assay was demonstrated with purified myelin, guinea pig basic protein or calf thymus histone. A positive response to OT and a normal response to PHA were noted. None of the animals developed clinical or histologic evidence of experimental allergic encephalomyelitis. TABLE 2 INDIRECT IMMUNOFLUORESCENCE SERUM Absorbed with

Liver Neuroblastoma Purified myelin Whole white matter Oligodendrocyte preparation

WITH RABBIT ANTI-OLIGODENDROGLIA

Target tissue oligoden- oligoden- oligodendroglia in droglia in droglia in suspension brain cerebellar sections cultures

liver

neurofibroblasts blastoma

++ ++ + ±

---

__ --

.

+ + + . .

+ .

. .

. .

__

164 TABLE 3 CELL-MEDIATED IMMUNITY IN GUINEA PIGS INJECTED WITH EITHER OLIGODENDROCYTE PREPARATION (OP) IN COMPLETE FREUNDS ADJUVANT (CFA), OR CFA ALONE Antigen tested ~

Lymphocyte transformation

Skin test

Mean SI 5: SEM

Incidence of positive (SI 3)

Incidence of positive (5 mm)

OP

OP

CFA

OP

CFA

10/12 1/12 0/10 0/4 10/12 11/12

0/5 0/5 0/5 0/5 4/5 4/5

13/16 2/16 0/12 0/4 4/4

0/5 0/5 0/5 0/5 5/5

0/3

0/3

Oligodendrocyte preparation 5.1 Purified bovine myelin 1.8 Guinea pig basic protein 1.6 Thymic histone 1.4 Old tuberculin 5.7 PHA 17.3 PBS

CFA :k 1.8 ± 0.3 ± 0.3 ± 0.2 ± 1.8 ± 6.0

1.4 ± 1.6 ± 1.2 ± 1.2 ± 6.1 ± 14.3 -

0.3 0.2 0.2 0.1 2.0 4.8

The highest response of lymphocyte proliferation was demonstrated by the use of 1 × l0 s oligodendrocytes, 50 pg purified myelin and 25 pg BP. Abbreviations: OP = oligodendrocyte preparation; CFA -- complete Freund's adjuvant; SI stimulation index ; PHA = phytohemagglutinin; PBS = phosphate buffer serum.

DISCUSSION In this study we have demonstrated the production o f specific h u m o r a l and cellmediated immune responses to oligodendroglia in experimental animals injected with oligodendrocyte preparation (OP) isolated from calf brain. As shown by indirect immunofluorescence (IF) technique, rabbit and guinea pig anti-oligodendroglia ( R A O ; G P A O ) sera reacted specifically with the surface of isolated oligodendrocytes in suspension and with oligodendroglia in brain sections and cerebellum cultures. The demonstration by I F o f R A O serum binding to isolated oligodendrocytes in suspension was previously reported by Poduslo et al. (1977) and by us in a preliminary report (Abramsky et al. 1978). The blocking experiments in this study, showing inhibition of the I F demonstrations o f R A O serum binding to oligodendroglia by nonfluorescein labeled goat anti-rabbit immunoglobulin, indicate that the binding is due to a c o m p o n e n t of the immunoglobulin fraction and represents an antigen-antibody reaction. The anti-oligodendrocyte serum did not react with other cells or with myelin, and the activity was absorbed out by incubation with OP or a whole white matter preparation containing oligodendrocytes, but not by other cell preparations or purified myelin. These findings suggest that the C N S myelin and the oligodendroglial m e m b r a n e which elaborates the C N S myelin are not a " s y n o n y m o u s unit" as was previously suggested (Bunge 1968), since O P contains determinants not present or not manifested in myelin. It seems that some o f the antigens responsible for the humoral immune response to our OP are n o t species-specific, do not exist or are not available for binding in myelin nor in other brain cells, and are unique to both the perineural

165 oligodendroglia of the grey matter and the interfacicular oligodendroglia of the white matter. An experimental autoimmune neurological disease, similar to experimental allergic encephalomyelitis (EAE), was recently induced in laboratory animals injected with OP in CFA (Raine et al. 1977, 1978; McDermott et al. 1977). It appears that the autoantigen in these animals was BP, since non-trypsinized human oligodendrocytes were more encephalitogenic than trypsinized oligodendrocytes which contained less BP. In the present study, animals injected with our OP in CFA did not show signs of EAE. The amount of BP in our preparation was apparently sufficient to induce low levels of anti-BP antibody in rabbits (but not guinea pigs), but probably insufficient to induce detectable cell-mediated immune response to BP. Without the induction of cellmediated immune response to BP, EAE does not develop (Shaw et al. 1965; Paterson 1966; Lisak and Zweiman 1974). Another antigen, galactocerebroside (GC) was probably present in our OP as well. However, significant titers of anti-GC antibody were demonstrated only in a few RAO samples, in spite of the fact that galactolipids exist in OP (Norton et al. 1975; Poduslo and McKhann 1977) and anti-galactocerebroside sera bind to oligodendroglia (Raffet al. 1978; Lisak et al. 1978, 1979). Antibody to oligodendroglia can be used to identify cell types in the brain and in CNS cultures at different stages of development. Identification of different antigens present in the oligodendroglial membrane and responsible for experimentally induced antioligodendroglia antibodies, can facilitate studies of myelination and demyelination. The use of antibodies specifically directed against oligodendroglia, oligodendroglial fractions, myelin, BP, GC, and other white matter antigens as immunological markers, may help explain some of the biological relationships between oligodendroglia and myelin in physiological and pathological conditions. For example, it was suggested that if antibodies are involved in the demyelination seen in multiple sclerosis (MS), they are probably anti-oligodendroglia rather than anti-myelin antibodies (Wolfgram and Duguette 1976). Anti-oligodendroglia antibodies were recently demonstrated by us in sera of patients with MS (Abramsky et al. 1977a, b), however, the significance of these antibodies in the pathogenesis of the disease and their relationship to the in vitro demonstration of the demyelination induced by antisera to whole white matter (Bornstein and Appel 1965; Saida et al. 1977), or GC (Fry et al. 1974; Saida et al. 1977;) is not yet known. The use of specific antibody markers can facilitate our understanding of the immunologic relationship between oligodendroglia and myelin antigens, and between demyelination and glial destruction in both experimentally and clinically demyelinating disorders. ACKNOWLEDGEMENTS We are grateful for the skilled technical assistance of Ms. Judy Parris, Ms. Joan George, Ms. Tova Tal and Ms. Margaret Manning, and we wish to thank Dr. Uri Littaur and Dr. F. Arthur McMorris for preparing the neuroblastoma cells and the fibroblasts.

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