Generation and biological properties of a monoclonal antibody to Galactocerebroside

Brain Research, 298 (1984) 203-208

203

Elsevier

Research Reports

Generation and Biological Properties of a Monoclonal Antibody to Galactocerebroside ABDOLMOHAMMAD ROSTAMI 1, P. ANN ECCLESTON l, DONALD H. SILBERBERG l, MIKIO HIRAYAMA l , ROBERT P. LISAKI, DAVID E. PLEASURE 1, S. MICHAEL PHILLIPS2

1Department of Neurology, and the 2Allergy and Immunology Section of the Department of Medicine, Universityof Pennsylvania School of Medicine, Philadelphia, PA 19104 (U.S.A.) (Accepted September 6th, 1983)

Key words: hybridoma - - anti-galactocerebrosideantibody - - Schwann cells - - oligodendrocytes- radioimmunoassay --immunofluorescence

Galactocerebroside (GalC) is a major glycolipid of myelin and myelin-forming cells. We have generated a mouse IgM monoclonal antibody to GalC (M-anti-GalC) which bound only to oligodendrocytes in rat and bovine central nervous system cultures as assessed by immunofluorescence. Double staining with rabbit anti-glial fibrillary acidic protein and anti-fibronectin antisera revealed no binding of M-anti-GalC to astrocytes or fibroblasts. Schwann cells, but not fibroblasts, were stained in short-term cultures of rat Schwann cells. M-anti-GalC exhibited in vitro cytotoxicity to rat and bovine oligodendrocytes in the presence of complement. This monoclonal antibody with its monospecificity, consistent titer, and capacity to induce cell lysis should be useful for in vitro and in vivo investigations concerning myelination and demyelination. INTRODUCTION G a l a c t o c e r e b r o s i d e (GalC) is the m a j o r glycolipid of myelin and cell m e m b r a n e s of myelin forming cells8, t2-14. Polyclonal rabbit antisera to G a l C (Ranti-GalC) specifically label the cell surface of cultured oligodendrocytes 5,8,t4 and bind to surface membrane of some cultured Schwann cells as well 5,7,13. Ranti-GalC has been shown to p r o d u c e experimental in vitro demyelination and myelin inhibition 1-3 and in vivo demyelination after intraneural injection 17. One-third of rabbits r e p e a t e d l y sensitized with G a l C develop a demyelinative p e r i p h e r a l n e u r o p a t h y 18. The studies with R - a n t i - G a l C have been limited due to: (a) the nature of polyclonal antisera which contain a family of antibodies with different antigenic specificities and biological function; (b) the inconsistency of the titers of the antisera; and (c) their limited supply. To circumvent these p r o b l e m s , we have produced a mouse monoclonal antibody to G a l C (M-

anti-GalC) which binds to the oligodendrocyte and Schwann cell surfaces and causes c o m p l e m e n t - m e diated cytotoxicity of cultured bovine and rat oligodendrocytes. MATERIALS AND METHODS

Animals B A L B / c mice and S p r a g u e - D a w l e y rats were obtained from Microbiological Associates (Walkersville, M D ) . Calf brains were o b t a i n e d from a local slaughter-house.

Cell line The non-secreting SP2/O mouse p l a s m a c y t o m a cell line was o b t a i n e d from the Cell C e n t e r of the University of Pennsylvania.

Hybridoma development B A L B / c mice were sensitized 9 times with 0.2 mg

Correspondence: A. Rostami, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. 0006-8993/84/$03.00 (~11984 Elsevier Science Publishers B.V.

204 of bovine galactocerebroside (lower spot cerebroside, Sigma, St. Louis, MO) mixed with bovine serum albumin (BSA; 1.0 mg; Sigma, St. Louis, MO) and complete Freund's adjuvant (CFA; 0.5 ml; Difco, Detroit, MI). All injections were made intraperitoneally except the last which was intravenous without CFA. Four days after the last injection, spleen cells from sensitized animals were fused with the myeloma line SP2/O using polyethylene glycol and the hybrids were cultured as described by Kennett 6. Supernatants from successful fusions were screened in duplicate for capacity to bind bovine GalC in a solid phase radioimmunoassay (RIA) (vide infra). Positive lines were then cloned in semisolid agarose in the presence of rabbit fibroblasts as feeders and retested after cloning. Ascitic fluid was obtained by intraperitoneal injection of BALB/c mice with 10 x 106 hybridoma cells. The antibody class was determined by double immunoprecipitate diffusion (Ouchterlony), using rabbit anti-mouse immunoglobulin antisera (Bionetics, Kensington, MD).

Radioimmunoassay (RIA ) for antigalactocerebroside antibodies Supernatants were assayed for the presence of antibodies against GalC using a modification of a published RIA 16. Wells of polyvinyl microtiter plates (Dynatech Laboratories, Alexandria, VA) were coated with bovine or synthetic GalC (N lignoceroyl dihydro galactocerebroside, Sigma) by adding 25 #1 of a 2 mg/ml solution of GalC in methanol to each well and evaporating to dryness at 60 °C. Plates were preincubated with 2% ovalbumin in PBS for 30 min at 37 °C to decrease non-specific binding of antibodies to the plastic surface and washed with the same solution followed by addition of 25 fll of hybridoma supernatants for 90 min at 37 °C. The plates were washed with PBS and 25 ,ul of the second antibody, 125I-rabbit anti-mouse F(ab')2 (Amersham Corp.) was added and was incubated for an additional 90 rain at 37 °C. After thorough washing and drying, the individual wells were assessed for the amount of bound radioactivity using a Packard Auto-Gamma Counter.

Indirect immunofluorescence for specificity of binding to cells in CNS and PNS cultures Purified bovine l0 and rat 4 oligodendrocytes were obtained using trypsinization in neutral buffered salts and Percoll gradient. These cells were cultured for up to 14 days as previously described. Corpus callosum was dissected from 5-6 day old Sprague-Dawley rats and dispersion cultures were maintained for up to 14 daysJ~. Schwann cells were cultured from newborn rats as previously reported omitting the step of differential adhesion in order to have fibroblasts in the cultures so that the specificity of binding to Schwann cells could be assessed 7. Live cultures were incubated at room temperature for 25 rain with: (a) supernatant or ascites of the mouse anti-GalC clone; (b) media or ascites of SP2/O line; (c) rabbit anti-GalCJ4; (d) rabbit anti-fibronectin (Cappel, Cochranville, PA) to identify fibroblasts and leptomeningeal cells; or (e) a mouse lgM monoclonal antibody to myoblast (M-anti-myoblast) with no activity against GalC in RIA (gift of Dr. Alan F. Horwitz, University of Pennsylvania, Philadelphia, PA) as a control for M-anti-GalC. After washing, the cultures were incubated with rhodamine (Rd) or fluorescein (FI) conjugated Ig fraction of the appropriate second antibody (goat anti-mouse Ig-Rd; goat anti-rabbit Ig-Rd or goat anti-rabbit Ig-F1 (Cappel)) for 25 min at room temperature. The cultures were then washed and the cells were post-fixed with acid alcohol (5% acetic acid-95% ethanol) for 15 min at 20 °C. To identify astrocytes, cultures of CNS origin were first fixed with acid-alcohol, washed and then incubated at room temperature for 25 rain with rabbit anti-glial fibrillary acid protein (rabbit-anti-GFAP) (gift of Dr. L. Eng, Palo Alto, CA). This was followed by incubation with goat anti-rabbit Ig-Fl. In double labeling experiments where both markers were surface determinants, cultures were incubated with both first antibodies or antisera followed after washing by both fluorochrome conjugated second antibodies. In these experiments the goat antimouse reagents were first incubated with rabbit Ig bound to agarose beads to remove any cross-reactivity to rabbit Ig. Goat anti-rabbit Ig F(ab')2-F1 (Cappel) was used as the other second antibody in double label experiments. When identification of GFAP was required, the cultures were first incubated with the antibody to the surface determinant and second anti-

205 body, then fixed and subsequently incubated with R-

cytes at all stages of culture. As noted (Table I), the

a n t i - G F A P followed by goat anti-rabbit Ig F(ab')2-F1.

percentage of cells in simultaneously studied aliquots of the same corpus callosum cultures binding mouse

Complement-mediated cytotoxicity assay To d e t e r m i n e c o m p l e m e n t - m e d i a t e d cytotoxicity, bovine oligodendrocyte cultures (I>95% rabbit antiG a l C +, plated at 1 x 105/12 m m coverslip) or rat olig o d e n d r o c y t e cultures (I>95% rabbit anti-GalC ÷ plated at 2.5 x 104/12 m m coverslip) were incubated with varying concentrations of supernatant for 30' at 20 °C in the presence of guinea pig serum as a source of c o m p l e m e n t (1:40 final concentration). Q u a n t i t a tion of cytotoxicity was achieved by exclusion of nigrosin dye 4. Controls consisted of cultures incubated with SPz/O supernatants, heat inactivated guinea pig serum with the mouse anti-GalC monoclonal antibody and M-anti-myoblast in the presence of complement. RESULTS

Screening for anti-GalC activity with R I A A m o n g the 432 wells in which the fused cells were distributed, 396 successfully fused cell lines developed. Supernatants from all of these were tested in the R I A for a n t i - G a l C activity. Five hybrid supernatants had significant binding to G a l C plates (more than 4 times binding c o m p a r e d to SP2/O media). Of these, one was successfully cloned. This supernatant reacted with rabbit anti-mouse IgM in an Ouchterlony i m m u n o p r e c i p i t a t e slide. Indirect immunofluorescence for specificity o f binding to cells in CNS and P N S cultures M - a n t i - G a l C b o u n d to rat and bovine oligodendroTABLE I

Comparison of percentage of galactocerebroside positive cells using immunofluorescence technique with mouse monoclonal and rabbit polyclonal anti-GalC antibodies Two hundred cells were counted per determination. DIV, days in vitro.

Oligodendrocytes

% Monoclonal anti-GalC +cells

% Rabbit antiGalC ÷cells

1 DIV

70%

71%

7 DIV 8 DIV 10 DIV

62% 50% 35 %

61% 48% 38%

Fig. 1. Double immunolabeling with M-anti-GalC and rabbit anti-GFAP in rat oligodendrocyte culture. A: phase contrast micrograph of 10 DIV oligodendrocyte culture from rat corpus callosum. A process bearing oligodendrocyte is in the center and two large flat cells are at the bottom. B: immunofluorescence staining of the same field with M-anti-GalC + goat antimouse Ig-Rd. Only the process bearing cell is stained. C: immunofluorescence staining of the same field after fixation with rabbit anti-GFAP + goat anti-rabbit-Ig-Fl. Only the flat cells show fibrillary cytoplasmic staining of astrocytes. (x422). The bright spot on left lower corner is the non-specific staining of debris.

206

Fig. 3. Surface labeling of Schwann cells with M-anti-GalC. A: phase contrast micrograph of 3 DIV rat Schwann cells. B: immunofluorescence staining of the same field with monoclonal anti-GalC + goat anti-mouse Ig-Rd. (× 281). anti-GalC and rabbit anti-GalC were the same, Flat cells with the appearance of astrocytes or fibroblasts did not bind mouse anti-GalC. In double label experiments all mouse anti-GalC + cells were G F A P and G F A P ÷ cells were mouse anti-GalC- (Fig. 1). The cells observed binding rabbit anti-fibronectin were always negative to mouse anti-GalC (Fig. 2). This was previously noted with rabbit anti-GalC. In Schwann cell cultures, only bipolar (Fig. 3) or round cells b o u n d mouse anti-GalC. This antibody b o u n d to many Schwann cells in 1-3 D I V cultures, but not in I>4 day Schwann cell cultures. No binding of IgM Manti-myoblast was noted to any CNS or PNS cells.

Fig. 2. Double immunofabelling with M-anti-GalC and rabbit anti-fibronectin in rat otigode~drocyte culture, A: phase contrast of micrograph of 14 DIV oligodendrocyte culture from rat corpus callosum with several process bearing cells and 4 large flat cells. B: immunofluorescence staining of the same field with M-anti-GalC + goat anti-mouse Ig-Rd. Two process bearing oligodendrocytes are stained. C: immunofluorescence

Cytotoxicity In the cytotoxicity assay using rat and bovine oligo-

staining of the same field with rabbit antifibronectin + goat anti-rabbit Ig-F1. The surface of large flat fibroblasts is stained. (x 539).

207 I00 90 8O 7O 60

Per Cent Cytotoxicify for

M-onh-GoIC

.50

Oh(Jodenro(jlio 4 0 50 SP2 Confrol

20 I0. 1/2

114

lifo

r/20

DduhOn of SupernotQnts

Fig. 4. Percent cytotoxicity of M-anti-OalC to bovine oligodendrocytes in the presence of complement. Quantitation of cytotoxicity was achieved by exclusion of nigrosin dye. The values are means of 3 experiments + S.D. dendrocytes culture and nigrosin dye exclusion test, the oligodendrocytes were susceptible to the cytotoxic effect of M-anti-GalC supernatant and ascites and complement when compared to SP2/O controls and feeding media (Fig. 4). When the complement source (guinea pig serum) was heated to 56 °C, the hybridoma showed activity similar to that of SP2/O control and the feeding media. M-anti-myoblast had activity similar to SP2/O control (data not shown in the graph). DISCUSSION The studies of GalC as a cell surface marker for oligodendrocytes and Schwann cells and the role of anti-GalC antibodies in the production of demyelination have resulted in increased understanding of some of the biological properties of myelin forming cells and myelin sheath as well as the complicated processes of myelination and demyelinationl-3, 8-14. To circumvent some of the problems in the use of polyclonal antisera, a monoclonal antibody to GalC was produced. This antibody, which is an IgM, shares many of the activities of the several polyclonal rabbit anti-GalC sera that have been tested so far. It bound

REFERENCES 1 Dorfman, S. H., Fry, J. M. and Silberberg, D. H., Antiserum induced myelination inhibition in vitro independent of the cytolytic effects of the complement system, Brain Re-

to oligodendrocytes and Schwann cells in CNS and PNS cultures and caused complement-mediated cytotoxicity in cultured rat and bovine oligodendrocytes. The fact that the percentage of cells in simultaneously studied aliquots of the same corpus callosum cultures binding mouse anti-GalC and rabbit anti-GalC were the same, provides strong evidence that the two antibodies recognize the same population of cells in the CNS culture. Ranscht et a1.15 produced a monoclonal antibody to synaptic plasma membrane from bovine hippocampi and the target antigen of this antibody was shown to be GalC. This monoclonal antibody bound to the oligodendrocyte and Schwann cell surfaces. Sommer et al. 21 produced several monoclonal antibodies to oligodendrocyte cell surface (01 to 04) by immunizing the mice with bovine corpus callosum. Two of these antibodies are believed to be against GalC 20. These reports demonstrate that GalC, although a small glycolipid, is of strong antigenic capacity and could be an important target antigen in the central and peripheral demyelinating diseases of man and experimental animals. The monoclonal antibodies to GalC can be used to further elucidate the role of this antigen in the normal process of myelination and in diseases of myelin sheath. Specifically, because of a theoretically unlimited supply, they can be used for separation of myelin forming cells in electronic cell sorters and be used to produce pure membrane preparations in affinity columns by binding the antibody to absorbant beads. The antibody is capable of both binding to cells and inducing cell lysis in vitro. The antibody and the cells producing the antibody could also be used in in vivo studies of myelin in BALB/c mice since problems related to allogenic or heterologous rejection would not be encountered. ACKNOWLEDGEMENTS This work was supported by NIH Grants NS08075 and NSl1037 and a grant from the Muscular Dystrophy Association.

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