Journal of Neuroscience Research 30:699-711 (1991)

Rapid Communication Galactocerebroside and Sulfatide Independently Mediate Ca2 t Responses in Oligodendrocytes C.A. Dyer and J.A. Benjamins Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan Galactocerebroside (GalC) and sulfated galactocerebroside (sulfatide) are sphingolipids highly enriched in myelin. The binding of antibodies reactive with either sulfatide or GalC to cultured oligodendrocytes causes a Cat+ influx, followed by microtubule depolymerization; however, antisulfatide is less effective than anti-GalC in altering cytoskeleton. Typical Cat+ responses are delayed for both antibodies but are transient for sulfatide-reactive antibodies in contrast to the sustained responses previously reported for anti-GalC (Dyer and Benjamins, J Cell Biol 111: 625-633, 1990). Approximately one-half as many oligodendrocytes respond to sulfatide-reactive antibodies (about 39%) as to anti-GalC (about 75%). Subpopulations of oligodendrocytes were identified that responded to neither antibody, only one antibody, or both antibodies, indicating that sulfatide and GalC independently mediate Cat+ responses. These results suggest that sulfatide and GalC have different physiologic roles in regulating elaboration of myelin membrane by oligodendrocytes in vivo and support the possibility that viral or immune attack via GalC or sulfatide on oligodendrocytes may mimic normal signals in a manner that disrupts the sequence of events that coordinates myelination or maintenance of myelin in vivo. Key words: cultured cells, microtubules, glycosphingolipids, antibodies, Indo-1 INTRODUCTION Glycosphingolipids are present in the outer leaflet of the membrane bilayers of essentially all eukaryotic cells (for review, see Hakomori, 1990). Their roles have not been clearly established in any cell type; however, several functions have been postulated by many investigators (Bansal and Pfeiffer, 1989; Facci et al., 1988; Nudelman et al., 1988; Okada et al., 1988; Hanai et al., 0 1991 Wiley-Liss, Inc.

1987, 1988; Curatalo, 1987; Fishman, 1982). Hakomori (1990) reviews evidence that these glycolipids may 1) regulate cell proliferation by modulating transmembrane signal transducers, 2) mediate cell-cell or cell-substratum recognition, and 3) serve as receptors for viral and bacterial toxins. There is increasing evidence strongly suggesting that the glycosphingolipids specifically found in myelinproducing cells, galactocerebroside and sulfatide, act as transmitters of environmental information. In such studies, antibodies to GalC and sulfatide have been useful tools in providing information about their functions. For example, the differentiation of oligodendrocyte progenitor cells is reversibly inhibited by the binding of a monoclonal antibody (the Ranscht monoclonal antibody; RmAb), which reacts with both galactocerebroside (GalC) and its sulfated form, sulfatide (Bansal and Pfeiffer, 1989). Similar experiments have been performed with Schwann cells; the binding of the R-mAb to Schwann cells removed GalC from Schwann cell surfaces and blocked elongation of the mesaxon (Ranscht et al., 1982). Thus the removal of GalC from the surface of the Schwann cell appears to arrest these cells in a premyelinating stage. These two examples suggest that the interaction of GalC or sulfatide with their environment is necessary for oligodendrocyte and Schwann cell differentiation and also support the hypothesis that these glycosphingolipids may function as receptors. Additional evidence that GalC may serve as a receptor comes from studies showing that antibodies to GalC blocked human immunodeficiency virus (HIV) internalization and infection in two CDCnegative, GalC-

Received August 26, 1991;revised October 7, 1991; accepted October 9, 1991. Address reprint requests to Dr. Charissa A. Dyer, Department of Neurology, Wayne State University, School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201.

700

Dyer and Benjamins

positive neural cell lines (Harouse et al., 1991). Furthermore, the HIV surface glycoprotein gp120 bound specifically to GalC on HPTLC plates. Since pathologic studies of brains from patients with HIV infections show demyelination (Gray et al., 1991; Berger et al., 1989), Harouse et al. (1991) speculate that GalC may be playing a role as a receptor for the entry of HIV into the oligodendrocyte. Harouse and coworkers also suggest that the gp 120-GalC interaction could affect oligodendrocyte function in the absence of full infection, based on our studies showing that prolonged exposure to IgG antibodies specific for GalC caused a reversible contraction of the membrane sheets elaborated by cultured murine oligodendrocytes (Dyer and Benjamins, 1988b). In our studies aimed at understanding the function of GalC and sulfatide in cultured murine oligodendrocytes, we have shown that antibodies specific for GalC induce an increase in intracellular Ca2’ in oligodendrocytes. The consequence of anti-GalC-mediated Ca2+ influx into oligodendrocytes is a cascade of events including microtubule disruption followed by reversible membrane sheet contraction (Dyer and Benjamins, 1988b, 1989, 1990). The anti-GalC-induced Ca2+ response does not appear to be mediated by voltage-operated Ca2+ channels (Dyer and Benjamins, 1990). In addition, the Ca2+ rise is due to an influx and not to release from intracellular stores; the cytoskeletal changes were shown to be dependent on this increase in intracellular Ca2+. The Ca2+ influx initiated by antibody binding suggests a mechanism by which ligands interacting with glycosphingolipids may initiate physiologic changes in oligodendroc ytes. Less is known about the role of sulfatide in myelinproducing cells. We have previously shown that antibodies reactive with sulfatide do not cause membrane sheet contraction, while anti-GalC does (Dyer and Benjamins, 1988b). The present study examines the effects triggered by the binding of antibodies reactive with sulfatide to mature murine oligodendrocytes. The aims of the present study were to determine whether sulfatide reactive antibodies initiate an influx of Ca2 into oligodendrocytes and, if so, whether the responses are independent of those initiated by anti-GalC. Unexpectedly, our results demonstrated that mature oligodendrocytes expressing both GalC and sulfatide are heterogeneous in their responses to the two antibodies, differing both in ability to respond and in the type of Ca2+ response initiated, +

MATERIALS AND METHODS Oligodendrocyte Cultures Murine oligodendroglial shake-off cultures were grown as previously described (Dyer and Benjamins, 1990), based on the method of McCarthy and DeVellis

(1980). Briefly, single cell suspensions were prepared from cerebra from 2-day-old mice and plated in 75-cm2 flasks. Cultures were grown in 10% newborn calf serum (Hyclone Laboratories, Logan, UT) in Dulbecco’s modified Eagle’s medium (DME) (Gibco Laboratories, Grand Island, NY) for about 7-10 days. When confluency was reached, the small, dark, process-bearing cells were shaken from the bed layer and plated on 25-mmdiameter coverslips. The shake-off cells were grown overnight in chemically defined medium (CDM) (Bottenstein, 1986), in nine-tenths CDM and one-tenth 10% calf serum DME for the next day, and then in four-fifths CDM and one-fifth 10%calf serum DME for the remaining days in culture. At the time used in experiments, about 24 days after birth, cultures contain about 30-50% oligodendrocytes .

Antibodies The anti-GalC antibodies used for the Ca2+ studies were polyclonal anti-GalC IgG. These antibodies were produced by injecting rabbits with GalC, keyhole limpet hernocyanin, and M . tuberculosis in Freund’s adjuvant as described by Benjamins et al. (1987). The IgG fraction was isolated from the serum using a protein ASepharose column (Pharmacia). The anti-GalC antibodies in the IgG fraction were specific for GalC and did not cross react with sulfatide. The antibody used for GalC/ sulfatide immunostaining was the R-mAb mouse IgG mAb (Ranscht et al., 1982; Bansal et al., 1989). The cell line was kindly given to us by Dr. Ranscht; ascites filtered through glass wool and centrifuged was used as the source of the antibody. The R-mAb reacts predominantly with GalC and sulfatide (Bansal and Pfeiffer, 1989; Benjamins, unpublished observations) and also with seminolipid, monogalactosyl diglyceride, and psychosine (Bansal et al., 1989). The hybridoma cell line that produces A007, a rat IgM mAb reactive with sulfatide, seminolipid, and proligodendrocyte antigen (Bansal et al., 1991), was a generous gift from Dr. Kari Stefansson. Hybridoma supernatants were used as the source of the antibody. Anti-tubulin mouse IgG mAb was a gift from Dr. Lester Binder. Second antibodies used for immunofluorescence were purchased from Organon Teknika (Malvern, PA). Calcium Studies Changes in intracellular Ca2+ were monitored using the Ca2+-sensitive dye Indo- 1 (Molecular Probes, Inc., Eugene, OR) (Dyer and Benjamins, 1990). Cultures were loaded with l p M Indo-1AM (Molecular Probes) in serum-free DME for 45-60 min at 37°C. Coverslips were placed in a holder that fits into the microscope stage of the ACAS laser cytometer (Meridian, Easl Lansing, MI). With phase optics, oligodendrocytes were

Galactocerebroside, Sulfatide, and Caz+ Responses

identified by their characteristic morphology, Murine oligodendrocytes have shiny cell bodies and elaborate extensive membrane sheets after 20 days in culture. We have found these unique characteristics to be reliable markers for identification of mature murine oligodendrocytes. For analysis of Ca2+ fluxes, each experiment involved examination of one cell per coverslip. The cells chosen for analysis were not in contact with surrounding cells. Line scans were performed to analyze changes in intracellular free Ca2' with time; a point on either side of the cell was chosen that defined the Iine through the cell that was repeatedly scanned during the analysis. Each cell was scanned 100 times at 1 sec per scan prior to addition of antibody to show that scanning itself does not cause an increase in intracellular Ca2' and to establish baseline and verify its stability. After another 15-20 scans, antibody was added and cells were scanned 300900 times at 2-3 sec per scan. The ratio of Ca2+-bound Indo- 1 to unbound Indo- 1 was determined for each scan and plotted against time; Indo-1 is excited at 388 nm and emits light at 405 nm when bound to Ca2+ and at 485 when Ca2+-free. Baselines and ratio changes are similar to those seen in our previous studies under identical conditions (Dyer and Benjamins, 1990); these represent average Ca2+ increases of between 300 and 500 nM above resting values of approximately 5-20 nM.

701

(GalC or sulfatide) was detected. This technique is able to detect 100 ng of either lipid.

Immunofluorescent Staining Since different batches of IgG fractions from rabbit sera, ascites fluid, and hybridoma supernatants vary significantly in antibody titer, concentrations of specific antibodies were determined by immunof Iuorescence. Various dilutions of A007 hybridoma supernatants, antiGalC IgG fractions, and R-mAb ascites were added to cultures to determine which resulted in solid or patched staining on live oligodendrocyte membrane sheets (see Dyer and Benjamins, 1990). These concentrations were then used as needed in the experiments described in this paper. For double labeling with R-mAb and A007, cultures were exposed to R-mAb followed by goat antimouse IgG conjugated to fluorescein (GAR-FITC), then A007 followed by goat anti-mouse IgM conjugated to rhodamine. In initial experiments, rhodamine and fluorescein patterns were identical whether or not the A007 antibody was present. This indicated that the rhodamineconjugated goat anti-mouse IgM (GAM-TRITC) contained antibodies that cross reacted with mouse IgG. The cross reactive antibodies were removed by absorption with a mouse IgG affinity column (Organon Teknika, Malvern, PA). When the above double label experiment was repeated without A007 with the mouse IgGImmuno-TLC absorbed rhodamine-conjugated goat anti-mouse IgM, Lipids were extracted by adding 2 ml chloroform: only background staining was observed for the rhomethanol (2:l) per coverslip followed by 0.5 ml 0.74% damine stain. Use of the mouse IgG-absorbed rhoKC1. The extracted lipids were washed with Folch's updamine-conjugated goat anti-mouse IgM allowed detecper phase (chloroform:methanol:0.74% KC1; 3:48:47). tion of different cell populations when double labering The organic phase was dried under nitrogen and then with R-mAb and A007. solubilized in 0.2 M sodium hydroxide in chloroform: Cultures double stained for either GalC or sulfatide methanol (2: 1) for alkaline methanolysis (Benjamins et and tubulin were first stained live with either anti-GalC al., 1976). After three washes with upper phase, the or A007 for 15 min, followed by the appropriate second lipids were separated on HPTLC plates (Fisher Scienantibody conjugated to fluorescein for 15 min at 37°C. tific, Springfield, NJ) in ch1oroform:methanol:acetone: The cultures were then fixed with 4% paraformaldehyde acetic acid:water (10:2:4:2: 1). Sulfatide and GalC stanfor 10 min, permeabilized with 0.05% saponin in PBS dards were purchased from Sigma (St. Louis, MO). for 10 min at room temperature, and treated with mouse Plates were fumed over concentrated ammonium hydrox- IgG anti-tubulin followed by GAM-TRITC. Cells were ide for 1 min to neutralize the pH; neutralization is im- viewed with a Leitz Orthoplan 2 fluorescent microscope portant for antibody binding in the blotting procedure. and were photographed with 400 ASA film. The silica was bound to the glass plates by dipping in 0.2% polyisobutylmethacrylate in hexane (Bansal et al., 1989). Immunoblotting was performed after blocking RESULTS . with 0.5% gelatin in phosphate-buffered saline (PBS). Plates were incubated with the primary antibody over- A007 and Anti-GalC. React With Sulfatide and night at 25"C, washed, then incubated with second anti- GalC, Respectively, on Oligodendrocytes With body conjugated to peroxidase (Sigma) for 2 hr at 37°C. Extensive Membrane Sheets Three methods were used to determine the speciThe plates were developed with 4-chloronaphthol and hydrogen peroxide. From each coverslip, representing ficity of anti-GalC and A007 on cultured murine oligoabout 30 pg of protein, approximately 1 pg of each lipid dendrocytes: 1) ELISA, 2) immunolTLC, and 3) immu-

702

Dyer and Benjamins

I

2

3

4

5

6

Fig. 1. Anti-GalC and A007 detect only GalC and sulfatide, respectively, via immuno-TLC. The plate on the left was reacted with A007 (150) and the plate on the right was reacted with anti-GalC (150). Lane 1: Lipids extracted from oligodendrocyte-enriched cultures. Lane 2: Five micrograms of a mixture of hydroxy sulfatide (arrow) and nonhydroxy sulfatide (arrowhead). Lane 3: One microgram of mixed sulfatides.

Lane 4: Lipids extracted from oligodendrocyte-enriched cultures. Lane 5: Five micrograms of mixed hydroxy GalC (arrow) and nonhydroxy GalC (arrowhead). Lane 6: One microgram of mixed GalC. Note the doublets for the hydroxyand nonhydroxy-GalC standards; these are likely due to different acyl chain lengths.

nocytochemistry. In ELISAs (Benjamins et al., 1987), anti-GalC reacts with GalC, and A007 with sulfatide; even at high concentrations of antibody, the rabbit antiGalC did not react with sulfatide and A007 did not react with GalC. Anti-GalC and A007 did not react with mixed gangliosides, ceramide, glucocerebroside, GM 1, o r asialo-GM1. The only exception was a minor reactivity of anti-GalC with psychosine, which is present in very low levels in oligodendrocytes; no reactivity with psychosine was observed with A007. Lipids were extracted from oligodendrocyte-enriched shake-off cultures, separated by HPTLC, and immunoblotted with anti-GalC or A007. The results showed that only GalC was detected by anti-GalC and that only sulfatide was detected by A007 (Fig. 1); any other immunoreactive lipid present at 10% or more of the levels of observed GalC and sulfatide would have been detected (see Materials and Methods). Finally, immunocytochemistry was performed to examine the specificity of anti-GalC and A007 on oligodendrocytes. A007 and second antibody were added to live oligodendrocytes; the same cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then treated with anti-GalC and second antibody. Under these conditions, permeabilization of membrane sheets does not occur (Dyer and Benjamins, 1988a) so that surface antigens on membrane sheets are detected. A007 was present in a patched distribution (Fig. 2a), while the membrane sheets were solidly stained for anti-GalC (Fig. 2b). This demonstrates that the binding of A007 in high concentrations does not

block the binding of anti-GalC. In addition, these results show that anti-GalC and A007 detect distinct antigens on oligodendrocytes with large membrane sheets. The reactivity of A007 appears to be identical to that of another monoclonal antibody 04, as shown by Bansal et al. (1991). The 04 antibody has been used to identify one stage of oligodendrocyte progenitors (Sommer and Schachner, 1982; Dubois-Dalcq, 1987; Gard and Pfeiffer, 1989); the nonlipid antigen on the oligodendrocyte progenitors reactive with A007 and 04 was originally called antigen “X” (Bansal et al., 1989), and then renamed proligodendrocyte antigen, or “POA” (Bansal and Pfeiffer, 1991). POA appears on the surface of GalC-negative oligodendrocyte progenitors that have not yet begun to synthesize sulfatide (Bansal et al.. 1991); no other cell type, including the type 2 astrocyte , has been demonstrated to express this antigen. We therefore performed double label immunocytochemical staining to determine whether A007 detects only sulfatide or sulfatide plus POA on sheet-bearing oligodendrocytes. In this and our previous study (Dyer and Benjamins, 1990), only membrane sheet-bearing oligodendrocytes were examined for Ca2+fluxes caused by antibody bincling. Both live and fixed cultures were used for the following immunocytochemical experiments; the same conclusions were drawn from both conditions. The R-mAb, which binds to sulfatide (and GalC) but not to POA, was added to cultures at a high concentration to saturate sulfatide. Cultures were then treated with A007 to detect

Galactocerebroside, Sulfatide, and Ca2+ ResDonses

703

Fig. 2 . Representative oligodendrocyte demonstrating that anti-GalC and A007 detect distinct antigens. Cultures were treated with A007 for 15 min, washed, and then treated with GAM-TRITC for 15 min at 37°C. Cultures were then fixed, exposed to anti-GalC for 15 min, washed, and exposed to

GAR-FITC for 15 min at room temperature. The oligodendrocyte in this figure has elaborated an extensive membrane sheet, which fills the entire field; the cell body is located at arrow. A007 is patched (a) while anti-GalC is solidly stained on the surface of this membrane sheet (b). x 300.

POA. Three types of cells that appear to reflect different stages of oligodendrocyte differentiation were identified based on their staining patterns. The first type consisted of process-bearing cells that were negative for R-mAb (GalC and sulfatide) and positive for A007 (POA antigen) (Fig. 3a,b). In view of the relationship of A007 to 04, these cells correspond to the 04+/GalC- oligodendrocyte progenitors previously described (Sommer and Schachner, 1982; Dubois-Dalcq, 1987; Gard and Pfeiffer, 1989); see also Knapp, 1991. The second type consisted of process-bearing oligodendrocytes positive for both A007 and R-mAb (Fig. 3c,d). These oligodendrocytes expressed GalC and/or sulfatide and POA and therefore are likely to be more differentiated than the above described progenitors. Finally, oligodendrocytes with large membrane sheets did not express POA at detectable levels but did express GalC and/or sulfatide (Fig. 3a,b and e,f); after R-mAb binding, no A007 binding was observed in these cells. Of the three types of cells described, these are morphologically the most differentiated (mature) oligodendrocytes. These immunocytochemical staining experiments clearly show that the POA antigen is present on immature, process-bearing oligodendrocytes and that expression of POA is lost on mature, membrane sheet-bearing oligodendrocytes (which are the cells examined in the study for changes in intracellular Ca2+). It is possible that these antibodies are detecting other glycolipids; for example, Bansal et al. (1989) concluded that seminolipid is present in small amounts in immature glial cultures. We do not know if seminolipid is expressed in our 22-26 day cultured oligodendrocytes, since our lipid samples were treated with alkaline methanolysis before immunoTLC. If any other cross-reactive alkali-stable lipids are

expressed in our cultures, then they are expressed below the limits of detection by immuno-TLC in our hands. GalC and sulfatide are clearly the major glycosphingolipid components in these cells and, based on our extensive characterization, are the most likely candidates for mediating the observed Ca2 responses initiated by anti-GalC and A007 in oligodendrocytes with large membrane sheets. +

A007 Causes Microtubule Depolymerization in a Subpopulation of Oligodendrocytes We have previously shown that mature membrane sheet-bearing oligodendrocytes contain lacy microtubular networks throughout their membrane sheets (Dyer and Benjamins, 1988b, 1990). In virtually all oligodendrocytes treated with anti-GalC, the sustained influx of Ca2+ mediated by GalC leads to depolymerization of microtubules, with the disappearance of the lacy networks (Dyer and Benjamins, 1990). The effect of A007 on microtubule stability was examined by indirect immunofluorescent staining (see Materials and Methods). Nearly all oligodendrocytes with large membrane sheets exposed to A007 before fixation had a patched surface distribution of sulfatide (Fig. 4a,c). Approximately one-half of these A007-treated mature oligodendrocytes exhibited the typical intact lacy microtubular networks throughout their membrane sheets (Fig. 4b), while the other half showed evidence of microtubular disruption, that is, absence of the lacy networks (Fig. 4d). Thus A007 was not as effective as anti-GalC in inducing microtubule depolymerization. These data suggest that the binding of these two antibodies to their respective glycolipids vary in their ability to elicit Ca2+ responses.

Fig. 3. GalC and/or sulfatide are present, but POA is not present on membrane sheet-bearing oligodendrocytes. Cultures were fixed with 4% paraformaldehyde for 10 min at room temperature, exposed to a concentration of R-mAb that resulted in intense solid staining, washed, and then exposed to GAM-FITC for 15 min each at 37°C. Cultures were then treated with A007; the concentration of A007 used resulted in solid staining of untreated oligodendrocytes. The cultures then were washed and treated with GAM-TRITC. The cells shown here are representative of the populations observed. a: Membrane sheet-bearing oligodendrocyte is intensely stained for R-mAb (GalC and/or sulfatide), but no staining is observed on process-bearing oligodendrocyte progenitor (arrow). b: A007

(POA antigen) brightly stains a process-bearing oligodendrocyte progenitor, but not the membrane sheet-bearing oligodendrocyte shown above (arrow). (Bleed-through of fluorescein does occur for intensely lit areas.) c: R-mAb intensely stains process-bearing oligodendrocyte (arrow). d: The same oligodendrocyte in c is also intensely stained for A007 (arrow). Also, a process-bearing oligodendrocyte is stained for A007 but not R-mAb (arrowhead). e: R-mAb solidly stains a membrane sheet-bearing oligodendrocyte and stains an immature oligodendrocyte in a punctate pattern (arrow). f: No A007 staining is observed on membrane sheet-bearing oligodendrocyte, but immature oligodendrocyte is brightly stained for A007 (arrow). X 300.

Galactocerebroside, Sulfatide, and Ca2+ Resvonses

705

Fig. 4. A007 causes microtubule depolymerization in a subpopulation of membrane sheet-bearing oligodendrocytes. a: Representative oligodendrocyte with redistributed, or patched, sulfatide on the membrane sheet (cell body at arrow). b: Same cell in a internally stained with anti-tubulin, showing intact lacy network of microtubules within the membrane sheet. c: Representative oligodendrocyte (cell body at arrow) with patched sulfatide on membrane sheets. d: Same cell in c

stained internally with anti-tubulin, showing the depolymerization of the lacy microtubular network in these membrane sheets; the large tubulin-positive veins remain. Another cell, which is not an oligodendrocyte (arrow), is also present in this field. Individual oligodendrocytes are shown instead of a field of several oligodendrocytes because a lower magnification does not adequately show the lacy network of microtubules that can be seen at this magnification. X 300.

Frequency and Type of Ca2+ Response Differ Due to A007 or Anti-GalC Treatment Ca2+ levels were analyzed fluorometrically in Indo- 1-loaded oligodendroglia using a Meridian ACAS laser cytometer (see Materials and Methods). Examination of individual oligodendrocytes for changes in intracellular Ca2+ due to A007 binding showed that a significant difference existed between the number of oligodendrocytes that responded to A007 and anti-GalC. Changes in intracellular Ca2+ were observed in 75% of the oligodendrocytes treated with anti-Gale (n = 29) while only 39% of oligodendrocytes treated with A007 responded (n = 53). This difference exists despite the fact that the majority of oligodendrocytes with membrane sheets stained intensely for both GalC and sulfatide. The other dramatic difference between these two antibodies is the types of Ca2+ responses they elicited.

Delayed, sustained Ca2+ rises were observed in 21 of 22 of the anti-GalC responding oligodendrocytes (Table I). However, heterogeneous Ca2 responses were observed due to A007 binding. Of the 21 cells responding to A007, 10 exhibited Ca2+ responses that are delayed and large, that is, similar in magnitude to anti-GalC, but transient (Fig. 5 , Table I). After reaching maximum values, Ca2+ levels dropped to near resting values within 5-12 min (Fig. 5). A sustained rather than a transient increase in Ca2+ was detected in six of 21 A007-responsive oligodendrocytes. Finally, five of 2 1 A007-responsive oligodendrocytes showed an immediate, small Ca2+ response, with kinetics similar to voltage-operated Ca2 channels. All together, it appears that A007 elicits and sustains a Ca2+ influx in many fewer oligodendrocytes than anti-GalC , and also causes microtubule depolymerization in fewer cells. The A007 response is unique and is not due to its antibody class. Preliminary data show +

+

706

Dyer and Benjamins

TABLE I. Sustained vs. Transient Calcium Responses Elicited by Anti-galactocerebrosideand A007 Cells treated with anti-GalC Types of responses No response Delayed sustained response Transient response Large, delayed Small, immediate”

Cells treated with A007

(%)

7 (25) 21 (72)

32 (61) 6 (11)

1(3)

lo} 5 (28)

aCa2+ levels peak in 40-50 sec at a ratio of approximately 0.1 units, which is equivalent to an increase of between 20 and 80 nM. Levels then fall to near resting values in about 150 sec. These small, immediate transient elevations in Ca’ mimic the voltage-operated Ca2+ channels in oligodendrocytes that are activated by 60 nm K + . +

that another IgM antibody reactive with myelin/oligodendrocyte-specific protein (MOSP) elicits a Ca2+ response in approximately 78% of oligodendrocytes tested (n = 18) (Dyer et al., 1991).

Anti-GalC IgG and A007 Ca2+ Responses Are Not Mediated by Nonspecific Pore Formation or Antibody Cross-Linking To examine whether nonspecific pore formation was the mechanism of Ca2+ entry, cultures were treated with propidium iodide (PI) and either anti-GalC or A007. PI is normally excluded from intact membranes; however, when nonspecific pores are formed, PI enters the cell and intercalates with DNA causing the nucleus to become fluorescent (Parks et al., 1986). T-cell perforin, a nonspecific pore-forming agent, causes swelling and rounding of oligodendrocyte cell bodies and disruption of processes; after perforin treatment, oligodendrocyte nuclei become brightly PI stained (Scolding et al., 1990). None of these effects were observed when PI was added with either anti-GalC or A007 to our cultures for 15 min at 37°C. For positive identification of oligodendrocytes, cultures were fixed after the 15 min exposure to PI and antibody and then were double labeled using the appropriate second antibody conjugated to fluorescein. These results indicate that oligodendrocytes exposed to anti-GalC or 007 are not “leaky” and that Ca2+ entry is occurring via a regulated mechanism. To determine if cross linking plays a role in these glycolipid-mediated Ca2+ responses, Fab fragments of the anti-GalC IgG antibodies were prepared according to Cayanis et al. (1986); it was not possible to prepare Fab fragments from A007 IgM that retained their immunoreactivity. When approximately 90 pg/ml of the antiGalC Fab fragments were added to oligodendrocytes, anti-GalC Fab fragment binding elicited a delayed, sustained influx of Ca2+ similar to the response induced by the intact anti-GalC IgG (Table 11). This response was

observed in four of the five oligodendrocytes examined, with the fifth oligodendrocyte not responding. A large amount of intact anti-GalC IgG is needed to prepare Fab fragments; thus our limited supply of IgG restricted the number of cells that we were able to examine with Fab fragments. However, it is clear from these results that the anti-GalC Fab fragment is capable of eliciting the same type of Ca2+ response as the intact IgG. Therefore, it is the specific binding of anti-GalC and A007 to their respective sphingolipids and not cross linking that initiates the Ca2 response. +

Initial Kinetics of Delayed, Large Anti-GalC- and A007-Induced Ca2+ Responses Are Similar: Both Appear To Be Due to Ca” Influxes Comparison of the delayed, large responses from A007-responsive cells and GalC-responsive cells show that, after a delay of 20 sec to 6 min, the initial phase of the response is similar for both antibodies (Table 11). The time taken to reach maximum Ca2+ levels averaged 205 sec for A007 and 99 sec for anti-GalC; however, these values are not significantly different, since the ranges are large. The ratio change, or magnitude of the Ca2+ increases, is similar for both antibodies (Table 11). Since these kinetics are similar, this may suggest that the same mechanism may be elicited by anti-GalC and A007 to increase intracellular Ca2 . Experiments with EGTA examined whether the A007-induced rise in intracellular Ca2+ is due primarily to an influx of extracellular Ca2+ or to release from internal stores. The anti-GalC-induced increase in intracellular Ca2+ has been shown to be due solely to influx: The response was blocked by adding EGTA prior to anti-GalC (Dyer and Benjamins, 1990). The majority of oligadendrocytes do not, respond to A007 (Table I); therefore, a large number of cells would need to be studied to obtain significant results. Since only one cell per coverslip is examined and the amount of antibody required for each analysis is considerable, this is an impractical approach. A more efficient experimental design was to add 2 mM EGTA to the bathing medium (which contained 1.8 mM Ca2+) after a response was initiated by A007. Three types of responses might occur. First, Ca2+ levels might continue to rise at the original rate, indicating that an influx was not occurring and that internal stores were being released. Second, the Ca2+ levels might plateau, or the rate of increase slow markedly. Assuming that the cells pump excess Ca2+ out, this result would suggest that both an influx and release from internal stores were contributing to the initial increase in Ca’ + . Third, the Ca2+ levels might rapidly decrease to near resting levels after the addition of EGTA, suggesting that the increase was due only to an influx of extracellular Ca2 . +

+

Galactocerebroside, Sulfatide, and Ca2+ Responses n

10

B

00

d 10

707

3-

\

0 d

2-

Anti-Sulfatide IgM

Anti-GalC IgG

W

-

0

c a a

1c

c

-I

0

200

400

0

200

400

600

800

T I M E (se c) Fig. 5. Marked increases in intracellular free Ca2+ are pre- taining that level for the remainder of the analysis. B: Followdominantly sustained for anti-GalC-treated oligodendrocytes ing addition of A007, a maximum increase in ratio between and transient for A007-treated oligodendrocytes. Antibodies three- and fourfold is reached in approximately 150 sec; intrawere used at concentrations resulting in a patched surface stain- cellular Ca2+ subsequently falls to near resting levels after ing pattern. A: Approximately 30 sec after addition of anti- about 12 min. These responses are representative of those sumGalC, intracellularCaZ begins to increase markedly, reaching marized in Table I. a fourfold increase in the 405/485 ratio by 150 see and main+

TABLE 11. Calcium Responses Induced by Anti-galactocerebrosideand A007* Time to response

Rise time

Agent

n

bet)

(set)

Ratio change

Anti-GalC IgG

15

99 2 35

1.0 k 0.5

Anti-GalC Fab

4

93 i 98 (20-360)a 137 ? 52 (50-180)

A007

10

180 ? 133 (40-390)

220 t 94

1.4

&

0.6

205 2 159

1.4

&

0.5

*Oligodendrocytes were exposed to antibody, and the changes in intracellular free Ca2+ were analyzed. A007 and anti-GalC were added to cultures at concentrations resulting in a patched surface staining pattern; more dilute antibody concentrations stain oligodendrocytes in a punctate pattern and do not elicit Ca2+ responses (Dyer and Benjamins, 1990). Problems in data collection prohibited the inclusion of six anti-GalC-responsive oligodendrocytes and one A007-responsive oligodendrocyte in these statistics. Of the five oligodendrocytes exposed to anti-GalC Fab fragments, four responded and one did not. The five Ca2+ responses observed that have characteristics similar to voltage operated CaZ+ channels are not included in the A007 responses. No responses were observed in astrocytes exposed to A007 or anti-GalC IgG. Nonimmune IgG did not trigger a Ca” response in oligodendrocytes. Values reported are the means -+ SD of the delayed CaZ responses. aRange of times to response observed.

dendrocytes examined in this manner, a similar rapid decrease in intracellular free Ca2’ was observed following addition of EGTA (Fig. 6), supporting the third possibility. It is unlikely that in all six cells the normal decrease would happen exactly at the time of addition of EGTA. Furthermore, attempts were made to add EGTA in early stages of the response before the maximum ratio changed occurred (compare Fig. 6b with 5b). In addition, Ca2+ returned to resting levels more rapidly in EGTA-treated cells than in cells treated with A007 alone (90-160 sec compared with 300-700 sec). Thus, in cells treated with A007 alone, the time to return to resting levels probably includes both influx and efflux. However, when EGTA is added, only the efflux is likely to be occurring, which may explain the shorter recovery time. Although it is impossible to rule out that some release of intracellular Ca2+ stores takes place, the majority of the A007-induced Ca2+ rise appears to be due to an influx of extracellular Ca2’

Sulfatide and GalC Independently Activate Ca2+ Responses: These Responses Identify Subpopulations of Oligodendrocytes As is shown in Table I and discussed above, about twice as many mature, membrane sheet-bearing oligoWhen two cells responding to anti-GalC were dendrocytes are responsive to anti-GalC as to the sultreated with EGTA, Ca2+ levels immediately decreased fatide reactive antibody, A007, despite the fact that these to near resting levels (Fig. 6). Since addition of EGTA two lipids are present in high concentrations on the maprior to anti-GalC completely blocks the rise in Ca2+ jority of membrane sheets. One hypothesis to explain (Dyer and Benjamins, 1990), the time taken for the anti- these data is that there may be subpopulations of oligoGalC Ca2+ response to fall to near resting is likely due dendrocytes that are responsive to A007 but not responto pumping Ca2+ out. In all six A007-responsive oligo- sive to anti-GalC and vice versa. To examine this possi+

708

Dyer and Benjamins

'.I I '

I

EGTA

i4-

1.o

B

1

0.9-

0.80.7-L 0.4

! '

0

I

I 1

100

200

300

400

I 0.61

Time Fig. 6. Anti-GalC- and A007-induced increases in Ca2+ levels rapidly return to near resting levels after addition of 2 mM EGTA. Antibodies were used at concentrations resulting in a patched staining pattern on oligodendrocytes. After an antibody-induced rise in Ca2+ was detected, 2 mM EGTA was added to the medium containing 1.8 mM Ca2+,and changes in bility , oligodendrocytes were exposed to an amount of A007 that resulted in intense surface staining and then, with A007 still present, were exposed to anti-GalC. Cells were observed for approximately 10 min after anti-GalC addition. Of the 13 oligodendrocytes examined, four were responsive to both antibodies (Fig. 7). These results indicate that anti-GalC and anti-sulfatide independently trigger influxes of Ca2+ in oligodendrocytes. Interestingly, after A007 binding, the anti-GalC response was transient rather than sustained in three of the four cells responding to both antibodies. Of the remaining cells, four were responsive to A007 only, two were responsive to anti-GalC only (transient responses), and three did not respond to either antibody. These results indicate that subpopulations of oligodendrocytes can be identified by their GalC- and sulfatide-mediated Ca2 responses. These subpopulations may represent I ) different stages of oligodendrocyte differentiation, 2) oligodendrocytes from different areas in the brain, or 3) a property unique to cultured oligodendrocytes. +

co -. 1 .od

v)

0

t

-

U

0

0.8-

t-

a

a

Anti-GalC

0.6-

0

200

400

600

900

TIME ( 8 e c) Fig. 7. GalC and sulfatide independently mediate Ca2+ responses in individual oligodendrocytes. Oligodendrocytes were exposed to high concentrations of each antibody that result in intense solid surface staining of membrane sheets. Following addition of A007, a typical delayed, marked increase in intracellular Ca2 was observed in this oligodendrocyte. As Ca2+ levels began to decrease with A007 still present, anti-GalC was added, and an immediate, large increase in intracellular Ca2 occurred. This increase was transient, with levels of free Ca2+ returning to pre-anti-GalC values about 5 min later (not shown). Ca2+ responses to anti-GalC were observed after A007 responses in three other oligodendrocytes. +

+

DISCUSSION This study demonstrates that antibodies to GalC and sulfatide elicit Ca2 responses in different populations of oligodendrocytes and that the Ca2+ responses themselves differ depending on which antibody is bound. The conclusion that GalC and sulfatide are the molecules mediating the observed Ca2+ responses is based on extensive analysis of antibody specificity by ELISA, immuno-TLC of glial lipids, and immunocytochemistry . Double label immunocytochemistry using the R-mAb (sulfatide-reactive antibody) and A007 (antibody reac+

tive with sulfatide and POA), demonstrated three stages of oligodendrocyte differentiation in our cultures: 1) process-bearing oligodendrocyte progenitors expressing POA but not GalC and/or sulfatide; 2) immature, process-bearing oligodendrocytes with high levels of both GalC and/or sulfatide and POA; and 3) mature, membrane sheet-bearing oligodendrocytes, which have lost expression of POA but continue to express GalC and/or

Galactocerebroside, Sulfatide, and Caz+ Responses

sulfatide. These data, taken together with our ELISA and immuno-TLC results, which show no cross-reactivity of A007 with GalC, indicate that A007 detects only sulfatide on mature oligodendrocytes. Since GalC and sulfatide are abundantly expressed on the surface of mature cultured oligodendrocytes, it is most likely that GalC and sulfatide are mediating the observed antibody-induced Ca2 responses. The number and type of anti-GalC- and A007-induced Ca2+ responses in membrane sheet-bearing oligodendrocytes differ. Nearly twice as many oligodendrocytes treated with anti-GalC respond with an influx of Ca2+ as do oligodendrocytes treated with A007. While both A007 and anti-GalC induced large, delayed Ca2+ influxes (Table I), typical Ca2 responses for A007 were transient, while anti-GalC responses were sustained. Thus, with A007, Ca2+ levels fell to resting values by 12 min after peaking, but with anti-GalC the response was sustained for at least 30 min. A minority of A007 induced Ca” responses were either 1) large, delayed, and sustained or 2) immediate, small, and transient. The reason for the heterogeneity of the A007 responses remains to be determined. The mechanisms involved in increasing the intracellular levels of Ca2+ in either the A007- or the antiGalC-treated oligodendrocytes are unknown. We can rule out the possibility that A007 or anti-GalC themselves induce de novo formation of nonspecific Ca2+permeable pores, since no nuclear staining was observed when cells were treated with propidium iodide and either A007 or anti-GalC. The initiation of the delayed GalC and sulfatide responses appears to be due to the specific interaction of the antibodies with their respective glycolipids and not to cross-linking of lipid molecules on the membrane surface since Fab fragments of the anti-GalC IgG elicit the same Ca2+ influx as intact IgG. This is not a unique phenomenon, since Fab fragments specific for either the T-cell receptor (Oettgen et al., 1985) or a mouse macrophage Fc receptor, elicit ion fluxes similar to their respective intact IgGs (Young et al., 1983a,b). Several mechanisms may account for the observed rise in intracellular Ca2 . First, the large, delayed A007 or anti-GalC Ca” responses may be due to second messenger-operated Ca2+ channels (SMOC) since 1) they appear to be due to an influx of Ca2+, and 2) activation of voltage-operated Ca2 channels (VOCs) do not mimic these responses. Indeed, second messengers have been shown to mediate Ca2’ influxes in cell types that do not use VOCs as a means of regulating Ca2+ entry (Exton, 1989; Penner et al., 1988; Hockberger and Swandulla, 1987; Meldolesi and Pozzan, 1987; Merrit and Rink, 1987). At least two possibilities may account for the delay in SMOC activity. One is that the delay reflects the time required to recruit Ca2 channels from intracellular +

+

+

+

+

709

membrane compartments. The second is that a threshold level of second messenger must accumulate for activation of channels. A recent study shows that, after muscarinic receptor stimulation of an astrocytoma cell line, there is a delay of several minutes before significant diacylglycerol accumulation is observed (Martinson et al., 1990). The possibility that a similar delay is operating in our system is supported by our preliminary data from 32P-labeling, which suggest that anti-GalC binding results in Ca2 -independent activation of a phosphatidylinositol-specific phospholipase. Two other mechanisms leading to a net increase in Ca2’ may be 1) that either A007 or anti-GalC might in some way inhibit efflux of Ca2+ while allowing influx to continue or 2) that Caz+ transporters are induced to work in reverse as a result of antibody binding (Dixon et al., 1987). Which ever of these possibilities is occurring, there must be some mechanism to check the rise in Ca2+, since Ca2+ falls to near resting levels with A007 and is maintained at a new steady-state level with anti-GalC. Our results suggest that both sulfatide and GalC play important roles in cellular responses of oligodendrocytes in vivo. The antibodies themselves may play a role in idiopathic diseases that affect central nervous system (CNS) myelin, such as multiple sclerosis. Antibodies to GalC were shown to cause increased birefringence and doubling of the periodicity between major dense lines and result in the production of disorganized layers of membrane in the extracellular space (Raine et al., 1981). Such results may be expected if an influx of Ca2+ occurred and cytoskeleton was altered, as observed in our anti-GalC-treated cultured oligodendrocytes. Since multiple sclerosis-like demyelination has been shown to occur as a result of HIV infection (Harouse et al., 1991), the binding of gp120 to Gale or sulfatide may elicit oligodendroglial responses similar to those of anti-GalC and A007. The sequence of events triggered by the binding of anti-GalC or A007 may mimic those of endogenous ligands on axonal membranes or on myelin itself. Two endogenous ligands in white matter have been identified for sulfatide (Law et al., 1988). These two proteins have been immunologically and functionally conserved during evolution; thus the authors speculate that their binding function plays an important role in the CNS. The modulation of cytoskeletal structures caused by anti-GalC and A007 binding to cultured oligodendrocytes may correspond to the normal in vivo conversion of noncompacted to compacted myelin elicited by endogenous signals. Indeed, after 6 hr of anti-GalC exposure, membrane sheets are devoid of the microtubule network and resemble unfurled compacted myelin (Benjamins and Dyer, 1990: Dyer and Benjamins, 1989). Our present results suggest that A007 binding does not lead to +

710

Dyer and Benjamins

mic free calcium in rat lymphocytes. J Cell Biol 105:1153membrane sheet contraction because it does not cause a 1161. sustained increase in Ca2+ like that caused by anti-GalC, Dyer CA, Benjamins JA (1988a): Redistribution and internalization of and the accompanying microtubule depolymerization is antibodies to galactocerebroside by oligodendroglia. J Neurosci less robust. Our data suggest that ligands binding to sul8:883-891. fatide produce signals that modulate the GalC response; Dyer CA, Benjamins JA ( I 988b): Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culprior exposure of membrane sheet-bearing oligodendroture. J Neurosci 8:4307-43 18. cytes to A007 results either an immediate rise in Ca2+, Dyer CA, Benjamins JA (1989): Organization of oligodendroglial which is transient or no response at all. We do not yet membrane sheets: 11. Galactocerebroside: antibody interactions know whether the transient and sustained Ca2+ increases signal changes in cytoskeleton and myelin basic protein. J Neulead to changes in gene expression, as are found in other rosci Res 24:212-221. systems (Morgan and Curran, 1988, 1989). However, Dyer CA, Benjamins JA (1990): Glycolipids and transmembrane signaling: Antibodies to galactocerebroside cause an influx of calour results lend support to the idea that sphingolipids cium in oligodendrocytes. J Cell Biol 111:625-633. play specific, major, and direct roles in initiating trans- Dyer CA, Geisert EE, Matthieu JM (1991): MOSP and MOG: TWO membrane signaling. specific oligodendrocyte proteins transduce different extracel-

ACKNOWLEDGMENTS This work was supported by NIH grant NS13143. The authors thank Drs. Rashmi Bansal and Stephen Pfeiffer for providing information on A007 specificity before publishing their manuscript and for helpful discussion of this manuscript. We also thank Kainette Jones for her expert secretarial skills in preparing this manuscript and Ken Thompson for preparation of graphs.

REFERENCES Bansal R, Pfeiffer SE (1989): Reversible inhibition of oligodendrocyte progenitor differentiation by a monoclonal antibody against surface galactolipids. Proc Natl Acad Sci USA 86:6181-6185. Bansal R, Stefansson K, Pfeiffer SE (1991): Proligodendrocyte antigen (POA): Monoclonal antibodies A007 and 04 both recognize late oligodendrocyte progenitors prior to the synthesis of sulfatide and galactocerebroside. J Neurochem 57:s 121. Bansal R, Warrington AE, Gard AL, Ranscht B, Pfeiffer SE (1989): Multiple and novel specificities of monoclonal antibodies 01, 04 and R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res 24:548-557. Benjamins JA, Callahan RE, Montgomery IN, Studzinski DM, Dyer CA (1987): Production and characterization of high titer antibodies to galactocerebroside. J Neuroimmunol 14:325-338. Benjamins JA, Miller SL, Morel1 P (1976): Metabolic relationships between myelin subfractions: Entry of galactolipids and phospholipids. J Neurocbem 27:565-570. Berger JR, Sheremata WA, Resnick L, Atherton S, Fletcher MA, Norenberg M ( 1989): Multiple sclerosis-like illness occurring with human immunodeficiency virus infection. Neurology 39: 324-329. Bottenstein JE (1986): Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes. Proc Natl Acad Sci USA 83:1955-1959. Cayanis E, Rajagopalan R, Cleveland WL, Edelman IS, Erlanger BF (1986): Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor. J Biol Chem 261:5094-5103. Curatolo W (1987): Glycolipid function. Biochim Biophys Acta 906: 137-160. Dixon SJ, Stewart D, Grinstein S, Speigel S (1987): Transmembrane signaling by the B subunit of cholera toxin: Increased cytoplas-

lular signals. J Neurochem 57:S42. Dubois-Dalcq M (1987): Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway. EMBO J 6:2587-2595. Exton JH (1988): Mechanisms of action of calcium-mobilizing agonists: Some variations on a young theme. Fed Am SOCExp Biol J 212670-2676. Facci L, Skaper SD, Favaron M, Leon A (1988): A role for gangliosides in astroglial cell differentiation in vitro. J Cell Biol 106: 821-828. Fishman PH (1982): Role of membrane gangliosides in the binding and action of bacterial toxins. J Membrane Biol 69:85-97. Gard A, Pfeiffer SE (1989): Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: Myelinogenic potential in a defined environment. Development 106:119-132. Gray F, Chimelli L, Mohr M, Clavelou P, Scaravilli F, Poirier J (1991): Fulminating multiple sclerosis-like leukoencephalopathy revealing hvman immunodeficiency virus infection. Neurology 41:105-109. Hakomori S (1990): Bifunctional role of glycosphingolipids. J Biol Chem 265: 18713-187 16. Hanai N, Dohi T, Nores GA, Hakomori S (1988): A novel ganglioside, de-N-acetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor receptor kinase and as a stimulator for cell growth. J Biol Chem 263:6296-6301. Hanai N, Nores G, Torres-Mendez CR, Hakomori S (1987): Modified ganglioside as a possible modulator of transmembrane signaling mechanism through growth factor receptors: a preliminary note. Biochem Biophys Res Commun 147:127-134. Harouse JM, Bhat S, Spitalnik SL, Laughlin M, Stefano K , Silberberg DH, Gonzalez-Scarano F (1991): Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceremide. Science 253:320-323. Hockberger PE, Swandulla D (1987): Direct ion channel gating: A new function for intracellular messengers. Cell Mol Neurobiol 7:299-235. Knapp PE (1991): Studies of glial lineage and proliferation in vitro using an early marker for committed oligodendrocytes. J Neurosci Res 30:336-345. Law H, Itkonnen 0 , Lingwood CA (1988): Sulfogalactolipid binding protein SLIP 1: A conserved function for a conserved protein. J Cell Physiol 137:462-468. Martinson EA, Trilivas I, Brown JH (1990): Rapid protein kinase C-dependent activation of phospholipase D leads to delayed 1,2-diglyceride accumulation. J Biol Chem 26522282-22287. McCarthy, KD, DeVellis J (1980): Preparation of separate astroglial

Galactocerebroside, Sulfatide, and Ca2+ Responses and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890-902. Meldolesi J, Pozzan T (1987): Pathways of Ca2+ influx at the plasma membrane: Voltage-, receptor-, and second messenger-operated channels. Exp Cell Res 171:271-283. Memtt JE, Rink TJ (1987): Regulation of cytosolic free calcium in fura-1-loaded rat parotid acinar cells. J Biol Chem 262: 1736217369. Morgan JI, Curran T (1988): Calcium as a modulator of the immediate-early gene cascade in neurons. Cell Calcium 9:303-311. Morgan JI, Cunan T (1989): Calcium and proto-oncogene involvement in the immediate-early response in the nervous system. Ann NY Acad Sci 568:283-290. Nudelman EC, Mandel U, Levery SB, Kaizu T, Hakomori S (1988): A series of disialogangliosides with binary 2-3 sialosyllactosamine structure, defined by monoclonal antibody NUH2, are oncodevelopmentally regulated antigens. J Biol Chem 263: 10915-18725. Oettgen HC, Terhorst C, Cantley LC, Rosoff PM (1985): Stimulation of the T3-T-cell receptor complex induces a membrane-potential-sensitive calcium influx. Cell 40583-590. Okada Y, Radin NS, Hakomori S (1988): Phenotypic changes in 3T3 cells associated with the change of sphingolipid synthesis by a ceramide analog, 2-decanoylamino-3-morpholino1-phenylpropanol (compound RV538). FEBS Lett 235:25-29. Parks DR, Lanier LL, Herzenberg LA (1986) Propidium iodide label-

71 1

ling of dead cells. In Wier DM (ed): “Handbook of Experimental Immunology, 4th ed, Vol. 1 .” Oxford: Blackwell Scientific, pp 13-14. Penner R, Matthews G, Neher E (1988): Regulation of calcium influx by second messengers in rat mast cells. Nature 334:499-504. Raine CS, Johnson AB, Marcus DM, Suzuki A, Bornstein MB (1981): Demyelination in Vitro. Absorption studies demonstrate that galactocerebroside is a major target. J Neurosci 52: 117-13 1. Ranscht B, Clapshow PA, Price J, Noble M, Seifert W (1982): Development of oligodendrocytes and schwann cells studied with a monoclonal antibody against galactocerebroside. Proc Natl Acad Sci USA 79:2936-2947. Scolding NJ, Jones J, Compston AS, Morgan BP (1990): Oligodendrocyte susceptibility to injury by T-cell perforin. Immunology 70:6-10. Sommer I, Schachner M (1982): Cells that are 04 antigen-positive and 01 antigen-negative differentiate into 01 antigen positive oligodendrocytes. Neurosci Lett 29: 183-188. Young JD-E, Unkeless JC, Kaback HR, Cohn ZA (1983a): Mouse macrophage Fc receptor for IgG 2blyl in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore. Proc Natl Acad Sci USA 80:1636-1640. Young JD-E, Unkeless JC, Young TM, Mauro A, Cohn ZA (1983b): Role for mouse macrophage IgG Fc receptor as ligand-dependent ion channel. Nature 306:186-189.

Galactocerebroside and sulfatide independently mediate Ca2+ responses in oligodendrocytes.

Galactocerebroside (GalC) and sulfated galactocerebroside (sulfatide) are sphingolipids highly enriched in myelin. The binding of antibodies reactive ...
3MB Sizes 0 Downloads 0 Views