The Journal of Neuroscience, October 1989, 9(10): 33714379 Type 1 Astrocytes Inhibit Myelination by Adult Rat Oligodendrocytes in vitro Charles L. Rosen,a Richard P. Bunge, March D. Ard,b and Patrick M. Wood Miami Project, University of Miami School of Medicine, Miami, Florida 33136 We have determined the effect of Type 1 astrocytes on the patients with multiple sclerosis. Whether this astroglial response myelination of dorsal root ganglion cell axons by oligoden- promotes or inhibits the recovery of normal oligodendrocyte drocytes obtained from adult animals. Experiments were ini- function is not clear. It has been observed (Blakemore, 1978) tiated by the addition of oligodendrocytes [purified either by in an animal model of chemically induced demyelination-re- density gradient centrifugation and treatment in culture with myelination that remyelination does not occur when astrocyte Sfluorodeoxyuridine (MU) or by fluorescence-activated cell numbers are low and when the myelin debris is not completely sorting after immunostaining with antigalactocerebroside removed. Since astrocytes are thought to participate in the antibody] to cultures of purified neurons. In control condi- phagocytosis of myelin debris during demyelination, these ob- tions, the added oligodendrocytes proliferate and, after 4 servations suggest that a weak astrocyte response might restrict weeks, accomplish substantial myelination of the sensory remyelination. On the other hand, there is a strong correlation axons. Type 1 astrocytes (purified from cultures of disso- between severe astrogliosis and the failure of effective remye- ciated newborn rat cerebral cortex by vigorous shaking to lination (Ludwin, 198 1; Prineas, 1985), implying that in some remove less adherent cells) or fibroblasts (purified from cul- situations, astrocytes might have negative effects on oligoden- tures of cranial periosteum by serial replating) were added drocyte function. to some of these cultures after the oligodendrocytes had Our view of astrocyte function has been expanded consid- attached and started to proliferate. We observed that the erably by recent demonstrations of two phenotypically distinct added Type 1 astrocytes, but not the added fibroblasts, forms (Raff et al., 1983). Type 1 astrocytes appear early in strongly inhibited myelination and caused decreased oli- development and are generated in large numbers when CNS godendrocyte proliferation or survival. These effects of added tissue from embryonic or newborn animals is dissociated and Type 1 astrocytes were reproduced with Type 1 astrocyte- cultured (Raff et al., 1984). They often display a flattened, ep- conditioned medium. We conclude that Type 1 astrocytes ithelial morphology in culture. Type 2 astrocytes are defined by can release soluble factors that inhibit oligodendrocyte mye- the expression on their surfaces of the antigen recognized by the lination. monoclonal antibody A2B5. They are believed to derive from multipotential cells, which also express the A2B5 antigen and The physical proximity of astrocytes and oligodendrocytes in which may also develop into oligodendrocytes. Type 2 astro- all regions of the CNS and their invariant sequential appearance cytes are generated after oligodendrocytes in cultures of early (first astrocytes and then oligodendrocytes) in development sug- postnatal optic nerve and are generated with a shorter delay in gest that astrocytes may influence oligodendrocyte function. Al- cultures of optic nerve from older animals (Raff et al., 1983). though the relationship between astrocytes and oligodendro- These astrocytes display a stellate morphology in culture. It has cytes undoubtedly changes as the tissue develops and then been suggested that they are also generated subsequent to oli- matures, it is generally accepted that the role of the astrocyte is godendrocytes in vivo, that they proliferate to eventually out- supportive of oligodendrocyte function. When this normal re- number the Type 1 astrocytes, and that they may play some lationship is disturbed, as in injury or in demyelination, a prom- role in nodal physiology (Miller et al., 1985; French-Constant inent astrocyte response often occurs. In some situations, this and Raff, 1986). However, this view conflicts with earlier studies response can produce astroglial scars, as in chronic lesions in (Skoff et al., 1976) that demonstrated that most of the astrocytes in the mature rat optic nerve are generated before oligodendro- cytes and should, therefore, be of the Type 1 phenotype. Thus, Received Sept. 29, 1988; revised Mar. 15, 1989; accepted Mar. 23, 1989. the relative abundance of Type 1 and Type 2 astrocytes in vivo We thank Dr. Junming Le for performing the assays for tumor necrosis factor, Mr. Alon Mogilner for assistance in computer programming, and Drs. L. Eng, B. remains controversial. In recent tissue culture experiments, Type Ranscht and M. Schachner for gifts of antibodies. In addition, we would like to 1 astrocytes have been shown to secrete a factor that promotes thank Ms. Susan Mantia for preparation of the manuscript and Mrs. Artree James the division of oligodendrocyte-Type 2 astrocyte progenitors for technical assistance. This work was supported by grant RGlIl8 from the National Multiple Sclerosis Society. M.D.A. was supported by a fellowship from and regulates the timing of their differentiation into oligoden- the National Multiole Sclerosis Societv. drocytes (Noble et al., 1988; Raff et al., 1988; Richardson et al., Correspondence ‘should be addressed to Dr. Patrick Wood, Miami Project, 1988). This factor has been shown to be similar or identical to University of Miami School of Medicine, 1600 N.W. 10th Avenue, R-48, Miami. FL 33136. platelet-derived growth factor. There is some evidence that the p Present address: Department of Cell Biology, New York University School of production of platelet-derived growth factor in the CNS parallels Medicine, 550 1st Avenue. New York, NY 10016. b Present address: Department of Anatomy, University of Mississippi Medical the development of astrocytes, suggesting that this platelet-de- Center, 2500 N. State Street, Jackson, MS 39216-4505. rived growth factor-mediated interaction may play a role in Copyright 0 1989 Society for Neuroscience 0270-6474/891103371-09$02.00/O gliogenesis in vivo; i.e., as the number of astrocytes increases, 3372 Rosen et al. * Astrocytes Inhibit Adult Oligodendrocyte Function more platelet-derived growth factor may become available to dium 1 with 1O-5 FdU on d 2-4, 6-8, and 11-13. Since axons are stimulate division of oligodendrocyte precursors (Richardson et mitogenic for oligodendrocytes (Wood and Bunge, 1986a), the FdU- containing medium was rinsed away before adding oligodendrocytes to al., 1988). These results indicate that Type 1 astrocytes may dorsal root ganglion neurons. Cell suspensions prepared from these exert a direct positive influence on normal oligodendrocyte de- cultures for replating onto neuronal cultures were >95% GalC-positive velopment in viva. There is some evidence that the astrocytes oligodendrocytes and ~2.5% GFAP-positive astrocytes. that proliferate in reaction to injury or demyelination also be- Oligodendrocytes were also purified by fluorescence-activated cell sorting. A cell suspension was prepared from‘3-month-old spinal cord long to the type 1 family (Miller et al., 1986). Thus, it might be as described above through the Percoll separation step. The cells (from expected, on the basis of the tissue culture experiments cited, one rat) were collected by centrifugation and resuspended in 0.5 ml of that the proliferation of Type 1 astrocytes in vivo would promote hybridoma supematant containing mouse monoclonal anti-GalC anti- oligodendrocyte proliferation and remyelination. body (gift of Dr. Barbara Ranscht). (The supematant was generated by In this report, we describe tissue culture experiments designed incubating confluent hvbridoma cultures in Dulbecco’s Modified Eagle’s Medium,buffered wiih 25 mM Hepes buffer and containing 5% heat- to ascertain the effect of Type 1 astrocytes on the myelination inactivated horse serum, for 48 hr at 37°C.) The cells were exposed to of sensory axons by oligodendrocytes obtained from adult an- the supematant for 30 min at room temperature; during this period imals. In these experiments, the oligodendrocytes, purified either they were resuspended at 5-min intervals. Fifty microliters of affinity by density gradient centrifugation and antimitotic treatment or purified fluorescein-conjugatedgoat antimouse IgG (Cappel, diluted 1:5) were added to the cell suspension, and the incubation was carried out by fluorescence-activated cell sorting, were added to cultures of for an additional 30 min with gentle trituration at 5-min intervals. The purified neurons to initiate myelination. Type 1 astrocytes, puri- suspension was then diluted to 3.0 ml with LS medium and filtered fied from cultures of dissociated newborn rat cerebral cortex by through 15-urn Nitex. This suspension was subjected to sorting on a the selective adhesion method of McCarthy and de Vellis (1980) Becton-Dickinson 440 cell sorter equipped with a 5-W argon laser. The as modified by Noble et al. (1984), were added to these cultures sorted cells were approximately 90-95% GalC+ oligodendrocytes as confirmed by cell counting in a hemocytometer. These cells were re- after the oligodendrocytes had attached and started to prolif- suspended in Medium 1 and plated directly onto neuronal cultures. erate. It was observed under these conditions