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Transformation of cells of astrocyte lineage into macrophage-like cells in organotypic cultures of mouse spinal cord tissue
Journal of the Neurological Sciences, 1986, 72:77-89
77
Elsevier
JNS 2595
Transformation of Cells of Astrocyte Lineage into Macrophage-like Cells in Organotypic Cultures of Mouse Spinal Cord Tissue H. Kusaka ~'*, A. Hirano t'2, M . B . Bornstein 2,3, G . R . W . Moore 3'4 and C.S. RaJne2, 4 ~Bluestone Laboratory, Division of Neuropathology, Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467; 2Department of Neuroscience, 3Saul R. Korey Department of Neurology, and 4Department of Pathology (Neuropathology), Rose F. Kennedy Center for Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) (Received 20 March, 1985) (Received, revised 22 August, 1985) (Accepted 22 August, 1985)
SUMMARY
Phagocytic cells on the surface of the explants and their relationships to the surface were examined morphologically and immunocytochemically in organotypic cultures of mouse spinal cord tissue. Phagocytic cells were rounded, had smooth cytoplasmic surfaces and were occasionally closely apposed to underlying cells by junctional complexes. These cells contained dense bodies, vacuoles, smooth and coated vesicles, a few microtubules and bundles of intermediate filaments similar to astroglial filaments. The superficial layer of the explant which usually consisted of astroglial cell bodies and their processes, sometimes contained immature neuroepithelial cells with numerous free ribosomes, centrioles, Golgi apparatus, microtubules and infrequently, intermediate filaments. Overall, the cells resembled poorly differentiated astrocytes. Numerous dense bodies and coated vesicles were observed in some of these immature cells as well as in astrocytes in the surface layer of the explant. Cytoplasmic bridges between immature cells within the explant and phagocytic cells on the surface were observed, Immunocytochemistry revealed the presence of glial fibrillary acidic protein within these surface phagocytic cells. It thus appears that immature neuroepithelial cells
Supported by grants from NIH NS-11605, NS-08952 and NS-11920 * Present address: Department of Neurology, Kitano Hospital, Osaka, Japan. Address correspondence to: Asao Hirano, M.D., Division of Neuropathology, Department of Pathology, Montefiore Medical Center, Bronx, NY 10467, U.S.A. Tel: (212)920-447. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
78 of astrocytic lineage are capable of transforming into macrophage-like cells in organotypic culture.
Key words:
A s t r o c y t e - E p i t h e l i a l cell - M a c r o p h a g e
- Mouse
organotypic culture -
Phagocytosis - Spinal cord
INTRODUCTION During demyelination in experimental allergic encephalomyelitis (EAE), monocytic macrophages are the major phagocytes of degenerating myelin (Raine 1984). However, in organotypic cultures of mouse spinal cord tissue in which demyelination has been induced by serum from animals with EAE, hematogenous macrophages are not involved in the demyelinating process but rather astrocytes perform the phagocytic activity and display distinct alterations (Raine and Bornstein 1970; Raine et al. 1973; Kusaka et al. 1985b). It is well known that on the surface of organotypic cultures of central nervous system (CNS) tissue rounded phagocytes containing dense bodies and vacuoles occur, the origin of which is unknown (Raine 1973). In this regard, we have examined these cells in CNS explants and the results suggest that these phagocytic cells are derived from transformed immature neuroepithelial cells which are presumably astrocytic precursors. MATERIALS AND METHODS Fragments of spinal cord from 13-14-day-old mouse embryos were dissected and cultured by standard techniques (Bronstein 1973). Twice a week, the culture medium was exchanged after complete washing of the explant. After 18-29 days in vitro (DIV), 25 explants were exposed to nutrient medium containing 25% serum from rabbits sensitized for EAE with whole white matter in complete Freund's adjuvant (Raine and Bornstein 1970), plus 10~o guinea pig serum as a source of complement. Cultures were sampled for electron microscopy (EM) after 15-360 min of exposure, As controls, 12 sister cultures were maintained in normal nutrient medium for 18-29 DIV. Fixation was performed by immersion of the explant while still on its collagen-coated coverslip, in phosphate-buffered 2.5~o glutaraldehyde for 1 h. The sample was then processed for EM by previously described techniques (Raine t973). From all 37 explants studied, 1.0-#m epon sections were cut and stained with 1 toluidine blue for light microscopy (LM). Selected areas were sectioned and examined by EM. For immunocytochemistry, selected 1-#m epon sections were stained for gliat fibrillary acidic protein (GFAP) using a modifmation of previously reported techniques (Sternberger et al. 1970; Baskin et al. 1979; DeArmond et al. 1981). Essentially, the sections were initially treated with a 1:4 solution of saturated sodium ethoxide in
79 absolute alcohol for 30 rain. After washing in absolute alcohol, distilled water and Tris-saline, they were immersed in 0.3 ~o hydrogen peroxide for 5 min and again rinsed in Tris-saline. The following were then serially applied to the sections for 45 min each: antiserum to GFAP (previously characterized by Chiu and Goldman 1984) at dilutions of 1:100 or 1:1000, swine anti-rabbit IgG (Accurate Chemical & Scientific Corp., Westbury, NY) at a 1 : 10 dilution, and 1 : 40 rabbit peroxidase anti-peroxidase (Copper Biomedical Inc., Malvern, PA). GFAP antiserum was omitted in the control sections. Each of the incubations was preceded by a 15-min application of 3 ~ normal swine serum (Flow Laboratories, McLean, VA) and followed by washing in Tris-saline. Staining was then carried out with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) in Tris buffer with hydrogen peroxide. Some sections were counterstained with hematoxylin.
RESULTS
The morphology of surface phagocytic cells and their relationship to the surface layer of the explants were examined in both control and experimental cultures. No essential differences were found between these groups. Therefore, for the purpose of this report the lrmdings are grouped together.
Light microscopy The overall organization of CNS explants has been reported previously (Raine 1973: Kusaka et al. 1984). Each explant of spinal cord tissue was dome-shaped in cross-section and rested fiat on the collagen substrate. When pia-arachnoid cells remained attached to the explant, they grew as a discontinuous layer along the CNS explant (Kusaka et al. 1985a). Cultures exposed to EAE serum showed an apparent loss of myelinated fibers in 1-/am sections stained with toluidine blue and an increase of densely-stained granules in astrocyte cell bodies and processes (Raine and Bornstein 1970; Kusaka et al. 1985b). In control and EAE serum-exposed cultures, several rounded phagocytic cells with dense toluidine-blue positive lipid inclusions were frequently observed on the surface of the explant (Fig. 1A).
Immunocytochemistry Sections treated with anti-GFAP serum occasionally showed small brown deposits of reaction product in the peripheral cytoplasm of phagocytic cells on the surface (Fig. 1B) and on bundles of glial filaments within the body of the explant. Neuronal elements did not stain. When counterstained with hematoxylin, the GFAPpositive material remained brown in contrast to basophilic nuclei and cytoplasmic lipid droplets (Fig. IC). In control sections (Fig. 1D) in which anti-GFAP antiserum was omitted, no reaction product was seen either in the explant or in the cytoplasm of rounded phagocytic cells on the culture surface.
80
Fig. 1. A: Rounded phagocytic cells are located on the surface of the explant. The cytoplasm contains many densely-staining lipid granules (arrows) around the nuclei and vacuoles of various sizes. Several mature astrocytes (small arrows) are observed near the surface of the explant. 26 DIV. I-FLmsection, toluidine blue stain, x 900.
B: The cytoplasm of a phagocytic cell on the surface of a culture stained for GFAP reactivity, contains many small rounded profiles (between arrows), seen as brown reaction product in the original preparation. The nucleus lies to the left. Several GFAP-positive fiber bundles in various planes are observed between neurons inside the explant. 26 DIV. l-/~m section, treated with anti-GFAP serum (1 : 100). × 900.
Electron microscopy T h e u p p e r surface layer o f the e x p l a n t generally c o n s i s t e d o f a c o n t i n u o u s layer o f a s t r o c y t e cell b o d i e s (Fig. 2 A ) or m o r e c o m m o n l y , their p r o c e s s e s ( K u s a k a et al, 1984). I n a d d i t i o n , small r o u n d e d i m m a t u r e cells w e r e o c c a s i o n a l l y o b s e r v e d in this region (Fig. 2B). T h e s e i m m a t u r e ceils h a d o v a l nuclei with a thin rim o f h e t e r o -
81
Fig. 1. C: Two phagocytic cells (arrows) on the surface contain many small deposits of reaction product (seen as brown in the original preparation), basophilic granules and vacuoles in the cytoplasm. 26 DIV 1-#m section, treated with anti-GFAP serum (1 : 1000) and counterstained with hematoxylin. × 900.
D: Phagocytic cells on the surface do not show brown reaction product but possess basophilic granules and large vacuoles. 26 DIV. l-#m section. Control section (anti-GFAP antiserum omitted) counterstained with hematoxylin. × 900.
chromatin, relatively small amounts of cytoplasm containing free ribosomes or polysomes, centrioles or microtubules and a few cisterns o f rough endoplasmic reticulum (RER). Other organelles, including intermediate filaments, were rare. These immature cells occasionally had cytoplasmic processes and contacted the processes of other cells by junctional complexes. Sometimes, they were partially covered by fragments o f basal lamina. On occasion, the immature cells rested on the surface o f the explant, still retaining epithelial properties such as junctional complexes, fragments o f basal lamina
82
Fig, 2. An astrocyte cell body (A) and an immature cell (B) are demonstrated in the upper surface layer of a culture after 20 DIV. A: An astrocyte contains a watery cytoplasm with glial filaments as well as microvillus-like projections and gap junctions (arrows). × 5400.
and microvillus-like projections. Cells with features intermediate between these immature cells and mature astrocytes were also found. Near the surface of the explant, ceils containing dense bodies were observed. Some of these contained bundles of intermediate filaments and glycogen granules and were identified as astrocytes. Some possessed a well-developed Golgi apparatus, numerous vesicles and only a few intermediate filaments. Others had the morphological features of the immature cells described above (Fig. 3). On rare occasions transitional appearances occurred where immature cells within the explant contained abundant dense bodies and vesicles, extended through the surface and displayed continuity with the cytoplasm of a phagocytic cell on the surface of the explant (Fig. 4). The surface phagocytic cell had a round or lobulated nucleus with aggregates of heterochromatin beneath the nuclear membrane. The abundant cytoplasm contained dense bodies, lysosomes, vacuoles, smooth and coated vesicles, as well as mitochondria, microtubules and occasional cisterns of R E R (Fig. 5). The outline of the cell was smooth with occasional pseudopodia and filopodia. Other phagocytic cells also occurred which contained less lysosomal material, but demonstrated more features in
83
Fig. 2. B: An immature cell with a large nucleus and a cytoplasm containing scattered free ribosomes, polyribosomes and RER shows primitivejunctional complexes(arrows). × 7300.
common with astrocytes, in particular glial filaments (Fig. 6). Presumably, the latter rounded cells derive from the immature stock and might differentiate further into the mature phagocytes shown in Fig. 5. Also, the glial filaments probably represent the ultrastructural counterpart of the G F A P reactivity (Fig. 1B). DISCUSSION The present study of myelinated organotypic cultures of mouse spinal cord tissue details the morphology of rounded phagocytic cells within or on the surface of the explant. On most occasions, these cells appeared indistinguishable from hematogenous macrophages (Van der Rhee et al. 1979). In addition, the upper layer of the explant, which generally consists of astrocyte cell bodies and their processes (Kusaka et al. 1984), also contained immature neuroepithelial cells, similar to ventricular cells (Henrikson and Vaughn 1974), glioblasts in vivo (Kitamura et al. 1984), or astrocyte precursor cells in colony cultures (Fedoroffet al. 1984). Cells with features intermediate between the immature cells and more-differentiated astrocytes were also observed. These immature cells occasionally contained numerous dense bodies, vacuoles and
84
Fig. 3. A cell with a poorly-differentiated cytoplasm contains many dense bodies (lysosomes). 26 DIV. × 7 300.
vesicles, suggestive of phagocytic and pinocytic activity. That these phagocytes were derived from immature cells was proven by the observation of cytoplasmic bridges between the two. Several types of cells are proposed in the origin of macrophages in the CNS, such as neuroectodermal cells (Vaughn et al. 1970; Brierley and Brown 1982), cells associated with blood vessels (Raedler and Raedler 1984), pial cells (Merchant and Low 1979), and blood monocytes (Fujita and Kitamura 1976). However, in myelinated organotypic cultures of spinal cord tissue, mesenchymal elements are usually absent, particularly endothelial cells, pericytes or hematogeneous ceils (Raine 1973), a feature probably related to the stage of development at the time of exptantation. When explanted together with cord tissue, pial cells always grow as a layer above the CNS explant (Bunge et al. 1965; Kusaka et al. 1985a). On the other hand, the phagoc~c activity of astrocytes in vivo is well-documented in a number of conditions (Hirano et al. 1965; Lampert and Cressman 1968; Lemkey-Jotmson et al. 1976; Fulcrand and Privat 1977; Noske et al. 1982; Raine et al. 1984), in addition to organotypic cultures of CNS tissue (Raine and Bornstein 1970; Raine et al. 1973; Kusaka et al. 1985b). Although containing features atypical of wandering hematogenous macrophages, immature g!ial cells are also known
85
Fig. 4. A portion of a cell similar to that described in Fig. 3 extends between the superficial astrocytic processes on the surface and connects with the cytoplasm of a phagocytic cell above. Control culture. 26 DIV. x 10000.
to possess phagocytic activity in vivo, particularly while they are actively proliferating (Vaughn et al. 1970; L e m k e y - J o h n s o n et al. 1976; Fulcrand and Privat 1977). Therefore, the observed primitive neuroepithelial cells, which are believed to have the potential to differentiate into astrocytes, might also express inherent phagocytic and pinocytic
86
Fig. 5. A phagocyticcellon the surfaceof the explantcontains numerousdense bodies and vesicles(arrows) in additionto vacuolesof various sizes, and is closelyapposed to the surfaceof the explant and an adjacent cell (arrowheads). 26 DIV. × 6000. activity, lose their epithelial features and transform into rounded phagocytic cells in organotypic CNS cultures. Epithelial cells have been reported to have similar features in different culture systems (Greenburg and Hay 1982). Adult and embryonic avian lens epithelia, when suspended and cultured inside gelling collagen, lose their epithelial characteristics, dissociate from the apical surface of the explant and migrate as individual cells which acquire the morphological characteristics reminiscent of mesenchymal cells. The immediate culture environment of the epithelial cell influences cell polarity as defined by the presence of basal and apical surfaces (Boulan and Sabatini 1978; Chambard etal. 1981). Dissociated epithelial cells redistribute macromolecules in the cell membrane and lose the differences between the apical and basolateral aspects of the cell body (Pisam and Ripoche 1976).
87
Fig. 6. A phagocyticcell contains a bundle of intermediate filaments (arrow), as well as dense bodies and is attached to the surface of the explant by a junctional complex (arrowhead). 26 DIV. × 10000.
The myelinated organotypic culture system studied here has a less complex organization than the same tissue in vivo and essentially lacks mesenchymal elements, particularly blood vessels and its upper layer has direct contact with the culture medium. In addition to the active proliferative ability of immature cells, these environmental factors conceivably favor the transformation of primitive neuroepithelial cells into macrophage-like cells in vitro, an inherent property which might later become manifest
88 d u r i n g pathological c o n d i t i o n s . H o w e v e r , in the p r e s e n t study, phagocytic cells freq u e n t l y s h o w e d close a p p o s i t i o n to n e i g h b o r i n g cells a n d p o s s e s s e d a s m o o t h cell surface with fewer p s e u d o p o d i a a n d rare j u n c t i o n a l c o m p l e x e s , features dissimilar to h e m a t o g e n o u s m a c r o p h a g e s a n d a p o i n t w o r t h y o f further exploration, p e r h a p s by monoclonal antibody technology and immunocytochemistry. Finally, it a p p e a r s t h a t along with the epithelial n a t u r e ( K u s a k a et al. 1985a) a n d phagocytic activity o f astrocytes ( K u s a k a et al. 1985b), yet a n o t h e r a s p e c t of glial cell b e h a v i o r , i.e., t r a n s f o r m a t i o n o f i m m a t u r e cells into m a c r o p h a g e - l i k e cells, has b e e n d i s s e c t e d with the aid o f o r g a n o t y p i c cultures o f C N S tissue.
REFERENCES Baskin, D.G., S.L. Erlandsen and J.A. Parsons (1979) Immunocytochemistry with osmium-fixed tissue, Part 1 (Light microscopic localization of growth hormone and prolactin with the unlabeled antibodyenzyme method), J. Histochem. Cytochem., 27: 867-872. Bornstein, M. B. (1973) Organotypic mammalian central and peripheral nerve tissue. In: P.F. Kruse, Jr. and M.D. Patterson, Jr. (Eds.), Tissue Culture, Methods and Application, Academic Press, New York, pp. 86-92. Boulan, E. R. and D. D. Sabatini (1978) Asymmetric budding of viruses in epithelial monolayers - - A model system for study of epithelial polarity, Proc. Nat. Acad. Sci. (USA), 75: 5071-5075. Brierley, J.B. and A.W. Brown (1982) The origin of lipid phagocytes in the central nervous system, Part 1 (The intrinsic microglia), J. Comp. Neurol., 211: 397-406. Bunge, R.P., M.B. Bunge and E.R. Peterson (1965) An electron microscope study of cultured rat spinal cord, J. Cell Biol., 24: 163-191. Chambard, M., J. Gabrion and J. Mauchamp (1981) Influence of collagen gel on the orientation of epithelial cell polarity - - Follicle formation from isolated thyroid cells and from preformed monolayers, J. Cell Biol., 91: 157-166. Chiu, F.-C. and J. E. Goldman (1984) Synthesis and turnover of eytoskeletal proteins in cultured astrocytes, J. Neurochem., 42: 166-174. DeArmond, S.J., M.W. Siegel, R.G. Dixon and L.F. Eng (1981) Post-embedding immunoperoxidase staining of glial fibrillary acidic protein for light and electron microscopy, J. Neuroimmunol., 1: 3-15. Fedoroff, S., J. Neal, M. Opas and V.I. Kalnins (1984) Astrocyte cell lineage, Part 3 (The morphology of differentiating mouse astrocytes in colony culture), J. NeurocytoL, 13: 1-20. Fujita, S. and T. Kitamura (1976) Origin of brain macrophages and the nature of the microglia. In: H.M. Zimmerman (Ed.), Progress in Neuropathology, 1Iol. 3, Grune and Stratton, New York, pp. 1-50. Fulcrand, J. and A. Privat (1977) Neurogtial reactions secondary to Wallerian degeneration in the optic nerve of the postnatal rat - - Ultrastructural and quantitative study, J. Comp. Neurol., ! 76:189-224. Greenburg, G. and E.D. Hay (1982) Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells, J. Cell Biol., 95: 333-339. Henrikson, C.K. and J.E. Vaughn (1974) Fine structural relationships between neurites and radial glial processes in developing mouse spinal cord, J. Neurocytol., 3: 659-675. Hirano, A., H. M. Zimmerman and S. Levine (1965) The fine structure of cerebral fluid accumulation, Part 7 (Reactions of astrocytes to cryptococcal polysaccharide implantation), J. Neuropath. Exp. Neurol., 24: 386-397. Kitamura, T., T. Miyake and S. Fujita (1984) Genesis of resting microglia in the gray matter of mouse hippocampus, J. Comp. Neurol., 226: 421-433. Kusaka, H., A. Hirano, M. B. Bornstein and C. S. Raine (1984) The organization of astrocytes in organotypic mouse spinal cord culture - - An electron microscope study, Neuropath. Appl. Neurobiot., 10:411-422. Kusaka, H., A. Hirano, M.B. Bornstein and C. S. Raine (1985a) Basal lamina formation by astrocytes hi organotypic cultures of mouse spinal cord tissue, J. Neuropath. Exp. Neurol., 44: 295-303. Kusaka, H., A. Hirano, M. B. Bornstein and C. S. Raine (1985b) Fine structure of astrocytic processes during serum-induced demyelination in vitro, J. Neurol. Sci., 69: 255-267. Lampert, P.W. and M.R. Cressman (1966) Fine-structural changes of myelin sheaths after axonal degeneration in the spinal cord of rats, Amer. J. Path., 49:1139-1155.
89 Lemkey-Johnston, N., V. Butler and W. A. Reynolds (1976) Glial changes in the progress of a chemical lesion - - An electron microscopic study, J. Comp. NeuroL, 167: 481-502. Merchant, R.E. and F.N. Low (1979) Scanning electron microscopy of the subarachnoid space in the dog - - Evidence for a non-hematogenous origin of subarachnoid macrophages, Amer. J. Anat., 156: 183-206. Noske, W., H. Lentzen and H. Herken (1982) Phagocytosis and morphological development of rat astrocytes in primary cultures, Cell MoL BioL, 28: 235-244. Pisam, M. and P. Ripoche (1976) Redistribution of surface macromolecules in dissociated epithelial cells, J. Cell Biol., 71: 907-920. Raedler, E. and A. Raedler (1984) Local proliferation of brain macrophages in central nervous system tissue cultures, J. Neuropath. Exp. NeuroL, 43: 531-540. Raine, C.S. (1973) Ultrastructural applications of cultured nervous system tissue to neuropathology. In: H. M. Zimmerman (Ed.), Progress in Neuropathology, 1Iol. 2, Grune and Stratton, New York, pp. 27-68. Raine, C. S. (1984) Biology of disease. Analysis of autoimmune demyelination - - Its impact upon multiple sclerosis, Lab. Invest., 50: 608-635. Raine, C. S. and M. B. Bornstein (1970) Experimental allergic encephalomyelitis - - An ultrastructural study of experimental demyelination in vitro, J. Neuropath. Exp. NeuroL, 29: 177-191. Raine, C. S., A. Hummelgard, E. Swanson and M.B. Bornstein (1973) Multiple sclerosis: Serum-induced demyelination in vitro - - A light and electron microscope study, J. NeuroL Sci., 20: 127-148. Raine, C. S., F. Mokhtarian and D. E. McFarlin (1984) Adoptively transferred chronic relapsing experimental autoimmune encephalomyelitis in the mouse - - Neuropathologic analysis, Lab. Invest., 51 : 534-546. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis and H.G. Meyer (1970) The unlabeled antibody enzyme method of immunohistochemistry - - Preparation and properties of solubld antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18: 315-333. Van der Rhee, H.J., C.P.M. Van der Burgh-De Winter and W.T. Daems (1979) The differentiation of monocytes into macrophages, epitheloid cells, and mononucleated giant cells in subcutaneous granulomas, Part 1 (Fine structure), Cell Tiss. Res., 197: 355-378. Vaughn, J. E., P. L. Hinds and R.P. Skoff (I 970) Electron microscopic studies of Wallerian degeneration in rat optic nerves, Part 1 (Multipotential glia), ,L Cornp. Neurol., 140: 175-206.
77
Elsevier
JNS 2595
Transformation of Cells of Astrocyte Lineage into Macrophage-like Cells in Organotypic Cultures of Mouse Spinal Cord Tissue H. Kusaka ~'*, A. Hirano t'2, M . B . Bornstein 2,3, G . R . W . Moore 3'4 and C.S. RaJne2, 4 ~Bluestone Laboratory, Division of Neuropathology, Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467; 2Department of Neuroscience, 3Saul R. Korey Department of Neurology, and 4Department of Pathology (Neuropathology), Rose F. Kennedy Center for Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) (Received 20 March, 1985) (Received, revised 22 August, 1985) (Accepted 22 August, 1985)
SUMMARY
Phagocytic cells on the surface of the explants and their relationships to the surface were examined morphologically and immunocytochemically in organotypic cultures of mouse spinal cord tissue. Phagocytic cells were rounded, had smooth cytoplasmic surfaces and were occasionally closely apposed to underlying cells by junctional complexes. These cells contained dense bodies, vacuoles, smooth and coated vesicles, a few microtubules and bundles of intermediate filaments similar to astroglial filaments. The superficial layer of the explant which usually consisted of astroglial cell bodies and their processes, sometimes contained immature neuroepithelial cells with numerous free ribosomes, centrioles, Golgi apparatus, microtubules and infrequently, intermediate filaments. Overall, the cells resembled poorly differentiated astrocytes. Numerous dense bodies and coated vesicles were observed in some of these immature cells as well as in astrocytes in the surface layer of the explant. Cytoplasmic bridges between immature cells within the explant and phagocytic cells on the surface were observed, Immunocytochemistry revealed the presence of glial fibrillary acidic protein within these surface phagocytic cells. It thus appears that immature neuroepithelial cells
Supported by grants from NIH NS-11605, NS-08952 and NS-11920 * Present address: Department of Neurology, Kitano Hospital, Osaka, Japan. Address correspondence to: Asao Hirano, M.D., Division of Neuropathology, Department of Pathology, Montefiore Medical Center, Bronx, NY 10467, U.S.A. Tel: (212)920-447. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
78 of astrocytic lineage are capable of transforming into macrophage-like cells in organotypic culture.
Key words:
A s t r o c y t e - E p i t h e l i a l cell - M a c r o p h a g e
- Mouse
organotypic culture -
Phagocytosis - Spinal cord
INTRODUCTION During demyelination in experimental allergic encephalomyelitis (EAE), monocytic macrophages are the major phagocytes of degenerating myelin (Raine 1984). However, in organotypic cultures of mouse spinal cord tissue in which demyelination has been induced by serum from animals with EAE, hematogenous macrophages are not involved in the demyelinating process but rather astrocytes perform the phagocytic activity and display distinct alterations (Raine and Bornstein 1970; Raine et al. 1973; Kusaka et al. 1985b). It is well known that on the surface of organotypic cultures of central nervous system (CNS) tissue rounded phagocytes containing dense bodies and vacuoles occur, the origin of which is unknown (Raine 1973). In this regard, we have examined these cells in CNS explants and the results suggest that these phagocytic cells are derived from transformed immature neuroepithelial cells which are presumably astrocytic precursors. MATERIALS AND METHODS Fragments of spinal cord from 13-14-day-old mouse embryos were dissected and cultured by standard techniques (Bronstein 1973). Twice a week, the culture medium was exchanged after complete washing of the explant. After 18-29 days in vitro (DIV), 25 explants were exposed to nutrient medium containing 25% serum from rabbits sensitized for EAE with whole white matter in complete Freund's adjuvant (Raine and Bornstein 1970), plus 10~o guinea pig serum as a source of complement. Cultures were sampled for electron microscopy (EM) after 15-360 min of exposure, As controls, 12 sister cultures were maintained in normal nutrient medium for 18-29 DIV. Fixation was performed by immersion of the explant while still on its collagen-coated coverslip, in phosphate-buffered 2.5~o glutaraldehyde for 1 h. The sample was then processed for EM by previously described techniques (Raine t973). From all 37 explants studied, 1.0-#m epon sections were cut and stained with 1 toluidine blue for light microscopy (LM). Selected areas were sectioned and examined by EM. For immunocytochemistry, selected 1-#m epon sections were stained for gliat fibrillary acidic protein (GFAP) using a modifmation of previously reported techniques (Sternberger et al. 1970; Baskin et al. 1979; DeArmond et al. 1981). Essentially, the sections were initially treated with a 1:4 solution of saturated sodium ethoxide in
79 absolute alcohol for 30 rain. After washing in absolute alcohol, distilled water and Tris-saline, they were immersed in 0.3 ~o hydrogen peroxide for 5 min and again rinsed in Tris-saline. The following were then serially applied to the sections for 45 min each: antiserum to GFAP (previously characterized by Chiu and Goldman 1984) at dilutions of 1:100 or 1:1000, swine anti-rabbit IgG (Accurate Chemical & Scientific Corp., Westbury, NY) at a 1 : 10 dilution, and 1 : 40 rabbit peroxidase anti-peroxidase (Copper Biomedical Inc., Malvern, PA). GFAP antiserum was omitted in the control sections. Each of the incubations was preceded by a 15-min application of 3 ~ normal swine serum (Flow Laboratories, McLean, VA) and followed by washing in Tris-saline. Staining was then carried out with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) in Tris buffer with hydrogen peroxide. Some sections were counterstained with hematoxylin.
RESULTS
The morphology of surface phagocytic cells and their relationship to the surface layer of the explants were examined in both control and experimental cultures. No essential differences were found between these groups. Therefore, for the purpose of this report the lrmdings are grouped together.
Light microscopy The overall organization of CNS explants has been reported previously (Raine 1973: Kusaka et al. 1984). Each explant of spinal cord tissue was dome-shaped in cross-section and rested fiat on the collagen substrate. When pia-arachnoid cells remained attached to the explant, they grew as a discontinuous layer along the CNS explant (Kusaka et al. 1985a). Cultures exposed to EAE serum showed an apparent loss of myelinated fibers in 1-/am sections stained with toluidine blue and an increase of densely-stained granules in astrocyte cell bodies and processes (Raine and Bornstein 1970; Kusaka et al. 1985b). In control and EAE serum-exposed cultures, several rounded phagocytic cells with dense toluidine-blue positive lipid inclusions were frequently observed on the surface of the explant (Fig. 1A).
Immunocytochemistry Sections treated with anti-GFAP serum occasionally showed small brown deposits of reaction product in the peripheral cytoplasm of phagocytic cells on the surface (Fig. 1B) and on bundles of glial filaments within the body of the explant. Neuronal elements did not stain. When counterstained with hematoxylin, the GFAPpositive material remained brown in contrast to basophilic nuclei and cytoplasmic lipid droplets (Fig. IC). In control sections (Fig. 1D) in which anti-GFAP antiserum was omitted, no reaction product was seen either in the explant or in the cytoplasm of rounded phagocytic cells on the culture surface.
80
Fig. 1. A: Rounded phagocytic cells are located on the surface of the explant. The cytoplasm contains many densely-staining lipid granules (arrows) around the nuclei and vacuoles of various sizes. Several mature astrocytes (small arrows) are observed near the surface of the explant. 26 DIV. I-FLmsection, toluidine blue stain, x 900.
B: The cytoplasm of a phagocytic cell on the surface of a culture stained for GFAP reactivity, contains many small rounded profiles (between arrows), seen as brown reaction product in the original preparation. The nucleus lies to the left. Several GFAP-positive fiber bundles in various planes are observed between neurons inside the explant. 26 DIV. l-/~m section, treated with anti-GFAP serum (1 : 100). × 900.
Electron microscopy T h e u p p e r surface layer o f the e x p l a n t generally c o n s i s t e d o f a c o n t i n u o u s layer o f a s t r o c y t e cell b o d i e s (Fig. 2 A ) or m o r e c o m m o n l y , their p r o c e s s e s ( K u s a k a et al, 1984). I n a d d i t i o n , small r o u n d e d i m m a t u r e cells w e r e o c c a s i o n a l l y o b s e r v e d in this region (Fig. 2B). T h e s e i m m a t u r e ceils h a d o v a l nuclei with a thin rim o f h e t e r o -
81
Fig. 1. C: Two phagocytic cells (arrows) on the surface contain many small deposits of reaction product (seen as brown in the original preparation), basophilic granules and vacuoles in the cytoplasm. 26 DIV 1-#m section, treated with anti-GFAP serum (1 : 1000) and counterstained with hematoxylin. × 900.
D: Phagocytic cells on the surface do not show brown reaction product but possess basophilic granules and large vacuoles. 26 DIV. l-#m section. Control section (anti-GFAP antiserum omitted) counterstained with hematoxylin. × 900.
chromatin, relatively small amounts of cytoplasm containing free ribosomes or polysomes, centrioles or microtubules and a few cisterns o f rough endoplasmic reticulum (RER). Other organelles, including intermediate filaments, were rare. These immature cells occasionally had cytoplasmic processes and contacted the processes of other cells by junctional complexes. Sometimes, they were partially covered by fragments o f basal lamina. On occasion, the immature cells rested on the surface o f the explant, still retaining epithelial properties such as junctional complexes, fragments o f basal lamina
82
Fig, 2. An astrocyte cell body (A) and an immature cell (B) are demonstrated in the upper surface layer of a culture after 20 DIV. A: An astrocyte contains a watery cytoplasm with glial filaments as well as microvillus-like projections and gap junctions (arrows). × 5400.
and microvillus-like projections. Cells with features intermediate between these immature cells and mature astrocytes were also found. Near the surface of the explant, ceils containing dense bodies were observed. Some of these contained bundles of intermediate filaments and glycogen granules and were identified as astrocytes. Some possessed a well-developed Golgi apparatus, numerous vesicles and only a few intermediate filaments. Others had the morphological features of the immature cells described above (Fig. 3). On rare occasions transitional appearances occurred where immature cells within the explant contained abundant dense bodies and vesicles, extended through the surface and displayed continuity with the cytoplasm of a phagocytic cell on the surface of the explant (Fig. 4). The surface phagocytic cell had a round or lobulated nucleus with aggregates of heterochromatin beneath the nuclear membrane. The abundant cytoplasm contained dense bodies, lysosomes, vacuoles, smooth and coated vesicles, as well as mitochondria, microtubules and occasional cisterns of R E R (Fig. 5). The outline of the cell was smooth with occasional pseudopodia and filopodia. Other phagocytic cells also occurred which contained less lysosomal material, but demonstrated more features in
83
Fig. 2. B: An immature cell with a large nucleus and a cytoplasm containing scattered free ribosomes, polyribosomes and RER shows primitivejunctional complexes(arrows). × 7300.
common with astrocytes, in particular glial filaments (Fig. 6). Presumably, the latter rounded cells derive from the immature stock and might differentiate further into the mature phagocytes shown in Fig. 5. Also, the glial filaments probably represent the ultrastructural counterpart of the G F A P reactivity (Fig. 1B). DISCUSSION The present study of myelinated organotypic cultures of mouse spinal cord tissue details the morphology of rounded phagocytic cells within or on the surface of the explant. On most occasions, these cells appeared indistinguishable from hematogenous macrophages (Van der Rhee et al. 1979). In addition, the upper layer of the explant, which generally consists of astrocyte cell bodies and their processes (Kusaka et al. 1984), also contained immature neuroepithelial cells, similar to ventricular cells (Henrikson and Vaughn 1974), glioblasts in vivo (Kitamura et al. 1984), or astrocyte precursor cells in colony cultures (Fedoroffet al. 1984). Cells with features intermediate between the immature cells and more-differentiated astrocytes were also observed. These immature cells occasionally contained numerous dense bodies, vacuoles and
84
Fig. 3. A cell with a poorly-differentiated cytoplasm contains many dense bodies (lysosomes). 26 DIV. × 7 300.
vesicles, suggestive of phagocytic and pinocytic activity. That these phagocytes were derived from immature cells was proven by the observation of cytoplasmic bridges between the two. Several types of cells are proposed in the origin of macrophages in the CNS, such as neuroectodermal cells (Vaughn et al. 1970; Brierley and Brown 1982), cells associated with blood vessels (Raedler and Raedler 1984), pial cells (Merchant and Low 1979), and blood monocytes (Fujita and Kitamura 1976). However, in myelinated organotypic cultures of spinal cord tissue, mesenchymal elements are usually absent, particularly endothelial cells, pericytes or hematogeneous ceils (Raine 1973), a feature probably related to the stage of development at the time of exptantation. When explanted together with cord tissue, pial cells always grow as a layer above the CNS explant (Bunge et al. 1965; Kusaka et al. 1985a). On the other hand, the phagoc~c activity of astrocytes in vivo is well-documented in a number of conditions (Hirano et al. 1965; Lampert and Cressman 1968; Lemkey-Jotmson et al. 1976; Fulcrand and Privat 1977; Noske et al. 1982; Raine et al. 1984), in addition to organotypic cultures of CNS tissue (Raine and Bornstein 1970; Raine et al. 1973; Kusaka et al. 1985b). Although containing features atypical of wandering hematogenous macrophages, immature g!ial cells are also known
85
Fig. 4. A portion of a cell similar to that described in Fig. 3 extends between the superficial astrocytic processes on the surface and connects with the cytoplasm of a phagocytic cell above. Control culture. 26 DIV. x 10000.
to possess phagocytic activity in vivo, particularly while they are actively proliferating (Vaughn et al. 1970; L e m k e y - J o h n s o n et al. 1976; Fulcrand and Privat 1977). Therefore, the observed primitive neuroepithelial cells, which are believed to have the potential to differentiate into astrocytes, might also express inherent phagocytic and pinocytic
86
Fig. 5. A phagocyticcellon the surfaceof the explantcontains numerousdense bodies and vesicles(arrows) in additionto vacuolesof various sizes, and is closelyapposed to the surfaceof the explant and an adjacent cell (arrowheads). 26 DIV. × 6000. activity, lose their epithelial features and transform into rounded phagocytic cells in organotypic CNS cultures. Epithelial cells have been reported to have similar features in different culture systems (Greenburg and Hay 1982). Adult and embryonic avian lens epithelia, when suspended and cultured inside gelling collagen, lose their epithelial characteristics, dissociate from the apical surface of the explant and migrate as individual cells which acquire the morphological characteristics reminiscent of mesenchymal cells. The immediate culture environment of the epithelial cell influences cell polarity as defined by the presence of basal and apical surfaces (Boulan and Sabatini 1978; Chambard etal. 1981). Dissociated epithelial cells redistribute macromolecules in the cell membrane and lose the differences between the apical and basolateral aspects of the cell body (Pisam and Ripoche 1976).
87
Fig. 6. A phagocyticcell contains a bundle of intermediate filaments (arrow), as well as dense bodies and is attached to the surface of the explant by a junctional complex (arrowhead). 26 DIV. × 10000.
The myelinated organotypic culture system studied here has a less complex organization than the same tissue in vivo and essentially lacks mesenchymal elements, particularly blood vessels and its upper layer has direct contact with the culture medium. In addition to the active proliferative ability of immature cells, these environmental factors conceivably favor the transformation of primitive neuroepithelial cells into macrophage-like cells in vitro, an inherent property which might later become manifest
88 d u r i n g pathological c o n d i t i o n s . H o w e v e r , in the p r e s e n t study, phagocytic cells freq u e n t l y s h o w e d close a p p o s i t i o n to n e i g h b o r i n g cells a n d p o s s e s s e d a s m o o t h cell surface with fewer p s e u d o p o d i a a n d rare j u n c t i o n a l c o m p l e x e s , features dissimilar to h e m a t o g e n o u s m a c r o p h a g e s a n d a p o i n t w o r t h y o f further exploration, p e r h a p s by monoclonal antibody technology and immunocytochemistry. Finally, it a p p e a r s t h a t along with the epithelial n a t u r e ( K u s a k a et al. 1985a) a n d phagocytic activity o f astrocytes ( K u s a k a et al. 1985b), yet a n o t h e r a s p e c t of glial cell b e h a v i o r , i.e., t r a n s f o r m a t i o n o f i m m a t u r e cells into m a c r o p h a g e - l i k e cells, has b e e n d i s s e c t e d with the aid o f o r g a n o t y p i c cultures o f C N S tissue.
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