- Email: [email protected]
Immunocytochemical localization of S-100 protein in interstitial cells of the monkey Macaca irus pineal gland
Neuroscience Letters, 57 (1985) 181-184 Elsevier Scientific Publishers Ireland Ltd.
181
NSL 03350
IMMUNOCYTOCHEMICAL LOCALIZATION OF S-100 PROTEIN IN INTERSTITIAL CELLS OF THE MONKEY M A C A C A I R U S PINEAL GLAND
C. GIROD* and N. D U R A N D Laboratoire d'Histologie, Facult~ de Mkdecine Alexis Carrel. 12 rue Guillaume Paradin, F-69372 Lyon COdex 08 (France)
(Received January 31st, 1985; Revised version received March 15th, 1985; Accepted March 16th, 1985)
Key words:
pineal gland - S-100 protein - interstitial cells - immunocytochemistry - monkey
Pineal interstitial cells of the monkey Macaca irus were shown to react with an anti-human S-100 protein antibody, using the indirect immunoperoxidase technique on sections o f paraplast-embedded pineal glands. Immunoreactivity was seen in the cytoplasm of the cells, stellate in shape and intermingled with pinealocytes; the latter did not stain with the antiserum against S-100 protein. Immunoreactivity was also present in the nuclei, as was reported in various other cell types immunostained with anti-S-100 protein antibodies. The present results support the view that interstitial cells of the monkey Macaca irus pineal gland may be of astroglial origin.
S-100 protein was originally isolated from the central nervous system of mammals by Moore [9]. This protein has a molecular weight of about 21,000 and was identified in brain extracts of mammals, birds, reptiles and fishes [18]. Using antibodies against S-100 protein, immunostaining of a large variety of cells was recently described (for review, see ref. 10). Through the use of immunocytochemical techniques for S-100 protein detection, Moller et al. [8] have obtained labelling of the interstitial cells of the rat pineal gland, and Highley et al. [4] have immunostained these cells in the goldfish pineal gland. The aim of the present study was to characterize the immunocytochemical localization of S-100 protein in the pineal gland of the monkey Macaca irus. Adult male and female monkeys were used. Under Nembutal anesthesia (10 mg/kg i.v.), brains were quickly dissected. The pineal glands and the surrounding nervous tissues were excised and immersed in G6rard's fixative (Bouin-Hollande sublimate without acetic acid) for 4-5 days. After washing in running water for 36 h, they were dehydrated in a graded ethanol series, cleared in butanol and embedded in paraplast. Serial frontal or sagittal sections (7 #m in thickness) were mounted on glass slides. After dewaxing in toluene, the sections were washed in ethanol, treated with iodized ethanol for removal of mercury pigment, rinsed in tap water and subsequently in 5~ sodium metabisulphite, then rinsed in distilled water. Antiserum against human S-100 protein, raised in rabbits, was purchased from *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
183
Ortho-Diagnostic Systems (Raritan, N J, U.S.A.). Tissue sections were processed for immunoenzymatic staining using the indirect reaction, as previously described [3], with or without counterstaining with Harris' haematoxylin. The sections were observed with a Zeiss photomicroscope and photographs were taken on Kodachrome 25 film. Specificity of S-100 protein immunoreactive staining was tested by substitution of normal rabbit serum or Tris-buffered saline for the specific antiserum. In addition, some sections were treated either with the sheep anti-rabbit IgG conjugate to peroxidase alone or with diaminobenzidine reagent alone to demonstrate possible endogenous peroxidase activity. S-100 protein immunoreactivity was found in a variable number of cells, scattered throughout the pineal gland. They were irregular in shape with an ovoid or triangular cell body and with 2-5 cytoplasmic processes enclosing isolated or clusters of pinealocytes (Figs. 1-4). Such processes often.approached the perNascular space around which they were able to form a barrier between these spaces and the pineal parenchyma. The peroxidase reaction product was distributed throughout the cytoplasm and was evident in the nucleus. Immunostained cells were few compared with the number of pinealocytes. No specific concentration of these cells was observed in one particular region of the pineal gland. No reaction product was seen in pinealocytes. After control treatments, sections were devoid of labelling. Immunostained cells correspond to the glial cells of the pineal gland, also called interstitial, dark or supporting cells. The results obtained in the monkey pineal gland are compatible with immunocytochemical results reported in pineal glands of other species using anti-S-100 and/or anti-glial fibrillary acidic (GFA) proteins [4, 5, 7, 8, 12-14]. A particular feature, the immunoreactivity of the nuclei, can be outlined. Using S-100 protein antibodies, such a result has been reported in various cell types [2, 6, 11, 15, 16]. Nevertheless, immunostaining of the nuclei in various cell types give rise to discussion. Indeed, if this reaction 'seems to depend heavily on the penetration of the antibodies into the tissue slices during the immunostaining procedure' [1], the use o f antibodies against the flsubunit of S-100 protein reveals, on the contrary, that 'the detection of only fl-subunit in nuclei suggests that S-100 protein may play some unknown role in genomic regulation' [17]. The presence of S-100 and G F A proteins in interstitial cells of the pineal gland supports the view that these cells may be of astroglial origin. But immunocytochemical arguments are in favor of the fact that interstital cells of the pineal gland are immature astrocytes persisting into adulthood [14]. We wish to thank Mrs. Mireille Raccurt for providing expert technical assistance.
Fig. 1-4. Various aspects of interstitial cells of the monkey Macaca irus pineal gland immunostained with an anti-human S-100 protein antibody. Fig. 1, no counterstaining; Figs. 2-4, counterstaining with Harris" haematoxylin. Figs. l 3, x 480: Fig. 4, x 640,
184 1 Cocchia, D. and Miani, N., lmmunocytochemical localization of the brain-specific S-100 protein in the pituitary gland of adult rat, J. Neurocytol., 9 (1980) 771 782. 2 Cocchia, D., Tibeno, G., Santarelli, R. and Michetti, F., S-100 protein in "follicular dendritic' cells of rat lymphoid organs. An immunochemical and immunocytochemical study, Cell Tiss. Res., 230 (t983) 95-103. 3 Girod, C., Immunocytochem~stry of the vertebrate adenohypophysis. In W. Graumann and K. Neumann IEds.), Handbuch der Histochemie, Vol. 8, Part 5, G. Fischer, Stuttgart, 1983, pp. 1 536. 4 Highley, H.R., McNulty, J.A. and Rowden, G,, Glial fibrillary acidic protein and S-100 protein in pineal supportive cells: an electron microscopic study, Brain Res., 304 (1984) 117-120. 5 Huang, S.-K., Nobiling, R., Schachner, M. and Taugner, R., Interstitial and parenchymal cells in the pineal gland of the golden hamster. A combined thin-section, freeze-fracture and immunofluorescence study, Cell Tiss. Res., 235 (1984) 327-337. 6 Kondo, H., lwanaga, T. and Nakajima, T., lmmunocytochemical study on the localization of neuronspecific enolase and S-100 protein in the carotid body of rats, Cell Tiss. Res., 227 (1982) 291 295. 7 L6wenthal, A., Flament-Durand, J., Karcher, D., Noppe, M. and Brion, J.P., Glial cells identified by anti-~-albumin (anti-GFA) in human pineal gland, J. Neurochem., 38 (1982) 863 -865. 8 Moiler, M., Ingild, A. and Bock, E., Immunohistochemical demonstration of S-100 protein and GFA protein in interstitial ceils of rat pineal gland, Brain Res., 140 (1978) 1-13. 9 Moore. B.W., A soluble protein characteristic to the nervous system, Biochem. Biophys. Res. Commun., 19 (1965) 739 744. 10 Nakajima, T., Kameya, T., Watanabe, S., Hirota, T. and Shimosato, Y., S-100 protein distribution in normal and neoplastic tissues. In R.A. DeLellis (Ed.), Advances in Immunohistochemistry, Masson. New York, 1984, pp. 141 158. 11 Nakajima, T., Yamaguchi, H. and Takahashi, K., S-100 protein in folliculo-stellate cells of the rat pituitary anterior lobe, Brain Res., 191 (1980) 523-531. 12 Papasozomenos, S.C., Glial fibrillary acidic (GFA) protein-containing cells in the human pineal gland, J. Neuropath. Exp. Neurol., 42 (1983) 391-408. 13 P~vet, P., Ultrastructure of the mammalian pinealocytes, In R.J. Reiter (Ed.), The Pineal Gland, Elsevier, New York, 1983, pp. 1-75. 14 Schachner, M., Huang, S.-K., Ziegelmfiller, P., Bizzini, B. and Taugner, R., Glial cells in the pineal gland of mice and rats. A combined immunofluorescence and electron-microscopic study, Cell Tiss. Res., 237 (1984) 245 252. 15 Shirasawa, N., Kihara, H., Yamaguchi, S. and Yoshimura, F., Pituitary folliculo-stellate cells immunostained with S-100 protein antiserum in postnatal, castrated and thyroidectomized rats, Cell Tiss. Res., 231 (1983) 235-249. 16 Shirasawa, N., Yamaguchi, S. and Yoshimura, F., Granulated folliculo-stellate cells and growth hormone cells immunostained with anti-S 100 protein serum in the pituitary glands of the goat, Cell Tiss. Res., 237 (1984) 7 14. 17 Takahashi. K., Isobe, T., Ohtsuki, Y., Akagi, T., Sonobe, H. and Okuyama, T., Immunohistochemical study on the distribution of ~ and/~ subunits of S-100 protein in human neoplasm and normal tissues, Virchows Arch. (Cell Pathol.), 45 (1984) 385-296. 18 Zomzely-Neurath, C.E. and Walker, W.A., Nervous system-specific proteins: 14-3-2 protein, neuronspecific enolase, and S-100 protein, in R.A. Bradshaw and D.M. Schneider (Eds.), Proteins of the Nervous System, Raven Press, New York, 1980, pp. 1 57.
181
NSL 03350
IMMUNOCYTOCHEMICAL LOCALIZATION OF S-100 PROTEIN IN INTERSTITIAL CELLS OF THE MONKEY M A C A C A I R U S PINEAL GLAND
C. GIROD* and N. D U R A N D Laboratoire d'Histologie, Facult~ de Mkdecine Alexis Carrel. 12 rue Guillaume Paradin, F-69372 Lyon COdex 08 (France)
(Received January 31st, 1985; Revised version received March 15th, 1985; Accepted March 16th, 1985)
Key words:
pineal gland - S-100 protein - interstitial cells - immunocytochemistry - monkey
Pineal interstitial cells of the monkey Macaca irus were shown to react with an anti-human S-100 protein antibody, using the indirect immunoperoxidase technique on sections o f paraplast-embedded pineal glands. Immunoreactivity was seen in the cytoplasm of the cells, stellate in shape and intermingled with pinealocytes; the latter did not stain with the antiserum against S-100 protein. Immunoreactivity was also present in the nuclei, as was reported in various other cell types immunostained with anti-S-100 protein antibodies. The present results support the view that interstitial cells of the monkey Macaca irus pineal gland may be of astroglial origin.
S-100 protein was originally isolated from the central nervous system of mammals by Moore [9]. This protein has a molecular weight of about 21,000 and was identified in brain extracts of mammals, birds, reptiles and fishes [18]. Using antibodies against S-100 protein, immunostaining of a large variety of cells was recently described (for review, see ref. 10). Through the use of immunocytochemical techniques for S-100 protein detection, Moller et al. [8] have obtained labelling of the interstitial cells of the rat pineal gland, and Highley et al. [4] have immunostained these cells in the goldfish pineal gland. The aim of the present study was to characterize the immunocytochemical localization of S-100 protein in the pineal gland of the monkey Macaca irus. Adult male and female monkeys were used. Under Nembutal anesthesia (10 mg/kg i.v.), brains were quickly dissected. The pineal glands and the surrounding nervous tissues were excised and immersed in G6rard's fixative (Bouin-Hollande sublimate without acetic acid) for 4-5 days. After washing in running water for 36 h, they were dehydrated in a graded ethanol series, cleared in butanol and embedded in paraplast. Serial frontal or sagittal sections (7 #m in thickness) were mounted on glass slides. After dewaxing in toluene, the sections were washed in ethanol, treated with iodized ethanol for removal of mercury pigment, rinsed in tap water and subsequently in 5~ sodium metabisulphite, then rinsed in distilled water. Antiserum against human S-100 protein, raised in rabbits, was purchased from *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
183
Ortho-Diagnostic Systems (Raritan, N J, U.S.A.). Tissue sections were processed for immunoenzymatic staining using the indirect reaction, as previously described [3], with or without counterstaining with Harris' haematoxylin. The sections were observed with a Zeiss photomicroscope and photographs were taken on Kodachrome 25 film. Specificity of S-100 protein immunoreactive staining was tested by substitution of normal rabbit serum or Tris-buffered saline for the specific antiserum. In addition, some sections were treated either with the sheep anti-rabbit IgG conjugate to peroxidase alone or with diaminobenzidine reagent alone to demonstrate possible endogenous peroxidase activity. S-100 protein immunoreactivity was found in a variable number of cells, scattered throughout the pineal gland. They were irregular in shape with an ovoid or triangular cell body and with 2-5 cytoplasmic processes enclosing isolated or clusters of pinealocytes (Figs. 1-4). Such processes often.approached the perNascular space around which they were able to form a barrier between these spaces and the pineal parenchyma. The peroxidase reaction product was distributed throughout the cytoplasm and was evident in the nucleus. Immunostained cells were few compared with the number of pinealocytes. No specific concentration of these cells was observed in one particular region of the pineal gland. No reaction product was seen in pinealocytes. After control treatments, sections were devoid of labelling. Immunostained cells correspond to the glial cells of the pineal gland, also called interstitial, dark or supporting cells. The results obtained in the monkey pineal gland are compatible with immunocytochemical results reported in pineal glands of other species using anti-S-100 and/or anti-glial fibrillary acidic (GFA) proteins [4, 5, 7, 8, 12-14]. A particular feature, the immunoreactivity of the nuclei, can be outlined. Using S-100 protein antibodies, such a result has been reported in various cell types [2, 6, 11, 15, 16]. Nevertheless, immunostaining of the nuclei in various cell types give rise to discussion. Indeed, if this reaction 'seems to depend heavily on the penetration of the antibodies into the tissue slices during the immunostaining procedure' [1], the use o f antibodies against the flsubunit of S-100 protein reveals, on the contrary, that 'the detection of only fl-subunit in nuclei suggests that S-100 protein may play some unknown role in genomic regulation' [17]. The presence of S-100 and G F A proteins in interstitial cells of the pineal gland supports the view that these cells may be of astroglial origin. But immunocytochemical arguments are in favor of the fact that interstital cells of the pineal gland are immature astrocytes persisting into adulthood [14]. We wish to thank Mrs. Mireille Raccurt for providing expert technical assistance.
Fig. 1-4. Various aspects of interstitial cells of the monkey Macaca irus pineal gland immunostained with an anti-human S-100 protein antibody. Fig. 1, no counterstaining; Figs. 2-4, counterstaining with Harris" haematoxylin. Figs. l 3, x 480: Fig. 4, x 640,
184 1 Cocchia, D. and Miani, N., lmmunocytochemical localization of the brain-specific S-100 protein in the pituitary gland of adult rat, J. Neurocytol., 9 (1980) 771 782. 2 Cocchia, D., Tibeno, G., Santarelli, R. and Michetti, F., S-100 protein in "follicular dendritic' cells of rat lymphoid organs. An immunochemical and immunocytochemical study, Cell Tiss. Res., 230 (t983) 95-103. 3 Girod, C., Immunocytochem~stry of the vertebrate adenohypophysis. In W. Graumann and K. Neumann IEds.), Handbuch der Histochemie, Vol. 8, Part 5, G. Fischer, Stuttgart, 1983, pp. 1 536. 4 Highley, H.R., McNulty, J.A. and Rowden, G,, Glial fibrillary acidic protein and S-100 protein in pineal supportive cells: an electron microscopic study, Brain Res., 304 (1984) 117-120. 5 Huang, S.-K., Nobiling, R., Schachner, M. and Taugner, R., Interstitial and parenchymal cells in the pineal gland of the golden hamster. A combined thin-section, freeze-fracture and immunofluorescence study, Cell Tiss. Res., 235 (1984) 327-337. 6 Kondo, H., lwanaga, T. and Nakajima, T., lmmunocytochemical study on the localization of neuronspecific enolase and S-100 protein in the carotid body of rats, Cell Tiss. Res., 227 (1982) 291 295. 7 L6wenthal, A., Flament-Durand, J., Karcher, D., Noppe, M. and Brion, J.P., Glial cells identified by anti-~-albumin (anti-GFA) in human pineal gland, J. Neurochem., 38 (1982) 863 -865. 8 Moiler, M., Ingild, A. and Bock, E., Immunohistochemical demonstration of S-100 protein and GFA protein in interstitial ceils of rat pineal gland, Brain Res., 140 (1978) 1-13. 9 Moore. B.W., A soluble protein characteristic to the nervous system, Biochem. Biophys. Res. Commun., 19 (1965) 739 744. 10 Nakajima, T., Kameya, T., Watanabe, S., Hirota, T. and Shimosato, Y., S-100 protein distribution in normal and neoplastic tissues. In R.A. DeLellis (Ed.), Advances in Immunohistochemistry, Masson. New York, 1984, pp. 141 158. 11 Nakajima, T., Yamaguchi, H. and Takahashi, K., S-100 protein in folliculo-stellate cells of the rat pituitary anterior lobe, Brain Res., 191 (1980) 523-531. 12 Papasozomenos, S.C., Glial fibrillary acidic (GFA) protein-containing cells in the human pineal gland, J. Neuropath. Exp. Neurol., 42 (1983) 391-408. 13 P~vet, P., Ultrastructure of the mammalian pinealocytes, In R.J. Reiter (Ed.), The Pineal Gland, Elsevier, New York, 1983, pp. 1-75. 14 Schachner, M., Huang, S.-K., Ziegelmfiller, P., Bizzini, B. and Taugner, R., Glial cells in the pineal gland of mice and rats. A combined immunofluorescence and electron-microscopic study, Cell Tiss. Res., 237 (1984) 245 252. 15 Shirasawa, N., Kihara, H., Yamaguchi, S. and Yoshimura, F., Pituitary folliculo-stellate cells immunostained with S-100 protein antiserum in postnatal, castrated and thyroidectomized rats, Cell Tiss. Res., 231 (1983) 235-249. 16 Shirasawa, N., Yamaguchi, S. and Yoshimura, F., Granulated folliculo-stellate cells and growth hormone cells immunostained with anti-S 100 protein serum in the pituitary glands of the goat, Cell Tiss. Res., 237 (1984) 7 14. 17 Takahashi. K., Isobe, T., Ohtsuki, Y., Akagi, T., Sonobe, H. and Okuyama, T., Immunohistochemical study on the distribution of ~ and/~ subunits of S-100 protein in human neoplasm and normal tissues, Virchows Arch. (Cell Pathol.), 45 (1984) 385-296. 18 Zomzely-Neurath, C.E. and Walker, W.A., Nervous system-specific proteins: 14-3-2 protein, neuronspecific enolase, and S-100 protein, in R.A. Bradshaw and D.M. Schneider (Eds.), Proteins of the Nervous System, Raven Press, New York, 1980, pp. 1 57.