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Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain
ELSEVIER
Neuroscience Letters 213 (1996) 193-196
N[UROSCI[NC[ IETT[RS
Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain M a s a h i r o N i s h i b o r i a, N a o k i N a k a y a a, A k i h i t o T a h a r a a, M a s a h i r o K a w a b a t a a, S h u j i M o r i b, K i y o m i S a e k i a'* aDepartment qf Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho,Okayama 700, Japan bSchool of Health Sciences, Okayama University, 2-5-1 Shikata-cho,Okayama 700, Japan
Received 16 May 1996; revised version received 27 June 1996; accepted 27 June 1996
Abstract
We investigated the immunohistochemical localization of a cytokine macrophage migration inhibitory factor (MIF) in the bovine brain. MIF was present in the ependymal cell linings of the cerebral ventricles throughout. Double immunostaining of the section with anti-glial fibrillary acidic protein (GFAP) antibody and with anti-MIF antibody showed that the astrocytes present in subependymal layer were immunoreactive, for MIF. In the hippocampus, the pyramidal cells in the CA3 and CA4 subfields and the granule cells of the dentate gyms were immunoreactive. The bundles of mossy fibers were stained along their projections to CA3 and CA4 regions. The nuclei of the subpopulation of these MIF-immunoreactive cells were also immunostained. These results indicated the widespread distribution of a cytokine, MIF, in the bovine brain and suggested the possibility that MIF might play additional roles than a proinflammatory mediator role in the brain. Keywords: Macrophage migration inhibitory factor; Ependyma; Astrocytes; Neurons; Bovine brain
Macrophage migration inhibitory factor (MIF), an initiator of the inflammatory response, was first characterized as a lymphokine preduced by activated T-lymphocyte [3,7]. The functional expression cloning from activated human T-lymphocytes identified cDNA encoding a protein with MIF activity [14]. Bucala et al. demonstrated that the monocyte/macrophage [6] and the pituitary [1] were another source of MIF. Upon stimulation with bacterial lipopolysaccharide (LPS), tumor necrosis factor-or (TNFc0 and interferon-% MIF was released from the lineage of monocytes/macrophages, and MIF in turn stimulated the release of TNF-a from l:hese cells through the autocrine and paracrine mechanisms [6]. Thus, MIF seems to be one of the critical cytokines in the host defense mechanism. MIF has also been suggested to be an anterior pituitary hormone which aggravated endotoxemia in mice [ 1,5,11 ]. Northern blot analysis showed that MIF was constitutively expressed in various tissues beyond the immune system [8,12,15], suggesting a widespread role of this cytokine. * Corresponding author. Tel.: +81 86 2357137; fax: +81 86 2233569.
Among the tissues, the high expression o f M I F mRNA was observed in the brain [8,12,15]. Recently, we purified MIF from bovine brain by using an affinity column with a synthetic peptide corresponding to the C-terminal region of a serpin (B-43) as a ligand [9,10]. In the present study, we investigated the presence of MIF-like immunoreactivity (MIF-LI) in the bovine brain to better understand the functional role of MIF in the brain. Bovine brain MIF was purified as described elsewhere [10]. High pressure liquid chromatography (HPLC)-purifled MIF (100/~g) in phosphate-buffered saline (PBS) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into the back of a rabbit. A booster injection was given 1 month later with Freund's incomplete adjuvant and additional injections were administered twice every 2 weeks. The rabbit serum was obtained 10 days after the final injection. Anti-MIF antiserum (1:2000) recognized a single band of about 10 kDa on Western blotting using the supernatant of the bovine brain homogenate (Fig. 1). Bovine brain
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12864-0
194
M. Nishibori et al. / Neuroscience Letters 213 (1996) 193-196
kDa 123 85 50 33 29 19 7.8
A
B
Fig. 1. Western blotting of MIF using anti-MIF antiserum. The supernatant of bovine brain homogenatewas electrnphoresed on a 13% polyacrylamide gel in the presence of sodium dodecyl sulfate. After electrophoresis, the proteins were transferred onto nitrocellulose membrane and blotted with anti-MIF antiserum (lane A, 1:2000) or preimmune serum (lane B, 1:2000). tissue pieces were excised and fixed in 10% formalin in 0.01 M PBS immediately after the removal of the brain from the skull. The fixed tissue pieces were dehydrated in an ethanol series, embedded in paraffin and cut into sections of 5 - 7 / z m thick. After blocking with 5% normal goat serum and 2% BSA, rabbit anti-MIF antiserum (diluted to 1:2000) or preimmune rabbit serum (1:2000) was applied to the sections overnight at 4°C. The sections were rinsed three times with PBS, incubated with biotinylated antirabbit IgG goat serum (Vector), washed and reacted with streptavidin-peroxidase (Sigma). Diaminobenzidine and hematoxylin were the substrates for the enzyme reaction and counterstain, respectively. Preimmune rabbit serum did not stain any structure in the tissue pieces examined. For double immunostaining, the section was incubated with anti-MIF rabbit antibody and anti-rat glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (5/~g/ ml, Boehringer) simultaneously. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG (Cappel, diluted to 1:60) and rhodamine-conjugated goat antimouse IgG (Cappel, diluted to 1:50), respectively. As shown in Fig. 2, MIF-LI were present in ependymal cell linings of the lateral ventricle, the third ventricle, the aqueduct of the midbrain and the fourth ventricle throughout. These immunoreactive cells were typical cuboid ciliated ependyma. The intensity of the staining of the cytoplasm was varied depending on the cells, especially in the aqueduct of the midbrain (Fig. 2C). The microvilli on the apical surface of the ependymal cells appeared not to be immunoreactive for MIF (Fig. 2A,B,D) while the protrusion-like structures were seen in the ependyma of the aqueduct of the midbrain (Fig. 2C). In addition to cytoplasm, the nuclei of the subpopulation of these cells
were also immunostained (arrowheads in Fig. 2B-D). The staining of the nuclei was often very marked compared to that of cytoplasm. These results indicated the cytoplasmic and nuclear localization of MIF-LI in the ependymal cells. In the hippocampus, the ependymal cell lining of the lateral ventricle was also immunoreactive for MIF (Fig. 3A). Numerous MIF-LI cells were observed in the alveus and the stratum oriens of the hippocampus. Double immunostaining with anti-MIF and anti-GFAP antibodies revealed that the cells with MIF-LI in this region were GFAP-positive astrocytes (Fig. 3A). We also identified MIF-LI astrocytes in subependymal regions of the lateral ventricle and the fourth ventricle by double immunostaining (data not shown). The pyramidal neurons in CA3 and CA4 but not in CA1 and CA2 were immunoreactive for MIF although the intensity of the staining was much weaker than those in ependymal cells and astrocytes (Fig. 3B). In addition to pyramidal cells, most of the granule cells in the dentate gyrus were immunoreactive (Fig. 3C). The molecular layer of dentate gyrus was uniformly stained. The bundles of mossy fibers were immunostained, enabling the pursuit of the projection of the fibers to pyramidal cells of CA3 and CA4 subfields (Fig. 3B). As in the case of ependymal cells, the subpopulation of the nuclei of astrocytes (the inset in Fig. 3A) and neurons (Fig. 3C) were stained to varied extent. In the present study, it was demonstrated that the ependymal cell linings of the cerebral ventricles had the strongest MIF-LI. The functional role of ependymal cells has been discussed [4,13]. Bleier et al. [2] proposed that ependymal cells constituted the resident phagocytotic system that provided the first line of defense against central nervous system invasion by various pathogens. Calandra et al. [6] reported that monocyte/macrophage was one of the major source of MIF in plasma. Thus, characteristic distribution of MIF-LI in the ependymal linings may suggest the existence of defense mechanism against the invasion of pathogens through cerebrospinal fluid. With this respect, it is important to determine whether MIF is released into surrounding structures including cerebrospinal fluid. The interesting finding was that MIF-LI was present in the nucleus as well as cytoplasm of three different types of cells and the intensity of the staining of the nuclei varied markedly depending on the cells. Therefore, it is possible that MIF not only exerts a cytokine function extracellularly but also plays a role in the nucleus as a signaling molecule. The fact that MIF cDNA was identified as the delayed early gene in response to growth factors [8] may support the idea. Recently, it was reported that MIF-LI was localized to secretory granules of the anterior pituitary cells in mice [13]. Bernhagen et al. [1] demonstrated the hormonal action of MIF released from the pituitary in response to the challenge of bacterial lipopolysaccharide in mice. The presence of MIF-LI in the specific neurons demonstrated in the present study implied that MIF may play additional
M. Nishibori et al. / Neuroscience Letters 213 (1996) 193-196
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Fig. 2. Photomicrographs of the sections immunostained with anti-MIF antiserum. The immunoreactive ependymal cell linings were shown. The lateral ventricle (LV) below the co~us callosum (CC) (A); the third ventricle (3V) at the periventricular area of the hypothalamus (B); the aqueduct of the midbrain (Aq) (C); and the fourth ventricle (4V) of the pons (D). Arrowheads in (B-D) indicate the strong MIF-LI nuclei of the ependymal cells. Scale bar, 20 #m.
Fig. 3. Photomicrographs of the hippocampal sections immunostained with anti-MIF antiserum. (A) Fluorescentmicrograph of the section immunostained with anti-MIF antibody and anti-GFAP antibody simultaneously. The section was incubated with anti-MIF rabbit antibody and anti-rat GFAP mouse monoclonal antibody. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti2mouse IgG, respectively. The MIF- and GFAP-immunofluorescence from the section was superimposed on the same film. Note that GFAP-positive astrocytes are all MIFpositive and that ependymal ceils (arrows) were immunoreactive for MIF but not for GFAP. Arrowhead in the inset indicates the M1F-LI nuclei of the astrocyte. (B) The pyramidal cells (as indicated by arrowheads) in CA3 subfield were shown. The bundles of mossy fibers (mf) were immunostained (arrows), enabling the pursuit of the projection of the fibers to pyramidal cells. (C) The granule cells in the dentate gyrus (DG) were shown. Arrowhead indicates the MIF-LI nuclei of the granule cell. Scale bar, 20/zm.
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M. Nishibori et al. /Neuroscience Letters 213 (1996) 193-I96
roles in the brain than a p r o i n f l a m m a t o r y mediator role p e r f o r m e d in the peripheral tissues and a hormonal action after the release f r o m the pituitary. W e gratefully a c k n o w l e d g e Shizuko Y o k o y a m a for her e x c e l l e n t technical assistance, This w o r k was partly supported by G r a n t - i n - A i d No. 0 6 6 7 0 1 1 4 f r o m the Japanese Ministry o f Education, Science, Sports and Culture, Japan and a Grant f r o m R y o b i t e i e n M e m o r i a l Foundation. [1] Bernhagen, J., Calandra, T., Mitchell, R.A., Martin, S.B., Tracey, K.J., Voelter, W., Manogue, K.R., Cerami, A. and Bucala, R., MIF is a pituitary-derived cytokine that potentiates lethal endotoxemia, Nature, 365 (1993) 756-759. [2] Bleier, R., Albrecht, R. and Crace, J.A.F., Supraependymal cells of the hypothalamic third ventricle: identification as resident phagocytes of the brain, Science, 189 (1975) 299-301. [3] Bloom, B.R. and Bennet, B., Mechanism of a reaction in vitro associated with delayed-type hypersensitivity, Science, 153 (1966) 80-82. [4] Bruni, J.E., Del Bigio, M.R. and Clattenburg, R.E., Ependyma: normal and pathological. A review of the literature, Brain Res. Rev., 9 (1985) 1-19. [5] Bucala, R., Identification of MIF as a new pituitary hormone and macrophage cytokine and its role in endotoxic shock, Immunol. Lett., 43 (1994) 23-26. [6] Calandra, T., Bernhagen, J., Mitchell, R.A. and Bucala, R., The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor, J. Exp. Med., 179 (1994) 1895-1902.
[7] David, J.R., Delayed hypersensitivity in vitro: its mediation by cell free substances formed by lymphoid cell-antigen interaction, Proc. Natl. Acad. Sci. USA, 56 (1966) 72-77. [8] Lanahan, A., Williams, J.B., Sanders, L.K. and Nathans, D, Growth factor-induced delayed early response genes, Mol. Cell. Biol., 12 (1992) 3919-3929. [9] Nishibori, M., Chikai, T., Kawabata, M., Ohm, J., Ubuka, T. and Saeki, K., Purification of a novel serpin-like protein from bovine brain, Neurosci. Res., 24 (1995) 47-52. [10] Nishibori, M., Nakaya, N., Mori, S., Kawabata, M., Tahara, A. and Saeki, K., Affinity purification of M1F/GIF from bovine brain by using a peptide ligand derived from a novel serpin, Jpn. J. Pharmacol., (1996) in press. [11] Nishino, T., Bernhagen, J., Shiiki, H., Caiandra, T., Dohi, K. and Bucala, R., Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland, Mol. Med., 1 (1995) 781-788. [12] Paralkar, V. and Wistow, G., Cloning the human gene for macrophage migration inhibitory factor (MIF), Genomics, 19 (1994) 4851. [13] Reichenbach, A. and Robinson, S.R., Ependymoglia and ependymoglia-like cells. In H. Kettenmann and B.R. Ransom (Eds.), Neuroglia, Oxford University Press, Oxford, 1995, pp. 58-84. [14] Weiser, W.Y., Temple, P.A., Witek-Giannoti, J.S., Remold, H.G., Clark, S.C. and David, J.R., Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor, Proc. Natl. Acad. Sci. USA, 86 (1989) 7522-7526. [15] Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J. and Zelenka, P.S., A migration inhibitory factor is expressed in the differentiating cells of the eye lens, Proc. Natl. Acad. Sci. USA, 90 (1993) 1272-1275.
Neuroscience Letters 213 (1996) 193-196
N[UROSCI[NC[ IETT[RS
Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain M a s a h i r o N i s h i b o r i a, N a o k i N a k a y a a, A k i h i t o T a h a r a a, M a s a h i r o K a w a b a t a a, S h u j i M o r i b, K i y o m i S a e k i a'* aDepartment qf Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho,Okayama 700, Japan bSchool of Health Sciences, Okayama University, 2-5-1 Shikata-cho,Okayama 700, Japan
Received 16 May 1996; revised version received 27 June 1996; accepted 27 June 1996
Abstract
We investigated the immunohistochemical localization of a cytokine macrophage migration inhibitory factor (MIF) in the bovine brain. MIF was present in the ependymal cell linings of the cerebral ventricles throughout. Double immunostaining of the section with anti-glial fibrillary acidic protein (GFAP) antibody and with anti-MIF antibody showed that the astrocytes present in subependymal layer were immunoreactive, for MIF. In the hippocampus, the pyramidal cells in the CA3 and CA4 subfields and the granule cells of the dentate gyms were immunoreactive. The bundles of mossy fibers were stained along their projections to CA3 and CA4 regions. The nuclei of the subpopulation of these MIF-immunoreactive cells were also immunostained. These results indicated the widespread distribution of a cytokine, MIF, in the bovine brain and suggested the possibility that MIF might play additional roles than a proinflammatory mediator role in the brain. Keywords: Macrophage migration inhibitory factor; Ependyma; Astrocytes; Neurons; Bovine brain
Macrophage migration inhibitory factor (MIF), an initiator of the inflammatory response, was first characterized as a lymphokine preduced by activated T-lymphocyte [3,7]. The functional expression cloning from activated human T-lymphocytes identified cDNA encoding a protein with MIF activity [14]. Bucala et al. demonstrated that the monocyte/macrophage [6] and the pituitary [1] were another source of MIF. Upon stimulation with bacterial lipopolysaccharide (LPS), tumor necrosis factor-or (TNFc0 and interferon-% MIF was released from the lineage of monocytes/macrophages, and MIF in turn stimulated the release of TNF-a from l:hese cells through the autocrine and paracrine mechanisms [6]. Thus, MIF seems to be one of the critical cytokines in the host defense mechanism. MIF has also been suggested to be an anterior pituitary hormone which aggravated endotoxemia in mice [ 1,5,11 ]. Northern blot analysis showed that MIF was constitutively expressed in various tissues beyond the immune system [8,12,15], suggesting a widespread role of this cytokine. * Corresponding author. Tel.: +81 86 2357137; fax: +81 86 2233569.
Among the tissues, the high expression o f M I F mRNA was observed in the brain [8,12,15]. Recently, we purified MIF from bovine brain by using an affinity column with a synthetic peptide corresponding to the C-terminal region of a serpin (B-43) as a ligand [9,10]. In the present study, we investigated the presence of MIF-like immunoreactivity (MIF-LI) in the bovine brain to better understand the functional role of MIF in the brain. Bovine brain MIF was purified as described elsewhere [10]. High pressure liquid chromatography (HPLC)-purifled MIF (100/~g) in phosphate-buffered saline (PBS) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into the back of a rabbit. A booster injection was given 1 month later with Freund's incomplete adjuvant and additional injections were administered twice every 2 weeks. The rabbit serum was obtained 10 days after the final injection. Anti-MIF antiserum (1:2000) recognized a single band of about 10 kDa on Western blotting using the supernatant of the bovine brain homogenate (Fig. 1). Bovine brain
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12864-0
194
M. Nishibori et al. / Neuroscience Letters 213 (1996) 193-196
kDa 123 85 50 33 29 19 7.8
A
B
Fig. 1. Western blotting of MIF using anti-MIF antiserum. The supernatant of bovine brain homogenatewas electrnphoresed on a 13% polyacrylamide gel in the presence of sodium dodecyl sulfate. After electrophoresis, the proteins were transferred onto nitrocellulose membrane and blotted with anti-MIF antiserum (lane A, 1:2000) or preimmune serum (lane B, 1:2000). tissue pieces were excised and fixed in 10% formalin in 0.01 M PBS immediately after the removal of the brain from the skull. The fixed tissue pieces were dehydrated in an ethanol series, embedded in paraffin and cut into sections of 5 - 7 / z m thick. After blocking with 5% normal goat serum and 2% BSA, rabbit anti-MIF antiserum (diluted to 1:2000) or preimmune rabbit serum (1:2000) was applied to the sections overnight at 4°C. The sections were rinsed three times with PBS, incubated with biotinylated antirabbit IgG goat serum (Vector), washed and reacted with streptavidin-peroxidase (Sigma). Diaminobenzidine and hematoxylin were the substrates for the enzyme reaction and counterstain, respectively. Preimmune rabbit serum did not stain any structure in the tissue pieces examined. For double immunostaining, the section was incubated with anti-MIF rabbit antibody and anti-rat glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (5/~g/ ml, Boehringer) simultaneously. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG (Cappel, diluted to 1:60) and rhodamine-conjugated goat antimouse IgG (Cappel, diluted to 1:50), respectively. As shown in Fig. 2, MIF-LI were present in ependymal cell linings of the lateral ventricle, the third ventricle, the aqueduct of the midbrain and the fourth ventricle throughout. These immunoreactive cells were typical cuboid ciliated ependyma. The intensity of the staining of the cytoplasm was varied depending on the cells, especially in the aqueduct of the midbrain (Fig. 2C). The microvilli on the apical surface of the ependymal cells appeared not to be immunoreactive for MIF (Fig. 2A,B,D) while the protrusion-like structures were seen in the ependyma of the aqueduct of the midbrain (Fig. 2C). In addition to cytoplasm, the nuclei of the subpopulation of these cells
were also immunostained (arrowheads in Fig. 2B-D). The staining of the nuclei was often very marked compared to that of cytoplasm. These results indicated the cytoplasmic and nuclear localization of MIF-LI in the ependymal cells. In the hippocampus, the ependymal cell lining of the lateral ventricle was also immunoreactive for MIF (Fig. 3A). Numerous MIF-LI cells were observed in the alveus and the stratum oriens of the hippocampus. Double immunostaining with anti-MIF and anti-GFAP antibodies revealed that the cells with MIF-LI in this region were GFAP-positive astrocytes (Fig. 3A). We also identified MIF-LI astrocytes in subependymal regions of the lateral ventricle and the fourth ventricle by double immunostaining (data not shown). The pyramidal neurons in CA3 and CA4 but not in CA1 and CA2 were immunoreactive for MIF although the intensity of the staining was much weaker than those in ependymal cells and astrocytes (Fig. 3B). In addition to pyramidal cells, most of the granule cells in the dentate gyrus were immunoreactive (Fig. 3C). The molecular layer of dentate gyrus was uniformly stained. The bundles of mossy fibers were immunostained, enabling the pursuit of the projection of the fibers to pyramidal cells of CA3 and CA4 subfields (Fig. 3B). As in the case of ependymal cells, the subpopulation of the nuclei of astrocytes (the inset in Fig. 3A) and neurons (Fig. 3C) were stained to varied extent. In the present study, it was demonstrated that the ependymal cell linings of the cerebral ventricles had the strongest MIF-LI. The functional role of ependymal cells has been discussed [4,13]. Bleier et al. [2] proposed that ependymal cells constituted the resident phagocytotic system that provided the first line of defense against central nervous system invasion by various pathogens. Calandra et al. [6] reported that monocyte/macrophage was one of the major source of MIF in plasma. Thus, characteristic distribution of MIF-LI in the ependymal linings may suggest the existence of defense mechanism against the invasion of pathogens through cerebrospinal fluid. With this respect, it is important to determine whether MIF is released into surrounding structures including cerebrospinal fluid. The interesting finding was that MIF-LI was present in the nucleus as well as cytoplasm of three different types of cells and the intensity of the staining of the nuclei varied markedly depending on the cells. Therefore, it is possible that MIF not only exerts a cytokine function extracellularly but also plays a role in the nucleus as a signaling molecule. The fact that MIF cDNA was identified as the delayed early gene in response to growth factors [8] may support the idea. Recently, it was reported that MIF-LI was localized to secretory granules of the anterior pituitary cells in mice [13]. Bernhagen et al. [1] demonstrated the hormonal action of MIF released from the pituitary in response to the challenge of bacterial lipopolysaccharide in mice. The presence of MIF-LI in the specific neurons demonstrated in the present study implied that MIF may play additional
M. Nishibori et al. / Neuroscience Letters 213 (1996) 193-196
195
Fig. 2. Photomicrographs of the sections immunostained with anti-MIF antiserum. The immunoreactive ependymal cell linings were shown. The lateral ventricle (LV) below the co~us callosum (CC) (A); the third ventricle (3V) at the periventricular area of the hypothalamus (B); the aqueduct of the midbrain (Aq) (C); and the fourth ventricle (4V) of the pons (D). Arrowheads in (B-D) indicate the strong MIF-LI nuclei of the ependymal cells. Scale bar, 20 #m.
Fig. 3. Photomicrographs of the hippocampal sections immunostained with anti-MIF antiserum. (A) Fluorescentmicrograph of the section immunostained with anti-MIF antibody and anti-GFAP antibody simultaneously. The section was incubated with anti-MIF rabbit antibody and anti-rat GFAP mouse monoclonal antibody. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti2mouse IgG, respectively. The MIF- and GFAP-immunofluorescence from the section was superimposed on the same film. Note that GFAP-positive astrocytes are all MIFpositive and that ependymal ceils (arrows) were immunoreactive for MIF but not for GFAP. Arrowhead in the inset indicates the M1F-LI nuclei of the astrocyte. (B) The pyramidal cells (as indicated by arrowheads) in CA3 subfield were shown. The bundles of mossy fibers (mf) were immunostained (arrows), enabling the pursuit of the projection of the fibers to pyramidal cells. (C) The granule cells in the dentate gyrus (DG) were shown. Arrowhead indicates the MIF-LI nuclei of the granule cell. Scale bar, 20/zm.
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M. Nishibori et al. /Neuroscience Letters 213 (1996) 193-I96
roles in the brain than a p r o i n f l a m m a t o r y mediator role p e r f o r m e d in the peripheral tissues and a hormonal action after the release f r o m the pituitary. W e gratefully a c k n o w l e d g e Shizuko Y o k o y a m a for her e x c e l l e n t technical assistance, This w o r k was partly supported by G r a n t - i n - A i d No. 0 6 6 7 0 1 1 4 f r o m the Japanese Ministry o f Education, Science, Sports and Culture, Japan and a Grant f r o m R y o b i t e i e n M e m o r i a l Foundation. [1] Bernhagen, J., Calandra, T., Mitchell, R.A., Martin, S.B., Tracey, K.J., Voelter, W., Manogue, K.R., Cerami, A. and Bucala, R., MIF is a pituitary-derived cytokine that potentiates lethal endotoxemia, Nature, 365 (1993) 756-759. [2] Bleier, R., Albrecht, R. and Crace, J.A.F., Supraependymal cells of the hypothalamic third ventricle: identification as resident phagocytes of the brain, Science, 189 (1975) 299-301. [3] Bloom, B.R. and Bennet, B., Mechanism of a reaction in vitro associated with delayed-type hypersensitivity, Science, 153 (1966) 80-82. [4] Bruni, J.E., Del Bigio, M.R. and Clattenburg, R.E., Ependyma: normal and pathological. A review of the literature, Brain Res. Rev., 9 (1985) 1-19. [5] Bucala, R., Identification of MIF as a new pituitary hormone and macrophage cytokine and its role in endotoxic shock, Immunol. Lett., 43 (1994) 23-26. [6] Calandra, T., Bernhagen, J., Mitchell, R.A. and Bucala, R., The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor, J. Exp. Med., 179 (1994) 1895-1902.
[7] David, J.R., Delayed hypersensitivity in vitro: its mediation by cell free substances formed by lymphoid cell-antigen interaction, Proc. Natl. Acad. Sci. USA, 56 (1966) 72-77. [8] Lanahan, A., Williams, J.B., Sanders, L.K. and Nathans, D, Growth factor-induced delayed early response genes, Mol. Cell. Biol., 12 (1992) 3919-3929. [9] Nishibori, M., Chikai, T., Kawabata, M., Ohm, J., Ubuka, T. and Saeki, K., Purification of a novel serpin-like protein from bovine brain, Neurosci. Res., 24 (1995) 47-52. [10] Nishibori, M., Nakaya, N., Mori, S., Kawabata, M., Tahara, A. and Saeki, K., Affinity purification of M1F/GIF from bovine brain by using a peptide ligand derived from a novel serpin, Jpn. J. Pharmacol., (1996) in press. [11] Nishino, T., Bernhagen, J., Shiiki, H., Caiandra, T., Dohi, K. and Bucala, R., Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland, Mol. Med., 1 (1995) 781-788. [12] Paralkar, V. and Wistow, G., Cloning the human gene for macrophage migration inhibitory factor (MIF), Genomics, 19 (1994) 4851. [13] Reichenbach, A. and Robinson, S.R., Ependymoglia and ependymoglia-like cells. In H. Kettenmann and B.R. Ransom (Eds.), Neuroglia, Oxford University Press, Oxford, 1995, pp. 58-84. [14] Weiser, W.Y., Temple, P.A., Witek-Giannoti, J.S., Remold, H.G., Clark, S.C. and David, J.R., Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor, Proc. Natl. Acad. Sci. USA, 86 (1989) 7522-7526. [15] Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J. and Zelenka, P.S., A migration inhibitory factor is expressed in the differentiating cells of the eye lens, Proc. Natl. Acad. Sci. USA, 90 (1993) 1272-1275.