Journal of the Neurological Sciences, 1990, 98:81-90
81
Elsevier JNS 03356
Transferrin receptors in rat central nervous system An immunocytochemical study Bruno Giometto, Felice Bozza, Vincenza Argentiero, Paolo Gallo, Silvana Pagni, M a r i a G r a z i a Piccinno and Bruno Tavolato Second Neurological Clinic, University of Padova, School of Medicine, 1-35137 Padova (Italy)
(Received 19 September, 1989) (Revised, received 19 March, 1990) (Accepted 19 March, 1990)
SUMMARY Using an immunocytochemical method we demonstrated the presence of TfR on adult rat neurons, particularly in the cerebral cortex and brain stem. The monoclonal antibody (mab) against rat TfR (clone OX 26) stained neurons of all cortical layers and in the brain stem where the reaction was most evident. Purkinje cells in the cerebellum and scattered neurons in the gray matter of the cervical spinal cord were weakly stained. Choroid plexus cells also reacted with the mab against TfR whereas oligodendrocytes in the cerebral white matter were faintly outlined by the mab. The presence of TfR on endothelial cells of brain capillaries was here confirmed.
Key words: Transferrin receptors; Immunocytochemistry; Central nervous system; Neuronal plasticity
INTRODUCTION Cell iron uptake is mediated by preliminary binding of transferrin (Tf), the iron-carrying protein, to a specific receptor (TfR) on the cell surface. The Tf/TfR complex is then internalized by endocytotic vesicles for the intracellular "transferrin Correspondence to: Prof. B. Tavolato,II NeurologicalClinic,Universityof Padova, Via Vendramini,
7, 1-35137 Padova, Italy. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
82 cycle", and the iron is finally released into the cytoplasm (Dautry-Varsat et al. 1983). TfR expression is restricted in normal human tissues: in fact it does not occur in resting cells, but has been demonstrated in rapidly proliferating cells such as the basal layer of squamous epithelium, and in a variety of different types of human malignancies (Gatter et al. 1983). Many studies suggest that Tf and iron play an important role in the central nervous system (CNS). Tf is synthesized by the brain (Levin et al. 1984), and is more concentrated in cerebrospinal fluid (CSF) than predicted by its molecular weight and hydrodynamic radius (Felgenhauer 1974; Gallo et al. 1985); it is required for the growth of some CNS cells (Saito et al. 1982). Neurons show a significant iron uptake in culture (Swaiman and Machen 1984). Nevertheless, only a few reports address the presence of TfR on CNS cells. Using immunocytochemical methods, TfR were demonstrated on brain capillaries (Jefferies et al. 1984) and on developing (not in adult) neurons (Oh et al. 1986); only autoradiographic methods have detected their presence on postnatal neurons (Hill et al. 1985) and oligodendrocytes in culture (Espinosa de los Monteros and Foucaud 1987). A morphological demonstration, however, of TfR on postnatal CNS cells is still
Fig. 1. Rat liversectionstainedby the mab OX26.The membraneofhepatocytes,and Kupffercells(arrow) are clearly outlinedby the mab. The section was counterstainedwith hematoxylin( x 400).
83 lacking. We employed an immunocytochemicalmethod with avidin-biotin amplification in an attempt to characterize the TfR presenting cells in the adult rat CNS.
MATERIALSAND METHODS Twelve adult Sprague-Dawley rats, 1-4 months old, anaesthetized with pentobarbitone were perfused through the ascending aorta with 100 ml cold saline followed by 500 ml 4 ~ paraformaldehyde (4 ° C) in 0.1 M phosphate buffer, pH 7.4. The cerebral and cerebellar hemispheres, brain stem and cervical spinal cord were dissected out and stored in cold fixative for 4 h; the tissues were then kept overnight in 10~o sucrose. Ten #m thick frozen sections of cerebral (motor area) and cerebellar cortex, pons and cervical spinal cord were cut, placed on poly-L-lysine coated slides and subsequently treated with 0.1~o H202 for 10 min, followed by 0.5~ trypsin for 10 min at 37 °C. The slides were then incubated overnight at 4 °C with an IgG2 mab against rat TfR (clone OX26, mab OX26, Seralab, U.K.) diluted 1 : 100 in phosphate-buffered saline (PBS), pH 7.4. The sections were washed 3 times in PB S, and then covered with an intermediate
Fig. 2. Controlsectionin whichthe first antibodywas omitted ( x 200).
84 layer of biotinylated rabbit anti-mouse Ig (Dakopatts, Denmark) diluted 1 : 200 in PBS for 1 h at room temperature. After further washing, a layer of horseradish-peroxidase conjugated avidin (Dakopatts, Denmark) diluted 1 : 500 was added for 30 min at room temperature. The substrate reaction was developed with 0.0570 diaminobenzidine tetrahydrochloride (Carlo Erba, Italy) and 0.01~o H202 in PBS. Finally, the slides were washed and mounted in glycerol gelatin (Merck, Darmstadt, F.R.G.). Rat liver was used as positive control tissue; 10-/~m frozen liver sections were processed and the reaction was developed as above described. These sections were counterstained with Mayer's hematoxylin. When negative control sections were processed the first antibody was omitted or substituted by other mabs of the same immunoglobulin class as OX26.
Fig. 3. Brain capillary stained by the mab against TfR in the cerebral white matter (arrow). Oligodendrocytes are also weakly recognized by the mat); groups of 4-5 cells are prevalent and a cluster of calls is present at the perivascular level (arrowhead). In these cells only the membrane is outlined by the reaction ( x 200).
85 RESULTS Rat Kupffer cells and hepatocytes were clearly outlined on the cell membrane by the mab OX26 (Fig. 1). No immunoreactivity was detected in the control sections where the first mab was omitted or substituted by other unrelated antibodies (Fig. 2). In the rat brain tissue, TfR immunoreactivity was detected on the endothelium of brain capillaries (Fig. 3). Choroid plexus cells were also positive with mab OX26 (Fig. 4). Oligodendrocytes were weakly stained in the cerebral white matter where they were present in pairs or cluster of 4 or 5 cells, and in the perivascular space (Fig. 3); the reaction was confined to the cell membrane and was quite weak. Oligodendrocytes were not recognizable in other brain regions. Nerve cells were clearly stained by the mab against TfR; however, a regional distribution of the pattern of staining was evidenced. Cerebral cortex
The neurons of all the cerebral layers of the motor cortex were stained by the mab OX26 (Fig. 5), especially those of layer V. Labelling was mainly confined to the cell
Fig. 4. Choroidplexus cells are positivewith the mab against TfR ( x 200).
86 membrane, but, in some cortical neurons, the whole cytoplasm was stained. In addition the soma, the proximal portion of the apical dendrites was also outlined by the antibody in the cortical neurons.
Brain stem and cerebellum The large pontine neurons showed intense immunoreactivity (Fig. 6), which was extended to the entire cytoplasm (Fig. 7). In the cerebellum, the Purkinje cells were faintly stained (data not shown); no TfR immunoreactivity was present in the cells of the granular and molecular layers. Spinal cord Scattered neurons in the cervical gray matter were faintly stained (data not shown), but not in relation to any particular laminar distribution. No specific reactivity was found in the cervical white matter.
Fig. 5. Cortical neurons stained by the mab OX26. Neurons of all cortical layers are recognized and the reactivityis prevalent around the cell membrane.The proximalportion of the apical dendrite is also positive ( × 200).
87 DISCUSSION In the present study we investigated the localization of TfR in rat brain using the mab OX26 (IgG2a) reported specific for TfR since it recognized a detergent-solubilized molecule that can bind 125I-labelled transferrin and precipitates a dimer ofglycoproteins with an apparent molecular weight of 95 000 (Jefferies et al. 1985). Sections of the rat liver showed, with the mab OX26, a staining confined to the sites where the presence of TfR is expected (Gatter et al, 1983; Jefferies et al. 1985) confirming the specificity of the mab. In this study we found that choroid plexus cells stained positively with the mab OX26; we also confirmed the report by Jefferies et al. (1984) that TfR were present on the endothelium of brain capillaries. Moreover, our study shows that in adult rat neurons TfR are largely represented. These however are variable in amount: they are numerous in cortical and brain stem neurons while Purkinje cells and spinal cord neurons were only faintly outlined by the mab OX26. Concerning these findings, our results are at variance with those obtained by Jefferies et al. (1984): these workers using the same mab observed weak reactivity in scattered neuronal cells. We believe that methodological differences could explain these discrepant data. In our investigation neuron reactivity was enhanced by treatment with trypsin, which showed TfR immunoreactivity mainly in the cytoplasm of neurons. This pattern of staining is not surprising, considering the intracellular cycle of Tf/TfR complex. Other authors have reported the presence of TfR particularly in the cytoplasm of CNS cells (Espinosa de los Monteros and Foucaud 1987). TfR present on the cell surface may be partially destroyed or masked by fixative or by postmortem modifications. On the other hand, the TfR epitope recognized by mab OX26 might become accessible to the antibody only after an increase in tissue and cell permeability induced by enzymatic digestion. Moreover, we exclude an aspecific staining induced by trypsin treatment since other mabs of the same immunoglobulin class, used as controls, failed to stain neurons. We are not able to explain the different pattern and degree of reactivity between cortex and brain stem neurons, and spinal cord neurons and Purkinje cells. Metabolic specificities might account for these findings. However, a regional brain distribution of TfR has been reported also by autoradiographic studies (Hill et al. 1985; Morris et al. 1989) that confirm a high level of TfR in cortex and pons. Although Connor and Fine (1986) clearly demonstrated Tf immunoreactivity in oligodendrocytes using a polyclonal antibody against rat Tf, we found a less distinct amount of TfR on these cells. In our opinion, these results are not conflicting. Indeed, using in situ hybridization techniques, oligodendrocytes have been found to produce Tf (Bloch et al. 1985), and it was demonstrated that they do not need Tfcontaining medium to growth in culture (Espinosa de los Monteros and Foucaud 1987). One possibility is that oligodendrocytes have a lower Tf uptake because they produce this protein; in this case, the presence of a surface receptor may not be relevant. We found weakly positive oligodendrocytes in the cerebral cortex only, particularly in the white matter where, as
Figs. 6 and 7. In the brain stem, pontine neurons show intense staining with the mab OX26 ( × 200); at higher magnification it is possible to recognize a neuron with its cytoplasm fully occupied by the reaction together with the apical dendrite. In this case, the membrane was deranged by enzymatic digestion which enabled good penetration of the mab ( × 1000).
89 previously reported by C o n n o r a n d F i n e (1986), they were arranged in clusters at the interfascicular a n d perivascular levels. The presence of large n u m b e r s of TfR in postnatal neurons, however, has several important implications: - The use of m a b against TfR as cytotoxic agents in brain tumors as suggested (Trowbridge 1983), could also destroy n o r m a l n e u r o n s leading to i m p o r t a n t neurological syndromes. - TfR appear to be implicated in cellular recognition (Lazarus a n d Baines 1985), and T f in morphogenesis ( P a r t a n e n et al. 1984); T f itself acts as a neurotrophic factor (Beach et al. 1983). Therefore, the presence of TfR in differentiated n e u r o n s could favor a late possibility of n e u r o n a l plasticity.
ACKNOWLEDGEMENTS We t h a n k Dr. F. Scaravilli for helpful c o m m e n t s on the manuscript.
REFERENCES Beach, R. L., H. Popiela and B.W. Festoff (1983) The identification of neurotrophic factor as a transferrin. FEBS Leg., 156: 151-156. Bloch, B., T. Popoviei, M.J. Levin, D. Tuil and A. Kahn (1985) Transferrin gene expression visualized in oligodendrocytesof the rat brain by using in situ hybridization and immunohistochemistry.Proc. Natl. Acad. Sci. USA, 82: 6706-6710. Connor, J.R. and R.E. Fine (1986) The distribution of transferrin immunoreactivity in the rat central nervous system. Brain Res., 368: 319-328. Dautry-Varsat, A., A. Ciechanover and H.F. Lodish (1983) pH and the recycling of transferrin during receptor-mediated endoeytosis. Proc. Natl. Aead. Sci. USA, 80: 2258-2262. Espinosa de los Monteros, A. and B. Foueaud (1987) Effect of iron and transferrin on pure oligodendrocytes in culture; characterization of a high-affinitytransferrin receptor at different ages. Dev. Brain Res., 35: 123-130. Felgenhauer, K. (1974) Protein size and eerebrospinal fluid composition. Klin. Wsehr., 52:1158-1164. Gallo, P., F. Bracco, S. Morara, L. Battistin and B. Tavolato (1985) The eerebrospinal fluid transferrin/tau proteins. A study by two-dimensionalpolyaerylamidegel eleetrophoresis (2D) and agarose isoelectrofocusing (IEF) followed by double-antibody peroxidase labelling and avidin-biotin amplification.J. Neurol. Sci., 70: 81-92. Gatter, K.C., G. Brown, I.S. Trowbridge, R.E. Woolston and D.Y. Mason (1983)Transferrin receptors in human tissues: their distribution and possible clinical relevance. 9". Clin. Pathol., 36: 539-545. Hill,J. M., M. R. Ruff,R. J. Weber and C. B. Pert (1985) Transferrin receptors in rat brain: Neuropeptide-like pattern and relationship to iron distribution. Proc. Natl. Acad. Sci. USA, 82: 4553-4557. Jefferies, W. A., M. R. Brandon, S. V. Hunt, A. F. Williams,K. C. Gatter and D.Y. Mason (1984) Transferrin receptors on endothelium of brain capillaries. Nature, 312:162-163. Jefferies, W. A., M. R. Brandon, A. F. Williamsand S. V. Hunt (1985) Analysisof lymphocyticstem cells with a monoclonal antibody to the rat transferrin receptor. Immunology, 54: 333-341. Lazarus, A. H. and M. G. Baines (1985) Studies on the mechanism of specificityof human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity. Cell. Immunol., 96: 255-266. Levin, M.J., D. Tuil, G. Uzan, J.C. Deyfus and A. Kahn (1984) Expression of the transferrin gene during development of nonhepatie tissues: high level of transferrin mRNA in fetal muscle and in adult brain. Biochem. Biophys. Res. Commun., 122: 212-217. Morris, C. M., J. M. Candy, A. E. Oakley, G. A. Taylor, S. Mountfort, H. Bishop, M. K. Ward, C. A. Bloxham
90 and J.A. Edwardson (1989) Comparison of the regional distribution of transferrin receptors and aluminium in the forebrain of chronic renal dialysis patients. J. Neurol. Sci., 94: 295-306. Oh, T.H., G.J. Markelonis, G.M. Royal and B.S. Bregman (1986) Immunocytochemical distribution of Transferrin and its Receptors in the Developing Chicken Nervous System. Dev. Brain Res.. 30: 207-220. Partanen, A.M., I. Thesleff and P. Ekblom (1984) Transferrin is required for early tooth morphogenesis. Differentiation, 27: 59-66. Saito, K., Y. Hogra, T. Hasegawa and E. Orawa (1982) Indispensability of iron for the growth of cultured chick cells. Dev. Growth Diff., 24: 571-580. Swaiman, K.F. and V.L. Machen (1984) Iron uptake by mammalian cortical neurons. Ann. Neurol.. 16: 66-70. Trowbridge, I. S. (1983) Therapeutic potential of monoclonal antibodies that block biological function. In: B.D. Boss, R. Langman, I. Trowbridge and R. Dulbecco (Eds.), Monoclonal Antibodies and Cancer, Academic Press, New York, pp. 53-61.
81
Elsevier JNS 03356
Transferrin receptors in rat central nervous system An immunocytochemical study Bruno Giometto, Felice Bozza, Vincenza Argentiero, Paolo Gallo, Silvana Pagni, M a r i a G r a z i a Piccinno and Bruno Tavolato Second Neurological Clinic, University of Padova, School of Medicine, 1-35137 Padova (Italy)
(Received 19 September, 1989) (Revised, received 19 March, 1990) (Accepted 19 March, 1990)
SUMMARY Using an immunocytochemical method we demonstrated the presence of TfR on adult rat neurons, particularly in the cerebral cortex and brain stem. The monoclonal antibody (mab) against rat TfR (clone OX 26) stained neurons of all cortical layers and in the brain stem where the reaction was most evident. Purkinje cells in the cerebellum and scattered neurons in the gray matter of the cervical spinal cord were weakly stained. Choroid plexus cells also reacted with the mab against TfR whereas oligodendrocytes in the cerebral white matter were faintly outlined by the mab. The presence of TfR on endothelial cells of brain capillaries was here confirmed.
Key words: Transferrin receptors; Immunocytochemistry; Central nervous system; Neuronal plasticity
INTRODUCTION Cell iron uptake is mediated by preliminary binding of transferrin (Tf), the iron-carrying protein, to a specific receptor (TfR) on the cell surface. The Tf/TfR complex is then internalized by endocytotic vesicles for the intracellular "transferrin Correspondence to: Prof. B. Tavolato,II NeurologicalClinic,Universityof Padova, Via Vendramini,
7, 1-35137 Padova, Italy. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
82 cycle", and the iron is finally released into the cytoplasm (Dautry-Varsat et al. 1983). TfR expression is restricted in normal human tissues: in fact it does not occur in resting cells, but has been demonstrated in rapidly proliferating cells such as the basal layer of squamous epithelium, and in a variety of different types of human malignancies (Gatter et al. 1983). Many studies suggest that Tf and iron play an important role in the central nervous system (CNS). Tf is synthesized by the brain (Levin et al. 1984), and is more concentrated in cerebrospinal fluid (CSF) than predicted by its molecular weight and hydrodynamic radius (Felgenhauer 1974; Gallo et al. 1985); it is required for the growth of some CNS cells (Saito et al. 1982). Neurons show a significant iron uptake in culture (Swaiman and Machen 1984). Nevertheless, only a few reports address the presence of TfR on CNS cells. Using immunocytochemical methods, TfR were demonstrated on brain capillaries (Jefferies et al. 1984) and on developing (not in adult) neurons (Oh et al. 1986); only autoradiographic methods have detected their presence on postnatal neurons (Hill et al. 1985) and oligodendrocytes in culture (Espinosa de los Monteros and Foucaud 1987). A morphological demonstration, however, of TfR on postnatal CNS cells is still
Fig. 1. Rat liversectionstainedby the mab OX26.The membraneofhepatocytes,and Kupffercells(arrow) are clearly outlinedby the mab. The section was counterstainedwith hematoxylin( x 400).
83 lacking. We employed an immunocytochemicalmethod with avidin-biotin amplification in an attempt to characterize the TfR presenting cells in the adult rat CNS.
MATERIALSAND METHODS Twelve adult Sprague-Dawley rats, 1-4 months old, anaesthetized with pentobarbitone were perfused through the ascending aorta with 100 ml cold saline followed by 500 ml 4 ~ paraformaldehyde (4 ° C) in 0.1 M phosphate buffer, pH 7.4. The cerebral and cerebellar hemispheres, brain stem and cervical spinal cord were dissected out and stored in cold fixative for 4 h; the tissues were then kept overnight in 10~o sucrose. Ten #m thick frozen sections of cerebral (motor area) and cerebellar cortex, pons and cervical spinal cord were cut, placed on poly-L-lysine coated slides and subsequently treated with 0.1~o H202 for 10 min, followed by 0.5~ trypsin for 10 min at 37 °C. The slides were then incubated overnight at 4 °C with an IgG2 mab against rat TfR (clone OX26, mab OX26, Seralab, U.K.) diluted 1 : 100 in phosphate-buffered saline (PBS), pH 7.4. The sections were washed 3 times in PB S, and then covered with an intermediate
Fig. 2. Controlsectionin whichthe first antibodywas omitted ( x 200).
84 layer of biotinylated rabbit anti-mouse Ig (Dakopatts, Denmark) diluted 1 : 200 in PBS for 1 h at room temperature. After further washing, a layer of horseradish-peroxidase conjugated avidin (Dakopatts, Denmark) diluted 1 : 500 was added for 30 min at room temperature. The substrate reaction was developed with 0.0570 diaminobenzidine tetrahydrochloride (Carlo Erba, Italy) and 0.01~o H202 in PBS. Finally, the slides were washed and mounted in glycerol gelatin (Merck, Darmstadt, F.R.G.). Rat liver was used as positive control tissue; 10-/~m frozen liver sections were processed and the reaction was developed as above described. These sections were counterstained with Mayer's hematoxylin. When negative control sections were processed the first antibody was omitted or substituted by other mabs of the same immunoglobulin class as OX26.
Fig. 3. Brain capillary stained by the mab against TfR in the cerebral white matter (arrow). Oligodendrocytes are also weakly recognized by the mat); groups of 4-5 cells are prevalent and a cluster of calls is present at the perivascular level (arrowhead). In these cells only the membrane is outlined by the reaction ( x 200).
85 RESULTS Rat Kupffer cells and hepatocytes were clearly outlined on the cell membrane by the mab OX26 (Fig. 1). No immunoreactivity was detected in the control sections where the first mab was omitted or substituted by other unrelated antibodies (Fig. 2). In the rat brain tissue, TfR immunoreactivity was detected on the endothelium of brain capillaries (Fig. 3). Choroid plexus cells were also positive with mab OX26 (Fig. 4). Oligodendrocytes were weakly stained in the cerebral white matter where they were present in pairs or cluster of 4 or 5 cells, and in the perivascular space (Fig. 3); the reaction was confined to the cell membrane and was quite weak. Oligodendrocytes were not recognizable in other brain regions. Nerve cells were clearly stained by the mab against TfR; however, a regional distribution of the pattern of staining was evidenced. Cerebral cortex
The neurons of all the cerebral layers of the motor cortex were stained by the mab OX26 (Fig. 5), especially those of layer V. Labelling was mainly confined to the cell
Fig. 4. Choroidplexus cells are positivewith the mab against TfR ( x 200).
86 membrane, but, in some cortical neurons, the whole cytoplasm was stained. In addition the soma, the proximal portion of the apical dendrites was also outlined by the antibody in the cortical neurons.
Brain stem and cerebellum The large pontine neurons showed intense immunoreactivity (Fig. 6), which was extended to the entire cytoplasm (Fig. 7). In the cerebellum, the Purkinje cells were faintly stained (data not shown); no TfR immunoreactivity was present in the cells of the granular and molecular layers. Spinal cord Scattered neurons in the cervical gray matter were faintly stained (data not shown), but not in relation to any particular laminar distribution. No specific reactivity was found in the cervical white matter.
Fig. 5. Cortical neurons stained by the mab OX26. Neurons of all cortical layers are recognized and the reactivityis prevalent around the cell membrane.The proximalportion of the apical dendrite is also positive ( × 200).
87 DISCUSSION In the present study we investigated the localization of TfR in rat brain using the mab OX26 (IgG2a) reported specific for TfR since it recognized a detergent-solubilized molecule that can bind 125I-labelled transferrin and precipitates a dimer ofglycoproteins with an apparent molecular weight of 95 000 (Jefferies et al. 1985). Sections of the rat liver showed, with the mab OX26, a staining confined to the sites where the presence of TfR is expected (Gatter et al, 1983; Jefferies et al. 1985) confirming the specificity of the mab. In this study we found that choroid plexus cells stained positively with the mab OX26; we also confirmed the report by Jefferies et al. (1984) that TfR were present on the endothelium of brain capillaries. Moreover, our study shows that in adult rat neurons TfR are largely represented. These however are variable in amount: they are numerous in cortical and brain stem neurons while Purkinje cells and spinal cord neurons were only faintly outlined by the mab OX26. Concerning these findings, our results are at variance with those obtained by Jefferies et al. (1984): these workers using the same mab observed weak reactivity in scattered neuronal cells. We believe that methodological differences could explain these discrepant data. In our investigation neuron reactivity was enhanced by treatment with trypsin, which showed TfR immunoreactivity mainly in the cytoplasm of neurons. This pattern of staining is not surprising, considering the intracellular cycle of Tf/TfR complex. Other authors have reported the presence of TfR particularly in the cytoplasm of CNS cells (Espinosa de los Monteros and Foucaud 1987). TfR present on the cell surface may be partially destroyed or masked by fixative or by postmortem modifications. On the other hand, the TfR epitope recognized by mab OX26 might become accessible to the antibody only after an increase in tissue and cell permeability induced by enzymatic digestion. Moreover, we exclude an aspecific staining induced by trypsin treatment since other mabs of the same immunoglobulin class, used as controls, failed to stain neurons. We are not able to explain the different pattern and degree of reactivity between cortex and brain stem neurons, and spinal cord neurons and Purkinje cells. Metabolic specificities might account for these findings. However, a regional brain distribution of TfR has been reported also by autoradiographic studies (Hill et al. 1985; Morris et al. 1989) that confirm a high level of TfR in cortex and pons. Although Connor and Fine (1986) clearly demonstrated Tf immunoreactivity in oligodendrocytes using a polyclonal antibody against rat Tf, we found a less distinct amount of TfR on these cells. In our opinion, these results are not conflicting. Indeed, using in situ hybridization techniques, oligodendrocytes have been found to produce Tf (Bloch et al. 1985), and it was demonstrated that they do not need Tfcontaining medium to growth in culture (Espinosa de los Monteros and Foucaud 1987). One possibility is that oligodendrocytes have a lower Tf uptake because they produce this protein; in this case, the presence of a surface receptor may not be relevant. We found weakly positive oligodendrocytes in the cerebral cortex only, particularly in the white matter where, as
Figs. 6 and 7. In the brain stem, pontine neurons show intense staining with the mab OX26 ( × 200); at higher magnification it is possible to recognize a neuron with its cytoplasm fully occupied by the reaction together with the apical dendrite. In this case, the membrane was deranged by enzymatic digestion which enabled good penetration of the mab ( × 1000).
89 previously reported by C o n n o r a n d F i n e (1986), they were arranged in clusters at the interfascicular a n d perivascular levels. The presence of large n u m b e r s of TfR in postnatal neurons, however, has several important implications: - The use of m a b against TfR as cytotoxic agents in brain tumors as suggested (Trowbridge 1983), could also destroy n o r m a l n e u r o n s leading to i m p o r t a n t neurological syndromes. - TfR appear to be implicated in cellular recognition (Lazarus a n d Baines 1985), and T f in morphogenesis ( P a r t a n e n et al. 1984); T f itself acts as a neurotrophic factor (Beach et al. 1983). Therefore, the presence of TfR in differentiated n e u r o n s could favor a late possibility of n e u r o n a l plasticity.
ACKNOWLEDGEMENTS We t h a n k Dr. F. Scaravilli for helpful c o m m e n t s on the manuscript.
REFERENCES Beach, R. L., H. Popiela and B.W. Festoff (1983) The identification of neurotrophic factor as a transferrin. FEBS Leg., 156: 151-156. Bloch, B., T. Popoviei, M.J. Levin, D. Tuil and A. Kahn (1985) Transferrin gene expression visualized in oligodendrocytesof the rat brain by using in situ hybridization and immunohistochemistry.Proc. Natl. Acad. Sci. USA, 82: 6706-6710. Connor, J.R. and R.E. Fine (1986) The distribution of transferrin immunoreactivity in the rat central nervous system. Brain Res., 368: 319-328. Dautry-Varsat, A., A. Ciechanover and H.F. Lodish (1983) pH and the recycling of transferrin during receptor-mediated endoeytosis. Proc. Natl. Aead. Sci. USA, 80: 2258-2262. Espinosa de los Monteros, A. and B. Foueaud (1987) Effect of iron and transferrin on pure oligodendrocytes in culture; characterization of a high-affinitytransferrin receptor at different ages. Dev. Brain Res., 35: 123-130. Felgenhauer, K. (1974) Protein size and eerebrospinal fluid composition. Klin. Wsehr., 52:1158-1164. Gallo, P., F. Bracco, S. Morara, L. Battistin and B. Tavolato (1985) The eerebrospinal fluid transferrin/tau proteins. A study by two-dimensionalpolyaerylamidegel eleetrophoresis (2D) and agarose isoelectrofocusing (IEF) followed by double-antibody peroxidase labelling and avidin-biotin amplification.J. Neurol. Sci., 70: 81-92. Gatter, K.C., G. Brown, I.S. Trowbridge, R.E. Woolston and D.Y. Mason (1983)Transferrin receptors in human tissues: their distribution and possible clinical relevance. 9". Clin. Pathol., 36: 539-545. Hill,J. M., M. R. Ruff,R. J. Weber and C. B. Pert (1985) Transferrin receptors in rat brain: Neuropeptide-like pattern and relationship to iron distribution. Proc. Natl. Acad. Sci. USA, 82: 4553-4557. Jefferies, W. A., M. R. Brandon, S. V. Hunt, A. F. Williams,K. C. Gatter and D.Y. Mason (1984) Transferrin receptors on endothelium of brain capillaries. Nature, 312:162-163. Jefferies, W. A., M. R. Brandon, A. F. Williamsand S. V. Hunt (1985) Analysisof lymphocyticstem cells with a monoclonal antibody to the rat transferrin receptor. Immunology, 54: 333-341. Lazarus, A. H. and M. G. Baines (1985) Studies on the mechanism of specificityof human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity. Cell. Immunol., 96: 255-266. Levin, M.J., D. Tuil, G. Uzan, J.C. Deyfus and A. Kahn (1984) Expression of the transferrin gene during development of nonhepatie tissues: high level of transferrin mRNA in fetal muscle and in adult brain. Biochem. Biophys. Res. Commun., 122: 212-217. Morris, C. M., J. M. Candy, A. E. Oakley, G. A. Taylor, S. Mountfort, H. Bishop, M. K. Ward, C. A. Bloxham
90 and J.A. Edwardson (1989) Comparison of the regional distribution of transferrin receptors and aluminium in the forebrain of chronic renal dialysis patients. J. Neurol. Sci., 94: 295-306. Oh, T.H., G.J. Markelonis, G.M. Royal and B.S. Bregman (1986) Immunocytochemical distribution of Transferrin and its Receptors in the Developing Chicken Nervous System. Dev. Brain Res.. 30: 207-220. Partanen, A.M., I. Thesleff and P. Ekblom (1984) Transferrin is required for early tooth morphogenesis. Differentiation, 27: 59-66. Saito, K., Y. Hogra, T. Hasegawa and E. Orawa (1982) Indispensability of iron for the growth of cultured chick cells. Dev. Growth Diff., 24: 571-580. Swaiman, K.F. and V.L. Machen (1984) Iron uptake by mammalian cortical neurons. Ann. Neurol.. 16: 66-70. Trowbridge, I. S. (1983) Therapeutic potential of monoclonal antibodies that block biological function. In: B.D. Boss, R. Langman, I. Trowbridge and R. Dulbecco (Eds.), Monoclonal Antibodies and Cancer, Academic Press, New York, pp. 53-61.
