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Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium
BRAIN RESEARCH ELSEVIER
Brain Research 712 (1996) 117-121
Research report
Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium Sylvia Rothenberger a, Michael R. Food a, Reinhard Gabathuler a Malcolm L. Kennard a Tatsuo Yamada b, Osamu Yasuhara b, Patrick L. McGeer b, Wilfred A. Jefferies a' * Biotechnology Laboratory and Departments of Medical Genetics, Microbiology and Immunology_, and Zoology, University of British Columbia, Vancouver, British Columbia V6T lZ3, Canada Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia V6T I Z3, Canada Accepted 24 October 1995
Abstract
One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium [7]. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form, We have recently demonstrated that GPl-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor [10]. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain. Kevwords: Blood brain barrier; Transferrin receptor; Capillary endothelium; Melanotransferrin; Human brain
I. Introduction
Iron is required for cellular energy processes [12]. It is generally thought that transferrin (Tf) delivers iron to cells by binding to cellular transferrin receptors (TRs) which then become internalized [11] and release the iron into the cell prior to iron utilization or storage of iron complexed with ferritin. The route of iron uptake into the brain is not clearly defined since iron acquisition must first involve translocation across the blood brain barrier (BBB). TRs are expressed on capillary endothelial cells and they are also sporadically and sparsely expressed on some neurons [7].
Abbreviations: BBB, blood brain barrier; TR, transferrin receptor; MTf, melanotransferrin; Tf, transferrin; GPI, glycosylphosphatidylinositol. * Corresponding author. Fax: (1) (604) 822-6780. /1006-8993/96/$15.00 © 1996 Elsevier Science B,V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 4 0 8 - X
In contrast, Tf and ferritin are found predominantly in association with glial cells [4,14]. It has been reported that iron transport into brain can be significantly inhibited by blockade of TRs with specific antibodies [18]. Nevertheless, hypotransferrinemic mice have been shown to have higher than normal iron uptake into the brain [18] and it has been recently reported that a significant amount of iron is transported into the brain by a route independent of Tf [18]. Melanotransferrin (MTf), or the p97 protein, may account for some of these discrepancies. It was first identified on the surface of melanoma cells [3,19,20] and has a structure which resembles serum Tf [15]. It binds iron [2], and has been shown to directly mediate cellular iron uptake [10]. It is found either attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor [1,5], or in the serum as a soluble form [3]. In addition to expression on melanoma cells, MTf has been reported to
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be expressed by a subset of liver cells [16] and in some cells of the fetal intestine [3]. There is growing interest in the transport of iron into the brain. Of special significance are models which utilize a monoclonal antibody, MRC-OX-26 [7,8], against the rat TR to deliver therapeutic compounds into the brains of rats [6]. It has been proposed that a viable human therapy for brain diseases could be based on the shuttling of compounds via specific receptor into the brain. Clearly, for this approach to reach its therapeutic potential an exact knowledge of the function of the TR and the routing of iron into and out of the brain must be derived. With our knowledge that p97 is directly able to transport iron into cells, we sought to analyze the expression of potentially important iron transport proteins in normal brains.
to immunoprecipitation. The primary antibodies used were the L235 and OKT9. Immunoprecipitation and SDS-PAGE were carried out as previously described [9].
2.3. Brain sections Seven brains from cases aged 54-82 were studied. The seven cases were all free from neurological signs and symptoms and standard neuropathological examination re-
2. Materials and methods
2.1. Sequential immunoprecipitation The human melanoma cell line SK-MEL-28 (HTB 72) and the CHO lines WTB and p97aWTBc3 were cultured as previously described [5]. The SK-MEL-28 cell line monolayers were washed with DMEM without methionine and then were biosynthetically labelled for 24 h with 150 /xCi/ml of [35S]methionine in supplemented DMEM. The cell lysates and supernatants were pre-cleared by centrifugation at 100,000 × g for 1 h. In the first immunoprecipitation, the primary antibodies used were the L235 (antihuman MTf, 1:1000 dilution, mouse monoclonal, IgG I, American Type Culture Collection (ATCC) HB8446); and the OKT9 (anti-human TR, 1:1000 dilution, mouse monoclonal, IgG 1, ATCC CRL 8021). The same antibodies were used on the same preparations in the second immunoprecipitation. For the third immunoprecipitation, the L235 was used on the sample which had previously been cleared of the TR, and the OKT9 was used on the sample which had previously been cleared of MTf. All samples were analyzed by SDS-PAGE under non-reduced conditions. The gels were subsequently fixed, dried and autoradiographed.
2.2. Pulse-chase labelling SK-MEL-28 cell line monolayers were maintained in medium lacking methionine for 1 h prior to labelling. Biosynthetic labelling of cells was done for 15 min with 2 ml of 150 /.LCi/ml of [35S]methionine per petri dish. Cells were then chased with normal medium containing an excess of cold methionine for the times indicated. A separate petri dish was used for each of the times. The cells were lysed in 20 mM Tris-HC1, pH 7.2, 150 mM NaC1, 2 mM EDTA, and 1% NP-40 with 20 /zg/ml phenylmethylsulfonyl fluoride (PMSF). The lysates and cell supernatants were then cleared by centrifugation prior
p97
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Fig. 1. A: sequential immunoprecipitation of MTf and TR from labelled SK-MEL-28 cells using anti-MTf (L235) and anti-TR (OKT9) antibodies. After 24 h of incubation with [35S]methionine, cells were lysed and both cell lysates and media were subjected to three sequential immunoprecipitations with these antibodies. The immunoprecipitates were analyzed on a 10-15% SDS-PAGE gel under non-reducing conditions, and an autoradiogram was developed after 24 h of exposure of the gel. The migration of the molecular mass markers is indicated on the left side of the gel. B: expression of human MTf and human TR in cultured cells of human SK-MEL-28 melanoma line, the CHO line WTB, and the MTf expressing CHO line p97aWTBc3. Each line was biosynthetically labelled with [35S]methionine and chased with cold methionine for 6 h. Cell lysates (lanes 1,2,5,6) and cell supernatants (lanes 3,4,7,8) were immunoprecipitated with the L235 MAb (lanes 1,2,3,4) or with the OKT9 MAb (lanes 5,6,7,8). Molecular mass markers are indicated on the left of the figure. Gels represented on the figure were exposed for 40 h.
S. Rothenberger et aL / Brain Research 712 (1996) 117-121
vealed no abnormalities in the brain tissue. Brains in all cases were obtained 2-32 h after death. Small blocks were dissected from various brain regions. These were the angular, midtemporal and precentrai cortices, the hippocampal formation including the entorhinal cortex, caudate, putamen, globus pallidus, substantia nigra, and the cerebral white matter and spinal cord, but not all regions were examined in all types of cases. The blocks were fixed for two days in phosphate-buffered 4% paraformaldehyde and then transferred to a maintenance solution of 15% sucrose in 0.1 M phosphate buffer pH 7.4, and kept in the cold until used. Sections were cut on a freezing microtome at 30 /xm thickness and stained by single or double immunohistochemical procedures [13]. The following antibodies were used in immunohistochemical studies: anti-human MTf, (L235); anti-human Tf (A-061, 1:10,000 dilution, rabbit polyclonal, DAKO); and three anti-human TR antibodies: OKT9; HTR-H684, 1 /xg/ml, purified mouse monoclonal; and HTR-Ro~S, 1:10,000 dilution, rabbit polyclonal. The last two antibodies were gifts of Dr. Ian Trowbridge (Salk Institute, San Diego). HTR-H684 was
119
raised against the TR cytoplasmic domain, while the HTRR a S antiserum was raised against the TR external domain.
2.4. Electron microscopy For electron microscopy, blocks of entorhinal cortex from two cases of Alzheimer's Disease (AD) were fixed in 1% glutaraldehyde/4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 24 h at 4°C, followed by immersion in 15% sucrose in 0.1 M phosphate buffer, pH 7.4, for several days at the same temperature. Sections were cut by vibratome at 50 /xm thickness and incubated with the L235 MAb (1:1000) for 5 days at 4°C. They were then treated with the appropriate Vectastain and ABC secondary antibody systems. After the diaminobenzidine (DAB) reaction, the sections were osmified, dehydrated and embedded in Epon. Ultra-thin sections were cut and examined with a Phillips EM201 electron microscope without counterstaining.
Fig. 2. lmmunohistochemical staining of brain for MTf (A,E), TR (B,C) and Tf (D). A: normal angular gyrus cortical gray matter stained with L235 MAb. Capillary endothelium is strongly positive. B: normal precentral cortical grey matter stained with rabbit anti-TR antiserum (HTR-Rt~S). Capillary endothelium is positive. C: normal angular gyrus from a nearby section to that shown in A stained with the OKT9 anti-TR MAb. In this section, only capillaries are stained although other sections show occasional weak neuronal staining. D: normal angular cortex stained with anti-Tf polyclonal antibody. The sparse cytoplasm of a few cells resembling oligodendrocytes are positive. E: electron micrograph of MTf expression in an endothelial cell. An ultrathin human brain section was labelled with the L235 MAb (see Section 2 for details). The resulting electron micrograph is shown at 1612 × magnification. Notice the unstained nucleus of the endothelial cell (n) and the heavy DAB reaction product in the cell cytoplasm (cy). A - D arc at the same magnification (bar in C = 50 /xm).
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3. Results
3.1. Sequential immunoprecipitation and pulse chase labelling In order to investigate the specificities of the antibodies used, the SK-MEL-28 cells were biosynthetically labelled with [35S]methionine and MTf and the TR were immunoprecipitated from both the cell lysate and the media (Fig. 1A). In the first immunoprecipitation, it is clear that the L235 antibody recognized a protein with a molecular mass of 95 kDa, corresponding to MTf, in both the cell lysate and media (lanes 1 and 3). Under similar conditions, the OKT9 antibody recognized a protein with a molecular mass of 180 kDa (lane 2) in the cell lysate and 85 kDa (lane 4) in the media, corresponding to the TR. In the second immunoprecipitation the same antibodies were again used to precipitate any remaining proteins. The residual MTf was removed from the samples (lanes 5 and 7). Similarly, the residual TR was also removed (lanes 6 and 8). In the third immunoprecipitation, the samples were exchanged and subjected to the OKT9 antibody in the case of the L 2 3 5 / M T f cleared preparation, and the L235 antibody in the case of the O K T 9 / T R cleared preparation. It is apparent that a protein with a molecular mass of 180 kDa is present in the cell lysate of the MTf depleted preparation (lane 9) and a protein with a molecular mass of 85 kDa is present in the media of the MTf depleted preparation (lane 11). These proteins correspond to the two forms of the human TR which have been described previously [17]. In a similar manner, a protein with a molecular mass of 95 kDa is present in both the cell lysate (lane 10) and the media (lane 12) of the TR depleted preparation. This protein corresponds to MTf. These results indicate conclusively that the L235 and OKT9 antibodies recognize MTf and the TR, respectively, and there is no apparent cross-reactivity. In addition, we have followed by pulse-chase, the fates of MTf and the TR after biosynthetic labelling of SKMEL-28, WTB and p97aWTBc3 cells. The L235 MAb (Fig. 1B, lanes 1, 2, 3, 4) recognized a protein with a molecular mass of 93 kDa (Fig. 1B, lane 1) that is processed to a higher molecular mass of 95 kDa after 6 h of chase (Fig. 1B, lanes 1, 2). This protein is not seen in WTB cells (Fig. 1B, lanes 1, 2). A considerably greater amount of this protein is present in the transfected cells, p97aWTBc3 (Fig. 1B, lanes 1, 2), identifying this protein as MTf. In addition, a soluble form of MTf is present in the cell supernatant after 6 h chase (Fig. 1B, lane 4). The presence of a 70 kDa band is likely to be due to a p97 degradation product and p97 appears to be more sensitive to degradation at earlier time points in the pulse-chase. The OKT9 MAb (Fig. 1B, lanes 5, 6, 7, 8) recognizes a protein with a similar mass in SK-MEL-28 cells corresponding to the reduced form of the human TR (Fig. 1B, lanes 5, 6). The human TR is not seen in the cell supernatant in this
figure (Fig. 1B, lanes 7, 8). It is clear that the L235 and OKT9 MAbs do not cross react with hamster MTf and TR in the CHO line WTB. These data separately and collectively establish the specificity of the anti-MTf and the principal anti-TR MAb used in this study. They confirm that MTf and the TR are synthesized and transported to the cell surface. Additionally, we can identify a soluble form of MTf in the medium [5].
3.2. Expression of MTf, Tf and the TR in brain tissues The cellular distributions of MTf, Tf and the TR were determined immunohistochemically in human brain tissue (Fig. 2). In brain cortex, capillary endothelium was strongly stained by antibodies to both MTf (Fig. 2A) and the three antibodies to the TR, two of which are illustrated (Fig. 2B,C). A few neurons showed relatively weak positive staining for the TR, as previously described [7], but none are visible in Fig. 2B. Furthermore, the anti-Tf MAb stained mostly glia (Fig. 2D) in agreement with a previous report [4]. These data establish the coincident expression of the TR and MTf on capillary endothelium and the lack of coincident expression of Tf with either the TR or with MTf. 3.3. Distribution of MTf in brain capillaries at the electron
microscopic level Electron microscopy was used to define the structures expressing MTf in capillaries. By this method the DAB reaction products in sections stained with the anti-MTf antibody were found in the cytoplasm of endothelial cells (Fig. 2E). These results suggest a role for MTf mediated metal transport through brain endothelium.
4. Discussion
The delivery of nutrients into the brain is well established. The nutrients must pass from the bloodstream through the capillary endothelium of the brain before being distributed to the cells within the brain. The detailed mechanism which allows crossing of the BBB remains unresolved. In principle, several alternative methods of traversing the brain endothelium could exist. However, the most likely of these involves the active transcytosis of ligands linked to receptors. The ligands may be delivered unaltered on the sublumenal surface and dissociate from the receptors. Alternatively, the ligands and possibly the receptors may in some way be altered during transcytosis resulting in the release of altered ligands at the sublumenal surface. In this study, we have shown that two proteins involved in iron binding and transport, MTf and the TR, are present in brain endothelial cells of all human brains studied. The expression of TR by brain endothelial cells and occasional
S. Rothenberger et al. / Brain Research 712 (1996) 117-121
neurons is consistent with previous work on its distribution within the brain [7]. A disparity between the localization of TR and Tf has previously been noted [4,7,8]. This difference in localization raises questions as to whether T f is exclusively responsible for mediating iron transport into and within the brain. Indeed, recent observations reveal the existence of mechanisms for iron transport across the b l o o d / b r a i n barrier which are exclusive o f Tf [18]. Our findings that: (i) MTf is expressed in capillary endothelium; (ii) that its expression is coincidental with the TR; and (iii) that M T f can act as an iron transport protein [10] are consistent with the hypothesis that M T f has a role in iron uptake in human brain. It is possible that M T f may directly deliver iron across the BBB in an analogous or competitive method to the TR. Alternatively, MTf may function in the excretion of iron from the brain into the blood stream [1], Finally, it is possible that M T f may function to limit the presence of free iron within the brain which would catalyze the formation of potentially harmful super oxide or hydroxyl radicals, which in turn could damage normal brain tissues. Substantial attention has been devoted to the development of compounds which transport therapeutics into the brain. Many of these are based on the use of drugs conjugated to antibodies against the TR. Likewise, antibodies against M T f may have utility in the deliverance of therapeutic compounds into the brain. The use of such compounds is presently under investigation but is hampered by the lack of specific reagents in species other than human.
Acknowledgements This work was supported by grants to W.A. Jefferies by the British Columbia Health Care Research Foundation, the Medical Research Council of Canada, the Vancouver Foundation and Synapse Technologies Inc. This work was also supported by grants to P.L. McGeer, Tatsuo Yamada, and Osamu Yasuhara by the A l z h e i m e r ' s Society o f British Columbia and individual British Columbians.
References [1] Alemany, R., Vila, M.R., Franci, C., Egea, G., Real, F.X. and Thomson, T.M., Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells, J. CellS ci., 104 (1993) 1155-1162. [2] Brown, J.P., Hewick, R.M., Hellstrtim, I., Hellstr/Sm,K,E., Doolittle, R.F, and Dreyer, W.J., Human melanoma-associated antigen p97 is structurally and functionally related to transferrin, Nature, 296 (1982) 171-173. [3] Brown, J.P~. Woodbury, R.G., Hart, C.E., Heltstrrm, I. and
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Hellstr~m, K.E,, Quantitative analysis of melanoma-associated antigen p97 in normal and neoplastic tissues, Proc. Natl. Acad. Sci, USA, 78 (1981) 539-543. [4] Connor, J.R., Menzies, S.L, St.Martin, SM. and Mufson, E.J., Cellular Distribution of Transferrin, Ferritin, and Iron in Normal and Aged Human Brains, J. Neurosci. Res., 27 (19901 595-611. [5] Food, M.R., Rothenberger, S, Gabathuler, R., Haidl, I.D., Reid, G. and Jefferies, W.A, Transport and Expression in Human Melanomas of a Transferrin-like Glycosylphosphatidylinositol-anchoredProtein, J. Biol. Chem., 269 (1994) 3034-3040. [6] Fridcn, P.M., Watus, L.R., Watson, P., Doctrow, S.R., Kozarich. J.W., Backman, C., Bergman, H., Hoffer, B., Bloom, F. and Granholm, A-C, Blood-Brain Barrier Penetration and in Vivo Activity of an NGF Conjugate, Science, 259 (19931 373-377. [7] Jefferies, W.A., Brandon, M.R, Hunt, S.V., Williams, A.F., Garter, K.C. and Mason, D.Y., Transferrin receptor on endothelium of brain capillaries, Nature, 312 (1984) 162-163. [8] Jefferies, W.A., Brandon, M.R., Williams, A.F. and Hunt, S.V., Analysis of lymphopoietic stern cells with a monoclonal antibody to the rat transferrin receptor, Immunology, 54 (19851 333-341. [9] Jefferies, W.A., Riither, U., Wagner, E.F. and Kvist, S., Cytolytic T cells recognize a chimeric MHC class I antigen in influenza A infected transgenic mice, EMBO J,, 7 (1988) 3423-3431. [10] Kennard, M.L, Richardson, D.R., Gabathuler, R, Ponka, P. and Jefferies, W.A., A novel iron uptake mechanism mediated by GPIanchored human p97, EMBOJ., 17 (19951 4178-4186. [11] Kiihn, LC., The transferrin receptor: a key function in iron metabolism, Schweiz. Med. Wschr., 119 (19891 1319-1326. [12] Kiihn, L.C, Schulman, H.M. and Ponka, P., Iron-Transferrin Requirements and Transferrin Receptor Expression in Proliferating Cells. In P. Ponka, H.M. Schulman and R.C. Woodworth (Eds.) Iron Tran~sport and Storage, CRC Press, Boca Raton, 1990, pp. 149-191. [13] McGeer, P.L., Akiyama, H., Kawamata, T., Yamada, T., Walker, D.G. and Ishii, T., Immunohistochemicallocalization of beta-amyloid precursor protein sequences in Alzheimer and normal brain tissue by hightand electron microscopy, J. Neurosci. Res, 31 (1992) 428-442. [14] Morris, C.M., Candy, J.M, Bloxham, C.A. and Edwardson, J.A., Immunocytochemical Localisation of Transferrin in the Human Brain, Acm Anat,, 143 (19921 14-18. [15] Rose, T.M., Plowman, G.D., Teplow, D.B.. Dreyer, W.J., Hellstrrm. K.E, and Brown, J.P., Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence, Pr~c'. NatL Acad. Sci. USA, 83 (1986) 1261-1265. [16] Sciot, R., De Vos, R., van Eyken, P., van der Steen, K., Moerman, P. and Desmet, V,J., In situ localization of melanotransferrin (melanoma-associated antigen P97) in human liver. A light- and electronmicroscopic immunohistochemical study. Li~:er, 9 (19891 110-119. [17] Shih, Y.J., Baynes, R.D., Hudson, B.G., Flowers, C.H., Skikne, B.S. and Cook, J.D., Serum Transferrin Receptor Is a Truncated Form of Tissue Receptor, J. Biol. Chem., 265 (19901 19077-19081. [18] Ueda, F., Raja, K.B., Simpson, R.J., Trowbridge, I.S. and Bradbury, M.W.B., Rate of 59Fe Uptake into Brain and Cerebrospinal Fluid and the Influence Thereon of Antibodies Against the Transferrin Receptor, J. Neurochem., 60 (19931 106--113. [19] Woodbury, R.G, Brown, J.P., Loop, S.M., Hellstrfm, K.E. and Hellstr/3m, I., Analysis of Normal Neoplastic Human Tissues for the Tumor-associated Protein p97, Int. J. Cancer, 27 (19811 t45-149. [20] Woodbury, R.G., Brown, J.P., Yeh, M.Y,. Hellstrrm, I. and ttellstr~im, K.E., Identification of a cell surface protein, p97, in human melanomas and certain other neoplasms. Proc. Natl. Acad. Sci. USA, 77 (19801 2183~2187.
Brain Research 712 (1996) 117-121
Research report
Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium Sylvia Rothenberger a, Michael R. Food a, Reinhard Gabathuler a Malcolm L. Kennard a Tatsuo Yamada b, Osamu Yasuhara b, Patrick L. McGeer b, Wilfred A. Jefferies a' * Biotechnology Laboratory and Departments of Medical Genetics, Microbiology and Immunology_, and Zoology, University of British Columbia, Vancouver, British Columbia V6T lZ3, Canada Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia V6T I Z3, Canada Accepted 24 October 1995
Abstract
One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium [7]. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form, We have recently demonstrated that GPl-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor [10]. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain. Kevwords: Blood brain barrier; Transferrin receptor; Capillary endothelium; Melanotransferrin; Human brain
I. Introduction
Iron is required for cellular energy processes [12]. It is generally thought that transferrin (Tf) delivers iron to cells by binding to cellular transferrin receptors (TRs) which then become internalized [11] and release the iron into the cell prior to iron utilization or storage of iron complexed with ferritin. The route of iron uptake into the brain is not clearly defined since iron acquisition must first involve translocation across the blood brain barrier (BBB). TRs are expressed on capillary endothelial cells and they are also sporadically and sparsely expressed on some neurons [7].
Abbreviations: BBB, blood brain barrier; TR, transferrin receptor; MTf, melanotransferrin; Tf, transferrin; GPI, glycosylphosphatidylinositol. * Corresponding author. Fax: (1) (604) 822-6780. /1006-8993/96/$15.00 © 1996 Elsevier Science B,V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 4 0 8 - X
In contrast, Tf and ferritin are found predominantly in association with glial cells [4,14]. It has been reported that iron transport into brain can be significantly inhibited by blockade of TRs with specific antibodies [18]. Nevertheless, hypotransferrinemic mice have been shown to have higher than normal iron uptake into the brain [18] and it has been recently reported that a significant amount of iron is transported into the brain by a route independent of Tf [18]. Melanotransferrin (MTf), or the p97 protein, may account for some of these discrepancies. It was first identified on the surface of melanoma cells [3,19,20] and has a structure which resembles serum Tf [15]. It binds iron [2], and has been shown to directly mediate cellular iron uptake [10]. It is found either attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor [1,5], or in the serum as a soluble form [3]. In addition to expression on melanoma cells, MTf has been reported to
118
S. Rothenberger et al. / Brain Research 712 (1990) 117-121
be expressed by a subset of liver cells [16] and in some cells of the fetal intestine [3]. There is growing interest in the transport of iron into the brain. Of special significance are models which utilize a monoclonal antibody, MRC-OX-26 [7,8], against the rat TR to deliver therapeutic compounds into the brains of rats [6]. It has been proposed that a viable human therapy for brain diseases could be based on the shuttling of compounds via specific receptor into the brain. Clearly, for this approach to reach its therapeutic potential an exact knowledge of the function of the TR and the routing of iron into and out of the brain must be derived. With our knowledge that p97 is directly able to transport iron into cells, we sought to analyze the expression of potentially important iron transport proteins in normal brains.
to immunoprecipitation. The primary antibodies used were the L235 and OKT9. Immunoprecipitation and SDS-PAGE were carried out as previously described [9].
2.3. Brain sections Seven brains from cases aged 54-82 were studied. The seven cases were all free from neurological signs and symptoms and standard neuropathological examination re-
2. Materials and methods
2.1. Sequential immunoprecipitation The human melanoma cell line SK-MEL-28 (HTB 72) and the CHO lines WTB and p97aWTBc3 were cultured as previously described [5]. The SK-MEL-28 cell line monolayers were washed with DMEM without methionine and then were biosynthetically labelled for 24 h with 150 /xCi/ml of [35S]methionine in supplemented DMEM. The cell lysates and supernatants were pre-cleared by centrifugation at 100,000 × g for 1 h. In the first immunoprecipitation, the primary antibodies used were the L235 (antihuman MTf, 1:1000 dilution, mouse monoclonal, IgG I, American Type Culture Collection (ATCC) HB8446); and the OKT9 (anti-human TR, 1:1000 dilution, mouse monoclonal, IgG 1, ATCC CRL 8021). The same antibodies were used on the same preparations in the second immunoprecipitation. For the third immunoprecipitation, the L235 was used on the sample which had previously been cleared of the TR, and the OKT9 was used on the sample which had previously been cleared of MTf. All samples were analyzed by SDS-PAGE under non-reduced conditions. The gels were subsequently fixed, dried and autoradiographed.
2.2. Pulse-chase labelling SK-MEL-28 cell line monolayers were maintained in medium lacking methionine for 1 h prior to labelling. Biosynthetic labelling of cells was done for 15 min with 2 ml of 150 /.LCi/ml of [35S]methionine per petri dish. Cells were then chased with normal medium containing an excess of cold methionine for the times indicated. A separate petri dish was used for each of the times. The cells were lysed in 20 mM Tris-HC1, pH 7.2, 150 mM NaC1, 2 mM EDTA, and 1% NP-40 with 20 /zg/ml phenylmethylsulfonyl fluoride (PMSF). The lysates and cell supernatants were then cleared by centrifugation prior
p97
B
TR
Cells Medium Cells Medium
kDa
1
2 3 4 5 6 7 8
0
6
97.4 ,.J UJ
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69
97.4 t--
97.4 0
r~
o~
69
Chase (hours)
0
6
0
6
0
6
Fig. 1. A: sequential immunoprecipitation of MTf and TR from labelled SK-MEL-28 cells using anti-MTf (L235) and anti-TR (OKT9) antibodies. After 24 h of incubation with [35S]methionine, cells were lysed and both cell lysates and media were subjected to three sequential immunoprecipitations with these antibodies. The immunoprecipitates were analyzed on a 10-15% SDS-PAGE gel under non-reducing conditions, and an autoradiogram was developed after 24 h of exposure of the gel. The migration of the molecular mass markers is indicated on the left side of the gel. B: expression of human MTf and human TR in cultured cells of human SK-MEL-28 melanoma line, the CHO line WTB, and the MTf expressing CHO line p97aWTBc3. Each line was biosynthetically labelled with [35S]methionine and chased with cold methionine for 6 h. Cell lysates (lanes 1,2,5,6) and cell supernatants (lanes 3,4,7,8) were immunoprecipitated with the L235 MAb (lanes 1,2,3,4) or with the OKT9 MAb (lanes 5,6,7,8). Molecular mass markers are indicated on the left of the figure. Gels represented on the figure were exposed for 40 h.
S. Rothenberger et aL / Brain Research 712 (1996) 117-121
vealed no abnormalities in the brain tissue. Brains in all cases were obtained 2-32 h after death. Small blocks were dissected from various brain regions. These were the angular, midtemporal and precentrai cortices, the hippocampal formation including the entorhinal cortex, caudate, putamen, globus pallidus, substantia nigra, and the cerebral white matter and spinal cord, but not all regions were examined in all types of cases. The blocks were fixed for two days in phosphate-buffered 4% paraformaldehyde and then transferred to a maintenance solution of 15% sucrose in 0.1 M phosphate buffer pH 7.4, and kept in the cold until used. Sections were cut on a freezing microtome at 30 /xm thickness and stained by single or double immunohistochemical procedures [13]. The following antibodies were used in immunohistochemical studies: anti-human MTf, (L235); anti-human Tf (A-061, 1:10,000 dilution, rabbit polyclonal, DAKO); and three anti-human TR antibodies: OKT9; HTR-H684, 1 /xg/ml, purified mouse monoclonal; and HTR-Ro~S, 1:10,000 dilution, rabbit polyclonal. The last two antibodies were gifts of Dr. Ian Trowbridge (Salk Institute, San Diego). HTR-H684 was
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raised against the TR cytoplasmic domain, while the HTRR a S antiserum was raised against the TR external domain.
2.4. Electron microscopy For electron microscopy, blocks of entorhinal cortex from two cases of Alzheimer's Disease (AD) were fixed in 1% glutaraldehyde/4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 24 h at 4°C, followed by immersion in 15% sucrose in 0.1 M phosphate buffer, pH 7.4, for several days at the same temperature. Sections were cut by vibratome at 50 /xm thickness and incubated with the L235 MAb (1:1000) for 5 days at 4°C. They were then treated with the appropriate Vectastain and ABC secondary antibody systems. After the diaminobenzidine (DAB) reaction, the sections were osmified, dehydrated and embedded in Epon. Ultra-thin sections were cut and examined with a Phillips EM201 electron microscope without counterstaining.
Fig. 2. lmmunohistochemical staining of brain for MTf (A,E), TR (B,C) and Tf (D). A: normal angular gyrus cortical gray matter stained with L235 MAb. Capillary endothelium is strongly positive. B: normal precentral cortical grey matter stained with rabbit anti-TR antiserum (HTR-Rt~S). Capillary endothelium is positive. C: normal angular gyrus from a nearby section to that shown in A stained with the OKT9 anti-TR MAb. In this section, only capillaries are stained although other sections show occasional weak neuronal staining. D: normal angular cortex stained with anti-Tf polyclonal antibody. The sparse cytoplasm of a few cells resembling oligodendrocytes are positive. E: electron micrograph of MTf expression in an endothelial cell. An ultrathin human brain section was labelled with the L235 MAb (see Section 2 for details). The resulting electron micrograph is shown at 1612 × magnification. Notice the unstained nucleus of the endothelial cell (n) and the heavy DAB reaction product in the cell cytoplasm (cy). A - D arc at the same magnification (bar in C = 50 /xm).
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3. Results
3.1. Sequential immunoprecipitation and pulse chase labelling In order to investigate the specificities of the antibodies used, the SK-MEL-28 cells were biosynthetically labelled with [35S]methionine and MTf and the TR were immunoprecipitated from both the cell lysate and the media (Fig. 1A). In the first immunoprecipitation, it is clear that the L235 antibody recognized a protein with a molecular mass of 95 kDa, corresponding to MTf, in both the cell lysate and media (lanes 1 and 3). Under similar conditions, the OKT9 antibody recognized a protein with a molecular mass of 180 kDa (lane 2) in the cell lysate and 85 kDa (lane 4) in the media, corresponding to the TR. In the second immunoprecipitation the same antibodies were again used to precipitate any remaining proteins. The residual MTf was removed from the samples (lanes 5 and 7). Similarly, the residual TR was also removed (lanes 6 and 8). In the third immunoprecipitation, the samples were exchanged and subjected to the OKT9 antibody in the case of the L 2 3 5 / M T f cleared preparation, and the L235 antibody in the case of the O K T 9 / T R cleared preparation. It is apparent that a protein with a molecular mass of 180 kDa is present in the cell lysate of the MTf depleted preparation (lane 9) and a protein with a molecular mass of 85 kDa is present in the media of the MTf depleted preparation (lane 11). These proteins correspond to the two forms of the human TR which have been described previously [17]. In a similar manner, a protein with a molecular mass of 95 kDa is present in both the cell lysate (lane 10) and the media (lane 12) of the TR depleted preparation. This protein corresponds to MTf. These results indicate conclusively that the L235 and OKT9 antibodies recognize MTf and the TR, respectively, and there is no apparent cross-reactivity. In addition, we have followed by pulse-chase, the fates of MTf and the TR after biosynthetic labelling of SKMEL-28, WTB and p97aWTBc3 cells. The L235 MAb (Fig. 1B, lanes 1, 2, 3, 4) recognized a protein with a molecular mass of 93 kDa (Fig. 1B, lane 1) that is processed to a higher molecular mass of 95 kDa after 6 h of chase (Fig. 1B, lanes 1, 2). This protein is not seen in WTB cells (Fig. 1B, lanes 1, 2). A considerably greater amount of this protein is present in the transfected cells, p97aWTBc3 (Fig. 1B, lanes 1, 2), identifying this protein as MTf. In addition, a soluble form of MTf is present in the cell supernatant after 6 h chase (Fig. 1B, lane 4). The presence of a 70 kDa band is likely to be due to a p97 degradation product and p97 appears to be more sensitive to degradation at earlier time points in the pulse-chase. The OKT9 MAb (Fig. 1B, lanes 5, 6, 7, 8) recognizes a protein with a similar mass in SK-MEL-28 cells corresponding to the reduced form of the human TR (Fig. 1B, lanes 5, 6). The human TR is not seen in the cell supernatant in this
figure (Fig. 1B, lanes 7, 8). It is clear that the L235 and OKT9 MAbs do not cross react with hamster MTf and TR in the CHO line WTB. These data separately and collectively establish the specificity of the anti-MTf and the principal anti-TR MAb used in this study. They confirm that MTf and the TR are synthesized and transported to the cell surface. Additionally, we can identify a soluble form of MTf in the medium [5].
3.2. Expression of MTf, Tf and the TR in brain tissues The cellular distributions of MTf, Tf and the TR were determined immunohistochemically in human brain tissue (Fig. 2). In brain cortex, capillary endothelium was strongly stained by antibodies to both MTf (Fig. 2A) and the three antibodies to the TR, two of which are illustrated (Fig. 2B,C). A few neurons showed relatively weak positive staining for the TR, as previously described [7], but none are visible in Fig. 2B. Furthermore, the anti-Tf MAb stained mostly glia (Fig. 2D) in agreement with a previous report [4]. These data establish the coincident expression of the TR and MTf on capillary endothelium and the lack of coincident expression of Tf with either the TR or with MTf. 3.3. Distribution of MTf in brain capillaries at the electron
microscopic level Electron microscopy was used to define the structures expressing MTf in capillaries. By this method the DAB reaction products in sections stained with the anti-MTf antibody were found in the cytoplasm of endothelial cells (Fig. 2E). These results suggest a role for MTf mediated metal transport through brain endothelium.
4. Discussion
The delivery of nutrients into the brain is well established. The nutrients must pass from the bloodstream through the capillary endothelium of the brain before being distributed to the cells within the brain. The detailed mechanism which allows crossing of the BBB remains unresolved. In principle, several alternative methods of traversing the brain endothelium could exist. However, the most likely of these involves the active transcytosis of ligands linked to receptors. The ligands may be delivered unaltered on the sublumenal surface and dissociate from the receptors. Alternatively, the ligands and possibly the receptors may in some way be altered during transcytosis resulting in the release of altered ligands at the sublumenal surface. In this study, we have shown that two proteins involved in iron binding and transport, MTf and the TR, are present in brain endothelial cells of all human brains studied. The expression of TR by brain endothelial cells and occasional
S. Rothenberger et al. / Brain Research 712 (1996) 117-121
neurons is consistent with previous work on its distribution within the brain [7]. A disparity between the localization of TR and Tf has previously been noted [4,7,8]. This difference in localization raises questions as to whether T f is exclusively responsible for mediating iron transport into and within the brain. Indeed, recent observations reveal the existence of mechanisms for iron transport across the b l o o d / b r a i n barrier which are exclusive o f Tf [18]. Our findings that: (i) MTf is expressed in capillary endothelium; (ii) that its expression is coincidental with the TR; and (iii) that M T f can act as an iron transport protein [10] are consistent with the hypothesis that M T f has a role in iron uptake in human brain. It is possible that M T f may directly deliver iron across the BBB in an analogous or competitive method to the TR. Alternatively, MTf may function in the excretion of iron from the brain into the blood stream [1], Finally, it is possible that M T f may function to limit the presence of free iron within the brain which would catalyze the formation of potentially harmful super oxide or hydroxyl radicals, which in turn could damage normal brain tissues. Substantial attention has been devoted to the development of compounds which transport therapeutics into the brain. Many of these are based on the use of drugs conjugated to antibodies against the TR. Likewise, antibodies against M T f may have utility in the deliverance of therapeutic compounds into the brain. The use of such compounds is presently under investigation but is hampered by the lack of specific reagents in species other than human.
Acknowledgements This work was supported by grants to W.A. Jefferies by the British Columbia Health Care Research Foundation, the Medical Research Council of Canada, the Vancouver Foundation and Synapse Technologies Inc. This work was also supported by grants to P.L. McGeer, Tatsuo Yamada, and Osamu Yasuhara by the A l z h e i m e r ' s Society o f British Columbia and individual British Columbians.
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