Western blotting analysis in patients with MS using human brain vessels as antigen Souberbielle BE, Swingler RJ, Davidson DLW, Cull RE, Atkinson S, Davison I, Anderson J, Bell JE, Russell WC. Western blotting analysis in patients with MS using human brain vessels as antigen. Acta Neurol Scand 1992: 86: 397-402. 0 Munksgaard 1992
B. E. Souberbielle”, R. J. Swingier', D. L. W. Davidson3, R. E. Cull2, S.Atkinson 1. Davison4, J. Anderson J. E. Bell6, W. C. Russell’
’,
’,
’
Departments of Biochemistry and Microbiology, Gatty laboratory, University of St Andrews, Neurology, Royal Infirmary, Edinburgh, Dundee Royal Infirmary, Pathology, Ninewells Hospital, Dundee, Neuropathology, Western General Hospital, Edinburgh, Scotland
I
Serum samples from patients suffering from multiple sclerosis, other neurological diseases and normal controls were screened by “western blotting” for antibody directed against proteins of human brain vessels purified from a post mortem brain. A small number of sera contained autoantibodies against some of the proteins of the brain vessels, particularly in patients suffering from MS, epilepsy and migraine. The significance of these results is discussed.
Multiple sclerosis (MS) has some features of an autoimmune disease but the target antigen (s) is still unknown. Possible autoantigens include myelin proteins (1) and components of the brain vessels (2) and auto-antibodies, with or without complement, may have a role in the autoimmune reaction (3). There are many features of MS which lead to the hypothesis that the BBB rather than myelin may play an important role in the aetiopathogenesis of MS. The typical early MS lesion is perivascular and serial sections have shown that plaques can extend for a considerable distance along the course of a vessel. MS does not just involve the myelin as plaques have also been described in the cortex and central grey matter (4). In addition, periphlebitis retinalis in MS patients is characterised by white cuffs surrounding segments of one or several retinal veins, although retinae lack myelin (5). MRI studies show that in the relapsing-remittingform of the disease, disruption of the BBB is an early event (6). Lastly, Adams and his colleagues have shown that in some MS lesions the cerebral venular wall is the site of lymphocytic infiltration that may initially be confined to the vessel wall alone (2). The only report of the presence of autoantibodies against brain endothelial cells in patients suffering from MS is by Tsukada et al., who found antibodies to rat brain endothelial cells in patients with MS
Key words: multiple sclerosis: epilepsy; migraine; blood brain barrier; immunoblotting B.E. Souberbielle, Department of Cellular and Molecular Sciences, Oncology Unit, Jenner Wing, St Georges Hospital Medical School, Cranmer Terrace, London SW17 ORE, England Accepted for publication March 1 1, 1992
and in patients suffering from HTLV 1 associated myelopathy using an ELISA assay (7) and then showed that sera from MS patients had antibodies directed to low molecular weight proteins (8, 11, 12.3 kd) (8). The “western blotting” technique has been used to define autoantigens in some autoimmune diseases and in paraneoplastic syndromes of suspected autoimmune origin (9-12). It is a useful technique for the detection of antibodies to proteins separated by molecular weight from an heterogeneous mixture. The use of this method with human brain vessels as antigens and with sera of patients suffering from multiple sclerosis as the source of potential autoantibodies has not been previously reported. If there are indeed antibodies against brain vessels in MS, this technique could help to identify and characterise further the autoantigen(s) at the molecular level. It may also be possible to relate potential autoantibodies to components specific for the BBB which have already been partially characterised (13, 14). Material and methods Preparation and characterisation of the human brain vessel antigens
Human brain vessels were prepared from a single brain < 36 h after death following a modified method described by Brendel et al. (15); all procedures were 397
Souberbielle et al. done at + 4 O C. A post mortem piece of central white and grey matter (about 10 g) stripped of the meninges and the surface vessels was cut into small pieces (2 mm’) in Hanks buffer solution containing 5 IU of heparin/ml, 15 mM glucose, penicillin and streptomycin. This was then homogenised using a hand teflon glass homogeniser of 300 pm aperture (20 up and down strokes) and then with a homogeniser of smaller aperture (150 pm) by the same number of strokes. The homogeneate was filtered through a 210 pm nylon mesh sieve (John Straniard, Manchester) and washed with Hanks buffer solution. Material trapped on the mesh was then processed again through the same cycle described above but the filtration was done through a 110 pm nylon mesh sieve. The vessels were collected on the 110 pm sieve and the purity of the preparation assessed by light microscopy using a cytocentrifuged preparation and staining by toluidine blue. By using these techniques it was possible to establish that the centrifugate contained more than 95% brain vessels of different size and the majority of the purified vessels were small ( < 20 pm in diameter - branched and non-branched) but bigger vessels and small arterioles were also present (Fig. 1). The cytocentrifuged purified vessels were positive for von Willebrand factor (an endothelial cell specific protein) with indirect immunofluorescence using a penetration buffer to permeabilise
Fig. 1. Purified brain vessels stained with toluidine blue ( x 100).
398
the cells and were also positive for y-glutamyl transpeptidase (yG.Tr.Pase) (an enzyme which is associated with brain endothelial cells) (16). SEM of the brain vessels‘ preparation
The vessels were also visualised by scanning electron microscopy (Fig. 2). Purified vessels in chilled Hanks buffer solution with heparin and antibiotics were centrifuged at 1000 g for 10 min. After change of medium by gentle aspiration the vessels were incubated at 4 ° C in 4% glutaraldehyde in phosphate buffer saline overnight. Immediately before preparation of the vessels for SEM, the vessels were dehydraded through increasing concentration of alcohol in PBS: 30 min at 50%, 15 min at 7 5 % , 15 min at 95 % , and twice for 15 min in absolute alcohol (between each incubation the purified vessels were allowed to sediment and the alcohol solution sucked off). The purified vessels were then allowed to sediment on circular 10 mm microcoverslips (Horwell A.R. Ltd). The preparation was then put into a Samdri critical point dryer with alcohol replaced with liquid C 0 2 and dryed. It was then fixed with tape on a stup and placed in a EMSCOPE sputter water where it was covered in 10 nanometers of gold and viewed in a JEOL JSM 35 C F scanning electronmicroscope.
Western blotting & MS in 20% (v/v) acetic acid, 20% (v/v) methanol) and then destained for 1 min with the same solution minus the naphthalene black. The unstained blots were blocked in 10%. Marvel in PBS for 1 h, incubated with the sera (each serum dilution contained 120 pg of IgG per strip in 400 p1 of 1% Marvel/PBS; this corresponded to dilutions ranging from 1/25 to 1/60 for the tested sera) for 1 h, washed and then incubated with the second antibody viz an anti human polyvalent immunoglobulin linked to horse radish peroxydase (1/50 in 400 pl of 1% Marvel/ PBS) (Sigma). The reaction was visualised by diaminobenzidine (20). Fig. 2. Scanning electron microscopy of purified brain vessels (white bar = 100 pm).
Clinical study
M S patients and neurological controls were selected
by the neurologists and their clinical and biochemical data recorded. Eighteen normal sera from laboratory personnel were also tested. The assay was carried out in a blind fashion. The diagnoses were as follow: MS 45 patients, epilepsy 20, Parkinson’s disease 2, myasthenia gravis 2, migraine/headache 5, stroke 2, neuropathy (1 diabetic) 2, syncopy 2; 1 each of: vertigo, carpal tunnel syndrome, hyperventilation, anoxic brain damage, vasculitis, pineal tumor, cervical spondylitis, prolapsed disc, poliomyositis, hysteria, cramp and motor neurone disease. The mean age of the MS group was 45 years (range 27-67), the neurological controls 45 years (range 16-84) and the normal controls 28 years (range 2260).
Results
Six (13%) MS patients and 8 (17%) neurological controls showed some reactivity above the background against the brain vessels. The diagnoses of the neurological controls with autoantibodies to brain vessels were as follows: prolapsed disc 1 patient, headache/migraine 2, vertigo l, epilepsy 4. Among the 18 normal controls, only one (5%) normal individual reacted against a protein of around 45 Kd (Fig. 3). However, although the proportion of normal controls with autoantibodies to brain vessels was lower in this group compared to the patients with neurological disease (MS and neurological controls), the difference did not reach statistical significance ( ~ ~ 0 .chi-square 5, = 1.23). The blots of the positive sera and other control sera are shown in Fig. 3. The molecular weights of the proteins to which reactivity of these patients was found is shown
Western blotting assay
IgG concentration was determined for each serum by an immunodotblot technique (17) using standard controls (ICL scientific, California) and thus the assay was standardised for the amount of IgG. Vessels were denatured in electrophoresis buffer (containing SD S and beta-mercaptoethanol), heated at 75 O C for 5 min, electrophorised as described (18) using a 15% SDS PAGE minigel. Molecular weight standard markers (BRL, Bethesda research laboratory) were run in parallel in order to characterise the vessel proteins. The proteins were then blotted onto nitrocellulosepaper (19) for 1 h at 0.8 mA/mm2using a semidry blotter (LKB). A strip was cut out at each side of the blotted nitrocellulose including the molecular weight marker strip and in order to localise the protein bands stained for 1 min with naphthalene black (0.01% (w/v) naphthalene black powder
Fig. 3. Blots of the purified brain vessels incubated with the sera from the MS patients (1-6) (MS), the neurological controls (714) [Neurol. Cont.] and the normal control (15) [N] containing autoantibodies to the brain vessels. Strips incubated with one MS patient (16) and one neurological control (17) sera [ - ] without such autoantibodies are also included. Track 18 [ R ] shows the reaction with the rabbit anti-brain vessels prepared in the laboratory. Tracks 19 and 20 [ - ] show the strips incubated with the conjugate alone, (anti-human antibody (track 19) and anti-rabbit antibody (track 20)).
399
Souberbielle et al. Table 1. Molecular weight (kd) of the bands recognised by autoantibodies in the sera of the 6 MS patients (MS1-MSG), the 8 neurological controls (Cl-C8), the normal individual (N) (in the same order as Fig. 3) and the rabbit anti-human brain vessels (R)
MSl
MS2
MS3
MS4
MS5
MS6
C1
c2
C3
C4
C5
300 250 190 110
190
190
C6
C7
C8
N
R
43
43
300 250 190 110
190 110
97
64
64 50
43 30
50
30 22
in Table 1. There was no non-specific background using the brain vessels as antigens and the negative control (conjugate alone) as shown in Fig. 3, Lane 19 (anti-human IgG) and lane 20 (anti-rabbit IgG). The positive antisera available was a rabbit antisera produced after injection of purified human brain vessels in complete Freund adjuvant. This antisera produced little reactivity with only 2 proteins recognised, one around 43 kd and one around 22 kd. The concentration of total IgG was not different between the different groups (MS: 11.6 mg/ml SD = 1.28; neurologcal controls: 11.86 mg/ml SD = 1.77; normal control: 11.55 mg/ml SD = 1.1) and when calculated for the patients with autoantibodies to brain vessel, it was also similar to the general population (1 1.4 mg/ml SD = 0.99). Discussion
There is some evidence in favour of the autoimmune theory of MS (1). Myelin has been the prime credible target but other components of the nervous system may be involved. In this report we have concentrated on the brain vessels - a component of the blood brain barrier (BBB) - as possible targets and investigated the humoral immune response using the “western blotting” technique. There was no statistical difference in the prevalence of autoantibodies to the brain vessels using this technique between the MS group and the other neurological patient or normal control groups although the proportion of individuals with autoantibodies to the brain vessels in the neurological patient groups was higher than in the normal control group. A report that patients suffering from MS and HTLV 1-associated myelopathy have autoantibodies to endothelial cells (7) (21) was based on ELISA tests using rat endothelial cells as antigen and with low dilution (< 1/10) of the tested sera. As these observations needed to be assessed using other methods we decided to use the immunoblotting technique.
400
50
43 22
26 22
22
22
The antibody detection system used in the present report is a relatively sensitive method but as proteins are denatured by the electrophoresis buffer it may be that in the process some critical conformational epitopes are lost. On the other hand, the vessels were prepared from a post-mortem brain less than 36 h after death and Choi et al. showed no difference in the protein pattern and functional integrity between rabbit brain capillaries isolated either at 0 or 42 h post mortem (22). Moreover, the purified vessels expressed von Willebrand factor and y-glutamyl transpeptidase indicating the integrity of the proteins of the vessels. As with other studies the source of antigens was from a single brain. Different brains may express different antigens and perhaps only brains of multiple sclerosis patients will express relevant antigens for the MS process. This was not explored in the present report. Bearing in mind the limitation of the technique, there was no strong evidence in the present study that patients suffering from MS are more prone to develop autoantibodies against the BBB than other neurological controls. The reaction detected in the positive sera was not directly demonstrated to be Fab specific and there is a possibility that some of the positive bands detected were due to the reaction of immunoglobulins with Fc receptors expressed on the brain vessels. However, this possibility seems unlikely as no bands was detected homogeneously across the majority of the positive sera and moreover a large proportion of the sera tested did not react with the brain vessels. The higher proportion of sera positive for autoantibodies in neurological patients compared to normal controls may be due to the relatively younger age of the normal group as the prevalence of autoantibodies may increase with age. However, the only normal individual who showed autoantibody against the brain vessels was only 25 years of age and there was no correlation with age among the neurological patients (MS and others) who showed autoantibodies against the brain ves-
Western blotting & MS sels: the mean age was 45 years - range: 29-67 (n = 14). The reaction seen in some individuals may in fact be a consequence of a neurological disease rather than a cause of the disorder. Proteins detected by “western blotting” are denatured before electroblotting. It may be that the autoantibodies corresponding to these proteins are particularly prevalent in neurological diseases. It has been hypothesised by Grabar that autoantibodies are physiologically normal, as natural autoantibodies may have a role in disposing of the product of metabolism and catabolism (23). Some researchers have shown that some autoantibodies occur in normal individuals, most of them being against cytoskeleton proteins (24,25). In the same way, Babikian and colleagues described the detection of autoantibody “bands” on “western blots” after incubation of sera from normal individuals with spinal cord extracts (12) and Newcombe and colleagues detected autoantibodies against a number of central nervous system proteins by western blotting in the sera of normal individuals and of neurological patients (26). In neurological diseases, proteins may be released into the circulation because of the disease process and these proteins could induce a humoral immune response. Although the numbers are very small, it appears that the neurological patients suffering from epilepsy or migraine/ headache and with autoantibodies to brain vessels detected on “western blotting” were overepresented: epilepsy (20 %), migraine/headache (40 %) but the significance of these findings is uncertain (27). It is also worth considering that the autoantibodies directed to brain vessels in some neurological patients may be related to vessel-specific components. Brain vessels are distinguished by the presence of tight junctions between endothelial cells which form the blood brain barrier (BBB) and may confer immunological “privilege” to the brain (28). Although little is known about the components responsible for the neural specificity of the brain vessels, some interesting features have already been uncovered and some proteins related to the BBB partially characterised. The exact location of the proteins detected by autoantibodies on “western blotting” is not known. It has been shown that the protein composition of the luminal membrane is different from that of the antiluminal membrane of the brain vessels (29). If autoantibodies to brain vessels are important in the aetiopathogenesis of MS, it is likely (but not necessary) that they will recognise antigens situated on the luminal side of the brain vessels. Pardrigde and colleagues raised an antisera against a 46 kd protein which seemed to be specific for bovine brain capillaries (13). In the same molecular weight region, an inducible BBB specific molecule called HT7 of around 45-52kd has been
characterised in chick brain microvessels (14). Sternberger & Sternberger raised a monoclonal antibody named anti-endothelial-barrier-antigen(EBA) which appeared to be specific for brain vessels including optic nerve, spinal cord and retina. The monoclonal antibody reacted also with some vessels in the spleen and some cells in the epidermis of the skin and recognised a 30/25/23.5 kd triplet (30). Other proteins have been located on the brain vessels e.g transferrin receptor (a 95 kd dimer) (31), insulin receptor (127 kd protein) (32), P-glycoprotein (170-180 kd) (33) and proteins involved in glucose transport and other transport systems (34-35). Pardrigde and colleagues have shown that low molecular weight proteins (14, 16, 17, 18 kd) in isolated brain capillaries are histones (36). Some of the proteins detected by autoantibodies in our study (Table 1) may correspond to these characterised brain vessels proteins. The histopathology of MS suggests that the MS lesions could be in part due to an immune response to brain vessels as part of an autoimmune reaction or because of a viral infection of the brain vessels. Detection of autoantibodies to brain vessels in patients suffering from neurological diseases especially MS has been difficult because of the limitations of the assays used. In this study antibodies against BBB epitopes were no more in MS patients than controls but there is a need for further studies looking at the prevalence of autoantibodies to brain vessels in patients with neurological diseases and new assays should be devised to address this question. Acknowledgements B.E.S. was supported by the Wellcome Trust and the Multiple Sclerosis Society of Great Britain.
References 1. WAKSMAN BH, REYNOLDSWE. Multiple sclerosis as a disease of immune regulation. Proc SOCExp Biol Med 1984: 175: 282-294. 2. ADAMSCWH, POSTONRN, BUKSJ, SIDHUYS, VIPOND H. Inflammatory vasculitis in multiple sclerosis. J Neurol Sciences 1985: 69: 269-283. 3. COMPTSON DAS, MORGANBP, CAMPBELL AK et al. Immunocytochemical localization of the terminal complement complex in multiple sclerosis. Neuropath App Neurobiol 1989: 15: 307-316. 4. ADAMSCWM. A colour atlas of multiple sclerosis & other myelin disorders. London: Wolfe Medical, 1989. 5. ENGELT, HUIDBERG A, UHRENHOLDT A. Multiple sclerosis, periphlebitis retinalis and cerebro-spinalis. A correlation between periphlebitis retinalis and abnormal technecium brain scintigraphy. Acta Neurol Scand 1984: 69: 293-297. 6. KERMODE AG, TOFTSPS, MACMANUS DB et al. Early lesion of multiple sclerosis. Lancet 1988: ii: 1203-1204. 7. TSAKUDA N, TANAKA Y, YANAGISAWA N. Autoantibodies to brain endothelial cells in the sera of patients with human T-lymphotropic virus type I associated myelopathy and other demyelinating disorders. J Neurol Science 1989: 90: 33-42.
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Souberbielle et al. 8. TSAKUDA N, TANAKAY, MIYAGIK, YANAGISAWA N, OKANOA. Autoantibodies to each protein fraction extracted from the cerebral endothelial cell membrane in the sera of patients with multiple sclerosis. J Neuroimmunol 1989: 24: 41-46. 9. TOWBIN H, GORDON J. Immunoblotting and dot immunobinding - current status and outlook. J Immunol Methods 1984: 72: 313-340. 10. LYNCHDM, HOWESE. Antigenic determinants in idiopathic thrombocytopenic purpura. Br J Haematoll986: 63: 30 1-308. 11. DE KEYSERF, VERBRUGGEN G , VEYSEM. Identification of a new antinuclear antibody by immunoblotting. Lancet 1989: ii: 927. 12. BABIKIAN VL, STEFANSSON K, DIEPERINK ME, ARNASON BGW, MARTONLS, LEVYBE. Paraneoplastic myelopathy: autoantibodies against protein in normal spinal cord and underlying neoplasm. Lancet 1985: ii: 49-50. 13. PARDRIGDE WM, YANGJ, EISENBERG J, MIETUSLJ. Antibodies to blood-brain barrier bind selectively to brain capillary endothelial lateral membranes and to 46K protein. J Cereb Blood Flow Metab 1986: 6: 203-211. 14. SEULBERGER H, LOTTSPEICH F, RISAUW. The inducible blood-brain barrier specific HT7 is a novel immunoglobulinlike cell surface glycoprotein. EMBO J 1990: 9: 2151-2158. 15. BRENDEL K, MEEZANE, CARLSON EC. Isolated brain microvessels: a purified, metabolically active preparation from bovine cerebral cortex. Sciences 1974: 185: 953-955. LE, CANCILLA PA. y-Glutamyl transpeptidase in 16. DEBAULT isolated brain endothelial cells: induction by glial cells in vitro. Sciences 1980: 207: 653-655. 17. RANDALLRE, NEWMANC, HONESSRW. Isolation and characterization of monoclonal antibodies to structural and nonstructural proteins of herpesvirus saimiri proteins. J Gen Virol 1985: 66: 2199-2213. 18. O’BRIENMER, SOUBERBIELLE BE, COWANM et al. Glycoprotein pattern in normal and malignant cervical tissue. Cancer let 1991: 58: 247-254. 19. BURNETTE WN. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Bioch 1981: 112: 195-203. 20. HARLOW E, LANED. Antibodies: a laboratory manual. Cold Spring Harbor NY: Cold Spring Harbor Laboratory, 1988: 508. 21. TANAKA Y, TSUKADA N, KOH CH-S, YANAGISAWA N. Anti-endothelial cell antibodies and circulating immune complexes in the sera of patients with multiple sclerosis. J Neuroimmunol 1987: 17: 49-59.
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22. CHOITB, PARDRIDGE WM. Phenylalanine transport at the human blood-brain barrier, studies with isolated human brain capillaries. J Biol Chem 1986: 261: 6536-6541. 23. GRABAR P. Autoantibodies and the physiological role of immunoglobulins. Immunol Today 1983: 4: 337-340. 24. GUILBERT B, DIGHIERO G, AVRAMEAS S. Naturally occurring antibodies against nine common antigens in human sera I. Detection, isolation, and characterisation. J Immunol 1982: 128: 2779-2187. 25. STEFANSSON K, MARTONL, DIEPERINK M, MOLNARG, SCHLAEPFER W, HEGALSON C. Circulating autoantibodies to the 200000 daltons protein of neurofilaments in the serum of healthy individuals. Sciences 1985: 228: 1117-1 119. 26. NEWCOMBE JIA, GAHAN S, CUZNERL. Serum antibodies against central nervous system proteins in human demyelinating disease. Clin Exp Immunol 1985: 59: 383-390. 27. BOLWIGTG. Blood-brain barrier studies with special reference to epilectic seizures. Acta Psychiatr. Scand suppl 1988: 345: 15-20. 28. GOLSTEIN GW. Endothelial cell-astrocyte interactions: a cellular model of the blood-brain barrier. Ann NY Aca Scien 1988: 529: 31-39. 29. LIDINSKY WA, DREWES LR. Characterization of the bloodbrain barrier: protein composition of the capillary endothelial cell membrane. J Neurochem 1983: 41: 1341-1348. NH, STERNBERGER LA. Blood-brain bar30. STERNBERGER rier protein recognized by monoclonal antibody. PNAS USA 1987: 84: 8169-8173. 31. JEFFERIES WA, BRANDON MR, HUNTSV, WILLIAMS AF, GATTERS KC, MASONDY. Transferrin receptor on endothelium of brain capillaries. Nature 1984: 312: 162-163. WM, EISENBERG J, YANGJ. Human blood32. PARDRIDGE brain barrier insulin receptor. J Neurochem 1985: 44: 17711778. 33. GORDON-CARDO C, O’BRIEN JP, CASALS D etal. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at the blod-brain barrier sites. PNAS 1989: 86: 695-698. 34. KALARIA RN, GRAVINA SA, SCHMIDLEY JW, PERRYG, HARIKSI. The glucose transporter of the human brain and blood-brain barrier. Ann Neurol 1988: 24: 757-764. 35. PARDRIDGE WM, OLDENDORF WH, CANCILLA P, FRANK HJL. Blood-brain barrier: interface between internal medicine and the brain. Ann Int Med 1986: 105: 82-95. 36. PARDRIDGE WM, NOWLINDM, CALAYCAY J, SHIVELY JE. Predominant low-molecular-weight proteins in isolated brain capillaries are histones. J Neurochem 1989: 53: 10141018.
B. E. Souberbielle”, R. J. Swingier', D. L. W. Davidson3, R. E. Cull2, S.Atkinson 1. Davison4, J. Anderson J. E. Bell6, W. C. Russell’
’,
’,
’
Departments of Biochemistry and Microbiology, Gatty laboratory, University of St Andrews, Neurology, Royal Infirmary, Edinburgh, Dundee Royal Infirmary, Pathology, Ninewells Hospital, Dundee, Neuropathology, Western General Hospital, Edinburgh, Scotland
I
Serum samples from patients suffering from multiple sclerosis, other neurological diseases and normal controls were screened by “western blotting” for antibody directed against proteins of human brain vessels purified from a post mortem brain. A small number of sera contained autoantibodies against some of the proteins of the brain vessels, particularly in patients suffering from MS, epilepsy and migraine. The significance of these results is discussed.
Multiple sclerosis (MS) has some features of an autoimmune disease but the target antigen (s) is still unknown. Possible autoantigens include myelin proteins (1) and components of the brain vessels (2) and auto-antibodies, with or without complement, may have a role in the autoimmune reaction (3). There are many features of MS which lead to the hypothesis that the BBB rather than myelin may play an important role in the aetiopathogenesis of MS. The typical early MS lesion is perivascular and serial sections have shown that plaques can extend for a considerable distance along the course of a vessel. MS does not just involve the myelin as plaques have also been described in the cortex and central grey matter (4). In addition, periphlebitis retinalis in MS patients is characterised by white cuffs surrounding segments of one or several retinal veins, although retinae lack myelin (5). MRI studies show that in the relapsing-remittingform of the disease, disruption of the BBB is an early event (6). Lastly, Adams and his colleagues have shown that in some MS lesions the cerebral venular wall is the site of lymphocytic infiltration that may initially be confined to the vessel wall alone (2). The only report of the presence of autoantibodies against brain endothelial cells in patients suffering from MS is by Tsukada et al., who found antibodies to rat brain endothelial cells in patients with MS
Key words: multiple sclerosis: epilepsy; migraine; blood brain barrier; immunoblotting B.E. Souberbielle, Department of Cellular and Molecular Sciences, Oncology Unit, Jenner Wing, St Georges Hospital Medical School, Cranmer Terrace, London SW17 ORE, England Accepted for publication March 1 1, 1992
and in patients suffering from HTLV 1 associated myelopathy using an ELISA assay (7) and then showed that sera from MS patients had antibodies directed to low molecular weight proteins (8, 11, 12.3 kd) (8). The “western blotting” technique has been used to define autoantigens in some autoimmune diseases and in paraneoplastic syndromes of suspected autoimmune origin (9-12). It is a useful technique for the detection of antibodies to proteins separated by molecular weight from an heterogeneous mixture. The use of this method with human brain vessels as antigens and with sera of patients suffering from multiple sclerosis as the source of potential autoantibodies has not been previously reported. If there are indeed antibodies against brain vessels in MS, this technique could help to identify and characterise further the autoantigen(s) at the molecular level. It may also be possible to relate potential autoantibodies to components specific for the BBB which have already been partially characterised (13, 14). Material and methods Preparation and characterisation of the human brain vessel antigens
Human brain vessels were prepared from a single brain < 36 h after death following a modified method described by Brendel et al. (15); all procedures were 397
Souberbielle et al. done at + 4 O C. A post mortem piece of central white and grey matter (about 10 g) stripped of the meninges and the surface vessels was cut into small pieces (2 mm’) in Hanks buffer solution containing 5 IU of heparin/ml, 15 mM glucose, penicillin and streptomycin. This was then homogenised using a hand teflon glass homogeniser of 300 pm aperture (20 up and down strokes) and then with a homogeniser of smaller aperture (150 pm) by the same number of strokes. The homogeneate was filtered through a 210 pm nylon mesh sieve (John Straniard, Manchester) and washed with Hanks buffer solution. Material trapped on the mesh was then processed again through the same cycle described above but the filtration was done through a 110 pm nylon mesh sieve. The vessels were collected on the 110 pm sieve and the purity of the preparation assessed by light microscopy using a cytocentrifuged preparation and staining by toluidine blue. By using these techniques it was possible to establish that the centrifugate contained more than 95% brain vessels of different size and the majority of the purified vessels were small ( < 20 pm in diameter - branched and non-branched) but bigger vessels and small arterioles were also present (Fig. 1). The cytocentrifuged purified vessels were positive for von Willebrand factor (an endothelial cell specific protein) with indirect immunofluorescence using a penetration buffer to permeabilise
Fig. 1. Purified brain vessels stained with toluidine blue ( x 100).
398
the cells and were also positive for y-glutamyl transpeptidase (yG.Tr.Pase) (an enzyme which is associated with brain endothelial cells) (16). SEM of the brain vessels‘ preparation
The vessels were also visualised by scanning electron microscopy (Fig. 2). Purified vessels in chilled Hanks buffer solution with heparin and antibiotics were centrifuged at 1000 g for 10 min. After change of medium by gentle aspiration the vessels were incubated at 4 ° C in 4% glutaraldehyde in phosphate buffer saline overnight. Immediately before preparation of the vessels for SEM, the vessels were dehydraded through increasing concentration of alcohol in PBS: 30 min at 50%, 15 min at 7 5 % , 15 min at 95 % , and twice for 15 min in absolute alcohol (between each incubation the purified vessels were allowed to sediment and the alcohol solution sucked off). The purified vessels were then allowed to sediment on circular 10 mm microcoverslips (Horwell A.R. Ltd). The preparation was then put into a Samdri critical point dryer with alcohol replaced with liquid C 0 2 and dryed. It was then fixed with tape on a stup and placed in a EMSCOPE sputter water where it was covered in 10 nanometers of gold and viewed in a JEOL JSM 35 C F scanning electronmicroscope.
Western blotting & MS in 20% (v/v) acetic acid, 20% (v/v) methanol) and then destained for 1 min with the same solution minus the naphthalene black. The unstained blots were blocked in 10%. Marvel in PBS for 1 h, incubated with the sera (each serum dilution contained 120 pg of IgG per strip in 400 p1 of 1% Marvel/PBS; this corresponded to dilutions ranging from 1/25 to 1/60 for the tested sera) for 1 h, washed and then incubated with the second antibody viz an anti human polyvalent immunoglobulin linked to horse radish peroxydase (1/50 in 400 pl of 1% Marvel/ PBS) (Sigma). The reaction was visualised by diaminobenzidine (20). Fig. 2. Scanning electron microscopy of purified brain vessels (white bar = 100 pm).
Clinical study
M S patients and neurological controls were selected
by the neurologists and their clinical and biochemical data recorded. Eighteen normal sera from laboratory personnel were also tested. The assay was carried out in a blind fashion. The diagnoses were as follow: MS 45 patients, epilepsy 20, Parkinson’s disease 2, myasthenia gravis 2, migraine/headache 5, stroke 2, neuropathy (1 diabetic) 2, syncopy 2; 1 each of: vertigo, carpal tunnel syndrome, hyperventilation, anoxic brain damage, vasculitis, pineal tumor, cervical spondylitis, prolapsed disc, poliomyositis, hysteria, cramp and motor neurone disease. The mean age of the MS group was 45 years (range 27-67), the neurological controls 45 years (range 16-84) and the normal controls 28 years (range 2260).
Results
Six (13%) MS patients and 8 (17%) neurological controls showed some reactivity above the background against the brain vessels. The diagnoses of the neurological controls with autoantibodies to brain vessels were as follows: prolapsed disc 1 patient, headache/migraine 2, vertigo l, epilepsy 4. Among the 18 normal controls, only one (5%) normal individual reacted against a protein of around 45 Kd (Fig. 3). However, although the proportion of normal controls with autoantibodies to brain vessels was lower in this group compared to the patients with neurological disease (MS and neurological controls), the difference did not reach statistical significance ( ~ ~ 0 .chi-square 5, = 1.23). The blots of the positive sera and other control sera are shown in Fig. 3. The molecular weights of the proteins to which reactivity of these patients was found is shown
Western blotting assay
IgG concentration was determined for each serum by an immunodotblot technique (17) using standard controls (ICL scientific, California) and thus the assay was standardised for the amount of IgG. Vessels were denatured in electrophoresis buffer (containing SD S and beta-mercaptoethanol), heated at 75 O C for 5 min, electrophorised as described (18) using a 15% SDS PAGE minigel. Molecular weight standard markers (BRL, Bethesda research laboratory) were run in parallel in order to characterise the vessel proteins. The proteins were then blotted onto nitrocellulosepaper (19) for 1 h at 0.8 mA/mm2using a semidry blotter (LKB). A strip was cut out at each side of the blotted nitrocellulose including the molecular weight marker strip and in order to localise the protein bands stained for 1 min with naphthalene black (0.01% (w/v) naphthalene black powder
Fig. 3. Blots of the purified brain vessels incubated with the sera from the MS patients (1-6) (MS), the neurological controls (714) [Neurol. Cont.] and the normal control (15) [N] containing autoantibodies to the brain vessels. Strips incubated with one MS patient (16) and one neurological control (17) sera [ - ] without such autoantibodies are also included. Track 18 [ R ] shows the reaction with the rabbit anti-brain vessels prepared in the laboratory. Tracks 19 and 20 [ - ] show the strips incubated with the conjugate alone, (anti-human antibody (track 19) and anti-rabbit antibody (track 20)).
399
Souberbielle et al. Table 1. Molecular weight (kd) of the bands recognised by autoantibodies in the sera of the 6 MS patients (MS1-MSG), the 8 neurological controls (Cl-C8), the normal individual (N) (in the same order as Fig. 3) and the rabbit anti-human brain vessels (R)
MSl
MS2
MS3
MS4
MS5
MS6
C1
c2
C3
C4
C5
300 250 190 110
190
190
C6
C7
C8
N
R
43
43
300 250 190 110
190 110
97
64
64 50
43 30
50
30 22
in Table 1. There was no non-specific background using the brain vessels as antigens and the negative control (conjugate alone) as shown in Fig. 3, Lane 19 (anti-human IgG) and lane 20 (anti-rabbit IgG). The positive antisera available was a rabbit antisera produced after injection of purified human brain vessels in complete Freund adjuvant. This antisera produced little reactivity with only 2 proteins recognised, one around 43 kd and one around 22 kd. The concentration of total IgG was not different between the different groups (MS: 11.6 mg/ml SD = 1.28; neurologcal controls: 11.86 mg/ml SD = 1.77; normal control: 11.55 mg/ml SD = 1.1) and when calculated for the patients with autoantibodies to brain vessel, it was also similar to the general population (1 1.4 mg/ml SD = 0.99). Discussion
There is some evidence in favour of the autoimmune theory of MS (1). Myelin has been the prime credible target but other components of the nervous system may be involved. In this report we have concentrated on the brain vessels - a component of the blood brain barrier (BBB) - as possible targets and investigated the humoral immune response using the “western blotting” technique. There was no statistical difference in the prevalence of autoantibodies to the brain vessels using this technique between the MS group and the other neurological patient or normal control groups although the proportion of individuals with autoantibodies to the brain vessels in the neurological patient groups was higher than in the normal control group. A report that patients suffering from MS and HTLV 1-associated myelopathy have autoantibodies to endothelial cells (7) (21) was based on ELISA tests using rat endothelial cells as antigen and with low dilution (< 1/10) of the tested sera. As these observations needed to be assessed using other methods we decided to use the immunoblotting technique.
400
50
43 22
26 22
22
22
The antibody detection system used in the present report is a relatively sensitive method but as proteins are denatured by the electrophoresis buffer it may be that in the process some critical conformational epitopes are lost. On the other hand, the vessels were prepared from a post-mortem brain less than 36 h after death and Choi et al. showed no difference in the protein pattern and functional integrity between rabbit brain capillaries isolated either at 0 or 42 h post mortem (22). Moreover, the purified vessels expressed von Willebrand factor and y-glutamyl transpeptidase indicating the integrity of the proteins of the vessels. As with other studies the source of antigens was from a single brain. Different brains may express different antigens and perhaps only brains of multiple sclerosis patients will express relevant antigens for the MS process. This was not explored in the present report. Bearing in mind the limitation of the technique, there was no strong evidence in the present study that patients suffering from MS are more prone to develop autoantibodies against the BBB than other neurological controls. The reaction detected in the positive sera was not directly demonstrated to be Fab specific and there is a possibility that some of the positive bands detected were due to the reaction of immunoglobulins with Fc receptors expressed on the brain vessels. However, this possibility seems unlikely as no bands was detected homogeneously across the majority of the positive sera and moreover a large proportion of the sera tested did not react with the brain vessels. The higher proportion of sera positive for autoantibodies in neurological patients compared to normal controls may be due to the relatively younger age of the normal group as the prevalence of autoantibodies may increase with age. However, the only normal individual who showed autoantibody against the brain vessels was only 25 years of age and there was no correlation with age among the neurological patients (MS and others) who showed autoantibodies against the brain ves-
Western blotting & MS sels: the mean age was 45 years - range: 29-67 (n = 14). The reaction seen in some individuals may in fact be a consequence of a neurological disease rather than a cause of the disorder. Proteins detected by “western blotting” are denatured before electroblotting. It may be that the autoantibodies corresponding to these proteins are particularly prevalent in neurological diseases. It has been hypothesised by Grabar that autoantibodies are physiologically normal, as natural autoantibodies may have a role in disposing of the product of metabolism and catabolism (23). Some researchers have shown that some autoantibodies occur in normal individuals, most of them being against cytoskeleton proteins (24,25). In the same way, Babikian and colleagues described the detection of autoantibody “bands” on “western blots” after incubation of sera from normal individuals with spinal cord extracts (12) and Newcombe and colleagues detected autoantibodies against a number of central nervous system proteins by western blotting in the sera of normal individuals and of neurological patients (26). In neurological diseases, proteins may be released into the circulation because of the disease process and these proteins could induce a humoral immune response. Although the numbers are very small, it appears that the neurological patients suffering from epilepsy or migraine/ headache and with autoantibodies to brain vessels detected on “western blotting” were overepresented: epilepsy (20 %), migraine/headache (40 %) but the significance of these findings is uncertain (27). It is also worth considering that the autoantibodies directed to brain vessels in some neurological patients may be related to vessel-specific components. Brain vessels are distinguished by the presence of tight junctions between endothelial cells which form the blood brain barrier (BBB) and may confer immunological “privilege” to the brain (28). Although little is known about the components responsible for the neural specificity of the brain vessels, some interesting features have already been uncovered and some proteins related to the BBB partially characterised. The exact location of the proteins detected by autoantibodies on “western blotting” is not known. It has been shown that the protein composition of the luminal membrane is different from that of the antiluminal membrane of the brain vessels (29). If autoantibodies to brain vessels are important in the aetiopathogenesis of MS, it is likely (but not necessary) that they will recognise antigens situated on the luminal side of the brain vessels. Pardrigde and colleagues raised an antisera against a 46 kd protein which seemed to be specific for bovine brain capillaries (13). In the same molecular weight region, an inducible BBB specific molecule called HT7 of around 45-52kd has been
characterised in chick brain microvessels (14). Sternberger & Sternberger raised a monoclonal antibody named anti-endothelial-barrier-antigen(EBA) which appeared to be specific for brain vessels including optic nerve, spinal cord and retina. The monoclonal antibody reacted also with some vessels in the spleen and some cells in the epidermis of the skin and recognised a 30/25/23.5 kd triplet (30). Other proteins have been located on the brain vessels e.g transferrin receptor (a 95 kd dimer) (31), insulin receptor (127 kd protein) (32), P-glycoprotein (170-180 kd) (33) and proteins involved in glucose transport and other transport systems (34-35). Pardrigde and colleagues have shown that low molecular weight proteins (14, 16, 17, 18 kd) in isolated brain capillaries are histones (36). Some of the proteins detected by autoantibodies in our study (Table 1) may correspond to these characterised brain vessels proteins. The histopathology of MS suggests that the MS lesions could be in part due to an immune response to brain vessels as part of an autoimmune reaction or because of a viral infection of the brain vessels. Detection of autoantibodies to brain vessels in patients suffering from neurological diseases especially MS has been difficult because of the limitations of the assays used. In this study antibodies against BBB epitopes were no more in MS patients than controls but there is a need for further studies looking at the prevalence of autoantibodies to brain vessels in patients with neurological diseases and new assays should be devised to address this question. Acknowledgements B.E.S. was supported by the Wellcome Trust and the Multiple Sclerosis Society of Great Britain.
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