monoclonal antibody binding to congophilic elements in human alzheimer … · neurofibrillary...

J Clin Pathol 1986;39:1199-1203

Monoclonal antibody binding to congophilic elementsin human Alzheimer brainS M MACDONALD,* MARGARET M ESIRIt

From the Departments of*Haematology and tNeuropathology, John Radcliffe Infirmary, Oxford

SUMMARY Immunohistochemical studies, using a monoclonal antibody, SMP, raised in a mouse

against aged human peripheral blood white cells, are described. This antibody reacts with dendriticreticulum cells in lymph node and tonsil, and, in Alzheimer brain, with argyrophilic plaques,neurofibrillary tangles, and cerebral vascular amyloid.

Alzheimer's disease, the commonest cause of senileand presenile dementia, is characterised patho-logically by the presence of large numbers ofargyrophilic plaques and neurofibrillary tangles in thehippocampus and neocortex of the brain.1 The stimu-lus to the development of these abnormal structures isunknown. One view proposes that argyrophilicplaque development may be attributable to disor-dered immune function.2 While evidence put forwardso far in support of this view is inconclusive, the pos-sibility that changes in the expression of cellular anti-gens with advancing age may have a role in the devel-opment of age related diseases such as Alzheimer'sdisease, is a reasonable one. An attempt was thereforemade to raise monoclonal antibodies that reactspecifically with cellular components from agedhumans, and we report here one result of such anattempt; the generation of a monoclonal antibodythat reacts intensely with the congophilic elements inhuman Alzheimer brain, identified as SMP.

Methods

GENERATION OF MONOCLONAL ANTIBODY SMPThe antibody was produced by cells formed by fusionof mouse myeloma cells with spleen lymphocytestaken from a mouse that had been immunised withpooled peripheral blood mononuclear cells fromseveral aged human donors (aged 71-82 years). Onemouse (BALB/C) was first hyperimmunised with 5 x107 tonsil cells from a young donor (aged 24 years),injected subcutaneously to render the mouse tolerantto non-aged peripheral blood mononuclear cells. Twomonths later the mouse was injected subcutaneouslywith 2 x 106 peripheral blood mononuclear cellsfrom three aged donors, followed two weeks later by

Accepted for publication 13 March 1986

an intravenous injection of 3 x 106 peripheral bloodmononuclear cells from a different group of six ageddonors, and a simultaneous intraperitoneal injectionof 1 x 106 peripheral blood mononuclear cells fromthe same aged donors. Cells from several donors wereused to increase the probability of raising antibodiesto an antigen common to all aged cells. The spleencells from this mouse were fused with a myelomamouse line (P3/NSl/I-Ag 4-1), and colonies werecloned out by limiting dilution. Supernatants wereassessed for antibody activity by screening on agedperipheral blood mononuclear cells and on Alzheimerbrain tissue.

IMMUNOHISTOLOGYTissue samples for detection of monoclonal antibodybinding from cerebral cortex of cases of Alzheimer'sdisease and elderly and young control necropsybrains were cut into 5-7mm cubes, covered in mount-ing medium (Tissue Tek II, Lamb, United Kingdom)and frozen in isopentane cooled in acetone and dryice. Cortical samples from four cases of Alzheimer'sdisease were examined. These showed numerousargyrophilic plaques and neurofibrillary tangles.Cryostat sections (5 pm) were cut on a freezing micro-tome and air dried or fixed in acetone at - 20°C. Sec-tions were treated with SMP monoclonal antibodyneat for 30 minutes at room temperature and thenwashed three times in minimal essential medium.Slides were then overlaid with sheep antimouse anti-serum diluted 1/20 for 30 minutes, and washed andlayered with a complex of monoclonal antibody withalkaline phosphatase and alkaline phosphatase(8 mg/ml, Sigma) for a further 30 minutes. Sectionswere washed again and substrate added (2 mg naph-thol As-Mx phosphate (Sigma) dissolved in 0-2 ml ofdimethyl formamide and made up to 10 ml with 0-1 MTris buffer, pH 8-2, to which was added I mg of

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1200levamisole (to block endogenous enzyme activity) andI mg of fast red TR salt). The substrate was filteredbefore use and applied directly to the slide. Positivereaction gave a red precipitate.

In some experiments application of neat SMPmonoclonal antibody for 30 minutes was followed byperoxidase conjugated antiserum to mouse immu-noglobulin and the diaminobenzidine reaction,resulting in a brown reaction product. Alternatively,in double labelling experiments binding sites of SMPwere detected using fluorescein labelled antiserum tomouse immunoglobulin following treatment withneat SMP monoclonal antibody for 30 minutesand reaction with monoclonal antibody RT97neurofilament antibody detected by layering on neatRT97 monoclonal antibody for 30 minutes followedby three washes with minimal essential medium andapplication of a peroxidase labelled antiserum tomouse immunoglobulin. This sequential treatment ofthe section with monoclonal antibodies SMP andRT97 did not result in non-specific binding, nor did itprevent binding of the second monoclonal antibodyto the section. For silver staining sections were fixedin 10% neutral formalin for 30 minutes before carry-ing out the standard von Braunmuhl technique. Amy-loid was detected with Congo red. The affinity ofSMP monoclonal antibody for peripheral bloodmononuclear cells was assessed using blood smearsfixed in acetone and stained with SMP monoclonalantibody followed by fluorescein conjugated anti-mouse immunoglobulin. Binding of SMP to periph-eral blood mononuclear cells was also studied using afluorescence activated cell sorter (FACS). Samples of

Macdonald, Esiri

Table Quantitation ofSMPpositive cells in peripheralblood mononuclear cellsfrom young, aged, and Alzheimergroups

Mean (SE) percentage of cells labelled

Young Aged Alzheimer

SMP positive 1 3 (0-3) 2-3 (0 6) 0-8 (0 18)

Antibody binding detected with fluorescence antisera to mouse IgGusing a FACS.

normal body tissues were screened for reactivity withSMP monoclonal antibody in frozen sections treatedin the same way as the sections of cerebral cortexdescribed above.

Results

Preliminary analysis of antibody containing super-natants showed that although no antibodies wereproduced which were specific for all aged peripheralblood mononuclear cells, a monoclonal antibody wasproduced (SMP) that reacted with about 2% of agedand young peripheral blood mononuclear cells (table)using FACS analysis and with an antigen present insections of Alzheimer brain. Using specific antisera(Birmingham University) in an Ouchterlony test,SMP was found to be of IgG2a class. We were notable to confirm staining of peripheral blood mono-nuclear cells in blood smears.

Treatment of normal brain with SMP gave negativeresults apart from slight staining of collagen contain-ing vascular elements. The results of treatment of

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Fig 1 Appearance ofAlzheimer cortex (cryostat sections) after treatment with SMP monoclonal antibody (a) and afterstaining with von Braunmuhl silver stain (b). Examples ofplaques are indicated by arrows. a x 130, b x 90.

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Fig 2 Higher power view ofcryostat section ofAlzheimercortex after reaction with SMP monoclonal antibody.Example ofplaque indicated by arrow. x 500.

Alzheimer brain sections with SMP were comparedwith the staining patterns obtained using silver stain-ing (von Braunmiihl) and staining for amyloid(Congo red) and neurofilaments (monoclonal anti-body RT973). The staining pattern of SMP reactivitywas very similar at low and high power magnificationto that obtained with silver staining of plaques (figs 1and 2). The number of senile plaques identified with

SMP and silver, counted at low power in 1-5mmdiameter circles, was very similar (35 with SMP, 37with silver). SMP binding in plaques showed someoverlap with amyloid as shown by Congo red stainingbut was more extensive. Leptomeningeal and corticalvessels in which amyloid was deposited in Alzheimerbrain, however, showed coextensive binding of SMP(fig 3). The neurofilament specific monoclonal anti-body RT97 did not stain all plaques demonstrablewith silver and SMP, but where plaques could beidentified with RT97 there was also staining withSMP (fig 4). SMP bound more extensively than RT97to those plaques that were doubly reactive.Neurofibrillary tangles, which are strongly reactivewith RT973, were also moderately reactive with SMP(fig 5).We investigated the affinity of SMP for other body

constituents in an attempt to further characterise theantigen. Sections of pituitary, pineal, and extracranialviscera from normal subjects were screened and didnot show binding of SMP, apart from very minimalstaining of connective tissue. Stronger binding ofSMP was seen in the germinal centres of lymphoidfollicles. In acetone fixed smears made from fresh ton-sil the pattern of SMP binding resembled that of cer-tain other monoclonal antibodies believed to reactwith dendritic reticulum cells: El 1 (M Slusarenko, PBeverley) and dendritic reticular cell antibody (DMason) (fig6). These monoclonal antibodies, how-ever, did not bind to Alzheimer brain. The possibilitythat SMP was identifying immunoglobulin lightchains in senile plaques3-5 was discounted as we

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Fig 3 (a) Adjacent sections ofAlzheimer cortex showing leptomeningeal arteries and cortical arteriole (*) andplaques(examples arrowed) after reaction with SMP monoclonal antibody (a) and Congo red (b). SMP binding and amyloiddeposits coincide in vessels, but SMP binding is more extensive in plaques. x 120.

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Fig 4 Argyrophilic plaque double stained by treatment with monoclonal antibody SMP detected withfluorescein (a) andmonoclonal antibody R797 detected with peroxidase (b). Reaction is more extensive with SMP than with R797. x 800.

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Fig 5 Cryosat section ofAlzheimer cortex treated withSMP monoclonal antibody with sites ofbinding detected bythe peroxidase reaction with diaminobenzidine.Neurofibrillary tangkes (*) bind monoclonal antibody.x500.

obtained no noticeable staining with antisera (Dako)to light chains.

Investigation of frozen sections of human cerebralcortex from cases of cerebral infarction and headinjury showed no noticeable staining using SMPmonoclonal antibody, apart from the slight stainingof vascular elements seen in normal brain.

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Fig 6 Smearfrom tonsil treated with SMP monoclonalantibody with site ofbinding detected by peroxidase reactionwith diaminobenzidine. Cells with appearances ofdendriticreticulum cells react with monoclonal antibody. x 550.

Discussion

These observations show that SMP binds to an epi-tope in Alzheimer brain, which has a distributionresembling that of the silver stained elements ofargyrophilic plaques, congophilic elements in lepto-meningeal and cortical blood vessels, and

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Monoclonal antibody binding to congophilic elements in human Alzheimer brain 1203

neurofibrillary tangles. The SMP antigen, therefore,seems to be common to all these pathological fea-tures. The staining pattern obtained in Alzheimerbrain differs from that obtained with theneurofilament antibody RT97 in that plaque stainingwas more extensive with SMP than with RT97 andthe vascular amyloid was stained by SMP but not byRT97. By electron microscopy plaques seem to becomposed of degenerate neuritic processes that con-tain mitochondria, dense bodies, and paired helicalfilaments resembling those of neurofibrillary tangles.'Astrocytic and microglial processes may also par-ticipate in plaque structure, and extracellular amyloiddeposits can be identified in plaques. Astrocytes andmicroglial cells in and around resolving cerebralinfarcts did not react with SMP. In normal brainSMP bound minimally and only to vascular elements.The observation of binding of SMP to a small pro-portion of peripheral blood mononuclear cells usingthe FACS could not be confirmed in blood smears.The SMP antigen, however, was clearly present ingerminal centres of lymphoid follicles where it seemedto react with dendritic reticulum cells. Many antigenicdeterminants common to lymphoid and central ner-vous system tissues have been detected using othermonoclonal antibodies,6 but to our knowledge noneof these has been related to follicular dendritic cells,nor to Alzheimer brain. The biochemical nature ofthe SMP antigen has proved difficult to determine,and so far attempts to immunoprecipitate or immu-noblot the antigen present in tonsil tissue andAlzheimer cerebral cortex have failed to establish themolecular weight of the antigen.The findings presented here of an antigen common

to cerebral amyloid, argyrophilic plaques, andneurofibrillary tangles in Alzheimer's disease support

the suggestion by Kidd et al7 that these features sharea common protein. Further investigations using thismonoclonal antibody may prove of value inattempting to understand the nature of the neuronaldegeneration that causes Alzheimer's disease.

MME received financial support from the MedicalResearch Council. We are grateful to Mr R Cross,who performed some silver staining of sections; Drs SLeibowitz and DY Mason, who provided valuableadvice and discussion; and Dr BH Anderton, whoprovided supplies of RT97 monoclonal antibody.

References

1 Tomlinson BE, Corsellis JAN. Ageing and the dementias. In:Hume Adams J, Corsellis JAN, Duchen LW, eds. Greenfield'sneuropathology. 4th ed. London: Arnold, 1984:951-1025.

2 Ishii T, Haga S. Identification of components of immunogobulinsin senile plaques by means of fluorescent antibody technique.Acta Neuropathol 1975;32:157-62.

3 Anderton BH, Breinberg D, Downes MJ, et al. Monoclonal anti-bodies show that neurofibrillary tangles and neurofilamentsshare antigenic determinants. Nature 1982;298:84-6.

4 Isersky C, Ein D, Horada M, Glenner GG. Immunochemicalcross-reactions of human amyloid protein with human immu-noglobulin light polypeptide chains. J Immunol 1972;108:486.

5 Kalyan-Raman UP, Kalyan-Raman K. Cerebral amyloid angio-pathy causing intracranial haemorrhage. Ann Neurol 1984;16:321-9.

6 Budka H, Majdic 0. Shared antigenic determinants betweenhuman hemopoietic cells and nervous tissues and tumours.Acta Neuropathol 1985;67:58-66.

7 Kidd M, Allsop D, Landon M. Senile plaque amyloid, pairedhelical filaments and cerebrovascular amyloid in Alzheimer'sdisease are all deposits of the same protein. Lancet 1985;i:278.

Requests for reprints to: Dr MM Esiri, Department ofNeuropathology, The Radcliffe Infirmary, OxfordOX2 6HE, England.



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