Annals of the Rheumatic Diseases 1991; 50: 776-781

Ankylosing spondylitis: an autoimmune disease?

H-J Lakomek, M Plomann, C Specker, M Schwochau

AbstractIdentification of several autoantibodies inserum samples from patients with ankylosingspondylitis or suspected ankylosing spondylitisis reported. Five antibodies associated withankylosing spondylitis were identified byapplying cytoimmunofluorescence andimmunoblotting techniques to antigen poolsfrom insect tissue. At least one of theseantibodies was found in 82% of serum samplesfrom patients with ankylosing spondylitis.A 36 kD drosophila antigen, which showed

the most common and most dominant re-action, was further purified and isolated.Thirty two (34%) of the serum samples from95 patients with definite ankylosing spondylitisand 12 (28%) of the serum samples from 43patients with suspected ankylosing spondylitisreacted with this antigen. Antibodies purifiedfrom the 36 kD antigen reacted specificallywith a 69 kD antigen present in separations oftotal protein preparations from humanlymphocytes and HeLa cells.The 36 kD antibody was not found in 29

patients with rheumatoid arthritis nor in 38apparently healthy controls. The prevalenceof the 36 kD antibody was comparable inHLA-B27 positive and negative patients. Inaddition, the same immunoreaction was foundin patients with so called 'seronegative'spondylarthropathies, particularly of theankylosing spondylitis-type, suggesting thatthis antibody is specific for ankylosingspondylitis or other 'seronegative' spondyl-arthropathies with the typical clinical andradiological changes of ankylosing spondylitis.

Department of Medicine,Endocrinology andRheumatology,Heinrich-Heine-Universitat Diusseldorf,D-4000 Dusseldorf,GermanyH-J LakomekC SpeckerInstitute for Genetics,Heinrich-Heine-Universitat Dusseldorf,D-4000 Dusseldorf,GermanyM PlomannM SchwochauCorrespondence to:Dr H-J Lakomek,Klinik fur Rheumatologie,Physikalische Medizin undEndokrinologie,Klinikum Minden,D-4950 Minden, Germany.Accepted for publication4 September 1990

Ankylosing spondylitis is the most common'seronegative' spondylarthropathy. The lowsensitivity of the New York criteria for thediagnosis of ankylosing spondylitis,' 2 especiallyin the early stages of the disease, and the lack ofspecific serological markers cause problemswith diagnosis.' Figure 1 shows a representa-tive spectrum of rheumatic diseases reported inthe Dusseldorfer Rheumaregister, a database com-prising about 2000 patients.9 In comparisonwith the other diseases ankylosing spondylitisshows the highest proportion of suspecteddiagnoses. Application of serological parametersspecific for ankylosing spondylitis could lead toan earlier diagnosis of the disease with higherconfidence and in greater number.

In 1984 an antibody specific for ankylosingspondylitis, which reacted with the 93 D heatshock puff of polythene chromosomes from

Rheumatoid 283arthritisSystemic lupus 79 2

erythematosus -Ankylosing _spondylitis 89 15

Psoriatic arthritis -Polymyositis/ 49 19Sjogren's syndromecleroderma 2Reactive 16 2arthritis 3351Osteoarthritis _

103

15

Other,rheumatic IOU_diseases ils gm

No rheumatic 340diseases L

* Suspected diagnosis Definite diagnosis

Figure I Spectrum ofdiagnoses in the Dusseldorfdatabasefor rheumatic diseases (1999 diagnoses in 1738 patients).

larval salivary glands of Drosophila melanogaster,was first described.'0 In 1987 four additionalinsect antigens were identified (36, 45, 52, and74 kD), which reacted specifically with anti-bodies present in the serum of patients withankylosing spondylitis. " Cytoimmunofluores-cence and immunoblotting techniques showedthat at least one of these antibodies was presentin 82% of all patients with definite ankylosingspondylitis and patients with suspected diseaseas well.

Since 19736 the strong correlation betweenankylosing spondylitis and the HLA-B27 haplo-type has been well recorded. No difference,however, was found between the nucleotidesequence of HLA-B27 genes from a healthysubject and from a patient with ankylosingspondylitis. 12 The structural identity of theHLA-B27 protein molecules in patients withankylosing spondylitis and normal subjects hasalso been reported.'3The prevalence of ankylosing spondylitis was

first estimated by De Blecourt in 19615 to be0-2% in men and 003% in women. In 1975Caln and Fries calculated that 1-6% of thewhite population probably develop ankylosingspondylitis, based on the fact that the HLA-B27haplotype is found in 8% of the white popu-lation.8 An improved diagnosis based on sero-logical data would reduce this large number ofapparently undiagnosed patients.Here we report the identification of xenotypic

and homotypic antigens reacting with antibodiesspecifically present in the serum of patients withankylosing spondylitis, thus proving ankylosingspondylitis to be an autoimmune disease.

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Materials and methodsSERUM SAMPLESAll serum samples were collected in the depart-ment of medicine C, endocrinology and rheu-matology at the Heinrich-Heine UniversityDusseldorf, FRG. Diagnosis of ankylosingspondylitis and seronegative spondylarthro-pathies was performed according to the NewYork criteria' 2 and of rheumatoid arthritisaccording to the American Rheumatism As-sociation criteria.'4 All patients' serum sampleswere tested by counterimmunoelectrophoresisfor the absence of antibodies to extractablenuclear antigens-for example, RNP, Sm, SSA,SSB. The serum samples were stored at -80°Cand diluted in phosphate buffered saline beforeuse.

CELL CULTUREThe stable KC cell line derived from Drosophilamelanogaster embryos was cultured at 250Cand suspended in medium D-22 free fromserum.'5 16 The stable H-33 cell line fromD hydei embryos was cultured at 25°C in amedium supplemented with 10% fetal calfserum.'7 18 HeLa S3 cells were cultured at 37°Csuspended in a modified minimum essentialmedium containing L-glutamine, non-essentialamino acids, fetal bovine serum, antibiotics, butno sodium bicarbonate.19 20

HARVESTING OF CELLSLymphocytes were isolated from heparinisedhuman venous blood provided by ProfessorBruster (Institut fir Blutgerinnungswesen undTransfusionsforshung, Heinrich-Heine-Univer-sitat, Dusseldorf) by differential centrifugationon a Ficoll-Paque solution (e= I. g/Ml).21 22After recovery from the interface they weresubjected twice to a short washing step withlymphocyte culture medium RPMI. HeLa,H-33, and KC cells were harvested by centri-fugation and washed twice in balanced saltsolution.

GENERAL PROCEDURE FOR TOTAL PROTEINPREPARATIONMost essential for reproducible isolation ofundegraded material is the immediate lysis ofthe cell suspension or tissue (pulverised inliquid nitrogen) in the presence of sarcosyl,urea, and ,B-mercaptoethanol in a tight fittingDounce homogeniser at 60-700C. Subsequentseparation from nucleic acids and polysac-charides is achieved by centrifugation in a CsClsolution with a final density of about 1-6 g/ml.The resulting float contains all peptides exceptfor a few dense glycoproteins and can be storedbelow -15°C undegraded for any length oftime. For good and reproducible electrophoreticseparations of the isolated antigens it is neces-sary to remove most of the sarcosyl and CsCl.This is achieved by quick (2 x 30 minutes)dialysis against a buffer (containing a lowconcentration of the ions defining the stackinggel conditions applied in the subsequent sodiumdodecylsulphate-polyacrylamide gel electro-

phoresis (SDS-PAGE)) at the lowest possibletemperature in the presence of a non-ionicdetergent in a flabby dialysis bag (measuring nomore than 2 mm in height when lying on a flatsupport).

PREPARATION OF TOTAL PROTEIN FROMLYMPHOCYTES, KC, H-33, OR HeLa CELLSThe following two buffers were used: buffer A:6 M urea, 0 I M NaCl, 5% ,-mercaptoethanol,1% sarcosyl, 10 mM TRIS/HCI pH 7'2; bufferB: as buffer A with sarcosyl replaced by 3%Nonidet P-40. After homogenisation in buffer Athe different lysates were heated to 60°C for 10minutes. The proteins were then collected as afloat after one hour's centrifugation at 30 000rpm (Beckman SW60 rotor= 120 000 g) in CsCl(final density 1-6 g/ml). The protein float wasdissolved in buffer B and dialysed againstTRIS/HCI buffer (25 mM, pH 7-4) and, finally,precipitated with ethanol.

IMMUNOBLOTSThe proteins were separated electrophoreticallyin linear pore gradient disc gels (100/o-20%polyacrylamide, 2 mm thick) in the presence ofurea (4 mol/l) and sodium dodecyl sulphate(0-1%).23 The protein separations were blottedto nitrocellulose sheets (semidry24); transferresults were visualised by staining with ponceauS. The sheets were then cut into strips andincubated with human serum samples diluted1:50 in phosphate buffered saline after blockingthe nitrocellulose with bovine serum albumin(0'1%) and Nonidet P40 (1%). Immuno-reactions were visualised by a biotinylatedantihuman immunoglobulin (RPN 1003;Amersham), a streptavidin-peroxidase complex(RPN 1051; Amersham), and diaminobenzidine/H202 or a fluorescein isothiocyanate conjugatedsheep antihuman immunoglobulin (MF 01,Wellcome).

PURIFICATION OF ANTIBODIESExcept for the visualisation the protein blotswere treated like immunoblots. The band con-taining the antibody-antigen complex was cutout. The antibodies were eluted for 15 minuteswith 3 M potassium thiocyanate and 0-1%bovine serum albumin in phosphate bufferedsaline and centrifuged in Centricon micro-concentrators (Amicon) for desalting andconcentration.25 26 The purified antibodysolution was stored at 8°C.

TWO DIMENSIONAL ELECTROPHORESISProteins were focused in either gel rods orhorizontal gels containing 9 M urea, 2%Nonidet P-40, and 2% ampholytes (pH 3 5-10)until the basic marker protein cytochrome creached the cathodic end. Before separationaccording to molecular weight using SDS-PAGEthe gels were equilibrated in a buffer containing10% glycerol, 2% sodium dodecyl sulphate, 5%13-mercaptoethanol, and 60 mM TRIS/HCI, pH6-8, for one hour.2730

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ResultsIn immunoblotting experiments we found astrong immune response with the 36 kDdrosophila protein in 32 (34%) serum samplesfrom patients with definite ankylosing spondy-litis and in 12 (28%) samples from patients withsuspected ankylosing spondylitis (table, fig 2).Immunoreactions of this type were identical inprotein preparations from serum free culturedKC cells and from fetal calfserum supplementedH-33 cells.To prove the specificity of the antibody to the

36 kD antigen we tested several serum samplesfrom patients with other 'seronegative' spondyl-arthropathies-for example, psoriatic arthritis,Crohn's disease-and other systemic rheumaticdiseases-for example, rheumatoid arthritis. Asshown in the table 15 (44%) of 34 serumsamples from patients with 'seronegative'spondylarthropathies with diagnosed ankylos-ing spondylitis and only two (13%) of 15samples from patients with other 'seronegative'arthropathies not of the ankylosing spondylitistype reacted with the 36 kD antigen, whereas

1 2 3 4 5 6 7 8 9 10 1 12 13 14 15

92Z5

45-

Figure 2 Staining ofa 36 kDKC cell protein specificforankylosing spoylitis. Western blot ofa separation ofa totalprotein preparationfrom the Drosophila melanogasterembryonicKC cell line incubated with serunfrom 13patients with ankylosing spondylitis (lanes 3-15) and poolednormal control serum (lane 2). The reactions were visualisedby a biotinylated secondary antibody, a streptavidin-peroxidase complex, and diaminobenzidine. Senrm samplesfrom the patients show a specific reaction with a 36 kDprotein (arrow). Lane 1, the control, shows the reaction ofthesecondary antibody alone. Thefaint bands at 36 kD visiblein lanes I and 2 resultfrom a cross reaction with thesecondary antibody. These as well as most ofthe other bandscannot be seen in reactions with afluorescein isothiocyanateconjugated sheep antihuman immumoglobulin antibody (MF01 Wellcome) as shown in ref 11.

Prevalence of antibodies to the Drosophila melanogaster 36 kD antigen in serum samplesfrom patients with various rheunatic diseases

Diagnoses Total number No (%) shoingof tested positive reactionserum samples with the

36 kD antigen

Definite AS * 95 32 (34)Suspected AS 43 12 (28)Definite and suspected ASHLA-B27 positive 120 42 (35)HLA-B27 negative 18 5 (28)

Seronegative spondylarthropathies with diagnosed AS 34 15 (44)Crohn's disease 23 10 (43)Psoriatic arthritis 11 5 (45)

Seronegative spondylarthropathies without diagnosed AS 15 2 (13)Rheumatoid arthritis 29 0Healthy controls 38 0

*AS=ankylosing spondylitis.

serum samples both from patients with rheuma-toid arthritis and from apparently healthycontrols did not show a positive reaction.To test for a possible correlation between the

presence of the antibody to the 36 kD antigenand the expression of HLA-B27 we screenedHLA-B27 positive and negative serum samples.In the patients with definite and suspectedankylosing spondylitis positive immune re-actions with the 36 kD antigen were found in 42(35%) of 120 HLA-B27 positive serum samplesand in five out of 18 (28%) HLA-B27 negativesamples (table).The presence of antibodies to the 36 kD

antigen in patients with ankylosing spondylitisseems to be independent of the patients' age,sex, and disease activity as indicated by erythro-cyte sedimentation rate and radiological changes.

For further characterisation of the 36 kDantigen the total KC protein was separated intwo dimensional gels with -non-equilibrium, pHgradient electrophoresis as first dimension andSDS-PAGE as second. This particular gel wasblotted one after the other to three differentnitrocellulose sheets. The first and third blotshowed a single spot at 36 kD and a basic pHreacting strongly with pooled ankylosingspondylitis serum samples, whereas the secondblot did not show any reaction at all whenincubated with a normal serum pool, thusproving the selective and highly specific reactionof the ankylosing spondylitis specific antibody(fig 3). Antibodies eluted from these spots inturn specifically reacted with the 36 kD band inWestern blots of total protein preparations ofKC cells (data not shown). The 36 kD proteinshowed an isoelectric point of 9 0 when focusedunder equilibrium conditions. When the serumof patients with ankylosing spondylitis andantibodies purified from the 36 kD drosophilaprotein were applied to Western blots of humanlymphocyte and HeLa proteins, a 69 kD humanantigen could be identified (figs 4A and B). Theeluted antibody was reincubated with a Western

_ NEPHGECa

UA-46

30

-46

Figure 3 Reaction ofthe 36 kD Drosphila protein selectveand specificfor anhylosing spondylitis. Two dimensionalseparation oftotal protein preparation ofKC cells: non-equilibrium, pH gradient electrophoresis (NEPHGE) andsodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE). The gel was blotted to three differentnitrocellulose filters (A, B, C)for 20 minutes each. BlotsAandC were reacted with pooled anhylosing spondylitts serumsamples, whereas blotB was incubated with a pool ofnormalserum samples as control. Detection ofthe bound humanantibodies occured with afluorescein isothiocyanateconjugated sheep antihunan itmwnoglobuilin secondaryantibody. BlotsA and C show a strong signal at 36 kD(arrows), whereas blotB shows no signal.

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1 2 3 4

K

925-

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29JI.

0Mr,

92-5

6.168' +....: :..

. ....

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Figure 4 Reaction with (A) a hwnan lymphocyte proteinand (B) a HeLa protein specificfor ankylosing spondylitis.Imnunoblots of(A) lymphocyte proteins and (B) HeLaproteins were incubated with pooled ankylosing spondylitisserum samples (lane 3), pooled normal control serum samples(lane 2), and antibodies elutedfrom the 36 kD Drosophilamelanogaster protein after reaction with pooled ankylosingspondylitis serum samples (lane 4). The reactions werevisualised by a biotinylated secondary antibody, astreptavidin-peroxidase complex, and diaminobenzidine. Thepooled ankylosing spondylitis serum samples and thepuiftedantibodies to the 36 kD antigen show specific reactions with a69 kD lymphocyte and a 69 kD HeLa protein (arrows).Other bands visible on lane 4 are reactions ofthe secondaryantibody. Lane 1, the control, shows the reaction ofthesecondary antibody alone.

blot identical to the one shown in fig 2. A singleband of 36 kD reacted, proving the quality andspecificity of the eluted antibody (fig 5). Elutionof the antibodies reacting with the 69 kDhuman lymphocyte antigen and reincubation onKC protein immunoblots showed a specificreaction with a 36 kD drosophila antigen (datanot shown).

DiscussionThe specificity and prevalence of the antibodiesto the 36 kD antigen, detectable in immuno-blots of D melanogaster and D hydei proteinpreparations, as well as those reacting with the93 D heat shock puff substantially improve thechances of diagnosing ankylosing spondylitisand thus help to differentiate it from othersystemic rheumatic diseases.

Introduction of these serological markers ofankylosing spondylitis aids the diagnosticprocess in the early stages and in women, whovery often have a milder form of disease.Furthermore, the antibody to the 36 kD antigensupports the identification of other 'sero-negative' spondylarthropathies, which arecharacterised by symptoms of ankylosingspondylitis. According to these results, the 36kD antibody could be used to identify patientswith ankylosing spondylitis or related diseases,like psoriatic arthritis or inflammatory boweldisease.

-29

-21

Figure S Quality ofthe eluted 36 kD specific antibody. Theimmunoblot ofDrosophila melanogaster proteins derivedfrom the embryonicKC cell line was incubated withantibodies purifiedfrom the reaction ofankylosing spondylitisserum samples with the 36 kD protein (lane 1), a stronglyreacting serum from a patient with ankylosing spondylitis(lane 2), a pool ofankylosing spondylitis serum samples (lane3), and pooled normal control serum samples (lane 4). Thereactins were visualised by a biotinylated secondaryantibody, a streptavidin-peroxidase comnplex anddiaminobenzidine. The serum samplesfrom the pateints withankylosing spondylitis and the purified antibodies to the 36kD antigen show a specific reaction with the 36 kD protein(arrow). LaneS, the control, shows the reaction ofthesecondary antibody alone (lane 5).

Ankylosing spondylitis and other 'sero-negative' spondylarthropathies are most tightlylinked to the major histocompatibility complex(MHC) class I locus, the HLA-B27 haplo-type.31 32 The molecule's function as a class Irestriction element for cytotoxic lymphocytes isnot altered by molecular variations of HLA-B27: the diseases are associated with differentvariants of HLA-B27.The 36 kD antibodies are not linked to HLA-

B27, as can be judged from the fact that HLA-B27 negative ankylosing spondylitis serumsamples also react with the 36 kD antigen. It hasbeen suggested, however, that the basis ofHLA-B27 disease association might be a non-functional contribution ofanHLA-B27 sequenceor a closely linked gene.3335 Using total proteinpreparations from human tissue, we were ableto show for the first time that antibodies elutedfrom the 36 kD drosophila antigen cross reacton immunoblots with lymphocyte and HeLaproteins. This means that the 36 kD antibody isan autoantibody and consequently, we feelankylosing spondylitis should correctly be

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classified as an autoimmune disease, though thissuggestion is still a matter for debate.3 39As HLA disease associations are not a mere

genetic coincidence, but rather a commonfeature of many immunologically mediateddisease processes, it is still possible that HLA-B27 in patients developing ankylosing spondy-litis undergoes structural modification owing tothe action of exterior agents (viruses, bacteria,etc). Cause of the disease may be multifactorial-HLA-B27 interacting with other factors, bothgenetic and environmental (low concordancerates for disease in monozygotic twins).Y2The cause of ankylosing spondylitis is not

necessarily an autoantibody, but might also be amalfunction of cellular immunity. We considerone possibility might be an immunoreactivelymphocyte clone producing the antibody to the36 kD antigen, which will be found only inpatients with ankylosing spondylitis. Accordingto this theory it is important that the 36 kDantibody shows strong immunostaining withlymphocyte proteins. In this context we wouldlike to point out that bacterially induced releaseof cytokines from T cells, especially interferon,can increase the expression of MHC codingclass I and II structures in such a way that cellsnormally unable to present an antigen are nowable to do so. A higher rate of foreign antigenpresentation leads to local activation andmaturation of T and B cells, and also ofcytotoxic cells, which are non-MHC restricted.Normal cell surface structures in the neighbour-hood of an MHC structure may now becomeimmunogenic and cause autoaggressive re-actions against normal cell antigens.43The structure and function of the human

antigens recognised by the antibodies specificfor ankylosing spondylitis are still unknown.Preliminary results'0" " show that the dros-ophila antigens are neither degradation productsof a larger entity nor heat shock proteins ormembers of the well known RNP-for example,snRNP, proteins. Furthermore, the auto-antibodies described here are definitely differentfrom those reported by Schwimmbeck et al,45which recognised the HLA-B27 antigens with amolecular weight of 45 kD and 12 kD.A prerequisite for the isolation of affinity

purified monospecific 36 kD antibodies is puri-fication of the 36 kD antigen by two dimensionalgel electrophoresis. The monospecific anti-bodies are used for screening human expressionvector libraries to obtain the respective humanantigen as fusion protein. Analysis of the genecoding for this antigen will open the way forinvestigation of the pathogenesis and cause ofankylosing spondylitis and other 'seronegative'spondylarthropathies with symptoms of anky-losing spondylitis.

We thank Gerlind Heimeroth and Jutta Schulten for theirtechnical assistance. Supported by a grant from the Bundesminis-terium fur Forschung und Technologie (01 ZU 8602/8).

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