Annals ofthe Rheumatic Diseases 1990; 49: 249-253

Serum lymphocytotoxic antibodies andneurocognitive function in systemic lupuserythematosus

A A Long, S D Denburg, R M Carbotte, D P Singal, J A Denburg

AbstractThe hypothesis that lymphocytotoxic anti-bodies are associated with neuropsychiatricinvolvement in systemic lupus erythematosus(NP-SLE) is re-evaluated in this study. In anunselected cohort of 98 women with SLE across-sectional study has been performedto analyse associations among standardisedclinical, neurological, and neuropsychologicalassessments and lymphocytotoxic antibodiesmeasured by microcytotoxicity assay. Fiftypatients showed objective clinical evidence ofcontinuing or past NP-SLE and 54 patientshad cognitive impairment. In accordance withprevious observations 44% (24/54) of thecognitively impaired group did not have clini-cally detectable evidence ofNP-SLE. Althoughlymphocytotoxic antibodies were found to beonly marginally more prevalent in thosepatients with a clinical diagnosis of NP-SLEthan in those without (32% v 23%), theseantibodies were significantly associated withcognitive impairment (X2=5*42; p<O0O2). Noassociation was detected between lympho-cytotoxic antibodies and either overallsystemic disease activity or other organsystem involvement, suggesting that theassociation between lymphocytotoxic anti-bodies and cognitive dysfunction in SLE isspecific.

Departments ofMedicine, Psychiatry andPathology,Chedoke-McMaster andSt Joseph's Hospitals,McMaster University,Hamilton, OntarioL8N 3Z5, CanadaA A LongS D DenburgR M CarbotteD P SingalJ A DenburgCorrespondence to:Dr J A Denburg,Room 4H21-HSC, McMasterUniversity, 1200 Main StreetWest, Hamilton,Ontario L8N 3Z5, Canada.

Accepted for publication16 May 1989

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterised bydiverse circulating autoantibodies.' The lattermay be defined functionally-for example,lymphocytotoxic antibodies,2 3 or immuno-chemically.4 Associations between centralnervous system disease in SLE and neuronereactive or lymphocyte/brain cross-reactive anti-bodies have been observed in a number ofstudies, suggesting a possible pathogenicrole for these autoantibodies in the neuropsych-iatric complications of SLE (NP-SLE). Evi-dence in support of this idea is the commondiffuse clinical presentation of NP-SLE, thevirtual absence of histopathologically docu-mentable vasculitis within the brain at necrospyof patients with NP-SLE.," 12 and the docu-mentation of an array of shared cell membraneantigens between lymphocytes and neurones(reviewed in ref 13).

Central nervous system involvement in lupusis manifested by a spectrum of neurological andpsychiatric disorders, ranging in severity fromflorid psychosis, hemiparesis, or chorea to mildparaesthesia or ill defined mood alterations. '"8

The neuropsychiatric complications of SLE aretypically diagnosed when such heterogeneousneurological or psychiatric features cannot beattributed to other causes." 19 20 Attempts toclassify NP-SLE systematically have beenmade,'4 15 21 but in general the variability ofthese complications and lack ofwidely recognisedspecific diagnostic criteria make both casedefinition and associations with putative aetio-logical factors, such as autoantibodies, prob-lematic. We have endeavoured to categorise ourpatients with SLE systematically according toneuropsychiatric involvement using a classifi-cation"-" which is an extension and modi-fication of that proposed by Kassan andLockshin. 4

This paper reports a cross-sectional analysisof unselected patients with SLE in whichassociations between circulating lymphocyto-toxic antibodies and clearly defined clinical orsubclinical NP-SLE (cognitive impairment) aresought.

Patients and methodsNinety eight female patients (mean age 35 years(range 16-66)) fulfilling the 1982 AmericanRheumatism Association revised criteria forSLE25 were studied after informed consent.The study population comprised consecutivereferrals as either inpatients or outpatients tothe lupus clinic at the McMaster UniversityMedical Centre. At the time of the study 62patients (63%) had active systemic disease asdetermined by the lupus activity criteria count,and of these, 36 patients were taking steroids.

Each patient underwent a comprehensiveclinical evaluation together with laboratorystudies to assess systemic disease activity.Laboratory evaluations included haematologicalevaluation (erythrocyte sedimentation rate,complete blood count, white cell differentialcount, prothrombin time, partial thromboplastintime), renal evaluation (serum urea, serumcreatinine, analysis of urine sediment, 24 hoururine protein measurement, creatinine clearancecalculation), and immunological evaluation(antinuclear antibodies, DNA antibodies,rheumatoid factor, quantitative immunoglobu-lins and serum protein electrophoresis, cryo-globulins, measurement of complement com-ponents C3 and C4, antibodies to extractablenuclear antigens, and immune complexmeasurement). Systemic disease activity wasscored according to the lupus activity criteriacount,26 modified to exclude central nervoussystem disease.

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CENTRAL NERVOUS SYSTEM DISEASE ASSESSMENTIn addition to detailed neurological history andphysical examination, each patient underwentelectroencephalography and brain scan (radio-isotope or computed tomography). Clinicalcriteria for NP-SLE were based on an exten-sion and modification of previous classifica-tions.'4 22-24 Briefly, neuropsychiatric mani-festations that were not attributable to causesother than SLE itself were divided into 'major'and 'minor' signs or symptoms. Major neuro-logical features included: cerebrovascular event,neuropathy (peripheral or cranial), movementdisorder, transverse myelitis, seizure, organicbrain syndrome, and meningitis. Major psychi-atric features included: major affective disorderor atypical psychosis. Minor neurological dis-orders included mood swings and adjustmentdisorder. Recent consensus studies on thedefinition of NP-SLE showed that most of theabove major categories were agreed upon by a

wide variety of specialists (Singer et al, manu-script submitted). The diagnosis of a majorneurological or psychiatric disorder was con-firmed by a neurologist or psychiatrist. Thepresence of minor signs or symptoms-forexample, adjustment disorder-was determinedfrom the patient's subjective reports or thediagnosis of a psychiatrist. Neuropsychiatricinvolvement in SLE was diagnosed if thepatient fulfilled (a) one major criterion; or (b)one or more minor criteria together with anabnormality in one of the following: electro-encephalography, brain scan, cerebrospinalfluid studies, or cerebral angiogram. Thepatients were classified into one of three groups;group 1: active NP-SLE, group 2: inactiveNP-SLE-that is, definite past history ofneuropsychiatric lupus, currently resolved, orgroup 3: never NP-SLE-that is, no past orpresent evidence of NP-SLE.

NEUROPSYCHOLOGICAL TESTINGNeuropsychological testing was performedwithin two weeks of the clinical evaluation,using a protocol, the administration, analysis,and interpretation of which have been describedin detail previously.22 23 In brief, a battery ofwell standardised psychological tests, selectedto cover a wide range of cognitive functions-for example, verbal reasoning, verbal memory,visual spatial function-was given to eachpatient. The test battery included: Wechsleradult intelligence scale-information, compre-hension, similarities, digit symbol substitution,picture completion, block design subtests;Wechsler memory scale-with one hour delayedrecall of stories, designs, and paired wordassociates; consonant trigrams; Rey auditoryverbal learning test; Rey-Osterreith complexfigure drawing-with one hour delayed recall;token test; trailmaking test; Stroop colour wordinterference test; design fluency test; Bentoncontrolled word association test; animal namingtest; finger tapping test; handedness question-naire. Testing took 21½2 to 3 hours to complete.To identify cognitive impairment the raw

scores from each test were converted to standard(Z) scores, with normal controls serving as agematched reference groups for the means and

standard deviations needed in these transfor-mations.22 23 The individual tests were groupedinto 17 summary scores or test groupings basedon a face-valid analysis of the possible cognitiveprocesses involved. The summary scores for anindividual were then compared with a derivedestimate of that individual's premorbid function.Any summary score more than two standarddeviations below the premorbid level was takento reflect significant impairment; the individual'stest profile was designated impaired if three ormore of the 17 summary scores met thiscriterion.27

MEASUREMENT OF LYMPHOCYTOTOXICANTIBODIESLymphocytes were separated from freshly drawnvenous blood by density gradient centrifugationusing Ficoll-Hypaque as described by Boyum2tand suspended in minimum essential medium.Fresh rabbit serum was used as a source ofcomplement. Lymphocytotoxicity was measuredby the microdroplet test described by Terasakiand McClelland. 9Positive controls (multiparousserum or commercial antilymphocyte globulin)and negative control wells (normal serum) wereincluded in each microtitre plate. Serum wascollected from each patient at the time ofclinical evaluation and was tested for lympho-cytotoxicity against lymphocytes from 30 panelmembers selected to cover a wide range ofHLAspecificities. Reaction of the serum and lympho-cytes before the addition of rabbit complementwas carried out at 4°C (one hour). Subsequentincubation with complement was carried out at22°C or 15°C for three hours and cell death wasmeasured by eosin dye exclusion. An individualserum was considered positive if 50% or morecell death occurred in at least 10% (>3 out of30) of normal panel donor cells. In a furthersubset of 15 patients, autoreactivity of thelymphocytotoxic antibodies was assessed usingthe patient's own peripheral blood lymphocytesas target cells. Reaction conditions were asdescribed above and a positive result wasrecorded if >50% cell death was found. Theindividual performing the lymphocytotoxicantibody assay was not aware of the results ofclinical and psychological evaluations.

ResultsASSESSMENT OF CENTRAL NERVOUS SYSTEMINVOLVEMENTTwenty six patients had clinically evident activecentral nervous system disease at the time ofstudy (group 1) (table 1). A further 24 patientshad a history of such involvement with subse-quent resolution (group 2). Neuropsychiatric

Table 1: Characteristics of neuropsychiatric involvement insystemic lupus erythematosus

Clinical (n=98) Focalldiffuse*

Group It 26Group 2t 24 22/28Group 3t 48

*Focal NP involvement/diffuse NP involvement (see text).tGroup l=active NP-SLE; group 2=inactive NP-SLE; group3=never NP-SLE (see text for details).

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involvement in SLE in these groups covered arange of diagnoses as previously reported,24with no patient having meningitis and nopatient being categorised on the basis of peri-pheral neuropathy alone. Clinical manifestationswere further categorised as 'diffuse' NP-SLE(n=28)-for example, major psychiatric dis-order, organic brain syndrome, or major non-focal neurological disease-or 'focal' NP-SLE(n=22)-for example, transverse myelitis, cere-bral vascular event, or isolated cranial neuro-pathy. Fifty three patients had isotope brainscans, all of which were normal. Of those whohad computed tomographic brain scans (n= 19),eight were abnormal: cortical atrophy in three;decreased density compatible with brain infarc-tion in three; single area of increased lucency ofindeterminate significance in one; features sug-gestive of subdural haematoma in one. Table 2shows the relation between the clinical categor-isation of NP-SLE and objectively definedcognitive impairment. Patients with clinicallydefined NP-SLE (groups 1 and 2) were no morelikely to show cognitive impairment than thosewithout clinical central nervous system involve-ment (group 3). This is in accordance withprevious data reporting that, in contrast with acontrol sample of patients with rheumatoidarthritis, there is a high prevalence of cognitiveimpairment in patients with SLE, irrespectiveof clinical status or other variables.22

LYMPHOCYTOTOXIC ANTIBODIES IN SLE SERUMLymphocytotoxic antibodies were present inthe serum samples of 27 of the 98 patients.Lymphocytotoxic activity was observed across arange ofHLA specificities with lymphocytotoxicsera killing an average of 13 of the 30 lymphocytepreparations (range 4-29). Three lymphocyto-toxic sera reacted (>50% cell death) with onlyfour of the panel lymphocyte preparations each,yet an analysis of the HLA phenotypes of thesepanel members failed to disclose a commonantigen for any HLA locus with respect to agiven serum (table 3), suggesting that auto-antibodies rather than alloantibodies are pro-bably being detected. Serum samples from 15

Table 2: Relation of cognitive impairment to clinicalNP-SLE* status

Cognitive Clinical NP-SLE No clinicalstatus (groups I and 2)t NP-SLE

(group 3)t

Impairedf 30 24Not impaired 20 24

*NP-SLE=neuropsychiatric involvement in systemic lupuserythematosus.tGroup l=active NP-SLE; group 2=inactive NP-SLE; group3=never NP-SLE (see text for details).tBy criteria previously detailed.22 23x2= 1-00, NS.

patients were tested against autologous lympho-cytes. Of these, five showed lymphocytotoxicactivity when tested against panel lymphocytesas well as cytotoxicity for autologous lympho-cytes; of the remaining 10 sera tested, which didnot show cytotoxicity against panel lymphocytes,eight were negative for autocytotoxicity (5/5 v2/10;) X2=8-57; p<0 01). Several serum samplesshowed degrees of cytotoxicity lower than ourcriteria for positivity, not being reliably dif-ferentiated from control sera and thus scored asnegative.

CORRELATIONS OF LYMPHOCYTOTOXICANTIBODIESLymphocytotoxic antibodies were found to beonly marginally more prevalent in patients withNP-SLE than in those without neuropsychiatricSLE (16/50 (32%) v 11/48 (23%)) and this wasnot statistically significant. Interestingly, whenclinical NP-SLE was observed in the presenceof lymphocytotoxic antibodies it generallyshowed a diffuse pattern (12/16 (75%) cases),contrasting with antibody negative patients whoshowed an equal prevalence of diffuse (16/34(47%)) and focal (18/34 (53%)) patterns of NP-SLE (x2=3A45; p<0Q10).

In contrast with the lack of correlation oflymphocytotoxic antibodies with overall clinicalNP-SLE, the presence of these antibodies wassignificanfly associated with cognitive impair-ment (table 4). Seventy four per cent (20/27) ofantibody positive patients were cognitivelyimpaired compared with 48% (34/71) ofantibodynegative patients (p<002). A detailed analysisof these neuropsychological data suggests that aparticular pattern of visual spatial cognitivedeficit is associated with the presence oflymphocytotoxic antibodies.27

Positive correlations of other pertinentlaboratory variables and either lymphocytotoxicantibodies or NP-SLE (defined clinically or bycognitive impairment) were not detected. Forexample, when other organ system involvement,at a clinical or laboratory level, or both, oroverall systemic disease activity was assessed inrelation to lymphocytotoxic antibodies, no

Table 4: Serum lymphocytotoxins in relation to cognitivestatus in systemic lupus erythematosus

Lymphocytotoxic Cognitive statusantibodies*

Impairedt Non-impaired

Present 20 7Absent 34 37

*Microcytotoxicity assay against lymphocytes from 30 paneldonors. Present implies that the serum shows >50% cell killingin more than 10%/o of donors (see text).tUsing criteria described in 'Patients and methods'.X2=5-42; p<0 02.

Table 3: HLA phenotypes of lymphocyte preparations killed at >500/o by individual lupus serum samples

Serum Donor NoNo

1 2 3 4

A2,26; B35; DR6 A2,3; Bw62; Cw3; Dw2,3 A1,2; B8,15; Cw4 A26,30; B7,13; Cw6A2,28; Bw62,w57; Cw3,6; Dw4,7 A2,3; Bw57,w60; Cw3; Dw7,8 A9,11; B5,w22 A2; B40,w46; Cw2; Dw2,3A2,26; B35; Cw6; Dw2,6 A1,2; B8,51; Cw4; Dw3,4 A26,30; B7,13; Cw6 Al; B7,8; Cw6

23

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Table 5: Serum lymphocytotoxins in relation to clinicalfeatures in systemic lupus erythematosus. Values are shown asnumber (%) ofpatients

LCA* LCAPresent Absent(n=27) (n=71)

Active systemic diseaset 17 (63) 45 (63)Organ system involvement*

Skin 11 (41) 25 (35) NSJoints 20 (74) 43 (61) NSKidneys 6 (22) 13 (18) NSSerosa 3 (11) 6 (8) NSHaematological 10 (37) 26 (37) NSOral ulcers 7 (26) 23 (32) NSAlopecia 9 (33) 23 (32) NSRaynaud's disease 8 (30) 25 (35) NS

*LCA=lymphocytotoxic antibodies.tLupus activity criteria count score ¢2 (see ref 26), excludingnervous system criterion.fPast or present involvement determined by clinical and labora-tory means with reference to American Rheumatism Associationrevised criteria25 where relevant.NS=not significant.

associations were detected (table 5). Cortico-steriod treatment, similarly, did not account forthe association between lymphocytotoxic anti-bodies and cognitive impairment (data notshown).

Seven of the eight patients with computedtomography abnormalities were classified ashaving either active or inactive NP-SLE and sixof these were cognitively impaired. Two of theeight patients had serum lymphocytotoxic anti-bodies. No significant associations among thesevariables were found.

DiscussionIn an unselected group of female patients withSLE we have used defined criteria to categor-ise patients systemically according to neuro-psychiatric involvement. To describe braininvolvement more fully we carried out a de-tailed neuropsychological evaluation of eachpatient." 23 This information allowed us toreport a specific association between serumlymphocytotoxic antibodies and cognitive dys-function in SLE, an association which occursindependently of other potential associationswith lymphocytotoxic antibodies in SLE, in-cluding overall disease activity, involvement ofother organ systems, or drug treatment.

Lymphocytotoxic antibodies in SLE serahave been reported repeatedly since 19702 3(reviewed in refs 30 and 31). Discrepancies inthe prevalence and associations of these anti-bodies have appeared in published work, duelargely to technical problems or inconsistenciesin criteria used for the determination of apositive lymphocytotoxic antibody test. Thus,for example, the prevalence of lymphocytotoxicantibodies has been estimated in SLE to be28-93%.32 3 These antibodies require incu-bation at cold (4°C) temperatures for optimaldetection,30 31 and most reports ascribe lym-phocytotoxic antibodies to sera which kill morethan 20% of a given panel lymphocyte prepara-tion in the presence of complement. In ourexperience, difficulty may arise in reliablydistinguishing such minor degrees ofcytotoxicityfrom control results and consequently we usedmore exacting criteria in the assessment oflymphocytotoxic antibody activity. Also, unless

the target lymphocytes killed in the assay showa broad range of HLA specificities it is likelythat alloantibodies, related to prior blood trans-fusion or pregnancies, will be detected andcontribute to an apparently high prevalence.With our criteria for lymphocytotoxic anti-bodies, we recorded a prevalence of 28% in 98consecutive unselected patients. Examination ofHLA specificities and autoreactivity (table 3)suggests that to a great extent autoantibodiesrather than alloantibodies are being detected inthis study.

Specific associations between lymphocytotoxicantibodies and central nervous system involve-ment in SLE have been reported.6 7 Suchobservations have also been inconsistent,34however, probably owing not only to differingmethods for lymphocytotoxic antibody detectionbut also to variability in the definition of NP-SLE. Case definition in NP-SLE has proveddifficult and until recently there has been nostandard of value. Using defined criteria forneuropsychiatric involvement together withsystematic neuropsychological categorisation ofour patients, we have reported nervous systeminvolvement in approximately 50% of our un-selected SLE population,22 yielding an overallprevalence similar to that of other reportedstudies (reviewed ref 16). Probably, such anapproach could generate information on theactual extent and type of nervous systeminvolvement in SLE.23 Although we havepreviously described correlations between seriallymphocytotoxic antibody assessments and overtneuropsychiatric events in a small group ofpatients with SLE,9 this does not hold in alarger group of unselected patients whose clini-cal categorisations have been standardised.Lymphocyte reactive autoantibodies cross

reactive with human brain constituents havebeen described,6 and others have shown notonly brain/lymphocyte cross reactivity of suchantibodies but also a higher incidence oflymphocytotoxic antibodies in NP-SLE.3sSeveral potential lymphocyte/brain cross-reactive antigens have now been identified,which may be the targets of SLE autoanti-bodies,'3 either through access to the brain afterblood-brain barrier damage,36 or as a result ofintrathecal immunoglobulin synthesis,37 38 orboth.39 40

In this study a specific positive associationbetween cognitive dysfunction and serum lym-phocytotoxic antibodies was found (table 4).The specificity of this association is emphasisedby the finding that it does not seem to be relatedto either overall systemic disease activity or toother organ system involvement in SLE (table5). Our findings are of interest as several groupsof autoantibodies have been linked with NP-SLE, often with specific patterns of involve-ment.41 Serum antineuronal antibodies measuredin a mixed haemadsorption assay using culturedneuroblastoma cells as targets are found chieflyin association with non-focal neuropsychiatricSLE24; a novel neuronal antigen is the target ofsome SLE autoantibodies.42 Focal disease, incontrast, such as cerebral vascular events orchorea, has been linked to antibodies withphospholipid specificity.43 4 Finally, a striking

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association between lupus psychosis and auto-antibodies directed at a ribosomal P proteinhas also recently been described.45 Thus an as-sociation between cognitive dysfunction andlymphocytotoxic antibodies may imply func-tional or anatomical localisation of lymphocyte/brain cross-reactive antigens in the brain, aconcept supported by our recent delineation of aspecific cognitive deficit in lymphocytotoxicantibody positive patients with SLE.27A large variety of non-specific cognitive and

personality problems have been noted in patientswith SLE, and some of these features may berelated to the high prevalence of cognitiveimpairment found in these subjects, particularlythose without clinically evident nervous systemdisease.22 The observations in this study suggestthat neurocognitive evaluation should beincluded together with neuronal autoanti-bodiesl' 13 35 42 45 as well as lymphocytotoxicantibodies in the assessment of patients withSLE. In view of the clinical heterogeneity ofNP-SLE it is probable that no single mechanismwill explain all the features satisfactorily. Iflymphocytotoxic antibodies or related autoanti-bodies are to be shown to be aetiological factorsin NP-SLE, however, prospective studies ofunselected groups of patients with standardisedclinical categorisations are necessary.

Drs C Barnes, W Bensen, F Bianchi, M A Blaichman, WBuchanan, C Craig, R Duke, W Kean, J Kelton, R Lo, PRooney, D Rosenthal, D Sauder, K Snith, A Upton, SWilkinson kindly allowed us to study their patients. We wish tothank Julie Schmidt for technical help, and Joel Singer forstatistical analysis. Janice Butera and Barb Lahie typed themanuscript. This study was supported by grants from theCanadian Arthritis Society, Physician's Services Incorporated,Lupus Society of Hamilton, St Joseph's Hospital Foundation,Lupus Foundation of Ontario, and the Regional MedicalAssociates, Hamilton.

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