Antibodies to Cellular Antigens in Sjogren's Syndrome

MARGAiRErA. ALSPAUGHand ENGM. TAN

From the Division of Allergy and Immunology, Scripps Clinic andResearch Foundation, Lajolla, California 92037

A B S T R A C T An extract of human lymphocytes fromcontinuous cell culture was used as the antigen sourceto detect antibodies in sera of patients with Sjdgren'ssyndrome (SS). Using double diffusion in agarose,85% of a selected group of patients had precipitatingantibodies. Three precipitating antigen-antibody systemswere detected and were shown to be different from thosedescribed previously in other systemic rheumatic dis-eases. The SS precipitating antibodies were temporarilyclassified as precipitins A, B, and C. SS patients withsicca syndrome but without clinical rheumatoid arthritishad precipitin systems A and/or B, and SS patientswith associated rheumatoid arthritis had precipitin sys-tem C. Serum reactants were demonstrated by immuno-electrophoresis to migrate in the y globulin region. Theprecipitating activity of the serum factors was not de-stroyed by treatment with 2-mercaptoethanol and wasnot removed by absorption of rheumatoid factor fromthe sera. The reactivity of the lymphocyte antigens wasdestroyed by treatment with trypsin but not by deoxy-ribonuclease or ribonuclease.

INTRODUCTIONSjdgren's syndrome (SS)' is a chronic inflammatoryautoimmune disease consisting of keratoconjunctivitissicca and/or xerostomia in association with a connectivetissue disease. It has been suggested that the clinical

Dr. Alspaugh is a National Institutes of Health post-doctoral trainee supported by U. S. Public Health ServiceGrant AI 00214.

Received for publication 23 August 1974 and in revisedform 13 November 1974.

l Abbreviations used in this paper: ANA, antinuclear anti-bodies; DLE, discoid lupus erythematosus; IEP, immuno-electrophoresis; MCTD, mixed connective tissue disease;2-ME, 2-mercaptoethanol; MEM, Earle's minimal essentialmedium; PBS, phosphate-buffered solution; PSS, progres-sive systemic sclerosis; RA, rheumatoid arthritis; RF, rheu-matoid factor; RNP, ribonucleoprotein; SLE, systemiclupus erythematosus; sNP, solubilized deoxyribonucleopro-tein; SS, Sjbgrens syndrome; Wil2, a continuously growingdiploid lymphocyte line obtained from a patient with heredi-tary spherocytosis.

diagnosis of SS can be made when any two of the threefeatures are found (1). Some of the more common con-nective tissue diseases found in association with SS arerheumatoid arthritis (RA), systemic lupus erythemato-sus (SLE), progressive systemic sclerosis (PSS), poly-myositis, and polyarteritis nodosa (1, 2). OccasionallySS may be associated with plasma cell dyscrasia andlymphoma (3).

In the past, diagnosis of the sicca syndrome reliedheavily on characteristic signs and physical tests suchas the Schirmer test for keratoconjunctivitis sicca (4)and the Lashley cup test for xerostomia (5). A recentadvance in the diagnosis of xerostomia is the evaluationof salivary glandular function by scintography (6).Confirmation of SS is often made by lip biopsy (6).Although these techniques are adequate, more informa-tive tests have been sought.

Various serological tests for SS have been evaluated.In studies where sera were tested for antinuclear anti-bodies, 48-68% were positive by immunofluorescenceon tissue substrates (1, 2). Nuclear staining patterns ofthe homogeneous, speckled, and nucleolar types havebeen described. However, sera from other connectivetissue diseases have also shown similar patterns of nu-clear staining, and SS cannot be differentiated by thistest alone (7). An immunofluorescent test measuringantisalivary duct antibodies has been used to test serafrom SS patients. 70% of the sera from SS-RA patientsreacted, but only 10% of the sera from patients withsicca syndrome alone were positive (6). Sera from pa-tients with SS have been tested against various tissueextracts by the complement fixation test (1). The inci-dence of positive tests was high when sera from patientswith sicca syndrome alone were tested (79%); however,the positives in the entire series were only 44%.

Immunodiffusion in gel has been used to detect serumprecipitating antibodies to tissue antigens (8-12). Inthese studies, extracts of human thyroid, spleen, and calfthymus nuclei have been used. A higher incidence ofprecipitating antibodies were reported in the sera ofpatients with sicca syndrome without RA (29-1%),

The Journal of Clinical Investigation Volume 55 May 1975 -1067-1073 1067

and a lower incidence of precipitins found in SS-RA(6-32%), so that the general incidence in all SS pa-tients varied from 31 to 43%. In the above studies, therelationship of tissue autoantibodies to disease processesin SS was not clarified. With the increasing awarenessthat humoral autoantibodies might be involved as par-ticipating factors in the causation of tissue injury (13),this study was carried out to re-examine and character-ize in greater detail the types of tissue autoantibodies inSS. Wehave employed extracts of a lymphocyte culturecell line as the source of tissue antigens. Patients withSS possessed autoantibodies which reacted with tissueantigens to give precipitin reactions which could be dif-ferentiated from autoantibodies found in other connec-tive tissue diseases. In addition, antibodies of two dif-ferent specificities found predominantly in SS withoutRA appeared to segregate from antibody of a thirdspecificity, which was present in SS with RAand absentin SS without RA.

METHODS

Patient selection. 20 patients with SS were screenedinitially; however, only 18 sera were available in sufficientquantities for repeated studies. The clinical diagnosis of SSfor each patient was established when at least two of thefollowing criteria were present: keratoconjunctivitis sicca,xerostomia, or a connective tissue disease. Confirmation ofthe clinical diagnosis required the following laboratory evi-dence. The ocular tests used were either a Schirmer test (4) ora rose bengal staining test (1). The tests used for xeros-tomia were either salivary gland scintigraphy (5) or a lipbiopsy (1) showing the abnormalities described in thesereports.

The patients with SS were divided into two major cate-gories, those with sicca complex without RA and thosewith sicca and associated clinical RA. The association withRA was made when symptoms and laboratory data met thecriteria for classical and probable RA, as defined by theAmerican Rheumatism Association (14). On the other hand,none of the patients with SS-sicca without RA had symp-toms satisfying criteria for classical or probable RA. Othersera which were examined were obtained from 27 patientswith SLE, 16 with PSS, 37 with classical and probable RA,12 with mixed connective tissue disease (MCTD), 22 withdiscoid lupus erythematosus (DLE), and 22 from apparentlyhealthy individuals. Patients with SLE met the preliminarycriteria for this disease (14) and did not have featuresdiagnostic of other connective tissue diseases. Patients wereclassified as having PSS on the basis of typical skin changesin the absence of polymyositis or diagnositic criteria forSLE. MCTDpatients were diagnosed on the basis of classi-cal symptoms and signs described previously (15) with theassociation of high titers of antibody to nuclear ribonucleo-protein (RNP). Patients with DLE had skin lesions whichwere considered to be compatible with this diagnosis, but, inaddition, did not have other features meeting criteria forSLE (14).

Tissue extracts. A human cell line (Wil2, donated byDr. R. Lerner, Scripps Clinic and Research Foundation, LaJolla, Calif.) was used as a source for tissue extraction.Wil, is a continuously growing diploid lymphocyte linewhich was obtained from a patient with hereditary sphero-

cytosis. The cells were seeded in Earle's minimal essentialmedium (MEM) supplemented with 2 mMeach of gluta-mine, sodium pyruvate, and nonessential amino acids (FlowLaboratories, Inc., Rockville, Md.). The supplemented me-dium also contained 10% fetal calf serum and 10 ml/literof antibiotic-antimycotic mixture (Grand Island BiologicalCo., Grand Island, N. Y.), and was adjusted to a final pHof 7.2 with 5.6% NaHCO&Cells were allowed to grow ona gyratory shaker to a concentration of 1.5 X 10Imm8.After this, they were centrifuged at 350 9 for 10 min, thepacked cells resuspended in MEMwithout calf serum, andwere centrifuged again at 900 g for 6 min. The washingprocess was repeated three times. After the final centrifuga-tion an equal volume of 0.25 M sucrose with 0.004 M cal-cium chloride at pH 6.2 (0.01 M phosphate) was added.The cells were then sonicated with a model W185 sonicator(Ultrasonic Systems Inc., Farmingdale, N. Y.) at a maxi-mumoutput for 30 s. The sonicate was then centrifuged at105,000 g for 20 min. The supernate was saved and used asa source of antigen. The entire procedure was performedat 40C.

Immunological techniques. For the demonstration of pre-cipitating antibodies in human sera, a modification of theOuchterlony double-diffusion method was used (16). Plasticpetri dishes (1 X 5 cm) containing 5 ml of 1% agarose wereused. Reactions were allowed to proceed at room tempera-ture, and precipitin lines were observed over a period of72 h. Early in these studies, it was apparent that reactivesera were segregating into two groups. For each group ofprecipitating sera, a prototype serum was chosen and usedas the reference serum at a dilution which gave optimumprecipitin lines with lymphocyte extract Sera were ana-lyzed in double diffusion by placing them in wells adjacentto reference sera and by examination of precipitin lines foridentity or nonidentity with these reference sera. In caseswhere precipitin lines were weak, manipulation of the sys-tem such as changing the dilution of sera or the concentra-tion of antigens in extract produced stronger precipitinlines and allowed definite identification with reference sera.

As a result of previous studies, other reference sera wereavailable which contained antibodies to DNA, solubilizeddeoxyribonucleoprotein (sNP), streptomycin (Sm) antigen,RNP antigen, and nucleolar RNA (17). Antibodies toDNA, sNP, and Sm antigens are present primarily inSLE, antibody to RNP in SLE and the MCTD, and anti-body to nucleolar RNA in scleroderma and SS (17). Withsera containing precipitating antibodies to Wilh extract, itwas possible to show that the antibodies in SS sera werenot identical to those previously described for other connec-tive tissue diseases.

Noble agar (1%) in 0.05 ionic strength barbital buffer,pH 8.5, was used for immunoelectrophoresis (IEP). Pa-tients' sera were placed in the center well and electro-phoresed for 1.5 h at 8 mA/slide. Lymphocyte extract wasadded to one trough and antiwhole human serum added tothe second trough. Reaction was allowed to proceed at roomtemperature. Precipitin lines were observed over a periodof 72 h. Appropriate control sera and antigens were treatedin the same manner.

Enzymatic treatment of extracts. Ribonuclease (RNase),deoxyribonuclease (DNase), and trypsin were obtained fromWorthington Biochemical Corp., Freehold, N. J. They werefirst tested in the appropriate buffers to determine that theenzymes were fully active in these buffers (18). These en-zymes were employed in the treatment of lymphocyte ex-tracts. The ratios by weight of enzyme to substrate usedwere as follows: RNase to substrate 1:1, 1:5, and 1: 30,

1068 M. A. Alspaugh and E. M. Tan

DNase to substrate 1: 2 and 1: 50, and trypsin to substrate1: 20 and 1: 100. All digestions were incubated at 370C for2 h. Trypsin digestions were performed in 0.04 M Trisbuffer at pH 8.1 in the presence of 0.0115 M calcium ions.DNase digestions were performed in 0.01 M phosphate buf-fered, 0.15 M NaCl at pH 7.4 (PBS) in the presence of0.006 M magnesium ions. The RNase digestions were per-formed also in PBS at pH 7.4. The treated extracts werethen titered out against prototype sera in the Ouchterlonytechnique and compared with controls.

Chemical treatment of patients' sera. Patients' sera weretreated with 2-mercaptoethanol (2-ME). Reactants wereadded to each other at volume of 1: 1 leaving the final mo-larity of 2-ME at 0.1 M and the sera at a dilution of 1: 2.After mixing, the reactants were left at room temperaturefor 15 h (19) before testing by the Ouchterlony technique.The treated sera were then titered out against the lympho-cyte extract and compared with controls.

Absorption of rheumatoid factor from patients' sera.Human y-globulins (fraction II) (Miles Laboratories Inc.,Miles Research Div., Elkhart, Ind.) at a concentration of25 mg/ml in PBS were heated at 630C for 20 min. Theaggregated globulins were then lyophilized and added to seraat 15 mg/ml and left overnight at 4VC. The samples werethen centrifuged at 36,000 g for 20 min, and the supernatewas tested for residual rheumatoid factor (RF) by thelatex agglutination test.

Chemical determinations. Protein concentrations were de-termined by the Lowry procedure (20). DNA was deter-mined by a modification of the diphenylamine reaction (21),and RNAwas determined by the orcinol reaction (22).

RESULTSCharacterization of crude extracts. Some of the char-

acteristics of the extracts have been determined by chem-ical methods. After the cells had been sonicated in asucrose-calcium chloride solution, the protein concentra-tion was 40 mg/ml. After centrifugation at 105,000 g,

-~~~~~~~~~~~~FIGURE 1 Immunodiffusion study showing the three dif-ferent precipitin systems found in SS sera reactive withWil2. Serum SS was from a patient with sicca syndromewhich produced two precipitins (A and B). Serum SS-RAwas from a patient with sicca syndrome and RA whichproduced one precipitin system with the extract and showedcomplete nonidentity with the precipitins of the SS serum.

FIGURE 2 Immunodiffusion study showing nonidentity ofprecipitating systems in SS and precipitins in other systemicrheumatic diseases. SLE1 was serum with antibody to Smantigen (the sharper of the two precipitin lines and closestto tissue extract well). MCTD1 and MCTD2 were serafrom MCTDwith precipitating antibody to RNP. This pre-cipitin line like the Sm precipitin was not related immuno-logically to the SS lines. SLE2 contained precipitating anti-body to sNP and was nonreactive because this antigen wasnot present in Wil2 extract.

the supernate contained 25 mg/ml protein, 5.3 mg/mlRNA, and 0.018 mg/ml DNA. This supernate was usedas a source of antigen for detecting antibodies in pa-tients' sera by double diffusion in agarose.

Preliminary screening of patients' sera by Ouchterlonytechnique. The sera from 20 SS patients were testedagainst lymphocyte extracts containing 25 mg/ml pro-tein in the Ouchterlony technique. 17 of these were posi-tive (85%). When the SS sera which were previouslypositive were titered out against the undiluted extract,precipitin lines were observed with some sera at 1/512dilution. When the extract was diluted out and testedagainst seven representative SS sera, the end pointsranged between 5 mg/ml and 1.2 mg/ml of protein.Some of the extract was dialyzed against PBS at pH7.4 and lyophilized. The lyophilized material was as re-active as unlyophilized extract and remained stable forat least 2 mo at 4GC.

Fig. 1 illustrates the three precipitin systems foundin sera from patients with SS. Serum SS, which con-tained precipitins A and B, was from a patient withsicca syndrome. The serum labeled SS-RA was from apatient with sicca syndrome and RA which containedprecipitin C. Wib extract was in the upper well. SerumSS produced two lines with extract, precipitin system A

Antibodies to Cellular Antigens in Sjigren's Syndrome 1069

GGAnti-Human Serum

WiI2 Extract

+

FIGURE 3 An SS serum (-y-globulin) containing precipi-tins A and B was placed in the center well and electro-phoresed. Antihuman serum was then added to the toptrough and Wil2 extract added to the bottom trough. Twoprecipitins developed against lymphocyte extract and boththese reactions were located in the IgG region. The irregu-lar lines in the a-,8-globulin region were artifacts and notprecipitin lines.

(closer to the Wih extract), and precipitin system B.Serum SS-RA produced one line with the extract(precipitin system C) that showed complete nonidentitywith systems A or B. It was important to establish thatthe precipitin systems observed with SS sera were dif-ferent from precipitating antibodies described in otherconnective tissue diseases. This was performed by usingpreviously analyzed sera containing known antibodies,and such a study is shown in Fig. 2. Serum SS wasfrom a patient with SS and reacted with Wih to givethe two precipitin systems described in Fig. 1. Precipitinsystem A was much stronger than precipitin system Bbecause the two tissue antigens were present in the ex-tract in different concentrations, with A predominatingover B. With this particular SS serum, antibody to theA antigen was present in higher titer than antibody tothe B antigen. SLE1 was serum from a patient withSLE and gave two precipitin lines with Wil2, thesharper line closer to the antigen well has been identi-fied in previous studies as the Sm system. The secondline further from the antigen well has not been identi-fied. It was clear that the Sm system of SLE was dif-

TABLE IPrecipitin Systems Found in Patients' Sera

Precipitins

Patient Disease Number of systems A B C

G. T. ss 1 + - -A. Z. ss 2 + + -G. G. ss 2 + + -J. M. ss 2 + + -T. H. ss 2 + + -P. C. sS 1 - + -M. F. ss-JRA 1 + - -M. C. ss-PSS 2 + - +P. F. ss-Raynauds 2 + - +M. M. ss-RA 1 - - +J. C. ss-RA 1 - - +H. O. ss-RA 1 - - +A. D. ss-RA 1 - - +M. G. ss-RA 1 - - +E. R. ss-RA 1 - - +

ss, sicca syndrome; JRA, juvenile rheumatoid arthritis.

TABLE I IIncidence of Antibodies to A, B, or C in

Connective Tissue Diseases

PrecipitinsPositive/

Disease tested A B C

RA 24/37 0 0 24 (65%)MCTD 0/12 0 0 0DLE 0/22 0 0 0PSS 0/16 0 0 0SLE 2/27 0 0 2NHS 1/22 0 0 1

ferent from the systems in SS. MCTD1and MCTD2were two sera from two patients with MCTD. Theyalso reacted with Wih extract, and this system has beenidentified as the RNP system of MCTD. The latterwas also not identical immunologically with the SSsystems. SLE2 was a serum with precipitating antibodyto deoxyribonucleoprotein but did not react with WiL,illustrating that the latter extract did not contain solubledeoxyribonucleoprotein. Studies not illustrated hereshowed that the SS-precipitating systems were not re-lated to precipitating antibodies to native or denaturedDNA. Similar studies were also conducted to analyzeprecipitin system C which showed that it was not re-lated to precipitating systems involving Sm, RNP, sNP,and native and denatured DNA.

Of 17 sera which were positive in early screeningstudies, only 15 remained for identification of precipitinsystems. Although only a limited number of SS serawere examined, the results appeared to show that pre-cipitins A and B were present mainly in patients withsicca syndrome, and precipitin C was present in patientswith SS-RA (see Table I). It was noted that the pa-tient with sicca and juvenile RA had precipitin A, andthe two patients with either SS-PSS or SS-Raynaud'shad precipitins A and C.

Sera from patients with diseases other than SS werealso studied for precipitins A, B, or C. The only groupwith a significant incidence was the RA group in which65% of the patients' sera contained precipitin C, sug-gesting that the presence or absence of RA may influ-ence which precipitins were detected (Table II).

IEP of patients' sera. IEP was performed to deter-mine if the serum factor(s) reacting with tissue anti-gens could be identified by this technique. Five sera(three from sicca patients and two from SS-RA pa-tients) with the highest titers of serum precipitins indouble diffusion also showed precipitins by IEP andin all five cases; serum precipitins were located in they-globulin region, and the precipitin arcs had the char-acteristic location and curvature of IgG. A representa-tive example (serum GG) is shown in Fig. 3, where

1070 M. A. Alspaugh and E. M. Tan

serum was electrophoresed in the center well and de-veloped with antiwhole human serum in the top troughand with tissue extract in the bottom trough. SerumaY-globulin demonstrated two precipitin arcs with gammamobility and in double diffusion; this serum had beenone of the reference sera with the two precipitin systemsA and B. The other two sera, containing precipitins Aand B, produced two arcs in IEP, but the two serafrom patients with SS-RA, containing precipitin C,produced one arc. Other sera with lower titers of pre-cipitins were also tested by IEP, but precipitin arcs werenot always detected, a feature which was thought to berelated to the lower sensitivity of the IEP techniquecompared to the Ouchterlony technique.

2-ME treatment of patients' sera. Sera from fivepatients with SS were treated with 2-ME, titered againstextracts, and the end points compared with controls.All five sera tested were resistant to 2-ME. Three ofthe sera were from SS patients with sicca syndromewhich contained precipitins A and B. The other twosera were from patients with SS-RA which containedprecipitin C.

Effects of physical or biochemical treatment of ex-tracts. Some of the physiobiochemical characteristicsof individual antigens have been determined. The ex-tract was treated with RNase, DNase, and trypsin andwas tested against various sera by the Ouchterlony tech-nique. In these studies sera from two patients were usedto represent systems A and B, and two sera from SS-RApatients were used to present system C in the Ouchter-lony technique. All the antigens in the A, B, and Csystems were rendered nonreactive by treatment withtrypsin at both enzyme-substrate ratios tested (1: 20and 1: 100), but were unaffected by treatment withnuclease enzymes at all ratios tested. The antigens werenot affected by 2-ME treatment or by heating at 560Cfor 1 h.

Absorption of sera with aggregated IgG. Sera fromseven patients with sicca syndrome alone and sera fromsix patients with SS-RA were tested by the Ouchterlonytechnique against heat-aggregated IgG. Sera from pa-tients with sicca syndrome were nonreactive with aggre-gated JgG. Five of the six sera from SS-RA patientsreacted. All five had high-titer RF by the latex agglu-tination test. Sera from these patients with SS-RA wereabsorbed with aggregated IgG until RF was reducedfrom a titer of 2,560 to 20 or less. The absorbed serawere negative for precipitins to aggregated IgG, butwere still positive against the lymphocyte extract, dem-onstrating that the precipitin reaction of the C systemwas not due to RF.

DISCUSSIONIn the present studies, we have described three types ofprecipitating autoantibodies in the sera of patients with

SS. With the limited number of SS sera examined, itappeared that two types of precipitating antibodies,temporarily identified as the A and B systems, werepresent predominantly in SS without RA, whereas thethird type of precipitating antibody was present in SSwith RA and not in SS without RA. This segregationof precipitating antibodies has been substantiated in astudy of a larger number of SS sera and will be re-ported in a later communication. In these studies 56patients with sicca syndrome and 33 patients withSS-RA were tested. 76% of the SS-RA patients hadprecipitin C and less than 9% had precipitins A and/orB. On the other hand, more than 79% of the patientswith sicca syndrome had A and/or B, whereas lessthan 5% had precipitin C. It should be noted that instudies by previous investigators, primarily those ofAnderson, Gray, Beck, and Kinnear (9), two types ofprecipitating antibodies, SiD and SiT, were described.It would appear to be more than coincidental that theseworkers also found that the two antibodies were presentin SS without RA but not in other forms of SS. Theyobserved that one of their antigens, SiT was destroyedby heating at 560C for 1 h and by digestion with tryp-sin, but SiD was not affected by either treatment. Inour studies, both the A and B antigens were stable toheat but both were destroyed by digestion with trypsin.At this time, it is difficult to explain the discrepanciesespecially if the two antigens were presumed to beidentical. However, we hope to obtain answers to thesequestions by exchange of sera and reagents with theseworkers.

In contrast to previous workers who have found lowincidence of precipitins to tissue extracts in SS withRA, we found precipitins to the C system in SS-RAin high incidence not only in the few selected sera re-ported here but also in a larger series.' This differencemay be due in part to the fact that our extracts wereobtained from tissue culture cells, a procedure whichpermitted immediate extraction of tissue antigens with-out undergoing autolysis. Alternatively, the C antigenmight be unique to lymphocytes or to certain tissue cul-ture cell lines, possibilities which we have not investi-gated. In any case, an interesting observation thatemerged was that serologically, SS-RA could be differ-entiated from the pure sicca syndrome or SS withoutRA, suggesting that these two forms of SS might notnecessarily be closely related. This was also suggestedby MacSween et al. (23) who proposed that a subclini-cal form of SS might be present in some patients withRA. This proposal was based on the finding that therewas a high incidence of salivary duct antibody in SS-RAbut a low incidence in SS without RA. Further work

'Alspaugh, M. A., N. Talal, and E. M. Tan. Unpub-lished observations.

Antibodies to Cellular Antigens in Sidgren's Syndrome 1071

showing differences in the forms of SS were studies(24, 25) that peripheral lymphocytes from SS-RA re-sponded less well to mitogenic stimulation with phyto-hemagglutinin, dinitrochlorobenezene, tetanus toxoid,and streptolysin, than lymphocytes from SS without RA.

The intracellular location of the tissue antigens whichreact with SS sera has not been clearly defined. In thestudy of Bloch, Buchanan, Wohl, and Bunim, the inci-dence of antinuclear antibodies (ANA) in a large seriesof SS patients was 68% (1). Studies in this laboratoryhave shown a 59% incidence of ANA in SS, but inthese same patients, 86% had precipitins A, B, or C.This suggests that at least one or more of the precipitinsmay be antibodies against cytoplasmic or nonnuclearcomponents. If normal human leukocytes were used assubstrate for ANA, in addition to nuclear staining,there was significant cytoplasmic staining with serawhich gave positive precipitin reactions, further sug-gesting that cytoplasmic antigens might be involved insome of the precipitin systems described here.

It is becoming clear from many recent reports thatautoantibodies of certain specificities are segregated intheir distribution. For example, antibodies to double-stranded DNAand to the Smnuclear antigen were de-tected predominantly in SLE (17, 26), and antibodiesto nuclear RNP were detected in the MCTDas wellas SLE (15, 17, 27). It would thus be important to beable to define the antigenic or biochemical specificitiesof the many autoantibodies found in certain diseases,since there might be distinct profiles of autoantibodyspecificities for each disease. The usefulness of such anapproach has already become apparent in SLE wheretests for anti-DNA antibodies have helped in diagnosis,as well as in management, of SLE. Furthermore, theseand other studies on the specificities of autoantibodiesin SS might be anticipated to lead to investigationsconcerning the relationship of these autoantibodies todisease processes. At the present time, it is not knownwhether these antibodies participate in causing tissueinjury or are epiphenomena of previous cellular damagefrom other causes. In any event it is clear that auto-antibodies in SS react with cellular antigens which areimmunochemically different from cellular antigens re-acting with autoantibodies in other systemic rheumaticdiseases. The processes which lead to the production ofautoantibodies of such diverse immunochemical speci-ficities and the reasons for the segregation of sometypes of autoantibodies in certain diseases are not under-stood at the present time.

ACKNOWLEDGMENTSWe are grateful to Doctors J. Johnson, L. Jacobs, P.Hench, and R. McKendry for donating sera and to Ms. C.Tifft for excellent technical assistance.

This work was supported by U. S. Public Health ServiceGrant AM12198.

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