Clin. exp. Immunol. (1990) 82, 509-514

Monoclonal process in primary Sjogren's syndrome andcross-reactive idiotype associated with rheumatoid factor

P. D. KATSIKIS*t, P. Y. YOUINOUt, V. GALANOPOULOU*, N. M. PAPADOPOULOS*,A. G. TZIOUFAS* & H. M. MOUTSOPOULOS*t *Laboratory of Clinical Immunology, Department of Medicine,

School of Medicine, loannina, Greece, and tLaboratory of Immunology, University of Brest, Brest, France

(Acceptedfor publication 21 June 1990)

SUMMARY

Monoclonal or oligoclonal B cell products have been described in the sera and urine of patients withprimary Sjogren's syndrome (PSS). In addition, monoclonal expansion of plasma cells has beenfound in the exocrine glands ofPSS patients with circulating monoclonal B cell products. The goal ofthis study was to raise an anti-idiotype to a cryoprecipitable monoclonal IgM kappa rheumatoidfactor (RF) from a PSS patient. Using the F(ab')2 fragments of the rabbit IgG anti-idiotype, anidiotype-specific ELISA was developed and sera from 32 patients with PSS (13 with monoclonalIgMK), 33 with rheumatoid arthritis, three with rheumatoid arthritis + Sjogren's syndrome (SS), 30with systemic lupus erythematosus, six with Waldenstr6m's macroglobulinaemia, and 20 healthycontrols were tested. The idiotype was primarily found in PSS patients with circulating monoclonalIgMK and more often in those who had a ratio of K: intracytoplasmically positive plasma cellsgreater than 3: 1 in the lymphocytic infiltrates of minor salivary glands, and systemic manifestations.The idiotype was also found in PSS and rheumatoid arthritis patients without circulating monoclonalcryoglobulins as well as in two of the six patients with Waldenstrom's macroglobulinaemia. Ourresults suggest that the monoclonal process observed in PSS could involve restricted idiotypic clonesthat are susceptible to malignant transformation.

Keywords idiotypes monoclonal cryoglobulinsSj6gren's syndrome

INTRODUCTION

Primary Sj6gren's syndrome (PSS) is an autoimmune disordercharacterized by dry mouth and eyes, the result of lymphocyticinfiltration of the salivary and lacrimal glands (Moutsopoulos etal., 1980). These patients have in their serum, in addition toother autoantibodies, high titres of rheumatoid factors (RFs)(Alspaugh & Whaley, 1982; Elkon et al., 1983). Furthermore,patients with the systemic form of the syndrome have in theirserum or urine monoclonal or oligoclonal immunoglobulinsand/or light chains (Moutsopoulos et al., 1983, 1985) as well asmixed monoclonal cryoglobulins (type II); the monoclonalimmunoglobulin of type II has RF activity and is usually IgMK(Tzioufas et al., 1986). Apart from the above, Sjdgren'ssyndrome (SS) patients are also at high risk for development ofnon-Hodgkin's lymphoma (Kassan et al., 1978). These findingssuggested an underlying monoclonal process in the course of thedisease.

Correspondence: Prof. H. M. Moutsopoulos, MD, FACP, Depart-ment of Medicine, School of Medicine, University of Ioannina, 451 10Ioannina, Greece.

rheumatoid factors monoclonal plasma cells

Recently, several investigators have reported the presence ofcross-reacting and private idiotypes on RF paraproteins (Car-son & Fong, 1983; Greenstein, Solomon & Abraham, 1984;Mageed et al., 1986b). While some of these RF idiotypes areshared between patients with rheumatoid arthritis (RA) andPSS (Gharavi et al., 1985; Mageed et al., 1986b; Pasquali,personal communication), this was not observed with others(i.e. the 17-109 idiotype) (Fox et al., 1986; Fong et al., 1986). InRA it has been demonstrated that although some RFs may carrycross-reactive determinants, these RFs may be only a smallpercentage of the polyclonal RF repertoire (Nelson et al., 1987).Taking into account the monoclonal process, which seems to bea feature of PSS, the idiotype repertoire of RF in PSS may behighly restricted. However, the extent to which these idiotypesare shared among PSS patients, as well as between PSS andother autoimmune patients, is not known.

In order to investigate such cross-reactive idiotypes in PSS,we raised polyclonal anti-idiotypes to a monoclonal RF derivedfrom a cryoprecipitable monoclonal IgMK RF from a PSSpatient. The presence of the idiotypes in the serum of patientswith autoimmune diseases or Waldenstrom's macroglobulinae-mia was also investigated.

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P. D. Katsikis et al.

MATERIALS AND METHODS

PatientsSera from 98 patients with autoimmune rheumatic diseases wereinvestigated.

Thirty-two patients had PSS (mean age 54+ 12 years).Diagnosis of PSS was based on the presence of two out of thethree following parameters: objective keratoconjunctivitis sicca(positive rose Bengal staining); subjective xerostomia anddecreased stimulated parotid flow rate (less than 05 ml/5 minper gland); and parotid gland enlargement. All patients had apositive minor salivary gland biopsy (Tarpley, Anderson &White, 1974). Thirteen of the 32 PSS patients had circulatingmixed monoclonal cryoglobulins (type II) (Tzioufas et al., 1986)with a total protein ranging from 0 3 to 11-5 mg/L. Thesecryoglobulins by high-resolution agarose electrophoresis tech-nique combined with immunofixation (Papadopoulos & Kint-zios, 1967; Pascali, Pezzoli & Chiarandini, 1982) were shown tocontain a monoclonal IgMK RF (Fig. 1). In 15 SS patients,paraffin-embedded labial minor salivary gland biopsy speci-mens were examined by a modified peroxidase-anti-peroxidase(PAP) method of Sternberger et al. (1970). The number and theproportion of plasma cells positively stained for individualintracytoplasmic K or i light chains were evaluated blindly bycounting the cells in 10 different non-overlapping fields/section.If the ratio K: A of intracytoplasmically stained plasma cellsexceeded 3: 1, it was considered that a clonally expanded plasmacell population existed in the infiltrates. This was decided sincethe K: A ratio of positive plasma cells in the minor salivary glandinfiltrates of PSS patients without circulating monoclonalcryoglobulins was found to be 1-5 + 0-2: 1 (Moutsopoulos et al.,1990); this was also suggested by Lennert (1981). In all PSSpatients serum RF was determined by latex agglutination(Rapitex, Behring, Mannheim, FRG); antibodies to extractablenuclear antigens were evaluated by counter-immunoelectro-phoresis (Moutsopoulos et al., 1984).

Thirty-six patients had RA (mean age 51 + 14 years). Threeof those also had SS and circulating polyclonal cryoglobulinswith total protein ranging from 0 3 to 0 42 mg/t. These threepatients were included among the 15 SS patients whose minorsalivary gland biopsy specimens were examined for monoclona-lity. Thirty patients had systemic lupus erythematosus (SLE)(mean age 43 + 12 years). All these patients fulfilled the criteriaof the American Rheumatism Association (Ropes et al., 1958;Tan et al., 1982). In addition, 20 healthy blood donors (meanage 24 + 5 years) and six patients who had Waldenstr6m'smacroglobulinaemia (mean age 59 + 7 years) were studied.Monoclonal gammopathies in the Waldenstrom's macroglobu-linaemia patients were determined by high-resolution agarosegel electrophoresis combined with immunofixation. All patientswith Waldenstrom's macroglobulinaemia had circulating IgMKparaprotein.

Preparation ofpolyclonal anti-idiotypesCryoglobulin was isolated and characterized as previouslydescribed (Tzioufas et al., 1986) from a patient (STE) with PSSwho had a known mixed monoclonal IgMK cryoglobulinaemia(Fig. 1).

STE monoclonal IgMK was prepared by gel filtrationchromatography on a 25 x 95-cm G-200 Sephadex column

a

b

c

d -.

'I

Anti -yAnti-p

Anti-yAnti-a

Anti-KAnti- X

Fig. 1. High-resolution agarose electrophoresis of a cryoprecipitatefrom a patient with primary Sjogren's syndrome. In the gammaglobulinregion a monoclonal band (a, arrow) is seen; upon fixation it proved tobe IgMK (b, c, d).

(Pharmacia Fine Chemicals, Uppsala, Sweden) in 0-1 M acetatebuffer, pH 3 5.

For the preparation of anti-idiotype II ss, part of the aboveisolated monoclonal IgMK was digested by trypsin at 65-Caccording to the method of Plaut & Tomasi (1970). The digestwas then applied to a 25 x 95-cm Sepharose 4B column(Pharmacia) and the Fab fragment was isolated and character-ized by immunodiffusion. A rabbit was then immunized with0-5 mg of Fab in Freund's complete adjuvant, followed bybooster injections in Freund's incomplete adjuvant at 2 and 4weeks.

Anti-idiotype I2ss was prepared by immunizing a rabbitwith whole STE monoclonal IgMKc following the same immuni-zation protocol as for Ilss. Raising anti-idiotype to twodifferent antigens was an attempt to produce anti-idiotypes ofhigher affinity or directed to different epitopes.

Rabbit IgG was purified by protein A chromatography andthen extensively adsorbed on an IgG column made with 100 mgofhuman Cohn's fraction II (Sigma, St Louis, MO) and an 1gMcolumn made with 50 mg of IgM prepared from pooled humansera by euglobulin precipitation and gel filtration of a G-200Sephadex column (Pharmacia). Rabbit IgG was then pepsin-digested (Nisonoff et al., 1960) and F(ab')2 fractionated on aG-200 Sephadex column (Pharmacia) and further purified byprotein A affinity chromatography. The F(ab')2 fragments wereconcentrated and tested for purity by a 10% SDS-PAGE(Laemmli, 1970) and immunodiffusion with goat antisera torabbit Fc and F(ab')2 fragments (Jackson Laboratories, BarHarbor, ME).

Anti-idiotypes were tested by ELISA and were found not tobind to 10 pg/ml human Cohn's fraction II (Sigma) or 10 yg/mlof pooled human IgM isolated as described above; they did bindstrongly to 1 pg/ml ofSTE monoclonal IgMK against which theywere raised.

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Idiotypes in Sjigren's syndrome

ELISA for total immunoglobulin idiotype activityFlat-bottomed, 96-well microtitre plates (Maxisorb, Nunc,Roskilde, Denmark) were coated for 2 h at 37 C with 5 yg/mlanti-idiotype in bicarbonate buffer 0-05 M, pH 9-6. Blocking wasperformed with 10% fetal calf serum (FCS) in PBS, pH 7-2, for2 h at 37 C. Subsequently, 1/20 dilutions of human sera wereadded for 2 h at 37 C. Goat F(ab')2 anti-human immunoglobu-lin conjugated to alkaline phosphatase (Jackson Labs) was

added at 1/1000 dilution for 1 h at room temperature. Subse-quently, p-nitrophenyl phosphate (p-NPP) in glycine buffer wasadded and the plates were incubated for 30 min at 37 C. Thereaction was stopped with 3 N NaOH and plates were read at405 nm on a spectrophotometer (Dynatech, Plochingen, FRG).Between all steps, washings were performed with 0.5% Tween20 in PBS, pH 7-2. The detection limit of the above ELISA was

100 pg/ml ofSTE monoclonal IgMK RF, when purified STE RFin PBS was assayed in ten-fold dilutions.

In order to exclude non-specific binding (e.g. pepsin aggluti-nator activity), all sera of SS and RA patients were tested againstnormal rabbit F(ab')2, with the ELISA described above.

00l<C:0.:t72_r_-

11C

100

90

80

70

60

50

40

30

20

10

0

3 6-125 12-5 25 50

Idiotype concentration (L4g/ml)100 200

Fig. 2. Inhibition of the idiotype binding (STE monoclonal IgMK) toIlIss anti-idiotype by IlIss (0) and I2ss (0) anti-idiotype.

1000roELISA for isotype distribution of idiotypesELISA plates as above were coated with 5 yg/ml goat F(ab')2anti-human IgM, IgG or IgA (Jackson Labs) in bicarbonatebuffer 0 05 M, pH 9-6, overnight at 4-C.

Plates were blocked with 10% FCS in PBS, pH 7-2, for 2 h at37 C. Sera were added at dilutions 1/50 and incubated for 2 h at37°C. Anti-idiotype at 50 ,g/ml was added for 2 h at 37°C. GoatF(ab')2 anti-rabbit F(ab')2, conjugated to horseradish peroxi-dase (Jackson Labs) at 1/1000 dilution was added for 2 h atroom temperature. Substrate ABTS was added for 30 min atroom temperature and absorbance at 405 nm was determined ona microplate reader.

In both ELISAs, since variation between plates did notexceed 15%, results were expressed directly as optical density(OD) values. The cut-off for positivity was set at the mean

+ 3 s.d. of the OD given by the sera of normal individuals.

Inhibition assays

Homologous and cross-inhibition assays were performed on

strongly positive sera using the. following ELISA: plates were

coated with 50 pg/ml anti-idiotype in 0-05 M bicarbonate buffer,pH 9-6, for 2 h at 37 C, blocked with 00 FCS in PBS for 2 h at37 C, incubated with 1/20 dilutions of sera in PBS for 2 h at37TC; then alkaline-phosphatase-conjugated goat F(ab')2 anti-human immunoglobulins, at 1/1000 dilution was added for 1 hat room temperature and p-NPP in glycine buffer as substratewas added for 30 min at 37-C. Reaction was stopped with 3 N

NaOH. For the inhibition studies sera with serial dilutions ofanti-idiotypes were incubated at 37-C for 2 h and then added tothe ELISA. Rabbit sera were used as controls. Inhibition (0/o)was expressed according to the formula

(OD Control) -(OD Inhibited) x 100(OD Control)

Statistical analysisResults were analysed using Student's t-test (where Fisher'sexact test was significant, the modified z-transformation was

applied), and Spearman's rank correlation.

800 _

E

cu)0

0c

a

600 _

400 _-

200 [

0

-it

-r t t APSS RA SLE C

Fig. 3. Serum idiotype level binding of 32 patients with primarySjogren's syndrome (PSS), 33 patients with rheumatoid arthritis (RA),30 patients with systemic lupus erythematosus (SLE) and 20 healthycontrol subjects (C).

RESULTS

Assay specificiti'In the homologous inhibition assays both anti-idiotypes inhi-bited in a dose-response manner the binding ofSTE monoclon-al cryoglobulin and positive sera to themselves.

Cross-inhibition between Ilss and I2ss was also clearlydemonstrated (Fig. 2), and together with the identical positivepattern in the sera tested suggested that both anti-idiotypesrecognized common or related determinants.

Sera from healthy individuals, and from patients with SSand RA, which were tested against normal rabbit F(ab')2 gave

511

.

P. D. Katsikis et al.

EC

LO)0

S 300 _

C

a)

~'200-

0B0.001100

a b c d

Fig. 4. Comparison of mean idiotype levels in patients with primarySjogren's syndrome with (U) and without (0) a, salivary glandsmonoclonacity; b, monoclonal cryoglobulins; c, extraglandular mani-festations; and d, antibodies to Ro(SSA)/La(SSB).

almost the same OD levels (62 + 21, 76+ 34 and 68 + 28,respectively). These levels were significantly lower than thoseobserved in the idiotype assay, suggesting that the non-specificbinding was minimal.

Idiotype expression in patients and healthy controlsReactivity of I lss and I2ss rabbit polyclonal anti-idiotypes toSTE monoclonal IgMK RF and patient sera was identical; we

therefore present results for Ilss only.Total immunoglobulin idiotype activity was found in 20 out

of 32 (62-5%) PSS patients (mean OD level 219+192 s.d.). Ofthe 20 positive sera, the idiotype was present on IgM in 20, IgGin 12, and IgA in 18.

Nine of the 33 patients (27%) with RA were positive for totalimmunoglobulin idiotype activity, (mean OD 117, s.d. 57). Allnine were positive for IgM isotype, six for IgG and for IgA.None of the SLE patients tested was found positive, (mean OD68, s.d. 27). The individual binding of the sera from autoimmunepatients and healthy controls is shown in Fig. 3.

The sera of the six patients with Waldenstrom's macro-

globulinaemia were diluted to a concentration of 5 pg IgM/mland then tested. Two patients gave in the ELISA a reading closeto that of 5 ,g/ml of purified STE monoclonal cryoglobulin andwere considered positive for the idiotype.

For the control group of healthy individuals, mean OD was

57 (s.d. 32). The mean OD idiotype level of the PSS patientswhen compared with that of the RA patients and the controlswas found to be significantly higher (P<0-02 and P<0-001,respectively). The average OD for the RA patients was higherthan for the controls in a statistically significant manner. The

SLE group did not differ significantly from the control subjects.

Correlation of idiotype with different parameters in PSS

Cryoglobulins and idiotype. The average OD level of PSSpatients with mixed monoclonal IgMK cryoglobulins differed

significantly from that of patients without cryoglobulins(P <0-02) (Fig. 4). The three patients with RA/SS and mixed

polyclonal cryoglobulins were all found negative for idiotype.Minor salivary gland biopsy and idiotype. Six of the 15 PSS

patients investigated for monoclonal plasma cells in the minor

salivary gland biopsy were found to have an infiltratingpopulation of plasma cells with a K: 2 ratio greater than 3: 1.More specifically two had a K: 2 ratio 99: 1 and on microscopyrevealed a picture of immunocytoma while in the rest the K:2ratio ranged from 3-6 to 5-8: 1. The average OD level of idiotypein these six patients when compared with that of the remainingnine in whom the ratio of K: 2 was 1-5 + 0-2: 1 was significantlyhigher (P < 0-05) (Fig. 4).

Extraglandular manifestations and idiotype. Of the 32 PSSpatients included in this study, 23 had extraglandular manifes-tations. The mean OD idiotype level of the 23 patients withextraglandular manifestations was significantly higher than thatof nine patients without (P < 0-01) (Fig. 4).

Anti-Ro(SSA)/La(SSB) and idiotype. Twenty-three of the32 PSS patients had antibodies to Ro(SSA)/LA(SSB) antigens.The mean OD idiotype level of these patients was significantlyhigher when compared with that of the remaining nine patients(P < 0 02) (Fig. 4).

RFand idiotype. Twenty-one of the PSS patients were foundpositive for RF, ranging from 0 to 10-240 U/ml.

RF levels and idiotype OD did not correlate significantly(P>0 1).

DISCUSSION

Idiotypic cross-reactivity of RF has been the subject of manyinvestigations. Monoclonal RF from unrelated individuals hadbeen originally classified into three cross-reactive idiotypegroups, Wa and Po, that include 60% and 20% of monoclonalRFs, respectively (Kunkel et al., 1973) and the Bla group, whichdefines a minor subgroup (Agnello et al., 1980). Other studiesusing monoclonal antibodies to determinants on K light chains(Carson & Fong, 1983; Mageed, Walker & Jefferis 1986a) orepitopes on heavy chains of monoclonal RF (Mageed et al.,1986b) have also shown an extensive cross-reactivity among theidiotypes of monoclonal RFs.

Patients with PSS have high titres of circulating RF (Elkonet al., 1983; Harley, 1987; Alspaugh & Whaley, 1989).

Cross-reactive idiotypes I lss and I2ss produced against STEmonoclonal IgMK RF isolated from the type II cryoglobulin ofa patient with PSS was found in 62 5% (20/32) of PSS patients.This idiotype was distributed in all isotypes, showing, however,a preference for IgM and IgA. The cross-reactive idiotypes werealso found in 27% of RA patients but in no patient with SLE.

Preliminary experiments with Western blotting have shownthat the K light chain of the STE monoclonal RF, against whichIlIss and I2ss were raised, bears the 17.109 idiotype (data notshown). We also have evidence that the STE monoclonal RFbears the G6 idiotype, since we have previously demonstratedthat RF activity in the sera of patient STE can be completelyinhibited by the G6 cross-reactive anti-idiotype (Shokri et al.,1989). Supporting the above is the finding of high levels of17.109 and G6 idiotype in the sera of patient STE (R. Jefferis,personal communication).

The levels of circulating cross-reactive idiotype correlatedwith a predominance of intracytoplasmically K-positive plasmacells in salivary gland infiltrations. This finding, taken togetherwith the observation that two of six patients with Walden-strom's macroglobulinaemia were positive for the idiotype,suggests that the clonal expansion in the salivary gland may risefrom B cells bearing immunoglobulins of a specific cross-

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Idiotypes in Sjogren's syndrome 513

reactive idiotype. The above observations are in accord with thefindings of Fox et al. (1986), who demonstrated that B cellsinfiltrating the salivary glands bear the cross-reactive idiotype17.109, which has been shown to be encoded by a germ linevKIIIb gene and frequently expressed by monoclonal RFs frompatients with Waldenstrdm's macroglobulinaemia. In addition,25% ofCD5+ B cell from chronic lymphocytic leukaemias bearthe 17.109 cross-reactive idiotype. CD5+ B cells have beenshown to be increased in SS (Dauphinee, Tovar & Talal, 1988;Youinou et al., 1988) and correlate with the presence ofcirculating monoclonal immunoglobulins in PSS patients(Youinou et al., 1988). Taken together these observations andthe present findings may imply that the monoclonal process thathas been well documented in PSS patients (Fishleder et al., 1987;Moutsopoulos et al., 1983, 1985) would arise from certainCD5 + B cell clones bearing specific idiotypes. In support of thisis our finding that patients with PSS and type II cryoglobulinshave significantly higher levels of idiotype than patients withoutcryoglobulins, while three patients with polyclonal cryoglobu-lins (type III) were all negative for the idiotype.

There is some dispute over the correlation of RF withextraglandular manifestations in SS. Some investigators havereported a correlation between IgM and IgA RF and thenumber of extraglandular manifestations (Muller et al., 1989);others have not found this association (Elkon et al., 1984). Herewe have shown that the levels of cross-reactive idiotype weresignificantly higher in PSS patients with extraglandular manifes-tations. This observation, taking into account that the idiotypedid not correlate with RF titre, suggests that a subset of RFsmay play some pathogenetic role in the extraglandular form ofthe disease. The correlation of antibodies to extractable nuclearantigens with the idiotype could reflect the result of polyclonalactivation or the expression of such idiotype by other autoanti-bodies (Painter, Monesteir & Bona, 1986).

It is not clear whether the cross-reactive idiotypes are theresult of clonal expansion or are derived from independentclones under selective pressures. Recently it has been proposedthat the 17.109 cross-reactive idiotype bearing B cells in PSS areof multi-clonal origin and are the result of an antigen or T cell-driven process which expands B cells that utilize particular Vregions (Kipps et al., 1989). The B cells utilizing these particularV regions could be especially prone to malignant transforma-tion.

We have shown that RFs in PSS share extensively cross-reactive idiotype, and that these are also shared to a lesser extentwith RA. The findings of this study suggest that the monoclonalprocess observed in PSS possibly involves cross-reactive idio-type-bearing B cells, which are also shared with B cell neo-plasias. The study also suggests that a subset of RF bearingcross-reactive idiotypes probably has a pathogenetic role in thedevelopment of extraglandular diseases in PSS patients. Anti-idiotypic intervention may be an alternative therapeuticapproach for PSS patients with extraglandular disease andmonoclonal B cell expansion.

ACKNOWLEDGMENTS

This work has been partly supported by the Association de R&cherchesur la Polyarthrite. We thank Ms Helen Prevezianou for secretarialassistance.

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