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Clin. exp. Immunol. (1986) 64, 570-580. The common occurrence of internal image type anti-idiotypic antibodies in rabbits immunized with monoclonal and polyclonal human IgM rheumatoid factors S. FONG, T. A. GILBERTSON, P. P. CHEN, J. G. KARRAS, J. H. VAUGHAN, & D. A. CARSON Department of Basic and Clinical Research, Division of Clinical Immunology (BCR4), Scripps Clinic and Research Foundation, La Jolla, California, USA. (Accepted for publication 23 January 1986) SUMMARY We have previously reported that rabbits immunized with a polyclonal human rheumatoid factor (RF) autoantibody preparation could induce anti-idiotypic antibodies bearing the 'internal image' of the Fc fragments of human IgG. The 'internal image' anti- idiotype have been shown to react with both the RF molecules as well as with the RF receptors on B lymphocytes. Under what conditions these anti-idiotypes occur is not known. Presently, we report that these anti-idiotypic antibodies occur more frequently than previously thought and could be isolated in sera of rabbits immunized with either monoclonal paraproteins with RF activity or other purified human polyclonal serum RFs. Immunization of rabbits with a peptide corresponding to the second complementar- ity-determining regions of a monoclonal RF did not induce this anti-idiotype. Immuniza- tion of goats with human RF similarly did not result in induction of such anti-idiotype. Induction of these anti-idiotypes thus depended upon immunization with the intact RF antigen as well as the species of animal immunized. The repeated isolation of 'internal image' anti-idiotypic antibodies from RF immunized rabbits suggests that the antigenic conformations recognized by human RF autoantibodies are restricted, and that 'internal image' anti-idiotypic species to RF may pre-exist within the rabbit immune network. Such broadly cross-reactive anti-idiotypic reagents provide unique reagents for studying the regulation of RF autoantibody synthesis. Keywords anti-idiotypic antibodies internal image rheumatoid factors INTRODUCTION The specific suppression of antibody synthesis by anti-idiotypic antibodies has been reported in animals and man (Rajewsky & Takemori, 1983). The potential use of these regulatory molecules to human autoantibodies has been limited by difficulties in preparing broadly cross-reactive anti- idiotypic antibodies and by the appearance of idiotype negative antibodies with the same antigenic specificity. Accumulating experimental evidence indicates that anti-idiotypic antisera may contain as many as four distinguishable antibody classes (Bona et al., 1982). The first two types are directed against Correspondence: Dr S. Fong, Department of Basic and Clinical Research, Division of Clinical Immunology (BCR4), Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. 570
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Page 1: The common occurrence of internal image type anti-idiotypic ...

Clin. exp. Immunol. (1986) 64, 570-580.

The common occurrence of internal image type anti-idiotypicantibodies in rabbits immunized with monoclonal and

polyclonal human IgM rheumatoid factors

S. FONG, T. A. GILBERTSON, P. P. CHEN, J. G. KARRAS, J. H. VAUGHAN, &D. A. CARSON Department of Basic and Clinical Research, Division of Clinical Immunology

(BCR4), Scripps Clinic and Research Foundation, La Jolla, California, USA.

(Acceptedfor publication 23 January 1986)

SUMMARY

We have previously reported that rabbits immunized with a polyclonal humanrheumatoid factor (RF) autoantibody preparation could induce anti-idiotypic antibodiesbearing the 'internal image' of the Fc fragments of human IgG. The 'internal image' anti-idiotype have been shown to react with both the RF molecules as well as with the RFreceptors on B lymphocytes. Under what conditions these anti-idiotypes occur is notknown. Presently, we report that these anti-idiotypic antibodies occur more frequentlythan previously thought and could be isolated in sera of rabbits immunized with eithermonoclonal paraproteins with RF activity or other purified human polyclonal serumRFs. Immunization of rabbits with a peptide corresponding to the second complementar-ity-determining regions of a monoclonal RF did not induce this anti-idiotype. Immuniza-tion of goats with human RF similarly did not result in induction of such anti-idiotype.Induction of these anti-idiotypes thus depended upon immunization with the intact RFantigen as well as the species of animal immunized. The repeated isolation of 'internalimage' anti-idiotypic antibodies from RF immunized rabbits suggests that the antigenicconformations recognized by human RF autoantibodies are restricted, and that 'internalimage' anti-idiotypic species to RF may pre-exist within the rabbit immune network. Suchbroadly cross-reactive anti-idiotypic reagents provide unique reagents for studying theregulation of RF autoantibody synthesis.

Keywords anti-idiotypic antibodies internal image rheumatoid factors

INTRODUCTION

The specific suppression of antibody synthesis by anti-idiotypic antibodies has been reported inanimals and man (Rajewsky & Takemori, 1983). The potential use of these regulatory molecules tohuman autoantibodies has been limited by difficulties in preparing broadly cross-reactive anti-idiotypic antibodies and by the appearance of idiotype negative antibodies with the same antigenicspecificity.

Accumulating experimental evidence indicates that anti-idiotypic antisera may contain as manyas four distinguishable antibody classes (Bona et al., 1982). The first two types are directed against

Correspondence: Dr S. Fong, Department of Basic and Clinical Research, Division of Clinical Immunology(BCR4), Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037,USA.

570

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Anti-idiotype to rheumatoidfactors 571conformation-dependent antigenic determinants associated with the antibody combining site, andthe variable region framework, respectively. A third class of anti-idiotype recognizes primarysequence associated idiotypic antigens, as revealed by synthetic variable region peptides. A fourthtype of anti-idiotype mimics the three dimensional structure of the antigen. These latter 'internalimage' anti-idiotypic antibodies have been postulated to play a role in the regulation of the immuneresponse (Jerne, 1974; Sege & Peterson, 1978; Schreiber et al., 1980; Homcy, Rockson & Haber,1982; Marasco & Becker, 1982; Wasserman et al., 1982; Jerne, Roland & Cazenave, 1982).We have recently reported that rabbits immunized with polyclonal human rheumatoid factor

(RF) autoantibodies from a single donor produced anti-idiotypic antibodies bearing the 'internalimage' of the Fc fragment of human IgG (Fong, Gilbertson & Carson, 1983). Because they reactwith multiple antibodies of the same specificity, 'internal image' anti-idiotypes potentially representideal reagent for the suppression of antibody synthesis in outbred populations (Fong et al., 1984).Hence, it was important to define the parameters that rendered feasible the induction of 'internalimage' of anti-idiotypes to human RF. Here we report the isolation and characterization of anti-idiotypic antibodies from different rabbits immunized with either monoclonal or polyclonal humanIgM RFs. The data suggests that anti-idiotypes bearing the 'internal image' of antigen occurcommonly among RF immunized rabbits. The elicitation of this class of anti-idiotype was highlydependent upon the immunizing antigen, and the species of animal immunized.

MATERIALS AND METHODS

Monoclonal and polyclonal RF. The monoclonal IgM RF preparations were obtained frompatients with cryoglobulinemia. These paraproteins have been previously described: Koh (Kaplanet al., 1971), Lay (Klapper & Capra, 1976), Sie (Stone, 1973), Pom (Capra & Kehoe, 1974), and Glo(Capra & Kehoe, 1975).

The polyclonal IgM RFs were obtained from patients with rheumatoid arthritis. The IgM RFswere isolated by precipitation with 40% saturated ammonium sulfate, followed by affinitychromatography on IgG-Sepharose 4B (Fong et al., 1984).

Rabbit Anti-RFantibodies. Rabbit antibodies against monoclonal and polyclonal IgM RF wereprepared in rabbits as previously described (Fong et al., 1983). Each animal received asubcutaneous injection with 0 5 mg of purified IgM RF emulsified in 1 ml of complete Freund'sadjuvant. The animals were boosted on day 21 with 0-5 mg ofRF in incomplete Freund's adjuvant.On day 42, they were boosted again with 0 5 mg ofRF in saline. Serum samples were collected 5, 7,and 9 days later. The antibodies were isolated by precipitation with 40% saturated ammoniumsulfate. Sera from normal rabbits were processed similarly.

Human IgG Fc fragments were prepared by papain digestion (Porter, 1959), from purifiedpooled IgG (Cohn Fraction II, Sigma, St Louis, Mo, USA), or from an IgGI, myeloma protein(War) and were isolated by sequential chromatography on DEAE cellulose and Ultrogel AcA34, aspreviously reported (Carson & Lawrance, 1978). The anti-Fc fragment specific antibodies wasobtained by immunization of rabbits or goats with purified Fc, following the same protocol used forthe preparation of anti-RF antibodies.

The anti-Fc antiserum was obtained by precipitation with 40% saturated ammonium sulfate anddialysed against 0-01 M phosphate, 0 15 M NaCl, pH 7-4 (phosphate buffered saline; PBS). The anti-Fc was conjugated to cyanogen bromide activated Sepharose-4B (Pharmacia, Piscataway, NJ,USA) as described (Fong et al., 1983). A ratio of 10 mg protein per ml agarose was used.

Fractionation of anti-idiotypes on rabbit anti-human IgG Fc sepharose-4B. The 40% saturatedammonium sulfate precipitate of rabbit anti-RF sera were dialysed against 0 01 M phosphate,0 15 M NaCl (pH 7-4). Ten to 12 ml of anti-RF (17 mg/ml to 40 mg/ml in PBS) or controlimmunoglobulins were recirculated over 10 ml of rabbit anti-human IgG Fc Sepharose-4B for 18 hat 4°C. The columns were washed with PBS containing 0.5% Tween 20 until the A280 was less than0 02-0-03. The adsorbed protein was eluted with 0 1 M glycine-HCI, pH 2 5, and was neutralized topH 7 4 with 1 M Tris base (pH 10-7). The preparation of F(ab')2 rabbit anti-idiotypes and F(ab')2normal rabbit IgG have been previously described (Fong et al., 1983; 1984).

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Enzyme linked immunosorbent assay (ELISA). Anti-idiotypic antibody activity was assayed byELISA. Microtitre plate wells (No. 3590, Costar, Cambridge, MA, USA) were incubated for 4 hwith 100 1d of a 60 ig/ml human IgG in PBS. The wells were washed three times with 0 1 M borate,0-2 M NaCl pH 8-0 containing 0 05% Tween 20 (borate buffered saline; BBS) and then werequenched with BBS supplemented with 10 mg/ml bovine serum albumin (BBS-BSA). RFpreparations or patient sera were incubated at room temperature for 1 h with equal volumes of anti-idiotype or normal rabbit IgG diluted in PBS. The mixtures (100 Pil) were then added to the IgGprecoated wells. After 2 h at room temperature, and 16 h at 4 C, the wells were washed three timeswith BBS, and then were incubated with alkaline phosphatase labelled goat anti-human IgM(Kirkegaard and Perry, Gaithersburg, MD, USA) diluted 1:800 in PBS. After 1 h incubation theplates were washed and the enzyme activity detected by addition of 100 pd of 1 mg/ml p-nitrophenylphosphate in 0 05 M sodium carbonate pH 9-8, 1 mm magnesium chloride. Anti-idiotypic antibodyactivity was monitored after 30 min of incubation at 37 C. The absorption at A405 was measured in aTitertek Multiscan Meter (Flow Laboratories, Rockville, MD, USA).

Enzyme-linked immunoassay for complement activation. An assay for complement activationbased upon the cleavage of C4 was used to assess reactivity of the F(ab')2 anti-idiotype to IgM-RF.The test was slightly modified from our previously described assay (Sabharwal et al., 1982).Polystyrene ELISA plates were precoated with 100 jil of 60 Mig/ml F(ab')2 anti-idiotype, with F(ab')2normal rabbit IgG or with reduced and alkylated human IgG (as a positive control) in BBS (pH 8 4)overnight at 4°C. The coated wells were quenched with 1% BSA-BBS. Serum globulins fromrheumatoid arthritis patients (Mei and Har) served as the source of RF. Control RF depletedpreparations were prepared by immunoadsorption on Sepharose-4B conjugated with human IgG at1O mg/ml beads. These RF preparations (100 pil/well) were added to the wells and incubatedovernight at 4°C. The wells were incubated with 100 ,ul of a 1:50 dilution complement in veronalbuffered saline. The complement source was a serum from a patient with acquired agammaglobuli-nemia which was depleted of immunoglobulins by adsorption with goat anti-human immunoglobu-lin antibodies conjugated Sepharose 4B and reconstituted with purified Clq to 50 ,ug/ml (a giftkindly provided by Dr J.G. Curd). The plates were incubated at 37 C for 1 h. The wells were washedand exposed to 100 iil per well fluorescein-conjugated goat anti-human C4 antiserum (5 pig/ml inPBS) provided by Dr John G. Curd. After overnight incubation at 4°C, the wells were exposed toalkaline phosphatase conjugated goat anti-fluorescine at 1:500 dilution in PBS for 1 h at room

temperature. The affinity purified goat anti-fluorescein antibodies were prepared as previouslydescribed (Fong et al., 1981). The enzyme activity was detected as described above for the RF assay.

RESULTS

Isolation of broadly cross-reactive anti-idiotypic antibodies from rabbits immunized with humanmonoclonal or polyclonal IgM RFRabbit anti-RF antibodies were recirculated over a rabbit anti-human IgG Fc-Sepharose-4Bcolumn. It was anticipated (a) that rabbit anti-idiotypic antibodies against human RFs wouldcontain a fraction bearing the 'internal image' of the human IgG Fc fragment, and (b) that only thisfraction would bind to rabbit anti-Fc antibodies bearing the same specificity as RFs.

As illustrated in Table 1, between 04-6-8% of the globulin in rabbit antisera to eithermonoclonal or polyclonal IgM RF bound to and was recovered from the anti-Fc affinity beads.Under the same conditions, rabbit antibodies to the peptide (PSH2) corresponding to the secondcomplementarity determining region (CDR) of the IgM heavy chain of Sie (Chen et al., 1984;Andrews & Capra, 1981) yielded 10% protein recovered, a value which lies within the test sera range.Less than 0-02% of the globulins in a control rabbit anti-burrow red blood cells anti-serum bound to

the anti-Fc affinity column. A monoclonal hybridoma mouse anti-idiotypic antibody (cross-reactive, non-internal image type) to human rheumatoid factor (17 109) served as an additionalcontrol yielded approximately 1% bound and eluted material. Thus, only a small fraction rabbit or

mouse antibody bound to the affinity-Sepharose 4B. The eluted immunoglobulins were dialyzedand then tested for anti-idiotypic antibody activity by inhibition of RF binding activity to humanIgG.

Sherman Fong et al.572

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Anti-idiotype to rheumatoidfactors 573Table 1. The amounts of rabbit anti-RF idiotype isolated by affinity chromatography on rabbit anti-human IgGFc specific antibody-Sepharose-4B

Protein* Protein boundt ProteinAntisera loaded (mg) and eluted (mg) recovered (%)

I. ANTI-MONOCLONAL RFt

Anti-Sie 435 4-80 1.1Anti-Glo 254 1 10 04Anti-Pom 367 3 17 09Anti-Lay 367 3-63 1.0Anti-Koh 262 2-00 0-8

II. ANTI-POLYCLONAL RFJAnti-Mei 168 2 66 1 6Anti-Ada 332 2 50 0 8Anti-Lyt 323 5 00 1 5Anti-Har 337 22 77 6 8

III. CONTROLS

Rabbit Anti-PSH2§ 324 3-36 1 0Rabbit Anti-BRBC¶T 52 <0 01 <0 02MouseAnti-RFId(I7 109)** 67 0-64 1.0

* Immunoglobulins isolated by precipitation with 40%O saturated ammoniumsulfate.

t Protein recovered by elution with 0-1 M glycine-HCl (pH 2 5).t Rabbit anti-sera against human monoclonal paraprotein or polyclonal IgM

RF.§ Rabbit anti-serum against a synthetic peptide corresponding to the second

complementarity-determining region of the heavy chain of the monoclonal IgMRF (Sie). The peptide sequence was Ser Pro Ala Lys Trp Thr Asp Pro Phe Gln GlyVal Tyr Ile Lys Trp Glu (Andrews & Capra, 1981). The antiserum was preparedaccording to the methods previously described (Chen et al., 1984).

' Rabbit anti-burro red blood cells (BRBC)** Control for non-specific binding of protein to Sepharose-4B column. 17 109

is a monoclonal anti-idiotype to the light chains of Sie and Glo (Carson & Fong,1983).

Anti-idiotypic antibodies derived from anti-polyclonal and anti-monoclonal IgM RF rabbit serainhibited polyclonal and monoclonal 1gM RF binding to human IgGAnti-idiotypic antibodies were purified from separate groups of rabbits immunized with ninedifferent monoclonal or polyclonal IgM-RF. Each anti-idiotypic preparation displayed detectableinhibitory activity against both monoclonal and polyclonal IgM-RF proteins. In Table 2 theinhibitory activity is expressed as the maximum percentage of inhibition achieved (plateau phase),relative to the initial IgM-RF activity (400 ng/ml) of purified monoclonal RFs or of serumpolyclonal RFs. When compared to controls, inhibition greater than 20% was consideredsignificant. The antibodies exerted only minimal inhibitory activity toward IgM anti-dinitrophenyl(Metzger, 1967) and IgM anti-tetanus toxoid antibodies. Normal rabbit IgG did not inhibit thebinding of RFs to human IgG. Inhibition of RF binding to IgG was not due to the binding of theanti-idiotypes to IgG. Preincubation of the anti-idiotypes with the IgG precoat in the ELISA didnot inhibit the binding of theIgM RFs (data not shown). In accord with our earlier results, none of'internal image' anti-idiotypes bound significantly to human IgG (A405 x 103 at 40 mig/ml anti-idiotype protein was 100 +43 and normal rabbit IgG was 51 + 7 after 30 min incubation).

The anti-idiotypic antibodies induced by polyclonal and monoclonal RFs inhibited only aproportion of the RF activity in the majority of samples tested. Some RFs were not blocked

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Sherman Fong et al.

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Anti-idiotype to rheumatoidfactors 575significantly. Polyclonal RF Har induced the most broadly cross-reactive anti-idiotype. As shownin Table 2, it inhibited 11 out of 15 different RFs. By comparison monoclonal RF Glo elicited anti-idiotypic antibodies that inhibited only six out of 13 RFs. There was no obvious tendency for thepolyclonal RF preparations to induce more broadly cross-reactive anti-idiotypic antibodies thanthe monoclonal RFs. Monoclonal RFs Sie, Glo and Lay and polyclonal RF Mei were eachinhibited by their homologous anti-idiotype preparation. In nearly all cases a RF sharing anti-idiotypic cross-reactivity with another RF can be blocked by anti-idiotypic antibodies preparedagainst the latter RF. The only exception in our studies was the inability of anti-Sie to inhibit LayRF while anti-Lay was capable of inhibiting Sie RF. A basis for this is unknown.

The inhibition patterns suggested that the 'internal image' anti-idiotypic reagents werecomposed of a restricted number of antibody species. To test this hypothesis, cross adsorptionstudies were performed with the anti-idiotypes to IgM RF Mei (Table 3). Pre-adsorption of the anti-

Table 3. Cross adsorption of anti-idiotypic activity

Net direct binding:(0 reduction)§

IgM RF usedAnti-idiotype for adsorption Sie Pal Mei

anti-Sie* IgM RF (Sie) 1341 (63)§ 218 (44) 101 (68)IgM RF (Pal) 217 (41)IgM RF (Mei) 203 (44)BSA control 365 (0) 391 (0) 324 (0)

anti-Meit IgM RF (Sie) 63 (60) 77 (50) 51(81)IgM RF (Pal) 28 (82)IgM RF (Mei) 26 (84)BSA control 158 (0) 153 (0) 389 (0)

* 'Internal Image' type rabbit anti-Sie (IgM-RF) isolatedby affinity chromatography on a rabbit anti-human Fc of IgGantibody conjugated Sepharose-4B column.

t F(ab')2 fragments of 'internal image' type rabbit anti-Mei IgM-RF was isolated by pepsin digestion and affinitychromatography.

T A405 X 103 obtained after 1 h incubation. The assay wasperformed by adsorbing 100 /1l of a 400 ng/ml anti-idiotypepreparation overnight at 4°C on polyvinyl chloride microtiterplates precoated with 100 ,ul of a 20 yg/ml IgM-RF Sie, IgM-RF Pal, IgM-RF Mei or 1% BSA-BBS. The unadsorbed anti-idiotype was then transfered to micro wells precoated witheither Sie RF, Pal RF, Mei RF or BSA, each identical inconcentration to the first adsorption step.

§ The per cent reduction of binding activity after adsorp-tion on RF as compared to adsorption on BSA.

¶ A405 X 103.

idiotype to RF Sie coated plates depleted much of the anti-idiotype binding activity to RFs Sie, Pal,and Mei. Adsorption to Pal and Mei only partially depleted the binding activity of anti-Sie to Siecoated plates. In a complementary fashion, pre-adsorption of anti-Mei with Mei RF depletedbinding activity towards Mei, Sie and Pal. Preadsorption to Sie and Pal RF similarly depleted theanti-idiotype.

Many human RFs will bind to aggregated rabbit IgG. However, the interaction ofRF with anti-idiotype was unrelated to the rabbit IgG binding capacity of the autoantibody. The monoclonal RFLay did not bind strongly to rabbit IgG, but was inhibited by the anti-idiotypes against Lay, Koh,

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Table 4. Inhibition by rabbit anti-idiotype is unrelated to whether the IgM RF can bind rabbit IgG

IgM RF bound(A4o5 x 103)

Inhibitionby Anti-Idiotype to RFt Hu IgG Rb IgG

Monoclonal IgM RF*Sie + 465 135Lay + 515 0

Polyclonal IgM RFMei + 700 700Rol + 550 306Tys 580 480Jac 520 290Hil 715 610

* Direct binding of 300-400 ng/ml of IgM RF (100p1) to poly-styrene plates precoated with either human IgG (Hu IgG) or rabbit IgG(Rb IgG).

t Each RF was tested with nine different anti-idiotypic antibodies(See Table 2).

Mei and Lys (Table 2). Similarly, the induction of the cross-reactive anti-idiotypic antibody activitydid not depend absolutely on whether the immunizing RF was capable of binding to rabbit IgG(Table 4). Thus, the IgM RF (Lay) successfully elicited the cross-reactive rabbit anti-idiotype.

Several control experiments tested the specificity of the immunization and purification scheme.Rabbits immunized with a synthetic peptide corresponding to the second CDR of the heavy chainsof the monoclonal IgM RF paraprotein Sie did not produce anti-idiotypic antibodies that bound torabbit-anti-human IgG Fc-Sepharose 4B. The mouse hybridoma 17 109, as reported previously,recognized a cross-reactive idiotype on RF kappa light chains including Sie ad Glo RF (Carson &Fong, 1983). Approximately 1% of mouse IgG anti-17 109 will bind nonspecifically to the rabbitanti-human IgG Fc-Sepharose 4B column (Table 1). The bound and eluted material did not display'internal image' type anti-idiotype activity. As shown in Table 2, the 17 109 anti-idiotype inhibitedthe IgG binding activity of Glo, but not Pal or Mei. The latter two RFs lack the 17 109 idiotype. Anormal mouse IgG preparation did not inhibit any of the RFs tested.

The reactivity of affinity-purified anti-idiotypic antibodies to RFs Sie and Mei with isolatedIgM-RF heavy and light chains was assessed by ELISA. Table 5 shows the direct binding of anti-Sieand anti-Mei to Ig Lay RF, and to free Lay kappa and y heavy chains. The anti-idiotypic antibodiesbound preferentially to intact RF and insignificantly to a 20-fold higher concentration of eitherisolated light or heavy chains. Under the same conditions polyvalent rabbit anti-Ig reacted with theseparated polypeptides.

Lack ofdetectable anti-idiotypic antibodies bearing the internal image ofIgG Fc in RF immune goatseraTo address the species specificity of 'internal image' anti-idiotypic antibodies against human RF, weimmunized two goats with different purified polyclonal IgM RF (Mei) and (Vie). The goat anti-RFsera were subjected to affinity chromatography on Sepharose-4B conjugated with goat anti-humanFc specific antibodies. This same goat anti-Fc Sepharose-4B column successfully absorbed broadlycross-reactive rabbit anti-idiotypic antibodies against human RF (results in reference: Fong,Gilbertson & Carson, 1983). Although the unfractionated goat anti-IgM-RF anti-sera inhibited RFactivity due to the presence of anti-human IgM antibodies, the 1% serum globulins that bound toand were eluted from the affinity column failed to inhibit 12 different IgM RFs and the IgMmonoclonal paraprotein (Wag) with anti-dinitrophenyl activity (Metzger, 1967).

576 Sherman Fong et al.

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Anti-idiotype to rheumatoidfactorsTable 5. The binding of anti-idiotypic antibody to intact IgM-RF (Lay) but not to isolated free heavy and lightchains

AQ5 x 101

Lay RFt Lay H-chainst Lay L-chains§

Rabbit anti-Sie RF* 158 + 12 8 +0 8 + 2Rabbit anti-Mei RF 372 + 8 84 + 4 70 + 24Normal rabbit IgG 56+4 12+ 1 9 + 5BSA 48+24 5+1 4+5Anti-human Ig¶ 894 + 37 421 + 16 463 + 12

* Rabbit anti-Sie RF, anti-Mei RF and normal rabbit IgG(50 jug/ml) binding to Lay RF, H-chains or L-chains weredetected with alkaline phosphatase labelled (AP)-F(ab')2 goatanti-rabbit Ig.

tLay RF (2x10-8M)t Lay H-chain (4 x 10-1 M)§ Lay L-chain (4 x 10-1 M)T Binding of Lay RF to polystyrene was detected with AP-

goat anti-human IgM. Binding of H-chains and L-chains weredetected with rabbit anti-human Ig followed by AP F(ab')2 goatanti-rabbit Ig.

Activation ofthe complement pathway by F(ab')2fragments ofanti-idiotypefollowing binding to 1gMrheumatoidfactorTo test whether the binding of F(ab')2 anti-idiotype to IgM-RF triggers the activation of thecomplement pathway, an assay based upon the cleavage of C4 was used (Sabharwal et al., 1982).The interaction of RF with the first complement component (Cl) can activate the molecule to CI.C I cleaves C4 to generate the C4b fragment, which has a metastable binding site that can react withplastic surfaces. As previously shown, the amount ofC4b deposited serves as a sensitive measure ofcomplement activation by RF (Sabharwal et al., 1982). As illustrated in Table 6, the F(ab')2 anti-idiotype was capable ofbinding to IgM RF while the normal rabbit IgG did not. The positve controlwas composed of RF binding to reduced and alkylated (R/A) human IgG. Partial depletion ofIgMRF (Har) by immunoabsorption resulted in reduced binding to F(ab')2 anti-idiotype. Figure 1

Table 6. Direct binding activity of F(ab')2 anti-Id to IgM RF

IgM RF bound(Net A405 x 103, t = 30 min)

IgM RF (Mei) + F(ab')2 anti-ida Mei 186 +0IgM RF (Mei) + F(ab')2 normal rabbit IgG 40 + 6IgM RF (Mei) + R/A human IgGt 733 ± 2IgM RF (Har) + F(ab')2 anti-id Mei 302 + 12IgM RF (Har)+ F(ab')2 normal rabbit IgG 37 + 2IgM RF (Har)+ R/A human IgG 640 + 3IgM RF (Har-IgG absorbed) + F(ab')2 Anti-id Mei 152 + 3IgM RF (Har-IgG absorbed)+ F(ab')2 normal rabbit IgG 5 + 2IgM RF (Har-IgG absorbed)+F(ab')2 R/A human IgG 440+ 19

* 'Internal image' anti-idiotype.t Reduced and alkylated human IgG (R/A human IgG).

577

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Sherman Fong et al.200

0 375300 150

050 200 100 5CI

25 100 50 / k

0 0 0 =

0.5 1-0 1-5 2*0 0.5 1-0 I*5 2-0 0.5 1-0 I*5 2-0

Time (h)

Fig. 1. The activation of complement, as measured by the binding of anti-C4 antibodies, by either (a) IgM RF(Mei) or (b) IgM RF (Har) following interaction with F(ab')2 rabbit anti-idiotype (Mei) (0) or with F(ab')2normal rabbit IgG (0). (&) denotes complement activation by IgM RF binding to reduced and alkylated humanIgG. (c) The complement activation achieved by IgM RF (Har) depleted of RF activity following interactionwith F(ab')2 anti-id (Mei) (v) or with F(ab')2 normal rabbit IgG (-).

shows that the binding of RFs Mei and Har to F(ab')2 anti-idiotype resulted in complementactivation. The triggering of C4 cleavage was immunospecific, since absorption of the RFpreparation on IgG-Sepharose-4B depleted partially both IgM-RF binding and complementactivation activity (Fig. 1, Table 6). These data are consistent with the notion that the 'internalimage' anti-idiotypes mimic the Fc portion ofIgG by complexing to the antigen binding site of RF.

DISCUSSION

Broadly cross-reactive anti-idiotypic antibodies against human RF autoantibodies were elicitedrepeatedly by immunization of rabbits with either polyclonal or monoclonal IgM-RF proteins. Thecross-reactive components represented only a minor part of the total anti-RF antibody response.The anti-idiotype could be isolated directly, by chromatography of the rabbit immune globulins onrabbit anti-human IgG-Fc affinity columns.

Several observations suggested that the affinity purified idiotypic antibodies displayed the'internal image' of the autoantigens recognized by RF. The anti-idiotypes bound to several differentIgM-RF preparations, and potently inhibited the binding of RF to human IgG. They did not alterthe activity of IgM antibodies against tetanus toxoid or the dinitrophenyl hapten, and did not binddirectly to human IgG. The anti-idiotypes reacted not only with human RF autoantibodies, but alsowith heterologous antibodies against human IgG-Fc fragments.

The interaction between F(ab')2 fragments of the anti-idiotypic antibodies and IgM-RFtriggered the activation of the classical complement pathway, as measured by the cleavage anddeposition of the C4 molecule. These data suggest that the anti-idiotype formed a complex at ornear the antigen binding site of IgM-RF and induced the structural change in the IgM pentamerrequired for complement fixation.

The induction of the 'internal image' anti-idiotypic antibodies was dependent upon theconformation of the antigen, and the species of animal immunized. Injection or rabbit with intactRF, but not with a RF-associated hypervariable region peptide, stimulated anti-idiotypeproduction. Importantly, all rabbits immunized with intact IgM-RF produced anti-idiotypes with'internal image' properties. In contrast, cross-reactive anti-idiotypes could not be isolated byfractionation ofgoat anti-RF antisera on a goat anti-human Fc ofIgG affinity column. Rabbit anti-RF antiserum passed over the same goat affinity column successfully yielded the 'internal image'anti-idiotype.

578

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Anti-idiotype to rheumatoidfactors 579Nine different rabbit antisera were induced by immunization with polyclonal or monoclonal

IgM-RF proteins from separate donors. Each antiserum was fractionated on the rabbit anti-Fcaffinity column. The cross-reactive patterns of the purified anti-idiotypic antibodies against abattery of RF preparations were remarkably similar. Thus, separate anti-idiotypes againstmonoclonal IgM-RFs with distinct primary structures nonetheless inhibited the binding to IgG ofpolyclonal RF autoantibodies from rheumatoid arthritis patients.

Altogether these results suggest: (a) that the number of antigenic determinants recognized by RFautoantibodies is limited, and (b) that 'internal image' anti-idiotypic antibodies may be elicitedfrom within the rabbit immune network by immunization with intact human RF, but not withsynthetic peptide fragments of RF. In this regard, IgM-RF autoantibodies have been reported torecognize a number of different epitopes in the Fc region of human IgG. However, a principalantigenic determinant has been localized to the C-terminal end of the Cy2 domain (Hunneyball &Stanworth, 1976a; Natvig, Gaarder & Turner, 1972; Hunneyball & Stanworth, 1976b). Thisdeterminant may be shared by human and rabbit IgG. Perhaps the 'internal image' anti-idiotypesmimic the conformation of the major RF-reactive antigens displayed by human and rabbit IgG.

IgM anti-IgG autoantibodies appear frequently in the sera of hyperimmunized humans andrabbits (Welch et al., 1983; Christian, 1963). It seems possible that the immune network of adultrabbits may include anti-idiotypic clones that recognize rabbit RF, and cross-react with human RF.Immunization with human RF would preferentially stimulate the synthesis of this broadly cross-reactive 'internal image' class of anti-idiotype.

It is interesting -to note that similar regiments of immunization in goats yielded no detectableanti-idiotypic antibodies. It is intriguing to speculate that such 'internal image' anti-idiotypicantibodies may be related or dependent upon the immunizing species' immune network. Somespecies may not maintain antibodies in their repertoire bearing properties that allow mimicry of theFc portions of human IgG.

Rabbit 'internal image' anti-idiotypes to human RF may represent a special phenomenonrelated to the physiologic role of anti-IgG antibodies, and the antigenic cross-reactions betweenhuman and rabbit IgG. Alternatively, 'internal image' anti-idiotypes may regularly appear duringthe immune response to many antigens. Anti-idiotypic antibodies with 'internal image' character-istics have been reported to occur as early as 5 days after secondary immunization of mice with asimple hapten (Cleveland et al., 1983). Furthermore, a recent study suggested that anti-idiotypicantibodies bearing the 'internal image' of tumour antigens could be elicited by immunization ofcancer patients with mouse monoclonal antibodies against the same antigens (Koprowski et al.,1984). Patients who developed such antibodies improved clinically.

'Internal image' anti-idiotypic antibodies potentially represent ideal reagents for immunemodulation, because they react with antibodies of the same specificity, independent of primarystructure. Although the concentration ofbroady cross-reactive anti-idiotypes to RF is low in typicalrabbit antisera, the antibodies can be isolated with relative ease. Moreover, if the number of RFreactive antigenic determinants is indeed restricted, it may be possible to generate a panel ofmonoclonal antibodies that together bear the 'internal image' of the human IgG-Fc fragment.

The authors wish to thank Drs J. D. Capra, H. Metzger and G. Abraham for providing RF-positive sera andisolated RF paraproteins. We also gratefully acknoweldge the excellent assistance of Ms Frances Kral, Ms JaneUhle and the BCR Word Processing Center in the preparation of this manuscript. Funding for this research wassupported in part by the following NIH grantsAG 04100, AM 25443 and RR 00833. Dr S. Fong is a recipient ofa Research Career Development Award from the NIH (AG 00279). Dr P. Chen is a recipient of a NewInvestigator Research Award (AM 35218). This is publication number 3227BCR from the Research Institute ofScripps Clinic, La Jolla, California.

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