Immunology 1990 71 586-591

IgG binding of monoclonal anti-nuclear antibodies from MRL-lpr/lpr mice D. S. PISETSKY, B. S. DARWIN & C. F. REICH Medical Service, Durham VA Hospital, Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, U.S.A. Acceptedfor publication 10 August 1990

SUMMARY with cross-reactive rheumatoid factor (RF) antibodies To assess the specificity of anti-nuclear activity, monoclonal anti-DNA and anti-Sm antibodies from MRL-lpr/lpr mice were tested for binding to a variety of IgG antigens. These antibodies had all been previously identified as binding heterologous IgG. By ELISA, antibodies in this panel all bound BALB/c myeloma proteins representing the different IgG subclasses, indicating broad reactivity with murine IgG as well as heterologous IgG. The determinant recognized by these antibodies was further investigated using the Fab, F(ab')2 and Fc fragments of both human as well as rabbit origin. All antibodies bound well to fragments as well as intact IgG antigens. These antibodies were further analysed by Western blotting, demonstrating that most bound to both heavy and light chains of human origin. Together, these observations suggest that some anti-nuclear antibodies bind a conserved antigenic determinant present widely on immunoglobulin chains. This determinant may represent a common sequence important in immunoglobulin domain structure.

INTRODUCTION Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antibodies to components of the cell nucleus (anti-nuclear antibodies, or ANA). These antibodies serve as markers of diagnostic and prognostic significance and in some instances have been directly implicated in the tissue injury characteristic of this disease (Tan et al., 1988). Because of the importance of ANA to the pathogenesis of SLE, the mechanisms underlying these responses have been intensively investigated in patients with SLE as well as murine models of this disease. These studies have demonstrated that ANA may arise by a variety of mechanisms, with the relative roles of polyclonal B-cell activation and antigen-specific induction likely varying among ANA of different specificity (Schwartz & Stollar, 1985; Pisetsky, 1987). To elucidate further the potential mechanisms for inducing ANA responses, recent studies have focused on the fine specificity of antibody binding using as models monoclonal antibodies obtained by somatic cell hybridization techniques. These studies have indicated that many monoclonal autoantibodies can bind antigens, both self and foreign, other than their nominal target (Lafer et al., 1981; Andre-Schwartz et al., 1984; Klinman et al., 1988). This property has been termed polyspecificity and, while it may not be unique to ANA, it is nevertheless

Abbreviations: ANA, anti-nuclear antibody; RF, rheumatoid factor; SLE, systemic lupus erythematosus. Correspondence: Dr D. S. Pisetsky, Box 151G. Durham VA Hospital, 508 Fulton St., Durham, NC 27705, U.S.A.

important in assessing the range of antigens which could stimulate an ANA response. In particular, polyspecificity suggests that a number of different antigens could induce an ANA response, with molecular mimicry a potential event in disease pathogenesis. Polyspecificity has also suggested a role of ANA as broadly reactive specificities in the pre-immune repertoire. Among polyspecific interactions demonstrated for ANA, rheumatoid factor (RF) activity is of interest because of the frequent co-existence of ANA and RF in connective tissue disease and the possibility that ANA arise as part of an ongoing RF response. Antibodies binding IgG as well as nuclear antigens have been identified among monoclonal antibodies derived from patients with SLE and rheumatoid arthritis as well as lupus mice (Hannestad & Johannesen, 1976; Hannestad & Stollar, 1978; Agnello et al., 1980; Rubin et al., 1984; Rauch et al., 1986). In a recent study we demonstrated that both monoclonal antiDNA and anti-Sm antibodies from MRL-lpr/lpr lupus mice bind IgG (Darwin et al., 1986). These studies did not define the precise antigenic specificity of these antibodies and the extent to which they resemble conventional RF antibodies from patients as well as antibodies from mice selected for IgG binding activity. We have therefore investigated in detail the specificity of a series of MRL-lpr/lpr monoclonal ANA for antigenic sites on IgG. In the studies presented herein, we demonstrate that these antibodies bind determinants present in the Fab as well as Fc portions of the IgG molecule. These antibodies resemble certain anti-Fab antibodies found in patient sera, and could arise as a component of an RF response.

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IgG binding of mAb 2.0

MATERIALS AND METHODS Antibodies Monoclonal anti-DNA antibodies 6N, 6/0, 6P and 6Q were prepared in this laboratory using the cell line NS1 and are all derived from a single MRL-lpr/lpr mouse (Jackson Lab., Bar Harbor, ME). Anti-Sm antibodies Y2 and Y12 were the gift of Dr Joan Steitz of Yale University, New Haven, CI, while 7.13 was the gift of Dr Sallie Hoch of the Agauron Institute, La Jolla, CA. The antigen specificity and IgG binding of these antibodies have been described in detail previously (Darwin et al., 1986). For these experiments, antibodies were purified from tissue culture fluid by affinity chromatography using Sepharose columns containing bound protein A (Sigma, St Louis, MO) or rabbit anti-mouse IgG (Miles Scientific, Naperville, IL). BALB/c myeloma proteins MOPC 21 (IgGl), UPC 10 (IgG2a), MOPC 195 (IgG2b), FLOPC 21 (IgG3) and RPC 5 (IgG2a) were purchased from Organon Teknika-Cappel (West Chester, PA). Human IgG (HIgG) and rabbit IgG (RIgG) and their Fab, F(ab')2 and Fc fragments were also purchased from Organon Teknika-Cappel. Some of the preparations used in these experiments were additionally purified by Sepharose

chromatography. Biotinylation of antibodies To obtain biotinylated antibody preparations, purified monoclonal antibodies were first dialysed overnight against 0-1 M NaHCO3, pH 81. Next, biotin succinimide ester (Biosearch, San Rafael, CA) dissolved in DMSO was added to a final concentration of 0 2 mg/ml to the antibody at 1 mg/ml and incubated for 1 hr at room temperature. The final step was dialysis against phosphate-buffered saline (PBS) containing sodium azide. ELISA assays Binding to IgG antigens was assayed as previously described (Darwin et al., 1986). For assay of binding to heterologous IgG, human or rabbit IgG or their fragments were coated to polystyrene microtitre plates (Dynatech, Chantilly, VA) by incubation of 0-5 ,g/well of antigen overnight at 40 in 8 M urea, 0-01 M Tris, pH 7-0; some wells were incubated only with urea to assess non-specific binding of the monoclonal antibodies to plates. Wells were then washed with PBS containing 0-05% Tween 20 (PBS-T) and then post-coated with 1% bovine serum albumin (BSA; Sigma) in PBS at room temperature for 1 hr. After additional washing, 100 ,l of dilutions of monoclonal antibodies in PBS-T were added to wells and incubated for 1 hr at room temperature. Wells were then washed and incubated with a 1:400 peroxidase goat anti-mouse IgG reagent (Organon Teknika-Cappel), that had been absorbed on HIgG and RIgG Sepharose columns to reduce background binding. The final step was incubation with 1:100 of 3,3',5,5'-tetramylbenzidine (TMB; Organon Teknika-Cappel) in 0 1 M citrate, pH 4-0, containing 1: 3000 H202. Optical densities were then determined using a Titertek Multiskan platereader. Assay of binding to mouse subclasses was performed using biotinylated antibody preparations, obtained as described. The assay was performed similarly as the determination of heterologous IgG binding except that coating of the myelomas was performed using 0 1 M phosphate buffer, pH 7-0. The presence of biotinylated antibody was determined by incubation with

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ND, not done. mouse IgG of different subclasses, heterologous IgG, Fab fragments as well as Fc fragments (Theofilopoulos et al., 1983; Aguado et al., 1987; Wolfowicz et al., 1988). Their reactivity to Fab fragments is not unique, however, since occasional MRLlpr/lpr antibodies selected on the basis of IgG binding also bind Fab fragments (Aguado et al., 1987). Furthermore, human antibodies binding both Fc and Fab fragments have been found among panels of hybridoma antibodies from patients with rheumatoid arthritis and SLE. These antibodies were not characterized in detail, however, since their cross-reactivity with Fab and Fc fragments was considered to preclude their categorization as classical RF (Rauch et al., 1986). Antibodies reacting with antigenic determinants present in either F(ab')2 or Fab fragments occur in a variety of clinical settings and appear to vary in specificity. Some of these antibodies react predominantly with determinants revealed during the generation of F(ab')2 fragments and have been termed pepsin agglutinators (Osterland, Harboe & Kunkel, 1963; Ling & Drysdale, 1981; Davey & Korngold, 1982; Heimer, Wolfe & Abruzzo, 1985). Other anti-F(ab')2 antibodies, present

in SLE sera, show apparent anti-idiotypic specificity (Abdou et al., 1981; Nasu et al., 1980, 1982), while a broadly reactive group without apparent anti-idiotypic binding has been identified in sera as well as cell cultures of patients with rheumatoid arthritis as well as normals (Birdsall & Rossen, 1982, 1983; Persselin, Louie & Stevens, 1984; Persselin & Stevens, 1985; Persselin et al., 1985; Gerardo et al., 1988); these antibodies react widely with human IgG without apparent specificity for individual determinants. Similar antibodies arise following immunization, possibly in response to immune complexes. Finally, a group of antibodies termed epibodies has been described in murine systems (Bona et al., 1986). These antibodies bind Fc fragments as well as idiotypic determinants. Specificity analysis of the type of RF we have described is difficult using serum as antibody source. Anti-Fab activity may be bound in immune complexes and hidden unless dissociated by agents such as urea (Birdsall & Rossen, 1983; Persselin et al., 1984, 1985). Furthermore, the heterogeneity of serum RF antibodies makes identification of a cross-reactive population difficult. Sequential affinity chromatography can be used to isolate molecules binding both Fc and Fab determinants; the use of monoclonal antibodies prepared from tissue culture obviates these difficulties, however. Although our antibodies might be expected to self-associate and form immune complexes, this potential has not obscured detection of their reactivity. Thus, our antibodies may preferentially bind conformational determinants of IgG revealed by binding to the solid phase or by binding to antigen; soluble or free IgG may not display the requisite sites for binding and complex formation. Alternatively, the affinity of these antibodies for IgG may be so low that unless stabilized by cross-linking on the solid phase, these interactions may be minimal. As a result, self-associating complexes may n6t readily form in solution. The determinant recognized by these antibodies is not yet known, although the reactivity with various IgG molecules as well as fragments suggests recognition of a conserved conformation of domain structure. Such structures have been postulated as a common feature of molecules in the immunoglobulin superfamily, which are related to IgG and involved in cell surface recognition (Williams & Barclay, 1988). Of the antibodies studied, all but one showed detectable binding to H and L chains by Western blotting under conditions in which control BALB/c myelomas were inactive; some human antibodies to

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Figure 3. Specificity of antibody 6/0 for RIgG and its fragments. Data are presented as described in Fig. 2.

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D. S. Pisetsky, B. S. Darwin & C. F. Reich polyspecific antibodies may vary and it is possible that certain sets of specificities are more likely to contribute to disease pathogenesis. We consider IgG binding by ANA an important cross-reactivity since it can be demonstrated in patient sera and could account for the induction of certain ANA. Because of their poor immunogenicity of DNA in animal models, alternative mechanisms for anti-DNA induction that do not require direct stimulation by self-DNA have been proposed. It can be postulated, therefore, that some anti-DNA as well as other ANA may arise as part of an RF response stimulated in vivo by immune complexes. Such antibodies may be especially common in patients with SLE because of their high level of immune complexes and impaired clearance mechanisms. These features would lead to an intense stimulation of IgG binding antibodies. Whether cross-reactivity of ANA with IgG is critical to the induction or an incidental feature of their binding specificity is being explored by analysis of the properties of antiDNA antibodies induced in normal animals by various means.

ACKNOWLEDGMENTS Figure 4. Immunoblot analysis of antibody 6/0. The binding of antibody 6/0 to isolated H and L chains was determined as described in the Materials and Methods. (a) MOPC 195; (b) 6/0; (c) peroxidaseconjugated rabbit anti-human IgG.

Fab also can bind separated IgG chains on blots (Gerardo et al., 1988). Unless there was substantial refolding of isolated and reduced chains on nitrocellulose to the native conformation, this result would suggest that our antibodies reacted preferentially with a linear determinant. Molecules in the immunoglobulin superfamily have sequence similarity and heavy and light chains in fact display regions of close homology (Williams & Barclay, 1988). Our antibodies may be directed to such conserved sequences. If cross-reactive ANA bound sequences important in Ig domain structure, this result could explain their binding to cell-surface antigens in this superfamily. In this regard, a BALB/c monoclonal anti-DR antibody bound IgG as well as other cross-reactive self and foreign antigens, supporting this model (Fernandez, Ternynck & Avrameas, 1989). Although all the antibodies we studied showed similar specificity, it is not clear how common such reactions are among either anti-DNA or anti-Sm antibodies from MRL-lpr/lpr mice. Four of the anti-DNA antibodies we studied were derived from the same mouse, two of which, 6N and 6Q, are clonally related (Shlomchik et al., 1990). Similarly, Y2 and Y12 come from the same mouse and display a common idiotype as well as pattern of reactivity with Sm polypeptides as well as DNA (Darwin et al., 1986; Pisetsky & Lerner, 1982). It is conceivable, therefore, that these antibodies represent a selective population of anti-Sm and anti-DNA antibodies which fortuitously share an unusual pattern of IgG cross-reactivity. Although the polyspecificity of ANA is now well documented, its precise relationship to their physiological function, mechanism of induction or potential role as mediators of immunopathological events is not yet clear. Polyspecificity has been demonstrated for non-autoantibodies, indicating that ANA and other autoantibodies are not unique to their ability to bind more than one biochemically dissimilar antigen (Klinman et al., 1988). However, the array of antigens bound by different

This work was supported by NIH grants AI-23308, AR-39162 and the Veterans Administration Medical Research Service.

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lpr mice.

To assess the specificity of anti-nuclear antibodies with cross-reactive rheumatoid factor (RF) activity, monoclonal anti-DNA and anti-Sm antibodies f...
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