Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.

Autoantibodies reactive with the different components small nuclear ribonucleoprotein (snRNP)3 complex and of the Ro/La antigenic complex are a characteristic feature of systemic lupus erythematosus (SLE) (1, 2). The U1-snRNP particle is composed of uridine-rich U1-RNA molecule that is complexed with U1-snRNP-specific 70-kDa A and C proteins and a core of common Sm proteins termed B/B′, D1, D2, D3, E, F, and G (3). Whereas the anti-RNP Abs target the 70-kDa A and C proteins, anti-Sm Abs predominantly recognize Smith B (SmB)/B′ and Smith D (SmD) (4, 5, 6). The Ro/La antigenic particle is composed of small cytoplasmic RNA molecules (hY1–hY5) that are complexed with the 60-kDa Ro and 48-kDa La proteins (7). Analysis of SLE sera often shows co-occurrence of anti-RNP Abs with anti-Sm Abs and that of anti-Ro Abs with anti-La Abs (8, 9). Longitudinal studies of sera from SLE patients and lupus-prone mice have demonstrated an ordered appearance of Abs to the different components of these antigenic particles (10, 11, 12, 13). Thus, Ab responses increased in complexity over a period of time by recognizing more Ags. This has led to the proposal that epitope spreading may account for the complexity of Ab responses in SLE (14). In this process, an immune response initiated against limited epitopes, diversifies to include other epitopes within the same protein (intramolecular epitope spreading) or other proteins within the same antigenic complex (intermolecular epitope spreading). Experimental animal model systems involving immunization of mice with synthetic peptides or whole proteins have provided evidence for this hypothesis (15, 16, 17, 18).

Our laboratory has been interested in studying the mechanisms of epitope spreading in SLE (19, 20). Previously, using Ro60 and its peptides as model Ags, we have clearly demonstrated intramolecular epitope spreading within Ro60. Surprisingly, in mice immunized with a Ro60 peptide, we detected Abs reactive with polypeptides from the snRNP complex (19). We demonstrated that these Abs were cross-reactive (20). Thus, in this model, the apparent intermolecular epitope spreading from Ro60 Ag to snRNPs can be accounted by cross-reactive Abs recognizing conformational epitopes. Our data and those from the literature have demonstrated the presence of conformational epitopes on SLE-related Ags (20, 21, 22, 23, 24). It has been postulated that these cross-reactive Abs may play a critical role in the pathogenesis of SLE (25, 26, 27). We have been interested in delineating the role of cross-reactive determinants in the diversification of autoantibody responses in SLE. For this study, we have focused our efforts on the polypeptides from the snRNP complex. Several reports have indicated the presence of cross-reactive determinants on different snRNPs (20, 21, 22, 23, 24, 28). Thus, we extended our model system utilizing recombinant polypeptides as the immunogens to the snRNP complex to determine whether cross-reactive Abs were induced and whether specific Abs to other components of snRNP were generated. Multiple peptides i.e., SmD, SmB, and the A ribonucleoprotein (A-RNP) were used as immunogens with alum as the adjuvant. Immune sera were analyzed in two different immunoassays. Our data show that in addition to cross-reactive Abs, specific Abs to other components of the snRNPs were generated. In all immunizations, Ab diversification through intermolecular epitope spreading had occurred. Interestingly, the differential patterns of epitope spreading were dependent on the immunogen initiating the immune response.

The cloning and expression of recombinant SmD and SmB have been described previously (19). The 6× histidine-tagged recombinant proteins were purified under denaturing conditions as described previously with a few modifications. Briefly, Escherichia coli cells expressing the recombinant proteins were resuspended in lysis buffer containing 0.1 M Tris (pH 8.0), 1 M NaCl, 1% Triton X-100, and 1% Tween 20 and lysed by sonication. The insoluble inclusion bodies, enriched in recombinant proteins, were separated by high speed centrifugation and washed in the same buffer by a cycle of sonication followed by centrifugation. This was followed by dissolving the inclusion bodies in 8 M urea buffer, containing, 10 mM 2-ME, 0.1 M sodium phosphate, and 0.01 M Tris, pH 8.0. The recombinant proteins were purified by Ni-NTA affinity chromatography following the manufacturer’s instructions (Qiagen, Valencia, CA). Purified proteins were dialyzed against distilled water, followed by dialysis against PBS. Purified proteins were concentrated by ultrafiltration and stored frozen at −70°C until use. The cDNA for mouse A-RNP, cloned in pET15 expression vector was a gift from Dr. J. Craft (Yale University, Hartford, CT). This protein does not have a His tag. Recombinant A-RNP was purified with a series of ion exchange columns as described previously (29).

Female A/J mice (H-2a) were obtained from the National Cancer Institute (Frederick, MD) and maintained in specific pathogen-free conditions at the University of Virginia vivarium. Purified recombinant proteins were adsorbed on to slurry of alum (Pierce Endogen, Rockford, IL) at a 1:1 ratio. Mice 8–10 wk old were immunized with 100 μg of recombinant protein by s.c. routes at two different sites. Animals received two additional immunizations with 50 μg of proteins adsorbed on to alum by the i.p. route at 2 and 4 wk post-initial immunization. Control mice were immunized with a mixture of alum and PBS. Mice were bled at regular intervals through the tail vein.

A cell extract of WEHI 7.1 cells was separated on 12% SDS-PAGE and transferred onto nitrocellulose paper overnight. The nitrocellulose paper was cut in strips, and nonspecific binding sites were blocked by incubation in PBS containing 5% milk proteins. The strips were probed with sera diluted in PBS containing 0.1% Tween 20 (PBST) and 5% milk. Bound Abs were detected with peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and ECL (Pierce Endogen). All incubations were for 1 h at room temperature, and in between steps the strips were washed three times with PBST. Human anti-Sm and human anti-RNP sera were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). Bound human Abs were detected with peroxidase-conjugated goat anti-human IgG (Southern Biotechnology Associates).

Two substrates were used for immunoprecipitation assays, the in vitro [35S]methionine-labeled recombinant proteins and cell extracts of metabolically labeled WEHI 7.1 cells. For in vitro labeling of recombinant proteins, the cDNAs encoding the open reading frames of SmD, SmB, and Ro60 were subcloned into the pGEM7Zf vector (Promega, Madison, WI) and used for in vitro transcription and translation. The pET15 vector containing the A-RNP cDNA was used to generate A-RNP. 35S-labeled A-RNP, SmB, SmD, and Ro60 were generated by performing in vitro transcription and translation reactions using the Quick TNT lysate kit (Promega) and L-[35S]methionine (PerkinElmer, Boston, MA), following the manufacturer’s instructions. The proteins generated by this method had no vector-encoded sequences or common epitope tags on either the N-terminal or C-terminal regions of the protein. WEHI 7.1 cells were metabolically labeled using the methods described by Satoh and Reeves (30). Briefly, WEHI 7.1 cells were grown overnight in methionine-deficient RPMI 1640, containing 10% FBS and supplemented with Express [35S] protein labeling mix (PerkinElmer). Cells were washed twice in PBS and lysed in 0.5 M NaCl, NET, Nonidet P-40 buffer (0.5 M NaCl, 2 mM EDTA, 50 mM Tris-HCl (pH 7.4), 0.3% Nonidet P-40) containing a mixture of protease inhibitors (leupeptin, aprotinin, PMSF). Cell debris was separated by high speed centrifugation, and labeled cell extracts were used immediately for immunoprecipitation.

For the precipitation of in vitro-translated proteins, immune sera were bound to 20 μl of GammaBind Plus Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature. Beads were washed once with the 0.5 M NaCl, NET, Nonidet P-40 buffer and used to immunoprecipitate 35S-labeled proteins. For precipitation of the snRNP particle, 5 μl of immune sera were mixed with 20 μl of protein A-Sepharose (Amersham Pharmacia Biotech) beads plus 12 μl of rabbit anti-mouse IgG (1 mg/ml). After 1 h incubation at room temperature and washing of beads with 0.5 M NaCl, NET, Nonidet P-40 buffer, labeled cell extract (equivalent of 2 million cells/sample) was added. Protein A-Sepharose beads were used due to high background binding of labeled cell extracts to GammaBind Plus Sepharose beads. No differences were observed in the ability of sera to immunoprecipitate the in vitro-translated recombinant proteins, whether GammaBind Plus Sepharose beads or a mixture of protein A-Sepharose beads plus rabbit anti-mouse IgG were used. After 2 h incubation in the cold room, the unbound proteins were removed by washing the beads three times with the 0.5 M NaCl, NET, Nonidet P-40 buffer, followed by a wash in the NET buffer (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl, pH 7.4). Immunoprecipitated proteins were separated on 12% SDS-PAGE and revealed by autoradiography.

Recombinant proteins were coupled with cyanogen bromide-activated Sepharose 4B beads (Amersham Pharmacia Biotech) following the manufacturer’s instructions. Immune sera were appropriately diluted and absorbed with different amount of Ag-coupled beads as described previously (19). Absorbed sera were used in Western blots and immunoprecipitation assays.

Sera from mice immunized with rSmD in alum were analyzed for their reactivity in Western blots with cell extract from a murine T cell lymphoma, WEHI 7.1 as substrate. Fig. 1 shows reactivity of sera obtained 2 mo after the initial immunization with rSmD in alum (lanes 1–8). All sera from mice immunized with rSmD contained IgG Abs that could recognize the SmD protein, the A-RNP, and the SmB/B′ proteins. In addition, several mice also generated Abs that were reactive with proteins between 16 and 21 kDa. Sera from mice immunized with alum did not show these reactivities (Fig. 1, lanes 9–14). The positive control human anti-Sm serum shows the characteristic pattern of anti-Sm/anti-snRNP Ab reactivity with dominant bands at 70 kDa (U1-RNP-associated 70-kDa protein), 31 kDa (A-RNP), 28/29 kDa (SmB/B′), 21 kDa (RNP-C), and 16 kDa (SmD). Similarly, the anti-RNP reference serum from CDC shows reactivity to the 70-kDa protein and A-RNP. Similar reactivity patterns to multiple snRNP peptides were obtained in another group of eight mice immunized with rSmD protein.

FIGURE 1.

Sera from mice immunized with SmD react with multiple cellular proteins in Western blot. WEHI 7.1 cell extract was separated by 12% SDS-PAGE, transferred on to nitrocellulose paper, and used as substrate to probe serum reactivity. Human anti-Sm and anti-RNP sera from CDC were used as positive controls and are marked as Sm (left) and RNP (right). Lanes 1–8, Reactivity of sera from different mice immunized with SmD. All sera react with SmD, A-RNP, and SmB to varying extents; lanes 9–14, reactivity of sera from control mice immunized only with adjuvant. No reactivity to SmD, A-RNP, and SmB is seen. The sera were used at 1/100 dilutions and obtained 2 mo postimmunization.

FIGURE 1.

Sera from mice immunized with SmD react with multiple cellular proteins in Western blot. WEHI 7.1 cell extract was separated by 12% SDS-PAGE, transferred on to nitrocellulose paper, and used as substrate to probe serum reactivity. Human anti-Sm and anti-RNP sera from CDC were used as positive controls and are marked as Sm (left) and RNP (right). Lanes 1–8, Reactivity of sera from different mice immunized with SmD. All sera react with SmD, A-RNP, and SmB to varying extents; lanes 9–14, reactivity of sera from control mice immunized only with adjuvant. No reactivity to SmD, A-RNP, and SmB is seen. The sera were used at 1/100 dilutions and obtained 2 mo postimmunization.

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In view of the previous findings in the Ro/La system that most if not all the reactivities to the nonimmunogens could be accounted for by a population of Abs, which are cross-reactive with the immunogen Ro60 and the other lupus-related proteins (20), detailed absorption analyses were conducted to determine whether specific Abs to the other components of the snRNP particles were generated as a result of immunization with rSmD. In addition, an immunoprecipitation assay was used to determine whether the identification of specific autoantibodies was assay dependent.

To determine whether Ab reactivity to other snRNP proteins was through cross-reactivity or through intermolecular epitope spreading, pooled immune sera at different time points were absorbed with Sepharose beads coupled with SmD, A-RNP, or SmB. Reactivity of absorbed sera with different snRNP proteins was then determined in Western blots with WEHI 7.1 cell extracts as the substrate. Sepharose beads coupled with a Ro60 peptide were used as the negative control. Fig. 2 shows results for a representative absorption experiment performed using pooled sera obtained 2 mo (lanes 1–5) and 3 mo (lanes 6–10) postimmunization. Untreated pooled sera, at both time points, reacted predominantly with SmD, A-RNP, and SmB. The pooled sera also reacted with several other proteins between 21 and 29 kDa (five bands) and to proteins between 16 and 21 kDa (two bands). Absorption of sera with the immunogen (rSmD)-coupled Sepharose beads depleted all Abs reactive with SmD and the proteins between 16 and 29 kDa. However, Abs reactive with A-RNP and SmB remained readily detectable, indicating intermolecular B epitope spreading. Similarly absorption of sera with A-RNP-coupled Sepharose beads resulted in complete depletion of Abs reactive with the A-RNP. Abs reactive with SmB and SmD were still detected. Absorption of sera with SmB-coupled Sepharose beads resulted in complete removal of Abs reactive with SmB/B′ and some reduction in Ab reactivity to A-RNP and SmD. In all absorptions, the Abs reactive to proteins between 16 and 29 kDa were depleted. These results suggest that immunization of mice with SmD resulted in the generation of different Ab populations, one of which recognizes cross-reactive epitope/epitopes between different snRNP proteins and other unidentified proteins whereas the others are specific to SmD, A-RNP, and SmB/B′ proteins. Similar results were obtained in absorption experiments performed with sera obtained 3 mo postimmunization. However, compared with the earlier time point, reactivity to SmD protein was significantly lowered, and the population of polyreactive Abs was not detected. A similar population of polyreactive Abs was observed in our earlier studies involving immunization with Ro60 peptides (20). The significance of this finding is not clear. Interestingly, by day 150 postimmunization, Ab reactivity to SmD protein was not detected in six of eight mice, but strong Ab reactivity to the A-RNP and SmB was still present (data not shown). Similar results were obtained with a second set of mice immunized with rSmD.

FIGURE 2.

Intermolecular epitope spreading to A-RNP and SmB occurs in mice immunized with SmD. Pooled sera from mice immunized with SmD were absorbed with different Ag-coupled Sepharose beads. The reactivity of unabsorbed and absorbed sera was analyzed in Western blots with the use of WEHI 7.1 cell extract. Lanes 1–5, Reactivity of sera obtained 2 mo postimmunization; lanes 6–10, reactivity of sera obtained 3 mo postimmunization.

FIGURE 2.

Intermolecular epitope spreading to A-RNP and SmB occurs in mice immunized with SmD. Pooled sera from mice immunized with SmD were absorbed with different Ag-coupled Sepharose beads. The reactivity of unabsorbed and absorbed sera was analyzed in Western blots with the use of WEHI 7.1 cell extract. Lanes 1–5, Reactivity of sera obtained 2 mo postimmunization; lanes 6–10, reactivity of sera obtained 3 mo postimmunization.

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Two groups of mice were immunized with either A-RNP or SmB, and sera at different time points were analyzed for reactivity to other snRNPs by Western blot. The reactivity patterns of sera obtained 2 and 3 mo postimmunization were similar. Fig. 3 shows reactivity for 3-mo-old sera. Sera from six mice immunized with the A-RNP (lanes 1–6) reacted strongly with the A-RNP. All mice showed varying degrees of reactivity to the SmB proteins, with sera in lanes 2 and 5 showing the strongest recognition. Sera in lanes 1 and 3 reacted with a protein that migrated similar to the 70-kDa protein. None of the sera reacted with the SmD protein. Sera from mice immunized with SmB (lanes 10–15) reacted predominantly with SmB and to varying degrees with A-RNP.

FIGURE 3.

Reactivity of sera from mice immunized with A-RNP and SmB in Western blots. All sera were obtained 2 mo postimmunization and used at 1/100 dilutions. Lanes 1–6, Reactivity of sera from mice immunized with A-RNP; lanes 7–9, reactivity of sera from control mice immunized with adjuvant only; lanes 10–15, reactivity of sera from mice immunized with SmB. Human anti-Sm and anti-RNP sera were used as positive controls.

FIGURE 3.

Reactivity of sera from mice immunized with A-RNP and SmB in Western blots. All sera were obtained 2 mo postimmunization and used at 1/100 dilutions. Lanes 1–6, Reactivity of sera from mice immunized with A-RNP; lanes 7–9, reactivity of sera from control mice immunized with adjuvant only; lanes 10–15, reactivity of sera from mice immunized with SmB. Human anti-Sm and anti-RNP sera were used as positive controls.

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To determine cross-reactivity vs intermolecular epitope spreading, pooled sera 3 mo postimmunization were absorbed with Ag-coupled Sepharose beads, and reactivity to different proteins was analyzed in Western blots. Fig. 4 shows a representative experiment. In mice immunized with the A-RNP (Fig. 4, left), the unabsorbed sera reacted strongly with the A-RNP, SmB/B′, and 70-kDa proteins. Absorption of sera with A-RNP removed Abs reactive with the A-RNP and SmB/B′, without affecting the Ab reactivity to the 70-kDa protein. Absorption with SmB-coupled beads completely removed Ab reactivity with the SmB with little effect on A-RNP and 70-kDa protein reactivity. Similar results were obtained with SmD-coupled beads although the pooled sera did not stain SmD. The likely explanation for this observation is the differential expression of the cross-reactive epitope by rSmD on the Sepharose beads. Absorption with control beads had no effect on the reactivities. These results suggest that immunization of mice with A-RNP results in the generation of a population of Abs reactive only with the A-RNP, a population of Abs cross-reactive with the A-RNP and SmB/B′ and some other proteins between 16 and 29 kDa, and a population of Abs specific for the 70-kDa protein. These results clearly demonstrate a different pattern of intermolecular epitope spreading in A-RNP-immunized mice in comparison with SmD-immunized mice.

FIGURE 4.

Differential patterns of intermolecular epitope spreading are observed in mice immunized with A-RNP and SmB. Pooled sera from A-RNP-immunized mice (left) and SmB-immunized mice (right) obtained 3 mo postimmunization were absorbed with different Ag-coupled Sepharose beads. Reactivity of unabsorbed and absorbed sera was analyzed in Western blots with the use of WEHI 7.1 cell extracts. Although specific Abs to 70-kDa protein are generated in the A-RNP-immunized mice, Abs reactive with SmB remain cross-reactive. In contrast, in SmB-immunized mice, specific Abs to A-RNP are generated.

FIGURE 4.

Differential patterns of intermolecular epitope spreading are observed in mice immunized with A-RNP and SmB. Pooled sera from A-RNP-immunized mice (left) and SmB-immunized mice (right) obtained 3 mo postimmunization were absorbed with different Ag-coupled Sepharose beads. Reactivity of unabsorbed and absorbed sera was analyzed in Western blots with the use of WEHI 7.1 cell extracts. Although specific Abs to 70-kDa protein are generated in the A-RNP-immunized mice, Abs reactive with SmB remain cross-reactive. In contrast, in SmB-immunized mice, specific Abs to A-RNP are generated.

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Analysis of pooled sera obtained 3 mo postimmunization from mice immunized with SmB is shown in Fig. 4, right. The unabsorbed sera reacted predominantly with A-RNP and SmB/B′. Absorption of sera with the immunogen-coupled Sepharose beads depleted all Ab reactivities except Abs reactive with A-RNP. Absorption of sera with A-RNP completely removed Abs reactive with the A-RNP and other proteins between 16 and 29 kDa but had no significant effect on the reactivity to SmB/B′ protein. Absorption of sera with SmD had no effect on reactivity to A-RNP or SmB/B′. Similar to the SmD and A-RNP immunizations, in SmB-immunized mice, multiple autoantibody populations are generated. The generation of Abs specific to A-RNP is clearly indicative of intermolecular B cell epitope spreading. Similar results were obtained with sera obtained 2 mo postimmunization (data not shown).

The reactivity patterns observed in Western blotting were confirmed by immunoprecipitation of in vitro-transcribed, translated, and [35S]methionine-labeled SmD, SmB, and A-RNP. Sera at different time points (1, 3, and 5 mo) after the initial immunization were analyzed for immunoprecipitating Abs. The strongest reactivity to these proteins was seen 5 mo postimmunization and is shown in Fig. 5. Abs reactive with SmD (eight of eight), A-RNP (seven of eight), and SmB (four of eight) were detected in SmD-immunized mice (Fig. 5,A). In A-RNP-immunized mice, only Abs to A-RNP (seven of seven) and SmB (three of seven) were detected (Fig. 5,B). In SmB-immunized mice, Abs reactive with SmB (six of six), A-RNP (five of six), and SmD (one of six) were detected (Fig. 5 C). Abs capable of immunoprecipitating Ro60 were not detected in any group of immunized mice (data not shown).

FIGURE 5.

Immunoprecipitation of in vitro-translated proteins by sera from mice immunized with SmD (A), A-RNP (B), and SmB (C). Reactivities to 35S-labeled A-RNP, SmB, and SmD are shown as separate panels. Five microliters of immune sera were used for immunoprecipitation, and all sera were obtained 5 mo postimmunization. Bound proteins were separated on a 12% SDS-PAGE and revealed by autoradiography. A, Pooled sera from MRL-lpr/lpr mice were used as positive control. Nonspecific binding of labeled proteins to the beads is shown in only the beads lane. Lanes 1–8, Reactivity of individual serum samples from SmD-immunized mice; lanes 9–12, representative reactivity of sera from control mice immunized with adjuvant. B, Reactivity of individual serum samples from seven mice immunized with A-RNP. C, Reactivity of individual serum samples from mice immunized with SmB.

FIGURE 5.

Immunoprecipitation of in vitro-translated proteins by sera from mice immunized with SmD (A), A-RNP (B), and SmB (C). Reactivities to 35S-labeled A-RNP, SmB, and SmD are shown as separate panels. Five microliters of immune sera were used for immunoprecipitation, and all sera were obtained 5 mo postimmunization. Bound proteins were separated on a 12% SDS-PAGE and revealed by autoradiography. A, Pooled sera from MRL-lpr/lpr mice were used as positive control. Nonspecific binding of labeled proteins to the beads is shown in only the beads lane. Lanes 1–8, Reactivity of individual serum samples from SmD-immunized mice; lanes 9–12, representative reactivity of sera from control mice immunized with adjuvant. B, Reactivity of individual serum samples from seven mice immunized with A-RNP. C, Reactivity of individual serum samples from mice immunized with SmB.

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Similar to the absorption experiments described above, pooled sera depleted of Abs reactive with their respective immunogens were able to immunoprecipitate other 35S-labeled proteins. This further confirmed the intermolecular epitope spreading in SmD and SmB immunized mice (Fig. 6, A and C). In the A-RNP-immunized mice (Fig. 6 B), absorption of sera with the immunogen depleted all Abs, thereby indicating that Abs reactive with SmB were cross-reactive.

FIGURE 6.

Analysis of differential patterns of intermolecular epitope spreading by immunoprecipitation assay. Pooled sera from mice immunized with SmD (A), A-RNP (B), and SmB (C) at 5 mo postimmunization were absorbed with different Ag-coupled Sepharose beads and used to immunoprecipitate a mixture of 35S-labeled A-RNP, SmB, and SmD proteins. Bound proteins were separated on 12% SDS-PAGE and revealed by autoradiography.

FIGURE 6.

Analysis of differential patterns of intermolecular epitope spreading by immunoprecipitation assay. Pooled sera from mice immunized with SmD (A), A-RNP (B), and SmB (C) at 5 mo postimmunization were absorbed with different Ag-coupled Sepharose beads and used to immunoprecipitate a mixture of 35S-labeled A-RNP, SmB, and SmD proteins. Bound proteins were separated on 12% SDS-PAGE and revealed by autoradiography.

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Thus, in mice immunized with SmD, Ab response spreads to A-RNP and SmB; and in mice immunized with SmB, Ab response spreads dominantly to A-RNP and rarely to SmD (in one of six mice). In A-RNP-immunized mice only cross-reactive Abs to SmB are generated. In these mice, we had detected Ab responses to the 70-kDa protein. However, we were unable to immunoprecipitate in vitro-transcribed and translated human 70 kDa protein. These results are summarized in Table I.

Table I.

Summary of intermolecular epitope spreading in A/J mice immunized with recombinant SmD, SmB, and A-RNP

ImmunogenAb Response Spread
SmD A-RNP, SmB 
SmB A-RNP, SmDa 
A-RNP 70 kDa,b cross-reactive Abs to SmB 
ImmunogenAb Response Spread
SmD A-RNP, SmB 
SmB A-RNP, SmDa 
A-RNP 70 kDa,b cross-reactive Abs to SmB 
a

Only by immunoprecipitation in one mouse.

b

Detected by Western blot analysis only.

Because of the immunogen-dependent differential epitope spreading as assayed by Western blot analysis and immunoprecipitation of in vitro-translated products, it would be of interest to determine whether the immune sera had Abs that were able to precipitate the whole snRNP particle. WEHI 7.1 cells were metabolically labeled with [35S]methionine. The whole cell extract prepared by sonication was used as a source of the snRNP particle. Analysis of individual sera obtained at different time points (1, 2, 3, and 5 mo post-initial immunization) for reactivity to the snRNP particle was conducted. Results for representative mice are shown in Fig. 7. A human reference anti-snRNP serum was used as control. Fig. 7, top, shows that sera from mice immunized with A-RNP (lanes 1–4) and SmB (lanes 5–8) precipitated the snRNP particle. Sera 2 and 3 mo postimmunization from mice immunized with the A-RNP predominantly react with the endogenous A-RNP as is evident from the higher intensity of the A-RNP band at these time points. An increase in intensity of other Sm proteins at 5 mo postimmunization as shown by lane 4 in Fig. 7, top, suggests that now Abs were reacting with A-RNP that was associated with other snRNP proteins. Similarly, sera from mice immunized with SmB predominantly react with the SmB protein at 1 mo postimmunization. By 2 mo, these Abs are capable of precipitating the whole snRNP particle. However, in these mice, the intensity of reactivity decreased by 5 mo. In contrast, a distinct pattern of reactivity is obtained in sera from mice immunized with SmD (Fig. 7, bottom, lanes 1–8). In these mice, Abs reactive with the snRNP particle peaked by 5 mo and were detected in seven of eight mice. Kinetics of this response from two representative mice is shown. In mouse 1 (lanes 1–4), these Abs appear 2 mo postimmunization and seem to increase by 5 mo. In this mouse, Abs reactive with A-RNP had appeared at the same time. In mouse 2 (lanes 5–8), Abs reactive with snRNP particle did not appear until 3 mo postimmunization and also coincided with Abs reactive with A-RNP. Abs reactive with the snRNP particle were not detected in any of the mice injected with adjuvant (Fig. 7, bottom, lanes 9–12).

FIGURE 7.

Kinetics of immunoprecipitation of intact snRNP particle by sera from mice immunized with A-RNP, SmB, and SmD. A cell extract of metabolically labeled WEHI 7.1 cells was used as a source of intact snRNP particle. Five microliters of immune sera were used for immunoprecipitation. Bound proteins were separated on 12% SDS-PAGE and revealed by autoradiography. Top, Characteristic reactivity pattern obtained with human anti-RNP antiserum. Lanes 1–4, Reactivity of sera obtained at different time points (1, 2, 3, and 5 mo) from a representative mouse immunized with A-RNP; lanes 5–8, serum samples at 1, 2, 3, and 5 mo from a representative mouse immunized with SmB. Bottom, lanes 1–4 and 5–8, reactivity of sera obtained at different time points (1, 2, 3, and 5 mo, respectively) from two representative mice immunized with SmD. Lanes 9–12 are sera at different time points from a representative control mouse immunized with adjuvant.

FIGURE 7.

Kinetics of immunoprecipitation of intact snRNP particle by sera from mice immunized with A-RNP, SmB, and SmD. A cell extract of metabolically labeled WEHI 7.1 cells was used as a source of intact snRNP particle. Five microliters of immune sera were used for immunoprecipitation. Bound proteins were separated on 12% SDS-PAGE and revealed by autoradiography. Top, Characteristic reactivity pattern obtained with human anti-RNP antiserum. Lanes 1–4, Reactivity of sera obtained at different time points (1, 2, 3, and 5 mo) from a representative mouse immunized with A-RNP; lanes 5–8, serum samples at 1, 2, 3, and 5 mo from a representative mouse immunized with SmB. Bottom, lanes 1–4 and 5–8, reactivity of sera obtained at different time points (1, 2, 3, and 5 mo, respectively) from two representative mice immunized with SmD. Lanes 9–12 are sera at different time points from a representative control mouse immunized with adjuvant.

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Our previous publications regarding autoantibody diversification with Ro60 and its peptides as immunogens (19, 20) demonstrate that cross-reactive Abs between the immunogens and various SLE-related ribonucleoproteins account for the apparent intermolecular epitope spreading and autoantibody diversification in the immunized mice. In this study, three components of the snRNP particle, SmD, SmB, and A-RNP, were adsorbed onto alum and used to immunize A/J mice. In all cases, Abs cross-reactive to the immunogen and certain polypeptides in the snRNP complex were detected in the course of the immune response. Western blot analysis showed that some of these Abs were cross-reactive to other unidentified cellular proteins. In contrast to our Ro60 peptide immunization model, in this study true intermolecular B cell epitope spreading was observed. With SmD as the immunogen, Abs specific for A-RNP and SmB were detected. Similarly, specific Abs to A-RNP were present in SmB-immunized mice. In the A-RNP-immunized mice, specific Abs to the 70-kDa protein were detected. In all three immunizations, Abs capable of immunoprecipitating the entire snRNP particle were induced. None of the groups had Abs reactive to native mouse Ro60 (data not shown).

The results of this serological analysis of the immune sera are very similar to those seen in patients with SLE in their complexities of autoantibody specificities. In SLE patients, Abs are directed against several proteins within the snRNP complex (1, 4, 5, 6). Analysis of patient sera over a period of time has demonstrated an ordered appearance of Abs reactive with an increasing number of proteins within the snRNP complex (11, 31, 32). The similarity in the evolution of Ab specificities provides some assurance that our mouse model is useful to study SLE-related autoantibody diversification. Unlike several other experimental models of epitope spreading relying on the use of CFA (15, 16, 17), we decided to use alum as an adjuvant. In preliminary experiments, A/J (H-2a), SJL/J (H-2s), and BALB/c (H-2d) mice were immunized with recombinant SmD in CFA. Although epitope spreading patterns in SJL/J and A/J mice were identical with those reported in this study, no spreading was observed in BALB/c mice (data not shown). However, the interpretation of Ab reactivities in SJL/J mice at later time points was complicated by high background reactivities to snRNPs in control mice immunized only with CFA. Thus, to rule out the possible role of CFA in anti-snRNP Ab responses, we used alum as an adjuvant. The use of alum as an adjuvant provides assurance that the immunogen was less likely to persist throughout the interval in which the immune response was studied. In addition, the immune responses were skewed to a Th2 type response, with the predominance of immunogen-specific IgG1 Abs (data not shown). Thus, the appearance of specific Abs to selected components of the snRNP particles and to the conformational epitope(s) in the intact snRNP particle over a period of time after the initial immunization suggests an important role for the endogenous Ag in our model.

In SmD-immunized mice, at earlier time points, the sera did not immunoprecipitate the snRNP particle despite having high titers of anti-SmD Abs. The ability to immunoprecipitate the snRNP particle coincided with the appearance of A-RNP-specific Abs (Fig. 7, bottom). These data suggests that B cells reactive with A-RNP recognize the entire snRNP particle. These B cells then process and present peptides from endogenous SmD to SmD-reactive T cells. The ensuing T cell-B cell interaction results in the generation of Abs reactive with A-RNP. A similar mechanism can explain the generation of anti-SmB Abs in these mice or the generation of anti-A-RNP Abs in mice immunized with SmB. These data support the hypothesis of interstructural T cell help proposed by Craft et al. (14, 33, 34) and the “particle hypothesis” proposed by Hardin (35).

However, our data from A-RNP- and SmB-immunized mice cannot be adequately explained by these two hypotheses. In mice immunized with A-RNP, the Ab response only spreads to the 70-kDa protein. Abs to SmB remain cross-reactive (by two independent immunoassays), even after 5 mo postimmunization. This is surprising considering the data from SmB- and SmD-immunized mice, which clearly demonstrates the presence of SmB specific B cells in A/J mice. Immunization with A-RNP abrogates tolerance to the endogenous Ag, which is evident from the generation of specific Abs to the 70-kDa protein and cannot be the reason for lack of epitope spreading to SmB. The restricted epitope spreading to 70-kDa protein in mice immunized with the A-RNP protein is not unique to our model system. Our data match the long known observation that patients with anti-Sm Abs almost always have anti-RNP Abs but some patients with anti-RNP Abs do not have anti-Sm Abs. In rabbits immunized with A-RNP purified from the rabbit thymus extract, the Ab response was only restricted to the immunogen (36). Similarly, in mice immunized with human A-RNP, only cross-reactive Abs to SmB were generated, with no Abs to SmD (15). A similar discussion can be made regarding the observation that in SmB-immunized mice, epitope spreading occurs predominantly to A-RNP. Several studies have shown that proteins within the snRNP complex are present in a cell in different combinations (37, 38, 39, 40). Apart from the intact snRNP particle, three stable complexes of Sm proteins, D3-B, D1-D2, and E-F-G, are found in the cell. This suggests the possibility that different forms of Sm protein complexes will be available to the immune system to drive an anti-snRNP Ab response. These data suggest that mechanisms additional to “particle hypothesis” and interstructural T cell help are operative in intermolecular B cell epitope spreading. We are currently investigating one such mechanism in SmD-immunized mice. In these mice, we have detected T cell responses to A-RNP (U. S. Deshmukh, manuscript in preparation). Thus, a T cell response to A-RNP in mice immunized with SmD might be contributing to better B cell spreading. As a corollary to this hypothesis, a lack of T cell response to SmB or SmD in A-RNP-immunized mice might be the reason for the lack of intermolecular B cell epitope spreading to these proteins.

Although other investigators have demonstrated epitope spreading within the snRNPs, using synthetic peptides from SmD (41, 42) and SmB (16), we have used a unique strategy of using three different proteins to study epitope spreading within the snRNP particle. This approach has allowed us to demonstrate that patterns of epitope spreading within multimeric antigenic complexes are dependent on the initiating Ag. In addition, lack of epitope spreading to A-RNP and SmB in BALB/c mice immunized with SmD in either CFA or alum as adjuvant (data not shown) or spreading to C-RNP in HLA-DR3-transgenic mice immunized with SmD (C. Jiang, manuscript in preparation) suggest that different strains of mice will have different outcomes of epitope spreading with the same initiating Ag.

These findings are highly significant toward understanding how autoantibody responses to lupus-associated Ags evolve in patients. Recently, based on Western blot analysis of human sera, it was suggested that A-RNP (13), or 70-kDa and SmB (11) initiate anti-Sm/snRNP Ab responses, with a minimal role for SmD. However, several studies have reported discrepancies in the anti-snRNP Ab reactivity patterns obtained in different immunoassays such as Western blotting, ELISA, and immunodiffusion (43, 44, 45, 46). Considering this and our data from the SmD-immunized mice, it can be stated that SmD might initiate anti-Sm/snRNP Ab responses in some SLE patients. Immune responses to self-Ags in SLE patients may be initiated through foreign molecular mimics or self-Ags modified through processes such as post-translational modifications (47). Although our data do not directly address the role of altered self-Ags in the initiation of anti-snRNP Ab responses, it has important implications for the generation of autoantibody responses through this pathway. Our data imply that there is no unique component of the snRNP particle that is responsible for the initiation of anti-snRNP Ab responses in SLE patients. Thus, different molecular mimics or different altered self-Ags would be responsible for the initiation of anti-Sm/snRNP Ab responses in different SLE patients.

We thank Sarah Poston and Poonam Sharma for providing technical assistance and Dr. Felicia Gaskin (University of Virginia) for help in the preparation of the manuscript.

1

This study is supported in part by Grants R01 AI-45199, R01 AR-42456, P50 AR-45222, and P30 Ca-45222 from the National Institutes of Health. U.S.D. is partly supported by a Scientist Development Grant from the American Heart Association. A part of this work was presented in abstract form in the 2001 Annual Scientific Meeting of the American College of Rheumatology, San Francisco, CA, November 10–15, 2001 (48 ).

3

Abbreviations used in this paper: snRNP, small nuclear ribonucleoprotein; A-RNP, A ribonucleoprotein; 70 kDa, U1-RNP-associated 70-kDa protein; SmB, Smith B; SmD, Smith D; SLE, systemic lupus erythematosus.

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