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* Division of Rheumatology and Immunology, Department of Internal Medicine and University of Virginia Specialized Center of Research in Systemic Lupus Erythematosus, and
Department of Microbiology and University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, VA 22908.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cloning and expression of recombinant SmD and SmB have been described previously (19). The 6x 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).
Mice and immunization
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.
Western blots
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).
Immunoprecipitation
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.
Ab absorptions
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Intermolecular epitope spreading to A-RNP and SmB/B' occurs in SmD-immunized mice
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.
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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.
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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.
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, 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). Immunogen dependence of distinct intermolecular epitope spreading patterns also shown by immunoprecipitation of in vitro transcribed and translated SmD, A-RNP, and SmB
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. 5A). In A-RNP-immunized mice,
only Abs to A-RNP (seven of seven) and SmB (three of seven) were
detected (Fig. 5B). In SmB-immunized mice, Abs reactive with
SmB (six of six), A-RNP (five of six), and SmD (one of six) were
detected (Fig. 5C). Abs capable of immunoprecipitating Ro60
were not detected in any group of immunized mice (data not shown).
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, A and
C). In the A-RNP-immunized mice (Fig. 6B),
absorption of sera with the immunogen depleted all Abs, thereby
indicating that Abs reactive with SmB were cross-reactive.
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Umesh S. Deshmukh, Box 800412, University of Virginia Health System, Charlottesville, VA 22908. E-mail address: usd7w{at}virginia.edu
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.
Received for publication January 18, 2002. Accepted for publication March 15, 2002.
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