Murine models of systemic lupus erythematosus: B and T cell responses to spliceosomal ribonucleoproteins in MRL/Faslpr and (NZB x NZW)F1 lupus mice
Fanny Monneaux,
Hélène Dumortier2,
Guenter Steiner1,
Jean-Paul Briand and
Sylviane Muller
Institut de Biologie Moléculaire et Cellulaire, UPR 9021 Centre National de la Recherche Scientifique, 15 rue René Descartes, 6700 Strasbourg, France
1 Division of Rheumatology, Department of Internal Medicine III, and Institute of Medical Biochemistry, University of Vienna, Dr Bohr-Gasse 9, A-1030 Vienna, Austria
2 Present address: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlands.
Correspondence to:
S. Muller
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Abstract
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(NZB x NZW)F1 and MRL/Faslpr lupus mice present a similar phenotype with a spectrum of autoantibodies associated with very severe nephritis. It is thought, however, that in contrast to other lupus-prone mice such as MRL/Faslpr mice, (NZB x NZW)F1 mice do not generate autoantibodies to ribonucleoproteins (RNP) Sm/RNP. In this study, we demonstrate that contrary to previous reports, the autoimmune response directed against Sm/RNP antigens also occurs in NZB x NZW mice. CD4+ T cells from unprimed 10-week-old NZB x NZW mice proliferate and secrete IL-2 in response to peptide 131151 of the U1-70K protein, which is known to contain a Th epitope recognized by CD4+ T cells from MRL/Faslpr mice. Peptide 131151, which was found to bind I-Ak and I-Ek class II MHC molecules, also bound both I-Ad and I-Ed molecules. This result led us to also re-evaluate longitudinally the anti-Sm/RNP antibody response in NZB x NZW mice. We found that 25-week-old mice do produce antibodies reacting with several small nuclear and heterogeneous nuclear (hn) RNP proteins, such as SmD1, U1-70K and hnRNP A2/B1 proteins. The fine specificity of these antibodies was studied with overlapping synthetic peptides. The same antigenically positive and negative peptides were characterized in MRL/Faslpr and NZB x NZW mice in the three proteins. This new finding can help to understand the mechanisms involved in the development of the anti-Sm/RNP antibody response and, particularly, the role played by non-MHC genes in this autoimmune response.
Keywords: autoimmunity, epitopes, lupus animal models, T lymphocytes
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Introduction
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One of the impediments in understanding systemic lupus erythematosus (SLE) is its marked heterogeneity. SLE is a complex chronic autoimmune disorder of uncertain origin characterized by the production of autoantibodies to a wide variety of self antigens. Hypergammaglobulinemia and circulating immune complexes are also often present in lupus. In addition, considerable evidence has been accumulated on the cellular immune defects and aberrations associated to lupus (1). Murine models, such as (NZBxNZW)F1, MRL/Faslpr (MRL/lpr) and BXSB mice, spontaneously develop SLE-like syndromes with a comparable heterogeneity and have provided a powerful tool to study the human disease (2). The NZBxNZW model was the first murine model described for lupus nephritis (3). The F1 hybrid between NZB and NZW mouse strains develops renal lesions that are remarkably similar to the pathology described in human lupus. During the last four decades, serological and histological consequences of the development of systemic autoimmunity in this model have been extensively studied (47). The renal dysfunction is accompanied by an increase in IgG autoantibodies, notably antinuclear and anti-DNA antibodies. Subsequently, the availability of other spontaneous, experimentally induced, transgenic and recombinant knockout lupus models has greatly enhanced the potential value of murine SLE as an experimental model (2,812). Both NZBxNZW and MRL/lpr mice develop a chronic spontaneous autoimmune disease involving different systems together with the appearance of hypergammaglobulinemia and pathogenic anti-DNA antibodies. Some properties of human SLE, such as its preponderance in females, are not found in the BXSB model. Other main characteristics of human lupus, such as the production of anti-small nuclear ribonucleoprotein (snRNP) autoantibodies are, however, features of the MRL (MRL+/+ and MRL/lpr mice) and (SWRxSJL)F1 mice (11), while NZB and NZW mice and the F1 hybrid NZBXNZW are known to be uniformly negative (2,13,14). The reasons for the absence of anti-Sm/RNP antibodies in NZBxNZW mice are still unclear (13).
We recently identified an epitope located within the peptide 131151 of the U1-70K RNP that is recognized by CD4+ T cells from 7- to 9-week-old MRL/lpr mice (15). This peptide also contains a B cell epitope recognized by IgG autoantibodies from these mice. In an attempt to study the contribution of the genetic background involved in the production of T cells and antibodies reacting with the 70K peptide 131151, we included NZBxNZW mice in our series of lupus mice and unexpectedly observed that mice gave a significant response to this peptide. The present study was thus carried out to re-examine the anti-Sm/RNP immune response in NZBxNZW mice. We studied longitudinally the development of the anti-snRNP response in NZBxNZW mice and found that as in the MRL/lpr model, CD4+ T cells from 10-week-old NZBxNZW mice do recognize the 70K peptide 131151. Moreover, the sera from 25-week-old NZBxNZW mice strongly reacted with SmD1, U1-70K and heterogeneous (hn) RNP A2/B1 proteins. The fine specificity of these autoantibodies was studied with synthetic peptides covering the three proteins and several B cell epitopes were identified. These findings have several implications, notably in the context of MHC and non-MHC genes involved in the antiSm/RNP autoimmune response in lupus mice.
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Methods
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Antigens
Extractable nuclear antigens (ENA) from rabbit thymus used in Western immunoblotting were purchased from Euromedex (Strasbourg, France; ref. 41009.1). No DNA or histones contamination was detected in the ENA preparation as determined by polyacrylamide and agarose gel electrophoresis. The His-tagged recombinant hnRNP A2/B1 protein (the B1 variant differs from hnRNP A2 only by a 12-amino-acid insert at the N-terminus) and the synthetic peptides corresponding to the sequence of mouse snRNP 70K, SmD1 and hnRNP A2 proteins have been described previously (1517). The identity of each purified peptide was assessed by matrix-assisted laser desorption ionization mass spectrometry using a Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany). Mononucleosomes were prepared from calf thymus as described previously (18) and purified on a 529% (w/v) sucrose gradient. They were characterized by 1.5% agarose gel electrophoresis and the content in histones was checked by 18% SDSPAGE. Native double-stranded (ds) DNA was purchased from Sigma (St Louis, MO; ref. D4764) and treated as described previously (18).
Mice
BALB/c (H-2d), NZBxNZW (H-2d/z) and MRL/lpr (H-2k) mice (all female) were purchased from Harlan (Gannat, France). Proteinuria was assessed using Albutix (Bayer Diagnostics, Basingstoke, UK) in fresh urine samples.
Western immunoblotting and ELISA for antibody measurements
The binding of mouse antibodies to the 70K and SmD1 proteins was tested by Western immunoblotting using ENA as previously described (16). The position of the bands corresponding to the 70K protein and SmD1 protein was revealed using the mouse IgG2a mAb 2.73 (19) and the mouse IgG3 mAb 7.13 (20) respectively. The reactivity of NZBxNZW mouse sera was also tested with recombinant hnRNP A2/B1 (rB1) protein as previously described (16). To test the binding of mouse sera to 70K, SmD1 and A2/B1 peptides, ELISA polyvinyl plates (Falcon, Oxnard, CA; ref. 3912) were coated overnight at 37°C with 2 µM of each peptide diluted in 0.05 M carbonate buffer, pH 9.6 (1517). The coating of plates with DNA and nucleosomes was as described previously (18) using 100 ng/ml dsDNA diluted in 25 mM citrate buffer, pH 5.4, and 300 ng/ml nucleosome (as expressed in DNA concentration) diluted in PBS, pH 7.4. In each assay, mouse sera were also tested in a non-coated well incubated with the respective coating buffers as a control. The subsequent steps of the respective tests were performed using mouse sera diluted 1:500 in PBS containing 0.05% Tween 20 (PBS-T) and goat anti-mouse IgG conjugated to horseradish peroxidase diluted 1:20,000 in PBS-T. The cut-off points of each assay were determined with a series of sera from 10 non-immunized BALB/c mice. Mouse sera were considered positive when the OD values were higher than the mean OD + 2 SD, i.e. OD
0.2 for all peptides tested in this study.
ELISA for cytokine measurements
IFN-
, IL-4, IL-6 and IL-10 secretion was evaluated by sandwich ELISA using commercial antibodies from PharMingen (San Diego, CA) and Falcon polyvinyl plates (15). Standard curves performed with known concentrations of recombinant cytokines (PharMingen) were used as internal controls.
Lymphocyte proliferation assays and measurement of cytokine secretion
The proliferation of lymph node cells (LNC) from NZBxNZW mice was as described previously (15,21). Briefly, CD4+ T cells were purified on magnetic beads coated with anti-CD4 mAb (Dynabeads; Dynal, Oslo, Norway), and the response to peptide 131151 of 70K (12.5200 µM) was measured using 5x105 CD4+ T cells/well and 105 mitomycin-treated autologous spleen cells as antigen-presenting cells (APC). After 24 h, 50 µl supernatant was taken off to test the production of IL-2 using CTL-L cells (21). For the detection of other cytokines (IFN-
, IL-4, IL-6 and IL-10), culture supernatants (50 µl) were collected after 24 h and tested in ELISA as described above. After 54 h, the cultures were pulsed during 18 h with [3H]thymidine (1 µCi/well). The cells were subsequently harvested on a filter and DNA-incorporated radioactivity was measured. The results are expressed as the arithmetic mean of thymidine uptake expressed as c.p.m. Proliferative responses were considered to be significantly positive when the [3H]thymidine uptake was equal to or above twice the uptake by LNC cultured in medium alone without peptide. The SD of triplicate cultures was always <20% of the mean. Control tests were performed by adding concanavalin A (Con A; 100 µl/well; 5 µg/ml; Sigma) to cells during the time (72 h) of the culture.
Binding of peptide analogues to I-Ad and I-Ed class II molecules
The MHC class II I-Ad and I-Ed restriction elements involved in the presentation of peptide analogues were determined by measuring the capacity of analogues to compete with the cI peptide 1226 for binding to I-Ad- or I-Ed-restricted APC (22). Mouse L fibroblasts transfected by either I-Ad (RT 2.3.3H) or I-Ed (RT 10.3H2; 5x104 cells/well) were incubated with various concentrations (0100 µM; 50 µl/well) of peptide. After 45 min at 37°C, 50 µl T cell hybridomas B26.1 or B26.2 (5x104 cells/well) and 50 µl peptide 1226 of cI (0.25 and 2 µM, for hybridomas B26.1 and B26.2 respectively) were added to I-Ed and I-Ad transfected cells respectively. Hybridomas B26.1 and B26.2 are derived from BALB/c mice immunized with the cI peptide 1226, and recognize this peptide in the I-Ed and I-Ad context respectively (23). After 24 h in culture, 50 µl aliquots of medium were removed from each well and tested for their content in IL-2 as described above.
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Results
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Spontaneous T cell response to the 70K peptide 131151 in unprimed NZBxNZW mice
We recently identified a Th epitope recognized by CD4+ T cells from young MRL/lpr mice (15). This T cell epitope is present in the 70K peptide 131151 which binds both I-Ak and I-Ek class II molecules. To better understand the origin, the functional characteristics and the specificity of both autoimmune APC and CD4+ T cells reacting in this context with the 70K peptide 131151, we tested the reactivity of autoreactive T cells from MRL/lpr mice and as a control included cells from NZBxNZW mice not known to mount an anti-snRNP response. To our surprise, we found that purified CD4+ T lymphocytes pooled from six unprimed 10-week-old NZBxNZW mice reacted specifically and in a dose-dependent manner with the 70K peptide 131151. As shown in Fig. 1
, this peptide induced proliferation with IL-2 secretion of unprimed CD4+ T cells from NZBxNZW mice and from MRL/lpr mice shown here for comparison. As in the MRL/lpr model (15), no IL-4, IL-6, IL-10 and IFN-
was detected in the cultures, neither in response to peptide 131151 nor after stimulation with Con A (data not shown).
We reproducibly observed a dramatic drop of the proliferation level when slightly higher concentrations of peptide 131151 (200 µM) were used ex vivo to stimulate CD4+ T cell from NZBxNZW mice (Fig. 1A
). It is well known that self-reactive T cells are activated in lupus (24) and this might explain why ex vivo, mature CD4+ T cells rapidly die in the presence of a small excess of activating peptide (25). We thus analyzed the expression of activation molecules at the surface of CD4+ T cells in response to peptide 131151. CD4+ T cells from NZBxNZW mice were collected after 24 h of culture, stained with labeled mAb and analyzed by flow cytometry. We found that expression of CD25 (IL-2 receptor
chain) and CD69 (early activation antigen) was significantly increased on NZBxNZW CD4+ T cells incubated with a high concentration (200 µM) of peptide 131151. Twenty-six percent of CD4+ T cells incubated with 200 µM of peptide 131151 versus 9% of cells incubated in the presence of an irrelevant peptide encompassing residues 7497 of the 70K protein expressed CD25. Under the same conditions, 26% of CD4+ T cells incubated with peptide 131151 expressed CD69 versus 6% of cells incubated with peptide 7497 (not shown). The proportion of T cells activated by peptide 131151 was thus estimated to 20%. Furthermore, we also observed that apoptosis, as evaluated by flow cytometry analysis using the classical Annexin VFITC staining test, occurred in ~14% of CD4+ T cells in the absence of peptide 131151 versus 40% of CD4+ T cells incubated in the presence of 200 µM of this peptide.
Binding of 70K peptide 131151 to I-Ad and I-Ed MHC class II molecules
We showed previously that peptide 131151 binds both the I-Ak and I-Ek MHC class II molecules (15). Since this peptide was recognized by T cells from H-2d/z NZBxNZW mice, we also tested whether it binds I-Ad and I-Ed class II molecules. Using the assay previously described (15,22) we found that pre-incubation with peptide 131151 of fibroblasts transfected by I-Ad and I-Ed reduced the IL-2 production level by 75 and 99% respectively at a molar excess of 50 compared to the parent peptide cI (Fig. 2
). This result indicates that peptide 131151 can be efficiently presented by both I-Ed and I-Ad molecules and seems therefore to behave as a universal epitope.

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Fig. 2. Binding of 70K peptide 131151 to I-Ed and I-Ad MHC class II molecules. Mouse L fibroblasts transfected by either I-Ed or I-Ad were first incubated with various concentrations of peptide 131151. After 45 min, cI peptide 1226 and T cell hybridomas that recognize this peptide in the I-Ed or I-Ad context were added to the cultures. After 24 h, the secretion of IL-2 was measured using CTL-L cells.
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Antibody reactivity of sera from NZBxNZW mice with snRNP and hnRNP proteins
The striking similarity between the T cell response observed in MRL/lpr and NZBxNZW mice prompted us to examine whether the antibody response in these two models also shows some resemblance. Sera from three independent groups of five NZBxNZW mice of different ages (14, 25 and 40 weeks; 15 mice in total) were first tested by Western immunoblotting for their reactivity with 70K and SmD1 proteins using ENA extracts. At week 14, anti-snRNP IgG antibodies were not detectable (Fig. 3
; mice 15). At week 25, reactivity to 70K and SmD1 proteins appeared in one (mouse 8) and two mice (mice 7 and 8) respectively, and no proteinuria was measurable. The number of NZBxNZW mice with a positive proteinuria and high levels of antibodies to 70K and SmD1 proteins increased at week 40 (three of five mice, Fig. 3
). The same three positive mice also possessed anti-dsDNA and anti-nucleosome IgG antibodies in their serum. It is noticeable that antibodies to nucleosomes were the first to appear in this series of mice (one of five mice at week 14 and two of five mice at week 25; Fig. 3
) and were present in certain mice in the absence of detectable DNA antibodies (mice 2, 6, 11 and 12). One mouse (mouse 8) possessed antibodies to dsDNA without antibodies to nucleosomes. Normal females BALB/c mice of the same age were completely negative.

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Fig. 3. Reactivity of sera from 15 NZBxNZW mice with 70K and SmD1 proteins. Sera tested were collected from 14-week-old mice (mice 15), 25-week-old mice (mice 610) and 40-week-old mice (mice 1115). mAb 2.73 and 7.13 were used to determine the position of the bands corresponding to the 70K and SmD1 proteins respectively. The identity of the protein labeled in samples 1 and 4 is not known. IgG antibodies reacting with nucleosomes and dsDNA were tested by ELISA. Mouse sera were diluted 1:500 and IgG antibodies only were tested. The proteinuria level was measured using Albutix in fresh NZBxNZW urine samples.
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In order to study more precisely the appearance of antibodies to snRNP proteins during the course of their disease, a longitudinal analysis with serial bleedings from nine NZBxNZW mice was performed between weeks 21 and 36. The samples were also tested for their reactivity to hnRNP A2/B1 (16). As illustrated in Fig. 4
with three representative mice (BW1, BW7 and BW9) and shown in Table 1
with the nine mice, depending on the mice, IgG antibodies reacting with the 70K, SmD1 and rB1 proteins in ELISA and Western blotting appeared between 21 and 30 weeks. If we compare the production of antibodies to the 70K, SmD1 and A2/B1 RNP, it is evident that the antibody response to SmD1 protein was the first to be detectable at 21 weeks (see BW4, BW5 and BW9 mice). At this age, however, elevated levels of IgG antibodies to nucleosome and dsDNA were present in almost every mice.

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Fig. 4. Longitudinal analysis in Western immunoblotting of antibodies to 70K, SmD1 and hnRNP A2/B1 proteins produced in NZBxNZW mice. Reactivity of serum samples from three to nine representative mice (BW1, BW7 and BW9) collected at 21, 24, 27, 30 and 36 weeks. The IgG response to hnRNP A2/B1 protein was tested using rB1. +, Positive control (mouse antiserum raised against rB1). See the legend of Fig. 3 for the description of conditions.
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Table 1. Longitudinal analysis of serological characteristics of nine NZB/W mice (the reactivity of the serum from two MRL/lpr mice tested at 12 and 20 weeks respectively is shown for comparison)
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Reactivity of IgG antibodies from NZBxNZW mice with 70K, SmD1 and hnRNPA2 peptides
In our previous report (15), we identified in the 70K protein a number of B cell epitopes recognized by antibodies from MRL/lpr mice. We found that IgG antibodies reacting with peptides 2141 and 131151 appeared very early in the serum of these mice, significantly before antibodies to the other 70K peptides and the 70K cognate protein. For an initial screening, we tested in the same ELISA conditions the sera collected from the 15 NZBxNZW mice described above (see Fig. 3
), with 20 and 12 overlapping peptides covering the whole sequence of the 70K protein and SmD1 respectively (15,17). In addition, we also studied the peptide 5070 of the hnRNP A2 protein, in which a major B cell epitope recognized by the antibodies from young MRL/lpr mice has been recently characterized (16). Among these 33 peptides tested, we observed that reactivity was largely confined to peptides 120 and 97119 of SmD1, 2141 and 131151 of the 70K protein, and 5070 of hnRNP A2 (data not shown). We then tested with these five peptides the serum samples collected longitudinally from the nine NZBxNZW mice described above (Fig. 4
). As shown in Table 1
, the results obtained in our first screening were confirmed. All mice produced IgG antibodies reacting with at least two of the five selected hn/snRNP peptides. In several animals, antibodies to peptides 131151 of the 70K protein, 120 of SmD1 and 5070 of hnRNP A2 were already observed in 21-week-old NZBxNZW mice, before or concomitantly with antibodies reacting in Western blotting with the respective cognate proteins, and in some cases they preceded anti-nucleosome and even anti-dsDNA antibodies (BW3, BW6 and BW7). Antibodies to the peptides occurred independently of each other.
The results described above are very similar in terms of specificity to those described previously in MRL/lpr mice (15,16; Fig. 5
and Table 1
). Moreover, when we examined the subclasses of antibodies reacting with the five peptides of SmD1, 70K and hnRNP A2 proteins, we found that IgG antibodies from NZBxNZW mice reacting with these peptides were mostly of the IgG2a subclass, less frequently of the IgG3 and IgG1 subclasses, and rarely of the IgG2b subclass (data not shown). The same pattern of IgG subclasses was found in the serum of 10 MRL/lpr mice tested in parallel for their reactivity with these five peptides (not shown).

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Fig. 5. Schematic representation of major autoepitopes recognized by IgG antibodies from lupus mice on the 70K, SmD1 and hnRNP A2/B1 antigens. The same peptides were recognized by IgG antibodies from NZBxNZW mice (open bars; this study) and MRL/lpr mice (shaded bars; 15,19 and Dumortier et al., unpublished) tested in the same ELISA conditions.
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Discussion
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Most of the work devoted to the immune response to snRNP in murine models of lupus has been performed using the MRL strain. MRL/lpr mice spontaneously develop a systemic autoimmune disease sharing significant features with human lupus. In addition to multiple unknown background genes that control their disease, MRL/lpr mice have a homozygous fas mutation, which accelerates autoimmunity (2,4,8,26). We recently identified an epitope located within the peptide 131151 of the U1-70K protein that is recognized by autoreactive CD4+ T cells from young MRL/lpr mice and examined some properties of these T cells (15). The nature of APC involved in the presentation of this peptide to CD4+ T cells is unknown, but it was clearly shown that MRL/lpr APC are required and that autologous B cells are necessary but not sufficient in this process (15). The current study was initially undertaken to assess the influence on T cell response to peptide 131151 of APC from mice of different haplotypes. Lupus-prone NZBxNZW mice were introduced in a series of preliminary experiments because although the genetic background of these two strains is very different, NZBxNZW and MRL/lpr mice develop lupus-like syndromes that are clinically and immunologically quite similar. Major differences among these two strains exist, however, and notably it has been repeatedly claimed that anti-snRNP antibodies are absent in NZBxNZW mice (2,8,13,14). Unexpectedly, the existence in unprimed 10-week-old NZBxNZW mice of CD4+ T cells proliferating and secreting IL-2 ex vivo in the presence of peptide 131151 was demonstrated. These cells produced no IL-4, IL-6, IL-10 and IFN-
in response to peptide 131151 or after stimulation with Con A.
Both our previous (15) and present studies show that peptide 131151 binds the four I-Ak, I-Ek, I-Ad and I-Ed MHC class II molecules, suggesting that this peptide might span a promiscuous Th epitope. We are currently testing this possibility. It is striking to observe that histone peptides that are specifically recognized by the spontaneously arising CD4+ T cells from lupus-prone I-Ad/q (SWRxNZB)F1 mice also contain non-MHC-restricted universal T epitopes (27). Promiscuous recognition of nucleosomal epitopes by T cells was found to be a property conferred by the TCR
chain. In light of this, it is interesting to note that the only MHC class II molecule expressed by non-obese diabetic mice, I-Ag7, which shares a common
chain with I-Ad but has a peculiar ß chain, has been reported recently, as I-Ad, to be very promiscuous in terms of peptide binding (28).
To further study the autoimmune response to snRNP in NZBxNZW mice, we analyzed the reactivity of sera collected from mice of different ages and found that 25-week-old NZBxNZW mice produce antibodies reacting with several well-characterized snRNP and hnRNP targeted by antibodies from MRL/lpr mice and lupus patients. High levels of IgG antibodies reacting in Western blotting and ELISA with the 70K, SmD1 and hnRNP A2/B1 proteins were detectable. In addition, some other antigens contained in the ENA extract were also recognized by these antibodies. They appeared generally simultaneously or after antibodies to nucleosome and dsDNA, and before or concomitantly to proteinuria. It appears evident that the previous failure to detect anti-Sm reactivity in NZB/W mice may be the `result of the insensitivity of the methods employed', as reported by Eisenberg et al. (13). It may be also argued that antibodies to 70K, SmD1 and hnRNP A2/B1 correspond to cross-reacting anti-DNA antibodies. It has been claimed that these proteins do bind DNA and proposed that sn/hnRNPDNA complexes might therefore interact with anti-DNA antibodies (2931). Although we cannot completely exclude this possibility, several observations (in the present work) can be put forward in this discussion: (i) in a few bleeds anti-sn/hnRNP were detected in the absence of anti-DNA reactivity (mice BW3, BW6, BW7 and BW9; Table 1
), (ii) it is not known if DNA interacts with short peptides of snRNP and hnRNP proteins, (iii) we found no cross-reaction in a competitive ELISA between dsDNA and antibodies reacting with peptide 131151, and (iv) it is difficult to understand why this feature would occur in NZB/W mice only and not in MRL/lpr mice.
To reinforce our results, we examined the fine specificity of these antibodies using overlapping peptides covering the whole sequence of the 70K and SmD1 proteins, as well as peptide 5070 of hnRNP A2 which contains a major B cell epitope recognized by IgG antibodies from young MRL/lpr mice (16). Because MRL/lpr and NZBxNZW mice possess distinct restriction MHC elements and a very different genetic background, fine differences in the specificity of antibodies generated in these two strains were initially expected. Again, and contrary to this assumption, the same antigenically positive and negative peptides were characterized in the three tested antigens using MRL/lpr and NZBxNZW antibodies. In both strains, IgG antibodies produced during the course of the disease reacted with peptides 2141 and 131151 of the 70K protein, 120 and 97119 of SmD1, and 5070 of hnRNP A2. In certain mice but not systematically in all mice (Table 1
), antibodies to SmD1 and A2 peptides were detected significantly before antibodies reacting in Western blotting with the whole respective proteins. This was not observed with the 70K peptides. This type of reactivity (positivity with a segment of a protein but not with the cognate protein itself) has been described by independent groups to occur in autoimmune sera. We extensively discussed the possible reasons that may explain this intriguing observation recently (16).
In this study we have demonstrated that, contrary to previous reports, the anti-sn and hnRNP immune response is not unique to the MRL strain, and also occurs in NZBxNZW mice. Although we have yet to study this question systematically, we found no difference in the fine specificity of antibodies to three different spliceosomal RNP, the U1-70K, SmD1 and hnRNP A2/B1 proteins. Antibodies reacting with the three whole proteins in Western blotting appeared in NZBxNZW mice at 2025 weeks (as compared to 1015 weeks in MRL/lpr mice), which correlates with their longer half-life (34 versus 22 weeks; 32). Moreover, CD4+ T cells from both strains recognized the same, promiscuous Th epitope located in residues 131151 of the 70K protein. Our results are important because they provide new information on the shared genetic regulation of the anti-snRNP autoimmune response. The mechanisms involved in this complex autoimmune response share possibly more common features than previously expected. In particular, we can argue that MHC genes at the H-2 locus, as well as some non-MHC genes, such as the gene encoding the Fas molecule, are not directly involved in this autoimmune response. The stochastic nature of the anti-Sm response in MRL mice has been previously discussed since only 25% of these mice develop anti-Sm antibodies (33,34). This observation may have implications to our understanding of defects leading to the breakdown of self-tolerance and production of snRNP autoantibodies in lupus. It may also provide the basis for novel experimental therapeutic strategies.
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Acknowledgments
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We thank Drs G. Fournié and D. Isenberg for their critical reading of the manuscript, Dr G. Pruijn for providing mAb 2.73 and 7.13, and Dr P. Decker for a gift of nucleosomes and mouse serum samples. This work was supported by a grant from the `Association de Recherche sur la Polyarthrite' and by the ICP program of the Austrian Federal Ministry for Science, Education and Culture. H. D. and F. M. were recipients of a pre-doctoral grant from the `Fondation pour la Recherche Médicale'.
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Abbreviations
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APC antigen-presenting cell |
Con A concanavalin A |
ds double stranded |
ENA extractable nuclear antigens |
hn heterogeneous nuclear |
LNC lymph node cell |
RNP ribonucleoprotein |
SLE systemic lupus erythematosus |
sn small nuclear |
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Notes
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Transmitting editor: S. Izui
Received 9 April 2001,
accepted 6 June 2001.
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