Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1–206 of A2 that contains most of the epitopes recognized by patients’ Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50–70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35–55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35–175 that generated an effective Th cell response with IL-2 and IFN-γ secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.

Among Abs directed against heterogeneous nuclear ribonucleoproteins (hnRNPs),4 those described as anti-RA33 refer to autoantibodies that target the 36-kDa hnRNP A2 as well as its alternatively spliced variants hnRNP-B1 (37 kDa) and B2 (38 kDa) present in spliceosomal hnRNP complexes (1). These complexes contain pre-mRNA and >20 different proteins but their structure is not known in detail nor are the functions of most hnRNPs fully understood. Apart from their established roles in packaging and splicing of pre-mRNAs, hnRNPs seem to be involved in diverse aspects of mRNA metabolism and regulation including mRNA transport, translation, and even transcription (2, 3, 4). Among the hnRNPs, the hnRNPs A/B form a distinct subgroup of highly related proteins that show the same structural features and presumably derive from an evolutionarily conserved ancestor gene. Their N-terminal half consists of two adjacent 80- to 90-residues-long conserved RNA-binding domains (RBDs), whereas their C-terminal half contains ∼45% glycine residues and is assumed to be involved in interactions with other hnRNPs and RNA (2, 5). hnRNP A1 is the best-studied hnRNP, and the crystal structure of a fragment of A1 containing both RBDs has been determined to a 1.9-Å resolution (6). There is no fine structural information available concerning hnRNPs A2 and B1 (which is identical with A2 except for an additional sequence of 12 residues inserted near the N terminus) (7), but it is generally assumed that their three-dimensional structure is very similar to that of A1. Although hnRNPs A/B are mostly located in the nucleus, they have also been found in the cytoplasm where they seem to play a role in nuclear export of mRNA and proteins (2, 8).

Spontaneously produced Abs against hnRNPs were first described >15 years ago to occur in 20–40% of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed-connective tissue disease (MCTD), and only occasionally in patients with other rheumatic diseases (9, 10). Subsequent studies showed hnRNPs A/B to form the major targets of these Abs, particularly hnRNP A2/B1 (11, 12, 13, 14, 15). Although anti-A2/B1 Abs are also present in SLE and MCTD, they might represent an important marker Ab for RA because Abs associated with SLE and/or MCTD, such as Abs to dsDNA and U1-small nuclear ribonucleoproteins (snRNPs), are absent in RA. Remarkably, in a recent clinical and serological study, anti-A2/B1 Abs were reported to be particularly frequent in lupus patients with erosive arthritis, otherwise a characteristic feature of RA (16).

Epitope mapping studies using overlapping hnRNP A2 tryptic and recombinant fragments have revealed that patients’ Abs preferentially reacted with domains that are located in RBDs I and II (17, 18). Interestingly, while the major epitopes recognized by most RA and SLE sera were located in residues 87–182, in which residues 91–105 seemed to be particularly important for patients’ Ab recognition, the region primarily targeted by Abs from patients with MCTD contained most of both RBDs (residues 1–170) (18). Similarly, Montecucco et al. (19) found that the N terminus of hnRNP A1 also contained the major epitopes recognized by the Abs from SLE and RA patients. It was further shown that although all anti-A1-positive sera from SLE patients reacted with the N-terminal fragment of the A1 protein, anti-A1-positive RA sera (9/27 sera tested) did not.

Abs reacting with A1 have been identified in lupus mice (12) but their fine specificity is not known. With the aim of investigating a potential pathogenic role of anti-A2/B1 autoimmunity, we have studied the Ab response to hnRNP A2/B1 in different murine models of lupus. The fine specificity of anti-A2/B1 Abs was tested in mouse sera with a series of 14 partially overlapping synthetic peptides covering the region 1–206 of hnRNP A2 that seems to contain most of the epitopes recognized by patients’ Abs. The concomitant occurrence of Abs reacting with hnRNPs A2/B1 and hnRNP A2 peptides, with SmBB′ and SmD1 snRNPs, and with dsDNA was followed during the course of the disease. The same panel of A2 peptides was also used to characterize T cell reactivity to the A2 protein in non-SLE-prone mice and to identify Th cell epitopes of this protein in normal mice.

The natural hnRNPs A2/B1 were prepared from HeLa nuclear extracts (20) and partially purified by heparin-Sepharose (Pharmacia, Uppsala, Sweden) chromatography as previously described (14). Recombinant Ags (rA2, rB1) were overexpressed in Escherichia coli either as a GST fusion protein (rA2) or as His-tagged protein (rB1) and affinity-purified using glutathione-Sepharose (Pharmacia) or nickel-chelate (Qiagen, Valencia, CA) chromatography, respectively. Both proteins were equally well recognized by human or murine autoimmune sera that were reactive with the natural Ags. HnRNP A2/B1 sequences are extremely conserved between mice/rats and humans (99%). Thus, human hnRNP A2/B1 can be considered as a valid Ag for the study of mouse autoantibodies. The 14 peptides covering residues 1–206 of the mouse hnRNP A2 described by Burd et al. (7) were synthesized using classical F-moc (N-[9-fluorenyl] methoxycarbonyl) solid-phase chemistry (21). Each peptide was purified by reversed-phase HPLC using a Perkin-Elmer (Roissy, France) preparative system on an Aquapore (Perkin-Elmer) ODS 20-μm column (10 × 100 mm). The elution was achieved by a linear gradient of aqueous 0.1% TFA (solvent A) and 0.08% TFA in 80% acetonitrile/20% water (solvent B) at a flow rate of 6 ml/min with UV detection at 220 nm. The homogeneity of each peptide was checked by analytical HPLC on a Nucleosil (Macherly-Nagel, Hoerdt, France) C18 5-μm column (4.6 × 150 mm), using a linear gradient of 0.1% TFA in water and acetonitrile containing 0.08% TFA. The identity of purified peptides was assessed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry using a Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany). Peptides of SmBB′ and SmD1 have been described previously (22, 23).

Four-week-old female MRL-lpr/lpr and MRL+/+ mice as well as female (NZB/NZW)F1, CBA/J, and C57BL/6 mice were obtained from Harlan (Gannat, France). Female BALB/c mice were obtained from Janvier (Le Genest, St. Isle, France). In addition, our study included 28 sera from 25 MRL-lpr/lpr female mice (aged 3–12 mo), 41 sera from five B6/lpr female mice (2–12 mo), 31 sera from 15 (NZB/NZW)F1 female mice (aged 4–14 mo), and 10 sera from BXSB male mice collected in Chapel Hill. The sera from 10 transgenic (Tg) mice expressing the Tax gene of human T cell lymphotropic virus type I were also tested. These sera were obtained from D. Saggioro (Padova, Italy) and were collected from Tax-Tg mice (six males and four females) that had a C57BL/6 genetic background and expressed the viral Tax protein under the control of the mouse metalloprotein promoter. These mice were found previously to display a high prevalence of arthropathy that resembles that of seronegative arthritis in humans (24). As control, we tested the sera from 10 Tax-negative littermates.

A series of nonlupus-prone mice (BALB/c, CBA, and C57BL/6) were immunized i.p. with 100 μg of rB1 protein in CFA for the first injection and IFA for the second and third ones and were bled after each injection. Another series of CBA mice were immunized against peptides 35–55, 50–70, 87–110, and 170–191 (two mice per peptide). They were injected four times s.c. with 100 μg of peptide emulsified in CFA for all injections.

Abs to hnRNPs A2/B1 were detected by Western blotting using HeLa nuclear extracts as well as a preparation of natural hnRNPs as described (14, 15, 20). Briefly, 200 μl of purified hnRNPs (1 mg/ml) were separated on a large preparative 12% polyacrylamide SDS gel and transferred to nitrocellulose membranes. The nitrocellulose was blocked for 1 h in PBS (10 mM sodium phosphate, 140 mM NaCl, pH 7.2) containing 3% dried milk and then incubated for 40 min with mouse sera at an initial dilution of 1:50 in blocking buffer. Sera showing strong reactivity were also tested at higher dilutions (up to 1:800). After washing, bound autoantibodies were detected by incubating for 30 min with alkaline phosphatase-coupled goat anti-mouse IgG (Accurate Chemical and Scientific, Westbury, NY). A human autoimmune serum from a patient with RA containing autoantibodies to hnRNP A2/B1 (and B2) was used as positive control. Mouse sera were also tested for their reactivity with rA2, rB1, and Sm Ags using this procedure. The presence of Abs to SmB/B′ and SmD1 proteins in MRL-lpr/lpr mice sera was alternatively detected by Western immunoblotting using extractable nuclear Ag from rabbit thymus (cat. no. 41009.1; Euromedex, Strasbourg, France). In this case, proteins were fractionated on 12.5% SDS-polyacrylamide gels and electrophoretically transferred to 0.45 μm nitrocellulose. The blotted strips were saturated in TBS containing 0.5% Tween (TBS-T) and 5% dried milk for 1 h at room temperature and then incubated with sera diluted 1:500 in TBS-T-milk for 1 h at room temperature. After washing in TBS-T, the membrane was incubated with goat anti-mouse IgG Abs conjugated to HRP (working dilution, 1:30,000 in TBS-T; cat. no. 115.035.008; Jackson ImmunoResearch, West Grove, PA). Enhanced chemiluminescent reagents (cat. no. RPN 2109; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) were used to reveal positive reactions. This procedure was also used as an alternative method to detect Abs reacting with rB1.

The binding of Abs to hnRNP A2 synthetic peptides was tested by ELISA. Microtiter plates (cat. no. 3912; Falcon, Oxnard, CA) were coated overnight at 37°C with 2 μM of each peptide diluted in 0.05 M carbonate buffer, pH 9.6. In each assay, mouse sera were also tested in an uncoated well incubated with coating buffer as a control. Saturation of plates was obtained by adding PBS containing 0.05% Tween 20 (PBS-T) and 0.5% BSA. The subsequent steps of the test were performed as described previously (25) using mouse sera diluted 1/1000 and goat anti-mouse IgG conjugated to HRP diluted 1/20,000 in PBS-T. All samples were systematically tested in at least two independent assays. Only the IgG Ab response was measured. The cut-off points of the assays as determined from a series of sera from 12 nonimmunized BALB/c mice were 0.1 OD unit (0.15 for peptide 50–70). When these threshold values were used, none of the sera from normal BALB/c mice was found positive. The same procedure was used for the test of Abs reacting with rB1 using 100 ng/ml of protein for coating ELISA plates and the mouse sera at a 1/500 dilution in PBS-T-BSA. The threshold value calculated as above for positive sera was kept at 0.25 OD unit. Subclasses were determined using HRP-conjugated anti-mouse Abs specific for IgG1, IgG2a, IgG2b, and IgG3 (Nordic, Tilburg, The Netherlands; working dilution, 1/5000 in PBS-T). IgG Abs to dsDNA were tested by ELISA (26).

A double Ab sandwich assay was used to measure the total IgG level in mouse sera. Goat anti-mouse IgG (0.1 μg/ml; cat. no. M1397; Sigma, St. Louis, MO) in PBS-T were first incubated overnight at 4°C in wells of microtiter plates. After repeated washings with PBS-T and blocking the remaining sites on the plastic by incubation with PBS-T and 0.5% BSA for 1 h at 37°C, plates were again washed three times and allowed to incubate for 1 h at 37°C with the serum to be tested (diluted 1/700,000 in PBS-T containing 0.4% BSA). After further washings, goat anti-mouse IgG conjugated to HRP (diluted 1/10,000 in PBS-T) was added for 30 min at 37°C. The final detection of bound IgGs was performed as described above. Purified mouse IgG (cat. no. I 5381; Sigma) was used to calibrate the test. For calculation, all OD values >3 were considered 3.0.

The procedures used in this work were essentially as described previously (27, 28). Inguinal, popliteal, and periaortic lymph nodes removed 10 days after immunization of CBA mice were washed in l-alanyl-l-glutamine-enriched RPMI 1640-Glutamax I (Life Technologies, Cergy-Pontoise, France) containing 10% FCS (DAP, Vogelgrun, France), 10 μg/ml gentamicin, 10 mM HEPES, and 5 × 10−5 M 2-ME. Cells were then resuspended at a concentration of 5 × 106 cells/ml in the same culture medium, and 100 μl of this suspension were added to microtiter wells (96-well flat-bottom culture plates; Costar, Cambridge, MA) containing 100 μl of medium with different concentrations of immunizing homologous peptides (0.03–100 μM). Each concentration was tested in triplicate, and tests were repeated at least three times in independent experiments. The cells were cultured at 37°C in 5.5% CO2. After 24 h, 50 μl of supernatant were taken off to test the production of IL-2 using CTL-L cells (27). Standard curves performed with known concentrations of recombinant IL-2 (PharMingen, San Diego, CA) were used for the test calibration. For the detection of IFN-γ, culture supernatants (50 μl) were collected after 24 h and tested in a double-sandwich ELISA using commercial Abs from PharMingen and the conditions recommended by the manufacturer. After 54 h, the cultures were pulsed for 18 h with tritiated thymidine [methyl-3H]thymidine, 2% ethanol, 6.7 Ci/mmol, 1 μCi/well; ICN, Orsay, France). The cells were subsequently harvested on a filter with an automatic cell-harvesting device (Packard, Meriden, CT), and DNA-incorporated radioactivity was measured using a Matrix 9600 direct beta counter (Packard). The results are expressed as the arithmetic mean of thymidine uptake expressed as cpm. The SD of triplicate cultures was always below 20% of the mean. Control tests were performed by adding Con A (100 μl/well; 5 μg/ml; Sigma) to cells during the time (72 h) of the culture.

Sera from MRL+/+, MRL-lpr/lpr, B6/lpr, and (NZB/NZW)F1 female mice as well as from BXSB male mice and control BALB/c and CBA female mice were tested by Western immunoblotting for their reactivity with natural hnRNP A2/B1 employing both nuclear extracts and a preparation of partially purified hnRNPs, as well as with rA2 and rB1. B1 is identical with A2 except for a 12-aa insertion 3 aa from the N terminus. These analyses revealed the presence of anti-A2/B1 autoantibodies in several MRL-lpr/lpr mice. As can be seen in Fig. 1 showing the reactivity of mouse sera with the natural proteins, a clear and pronounced IgG Ab reactivity was observed in these autoimmune sera. All sera reacting with natural A2 also reacted with natural B1 and B2, which is assumed to be another alternatively spliced variant of hnRNP A2/B1 (1, 7). Most of these sera were also weakly reactive with hnRNP A1, presumably due to cross-reactivity between the two closely related hnRNPs as described for human autoimmune sera (15). Thus, anti-A2/B1 Abs were detected in 7 of 25 MRL-lpr/lpr mice aged between 20 and 48 wk from Chapel Hill and 4 of 7 MRL lpr/lpr mice aged between 6 and 42 wk from Strasbourg. These reactivities were also seen when recombinant Ags were blotted (see below). In contrast, anti-A2/B1 Abs were not detectable in the sera collected from MRL+/+ (aged between 10 and 29 wk), B6/lpr, (NZB/NZW)F1 (aged between 12 and 32 wk), and BXSB mice and also not in sera from BALB/c, C57BL/6, and CBA control mice.

FIGURE 1.

Reactivity of sera from MRL lpr/lpr mice with natural hnRNPs A2/B1. Immunoblot reactivity of 20 MRL-lpr/lpr sera probed against partially purified natural hnRNP A2/B1. +, Represents a human anti-A2/B1 (anti-RA33) reference serum. Note that all positive mouse sera (n = 7) stain not only the A2 band but also the B1/B2 double band above. Weak staining of the A1 band (below the A2 band) can also be seen. Mouse sera were diluted 1/50 and IgG Abs only were tested.

FIGURE 1.

Reactivity of sera from MRL lpr/lpr mice with natural hnRNPs A2/B1. Immunoblot reactivity of 20 MRL-lpr/lpr sera probed against partially purified natural hnRNP A2/B1. +, Represents a human anti-A2/B1 (anti-RA33) reference serum. Note that all positive mouse sera (n = 7) stain not only the A2 band but also the B1/B2 double band above. Weak staining of the A1 band (below the A2 band) can also be seen. Mouse sera were diluted 1/50 and IgG Abs only were tested.

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To identify the linear epitopes recognized by Abs from lupus mice, the serum from 19 MRL+/+ mice, 21 MRL-lpr/lpr mice (positive or negative with A2/B1 proteins), 17 NZB/NZW mice, and 10 normal BALB/c mice used as control were then tested for the presence of IgG Abs reacting in ELISA with 14 overlapping (21–27 residue-long) synthetic peptides covering the region 1–206 of the A2 protein known to contain the major antigenic regions (17). The sequence of these peptides is shown in Fig. 2. Surprisingly, virtually all MRL-lpr/lpr mice showed pronounced reactivities that were preferentially directed to 3 of the 14 peptides, namely peptides 35–55 (15/21 mice), 50–70 (20/21 mice), and 87–110 (10/21 mice; Table I). All sera reacting with peptides 35–55 and 87–110 also reacted with peptide 50–70. Positive reactions with other peptides were seldom found (as shown with a few peptides in Table I). No difference was seen between animals with or without signs of arthropathy (data not shown). Only 3 of 19 sera from MRL+/+ mice (including the serum from relatively old mice of 27–29 wk) were positive with peptide 50–70, and no reaction was seen with the other A2 peptides. The sera from female (NZB/NZW)F1 lupus mice and Tax-Tg mice were negative or only very weakly reactive with all peptides as were sera from BALB/c mice (Table I).

FIGURE 2.

Primary structure of mouse hnRNPA2 and sequence of A2 synthetic peptides. Compared with A2, its alternatively spliced variant hnRNP B1 contains a 12-aa residue (KTLETVPLERKK) insertion close to the N terminus (after residue E2 of A2).

FIGURE 2.

Primary structure of mouse hnRNPA2 and sequence of A2 synthetic peptides. Compared with A2, its alternatively spliced variant hnRNP B1 contains a 12-aa residue (KTLETVPLERKK) insertion close to the N terminus (after residue E2 of A2).

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Table I.

Occurrence of IgG Abs reacting with hnRNPA2 peptides in lupus mice

MiceNo. of Sera Positive with hnRNP A2 Peptidesa
StrainsAge (wk)n35-5550-7087-11090-116125-146140-160155-175
MRL-lpr/lpr 12–20 21 15 20 10 
MRL+/+ 10–12 14 
 27–29 
NZB/NZW 12–14 12 
 30–32 
Tg Tax + 10 
BALB/c 12 10 
MiceNo. of Sera Positive with hnRNP A2 Peptidesa
StrainsAge (wk)n35-5550-7087-11090-116125-146140-160155-175
MRL-lpr/lpr 12–20 21 15 20 10 
MRL+/+ 10–12 14 
 27–29 
NZB/NZW 12–14 12 
 30–32 
Tg Tax + 10 
BALB/c 12 10 
a

Cut-off OD value for positivity = 0.10 except for peptide 50-70 (= 0.15); serum dilution, 1/1000.

IgG Abs from MRL-lpr/lpr mice reacting with peptide 50–70 were mostly of the IgG1 and IgG2a subclasses and infrequently of the IgG2b and IgG3 subclasses. The highest level of total IgG during the course of the lupus disease in MRL-lpr/lpr mice varied from 11 to 30 mg/ml. The degree of correlation in the same serum samples between the levels of IgG Abs reacting with peptide 50–70 and those of total IgG was r2 = 0.690 (n = 35 from eight mice). In the same mice, the r2 correlation factor between the levels of IgG Abs reacting with peptide 50–70 and dsDNA was 0.494.

A longitudinal analysis performed with serial bleedings from seven MRL-lpr/lpr mice showed that anti-A2/B1 IgG Abs, as detected by Western immunoblotting with the natural and recombinant A2 and B1 proteins, were present in the sera of these mice between 8 and 26 wk after birth (Fig. 3). In general, anti-A2/B1 Abs were found concomitantly to or preceded by a few weeks IgG Abs reacting with SmB/B′, SmD1, and dsDNA (Table II).

FIGURE 3.

Reactivity with rB1 of sera collected serially from five MRL-lpr/lpr mice. The sera tested were: 1, sera from mouse 1 collected at 6, 10, 15, and 26 wk; 2, sera from mouse 2 at 6, 8, and 12 wk; 3, mouse 3 at 8 and 15 wk; 4, mouse 4 at 6, 10, 18, 24, 28, and 42 wk; and 9, mouse 9 at 6, 12, 17, 22, and 26 wks. Positive control (+), serum obtained from a BALB/c mouse immunized with rB1. Negative control (−), test with the peroxidase-conjugated second Ab and substrate only. Mouse sera were diluted 1/500, and IgG Abs only were tested.

FIGURE 3.

Reactivity with rB1 of sera collected serially from five MRL-lpr/lpr mice. The sera tested were: 1, sera from mouse 1 collected at 6, 10, 15, and 26 wk; 2, sera from mouse 2 at 6, 8, and 12 wk; 3, mouse 3 at 8 and 15 wk; 4, mouse 4 at 6, 10, 18, 24, 28, and 42 wk; and 9, mouse 9 at 6, 12, 17, 22, and 26 wks. Positive control (+), serum obtained from a BALB/c mouse immunized with rB1. Negative control (−), test with the peroxidase-conjugated second Ab and substrate only. Mouse sera were diluted 1/500, and IgG Abs only were tested.

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Table II.

Longitudinal study of IgG Abs from MRL-lpr/lpr mice

MRL-lpr/lprAge (wk)Reactivity in Western Immunoblottinga withReactivity in ELISAb with dsDNA
A2/B1Sm B/B′SmD1
− − − − 
 10 − − − − 
 15 − 
 26 
− − − − 
 Weak − 
 12 
− − − − 
 10 − − 
 15 
− − − − 
 10 − − − 
 18 
 24 ND ND 
 28 − 
 42 
− − − − 
 ND ND − 
 10 − − Weak 
 12 ND ND 
 24 
 32 
− − − − 
 12 − − − − 
 15 ND ND 
 17 
− − − − 
 12 − − − Weak 
 17 − 
 22 
 26 Weak 
MRL-lpr/lprAge (wk)Reactivity in Western Immunoblottinga withReactivity in ELISAb with dsDNA
A2/B1Sm B/B′SmD1
− − − − 
 10 − − − − 
 15 − 
 26 
− − − − 
 Weak − 
 12 
− − − − 
 10 − − 
 15 
− − − − 
 10 − − − 
 18 
 24 ND ND 
 28 − 
 42 
− − − − 
 ND ND − 
 10 − − Weak 
 12 ND ND 
 24 
 32 
− − − − 
 12 − − − − 
 15 ND ND 
 17 
− − − − 
 12 − − − Weak 
 17 − 
 22 
 26 Weak 
a

Serum dilution, 1/500.

b

Serum dilution, 1/1000; cut-off OD value for positivity, 0.20.

The 72 serum samples collected from these seven MRL-lpr/lpr mice were then systematically assayed by ELISA with the two A2 peptides that had shown the highest reactivity in our initial screening (i.e., peptides 35–55 and 50–70). As illustrated in Fig. 4 with four mice as an example, IgG Abs reacting with peptide 50–70 preceded Abs to peptide 35–55 and, remarkably, also Abs to dsDNA. In general, Abs reacting with peptide 50–70 were detected in bleeds collected as early as week 6. The Ab levels reached a maximum at different times in individual mice, around weeks 12–15. In four mice (no. 1, 2, 3, and 7), the Ab levels remained high when the animals died from their disease at weeks 15–28. It is noticeable that in the three other longer-lived mice (no. 4, 5, and 9), we observed first a decline of Ab reactivity around weeks 24–29 and then again an increase just preceding the death of the animals (at weeks 26–42). This fluctuation (drop and increase) was also found with anti-DNA Abs (see mouse 4).

FIGURE 4.

Longitudinal analysis by ELISA of IgG Abs to hnRNP A2 peptides 35–55 and 50–70 and to dsDNA in four MRL-lpr/lpr mice. Sera were diluted 1/1000 and allowed to react with A2 peptides 35–55 (▪) and 50–70 (▾) and with dsDNA (•).

FIGURE 4.

Longitudinal analysis by ELISA of IgG Abs to hnRNP A2 peptides 35–55 and 50–70 and to dsDNA in four MRL-lpr/lpr mice. Sera were diluted 1/1000 and allowed to react with A2 peptides 35–55 (▪) and 50–70 (▾) and with dsDNA (•).

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In addition, we studied by ELISA the reactivity of these 72 sera (collected serially from seven MRL-lpr/lpr mice) with a panel of SmB/B′- and SmD1-derived synthetic peptides. The 12 overlapping SmD1 peptides tested covered the whole sequence of the protein (23), whereas the three SmB/B′ peptides corresponded to sequences known to contain dominant epitopes of the B/B′ proteins (29). Abs reacting with SmB/B′ peptides were seldom observed in the bleedings. However, as early in time as Abs to hnRNP A2 peptide 50–70, high levels of Abs reacting with SmD1 peptides 1–20 and 97–119 (but not with any of the other 10 peptides tested) were found in all MRL-lpr/lpr mice (Fig. 5). The fluctuation “drop and increase” of these Abs was found to parallel the levels of Abs to peptide 50–70 and dsDNA (Figs. 4 and 5). Among the 35 serum samples studied, many contained Abs reacting with both peptide 50–70 of A2 and peptide 1–20 of SmD1 (r2 = 0.564) or peptide 97–119 of SmD1 (r2 = 0.692).

FIGURE 5.

Longitudinal analysis in ELISA of IgG Abs to hnRNPA2 peptide 50–70 and SmD1 peptides 1–20 and 97–119 in four MRL-lpr/lpr mice. Sera were diluted 1/1000 and allowed to react with A2 peptide 50–70 (▾) and with SmD1 peptides 1–20 (□) and 97–119 (▪).

FIGURE 5.

Longitudinal analysis in ELISA of IgG Abs to hnRNPA2 peptide 50–70 and SmD1 peptides 1–20 and 97–119 in four MRL-lpr/lpr mice. Sera were diluted 1/1000 and allowed to react with A2 peptide 50–70 (▾) and with SmD1 peptides 1–20 (□) and 97–119 (▪).

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Because in MRL-lpr/lpr mice the dominant anti-A2/B1 response was directed mainly to the regions 35–55 and 50–70, we sought to determine whether Abs raised in nonautoimmune mice against rB1 recognized the same epitopes. The protein was injected into BALB/c (H-2d), CBA/J (H-2k), and C57BL/6 (H-2b) mice (three mice per group) in the presence of CFA. The panel of A2 peptides described above was used in ELISA to map the B cell epitopes recognized by IgG Abs from immunized mice. As shown in Fig. 6, although all antisera reacted very strongly with rB1, they recognized very few peptides. Abs from BALB/c and CBA/J mice reacted only with peptide 65–85 (2/3 and 1/3 mice, respectively), and Abs from C57BL/6 mice reacted with peptides 17–38, 80–100, and 125–146 (1/3 mice; reactivity observed with the serum from the same mouse). Remarkably, none of the immunized mice was found to react with peptides 35–55 or 50–70, respectively.

FIGURE 6.

Mapping of B cell epitopes of hnRNP A2/B1 in mice immunized with rB1. Sera from BALB/c, CBA/J, and C57BL/6 mice (three mice per group) were allowed to react in ELISA with the 14 overlapping peptides (2 μM) and rB1 (100 ng/ml). Sera were diluted 1/500. The results are shown for the three individual mice of each group (□, ▪, and ▦).

FIGURE 6.

Mapping of B cell epitopes of hnRNP A2/B1 in mice immunized with rB1. Sera from BALB/c, CBA/J, and C57BL/6 mice (three mice per group) were allowed to react in ELISA with the 14 overlapping peptides (2 μM) and rB1 (100 ng/ml). Sera were diluted 1/500. The results are shown for the three individual mice of each group (□, ▪, and ▦).

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We then examined whether any of the 14 overlapping A2 peptides were able to generate an effective Th cell response in nonautoimmune mice. CBA mice, selected for this study because they share the same MHC haplotype as MRL/lpr mice, were primed s.c. with the 14 A2 peptides (two mice per peptide) in CFA. After 10 days, draining lymph nodes were removed and each cell suspension was tested ex vivo with the original priming peptide for its ability to proliferate and produce IL-2 and IFN-γ. Significant proliferative responses with IL-2 and IFN-γ secretion were reproducibly observed with several overlapping peptides (Fig. 7, AC). The strongest proliferative responses were consistently found with the overlapping peptides covering the region 35–175. The peptides encompassing residues 87–160 (namely peptides 87–110, 90–116, 110–130, 125–146, and 140–160) also induced the production of IFN-γ in the cultures primed in vivo with the homologous peptides. No proliferation or IL-2 and IFN-γ secretion was observed when the A2 peptides were added to cultures of control mice injected with CFA alone.

FIGURE 7.

Mapping of T cell epitopes on hnRNP A2/B1. CBA mice were immunized with the 14 overlapping A2 peptides (AC; two mice per peptide) or with rB1 (D–F). Lymph node cells were recalled ex vivo in the presence of the 14 overlapping peptides covering the sequence 1–206 of hnRNP A2. The proliferative response (A and D), and IL-2 (B and E) and IFN-γ secretion (C and F) were measured ex vivo in the presence of various peptide concentrations (only the response with 100 μM peptide is shown). T cell proliferation and IL-2 secretion are expressed as stimulation indices corresponding to cpm in cultures with peptide per cpm in cultures without peptide. A mean stimulation index >2.0 in the proliferation test was considered positive. The average [H3]thymidine incorporation in the absence of peptide was 2000 and 600 cpm in the proliferation and IL-2 secretion assays, respectively. The maximal response of T cells measured in the presence of Con A corresponded to average stimulation index values of 15 (proliferation) and 200 (IL-2 secretion). IFN-γ secretion was measured using a double-sandwich ELISA. Results are expressed as the mean OD value of duplicate measurements at 450 nm. This experiment is representative of three independent experiments with similar results.

FIGURE 7.

Mapping of T cell epitopes on hnRNP A2/B1. CBA mice were immunized with the 14 overlapping A2 peptides (AC; two mice per peptide) or with rB1 (D–F). Lymph node cells were recalled ex vivo in the presence of the 14 overlapping peptides covering the sequence 1–206 of hnRNP A2. The proliferative response (A and D), and IL-2 (B and E) and IFN-γ secretion (C and F) were measured ex vivo in the presence of various peptide concentrations (only the response with 100 μM peptide is shown). T cell proliferation and IL-2 secretion are expressed as stimulation indices corresponding to cpm in cultures with peptide per cpm in cultures without peptide. A mean stimulation index >2.0 in the proliferation test was considered positive. The average [H3]thymidine incorporation in the absence of peptide was 2000 and 600 cpm in the proliferation and IL-2 secretion assays, respectively. The maximal response of T cells measured in the presence of Con A corresponded to average stimulation index values of 15 (proliferation) and 200 (IL-2 secretion). IFN-γ secretion was measured using a double-sandwich ELISA. Results are expressed as the mean OD value of duplicate measurements at 450 nm. This experiment is representative of three independent experiments with similar results.

Close modal

To pursue this issue further, we then tested whether A2 peptides were able to recall the proliferative response in CBA/J mice that had been immunized with rB1. As shown in Fig. 7, DF, none of the A2 peptides was able to induce proliferation or IL-2 and IFN-γ secretion in the cultures containing T lymphocytes from mice primed in vivo with rB1 in the presence of CFA. However, the whole hnRNP B1 was recognized ex vivo by these T cells. Although IL-2 secretion was weak, we observed a significant proliferation and a strong IFN-γ production.

The results described above led us to study the antipeptide Ab response in normal mice and to determine whether these Abs react with rB1. The Ab response to peptides 35–55, 50–70, 87–110, and 170–191 was studied in CBA mice immunized s.c. with these peptides in the presence of CFA (two mice per peptide). Peptides 35–55, 50–70, and 87–110 were selected because they were recognized by Abs from MRL/lpr mice (Table I). These three peptides generated a strong IgG Ab response, in general, after two injections (antisera diluted 1/1000; not shown). The generated antipeptide Abs were very specific, and no cross-reaction was found between these antisera and unrelated A2 peptides or SmD1 peptides. Interestingly, no Ab response was found in CBA mice immunized with peptide 170–191, which was also unable to generate a T cell response in the same mice (Fig. 7) and was not recognized by Abs from MHC-related MRL/lpr mice (Table I). Abs raised against peptides 35–55, 50–70, and 87–110 reacted in ELISA with rB1 (2/2 mice immunized against peptide 35–55 and 1/2 mice immunized against peptides 50–70 and 87–110; Table III). This result suggests that these three short regions are accessible at the surface of rB1 used as Ag in an indirect ELISA test.

Table III.

Reactivity of mouse antipeptide Abs with hnRNP B1 proteina

MiceImmunization with PeptidesReactivity of Antipeptide Abs in ELISA with
Homologous peptidehnRNP B1 protein
 35-55 >3b 0.93 
 35-55 >3 0.48 
 50-70 1.14 0.49 
 50-70 1.84 0.20 
 87-110 >3 1.34 
 87-110 >3 0.23 
170-191 0.01 0.01 
170-191 0.01 0.01 
MiceImmunization with PeptidesReactivity of Antipeptide Abs in ELISA with
Homologous peptidehnRNP B1 protein
 35-55 >3b 0.93 
 35-55 >3 0.48 
 50-70 1.14 0.49 
 50-70 1.84 0.20 
 87-110 >3 1.34 
 87-110 >3 0.23 
170-191 0.01 0.01 
170-191 0.01 0.01 
a

Mouse antisera were diluted 1:500 and allowed to react with homologous peptide and rB1. Only IgG Abs were tested.

b

OD values at 450 mm.

Autoantibodies directed against spliceosomal proteins are a typical feature of SLE. Thus, Abs to Sm and U1 snRNP Ags were described already many years ago to occur in up to 50% of the patients. More recently, Abs to the spliceosome-associated hnRNPs A1 and A2/B1 were found in SLE patients but also in other rheumatic autoimmune disorders, particularly in RA (11, 12, 13, 14, 15, 16). To study the pathological significance of the anti-hnRNP autoimmunity in SLE, we investigated several murine models for the presence of anti-A2/B1 Abs. We show first that among the different lupus mouse models tested, only MRL-lpr/lpr female mice, and not mice with either the MRL or lpr genetic background, produced IgG Abs that reacted with the A2/B1 proteins. We found no reactivity in NZB/NZW mice of 12–32 wk. We further show in this study that the major linear A2 epitopes recognized by the Abs from MRL-lpr/lpr mice were located in residues 35–55 and 50–70. It is no-ticeable that the peptide 50–70 contains the highly conserved sequence 50RGFGFVTF57 (the RNP1 motif) that is also present in peptide 140–160 (residues 141–148) located in RBD II. However, this peptide was seldom recognized by the Abs from MRL-lpr/lpr mice. Most interestingly, IgG Abs reacting with peptide 50–70 were detected before IgG Abs to dsDNA, which are typical marker Abs of pathological significance in SLE. Abs reacting with peptide 35–55 were detectable several weeks after those reacting with peptide 50–70, suggesting an intramolecular B cell epitope spreading process during the course of the disease, as described in patients and mice with other nuclear RNPs such as U1A and SmB/B′ proteins (30, 31). Preliminary data obtained with the serum from patients with SLE, RA, and other rheumatic diseases indicate that peptide 35–55 is also recognized, though at a relatively low frequency, by lupus Abs.

In most cases, Abs to peptide 50–70 were detected significantly before Abs reacting by Western blotting with the whole (natural or recombinant) A2/B1 protein (compare the results in Fig. 4 and Table II). In fact, while virtually all sera showed moderate to very strong reactivities with peptide 50–70 and/or 35–55, only about one-third was also reactive with the full-length protein. This type of reactivity in certain autoimmune sera (positivity with a segment of the protein but not with the cognate protein itself) is not unique and was in this work also observed with SmD1 peptides (compare the results in Fig. 5 and Table II). It has been reported with different self-proteins specifically recognized by Abs from patients and mice with systemic autoimmune diseases (32, 33, 34, 35, 36, 37, 38, 39). We obviously cannot exclude that this observation may be simply explained by a difference in the inherent sensitivity of the respective autoantibody tests (this is also true when we compare the kinetics of appearance of different Ab subsets). However, this is unlikely because all of the assays were optimized for maximum sensitivity/specificity. Thus, as discussed recently (40), this reactivity is more likely to reflect the fact that certain reactions are better visualized when peptides bearing a major epitope, rather than whole purified or recombinant proteins, are assayed under different test conditions. One has to bear in mind that most Ags (including Ags in apoptotic bodies) are complexed in vivo with other proteins and/or nucleic acids, which may lead to exposure of epitopes that might not be readily accessible when the isolated (pure) protein is used in ELISA or immunoblotting. An alternative hypothesis would be that Abs in certain autoimmune diseases show stronger reactivity with denatured, rather than native, proteins. It is indeed possible that in these diseases non-native proteins may have pathogenic significance either in the initiation or propagation of the autoimmune response. It has to be noted that this type of reactivity was not observed when mice of different MHC haplotypes were immunized with rA2/B1. In the latter case, the IgG Ab response was strikingly restricted to a very small number of peptides and the epitopes characterized after immunization with rA2/B1 differed totally from those identified in autoimmune mice. Thus, among the four peptides recognized by anti-A2/B1 Abs from H-2d, H-2k, and H-2b mice, only peptide 125–146 sometimes showed positive reaction with Abs from H-2k MRL-lpr/lpr mice, and we found no clear MHC associations. This result may reflect the tolerance status of normal mice. It may also indicate that the antigenic stimulus giving rise to these autoantibodies is not the free native protein.

We also examined whether any of the 14 overlapping peptides of the A2 protein (spanning the region 1–206) were able to generate an effective Th cell response in nonautoimmune mice sharing the same MHC haplotype H-2k as MRL/lpr mice. Within the region 50–175, we identified a series of T cell epitopes present in peptides that recalled ex vivo lymph node cells generated in vivo with the homologous peptides. However, we found that none of these peptides was able to stimulate T cells from CBA mice immunized against rA2/B1, suggesting that they may correspond to cryptic epitopes.

In summary, a major epitope of hnRNP A2/B1 recognized very early during the course of the lupus disease by Abs from most of MRL-lpr/lpr mice has been identified in residues 50–70. This sequence was very specifically recognized by IgG Abs from MRL-lpr/lpr but not from other lupus mice and not by Abs from mice of different MHC haplotypes immunized against rB1. Interestingly, it is not present in the region known to contain the major (probably discontinuous) epitopes recognized by Abs from patients with SLE and RA. Because in our test conditions Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that the region 50–70 contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response. Abs to other peptides in SmD1 (i.e., peptides 1–20 and 97–119) were also detectable at the same early stage. Because snRNP Ags and hnRNPs-A1 and A2/B1 are in close neighborhood in the presplicing complex (at the 5′ splice site) (41, 42), this spliceosomal subcomplex may thus form one of the first targets of the autoimmune response in lupus. Thus, we might hypothesize that in the MRL-lpr/lpr model lupus autoimmunity starts with such antipeptide reactivities, which then spread to other epitopes and later on also to other Ags, including dsDNA, as has been observed in rabbits immunized with SmB-derived peptides (31). Other nuclear complexes, such as the nucleosome, may also play a key role (43), although additional work is required to know whether antihistone peptide Ab subsets occur before antinucleosome and anti-DNA Abs in MRL-lpr/lpr mice. This was not found in lupus mice with graft-vs-host disease (26). Although the mechanisms leading to loss of tolerance against such ribonucleoprotein complexes are still obscure, the occurrence of a broad autoimmune response toward the spliceosome in this particular model of SLE may allow investigations of possible pathways, such as aberrant expression and/or modifications of the primary target Ag(s) both in normal and apoptotic cells. It will be interesting in this context to study the T cell response to A2/B1 and SmD1 proteins in MRL-lpr/lpr mice, and see whether the same or distinct regions play a triggering role in the induction of a specific CD4+ T cell response. The comparison of the T cell repertoire of nonlupus-prone and MRL/lpr mice could provide further insights into the mechanisms involved in the production of anti-hnRNP and also anti-snRNP Abs and may lead to a better understanding of the pathogenesis SLE.

We thank Faïza Rharbaoui (Strasbourg) for her participation in the project and Elisabeth Hoefler (Vienna) for expert technical assistance with autoantibody determination.

1

This work was supported by a grant from the Association de Recherche sur la Polyarthrite (to S.M.) and by Grant 7021 from the Jubiläumsfonds der Österreichischen Nationalbank (to G.S.). Part of the work was performed in the context of the Interdisciplinary Cooperative Project of the Medical Faculty of the University of Vienna supported by the Austrian Ministry of Science. H.D. was a recipient from a predoctoral grant from the Fondation pour la Recherche Médicale.

4

Abbreviations used in this paper: hnRNP A2/B1, heterogeneous nuclear ribonucleoproteins A2 and B1; MCTD, mixed-connective tissue disease; RA, rheumatoid arthritis; RBD, RNA-binding domain; SLE, systemic lupus erythematosus; snRNP, small nuclear ribonucleoprotein; Tg, transgenic; PBS-T, PBS containing 0.05% Tween 20.

1
Steiner, G., K. Skriner, J. S. Smolen.
1996
. Autoantibodies to the A/B proteins of the heterogeneous nuclear ribonucleoprotein complex: novel tools for the diagnosis of rheumatic diseases.
Int. Arch. Allergy Immunol.
111
:
314
2
Nakielny, S., G. Dreyfuss.
1997
. Nuclear export of proteins and RNAs.
Curr. Opin. Cell Biol.
9
:
420
3
Carson, J. H., S. Kwon, E. Barbarese.
1998
. RNA trafficking in myelinating cells.
Curr. Opin. Neurobiol.
8
:
607
4
Biamonti, G., C. Ghigna, R. Caporali, C. Montecucco.
1998
. Heterogeneous nuclear ribonucleoproteins (hnRNPs): an emerging family of autoantigens in rheumatic diseases.
Clin. Exp. Rheumatol.
16
:
317
5
Burd, C. G., G. Dreyfuss.
1994
. Conserved structures and diversity of functions of RNA-binding proteins.
Science
265
:
615
6
Xu, R. M., L. Jokhan, X. Cheng, A. Mayeda, A. R. Krainer.
1997
. Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs.
Structure
5
:
559
7
Burd, C. G., M. S. Swanson, M. Görlach, G. Dreyfuss.
1989
. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts.
Proc. Natl. Acad. Sci. USA
86
:
9788
8
Kamma, H., H. Horiguchi, L. Wan, M. Matsui, M. Fujiwara, M. Fujimoto, T. Yazawa, G. Dreyfuss.
1999
. Molecular characterization of the hnRNP A2/B1 proteins: tissue-specific expression and novel isoforms.
Exp. Cell Res.
246
:
399
9
Fritzler, M. J., R. Ali, E. M. Tan.
1984
. Antibodies from patients with mixed-connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid of the nuclear matrix.
J. Immunol.
132
:
1216
10
Zouali, M., A. Eyquem.
1984
. Antibodies to heterogeneous nuclear ribonucleoproteins in sera from patients with rheumatic autoimmune diseases.
J. Clin. Immunol.
4
:
209
11
Dangli, A., A. Guialis, E. Vretou, C. E. Sekeris.
1988
. Autoantibodies to the core proteins of hnRNPs.
FEBS Lett.
231
:
118
12
Jensen, L. A., E. L. Kuff, S. H. Wilson, A. D. Steiner, D. M. Klinman.
1988
. Antibodies from patients and mice with autoimmune diseases react with recombinant hnRNP core protein A1.
J. Autoimmun.
1
:
73
13
Astaldi Ricotti, G. C. B., M. Bestagno, A. Cerino, C. Negri, R. Caporali, F. Cobianchi, M. Longhi, C. M. Montecucco.
1989
. Antibodies to hnRNP core protein A1 in connective tissue diseases.
J. Cell. Biochem.
40
:
43
14
Steiner, G., K. Hartmuth, K. Skriner, I. Maurer-Fogy, A. Sinski, E. Thalmann, W. Hassfeld, A. Barta, J. S. Smolen.
1992
. Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.
J. Clin. Invest.
90
:
1061
15
Hassfeld, W., G. Steiner, A. Studnicka-Benke, K. Skriner, W. Graninger, I. Fischer, J. S. Smolen.
1995
. Autoimmune response to the spliceosome: an immunological link between rheumatoid arthritis, mixed connective tissue disease and systemic lupus erythematosus.
Arthritis Rheum.
38
:
777
16
Richter Cohen, M., G. Steiner, J. S. Smolen, D. A. Isenberg.
1998
. Erosive arthritis in systemic lupus erythematosus: analysis of a distinct clinical and serological subset.
Br. J. Rheumatol.
37
:
421
17
Skriner, K., G. Steiner, W. H. Sommergruber, A. Sinski, J. S. Smolen.
1994
. Anti-RA33 autoantibodies may recognize epitopes in the N-terminal region of hnRNP-A2 (RA33).
Clin. Exp. Rheumatol.
12
: (Suppl. 11):
S79
18
Skriner, K., W. H. Sommergruber, V. Tremmel, I. Fischer, A. Barta, J. S. Smolen, G. Steiner.
1997
. Anti-A2/RA33 autoantibodies are directed to the RNA binding region of the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.
J. Clin. Invest.
100
:
127
19
Montecucco, C., R. Caporali, C. Negri, F. de Gennaro, A. Cerino, M. Bestagno, F. Cobianchi, G. C. B. Astaldi-Ricotti.
1990
. Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein.
Arthritis Rheum.
33
:
180
20
Hassfeld, W., G. Steiner, K. Hartmuth, G. Kolarz, O. Scherak, W. Graninger, N. Thumb, J. S. Smolen.
1989
. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis.
Arthritis Rheum.
32
:
1515
21
Neimark, J., J.-P. Briand.
1993
. Development of a fully automated multichannel peptide synthesizer with integrated TFA cleavable capability.
Pept. Res.
6
:
219
22
Hoet, R. M., J. Raats, R. De Wildt, H. Dumortier, S. Muller, F. Van Den Hoogen, W. J. van Venrooij.
1998
. Human monoclonal autoantibody fragments from combinatorial antibody libraries directed to the U1 snRNP associated U1C protein; epitope mapping, immunolocalization and V-gene usage.
Mol. Immunol.
35
:
1045
23
Dumortier, H., M. Abbal, M. Fort, J.-P. Briand, A. Cantagrel, S. Muller.
1999
. MHC class II gene association with antibodies to U1A and SmD1 proteins.
Int. Immunol.
11
:
249
24
Saggioro, D., A. Rosato, G. Esposito, M. P. Rosenberg, J. Harrison, B. K. Felber, G. N. Pavlakis, L. Chieco-Bianchi.
1997
. Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.
J. Acquir. Immune Defic. Syndr. Hum. Retrovirol.
14
:
272
25
Benkirane, N., M. Friede, G. Guichard, J.-P. Briand, M. H. V. Van Regenmortel, S. Muller.
1993
. Antigenicity and immunogenicity of modified synthetic peptides containing d-amino acid residues.
J. Biol. Chem.
268
:
26279
26
Mézière, C., F. Stöckl, S. Batsford, A. Vogt, S. Muller.
1994
. Antibodies to DNA, chromatin core particles and histones in mice with graft-versus-host disease and their involvement in glomerular injury.
Clin. Exp. Immunol.
98
:
287
27
Mézière, C., M. Viguier, H. Dumortier, R. Lo-Man, C. Leclerc, J.-G. Guillet, J.-P. Briand, S. Muller.
1997
. In vivo T helper cell response to retro-inverso peptidomimetics.
J. Immunol.
159
:
3230
28
Decker, P., A. Le Moal, J.-P. Briand, S. Muller.
2000
. Identification of a minimal T cell epitope recognized by anti-nucleosome Th cells in the C-terminal region of histone H4.
J. Immunol.
165
:
654
29
Hoch, S. O..
1994
. The Sm antigens. W. J. van Venrooij, and R. N. Maini, eds.
Manual of Biological Markers of Disease
Kluwer Academic Publishers, Dordrecht. p. B2.41.
30
Fatenejad, S., M. J. Mamula, J. Craft.
1993
. Role of intramolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles.
Proc. Natl. Acad. Sci. USA
90
:
12010
31
James, J. A., T. Gross, R. H. Scofield, J. B. Harley.
1995
. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: SmB/B′-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity.
J. Exp. Med.
181
:
453
32
Barakat, S., J.-P. Briand, J.-C. Weber, M. H. V. Van Regenmortel, S. Muller.
1990
. Recognition of synthetic peptides of SmD autoantigen by lupus sera.
Clin. Exp. Immunol.
81
:
256
33
Hines, J. J., W. Danho, K. B. Elkon.
1991
. Detection and quantification of human anti-Sm antibodies using synthetic peptide and recombinant SmB antigens.
Arthritis Rheum.
34
:
572
34
Sabbatini, A., M. P. Dolcher, B. Marchini, S. Bombardieri, P. Migliorini.
1993
. Mapping of epitopes on the SmD molecule: the use of multiple antigen peptides to measure autoantibodies in systemic lupus erythematosus.
J. Rheumatol.
20
:
1679
35
Ricchiuti, V., D. A. Isenberg, S. Muller.
1994
. HLA association of anti-Ro60 and anti-Ro52 antibodies in Sjögren’s syndrome.
J. Autoimmun.
7
:
611
36
Stemmer, C., J.-P. Briand, S. Muller.
1994
. Mapping of linear epitopes of human histone H1 recognized by rabbit anti-H1/H5 antisera and antibodies from autoimmune patients.
Mol. Immunol.
31
:
1037
37
Petrovas, C. J., P. G. Vlachoyiannopoulos, A. G. Tzioufas, C. Alexopoulos, V. Tsikaris, M. Sakarellos-Daitsiotis, C. Sakarellos, H. M. Moutsopoulos.
1998
. A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.
J. Immunol. Methods
220
:
59
38
Riemekasten, G., J. Marell, G. Trebeljahr, R. Klein, G. Hausdorf, T. Häupl, J. Schneider-Mergener, G. R. Burmester, F. Hiepe.
1998
. A novel epitope on the C-terminus of SmD1 is recognized by the majority of sera from patients with systemic lupus erythematosus.
J. Clin. Invest.
102
:
754
39
Decker, P., J.-P. Briand, G. de Murcia, R. W. Pero, D. A. Isenberg, S. Muller.
1998
. Zinc is an essential co-factor for recognition of the DNA-binding domain of poly(ADP-ribose) polymerase by antibodies in autoimmune rheumatic and bowel diseases.
Arthritis Rheum.
41
:
918
40
Muller, S..
1999
. Peptides in diagnosis of autoimmune diseases. S. Pillai, and P. C. Van Der Vliet, eds. In
Synthetic Peptides as Antigens: Laboratory Techniques in Biochemistry and Molecular Biology
Vol. 28
:
247
-280. Elsevier, Amsterdam. Chapter 7.
41
Buvoli, M., F. Cobianchi, S. Riva.
1992
. Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly.
Nucleic Acids Res.
20
:
5017
42
Mayeda, A., S. H. Munroe, J. F. Caceres, A. R. Krainer.
1994
. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins.
EMBO J.
13
:
5483
43
Amoura, Z., J.-C. Piette, J.-F. Bach, S. Koutouzov.
1999
. The key role of nucleosomes in lupus.
Arthritis Rheum.
42
:
833