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,§,§
*
Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche, Centre National de la Recherche Scientifique, Strasbourg, France;
Division of Rheumatology, Department of Internal Medicine III, University of Vienna, Vienna, Austria;
Division of Rheumatology and Immunology, University of North Carolina, Chapel Hill, NC 27599; and
§
Ludwig-Boltzmann-Institute for Rheumatology, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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).
Mice and sera
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.
Ab analysis by Western immunoblotting
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.
Ab analysis by ELISA
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.
Lymphocyte proliferation assays and measurement of cytokine secretion
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 x 10-5 M 2-ME. Cells were then
resuspended at a concentration of 5 x 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-, 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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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).
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Longitudinal pattern in MRL-lpr/lpr mice of anti-hnRNP and snRNP and peptide responses
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).
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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).
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). 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).
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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.
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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-. Significant proliferative responses with IL-2 and
IFN- secretion were reproducibly observed with several overlapping
peptides (Fig. 7, A–C). 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- in the cultures primed in vivo with the
homologous peptides. No proliferation or IL-2 and IFN- secretion was
observed when the A2 peptides were added to cultures of control mice
injected with CFA alone.
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, D–F,
none of the A2 peptides was able to induce proliferation or IL-2 and
IFN- 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- production. Analysis of the antipeptide Ab response in normal mice
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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
3 Address correspondence and reprint requests to Dr. Sylviane Muller, Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9021 Centre National de la Recherche Scientifique, 15 rue R. Descartes, 67000 Strasbourg, France.
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.
Received for publication February 24, 2000. Accepted for publication June 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C.-H. Suh, J. H. Freed, and P. L. Cohen T Cell Reactivity to MHC Class II-Bound Self Peptides in Systemic Lupus Erythematosus-Prone MRL/lpr Mice J. Immunol., February 15, 2003; 170(4): 2229 - 2235. [Abstract] [Full Text] [PDF]
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R. Fritsch, D. Eselbock, K. Skriner, B. Jahn-Schmid, C. Scheinecker, B. Bohle, M. Tohidast-Akrad, S. Hayer, J. Neumuller, S. Pinol-Roma, et al. Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis J. Immunol., July 15, 2002; 169(2): 1068 - 1076. [Abstract] [Full Text] [PDF]
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 F. Monneaux, H. Dumortier, G. Steiner, J.-P. Briand, and S. Muller Murine models of systemic lupus erythematosus: B and T cell responses to spliceosomal ribonucleoproteins in MRL/Faslpr and (NZB NZW)F1 lupus mice Int. Immunol., September 1, 2001; 13(9): 1155 - 1163. [Abstract] [Full Text] [PDF]
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)
and 50–70 (
) and with dsDNA (•).
) and 97–119
(
).