MHC class II gene associations with autoantibodies to U1A and SmD1 proteins

Hélène Dumortier, Michel Abbal1, Marylise Fort1, Jean-Paul Briand, Alain Cantagrel2 and Sylviane Muller

Institut de Biologie Moléculaire et Cellulaire, CNRS UPR 9021, Strasbourg, France
1 Service de Rhumatologie and
2 Laboratoire d'Immunologie, CHU Rangueil, Toulouse, France

Correspondence to: S. Muller, UPR 9021 CNRS, Institut de Biologie Moléculaire et Cellulaire, 15, rue Descartes, 67084 Strasbourg, France


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Autoantibodies against U small nuclear ribonucleoproteins (snRNP) are frequently present in the serum of patients with systemic rheumatic diseases, and have been reported to be associated with HLA-DR and -DQ genes. To better define the role of HLA genes in the production of such antibodies, we studied immunogenetic associations with autoantibodies reacting with U1 RNP, U1A and SmD1 proteins, and synthetic peptides containing immunodominant linear epitopes of these proteins. Only two out of the 15 overlapping peptides of U1A (i.e. peptides 35–58 and 257–282) and three of 11 peptides of SmD1 (i.e. peptides 1–20, 44–67 and 97–119) were significantly recognized by patients' sera selected on the basis of their antibody positivity with RNP in immunodiffusion. The distribution of DRB1, DQB1 and DPB1 alleles among the anti-RNP antibody-positive patients (n = 28) and healthy control subjects was similar. Antibodies against U1A (tested in Western immunoblotting with HeLa cell extracts) were positively associated to DRB1*06 allele; antibodies reacting with SmD1 peptide 44–67 were negatively associated to DRB1*02 and DQB1*0602 alleles. No association was found between DPB1 alleles and antibodies reacting with U1A and SmD1 antigens. This first study reporting an association between autoantibodies reacting with U1A and SmD1 proteins (and peptides of these proteins), and immunogenetic markers suggest that the production of antibody subsets directed against different components (or regions of these proteins) bound to the same snRNP particle is associated with distinct MHC class II alleles.

Keywords: autoantibody, MHC class II, ribonucleoprotein, SmD1, U1A


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients with rheumatic autoimmune diseases such as systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) often produce IgG antibodies against components of the U1 spliceosomal small nuclear ribonucleoprotein (snRNP) particle. The U1 snRNP contains one U1 RNA molecule complexed with eight so-called Sm or core proteins (B, B', D1, D2, D3, E, F, G) and three U1-specific proteins (70K, A and C). U1 RNP antibodies are found in 100% of patients with MCTD and 40% of patients with SLE. They usually recognize the U1-specific proteins 70K, U1A and U1C (1). Sm antibodies are markers for SLE. They are found in 5% of non-selected European lupus patients and 30% North American lupus patients (2,3). Sm antibodies are directed to the B/B', D1, D2, D3, E, F and G core proteins that are present in most U-RNP particles (notably U1, U2, U4, U5 and U6).

Several B cell epitopes on U1A and SmD1 proteins have been identified (4,5). In previous studies (68), we reported that peptides 1–20, 44–67 and 97–119 of SmD1, and 1–11, 35–58 and 257–282 of U1A mimicked major linear epitopes of these proteins. The region 201–241 as well as the C-terminal sequence 242–282 of U1A were subsequently reported to contain major T epitopes recognized by T cells from MCTD patients (9).

The molecular mechanism underlying the development of SLE and MCTD and the production of snRNP antibodies is unknown, but genetic factors are probably implicated in the pathogenesis. Since the HLA class II molecules present processed antigen to the TCR and result in an antigen-specific immune response, studies clarifying the possible associations between HLA class II antigens and particular antibodies may help to understand and control the autoimmune response in these diseases. Sm/RNP antibodies (characterized by immunodiffusion, Western immunoblotting or counter-current immunoelectrophoresis) have been associated with HLA-DR4 (10,11) or HLA-DR7 (2) in Caucasian and black patients with SLE. Analyzing black and white patients with either anti-Sm or RNP precipitin autoantibodies, Olsen et al. (12) have shown that there are distinct patterns of major HLA class II allele associations according to the racial origin of patients and the specificity (anti-Sm or anti-RNP) of antibodies. They found that HLA-DQ associations may be more primary than HLA-DR associations.

In the present study, Caucasian subjects were selected on the basis of the presence of RNP precipitin antibodies in their serum. The reactivity of patients' sera was tested by Western immunoblotting, and, in ELISA, with synthetic peptides of U1A and SmD1 proteins. Potential HLA associations with antibody subsets reacting with RNP (in immunodiffusion), U1A and SmD1 proteins (in immunoblotting experiments), and U1A and SmD1 peptides (in ELISA) were examined. The sequence-specific oligonucleotide (SSO) typing method was utilized to analyze HLA-DR, -DQ and -DP genes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Synthetic peptides
Eleven peptides (16–24 residues long) covering the entire sequence of the SmD1 protein described by Rokeach et al. (13) and 15 peptides (12–32 residues long) covering the sequence of the U1A protein described by Sillekens et al. (14) were tested in this study (Fig. 1Go). The synthesis and purification of some of these peptides were described previously (6,7). Additional peptides were synthesized using Fmoc chemistry as described (15). Homogeneity of all peptides was assessed by analytical HPLC on a 5 µ Nucleosil column (4.6x150 mm), using a triethylammonium phosphate buffer system. Peptides were purified using a medium pressure chromatography apparatus and their final purity was at least 60%. Verification of peptide identity was performed by matrix assisted laser desorption/ionization mass spectrometry using a Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany). An additional cysteine residue was added at the C-terminus of peptide 1–11 of U1A to allow its selective conjugation to BSA. For coupling, m-maleimido benzoyl-N-hydroxysuccinimide ester was used (16).



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Fig. 1. SmD1 and U1A synthetic fragments.

 
Human sera
A panel of 28 anti-snRNP positive sera was selected based on results of the Ouchterlony test. The main clinical and biological characteristics of 28 patients have been described in detail elsewhere (17). These patients were followed in the hospitals of Toulouse (south-west France), they were Caucasian, and had MCTD, SLE, rheumatoid arthritis (RA), Sjögren's syndrome (SS) and scleroderma (Scl) (Table 1Go). As control, 25 sera from healthy volunteers were used.


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Table 1. Clinical and serologic characteristics of 28 Caucasian patients with anti-snRNP antibodies
 
ELISA
The ELISA test of human sera was essentially as described previously (7). The sera diluted in PBS containing 0.05% Tween and 1% BSA (PBS-T-BSA) were directly incubated in peptide-coated wells. In general 2 µM peptide diluted in 0.05 M carbonate buffer, pH 9.6, was incubated in microtiter plates. The only exceptions were for peptides 1–20 SmD1 (0.25 µM), 35–58 U1A (0.5 µM) and 257–282 U1A (0.2 µM). The cut-off point of the various assays was defined by using 25 normal human sera (NHS) that were tested with the 26 peptides. From the binding OD values obtained with these sera, the threshold value for positive sera was kept at 0.3 OD units (mean OD value + 2 SD). None of the NHS was found positive with any of the antigens tested when this threshold OD value was applied. All samples were tested systematically in at least two independent assays. For calculation, all OD values >3 were considered as 3.0. Average values corresponded to the arithmetical mean of both positive and negative OD values in the series. The correlation between antibody reactivity and clinical symptoms or other biological parameters was determined by {chi}2 analysis, with Yates' correction when necessary.

Western immunoblotting
Western blotting analysis of patients' sera was performed using the ANA-bioblot kit from Biocode (Sclessin, Belgium). In this kit, nuclear antigens from human HeLa cells are electroblotted onto strips. A reference strip provided with each kit allows interpretation of results. Molecular weight markers appear in red and blue bands correspond to positive reactions. The test was performed following the instructions recommended by the supplier.

PCR-based DNA typing of HLA-DR, -DQ and -DP class II genes
For HLA class II genotyping, genomic DNA was extracted from peripheral blood cells using the salting out technique (18). The DRB1*, DQB1* and DPB1* genes were amplified by PCR with primers specific for framework sequences flanking the polymorphic region of exon 2. The reagents and procedures were those recommended by the XIIth Workshop of Histocompatibility (19). Amplified DNA was subsequently blotted onto nylon membrane (Hybond; Amersham, Amersham, UK) and hybridized to labeled SSO probes. After washing the membranes in stringent conditions, the chemiluminescent signal was detected. The set of probes used for DQB1* loci polymorphism study did not allow us to discriminate between DQB1*0201 and *0202 alleles. These alleles are therefore referred below as /201//202.

The normal control group used for DRB1* and DQB1* loci studies consisted of respectively 384 and 110 unrelated volunteer bone marrow donors from south-west France. Because HLA DPB1* typing was not available for these control groups, we built a specific group of 28 individuals matching for DRB1* and DQB1* alleles with the 28 RNP antibody-positive patients.

Statistical analyses
Statistical analysis of HLA alleles distribution between groups and subgroups of patients and controls was performed using the {chi}2 test without or with Yates' correction when necessary. Bonferroni's correction was also used when it was appropriate (20).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reactivity of patients' sera with U1A and SmD1 in Western blotting and with U1A and SmD1 peptides in ELISA
Sera from 28 patients reacting positively with snRNP in the Ouchterlony test (17) were systematically tested in Western blotting with HeLa cell nuclear extracts and in ELISA with 26 overlapping peptides (Fig. 1Go) covering the entire sequence of the U1A and SmD1 proteins. As shown in Table 1Go, 16 of 28 patients' sera (57.1%) reacted in Western blotting with the 70K protein, 12 (42.9%) with A, 17 (60.7%) with B/B', 10 (35.7%) with C and eight (28.6%) with the D1 protein. The reactivity of some individual sera is illustrated in Fig. 2Go. Among the 28 sera, 13 contained antibodies reacting with at least two proteins of the U1 RNP antigen (70K, A or C), and 19 contained antibodies reacting with the Sm proteins B/B' and D1. Five sera reacted with 70K, A and/or C proteins but not with B/B' and/or D1. Four sera reacted with B/B' and/or D1 proteins but not with the 70K, A and C proteins. As cross-reactive epitopes on the A and D1 unfolded proteins have been previously reported (21,22), we have also examined the prevalence of anti-A and -D1 protein autoantibodies in our serum samples. Four sera reacted with A and D1 proteins, and 12 reacted with A or D1 but not with both proteins (Table 1Go).



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Fig. 2. Immunoblotting of components present in HeLa nuclear extracts (Biocode) with six snRNP+ patients' sera. Sera were diluted 1:100. On the left, the position of positive reactions obtained with a reference serum is provided by Biocode for each kit. The position of mol. wt markers appears in red. Positive antibody reactions appear in blue.

 
In ELISA, only two out of the 15 overlapping peptides of U1A (i.e. peptides 35–58 and 257–282) and three of 11 peptides of SmD1 (i.e. peptides 1–20, 44–67 and 97–119) were significantly recognized by RNP+ sera. Thus, although six new peptides were included in the present study, compared to our previous analyses (6,7), and that subjects of the present study were selected using very different criteria, we could not identify additional linear antigenic regions recognized by patients' antibodies. The peptides most frequently recognized by RNP+ sera encompassed residues 44–67 of SmD1 (11 of 28; 39.3%; mean OD value 0.33; SD 0.27), 1–20 of SmD1 (eight of 28; 28.6%; mean OD value 0.24; SD 0.20), 257–282 of U1A (six of 28; 21.4%; mean OD value 0.21; SD 0.10) and 97–119 of SmD1 (six of 28; 21.4%; mean OD value 0.18; SD 0.14). Fourteen percent of sera reacted with peptide 35–58 of U1A; the other peptides were recognized by only one or none of the sera. All sera positive with peptide 97–119 of SmD1 also reacted with peptide 1–20 SmD1 and all sera reacting with peptide 1–20 SmD1 reacted with peptide 44–67 of SmD1 (Table 1Go). Out of the 28 RNP+ sera, four contained antibodies reacting with SmD1 in Western blotting and with peptide 44–67 of SmD1 in ELISA. Four sera were positive with SmD1 but not with peptide 44–67. Seven sera reacted with peptide 44–67 but not with SmD1 in Western blotting. Among the 12 sera reacting with U1A, four reacted with peptide 257–282 U1A, whereas among the six sera positive in ELISA with the peptide 257–282, two did not react with U1A in Western blotting.

Among the patients studied, 11 had an hypergammaglobulinemia >=18 mg IgG/ml. We found no significant correlation between the level of total IgG in serum of these patients and the presence or level of specific antibodies reacting with U1A and SmD1 proteins, and U1A and SmD1 peptides. Likewise, although antibodies reacting with SmD1 and with SmD1 peptide 44–67 were more frequently found in certain forms of arthritis (0.05 <= corrected P < 0.1), no statistically significant differences concerning clinical data could be detected in the groups of 28 patients with or without antibodies to U1A and SmD1 proteins, and peptides 257–282 of U1A and 44–67 of SmD1.

Genotyping of HLA-DRB1, -DQB1 and -DPB1 alleles
We determined the genotypes of HLA class II alleles by the PCR-SSO method and listed their allelic frequencies among the various groups of patients (with or without antibodies reacting with U1A and SmD1 proteins, U1A peptide 257–282 and SmD1 peptide 44–67) and control subjects. As shown in Fig. 3Go, the distribution of HLA-DRB1, -DQB1 and -DPB1 alleles among the 28 anti-RNP autoantibody-positive patients and healthy control subjects was similar except for DPB1*0301 (Fig. 3CGo). However, as is usual when an association is reported for the first time, we applied the Bonferroni's correction to these values and then found that the differences in frequencies became not significant (P < 0.16, for 16 alleles).




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Fig. 3. HLA-DRB1, -DQB1 and -DPB1 alleles in snRNP+ patients and control healthy subjects. *Statistically significant with corrected P < 0.05.

 
With respect to U1A antigens (Table 2Go), the DRB1*06 allele was significantly more frequent (corrected P < 0.05) in anti-RNP+ patients with antibodies reacting with U1A protein in Western blotting than in anti-RNP+ patients without antibodies reacting with U1A protein. No statistically significant differences in the frequencies of any other subtypes of HLA class II antigens (including DPB1 alleles, not shown) were observed between these two groups of patients, as well as between the groups of patients with or without IgG antibodies reacting in ELISA with U1A peptide 257–282 (Table 2Go).


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Table 2. HLA-DR and -DQB1 specificities in RNP+ patients with and without U1A antibodies
 
With respect to SmD1 antigens (Table 3Go), no positive or negative associations were observed between the presence of antibodies reacting with SmD1 in Western blot and any of the HLA-DRB1, -DQB1 and -DPB1 alleles. On the other hand, anti-44–67 SmD1 antibodies were negatively associated with DRB1*02 and DQB1*0602 alleles (corrected P < 0.05). No association was found with DPB1 alleles (not shown).


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Table 3. HLA-DR and -DQB1 specificities in RNP+ patients with and without SmD1 antibodies
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Assays based on the use of synthetic peptides have proved useful not only for the detection, but also in some cases for the discrimination of antibodies present in patients with related autoimmune diseases. This observation supports the view that different processes may govern the emergence of different antibody subsets cross-reacting with the same protein. We have previously shown (23) that in patients with SS, antibodies reacting with the whole 52 kDa Ro/SSA (Ro52) protein and with particular immunodominant domains of this protein were independently associated with distinct HLA haplotypes. A similar conclusion was also reached by Scofield et al. who analyzed antibodies reacting with the Ro particle (24). The aim of our study was to determine whether this finding could be extended to other autoantigens and thus to better understand the fine regulation of antibody production in autoimmune patients. We studied a series of patients satisfying well-established diagnostic criteria (17). The initial inclusion criteria were the presence in the serum of these patients of anti-RNP antibodies determined by immunodiffusion, the existence of records as complete as possible and the availability of HLA typing of patients.

In this study, we first carefully analyzed the fine specificity of selected patients' sera and confirmed our previous results (6,8) showing that the autoimmune response against U1A and SmD1 proteins targets several linear regions of these molecules located in residues 35–58 and 257–282 of U1A, and 1–20, 44–67 and 97–119 of SmD1. In our series of 28 patients, 12 had antibodies positive with U1A and six of them possessed antibodies positive with peptide 257–282 of U1A. Eight patients had antibodies reacting with the whole human SmD1 protein in Western blotting and 11 (not necessarily the same) possessed antibodies reacting in ELISA with the SmD1 peptide 44–67. The striking reactivity of certain patients' sera with SmD1 peptides while they show no apparent reactivity with the parent protein has been previously described in the case of SmD1 (6,25). This type of reactivity was also observed with other autoantigens, such as histones, the Ro52 protein and poly(ADP ribose)polymerase (2629).

We explored the possible associations between HLA genes and various anti-Sm/RNP antibody populations. The distribution of DRB1, DQB1 and DPB1 alleles among the 28 anti-RNP autoantibody-positive patients and healthy control subjects was similar. In our series of RNP+ patients, antibodies reacting with U1A in Western blotting were positively associated to DRB1*06 and antibodies reacting with SmD1 peptide 44–67 were negatively associated to DRB1*02. It is noticeable that Olsen et al. (12) found no specific HLA-DR2 or -DR4 subtype associations with either anti-Sm or -RNP precipitin autoantibodies in Caucasian lupus patients. More recently, Kuwana et al. (30, 31) reported that Japanese MCTD patients with DR2 had a higher titer of anti-U1 RNP antibody (both anti-70K and anti-U1C proteins) and pleuritis, and patients with DR4 had a higher titer of anti-U1 RNP antibodies (especially those with DRB1*0401), anti-70K reactivity, swollen hands and arthritis (mostly those with DRB1*0405). It was also found by others that the frequency of HLA-DR2/DR4 was increased among children and adults with MCTD and anti-70K antibodies (32,33). It is obviously difficult to explain the origin of some of these discrepancies. We can only speculate that several parameters evidently affect the results, such as the size of the cohort, the statistical stringency used to interpret the results, and the nature and sensitivity of tests used to define the specificity of patients' antibodies. Comparison of the amino acid sequence of the outermost domain of DRB1*02 with that of the other DRB1* alleles showed one interesting sequence feature which clearly discriminates DRB1*02 from the other DRB1* alleles, i.e. a proline (P)–lysine (K)–arginine (R) sequence (within this sequence, P and R residues are characteristic for DRB1*02, while K is conserved in all DRB1* alleles) present at positions 11–13. It is also noticeable that with the exception of the DRB1*1001 allele, only DRB1*02 alleles contain a glutamine (Q) residue at position 96. It is possible that DRB1*02 patients cannot mount a specific immune response to SmD1 peptide 44–67 because this HLA class II molecule efficiently presents this self peptide leading to an early deletion of autoreactive Th clones. Comparison of the amino acid sequence of DRB1*06 with that of the other DRB1* alleles showed no sequence particularities.

At the DQB1* locus, we did not find any positive or negative associations with DQB1*0201/0202, *0302 and *0501, which were previously observed in Caucasian lupus patients with anti-RNP precipitin antibodies (12) or with DQB1*0302, which has been reported in a series of 49 Japanese patients with anti-U1 RNP (29). At this locus, however, we found a negative association between DQB1*0602 and antibodies reacting with SmD1 peptide 44–67. It is interesting to note that black patients with anti-Sm antibodies were previously reported to have significantly increased frequencies of the DQB1*0602 chain of DQw6, compared with healthy controls or SLE patients without anti-Sm or RNP antibodies (12). This association was not observed in Caucasian SLE patients. Comparison of the amino acid sequence of DQB1*0602 with that of the other DQB1* alleles showed no particular sequence features.

At the DPB1* locus, the only feature initially observed was a negative association with the DPB1*0301 allele in the group of patients with anti-RNP precipitin antibodies compared to the group of healthy subjects (corrected P < 0.01). The absence of a strong linkage disequilibrium between the DPB1* locus and the DRB1* or DQB1* loci has no incidence on DPB1* allele distribution, in contrast to what was reported regarding DRB1*03/07 and DQB1*201/202, for example. The observed decreased frequency of DPB1*0301 allele could thus be considered as totally significant. However, when Bonferroni's correction was applied to these values, the differences in frequencies became not significant.

It is notable that we found no association between antibodies to U1A peptide 257–282 and any subtype of HLA class II antigen. The 257–282 sequence is present in the peptide 242–282 which was found previously to be recognized by T cells from four of 10 MCTD patients analysed (9).

This study is the first report of an association between autoantibodies directed against U1A and SmD1 proteins/peptides with immunogenetic markers. Previous studies described genetic association with Sm, RNP or Sm/RNP antibodies generally detected using the Ouchterlony technique or counter-current electrophoresis (see 34 for review). Although further investigation in a much larger number of patients is certainly required, our data suggest that the production of anti-RNP precipitin antibodies, that of antibodies reacting with the whole U1A and SmD1 proteins or immunodominant peptides of these proteins are, at least in part, influenced immunologically by different HLA haplotypes. To some extent, this may explain certain serological heterogeneity in anti-U1 RNP antibody-positive patients. Other important susceptibility factors may obviously be involved in the antibody production in autoimmune patients. For example, evidence for an interplay of the MHC class II and TCR Vß alleles in the control of specific autoantibody response to U1A peptide 35–58 and SmD1 peptide 1–20 has been recently shown (35). Information obtained from the T cell epitope mapping of these proteins should greatly help to further establish the immunogenetic basis of the autoimmune response to U snRNP (9,36).


    Acknowledgments
 
We thank the members of the Autoimmunity Group of the Hospitals of Toulouse for their help in this study, as well as J. M. Amigues who was involved in the initial stages of this work.


    Abbreviations
 
MCTDmixed connective tissue disease
NHSnormal human serum
snsmall nuclear
RArheumatoid arthritis
RNPribonucleoprotein
Sclscleroderma
SLEsystemic lupus erythematosus
SSSjögren's syndrome
SSOsequence-specific oligonucleotide

    Notes
 
Transmitting editor: J.-F. Bach

Received 20 May 1998, accepted 22 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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