Infection and Immunity Research Group, Division of Life Sciences and
2 Vascular Biology Research Centre, King's College London, UK,
1 Kitasato University School of Medicine, Japan and
3 Bloomsbury Rheumatology Unit, University College London, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. AECA in sera of 15 SLE patients were measured by ELISA and Western blot analysis was used to examine the diversity of autoantigen targets in two clinically active patients. A human umbilical vein endothelial cell cDNA expression library was immunoscreened with sera from these two patients to identify their autoantigen targets. An anti-ribosomal P peptide antibody ELISA was used to assess the clinical significance of anti-ribosomal P protein antibodies in the sera of one patient.
Results. Significantly higher AECA levels were found in five patients with active disease and nephritis than in five patients with clinically inactive disease. Sera from two clinically active patients were found to recognize distinct spectra of autoantigens. The candidate autoantigens that were identified included (1) endothelial cell-specific plasminogen activator inhibitor; (2) the classical lupus antigen, i.e. ribosomal P protein P0; and (3) proteins never before described as putative autoantigens in SLE, including ribosomal protein L6, elongation factor 1, adenyl cyclase-associated protein, DNA replication licensing factor, profilin II and the novel proteins HEAPLA 1 and HEAPLA 2 (human endothelial associated putative lupus autoantigens 1 and 2). In one patient, antibodies against ribosomal P protein P0 were predominant and levels of these antibodies correlated with total AECA levels, anti-DNA antibody titres, overall clinical score and renal disease in a longitudinal study.
Conclusions. A panel of candidate endothelial autoantigens in SLE, which includes previously described autoantigens and novel targets, has been identified by a molecular cloning strategy. This novel molecular approach could also be applied to the identification of autoantigens in other autoimmune vascular diseases.
KEY WORDS: Systemic lupus erythematosus, cDNA expression library, Endothelial cell, Ribosomal protein P0, Anti-endothelial cell antibody.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Until recently, very few published data were available on the identity of endothelial cell autoantigens in any immune disorder. In SLE, it is known that the epitopes recognized by AECA appear to vary between patients [3]. Van der Zee et al. [4] have demonstrated different proteins recognized by AECA in SLE using Western blotting techniques. Antibodies against 38-, 41- and 150-kDa proteins were closely linked with the presence of lupus nephritis. Our own studies have demonstrated that AECA in subjects with lupus bind to at least 10 endothelial cell antigens [5]. We have also found that patients with nephritis had notable antibody reactivity with antigens of about 40 and 58 kDa, suggesting that AECA of particular specificities might play a role in the pathogenesis of lupus nephritis. In systemic vasculitis, Brasile et al. [5] suggested that five distinct antigens on endothelial cells and monocytes are recognized by AECA, and they found by indirect immunofluorescence that these endothelial cell targets were present in some, but not all, organs [6]. We have shown that AECA from patients with systemic vasculitis recognize antigens of molecular weights between 30 and 35 kDa [2]. Finally, an 18-kDa endothelial cell membrane antigen was identified by autoantibodies from patients with SSc which strongly associated with CREST syndrome, a variant of this disorder [7]. Together, all these data suggest that a characteristic and limited spectrum of distinct membrane antigens are the targets of the AECA found in different patient groups.
To generate large amounts of endothelial cell membrane lysate proteins for two-dimensional electrophoresis is technically difficult, time-consuming and expensive. However, by using this procedure we have been successful in obtaining the NH2-terminal sequence of one endothelial-associated putative autoantigen. We found a systemic vasculitis-associated 28-kDa protein which had 93% amino acid identity with triose phosphate isomerase (TPI) [8]. Interestingly, Wheeler et al. [9] reported the presence of anti-TPI antibodies together with anti-vimentin antibodies in human transplant-associated coronary artery disease. In the present study, we used a novel molecular cloning approach to identify endothelial cell autoantigens in SLE by immunoscreening a HUVEC cDNA expression library with sera from two SLE patients. We report the first description of a panel of candidate endothelial autoantigens in SLE. We have also shown that in one patient anti-ribosomal P peptide antibodies correlated with disease activity, suggesting that autoantibodies against ribosomal P protein may indeed be important in the pathogenesis of lupus.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patient 1 from Group 1 was of mixed Indian/West Indian ethnic origin and was diagnosed as having SLE 18 yr ago. She currently has severe disease with antinuclear antibodies (ANA), high titres of anti-DNA antibodies, anti-Ro, anti-RNP and the lupus anticoagulant, and she has joint, skin, renal and neurological involvement. As a result of Raynaud's phenomenon, she has lost several fingers. Patient 10 from Group 2, on the other hand, does not have kidney disease but does have active arthritis. She is of Caucasian extraction and was diagnosed with lupus/rheumatoid arthritis overlap 20 yr ago. She has ANA, elevated anti-DNA antibodies, pleuritis, leucopenia and lymphopenia.
Measurement of antibodies to endothelial cells
Endothelial cells were harvested from HUVEC and grown under established conditions. A cellular ELISA incorporating HUVEC was used to detect AECA as previously described [12]. Briefly, second-passage HUVEC were seeded at 2 x 104 in 100 µl of RPMI 1640 containing 20% fetal calf serum (Gibco, Paisley, UK), 10 U/ml heparin (Leo Laboratories, Princes Risborough, UK) and 30 µl/ml endothelial growth factor (Sigma, Poole, UK) on sterile polystyrene 96-well plates (well diameter 6 mm) (Nunc, Roskilde, Denmark) precoated with 100 µl of 1% gelatin (Sigma). After 48 h of culture at 37°C, the plates were washed and then blocked with 2% bovine serum albumin (BSA) (Sigma) in 0.15 M phosphate-buffered saline (PBS). After three washes with PBS, 100 µl of triplicate samples of test serum diluted 1/400 in 2% BSA/PBS were added to each well and incubated at room temperature. After three washes, 100 µl goat anti-human IgG alkaline phosphatase conjugate (Tago, Burlingame, California, USA) diluted in 2% BSA/PBS was added to each well for 1 h at room temperature. After four washes with PBS, the plates were developed with 100 µl of the substrate p-nitrophenyl phosphate (Sigma). IgG AECA results were expressed as percentage of the reaction of a known positive control serum. We established previously that 95% of normal negative control sera gave absorbance values of <10% of the positive control serum from a patient with SLE.
Western blotting
Plasma membranes from second- or third-passage HUVEC were prepared as follows: 50 x 106 cells were scraped off the flasks, washed in Hanks buffered salt solution at 4°C and pelleted twice at 600 g for 10 min at 4°C, resuspended in 5 mM HEPES, pH 7.4, at 4°C for 10 min and homogenized mechanically until approximately 90% were disrupted, and the nuclei were removed by centrifugation. The supernatant was centrifuged at 27 000 g for 20 min and the pellet containing the plasma membranes resuspended in 0.5% CHAPS detergent (Sigma) containing 1 mM PMSF, was incubated at 4°C overnight. Next day a further centrifugation at 11 000 g was performed for 15 min at 4°C and the supernatant, containing endothelial cell membrane proteins, was collected.
The solubilized proteins were separated using 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Sigma). Blocking was done using 2% BSA/PBS-Tween for 1 h at 37°C. The patient's serum was applied (1/20 diluted in 2% BSA/10% goat serum in PBS/Tween) overnight at 4°C. Next day, after washing with PBS, goat anti-human IgG peroxidase conjugate 1/500 diluted in 2% BSA/PBS-Tween was applied for 1 h, and substrate was added (PBS, hydrogen peroxide, diaminobenzidine).
Immunoscreening a recombinant endothelial cell expression library with sera from SLE patients
A human HUVEC cDNA expression library was obtained commercially (Stratagene, Cambridge, UK) and used to infect plated Y1090 Escherichia coli. Expression of ß-galactosidase fusion proteins was induced by overlaying with nitrocellulose filters (Stratagene) saturated with 10 mM isopropyl thiogalactoside. After the transfer of proteins (3 h), immunological screening was performed on the nitrocellulose, using the serum of an SLE patient. The filters were blocked with 10% goat serum (Sigma) and 5% Marvel milk powder (Chives and Whithers, Dublin, Ireland), and incubated first with a 1:500 dilution of patients serum. A 1:500 dilution was chosen because two normal control sera gave a negative signal at this concentration. Then peroxidase-conjugated anti-human IgG was added together with the diaminobenzidine substrate. Positive plaques were rescreened with the same lupus serum until all daughter plaques revealed positive signals. Next, using a helper phage, we performed in vivo excision of the phagemid into SolR cells, which allowed the insert to be characterized in a plasmid system. DNA sequencing of the positive clones was performed using the automated DNA sequencer in the Molecular Biology Unit at King's College, London. Database searches for matches of DNA sequences of positive clones were performed using the University of Wisconsin GCG Package, to establish whether the cDNAs encoded known or novel proteins.
Synthesis of ribosomal P peptide
The immunodominant epitope of human ribosomal P0 protein is contained in the carboxyl terminal 20 amino acid sequence (residues 298317) [13]. A peptide of this sequence was made using an automated PS3 peptide synthesizer (Rainin Instrument Company, Emeryville, CA, USA) and standard Fmoc methodology with a Rink amide MBHA resin to yield a peptide amide. The peptide was analysed and purified (90% pure) using reverse-phase high-performance liquid chromatography against a C18 column. Mass spectrometry analysis of the purified product confirmed the correct mass of the peptide.
Immunoassay
This was based on that described by Ward et al. [14]. Briefly, ribosomal P peptide at 10 µg/ml in 50 µl deionized water was incubated overnight at 37°C in Falcon Microtest III flexible plates (Marathon Laboratory Supplies, Oxard, California, USA) without a lid so that the water evaporated. Next day, the plates were washed twice with PBS and then blocked with 50 µl/well of 2% Marvel milk powder for 1 h at 37°C. The plates were washed twice with PBS-Tween (PBS containing 0.1% Tween). Next, we added 50 µl of the patient's serum diluted 1:200 in PBS/Marvel/Tween (PBS containing 0.05% Tween 20 and 1% Marvel) and incubated for 1 h at 37°C. After four washes with PBS-Tween, we added 50 µl/well of alkaline phosphatase-conjugated goat anti-human IgG (Tago, TCS Biological Ltd, Buckingham, UK) diluted 1:1000 in PBS/Marvel/Tween and incubated the plates for 1 h at 37°C. After six washes with PBS-Tween the plates were developed with 50 µl of substrate per well.
Statistical analysis
The MannWhitney U-test was used to compare levels of AECA in sera of patients from different groups (Groups 1, 2, 3 detailed above). The relationship between anti-ribosomal P peptide antibody levels and other measures of SLE disease activity was analysed using Spearman's rank correlation test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Western blot analysis of autoantigens recognized by antibodies in two sera with high levels of AECA
The molecular weight and diversity of proteins recognized by AECA from two patients (patients 1 and 10) were estimated by Western blot analysis. The spectra of proteins recognized by AECA from these two patients were quite distinct. Antibodies from patient 1 recognized at least 10 proteins from 35 to 110 kDa, whereas antibodies from patient 10 recognized more than 16 proteins covering the molecular weight range from 30 to 200 kDa. The most prominent protein bands for each patient are indicated by arrows. No protein bands were demonstrated with the control serum (Fig. 2). These results show that AECA in different SLE patients may recognize a distinct and limited number of autoantigens.
|
Immunoscreening a recombinant endothelial cell expression library with sera from SLE patients
In order to determine the identity of autoantigens recognized by AECA from patients 1 and 10, we screened a HUVEC cDNA expression library with their sera. Plaques (5 x 104) were screened with sera from each of two SLE patients, resulting in the isolation of 19 and 4 antigen clones respectively. Clones identified as positive in this first round of screening were subjected to two further rounds of screening to ensure that they were positive for reactivity to patient sera and to purify them to homogeneity. DNA sequencing demonstrated that, for patient 1, 14 of the clones encoded the ribosomal P protein P0 [15], one encoded elongation factor 1 [16], one encoded adenylyl cyclase-associated protein (CAP 1) and one encoded profilin II (Table 1
) [17]. Two clones did not have any significant homology to any known proteins as determined by DNA and searching a protein database (GenBank release 113.0, August 1999), and are therefore novel: they are referred to as human endothelial-associated putative lupus autoantigens (HEAPLA 1, HEAPLA 2) (Table 1
). Sera from patient 10 had antibodies which bound plasminogen activator inhibitor [18], fibronectin [19], ribosomal protein L6 [20] and DNA replication licensing factor [21] (Table 1
).
|
Longitudinal study of ribosomal P peptide antibodies in a patient with lupus nephritis
Serial profiles of anti-ribosomal P peptide antibody in sera from patient 1, which had been collected over a 12-yr period, were measured. The levels of anti-ribosomal P protein antibody over this time were related to anti-endothelial cell antibody, anti-DNA antibody levels, global clinical scores and renal function. The patient was assessed using the BILAG disease activity index. This index scores the patient from A (most active) to E (no activity) in each of eight organs or systems. The score was converted to a global index using the following notation: A = 9, B = 3, C = 1, D/E = 0. The results are shown in Fig. 3. In general, fluctuations in anti-ribosomal P peptide levels were concomitant and were statistically correlated, as assessed by Spearman's correlation coefficient, with changes in AECA (P < 0.001), anti-DNA antibody levels (P < 0.001) and global clinical score (GCS) (P < 0.001). With regard to the correlation of anti-ribosomal P peptide levels with renal disease score, the Spearman's correlation coefficient revealed a weakly significant (P < 0.05) correlation.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fourteen of 19 independent positive clones derived from an endothelial cell cDNA expression library screened with sera from a lupus patient with active nephritis encoded ribosomal P protein. Our data suggest, therefore, that this 38-kDa ribosomal P protein is an important endothelial autoantigen in this particular SLE patient. In support of this, antibodies from this patient were also shown to recognize a 38-kDa protein by Western blot analysis. We [2] and others [4] have previously reported the association of an endothelial autoantigen of 3840 kDa with lupus nephritis.
Using sequential serum samples from the patient with active nephritis, we found that anti-ribosomal P protein peptide antibody concentrations corresponded with general markers of disease activity. Our data also showed a weakly significant association between nephritis and anti-ribosomal P protein peptide antibody concentrations in this one patient. In a more extensive analysis in which we examined all our original patient group sera (Groups 13), we found no significant correlation between anti-ribosomal P protein peptide antibody concentrations and renal disease (data not shown). Moreover, we have shown in a cross-sectional study that 15% of 55 SLE patients had elevated anti-ribosomal P peptide levels but no significant association with renal involvement was found (G. Frampton et al., unpublished results). Martin and Reichlin [22] showed that fluctuations of antibody to ribosomal P protein correlated with disease activity, including the appearance and remission of nephritis in SLE. Recently, Chindalore et al. [23] have shown the presence of anti-ribosomal P antibodies, retrospectively studied, in 30% of 69 lupus patients with active disease. Moreover, they found that 75% (15/20) of these had active nephritis. They concluded that anti-ribosomal P antibodies, like anti-DNA antibodies, may also contribute to renal pathology in lupus. These studies, showing a correlation between anti-ribosomal P proteins, autoantibodies and renal disease, suggest an immunopathogenic role for anti-ribosomal P antibodies. However, there have been no reports of recovery of anti-ribosomal P antibodies from nephritic kidneys of SLE patients [23]. Autoantibodies to ribosomal P proteins in patients with SLE have been recognized for more than a decade [24], and various studies have demonstrated their association with neuropsychiatric disease [2527]. However, other studies have failed to show a relationship [2830]. There is also wide variation in the reported prevalence of these antibodies in patients with SLE. Initial studies reported a frequency of 510% [24] or 20% [31]. However, recent studies from Japan found a higher prevalence of anti-P antibodies in unselected SLE patients, of 42% [30].
Autoantibodies to ribosomal P protein have been reported to react with plasma membrane components, including those of mesangial cells [32], endothelial cells [33] and T-cells [34], suggesting that autoantibodies to ribosomal P proteins bind not only to intracellular antigen but also to a plasma membrane-associated target on human cells [14]. Therefore, there are two likely explanations for these results: (1) the protein is located both inside the cell and on the plasma membrane; (2) there are two different but antigenically related proteins at these locations. It has been suggested that antibodies to ribosomal P protein exert their pathogenicity by augmenting apoptosis [32]. We have recently demonstrated direct binding of rat IgG anti-ribosomal peptide antibodies to cultured HUVEC by immunofluorescence and FACS (fluorescence-activated cell sorter) analysis (G. Frampton et al., unpublished results). Our data are therefore consistent with those of Yoshio et al. [33], who showed that anti-ribosomal P antibodies, purified from lupus sera using recombinant ribosomal P protein affinity columns, reacted with the surface of human umbilical vein endothelial cells. These observations, taken together, suggest that anti-ribosomal P antibodies bind to the vasculature and may thus be involved in the vascular damage seen in SLE.
Anti-dsDNA antibodies are a hallmark of SLE and are involved in the pathogenesis of lupus nephritis, but the mechanism by which they cause disease is uncertain. In addition, in a minority of patients, titres of anti-DNA antibodies are consistently normal and therefore of dubious clinical relevance. In our sequential study, we found a good correlation between anti-dsDNA antibodies and anti-ribosomal P peptide antibodies, but we did not observe a significant reduction in the anti-DNA antibody binding activity in ELISA after preincubation with ribosomal P peptide, or in the anti-ribosomal P peptide ELISA after preincubation with dsDNA (data not shown). The lack of inhibition in either of these assays suggests the coexistence of two distinct autoantibody populations with separate binding specificities for ribosomal P protein and dsDNA respectively.
In this study, we have used a novel molecular cloning strategy in attempts to identify previously unidentified endothelial cell autoantigens in SLE patients. The isolation of ribosomal P protein, a previously identified autoantigen, is encouraging and provides proof of principle for this molecular cloning approach. At this stage, we can only speculate on the significance of the other candidate endothelial cell autoantigens identified in this study. Antibody binding to the endothelial cell-specific protein plasminogen activator inhibitor could clearly lead to a prothrombotic condition through altering the fibrinolytic system, which could contribute to the thrombotic complications commonly observed in lupus patients. In addition, antibodies binding to the other candidate autoantigens would conceivably alter endothelial cell function by affecting protein synthesis, signal transduction or cell attachment to the basement membrane. Having established the prevalence of autoantibodies to these proteins in SLE patients, it will be possible to carry out studies to see whether these antibodies affect endothelial cell functions. Thus, it will be possible to establish whether autoantibodies to this newly identified panel of AECA contribute to the pathology of SLE.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|