Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: Application to enzyme‐linked immunosorbent assay

Objective

To characterize an immunodominant epitope on RNA polymerase III (RNAP III) recognized by systemic sclerosis (SSc) sera and to develop an enzyme‐linked immunosorbent assay (ELISA) for the detection of serum anti–RNAP I/III antibodies.

Methods

RNAP III–specific subunits RPC62 and RPC155 were generated in a bacterial expression system as a series of recombinant fragments. Reactivities to these recombinant fragments were examined by immunoblots and/or ELISA in 16 SSc sera containing anti–RNAP I/III antibodies, 89 SSc sera lacking anti–RNAP I/III antibodies, 61 systemic lupus erythematosus (SLE) sera, and 61 healthy control sera.

Results

Anti–RNAP I/III–positive SSc sera recognized several distinct epitopes on RPC62 and RPC155 in various combinations, but the fragment encoding amino acids at positions 732–1166 of RPC155 was recognized by all 11 anti–RNAP I/III–positive SSc sera tested. Carboxyl‐ and amino‐terminal deletion studies showed that at least 130 amino acids at positions 891–1020 of RPC155 were necessary for the antibody binding, but strong reactivity required an additional amino‐terminal extension. When a purified recombinant fragment containing the immunodominant epitope was used as the antigen source in an ELISA, elevated antibody reactivity was detected in all 16 anti–RNAP I/III–positive SSc sera, but in no anti–RNAP I/III–negative SSc, SLE, or healthy control sera, representing a sensitivity of 100% and a specificity of 100%.

Conclusion

A major epitope commonly recognized by SSc sera containing anti–RNAP I/III antibodies was identified on RPC155. The ELISA using a recombinant fragment expressing the immunodominant epitope should be a valuable tool for routine screening for anti–RNAP I/III antibodies in clinical diagnostic laboratories.

     

Enzyme‐linked immunosorbent assay for detection of Anti–RNA polymerase III antibody: Analytical accuracy and clinical associations in systemic sclerosis

Objective

We have recently developed an enzyme‐linked immunosorbent assay (ELISA) for detection of anti–RNA polymerase III (anti–RNAP III) antibody, using a recombinant fragment containing the immunodominant epitope as the antigen source. This study was conducted to assess the analytical accuracy and clinical associations of the anti–RNAP III ELISA in patients with systemic sclerosis (SSc).

Methods

To evaluate analytical sensitivity and specificity of the ELISA, both immunoprecipitation tests and ELISA were used to detect anti–RNAP III antibody in 534 SSc sera from patients at 3 medical centers. Sera from 522 SSc patients and 516 controls, including patients with other connective tissue diseases and blood bank donors, were also evaluated to assess the clinical sensitivity and specificity of the ELISA. Clinical findings in anti–RNAP III antibody–positive SSc patients were compared between patient groups stratified according to anti–RNAP III antibody levels determined by the ELISA.

Results

In SSc patients, our ELISA showed analytical sensitivity of 91% and analytical specificity of 99% compared with the immunoprecipitation assay (a gold standard for detection of anti–RNAP III antibody). The additional analysis using a large series of SSc and control sera showed that clinical sensitivity and specificity of the ELISA with respect to the diagnosis of SSc were 17% and 98%, respectively. A high level of anti–RNAP III antibody was associated with diffuse cutaneous SSc, higher maximum total skin score, and increased frequency of tendon friction rubs.

Conclusion

The anti–RNAP III ELISA is analytically accurate and clinically specific. With this assay, testing for anti–RNAP III antibody can be made routinely available.

     

A new conformational epitope generated by the binding of recombinant 70‐kd protein and U1 RNA to anti–U1 RNP autoantibodies in sera from patients with mixed connective tissue disease

Objective

To establish an enzyme‐linked immunosorbent assay (ELISA) using a complex of in vitro–transcribed U1 RNA and recombinant 70‐kd, A, and C proteins (C‐ELISA) to detect anti–U1 RNP antibodies reactive in double immunodiffusion (DID), but not in ELISA using the proteins alone (P‐ELISA).

Methods

Sera from 196 patients with mixed connective tissue disease were used to test reactivity in P‐ and C‐ELISAs, and the specificity of the sera was also tested by DID and immunoprecipitation (IP).

Results

In P‐ELISA, 15 of 196 sera positive for anti–U1 RNP in DID did not react, while all sera reacted in C‐ELISA. The reactivity of 15 sera to the U1 RNA was tested by IP and ELISA, and only 3 sera reacted with the U1 RNA. These results indicated that the increased reactivity in C‐ELISA was not due to the U1 RNA itself. We confirmed that the 70‐kd and A proteins were bound directly to the U1 RNA by IP using antibodies to His‐tag, and we tested the reactivity of the sera to the U1 RNA–70‐kd protein complex and the U1 RNA–A protein complex by IP. All sera reacted with the U1 RNA–70‐kd protein complex, and 1 sample reacted with the U1 RNA–A protein complex.

Conclusion

These results suggest that some anti–U1 RNP–positive sera specifically recognize the conformational structure altered by the binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is as useful as the DID method for detecting anti–U1 RNP antibodies.

     

Autoantibodies against B23, a nucleolar phosphoprotein,occur in scleroderma and are associated with pulmonary hypertension

Objective

To determine whether the abundant nucleolar phosphoprotein B23 is a target of autoantibodies in scleroderma, and to examine the clinical phenotype associated with these antibodies.

Methods

Ninety‐two randomly selected scleroderma sera were screened by enzyme‐linked immunosorbent assay against recombinant human B23. Demographic, clinical, and serologic parameters associated with B23 autoantibody status were examined.

Results

We demonstrated that autoantibodies against B23 occur in ～11% of sera obtained from patients with scleroderma. B23 seropositivity was related to pulmonary hypertension, antifibrillarin antibody, anti‐RNP antibody, and decreased lung capacity. In multivariate analysis, B23 autoantibodies remained strongly associated with moderate‐to‐severe pulmonary hypertension and antifibrillarin antibodies.

Conclusion

These data unite B23 with the group of nucleolar autoantigens targeted in scleroderma and thus focus attention on changes in the nucleolus that render its components immunogenic in this disease. The demonstration that antibodies to B23 are associated with an increased prevalence of pulmonary hypertension points to anti‐B23 antibodies as a possible marker of a specific phenotype in scleroderma.

     

Close temporal relationship between onset of cancer and scleroderma in patients with RNA polymerase I/III antibodies

Objective

This study was undertaken to examine the temporal relationship between scleroderma development and malignancy, and to evaluate whether this differs by autoantibody status among affected patients.

Methods

Study participants had a diagnosis of scleroderma, a diagnosis of cancer, cancer, an available serum sample, and a cancer pathology specimen. Sera were tested for autoantibodies against topoisomerase I, centromere, and RNA polymerase I/III by immunoprecipitation and/or enzyme‐linked immunosorbent assay. Clinical and demographic characteristics were compared across autoantibody categories. Expression of RNA polymerases I and III was evaluated by immunohistochemistry using cancerous tissue from patients with anti–RNA polymerase antibodies.

Results

Twenty‐three patients were enrolled. Six patients tested positive for anti–RNA polymerase I/III, 5 for anti–topoisomerase I, and 8 for anticentromere, and 4 were not positive for any of these antigens. The median duration of scleroderma at cancer diagnosis differed significantly between groups (−1.2 years in the anti–RNA polymerase I/III group, +13.4 years in the anti–topoisomerase I group, +11.1 years in the anticentromere group, and +2.3 years in the group that was negative for all antigens tested) (P = 0.027). RNA polymerase III demonstrated a robust nucleolar staining pattern in 4 of 5 available tumors from patients with antibodies to RNA polymerase I/III. In contrast, nucleolar RNA polymerase III staining was not detected in any of 4 examined tumors from the RNA polymerase antibody–negative group (P = 0.048).

Conclusion

Our findings indicate that there is a close temporal relationship between the onset of cancer and scleroderma in patients with antibodies to RNA polymerase I/III, which is distinct from scleroderma patients with other autoantibody specificities. In this study, autoantibody response and tumor antigen expression are associated. We propose that malignancy may initiate the scleroderma‐specific immune response and drive disease in a subset of scleroderma patients.

     

Direct binding of anti–DNA topoisomerase I autoantibodies to the cell surface of fibroblasts in patients with systemic sclerosis

Objective

Fibroblasts play a crucial role in the development of systemic sclerosis (SSc), and antifibroblast antibodies (AFAs) capable of inducing a proinflammatory phenotype in fibroblasts have been detected in the sera of SSc patients. This study examined the prevalence of AFAs in SSc and other diseases and the possible correlation between AFAs and known antinuclear antibody specificities in SSc patients.

Methods

Sera from 99 patients with SSc, 123 patients with other autoimmune and nonautoimmune diseases, and 30 age‐ and sex‐matched healthy controls were examined. AFA prevalence was assessed by flow cytometry and further characterized by indirect immunofluorescence, enzyme‐linked immunosorbent assay (ELISA), and immunoblotting. Anti–topoisomerase I (anti–topo I) from SSc sera were purified by affinity chromatography on topo I.

Results

AFAs were more common in SSc patients (26.3%) than in any other disease groups studied. The presence of AFA was significantly associated with pulmonary involvement and death. AFA‐positive sera from SSc patients bound to all human and rodent fibroblasts tested, but not to human primary endothelial cells or smooth muscle cells. All SSc AFAs strongly reacted with topo I by ELISA and immunoblotting. The binding intensity of SSc AFAs correlated strongly with reactivity against topo I on immunoblots of fibroblast extracts and with the immunofluorescence pattern typical of anti–topo I on permeabilized cells. Total IgG and affinity‐purified anti–topo I from AFA‐positive SSc sera were found to react with the surface of unpermeabilized fibroblasts by flow cytometry as well as by immunofluorescence and confocal microscopy.

Conclusion

This is the first report establishing that AFAs in SSc are strongly correlated with anti–topo I and, furthermore, that anti–topo I antibodies themselves display AFA activity by reacting with determinants at the fibroblast surface.

     

Association of α‐actinin–binding anti–double‐stranded DNA antibodies with lupus nephritis

Objective

Anti–double‐stranded DNA (anti‐dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross‐reacting with α‐actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity.

Methods

One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti‐DNA component). Anti‐dsDNA antibodies were detected by conventional enzyme‐linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti–α‐actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity‐purified for cross‐reactivity studies and measurement of antibody avidity.

Results

Sera from 62 of the SLE patients had anti‐dsDNA antibodies; 21 of these sera also had anti–α‐actinin antibodies, as compared with 1 of the 38 sera without anti‐dsDNA antibodies. Of the 22 patients with anti–α‐actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti–α‐actinin antibodies (P < 0.01). In patients with GN, anti–α‐actinin, but not anti‐dsDNA, antibodies correlated with the SLEDAI score (minus the anti‐DNA component) and with treatment. The fraction of serum anti‐dsDNA antibodies that cross‐reacted with α‐actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high‐avidity anti‐dsDNA antibodies and an in‐house conventional ELISA.

Conclusion

The α‐actinin–binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.

     

Anti‐DNA topoisomerase IIα autoantibodies in localized scleroderma

Objective

To determine the prevalence and clinical correlation of anti‐DNA topoisomerase IIα (anti–topo IIα) antibody in patients with localized scleroderma.

Methods

Anti–topo IIα antibodies or anti‐DNA topoisomerase I (topo I) antibodies were determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. Inhibition of topo IIα enzymatic activity by the antibodies was evaluated by decatenation assays using kinetoplast DNA as a substrate.

Results

IgG or IgM anti–topo IIα antibody was detected in 76% (35 of 46) of patients with localized scleroderma, and in 85% (11 of 13) of patients with generalized morphea, the severest form of localized scleroderma. This prevalence of the antibody in patients with localized scleroderma was much higher than that found in patients with systemic sclerosis (SSc) (5 of 37 [14%]), systemic lupus erythematosus (2 of 26 [8%]), dermatomyositis (2 of 20 [10%]), and in healthy controls (3 of 42 [7%]). Immunoblotting confirmed the presence of IgG anti–topo IIα antibody in sera from patients with localized scleroderma and showed no cross‐reactivity of anti–topo IIα antibody with topo I. Anti–topo I antibody was not detected by ELISA in any sera from patients with localized scleroderma. In addition, anti–topo I antibody from SSc patients did not cross‐react with topo IIα. The presence of anti–topo IIα antibody was associated with a greater total number of sclerotic lesions and number of plaque lesions in patients with localized scleroderma. Furthermore, anti–topo IIα antibody was able to inhibit topo IIα enzymatic activity.

Conclusion

The results of the present study indicate that anti–topo IIα is a major autoantibody in localized scleroderma, and is distinct from anti–topo I antibody in SSc.

     

Critical comparative analyses of anti–α‐actinin and glomerulus‐bound antibodies in human and murine lupus nephritis

Objective

Although anti–double‐stranded DNA (anti‐dsDNA) antibodies are important in lupus nephritis, the question regarding which glomerular structures (α‐actinin, nucleosomes, or others) are recognized by nephritogenic anti‐dsDNA antibodies is still controversial. In this study, we determined which glomerular structures are recognized by monoclonal and in vivo–bound nephritogenic antibodies.

Methods

Western blotting was used to analyze the ability of nephritogenic anti‐dsDNA antibodies to recognize glomerular and nucleosomal structures. Sera from patients with lupus nephritis, sera from random antinuclear antibody–positive patients, and paired antibodies from sera and kidney eluates from nephritic (NZB × NZW)F₁ mice were analyzed for activity against proteins identified by monoclonal nephritogenic antibodies, and against α‐actinin, dsDNA, nucleosomes, histone H1, heparan sulfate, DNase I, and type IV collagen. Immunoelectron microscopy was used to determine the glomerular localization of α‐actinin and in vivo–bound autoantibodies in nephritic (NZB × NZW)F₁ mouse kidneys.

Results

Anti–α‐actinin antibodies were observed in human and murine lupus nephritis sera and in sera from patients without systemic lupus erythematosus and were not detected in kidney eluates from nephritic mice. Antibodies to dsDNA and histone H1 were detected in all eluates. Western blot analyses revealed that nephritogenic anti‐dsDNA antibodies recognized a 32‐kd band, identified as histone H1. Competitive enzyme‐linked immunosorbent assay demonstrated that nephritogenic monoclonal antibodies, and dominant antibodies eluted from nephritic kidneys, cross‐reacted with dsDNA and H1. This cross‐reactive anti‐H1 specificity was largely absent in sera from those mice. Immunoelectron microscopic analysis of nephritic (NZB × NZW)F₁ mouse kidneys revealed that antibodies eluted from kidneys, but not anti–α‐actinin antibodies, bound to distinct nephritis‐associated electron‐dense structures linked to glomerular basement membranes.

Conclusion

Cross‐reactive anti‐dsDNA/anti–histone H1 antibodies, but not anti–α‐actinin antibodies, are central among those deposited in nephritic glomeruli.

     

DNA topoisomerase I binding to fibroblasts induces monocyte adhesion and activation in the presence of anti–topoisomerase I autoantibodies from systemic sclerosis patients

Objective

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis due to excessive and dysregulated collagen production by fibroblasts. Previously, we reported that anti–DNA topoisomerase I (anti–topo I) antibodies bound specifically to fibroblast surfaces; however, we had not identified their antigenic target. We undertook this study to characterize the target of anti–topo I antibodies on fibroblasts and the effects of their binding.

Methods

Purified topo I or topo I released from apoptotic cells was tested for surface binding to a number of human cell types by cell‐based enzyme‐linked immunosorbent assay, flow cytometry, and indirect immunofluorescence. Antibodies purified from SSc patient and normal control sera were used to detect topo I binding. The consequences of topo I and anti–topo I binding to fibroblasts were assessed by coculture with THP‐1 monocytes.

Results

The autoantigen topo I itself was found to bind specifically to fibroblasts in a dose‐dependent and saturable manner, where it was recognized by anti–topo I from SSc patients. The binding of anti–topo I subsequently stimulated adhesion and activation of cocultured monocytes. Topo I released from apoptotic endothelial cells was also found to bind specifically to fibroblasts.

Conclusion

The findings of this study thus confirm and extend the findings of our previous study by showing that topo I binding to fibroblast surfaces is both necessary and sufficient for anti–topo I binding. Second, topo I–anti–topo I complex binding can then trigger the adhesion and activation of monocytes, thus providing a plausible model for the amplification of the fibrogenic cascade in anti–topo I–positive SSc patients.

     

RNA helicase encoded by melanoma differentiation–associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease

Objective

To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C‐ADM) and rapidly progressive interstitial lung disease (ILD).

Methods

An anti–CADM‐140 antibody–positive prototype serum sample was used to screen a HeLa cell–derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro–transcribed and in vitro–translated products using anti–CADM‐140 antibody–positive and anti‐CADM‐140 antibody–negative sera. The lysates of COS‐7 cells transfected with the putative antigen were similarly tested. An enzyme‐linked immunosorbent assay (ELISA) to detect the anti–CADM‐140 antibody was established using a recombinant CADM‐140 antigen, and its specificity and sensitivity for C‐ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases.

Results

By cDNA library screening and immunoprecipitation of in vitro–transcribed and in vitro–translated products, we obtained a cDNA clone encoding melanoma differentiation–associated gene 5 (MDA‐5). The anti–CADM‐140 antibodies in patients' sera specifically reacted with MDA‐5 protein expressed in cells transfected with full‐length MDA‐5 cDNA, confirming the identity of MDA‐5 as the CADM‐140 autoantigen. The ELISA, using recombinant MDA‐5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the “gold standard” immunoprecipitation assay, and was useful for identifying patients with C‐ADM and/or rapidly progressive ILD.

Conclusion

Given that RNA helicase encoded by MDA‐5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C‐ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA‐5 protein makes detection of the anti–CADM‐140 antibody routinely available.

     

Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies

OBJECTIVE: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay. METHODS: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. RESULTS: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. CONCLUSION: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.     

Deiminated Epstein‐Barr virus nuclear antigen 1 is a target of anti–citrullinated protein antibodies in rheumatoid arthritis

Objective

To test the hypothesis that deimination of viral sequences containing Arg–Gly repeats could generate epitopes recognized by anti–citrullinated protein antibodies (ACPAs) that are present in rheumatoid arthritis (RA) sera.

Methods

Multiple antigen peptides derived from Epstein‐Barr virus (EBV)–encoded Epstein‐Barr nuclear antigen 1 (EBNA‐1) were synthesized, substituting the arginines with citrulline, and were used to screen RA sera. Anti–cyclic citrullinated peptide antibodies were purified by affinity chromatography and tested on a panel of in vitro deiminated proteins. Their ability to bind in vivo deiminated proteins was evaluated by immunoprecipitation, using EBV‐infected cell lines.

Results

Antibodies specific for a peptide corresponding to the EBNA‐1^35–58 sequence containing citrulline in place of arginine (viral citrullinated peptide [VCP]) were detected in 50% of RA sera and in <5% of normal and disease control sera. In addition, affinity‐purified anti‐VCP antibodies from RA sera reacted with filaggrin‐derived citrullinated peptides, with deiminated fibrinogen, and with deiminated recombinant EBNA‐1. Moreover, anti‐VCP antibodies immunoprecipitated, from the lysate of calcium ionophore–stimulated lymphoblastoid cell lines, an 80‐kd band that was reactive with a monoclonal anti–EBNA‐1 antibody and with anti–modified citrulline antibodies.

Conclusion

These data indicate that ACPAs react with a viral deiminated protein and suggest that EBV infection may play a role in the induction of these RA‐specific antibodies.

     

Is α‐actinin a target for pathogenic anti‐DNA antibodies in lupus nephritis?

Objective

Following recent reports that pathogenic murine anti‐DNA antibodies bind to α‐actinin, it was obviously of interest to assess the ability of human pathogenic anti–double‐stranded DNA (anti‐dsDNA) antibodies to bind this antigen. Both human monoclonal anti‐DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated.

Methods

An enzyme‐linked immunosorbent assay was established to measure immunoglobulin binding to α‐actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high‐affinity human anti‐dsDNA IgG hybridomas (RH14, B3, and DIL‐6) and 7 human IgM anti‐DNA hybridomas was also investigated.

Results

A greater proportion of anti‐dsDNA IgG–binding antibodies purified from patients with renal disease bound to α‐actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100‐kd α‐actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to α‐actinin, while nonpathogenic DIL‐6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to α‐actinin.

Conclusion

The results of our study support the findings of previous studies using murine anti‐DNA monoclonal antibodies, which suggest that pathogenic anti‐dsDNA antibodies cross‐react with α‐actinin.

     

Sera from anti–Jo‐1–positive patients with polymyositis and interstitial lung disease induce expression of intercellular adhesion molecule 1 in human lung endothelial cells

Objective

To investigate whether sera or purified IgG from patients with polymyositis (PM) and patients with dermatomyositis (DM), with or without interstitial lung disease (ILD), can activate endothelial cells (ECs).

Methods

Patients' sera were selected based on the presence or absence of anti–Jo‐1, anti‐SSA, or anti–U1 small nuclear RNP autoantibodies. The presence of autoantibodies was determined by line blot assays. Cultured human microvascular ECs derived from lung tissue (HMVEC‐L) were incubated with sera or purified IgG from 22 patients with PM, 7 patients with DM, and 10 healthy individuals as controls. Assessment of intercellular adhesion molecule 1 (ICAM‐1) expression was conducted by immunofluorescence (n = 22) and by cell‐based enzyme‐linked immunosorbent assay (ELISA) (n = 20). Serum levels of soluble ICAM‐1 (sICAM‐1) were determined by ELISA.

Results

Sera from PM patients with ILD who were positive for anti–Jo‐1 autoantibodies had a significantly stronger effect on the expression of ICAM‐1 by HMVEC‐L in comparison with sera from healthy controls and patients with other autoantibodies. Purified IgG did not induce ICAM‐1 expression. Higher serum levels of sICAM‐1 were found in patients with myositis compared with healthy controls.

Conclusion

EC activation with ICAM‐1 expression could contribute to the multiorgan involvement, including the development of myositis and ILD, in patients carrying anti–Jo‐1 autoantibodies. The EC‐activating factors are not the autoantibodies themselves, but might be systemic factors associated with these autoantibodies.

     

Benefit of early treatment in inflammatory polyarthritis patients with anti–cyclic citrullinated peptide antibodies versus those without antibodies

Objective

To compare the clinical utility of anti–cyclic citrullinated peptide (anti‐CCP) antibodies and rheumatoid factor (RF) testing in predicting both functional outcome and response to treatment in early inflammatory polyarthritis (IP) patients.

Methods

A total of 916 IP subjects from a primary care incidence registry (1990–1994) had anti‐CCP antibody and RF status determined at baseline. Mean change in Health Assessment Questionnaire (HAQ) score between baseline and 5 years was compared by antibody status. The effect of treatment with disease‐modifying antirheumatic drugs and/or steroids over 5 years, early (<6 months of symptom onset) versus late initiation, and duration of treatment were also compared by anti‐CCP antibody status. The analysis was adjusted for treatment decisions and censoring over the followup, using marginal structural models.

Results

Anti‐CCP antibody–positive patients (n = 268) had more severe disease both at presentation and 5 years of followup, and this was independent of RF. On adjustment, anti‐CCP antibody–negative patients treated early experienced a significant improvement in functional disability compared with anti‐CCP antibody–negative patients who were never treated (?0.31; 95% confidence interval [95% CI] ?0.53, ?0.08), and experienced additional benefit for each additional month of early treatment. Anti‐CCP antibody–positive patients treated early did not have a significant improvement in HAQ score compared with those not treated (?0.14; 95% CI ?0.52, 0.24).

Conclusion

In this first observational study to examine the influence of anti‐CCP antibody status on treatment response, anti‐CCP antibody–positive IP patients showed less benefit from treatment, particularly early treatment, than anti‐CCP antibody–negative patients. This provides support for the inclusion of anti‐CCP antibodies as well as RF in the classification criteria for rheumatoid arthritis and for stratification by anti‐CCP antibody status in clinical trials.

     

An altered immune response to Epstein‐Barr nuclear antigen 1 in pediatric systemic lupus erythematosus

Objective

New examples support the concept that host immune responses to pathogenic organisms can act as the nidus for autoimmunity. Two such examples implicate the Epstein‐Barr virus (EBV) in systemic lupus erythematosus (SLE), i.e., data consistent with SLE anti‐Sm and anti–60‐kd Ro autoantibodies emerging from distinct humoral immune responses to Epstein‐Barr nuclear antigen 1 (EBNA‐1). We undertook this study to further test whether the humoral immune response to EBNA‐1 is a risk factor for pediatric SLE.

Methods

Sera from pediatric lupus patients and healthy matched controls were tested for anti–EBNA‐1 by Western blotting and enzyme‐linked immunosorbent assay (ELISA). To define the fine specificity of their anti–EBNA‐1 humoral immune response, fragments of EBNA‐1 and the maximally overlapping unique octapeptides of EBNA‐1 were tested by modified ELISAs.

Results

All 36 pediatric SLE patient sera tested recognized EBNA‐1, while sera from only 25 of 36 matched EBV‐positive controls targeted EBNA‐1 (P < 0.005). Epitope mapping revealed that the humoral anti–EBNA‐1 response in pediatric SLE was distinct from and less restricted than that in matched normal individuals. Meanwhile, no significant differences between SLE patient sera and control sera were observed in the responses to other herpesviruses or in binding to sequential epitopes from cytomegalovirus immediate‐early antigen or EBNA‐2.

Conclusion

Anti–EBNA‐1 antibodies are associated with pediatric‐onset SLE. Furthermore, an altered humoral immune response to EBNA‐1, characteristic of SLE, has been found and may be an important SLE susceptibility factor.

     

Brief Report: Anti–Carbamylated Protein Antibodies Are Present in Arthralgia Patients and Predict the Development of Rheumatoid Arthritis

Objective

Recently, we discovered a new autoantibody system in rheumatoid arthritis (RA): anti–carbamylated protein (anti‐CarP) antibodies. These antibodies have value in predicting joint destruction; however, it is not clear whether they are present before the diagnosis of RA and whether they have value as predictors of RA development. Therefore, we studied whether anti‐CarP antibodies are present in patients with arthralgia and whether their presence is associated with the development of RA.

Methods

Sera from 340 arthralgia patients who did not have clinical signs of arthritis but who were positive for IgM rheumatoid factor (IgM‐RF) and/or anti–cyclic citrullinated peptide 2 (anti–CCP‐2) and 32 healthy controls were tested for anti‐CarP IgG antibodies. Of the patients with arthralgia, 111 were IgM‐RF positive/anti–CCP‐2 antibody negative and 229 were anti–CCP‐2 antibody positive. Patients were observed for the development of RA (based on the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria) during a median followup period of 36 months. Cox proportional hazards regression analysis was performed to compare the risk of developing RA between arthralgia patients who were positive for anti‐CarP antibodies and those who were negative for anti‐CarP antibodies during followup.

Results

Anti‐CarP antibodies were present in the sera of 39% of the patients. One hundred twenty patients developed RA, after a median of 12 months (interquartile range [IQR] 6–24). The presence of anti‐CarP antibodies was associated with the development of RA in the entire arthralgia cohort after correction for RF and anti–CCP‐2 antibody status (hazard ratio 1.56 [95% confidence interval 1.06–2.29], P = 0.023), as well as in the anti–CCP‐2 antibody–positive subgroup (odds ratio 2.231 [95% confidence interval 1.31–3.79], P = 0.003).

Conclusion

Anti‐CarP antibodies are present in patients with arthralgia, and their presence predicts the development of RA independent of anti–CCP‐2 antibodies.