IgG anti–NR2 glutamate receptor autoantibodies from patients with systemic lupus erythematosus activate endothelial cells

Objective

To investigate the possibility that IgG anti–NR2 glutamate receptor antibodies (anti‐NR2) derived from patients with systemic lupus erythematosus (SLE) cause an immunologic interaction with endothelial cells (ECs) in the blood–brain barrier, resulting in inflammation of the blood–brain barrier, allowing the entrance of these autoantibodies into the cerebrospinal fluid.

Methods

Purified IgG anti‐NR2 antibodies from 14 patients with SLE were tested for their ability to bind to double‐stranded DNA (dsDNA) and ECs, to modulate endothelial adhesion molecule expression and cytokine production by ECs, and to activate the NF‐κB pathways in the ECs. Purified IgG from 5 normal subjects was used as a negative control.

Results

Purified IgG anti‐NR2 antibodies bound to dsDNA in an IgG‐dose–dependent manner. This interaction up‐regulated the expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on the EC surface and increased the production of interleukin‐6 (IL‐6) and IL‐8, but not tumor necrosis factor α or IL‐1β, by ECs. Purified IgG anti‐NR2 also activated the degradation of cytoplasmic IκB, indicating the activation of NF‐κB in the ECs.

Conclusion

EC activation through the NF‐κB signaling pathway induced by IgG anti‐NR2 antibodies in the central nervous system of SLE patients may lead to inflammation of the blood–brain barrier, initiating the pathogenesis of neuropsychiatric SLE.

     

In vivo analysis of the apoptosis‐inducing effect of anti–endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay

Objective

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti–endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis‐inducing effect of AECAs in vivo.

Methods

The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD‐200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA‐positive serum samples from UCD‐200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1‐week‐old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL.

Results

Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA‐positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls.

Conclusion

This study is the first to demonstrate the in vivo apoptosis‐inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

     

Platelet‐activating factor–acetylhydrolase and other novel risk and protective factors for cardiovascular disease in systemic lupus erythematosus

Objective

There is an important inflammatory component to atherosclerosis and cardiovascular disease (CVD). It is therefore interesting that the risk of CVD is high in inflammatory diseases such as systemic lupus erythematosus (SLE). In this study, we investigated nontraditional risk factors for the development of CVD in patients with SLE.

Methods

Twenty‐six women (mean age 52 years) with SLE and a history of CVD were compared with 26 age‐matched women with SLE and no clinical manifestations of CVD (SLE controls) and 26 age‐matched healthy women (population controls). Serum levels of several novel nontraditional risk and protective factors were determined: heat‐shock protein (HSP)–related factors (Hsp60, Hsp70, anti–human Hsp60, anti–human Hsp70, and anti–mycobacterial Hsp65), platelet‐activating factor–acetylhydrolase (PAF‐AH) activity, secretory phospholipase A₂ GIIA (sPLA₂), and anti–endothelial cell antibody (AECA). The intima‐media thickness and the presence of plaques in the common carotid arteries were determined by B‐mode ultrasound as a surrogate measure of atherosclerosis.

Results

Levels of PAF‐AH, but not HSP‐related factors, AECA, or sPLA₂, were significantly increased in SLE cases. Only PAF‐AH discriminated between SLE cases and SLE controls (P = 0.005). PAF‐AH was significantly associated with low‐density lipoprotein (LDL) cholesterol and total cholesterol in the SLE cases (r = 0.50, P = 0.0093 and r = 0.54, P = 0.0045), but not in either control group.

Conclusion

The increased levels of PAF‐AH in SLE cases and the association between PAF‐AH and LDL cholesterol adds support to the notion that PAF‐AH may promote atherothrombosis in SLE. The role of HSPs in CVD is complex, since anti‐Hsp65 appears to be associated with the presence of CVD, whereas Hsp70 might protect against it. In this cross‐sectional study, levels of HSP‐related factors, AECA, and sPLA₂ were not associated with CVD in SLE.

     

Mouse monoclonal anti–human thrombomodulin antibodies bind to and activate endothelial cells through NF‐κB activation in vitro

Objective

To clarify whether mouse monoclonal antibodies (mAb) against human thrombomodulin (TM), which react with human TM present on the endothelial cell (EC) surface, have anti–endothelial cell antibody (AECA) activity and influence antiinflammatory properties of human TM expressed on the EC surface in vitro.

Methods

Three preparations of mouse mAb against human TM that react with different sites of the human TM epidermal growth factor–like domain were tested for their ability to 1) bind to ECs, 2) modulate cytokine secretion from ECs, EC adhesion molecule expression, and neutrophil adhesion to ECs, and 3) stimulate nuclear translocation of NF‐κB through the degradation of cytoplasmic IκB in ECs. Recombinant human interleukin‐1β (IL‐1β) was used as a positive control, and mouse IgG1 and mouse IgG2a were used as negative controls.

Results

The 3 preparations of mouse mAb against human TM that bind to unfixed EC monolayers enhanced IL‐6 and IL‐8 secretion from ECs, up‐regulated expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on EC monolayers, and enhanced neutrophil adhesion to ECs to a degree similar to that observed with IL‐1β stimulation, but they did not induce the secretion of tumor necrosis factor α or IL‐1β from ECs throughout the incubation period. The 3 preparations stimulated nuclear translocation of NF‐κB through the degradation of cytoplasmic IκB. Mouse IgG1 and mouse IgG2a did not exhibit such effects.

Conclusion

These results suggest the possibility that AECA can react with antigens such as TM that are present on the EC surface and activate ECs. Such events on ECs may lead to vascular inflammation and damage in patients with connective tissue diseases and vasculitis in which AECA are present.

     

Reactivity of γ/δ T cells to human 60‐kd heat‐shock protein and their cytotoxicity to aortic endothelial cells in Takayasu arteritis

Objective

Increased numbers of circulating γ/δ T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat‐shock protein Hsp60/65 and γ/δ T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that γ/δ T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of γ/δ T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA.

Methods

Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating γ/δ T cells to human Hsp60 were detected by flow cytometry–based bromodeoxyuridine incorporation assay. Cytotoxicity of the γ/δ T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay.

Results

The γ/δ T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the γ/δ T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean ± SD proliferative response of γ/δ T cells in patients with TA was 21.4 ± 11.3%, compared with 4.2 ± 1.2% in patients with SLE and 4.01 ± 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the γ/δ T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 ± 15.0% versus 9.6 ± 2.13% in SLE patients and 8.1 ± 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of γ/δ T cells with Hsp60. The cytotoxicity of the γ/δ T cells was significantly inhibited by treatment of these cells with concanamycin A and anti–Fas ligand–blocking antibodies.

Conclusion

The results show that γ/δ T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and γ/δ T cells in the pathogenesis of TA.

     

Human α‐enolase from endothelial cells as a target antigen of anti–endothelial cell antibody in Behçet's disease

Objective

To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti–endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD.

Methods

The proteomics technique, with 2‐dimensional gel electrophoresis and matrix‐assisted laser desorption ionization−time‐of‐flight (MALDI‐TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme‐linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis).

Results

Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be α‐enolase by 2‐dimensional gel electrophoresis followed by immunoblotting and MALDI‐TOF mass spectrometry. Recombinant α‐enolase protein was isolated and refined by gene cloning. On Western blots, AECA‐positive IgM from the sera of patients with active BD reacted strongly with recombinant human α‐enolase. BD patient sera positive for anti–α‐enolase did not react with human γ‐enolase. On dot‐blotting, reactivity to human α‐enolase was detected only in the IgM‐positive group. Fifteen of the 18 AECA‐positive sera that were positive for the HDMEC antigen showed reactivity to recombinant α‐enolase IgM antibody by ELISA.

Conclusion

The α‐enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to α‐enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, α‐enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

     

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.

     

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.

     

Association of autoantibodies to heat‐shock protein 60 with arterial vascular events in patients with antiphospholipid antibodies

Objective

Anti–heat shock protein 60 autoantibodies (anti‐Hsp60) are associated with cardiovascular disease and are known to affect endothelial cells in vitro, and we have recently shown that anti‐Hsp60 promote thrombosis in a murine model of arterial injury. Based on those findings, we undertook the present study to investigate the hypothesis that the presence of anti‐Hsp60, alone or in combination with other thrombogenic risk factors, is associated with an elevated risk of vascular events.

Methods

The study population was derived from 3 ongoing cohort studies: 2 independent systemic lupus erythematosus (SLE) registries and 1 cohort comprising SLE patients and non‐SLE patients. Data from a total of 402 participants were captured; 199 of these participants had had confirmed vascular events (arterial vascular events in 102, venous vascular events in 76, and both arterial and venous vascular events in 21). Anti‐Hsp60 were detected by enzyme‐linked immunoassay, and association with vascular events was assessed by regression analysis.

Results

Multiple regression analysis revealed that arterial vascular events were associated with male sex, age, and hypertension. Analyses of the vascular events according to their origin showed an association of anti‐Hsp60 with arterial vascular events (odds ratio 2.26 [95% confidence interval 1.13–4.52]), but not with venous vascular events. Anti‐Hsp60 increased the risk of arterial vascular events (odds ratio 5.54 [95% confidence interval 1.89–16.25]) in antiphospholipid antibody (aPL)–positive, but not aPL‐negative, individuals.

Conclusion

We demonstrate that anti‐Hsp60 are associated with an increased risk of arterial vascular events, but not venous vascular events, in aPL‐positive individuals. These data suggest that anti‐Hsp60 may serve as a useful biomarker to distinguish risk of arterial and venous vascular events in patients with aPL.

     

Anti–topoisomerase I (Anti–Scl‐70) antibodies in patients with systemic lupus erythematosus

Objective

To investigate the presence and clinical significance of anti–Scl‐70 antibodies in patients with systemic lupus erythematosus (SLE).

Methods

Levels of antibodies against Scl‐70 were determined by a commercial clinical enzyme‐linked immunosorbent assay (ELISA) during routine evaluation. Results were verified by an additional ELISA with a characterized bovine Scl‐70, by ELISA with a recombinant human topoisomerase I, by Western blot, and by double diffusion in agar gel. Disease activity was estimated retrospectively by the Systemic Lupus Activity Measure (SLAM).

Results

Of 128 consecutive SLE patients, 25% were positive for anti–Scl‐70 antibody; this antibody activity was cognate in nature. No SLE patient could be classified as also having systemic sclerosis. The levels of anti–Scl‐70 were significantly correlated with the SLAM score for the entire cohort (r = 0.563, P < 0.001) and for 7 individual patients with multiple longitudinal measurements (r = 0.755–0.951, P < 0.001; n = 6) (r = 0.378, P < 0.05; n = 1). A significant correlation was also found between the levels of anti–Scl‐70 and anti–double‐stranded DNA antibodies (r = 0.558, P < 0.001). Patients with anti–Scl‐70 had significantly higher risk of pulmonary hypertension (P < 0.01) and renal involvement (P < 0.001) than patients without this antibody.

Conclusion

Anti–Scl‐70 antibody is present in a significant subset of patients with SLE. For this subset, it offers a good correlate of disease activity and suggests increased risk for pulmonary hypertension and nephritis.

     

Anti–N‐methyl‐D‐aspartate receptor antibodies,cognitive dysfunction,and depression in systemic lupus erythematosus

Objective

To assess the association of cognitive dysfunction and depression with serum antibodies to N‐methyl‐D ‐aspartate (NMDA) receptor (anti‐NR2) and analyze clinical and neuroimaging correlates in patients with systemic lupus erythematosus (SLE).

Methods

Sixty patients underwent neurocognitive assessment, evaluation for depression with the Beck Depression Inventory II (BDI‐II) and psychiatric interview (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition [DSM‐IV] criteria), brain magnetic resonance imaging, and proton magnetic resonance spectroscopy imaging (¹H‐MRSI). Cognition was assessed in 5 domains: memory, attention/executive, visuospatial, motor, and psychomotor, and adjusted to each individual's best level of prior cognitive functioning estimated from the reading subtest of the Wide Range Achievement Test–3 (WRAT‐3). Serum anti‐NR2 antibodies were measured by enzyme‐linked immunosorbent assay using a pentapeptide from the human NMDA receptor.

Results

Cognitive dysfunction was found in 28 of 60 patients (mild in 8, moderate in 20) before adjustment for WRAT‐3 and in 35 of 60 patients (mild in 15, moderate in 11, and severe in 9) after adjustment for WRAT‐3. The changes were most pronounced in the memory and visuospatial domains. There was no significant association between anti‐NR2 antibody levels and cognition. On ¹H‐MRSI, patients with moderate or severe cognitive dysfunction had significantly higher choline:creatine ratios in the dorsolateral prefrontal cortex and the white matter, compared with patients with mild or absent cognitive dysfunction. Anti‐NR2 antibodies were significantly correlated with BDI scores; patients with BDI‐II scores of ≥14 had higher serum levels of anti‐NR2 antibodies (P = 0.005, 95% confidence interval 0.83, 4.31), and there was a trend toward higher anti‐NR2 antibody levels among patients who fulfilled the DSM‐IV criteria for major depression.

Conclusion

Serum anti‐NR2 antibodies are associated with depressive mood but not with cognitive dysfunction in SLE at a given time point. Larger longitudinal studies are needed to address the possible association between anti‐NR2 antibodies and depression in SLE.

     

Autoantibody to c‐Mpl (thrombopoietin receptor) in systemic lupus erythematosus: Relationship to thrombocytopenia with megakaryocytic hypoplasia

Objective

To examine the prevalence, clinical associations, and pathogenic role of autoantibodies to c‐Mpl, the thrombopoietin (TPO) receptor, in patients with systemic lupus erythematosus (SLE).

Methods

Sera from 69 SLE patients, 84 patients with idiopathic thrombocytopenic purpura (ITP), and 60 healthy individuals were screened for anti–c‐Mpl antibodies by enzyme‐linked immunosorbent assay using recombinant c‐Mpl as an antigen. Clinical findings, autoantibody profiles, and serum TPO levels were compared between SLE patients with and without anti–c‐Mpl antibodies. A pathogenic role for the anti–c‐Mpl antibody was evaluated by examining its inhibitory effect on TPO‐dependent cell proliferation and megakaryocyte colony formation.

Results

Serum anti–c‐Mpl antibody was detected in 8 SLE patients (11.6%) and 7 ITP patients (8.3%), but in none of the healthy controls. Anti–c‐Mpl antibody was associated with thrombocytopenia (P = 0.0002) and a decrease in bone marrow megakaryocytes (P = 0.02) in SLE patients. Serum TPO levels in thrombocytopenic SLE patients with anti–c‐Mpl antibodies were significantly elevated compared with levels in those without the antibodies (P = 0.007). IgG fractions purified from anti–c‐Mpl antibody–positive sera bound to c‐Mpl expressed on the cell surface and inhibited TPO‐dependent cell proliferation and megakaryocyte colony formation.

Conclusion

Autoantibody to c‐Mpl is present in a subset of SLE patients with thrombocytopenia and megakaryocytic hypoplasia. It is likely that the impaired thrombopoiesis in these patients is mediated by the anti–c‐Mpl antibody, which functionally blocks an interaction between TPO and c‐Mpl.

     

Inhibition of lupus disease by anti–double‐stranded DNA antibodies of the IgM isotype in the (NZB × NZW)F1 mouse

Objective

In systemic lupus erythematosus (SLE), immune complexes (ICs) containing pathogenic IgG anti–double‐stranded DNA (anti‐dsDNA) autoantibodies are deposited in renal capillaries and initiate glomerulonephritis (GN) by the activation of complement and effector cells. In contrast, it has been demonstrated that the presence of IgM anti‐dsDNA antibodies correlates negatively with the development of GN in SLE. The aim of this study was to determine whether anti‐dsDNA antibodies of the IgM isotype protect against IC‐mediated organ damage in SLE.

Methods

Lupus‐prone (NZB × NZW)F₁ mice (females) were treated with murine monoclonal IgM anti‐dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 μg/week starting at 16 weeks of age (prophylactic) or at 24 weeks of age (therapeutic).

Results

Mice treated with IgM anti‐dsDNA exhibited a delayed onset of proteinuria and a reduced degree of renal pathology, which resulted in significantly improved survival as compared with control mice. Serum concentrations of IgG anti‐dsDNA antibodies were not significantly modified. However, glomerular deposition of ICs was markedly reduced in both treatment protocol groups. In contrast, higher amounts of IgG and IgM and increased expression of Fcγ receptor were demonstrated in liver sections from the treated mice compared with the untreated mice, suggesting an enhanced clearance of soluble ICs from phagocytic cells of the reticuloendothelial system.

Conclusion

These data demonstrate the efficacy of IgM anti‐dsDNA treatment in inhibiting the pathologic changes of lupus in (NZB × NZW)F₁ mice. Lower glomerular IC deposition is associated with a reduced inflammatory response and impaired organ damage. The reduced frequency of GN in SLE patients who have IgM anti‐dsDNA antibodies may therefore reflect a disease‐modifying effect of this class of autoantibodies that has potential therapeutic implications. Our findings should encourage the development of new therapeutic modalities using IgM anti‐dsDNA antibodies in humans with SLE.

     

Cross‐reactivity of human lupus anti‐DNA antibodies with α‐actinin and nephritogenic potential

Objective

Cross‐reactivity with kidney antigens is believed to be a critical determinant in the renal pathogenicity of anti–double‐stranded DNA (anti‐dsDNA) antibodies. Murine nephritogenic anti‐dsDNA antibodies have been shown to cross‐react with α‐actinin, and anti–α‐actinin antibodies have been found to be deposited in the kidneys of lupus mice with active nephritis. Furthermore, in humans with systemic lupus erythematosus (SLE), it has been found that a greater proportion of polyclonal IgG anti‐dsDNA antibodies from patients with renal involvement bind to α‐actinin than do those from patients without renal disease. We undertook this study to substantiate a direct link between cross‐reactive anti‐dsDNA/anti–α‐actinin antibodies and the pathogenesis of lupus nephritis in humans.

Methods

A panel of 10 anti‐dsDNA and/or anti–α‐actinin antibodies was generated by Epstein‐Barr virus transformation of lymphocytes from patients with SLE and was extensively characterized. Antibody binding was studied by enzyme‐linked immunosorbent assay and Western blotting. Antibody potential for pathogenicity was assessed by measuring binding to isolated glomeruli and mesangial cells and by evaluation of histologic features of the kidney following injection in vivo.

Results

All anti‐dsDNA antibodies isolated also bound α‐actinin. Cross‐reactive antibodies bound to mesangial cells and to isolated glomeruli ex vivo. Binding to glomeruli was not inhibited by DNase treatment, but could be abrogated by α‐actinin. Furthermore, histopathologic abnormalities seen in mice injected intraperitoneally with a cross‐reactive cell line included fusion of podocyte foot processes and subepithelial and subendothelial deposition.

Conclusion

These studies provide strong support for the hypothesis that α‐actinin is a major cross‐reactive target for anti‐dsDNA antibodies in SLE patients. Cross‐reactive anti‐dsDNA/anti–α‐actinin antibodies from SLE patients are pathogenic and may contribute to the kidney lesions in lupus nephritis.

     

Induction of endothelial cell apoptosis by heat-shock protein 60-reactive antibodies from anti-endothelial cell autoantibody-positive systemic lupus erythematosus patients

OBJECTIVE: To determine whether anti-endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted. METHODS: AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity-purified on this antigen and incubated with ECs to determine their physiologic effects. Anti-Hsp60 antibody titers were determined by enzyme-linked immunosorbent assay. The relationship of anti-Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed. RESULTS: Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60-kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti-Hsp60 antibodies from commercial sources or affinity-purified from SLE sera that bound ECs. Incubation of ECs with these anti-Hsp60 antibodies induced apoptosis in a time- and dose-dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti-Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti-Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients. CONCLUSION: The presence of Hsp60 at the surface of ECs serves as a target for the anti-Hsp60 antibodies in SLE sera. These anti-Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.     

Anti–ribosomal P protein antibody in human systemic lupus erythematosus up‐regulates the expression of proinflammatory cytokines by human peripheral blood monocytes

Objective

Autoantibodies to ribosomal P proteins (anti‐P antibodies) are detected in 12–16% of patients with systemic lupus erythematosus (SLE) and have been found to be associated with some manifestations of the disease, including lupus psychosis and hepatitis. Recent studies have disclosed that anti‐P antibodies react with activated T cells but not with B cells, suggesting possible direct effects of anti‐P antibodies on immune regulation. The present study was designed to explore the presence of the epitope recognized by anti‐P antibodies on human peripheral blood monocytes.

Methods

Highly purified peripheral blood monocytes obtained from healthy donors were cultured with or without interferon‐γ (IFNγ) in the presence of either anti‐P antibodies purified by affinity chromatography from the sera of patients with SLE or control IgG.

Results

Flow cytometry analysis disclosed that fresh (day 0) monocytes did not express the ribosomal P epitope, whereas expression of the ribosomal P epitope was induced on annexin V–negative monocytes after activation through plastic adherence for 48 hours. More important, anti‐P antibodies (compared with normal IgG or IgG from SLE patients devoid of anti‐P antibodies) enhanced the production of tumor necrosis factor α (TNFα) and interleukin‐6 (IL‐6) by activated monocytes. Accordingly, anti‐P antibodies also up‐regulated the expression of TNFα and IL‐6 messenger RNA in activated monocytes. Of note, F(ab′)₂ fragments of anti‐P antibodies, which do not result in Fcγ receptor (FcγR) crosslinking, also effectively up‐regulated the expression of TNFα and IL‐6.

Conclusion

These results indicate that human peripheral blood monocytes express the ribosomal P epitope upon activation, irrespective of induction of apoptosis. Moreover, the data suggest that anti‐P antibodies might modify a variety of inflammatory responses through up‐regulation of the expression of proinflammatory cytokines in monocytes, in a manner that does not involve FcγR crosslinking.

     

Association of anti–citrullinated vimentin and anti–citrullinated α‐enolase antibodies with subsets of rheumatoid arthritis

Objective

To determine whether the anti–citrullinated vimentin peptide 60–75 (anti–Cit‐vimentin) and the immunodominant anti–citrullinated α‐enolase peptide 1 (anti–CEP‐1) antibodies are associated with subsets of patients with rheumatoid arthritis (RA) independently of the associations between anti–cyclic citrullinated peptide (anti‐CCP) antibodies and clinical features of RA.

Methods

The 3 antibody types were quantified by enzyme‐linked immunosorbent assay (ELISA) in serum samples from 521 patients with RA and 173 healthy controls of Spanish ancestry. Genotypes for HLA–DRB1 alleles and rs2476601 in PTPN22 were available for these patients and controls plus an additional 106 healthy controls. A combined analysis of the 3 antibodies was conducted using stratified contingency tables and logistic regression models.

Results

A differential, particularly strong, and independent association was observed between the presence of anti–Cit‐vimentin antibodies and the presence of shared epitope (SE) alleles, specifically in patients carrying 2 SE alleles, and between the presence of anti– Cit‐vimentin antibodies and the prevalence of joint erosion. Associations were observed between anti–CEP‐1 positivity and the presence of HLA–DRB1 and PTPN22 risk alleles and their additive interaction. These associations were not accounted for by the anti‐CCP status.

Conclusion

Our results indicate that the 2 antibodies against citrullinated peptides analyzed in this study add specific information beyond that obtained with the anti‐CCP status. They define subgroups of patients with RA in which genetic factors have different weight and there is an observed difference in the prevalence of erosions.