Enhanced recognition of reactive oxygen species damaged human serum albumin by circulating systemic lupus erythematosus autoantibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore, we hypothesized that oxidative by-products, such as hydroxyl radical (√OH), could lead to neoantigens like √OH damaged human serum albumin (HSA), which could in turn initiate autoimmunity in SLE. In the present study, the binding characteristics of SLE autoantibodies with native and √OH damaged HSA were assessed. SLE patients (n = 74) were examined by direct binding ELISA and the results were compared with healthy age- and sex-matched controls (n = 44). High degree of specific binding by 52.7% of patients sera towards √OH damaged HSA, in comparison to its native analogue (p < 0.05) was observed. Normal human sera showed negligible binding with either antigen. Competitive ELISA and gel retardation assays reiterate the direct binding results. The increase in total serum protein carbonyl levels in the SLE patients was largely due to an increase in oxidized albumin. HSA of SLE patients (SLE-HSA) and normal subjects (normal-HSA) were purified. Spectroscopic analysis confirmed that the SLE-HSA samples contained higher levels of carbonyls than normal-HSA (p < 0.01). SLE-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in SLE patients.     

Natural autoantibodies in systemic lupus erythematosus.

We have tested the sera of 25 patients with systemic lupus erythematosus (SLE) for antibody activity against a panel of six antigens: DNA, TNP, actin, tubulin, myosin, albumin. Eluates from renal biopsy tissue were also tested. Sera from patients with lupus nephritis were found to contain high titres of IgA antibodies directed against the antigens of the panel, and marked IgG anti-DNA and anti-TNP antibody activity. The IgG anti-TNP antibodies isolated from SLE serum by affinity chromatography on a TNP-immunoadsorbent, were also found to possess anti-DNA activity. Kidney eluates obtained from biopsy specimens of SLE patients contained IgG antibodies strictly specific for DNA in three out of the nine patients tested, while three eluates from the remaining six patients reacted with DNA and TNP and three with DNA and all the other antigens of the panel. These results strongly suggest that in SLE sera there are at least three populations of circulating anti-DNA antibodies: those strictly specific for DNA, those recognizing DNA and TNP and those recognizing DNA and other macromolecules. Furthermore, because six out of nine of the eluates contained antibodies with an absolute or restricted specificity for DNA, this suggests that these antibodies are more often pathogenic than the polyspecific ones recognizing DNA and other macromolecules.     

Anti-ribosomal ribonucleoprotein autoantibodies in systemic lupus erythematosus

Sera from systemic lupus erythematosus (SLE) patients giving a fluorescent ribosomal pattern on tissue and cell preparations also showed precipitating autoantibodies against purified rat liver ribosomes. Ribosomal antigen is also present in rabbit thymus cellular extract (RTE), since the same sera gave precipitin lines against RTE in identity with ribosomes. Immunofluorescent staining was completely inhibited by serum absorption with ribosomes or with RTE. However ribosomal RNA and RNase or trypsin-treated ribosomes failed to react with these autoantibodies as demonstrated in immunoabsorption and immunodiffusion studies. These data suggest that these sera contain autoantibodies directed against some antigenic site composed of a portion of both RNA and ribosomal protein. Ribosomal autoantibodies were detectable at a low frequency in SLE patients characterized by an active disease and renal involvement.     

IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus

We describe the production of six mouse-human heterohybridomas secreting human IgG anti-dsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of anti-DNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay. Inhibition studies revealed a remarkable specificity for certain polynucleotide structures. To our knowledge these are the first hybridomas described in the human system that secrete anti-dsDNA antibodies of the IgG class.     

Increased binding of circulating systemic lupus erythematosus autoantibodies to recombinant interferon alpha 2b

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various types of immunological abnormalities including circulating and tissue‐fixed autoantibodies reactive with autoantigens. The mechanism that can explain the production of these antibodies is unclear. Here we address the binding specificity of SLE autoantibodies with recombinant alpha interferon 2b (hrIFN α‐2b), commercially available interferon (IFN α‐2b), and the gene (cIFN α‐2b) encoding this interferon. hrIFN α‐2b showed higher binding with naturally occurring SLE autoantibodies as compared to IFN α‐2b (p < 0.05) or cIFN α‐2b gene (p < 0.001) as assessed by direct binding, inhibition ELISA, and quantitative precipitin titration. The relative affinity of SLE autoantibodies for hrIFN α‐2b, IFN α‐2b, and cIFN α‐2b gene was in the order of 1.13 × 10^?7, 1.38 × 10^?6, and 1.22 × 10^?6, respectively. hrIFN α‐2b is shown to have unique epitopes that would explain the possible antigenic role of hrIFN α‐2b in the generation of SLE autoantibodies. Anti‐hrIFN α‐2b antibodies have been shown to represent an alternative immunological probe for the estimation of interferon alpha 2b in the serum of SLE patients.     

Enhanced tryptophan degradation in systemic lupus erythematosus

In vitro and in vivo, tryptophan degradation was found to be associated with T cell functional loss and tolerance induction. In systemic lupus erythematosus (SLE) besides the Th2-type cytokine interleukin-10, Th1-type cytokines including interferon-gamma (IFN-gamma) are expressed especially during exacerbation of the disease. IFN-gamma stimulates the enzyme indoleamine (2,3)-dioxygenase (IDO) converting tryptophan to the metabolite kynurenine which in macrophages is subsequently degraded to other, partly neurotoxic compounds like quinolinic acid, and finally to nicrotinamides. We measured kynurenine and tryptophan concentrations in the sera of 55 SLE patients. In these patients, the concentrations of tryptophan (median, interquartile range: 53.9, 45.7-64.1 microM) were lower (p < 0.0001), and the kynurenine concentrations (2.45, 1.75-3.40 microM) were increased (p < 0.0005) compared to healthy blood donors (70.0, 63.8-80.6; 1.80, 1.45-2.27 microM, respectively). Also the kynurenine per tryptophan quotients (K/T), which allow to estimate IDO activity, were significantly higher in patients than in normals (0.043, 0.033-0.062 vs. 0.027, 0.021-0.030; p < 0.0001), indicating enhanced IDO-induced tryptophan degradation in SLE. There was no significant relationship between tryptophan, kynurenine and the SLEDAI, and also the correlation of K/T with SLEDAI was rather weak (rs = 0.243, p < 0.05). Higher K/T was found in patients presenting with serositis (p = 0.01), decrease of complement (c3, c4; p < 0.01) and blood count change (anemia, leucopenia, lymphopenia; p = 0.032) than in patients without such disease manifestations. The significant correlation found between K/T and neopterin (rs = 0.808, p < 0.001), a marker of immune activation, points to a role of immune activation to be responsible for tryptophan degradation in SLE patients.     

A solid phase radioimmunoassay for detection of serum autoantibodies in systemic lupus erythematosus.

Solid phase radioimmunoassay (RIA) methods for measuring autoantibodies in systemic lupus erythematosus (SLE) patients' serum were developed to improve the sensitivity and quantitative precision of these determinations. Two mechanical systems were studied: (1) acetone fixed cell monolayers in glass tubes and (2) antigen coated plastic beads. Both systems were sensitive and reproducible, giving serum dilution end-points between two and four orders of magnitude (100-10,000 times) greater than those obtained by fluorescence microscopy. The most sensitive, versatile system involves the coating of plastic beads with nuclear antigen(s), incubation overnight with sera and labelling with 125I conjugated antihuman globulin. Linear binding of this radioactive tag is obtained over a wide range of SLE serum dilutions and the slopes of the serum dilution titration curves are almost identical for all SLE patients' sera we have tested. Therefore, a standard titration curve can be constructed from the results with a positive serum, and end-point dilutions of unknown sera estimated from results obtained with a single serum dilution. Alternatively, binding ratios of unknown sera can be usefully compared at fixed dilutions with standard positive and negative sera. For example, high binding ratios (greater than 3.0) were obtained with 19/20 SLE sera and 0/20 control sera. Antigens used in these systems include crude, whole-cell lysates and lysates from purified nuclei. These RIA methods appear to provide certain advantages over existing autoantibody assay methods because they are relatively simple, sensitive, reproducible and potentially capable of measuring a variety of autoantibody specificities.     

Binding of circulating SLE autoantibodies to oxygen free radical damaged chromatin

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various immunologic disorders, including production of autoantibodies, formation of immune complexes, decreased serum complement levels, and lymphocytopenia. One of the hallmarks of this disease is the loss of tolerance to nuclear antigens. The dominant presence of antibodies against the exposed conformational epitopes on chromatin strongly suggests that the pathogenic immune response in lupus is driven by chromatin. In the present study, the binding of SLE autoantibodies with native chromatin and oxygen free radical damaged chromatin was studied. As assessed by direct binding and inhibition ELISA, circulating SLE autoantibodies exhibited a high degree of specificity towards the reactive oxygen species (ROS)-modified chromatin in comparison to native chromatin and this binding specificity was reiterated visually by gel retardation assay. The data suggested possible role of modified chromatin in the induction of SLE autoantibodies and higher recognition of oxidatively damaged chromatin by antibodies in sera of SLE patients. It is indicated that free radical modified chromatin or nucleosomes might be the antigen for the production of circulating autoantibodies in SLE.     

Binding of circulating SLE autoantibodies to oxygen free radical damaged chromatin

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various immunologic disorders, including production of autoantibodies, formation of immune complexes, decreased serum complement levels, and lymphocytopenia. One of the hallmarks of this disease is the loss of tolerance to nuclear antigens. The dominant presence of antibodies against the exposed conformational epitopes on chromatin strongly suggests that the pathogenic immune response in lupus is driven by chromatin. In the present study, the binding of SLE autoantibodies with native chromatin and oxygen free radical damaged chromatin was studied. As assessed by direct binding and inhibition ELISA, circulating SLE autoantibodies exhibited a high degree of specificity towards the reactive oxygen species (ROS)-modified chromatin in comparison to native chromatin and this binding specificity was reiterated visually by gel retardation assay. The data suggested possible role of modified chromatin in the induction of SLE autoantibodies and higher recognition of oxidatively damaged chromatin by antibodies in sera of SLE patients. It is indicated that free radical modified chromatin or nucleosomes might be the antigen for the production of circulating autoantibodies in SLE.     

Polyreactive autoantibodies in systemic lupus erythematosus have pathogenic potential

The present study was undertaken to determine whether germline encoded and polyreactive antibodies might be pathogenic and whether the breach of early tolerance checkpoints in systemic lupus erythematosus (SLE) might lead to a population of B cells expressing germline encoded antibodies that become pathogenic merely by class switching to IgG in a pro-inflammatory milieu.We demonstrate here that IgM, DNA-reactive antibodies obtained from lupus patients that are unmutated and display polyreactivity can bind to isolated glomeruli and exhibit neurotoxic potential.Thus, the IgM polyreactive repertoire in SLE includes antibodies that may acquire pathogenic function merely by undergoing class-switch recombination to become IgG antibodies.     

High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus

Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H₂O₂, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice.     

Experimental induction of systemic lupus erythematosus by recognition of foreign Ia

A chronic GVH reaction induced in normal mice results in a syndrome that closely resembles SLE. In this study, we compared the autoimmune GVH syndrome induced in parent (C57BL/6Kh [B6] and B6.C-H-2bm12 [bm12]) and F1 [( B6 x bm12]F1) mice by transfer of parental spleen cells. A majority of the mice in all groups developed autoantibodies to chromatin and erythrocytes, and some of the mice also produced anti-dsDNA antibodies. The predominant isotype of the anti-chromatin autoantibodies was found to be IgG2a, although high levels of IgG2b antibodies were also present. Autoantibody production was in general more intense and more prevalent in parent----F1 hybrid combinations, compared to parent----parent infusions. No influence of host or graft gender was observed. These studies show that a chronic GVH reaction can be induced by both parent----parent and parent----hybrid combinations.     

系统性红斑狼疮自身抗体研究进展

经化学修饰的组织抗原,与正常组织成分有交叉反应的外来抗原、隔绝的体内自身成分及低分化的组织抗原等在一定条件下可刺激机体组织产生自身抗体.某些自身抗体因对系统性红斑狼疮(SLE)的判断具有高度特异性,已成为诊断SLE的血清指标或特异性抗体,有些自身抗体与疾病的活动性有相关性.因此,测定自身抗体有助于SLE的诊断,并对疾病的活动程度,观察治疗效果,指导临床用药具有重要的临床意义.     

The role of autoantibodies in pediatric neuropsychiatric systemic lupus erythematosus

Neuropsychiatric syndromes are prevalent in pediatric patients with systemic lupus erythematosus (SLE) and often manifest early in disease course and with significant associated morbidity. Postulated pathogenic mechanisms of peripheral and central nervous system events include vasculopathy, autoantibody effects and systemic inflammation. The pathogenic roles of anti-phospholipid, anti-ribosomal-P and anti-neuronal autoantibodies have been examined in both focal and diffuse adult neuropsychiatric syndromes. Few studies have probed associations between these autoantibodies and pediatric neuropsychiatric SLE (NP-SLE). Retrospective review of a large ethnically diverse pediatric SLE cohort revealed anti-phospholipid, anti-ribosomal P, and anti-neuronal antibodies to be more prevalent than in many adult studies. Rates of anti-phospholipid and anti-ribosomal P antibody positivity were similar to those of other pediatric reports. Association between anti-neuronal antibodies and NP-SLE events appeared statistically significant in this cohort. Prospective inception cohort studies will need to be undertaken to investigate the significance and utility of autoantibody testing in pediatric NP-SLE.     

Elevated serum anti-endothelial cell autoantibodies titer is associated with lupus nephritis in patients with systemic lupus erythematosus.

BACKGROUND AND PURPOSE: Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease associated with endothelial dysfunction and the existence of multiple species of autoantibodies. However, the association between endothelial dysfunction and renal manifestations remains unclear in Taiwanese SLE patients. METHODS: Serum samples were collected from SLE patients with biopsy-proven lupus nephritis (n = 32), stable SLE patients (n = 32) and healthy controls (n = 32). The SLE Disease Activity Index (SLEDAI) of SLE patients was scored, and levels of anti-endothelial cell antibodies (AECA) and anti-endothelial activities in serum samples were measured by cell-enzyme-linked immunosorbent assay and crystal violet assay, respectively, using cultured human endothelial EA.hy926 cells. RESULTS: Significantly higher AECA (p<0.001) and anti-endothelial activities (p<0.001) were found in sera from patients with lupus nephritis compared with that from stable SLE patients or controls. Moreover, AECA titers (p<0.001) and anti-endothelial activities (p<0.001) were strongly correlated with SLEDAI scores in these patients. CONCLUSION: The strong correlations of AECA and anti-endothelial activity with lupus nephritis activity support an endothelial origin for renal complications in Taiwanese SLE patients.     

Collagen autoantibodies in patients with vasculitis and systemic lupus erythematosus

In order to determine whether collagen autoantibodies are present in sera from patients with vascular diseases, a highly sensitive solid-phase radioimmunoassay was utilized to evaluate the presence of antibodies against collagen types I to VI and laminin in sera of 20 patients with vasculitis and 20 patients with systemic lupus erythematosus (SLE). Autoantibodies to interstitial collagen types I and II were noted in 20 and 35% of the patients, respectively. Most significantly, 70% of the polyarteritis nodosa patients and 55% of the total vasculitis group had autoantibodies to collagen type IV. In SLE, autoantibodies to collagen type V were detected in 70% and to collagen type IV in 85% of the patient sera. These data indicate that basement membrane and basement membrane-associated collagens may become immunogenic in patients with vasculitis and SLE. The role of these autoantibodies is most likely a result of endothelial damage secondary to the initial inflammatory disease process. However, it can be concluded from the present studies that collagen types IV and V are involved in the immune response and thereby may perpetuate further vascular damage.     

IgG4 autoantibodies to DNA in systemic lupus erythematosus patients

The presence of DNA-specific IgG4 antibodies was demonstrated in the sera of patients with systemic lupus erythematosus (SLE) by a microtiter solid-phase radioimmunoassay. A patient with distal interphalangeal swelling and extensive ulcers in the oral cavity, seronegative for anti-DNA antibodies of the IgG isotype, was found to have anti-DNA autoantibodies exclusively of the IgG4 subclass. These autoantibodies directed against the dsDNA conformation cross-reacted with chondroitin sulfate, dermatan sulfate and heparin.     

The clinical utility of anti-ribosomal P autoantibodies in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that can affect multiple organs and thus has a large spectrum of clinical presentations. Assessment of the autoantibody profile is fundamental for the clinical management of SLE patients, providing important data for diagnosis, clinical characterization and disease activity evaluation. Anti-ribosomal P protein (anti-Rib-P, anti-P) antibody, described in the 1980s, is a serological marker for SLE that is present in 13–20% of cases. This reactivity was initially thought to be associated with neuropsychiatric involvement in SLE, with certain conflicting results. Subsequently, associations of anti-Rib-P with liver and renal involvement in lupus were reported. Recently, anti-Rib-P was detected in autoimmune hepatitis patients. Anti-Rib-P reactivity to Trypanosoma cruzi ribosomal target antigens in patients with Chagas heart disease has also been described. This review focuses on the usefulness of the determination of anti-Rib-P in SLE and in other autoimmune and non-autoimmune disorders in clinical practice.