IgG subclasses of autoantibodies in systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity

The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren's syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.     

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.     

The inhibition of protein synthesis by IgG containing anti-ribosome P autoantibodies from systemic lupus erythematosus patients

Autoantibodies found in approximately 15% of patients with systemic lupus erythematosus recognize three 60 S ribosomal phosphoproteins P0, P1, and P2. Fab fragments obtained from sera of these patients inhibited globin mRNA translation in an in vitro protein synthesizing system which was reversed by the addition of excess ribosomes. Further studies suggested that these antibodies bind to ribosomes in the intact cell. Thus, when IgG fractions from these sera were microinjected into cultured human fibroblasts [35S]methionine incorporation into cellular proteins was inhibited.     

Effect of sera from women with systemic lupus erythematosus or antiphospholipid syndrome and recurrent abortions on human placental explants in culture

BACKGROUND: Systemic lupus erythematosus (SLE) with or without evidence of antiphospholipid antibodies (aPA) and antiphospholipid syndrome (APS) is associated with a high rate of spontaneous abortions. The placenta is thought to be the site of pathological damage in many of these abortions. To test this hypothesis, we studied the effects of sera obtained from women with SLE with or without treatment on human placental explants in culture. METHODS: We cultured 5.5- to 7.5-week-old human placental explants in a culture medium containing F-12 DMEM and 10% FCS or in 90% human serum obtained from nonpregnant women with SLE prior to or after treatment. Culture was carried out for 96 hr. At the end of the culture period, we studied the secretion of the placental hormones estrogen (E2), progesterone (PGN), and human chorionic gonadotropin (hCG). In addition, we studied the proliferation rate (using PCNA staining) and the rate of apoptosis (using ApoTag) of the trophoblastic cells. RESULTS: Placentae grew better in normal human serum than in a chemically defined medium of F-12 DMEM and 10% FCS. Enhanced growth and higher secretion rates for hCG and estradiol (E2) were manifested in placentae cultured in control sera with no change in PGN secretion. Secretion rates of hCG and PGN (but not of E2 in the treated group) by placental explants were similar to that of controls. However, the serum levels prior to culture were not measured. Further, explants in serum from untreated women with SLE produced a significant decrease in the proliferation rate of the trophoblastic cells and an increase of apoptosis. Treatment significantly reduced the apoptotic rate and increased cell proliferation, but the cell proliferation rate was still lower than that noted in controls. CONCLUSIONS: We conclude that sera from women with SLE may directly damage the developing placenta reducing proliferation and enhancing apoptosis. Successful treatment of the women reduces that damage.     

IgG subclasses of anti-tetanus toxoid antibodies in adult and newborn normal subjects and in patients with systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity

The IgG subclasses of anti-tetanus toxoid (anti-TT) antibodies were quantitated in normal sera and sera from patients with rheumatic disease. Detection relied on a set of four mouse monoclonal antibodies, each of which showed specificity for the respective isotype, independent of gamma-chain allotype or light chain class of the human antibody. Approximately 90% of the total anti-TT activity in normal adults and patients with Sjogren's syndrome was IgG1. In addition, IgG4 antibodies were detected in one-half the samples, but IgG2 and IgG3 antibodies were observed in only two out of 36 sera. However, antibodies elicited in children immunized with TT were exclusively IgG1 and IgG3, with IgG4 antibodies detectable only at birth (presumably due to transplacental passage of antibody) in three of 12 children. In contrast to normal adults, patients with systemic lupus erythematosus (SLE) and drug-induced autoimmunity (DIA) had a more promiscuous isotype profile. IgG2 and/or IgG3 anti-TT antibodies were detected in 13 of 22 SLE patients and IgG3 antibodies in six of 11 patients with DIA. IgG4 anti-TT antibodies were predominant in seven of these 33 patients. These findings suggest that IgG isotypes may depend on the frequency of the stimulus, but global alterations in immunologic status as reflected in systemic autoimmune disease may override the homeostatic mechanisms that control isotype restriction.     

One-step purification of rabbit histidine rich glycoprotein by dye-ligand affinity chromatography with metal ion requirement

A simple method for purification of the histidine rich glycoprotein (rHRG) from rabbit sera was developed. The rHRG was purified by one-step affinity chromatography using the triphenylmethane dye "acid fuchsin" as a specific ligand, which gave an overall yield above 80%. Interestingly, the binding of rHRG to the ligand required the divalent transition-metal ions such as Zn2+, Ni2+, and Co2+ at pH 9.5. In the presence of 0.5 mM ZnCl2, the binding was enhanced 15 times compared with that in the absence of ZnCl2. Bound rHRG was efficiently eluted from the affinity absorbent with 100 mM imidazole or histidine. Purified rHRG was homogeneous with an Mr of 94 kDa when analyzed by SDS-PAGE, whereas isoelectric focusing revealed microheterogeniety with pI values ranging from 6.3 to 6.8. Blotting analysis with lectins specific for carbohydrate moieties and treatment with glycosidases demonstrated that rHRG is a highly N-glycosylated protein with diverse carbohydrate structures.     

Anti-plasminogen autoantibodies from plasma of patients with systemic lupus erythematosus having anti-phospholipid antibody syndrome: isolation and some immunochemical properties

Blood plasma samples from patients with systemic lupus erythematosus having the anti-phospholipid antibody syndrome were found to contain anti-plasminogen antibodies of the IgG class. The titers of anti-plasminogen autoantibodies of the IgG class were elevated in these patients compared with normal controls. Part of the pool of IgG anti-plasminogen antibodies reacts with an epitope in the lysine-binding sites of plasminogen. Anti-plasminogen IgG isolated from patients' blood plasma is specific only for a native epitope of human plasminogen passively adsorbed on immunosorbent micro-titration plate. As shown by enzyme immunoassay, autoantibodies to plasminogen of the IgG class cross-react with human fibrinogen.     

Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients

Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.     

系统性红斑狼疮患者血清中CD83与自身抗体的表达及其相互关系

目的：检测系统性红斑狼疮（systemic lupus erythematosus,SLE）患者血清中CD83（soluble CD 83,sCD 83）和多种自身抗体的表达水平,并探讨其相互关系。方法：ELISA检测患者可溶性CD 83和AnuA的表达,应用间接免疫荧光的方法检测抗cmDNA抗体,应用乳凝法检测血清中的DNP,采用胶体金标记和快速膜渗滤技术测定血清中的抗dsDNA抗体。结果：对照组患者血清中可溶性CD 83的表达为（0.26±0.10）ng/ml,实验组患者血清中可溶性CD 83的表达为（5.56±0.72）ng/ml。与对照组相比,实验组患者血清中可溶性CD 83的平均浓度明显升高。在抗dsDNA抗体阴性的51例系统性红斑狼疮患者中AnuA的阳性率明显高于抗DNP抗体和抗cmDNA抗体,同样在抗DNP抗体阴性的58例系统性红斑狼疮患者中AnuA的阳性率明显高于dsDNA抗体和抗cmDNA抗体。系统性红斑狼疮患者中可溶性CD83的水平（〈2.68 ng/ml）与各种自身抗体（抗dsDNA抗体、AnuA、抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.542,0.613,0.489和0.367）。具有高水平可溶性CD83的系统性红斑狼疮患者（≥2.68 ng/ml）,与各种自身抗体（抗dsDNA抗体,AnuA,抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.711,P〈0.05）、（r=0.845,P〈0.01）、（r=0.862,P〈0.01）和（r=0.724,P〈0.051）。结论：可溶性CD83通过活化DC细胞并激活补体系统,参与系统性红斑狼疮的发生发展,联合可溶性CD83和多种自身抗体的检测,能更明确系统性红斑狼疮患者病情的严重程度,有利于SLE的诊断和治疗。     

系统性红斑狼疮患者血清中CD83 与自身抗体的表达及其相互关系

目的:检测系统性红斑狼疮(systemic lupus erythematosus,SLE)患者血清中 CD83(soluble CD 83,sCD 83)和多种自身抗体的表达水平,并探讨其相互关系.方法:ELISA 检测患者可溶性 CD 83 和AnuA的表达,应用间接免疫荧光的方法检测抗cmDNA 抗体,应用乳凝法检测血清中的DNP,采用胶体金标记和快速膜渗滤技术测定血清中的抗 dsDNA 抗体.结果:对照组患者血清中可溶性 CD83 的表达为(0.26±0.10)ng/ml,实验组患者血清中可溶性 CD83 的表达为(5.56±0.72)ng/mI.与对照组相比,实验组患者血清中可溶性CD 83的平均浓度明显升高.在抗dsDNA抗体阴性的 51 例系统性红斑狼疮患者中 AnuA 的阳性率明显高于抗DNP 抗体和抗 cmDNA 抗体,同样在抗 DNP 抗体阴性的 58 例系统性红斑狼疮患者中 AnuA 的阳性率明显高于 dsDNA 抗体和抗 cmDNA 抗体.系统性红斑狼疮患者中可溶性 CD83 的水平(<2.68 ng/ml)与各种自身抗体(抗 dsDNA 抗体、AnuA、抗DNP抗体和抗 cmDNA 抗体) 水平的相关系数分别为(r＝0.542,0.613,0.489和0.367).具有高水平可溶性CD83的系统性红斑狼疮患者( ≥2.68 ng/ml),与各种自身抗体(抗dsDNA抗体,AnuA,抗 DNP 抗体和抗cmDNA 抗体)水平的相关系数分别为(r＝0.711,P<0.05)、(r＝0.845,P<0.01)、(r=0.862,P<0.01)和(r=0.724,P<0.051).结论:可溶性CD83通过活化DC细胞并激活补体系统,参与系统性红斑狼疮的发生发展,联合可溶性 CD83 和多种自身抗体的检测,能更明确系统性红斑狼疮患者病情的严重程度,有利于 SLE 的诊断和治疗.     

High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus

Introduction

Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.

Methods

Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of ³⁵S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, β2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.

Results

Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.

Conclusions

Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.     

Association of a polyuridylate-specific endoribonuclease with small nuclear ribonucleo-proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus

Immunoglobulins, containing antibodies against U1-snRNP, have been prepared from a patient with systemic lupus erythematosus. After coupling these antibodies to a Sepharose matrix, U-snRNPs have been isolated and purified from rat liver nuclei by use of immunoaffinity chromatography. The resulting RNPs had the typical protein pattern of U-sn RNPs and a sedimentation coefficient of 12 S. The U-snRNP preparation was associated with an endoribonuclease which required Mg2+ for optimal activity. The enzyme, with an pH optimum of 6.2, degraded only poly(U). Other single-stranded polyribo- and polydeoxyribonucleotides, tRNA, as well as double-stranded RNA and DNA were not digested. The products of a terminal digestion are (U)6-12 with 3'-OH and 5'-P termini. The possible involvement of this endoribonuclease in the splicing of hnRNA is discussed.     

Serum nitrated nucleosome levels in patients with systemic lupus erythematosus: a retrospective longitudinal cohort study

Introduction

Circulating nucleosomes released from apoptotic cells are important in the pathogenesis of systemic lupus erythematosus (SLE). Both nucleosomes and anti-nucleosome antibodies are deposited in inflamed tissues in patients with SLE. Active inflammation promotes nitration of tyrosine residues on serum proteins. Our hypothesis was that levels of nitrated nucleosomes would be elevated in patients with SLE and could be associated with disease activity. We therefore carried out a retrospective longitudinal study to investigate factors affecting levels of nitrated nucleosomes (NN) in patients with SLE.

Methods

A novel serum ELISA was developed to measure serum NN and modified to measure serum nitrated albumin (NA). Levels of both NN and NA were measured in 397 samples from 49 patients with SLE followed through periods of disease flare and remission for a mean of 89 months. Anti-nucleosome antibody (anti-nuc) levels were measured in the same samples. The effects of 24 different clinical, demographic and serological variables on NN, NA and anti-nuc levels were assessed by univariable and multivariable analysis.

Results

Patients with SLE had higher mean NN than healthy controls or patients with other autoimmune rheumatic diseases (P =0.01). Serum samples from 18 out of 49 (36.7%) of SLE patients were never positive for NN. This group of 18 patients was characterized by lower anti-double stranded DNA antibodies (anti-dsDNA), disease activity and use of immunosuppressants. In the remaining 63.3%, NN levels were variable. High NN was significantly associated with anti-Sm antibodies, vasculitis, immunosuppressants, hydroxychloroquine and age at diagnosis. NN levels were raised in neuropsychiatric flares. NN levels did not completely parallel NA results, thus providing additional information over measuring nitration status alone. NN levels were not associated with anti-nuc levels.

Conclusions

NN are raised in a subset of patients with SLE, particularly those who are anti-Sm positive. Elevated NN may be a marker of vascular activation and neuropsychiatric flares in these patients.     

IgG anti-apolipoprotein A-1 antibodies in patients with systemic lupus erythematosus are associated with disease activity and corticosteroid therapy: an observational study

IntroductionIgG anti-apolipoprotein A-1 (IgG anti-apoA-1) antibodies are present in patients with systemic lupus erythematosus (SLE) and may link inflammatory disease activity and the increased risk of developing atherosclerosis and cardiovascular disease (CVD) in these patients. We carried out a rigorous analysis of the associations between IgG anti-apoA-1 levels and disease activity, drug therapy, serology, damage, mortality and CVD events in a large British SLE cohort.MethodsSerum IgG anti-apoA-1 levels were measured in 100 healthy controls to define a cut-off for positivity. In 499 patients with SLE we obtained the earliest stored serum sample from their disease course and measured IgG anti-apoA-1 level. We then examined associations between IgG anti-apoA-1 positivity in early disease and the development of damage, CVD or death over a mean follow-up period of 12.1 years in these patients. In a separate study, we measured IgG anti-apoA-1 levels in 397 samples taken longitudinally from 49 patients with SLE over a mean period of 89 months of fluctuating disease activity and carried out multi-variable analysis to examine the demographic, serological, disease activity and treatment factors associated with IgG anti-apoA-1 level over time.ResultsIn the longitudinal study, IgG anti-apoA-1 levels were significantly higher in patients with persistently active disease, those on high dose corticosteroid and those not taking hydroxychloroquine. Of the 499 subjects who had early disease IgG anti-apoA-1 levels measured, 135 (27%) were positive. However, we found no convincing associations between early IgG anti-apoA-1 positivity and development of damage, mortality or CVD.ConclusionsIgG anti-apoA-1 developed early in a quarter of our patients with SLE, but this had no major impact on subsequent clinical outcomes. However, levels of IgG anti-apoA-1 vary over time and are associated with disease activity, treatment with high dose corticosteroid and not taking hydroxychloroquine.     

Impaired expression of high affinity interleukin 2 receptor on activated lymphocytes from patients with systemic lupus erythematosus

Peripheral lymphocytes stimulated with phytohemagglutinin (PHA-blasts) were examined for their responsiveness to exogenous interleukin 2 (IL-2). The proliferative response of PHA-blasts to IL-2 was significantly lower in patients with systemic lupus erythematosus (SLE) than in normal subjects. To clarify the reason for this defect, the expression of IL-2 receptor (IL-2R) on PHA-blasts was investigated using anti-Tac antibody and purified IL-2. Cytofluorometric analysis showed no statistical differences in the Tac positivity of PHA-blasts among normal subjects and patients with active and inactive SLE. Scatchard analysis using 125I-labeled anti-Tac monoclonal antibody revealed that the number of Tac epitopes on PHA-blasts was also not different among them. Next, the affinity of IL-2R expressed on PHA-blasts was determined by Scatchard analysis using radiolabeled IL-2 as a ligand. The number of high affinity IL-2R on the PHA-blasts was significantly decreased in patients with active and inactive SLE, as compared with normal subjects. The responsiveness of PHA-blasts to exogenous IL-2 was well correlated to the number of high affinity IL-2R, but not to the number of Tac epitopes or total IL-2R. Inasmuch as high affinity components of IL-2R are functionally active, the defective expression of high affinity IL-2R may be responsible for the T cell dysfunctions in SLE.     

Reactivity of autoantibodies and DNA/anti-DNA complexes with a novel 110-kilodalton phosphoprotein in systemic lupus erythematosus and other diseases.

Utilizing nonionic detergent lysates of human lymphoid and non-lymphoid cells as substrate, IgM and/or IgG antibodies to a 110-kDa/isoelectric point 5.4 phosphoprotein (110K) was demonstrated in serum from patients with SLE or certain other systemic autoimmune disorders by immunoblotting and immunoprecipitation. Ig of this specificity was not demonstrable in serum from normal individuals, but, in a limited survey, was detected in serum from patients with acute hepatitis A or infectious mononucleosis. 110K shares a number of properties with nucleolin, i.e., identical Mr and isoelectric point, localization in both the nucleus and the cytosol, increased expression in rapidly dividing cells, and shown to be distinct from already defined autoantigens of similar size, i.e., topoisomerase I, PM-Scl, and RNA polymerase I. Because 110K could bind denatured DNA, as demonstrated by its specific absorption by DNA-cellulose and by its reactivity with monoclonal anti-ssDNA antibody in the presence of denatured DNA, special efforts were made to distinguish reactivity of pre-formed DNA/anti-DNA complexes in SLE serum from that due to specific anti-110K autoantibodies. Although binding to 110K could be mediated by DNA and anti-DNA in some SLE sera, the accumulated evidence supports the existence of a major new autoantibody system in SLE, other autoimmune diseases, and certain virus infections.     

Monthly plasmapheresis for systemic lupus erythematosus with diffuse proliferative glomerulonephritis: a pilot study

Twelve patients with systemic lupus erythematosus and biopsy-proved diffuse proliferative glomerulonephritis were randomly allocated to a control group (to continue receiving conventional therapy only) or to a plasmapheresis group (to receive conventional therapy along with one 4-I plasma exchange a month). The six patients treated with plasmapheresis had better preservation of renal function, reduced disease activity, fewer admissions to hospital and less need for steroid and immunosuppressive therapy than the six control patients. The patients treated with plasmapheresis also showed evidence of reduced immunologic activity and had no side effects attributable to the plasma exchange. These results suggest that monthly plasma exchange should be assessed in a controlled randomized trial as a possible therapeutic adjunct in patients with systemic lupus erythematosus and diffuse proliferative glomerulonephritis.