Electrophysiological dysfunction induced by anti-ribosomal P protein antibodies injection into the lateral ventricle of the rat brain.

Objective Anti-ribosomal P antibodies (anti-P) are strongly associated with neuropsychiatric lupus. This study was designed to determine whether these antibodies are capable of causing electro-oscillogram (EOSG) and behavior alterations in rats. Methods IgG fraction anti-P positive and affinity-purified anti-P antibodies were injected intraventricularly in rats. Sequential cortical and subcortical EOSGs were analyzed during 30 days. IgG anti-Ro/SS-A and normal IgG were used as controls. Results All 13 animals injected with IgG anti-P demonstrated a high prevalence of polyspikes, diffusely distributed in hippocampal fields and cerebral cortex. These abnormalities persisted approximately a month. Remarkably, an identical electrical disturbance was observed with the inoculation of affinity-purified anti-P antibodies. The EOSG alterations were associated with behavioral disorders with varying degrees of severity in every animal injected with anti-P. In contrast, no changes in EOSG or behavioral disturbances were observed in the control group. Conclusion Our study indicates that anti-P antibodies can directly induce electrophysiological dysfunction in central nervous system particularly in hippocampus and cortex associated with behavior disturbances.

Idiotypic cross-reactivity between antibodies of different specificities.

Cross-idiotypic specificity has been demonstrated between antibody populations of different specificities using antibodies directed toward human sickle cell hemoglobin (HbS). A site-specific antibody directed toward the beta6-position of HbS, anti-Val, was used to elicit an anti-idiotypic response in rabbits. Using this anti-idiotypic serum, idiotypic cross-reactivity was demonstrated between antibody populations that bind to human adult hemoglobin (HbA). It was demonstrated that in the case of the goat antibodies, these idiotypically cross-reacting antibodies are directed towards the beta6-position of the hemoglobin molecule. However, they differ in their specificity, binding to this site on HbA, whereas anti-Val binds only to HbS. The sheep antibody populations directed toward HbS differ qualitatively from those of the goat. Using rabbit anti-idiotypic serum specific for sheep anti-Val, cross-reactivity could be demonstrated with antibodies directed toward the alpha-chain of the hemoglobin molecule, as well as the beta-chain. There was also a low level of cross-reactivity with antibodies from a goat immunized with HbA.

Evolution of an idiotypic determinant: anti-Val.

An antibody population which reacts only with human sickle cell hemoglobin (HbS) and not with normal human hemoglobin, has been isolated from goat, sheep, and guinea pig antisera. These antibody populations termed anti-Val (Val), isolated from an individual goat (no. 6) and sheep (no. 26) have been used to elicit anti-idiotypic responses in rabbits. These anti-idiotypic sera were used to study the idiotypic cross-reactions between the goat and sheep anti-Val. Strong cross-reactions were present using either Ra anti-goat anti-Val or Ra anti-sheep anti-Val. Guinea pig anti-Val did not cross-react with these anti-idiotypic sera. Binding of HbS to the anti-Val of the goat and sheep could be blocked by the anti-idiotypic sera, but the binding of HbS to the guinea pig anti-Val could not. These data demonstrate idiotypic cross-reactivity between two closely related species.

Anti-threonyl-tRNA synthetase, a second myositis-related autoantibody.

An autoantibody known as PL-7 was found in the serum of four patients with myositis and one with a systemic lupus erythematosus-like syndrome. The PL-7 antigen is an 80,000 dalton polypeptide that coprecipitates with transfer RNA. In aminoacylation reactions, PL-7 IgG inhibited the charging of tRNA with threonine but had little or no effect on charging with other amino acids. Experimental antibodies raised against purified threonyl-tRNA synthetase recognized the same 80,000 dalton polypeptide, but tRNA was not coprecipitated. We conclude that PL-7 antibody is directed at threonyl-tRNA synthetase, and that different antigenic sites are recognized by the human and experimental autoantibodies. Our findings emphasize the link between myositis and autoimmunity to tRNA-related structures.

Alteration by heat shock and immunological characterization of Drosophila small nuclear ribonucleoproteins.

Sera from human patients with systemic lupus erythematosus (SLE) have been shown to react with snRNP particles of both mammals and Drosophila (Mount, S. M. and J. A. Steitz. 1981. Nucleic Acids Res. 9:6351-6368). We have utilized fully characterized monospecific sera and specifically purified antibodies to carry out indirect immunofluorescence experiments with frozen sections of Drosophila embryos. Embryos subjected to severe heat shock before sectioning showed reduced binding of anti-Sm sera. Anti-nRNP sera reacted identically with antigens of heat shocked and non-heat-shocked sections. The reduction in anti-Sm fluorescence was restored by a brief salt wash. These results imply a noncovalent alteration in the conformation of Sm antigens with the administration of heat shock that can revert with exposure to salt. Drosophila antigens have been compared to mammalian standards, showing partial identity with bovine spleen extract (BSE) antigens when reacted with anti-Sm sera. The antigenic relatedness between affinity-purified heat-shocked and non-heat-shocked Drosophila antigens and their mammalian homologues was examined by quantitative ELISA methodology. In all cases, the Drosophila antigens from heat-shocked and non-heat-shocked embryos were identical. We theorize that the heat shock-induced alteration of Sm antigen reverst during extraction. Because the snRNP antigens have been shown to be involved in splicing, and because splicing is inhibited during heat shock (Yost, H. J., and S. Lindquist. 1986. Cell. 45:185-193), our results provide information on the nature and stability of a change in these antigens which may be a central element in control of the heat shock response.

Antibodies to rabbit cytochrome c arising in rabbits.

Antibodies reactive with rabbit cytochrome c have been observed in rabbits immunized with several heterologous cytochromes. Such antibodies have also been observed in rabbits immunized with rabbit cytochrome c conjugated to bovine gamma globulin. The serum of a rabbit immunized with human cyto chrome c reacted with the cytochrome c of the same rabbit.

Gene interaction at HLA-DQ enhances autoantibody production in primary Sjögren's syndrome.

Primary Sjögren's syndrome is an autoimmune disorder characterized by dryness of the mouth and eyes. The human leukocyte antigen (HLA) locus DQ is related to the primary Sjögren's syndrome autoantibodies that bind the RNA proteins Ro/SSA and La/SSB. Both DQ1 and DQ2 alleles are associated with high concentrations of these autoantibodies. An analysis of all possible combinations at DQ has shown that the entire effect was due to heterozygotes expressing the DQ1 and DQ2 alleles. These data suggest that gene interaction between DQ1 and DQ2 (or alleles at associated loci), possibly from gene complementation of trans-associated surface molecules, influences the autoimmune response in primary Sjögren's syndrome.

Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells.

Autoantibodies to ribosomal P-proteins are present in 12-16% of patients with systemic lupus erythematosus and are associated with neuropsychiatric disease. As the ribosomal P proteins are located in the cytoplasm, the pathogenic effects of their cognate autoantibodies are unclear. In this study affinity-purified anti-P autoantibodies were used to explore the cell surface of several types of human and animal cells. Immunofluorescence as well as EM immunogold analysis demonstrated, on the surface of human hepatoma cells, the presence of an epitope that is antigenically related to the immunodominant carboxy terminus of P-proteins. The presence of this epitope was also demonstrated on the surface of human neuroblastoma cells and, to a lesser extent, on human fibroblasts. Furthermore, the Western blot technique revealed in purified human and animal plasma membranes a 38-kD protein that is closely related or identical with ribosomal P0 protein. The availability of reactive P peptide on the surface of cells makes possible the direct effect of autoantibodies on the function and viability of cells that express this antigenic target. This delineates one of the possible impacts of anti-P antibodies in disease expression.

Molecular properties of the Ro/SSA antigen and enzyme-linked immunosorbent assay for quantitation of antibody.

Antibodies to the Ro/SSA antigen occur in patients with systemic lupus erythematosus and Sjögren's syndrome. An immunoaffinity method for the preparation of electrophoretically homogeneous Ro/SSA antigen is described. Several molecular properties of the antigen have been determined. The native RNA protein particle has a molecular weight of approximately 100,000 D determined by gel filtration. Sodium dodecyl sulfate-analysis of the purified Ro/SSA antigen and analysis by staining of bands with silver and Coomassie Blue, Western blotting, and RNAase treatment leads to a hypothesis for the structure of the particle in which an antigenic 60,000 protein is bound to 24,000-27,000 RNA molecules which are not antigenic. An enzyme-linked immunoabsorbent assay method for assay of anti-Ro/SSA is also described which sensitively measures antigen binding at dilutions of sera containing anti-Ro/SSA precipitins up to 10(7) fold. Normal sera on average have 10(3) less binding activity.

Clinical and serological differences between systemic lupus erythematosus patients with antibodies to Ro versus patients with antibodies to Ro and La.

Among 55 systemic lupus erythematosus patients having antibodies to Ro and/or La, two major groups were distinguished by titration of sera in counterimmunoelectrophoresis. The first group (30 patients) had antibodies to Ro alone. This was associated with a high incidence of antibodies to DNA (77%) and serious renal disease (53%). The second group (23 patients) had antibodies to Ro and La, and this was associated with a lower incidence of antibodies to DNA (30%) and a very low incidence of nephritis (9%). In this group a phenomenon of linkage of anti- Ro and anti-La titers was observed. Additionally two patients with only anti-La were found. Neither had clinically apparent renal disease. Thus, systemic lupus erythematosus patients with anti-Ro fall into two subgroups that differ considerably in their prevalence of anti-DNA and serious renal disease.

Anti-KJ: a new antibody associated with the syndrome of polymyositis and interstitial lung disease.

Antibodies to aminoacyl-tRNA synthetases (anti-Jo-1, anti-PL-7, anti-PL-12) have been found in the serum of some patients with polymyositis (PM). Patients with these antibodies have an unusually high rate of interstitial lung disease (ILD) in association with their PM. Two patients (K.J. and B.T.) with severe ILD and PM were found to have antibodies to a cytoplasmic antigen, but tests to determine whether the antigen was an aminoacyl-tRNA synthetase were negative, including tests of KJ serum for inhibitory effects on the 20 synthetases. KJ immunoprecipitates did not contain tRNA, in contrast to antisynthetase sera. When IgG samples were added to a reticulocyte in vitro translation system at a concentration of 0.3 mg/ml, KJ IgG inhibited globin mRNA translation by 98%, while anti-Jo-1 IgG inhibited 62% and normal IgG had little effect. Thus, both anti-KJ and the antisynthetases are directed at antigens that are involved in translation and protein synthesis, and both are associated with the syndrome of lung disease and PM. This syndrome may be associated with antibodies to translation-related proteins in general, which may have implications for the link of PM and enteroviruses, which are mRNA viruses.

HLA-DQ gene complementation and other histocompatibility relationships in man with the anti-Ro/SSA autoantibody response of systemic lupus erythematosus.

A strong gene interaction between HLA-DQ1 and DQ2 alleles has been associated with anti-Ro/SSA autoantibodies (Harley, J.B., M. Reichlin, F. C. Arnett, E. L. Alexander, W. B. Bias, and T. T. Provost. 1986. Science [Wash. DC]. 232:1145-1147; Harley, J. B., A. S. Sestak, L. G. Willis, S. M. Fu, J. A. Hansen, and M. Reichlin. 1989. Arthritis Rheum. 32:826-836; Hamilton, R. G., J. B. Harley, W. B. Bias, M. Roebber, M. Reichlin, M. C. Hochberg, and F. C. Arnett. 1988. Arthritis Rheum. 31:496-505). To test a gene complementation mechanism for these results, restriction fragment length polymorphisms (RFLP) of the DQ alpha and DQ beta genes have been related to Ro/SSA precipitins in patients with systemic lupus erythematosus. In this study Ro/SSA precipitins are related to the simultaneous presence of a particular pair of RFLPs. A DQ alpha RFLP associated with HLA-DQ1 and a DQ beta RFLP associated with HLA-DQ2 predict that the alpha beta heterodimer in HLA-DQ1/DQ2 heteroxygotes is most closely related to anti-Ro/SSA autoantibodies, thereby supporting a gene complementation mechanism. Beyond this effect, an RFLP associated with HLA-DQ2 and/or DR7 is also related to Ro/SSA precipitins. Multiple molecular histocompatibility mechanisms are implicated, therefore, in the production of anti-Ro/SSA autoantibodies in autoimmune disease. For anti-Ro/SSA autoantibodies in SLE, and perhaps more generally, these data show that the histocompatibility antigens are among the elements that confer autoimmune response specificity and restrict the production of particular autoantibodies among patients with systemic lupus erythematosus.

Lupus/Sjögren's autoantibody specificities in sera with paraproteins.

Antinuclear antibody and anti-RNA-protein autoantibodies were determined in 143 sera containing paraproteins and 39 control sera. Antinuclear antibodies were commonly present in the paraprotein sera by indirect immunofluorescence. 19 of 143 sera (13%) had elevated anti-Ro/SSA activity in a solid phase Ro/SSA binding assay, and 5 (3.5%) had Ro/SSA precipitating autoantibody. Eighteen sera had La/SSB binding autoantibodies (12%) but only one had an anti-La/SSB precipitin. Anti-nRNP(Sm) was not detected in any of these sera. The solid phase anti-RNA protein assays were repeated using anti-lambda and anti-kappa conjugates. Both lambda and kappa light chain autoantibodies were found in all positive sera consistent with polyclonal anti-Ro/SSA and anti-La/SSB responses. Paraprotein sera containing Ro/SSA precipitins were analyzed by isoelectric focusing followed by exposure to 125I-labeled Ro/SSA and autoradiography. All sera with anti-Ro/SSA binding paraproteins also contained polyclonal anti-Ro/SSA. Our data are consistent with the hypothesis that anti-Ro/SSA paraproteins are common and arise from a previously present polyclonal anti-Ro/SSA response.

Lupus autoantibodies to native DNA cross-react with the A and D SnRNP polypeptides.

Antibodies to native DNA (nDNA) in sera from patients with systemic lupus erythematosus have been found to frequently correlate with antibodies to the A and D SnRNP proteins measured in Western blot assays. 40 of 54 SLE (74.1%) sera with anti-nDNA bound to A and D proteins, while 9 of 113 sera (8%) without anti-nDNA bound the A and D proteins, P < 10(-8) by Fisher's exact test. Antibodies to nDNA correlated closely with anti-A and anti-D in seven of eight patients followed sequentially, r = 0.7865. Nine human polyclonal anti-nDNA populations were isolated from DNA cellulose columns. Seven reacted equally with A and D, and two reacted predominantly with D. Two of three murine monoclonal anti-DNA antibodies isolated from NZB/NZW F1 hybrid mice bound A and D equally in Western blot with a titer > 1/40,000. These reactions were directed to the unfolded A and D proteins measurable in Western blot since these monoclonals (and several of the human anti-nDNA populations) failed to react with native U1RNP in ELISA or in RNA immunoprecipitation experiments. These newly recognized cross reactions of anti-nDNA may amplify the immune response to DNA and be part of the original immunogenic drive.

Heterogeneity of the Ro/SSA antigen. Different molecular forms in lymphocytes and red blood cells.

Ro(SSA) is an intracellular ribonucleoprotein against which autoantibodies are found in a portion of patients with Sjögren's syndrome and systemic lupus erythematosus. A form of Ro(SSA) is described in red blood cells that shares a line of identity with purified Ro(SSA) from bovine spleen and human lymphocytes in counterimmunoelectrophoresis, but has different molecular properties. Ro(SSA) from red blood cells exists in association with only two small RNAs as opposed to four in other cell types, as determined by RNA extraction of protein A-assisted immunoprecipitates. In addition to the common 60-kD Ro(SSA) protein, Western blot analysis revealed an additional 52-kD protein in lymphocytes and a 54-kD protein in red blood cells. The 60-kD form of Ro(SSA) in red cells was found to be antigenically distinct from that in the lymphocyte, because sera were identified that bound each exclusively. Finally, a rabbit antibovine Ro(SSA) serum distinguished red cell from lymphocyte Ro(SSA). These results suggest two distinctive populations of Ro(SSA) proteins and distributions of Ro(SSA) RNAs in the lymphocyte and red blood cell.

Implications of anti-Ro/Sjögren's syndrome A antigen autoantibody in normal sera for autoimmunity.

We have applied a sensitive assay to analyze lupus and Sjögren's syndrome autoantibodies in 40 normal sera. Seven of these bound Ro/Sjögren's syndrome A antigen (SSA). Although this binding was 1,000-fold lower than the highest anti-Ro/SSA level measured from patients, it was inhibited by human Ro/SSA. Positive normal serum-bound Ro/SSA in Western immunoblots and binding activity was demonstrated in the F(ab')2 fragment of IgG. Affinity purification of normal anti-Ro/SSA IgG increased the specific anti-Ro/SSA binding by greater than 17-fold. This purified antibody formed a Ro/SSA precipitin and had a relative affinity for Ro/SSA identical to that of Ro/SSA precipitin-positive patients. These data demonstrate that the anti-Ro/SSA present in healthy normal donors is true autoantibody. Anti-La/Sjögren's syndrome B antigen (SSB) autoantibodies were found in 3 of the 40 normal sera, while none bound nuclear ribonucleoprotein (Sm). Finding low levels of anti-Ro/SSA and anti-La/SSB among normals may indicate that anti-Ro/SSA and anti-La/SSB occur in disease by enhancement of a preexisting immune response.

Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen.

Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor.

Autoantibodies to the ribosomal P proteins in systemic lupus erythematosus.

This paper describes the clinical significance of antibodies to the ribosomal P proteins in systemic lupus erythematosus. It appears that liver disease due to the lupus process and not attributable to viral infection, alcohol or drugs is associated with anti-ribosomal P. In addition, there is a strong relationship to central nervous system disease and nephritis of antibodies to ribosomal P proteins. The prevalence of the anti-P antibodies is strongly related to disease activity wherein disease remission is associated with disappearance of anti-P antibodies. These phenomena taken together suggest an immunopathogenic role for anti-P antibodies. This idea is strongly supported by the observation that immunoglobulin G containing antiribosomal P activity binds and penetrates living cells with profoundly inhibitory effects on protein synthesis. Finally, a new era of research has been uncovered by the observation that in 54 of 55 instances normal sera passed over a ribosome-sepharose column unmasks anti-P antibodies, which can be eluted from the ribosome column with 3.0 M magnesium chloride. This suggests that anti-idiotypes regulate the expression of anti-P antibodies in normal persons and in lupus patients this regulation is ineffective, with the development of free anti-P antibodies in a proportion of patients with active disease.

Autoimmune-associated congenital heart block: demographics, mortality, morbidity and recurrence rates obtained from a national neonatal lupus registry.

OBJECTIVES: The present study describes the demographics, mortality, morbidity and recurrence rates of autoimmune-associated congenital heart block (CHB) using information from the Research Registry for Neonatal Lupus. BACKGROUND: Isolated CHB detected at or before birth is strongly associated with maternal autoantibodies to 48-kD SSB/La, 52-kD SSA/Ro and 60-kD SSA/Ro ribonucleoproteins and is a permanent manifestation of the neonatal lupus syndromes (NLS). Available data are limited by the rarity of the disease. RESULTS: The cohort includes 105 mothers whose sera contain anti-SSA/Ro or anti-SSB/La antibodies, or both, and their 113 infants diagnosed with CHB between 1970 and 1997 (56 boys, 57 girls). Of 87 pregnancies in which sufficient medical records were available, bradyarrhythmia confirmed to be CHB was initially detected before 30 weeks of gestation in 71 (82%) (median time 23 weeks). There were no cases in which major congenital cardiac anatomic defects were considered causal for the development of CHB; in 14 there were minor abnormalities. Twenty-two (19%) of the 113 children died, 16 (73%) within 3 months after birth. Cumulative probability of 3-year survival was 79%. Sixty-seven (63%) of 107 live-born children required pacemakers: 35 within 9 days of life, 15 within 1 year, and 17 after 1 year. Forty-nine of the mothers had subsequent pregnancies: 8 (16%) had another infant with CHB and 3 (6%) had a child with an isolated rash consistent with NLS. CONCLUSIONS: Data from this large series substantiate that autoantibody-associated CHB is not coincident with major structural abnormalities, is most often identified in the late second trimester, carries a substantial mortality in the neonatal period and frequently requires pacing. The recurrence rate of CHB is at least two- to three-fold higher than the rate for a mother with anti-SSA/Ro-SSB/La antibodies who never had an affected child, supporting close echocardiographic monitoring in all subsequent pregnancies, with heightened surveillance between 18 and 24 weeks of gestation.

Acute renal failure secondary to interstitial lupus nephritis.

A patient with systemic lupus erythematosus (SLE), followed up over a six-month period, exhibited numerous immunologic abnormalities and varied renal pathologic features. Initial findings included minimal glomerular lesions, serum antibodies directed solely against nuclear RNA protein, and lupus band test showing pure IgM deposition. These findings suggested a good prognosis. Subsequently, the patient developed acute renal failure secondary to an interstitial lupus nephritis, without progression of the glomerular abnormality. Serum antibodies to the nuclear non-nucleic acid macromolecule and single stranded and native DNA were demonstrated concurrently. New skin deposits of IgG and IgA in addition to IgM also were observed. This patient demonstrates the potential progression of lupus renal disease despite the initial favorable prognostic indicators.