Autoantibodies to intracellular proteins. Clinical and biologic significance.
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Antibodies, Antinuclear↗
Biomedical subjects
K B Elkon
Publications and source records attributed to K B Elkon.
Spectrotypic analysis of IgM and IgA rheumatoid factors.
The spectrotypes of IgA and IgM rheumatoid factors (RF) were analysed in whole serum as well as immunoglobulin fractions and purified RF from patients with one of three autoimmune disorders. As predicted from the pI ranges of normal human serum IgM and IgA in agarose, IgM RF had near neutral pIs, whereas IgA RF showed more acidic (lower) pI values. Serum IgA and IgM RF from patients with rheumatoid arthritis or the sicca syndrome showed considerable charge heterogeneity whereas IgM RF from mixed cryoglobulinaemia or rheumatoid vasculitis patients showed monoclonal and oligoclonal banding patterns respectively. Clonotypic analysis was best achieved with isolated light and heavy chains from highly purified RF. The IgA RF spectrotypes from unmodified paired serum and saliva IgA were clearly different whereas after desialation, similar pI values and, in one case, similar spectrotypes were observed. These observations are compatible with the hypothesis that serum and saliva IgA RF are derived from similar clonal precursors. The methods used in these studies may also be of use in the analysis of IgA and IgM antibody diversity in a number of other situations.
Antibody Diversity↗
Lupus autoantibodies target ribosomal P proteins.
All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.
Antibody Specificity↗
Lupus pregnancy. II. Unusual pattern of hypocomplementemia and thrombocytopenia in the pregnant patient.
To explore the causes of complications in pregnant women with systemic lupus erythematosus (SLE), we prospectively evaluated 34 pregnancies in 28 SLE patients, and 2 additional pregnancies in patients with lupus anticoagulant and positive antinuclear antibody, but no other manifestations of SLE. Nineteen pregnancies (55%) were complicated by marked proteinuria, thrombocytopenia, and/or lupus anticoagulant. Hypocomplementemia occurred in 18 pregnancies (52%). Neither thrombocytopenia-anticoagulant nor proteinuria was accompanied by an increase in antibody to double-stranded DNA or by clinical signs of active SLE. Antibody to Ro antigen did not predict fetal death. Both thrombocytopenia and proteinuria appeared abruptly during pregnancy and disappeared quickly after delivery. Fetal death was the result in 7 of 9 (77%) pregnancies in patients with anticoagulant, 6 of 10 (60%) in patients with thrombocytopenia, 6 of 18 (33%) in patients with hypocomplementemia, and 3 of 11 (27%) in patients with proteinuria. Twenty of 29 (68%) children were identified as male. The pathogenesis of hypocomplementemia was evaluated by a new assay, C1s-C1 inhibitor complex, which is thought to measure rate of complement activation by the classical pathway. Most pregnant patients with low CH50 levels and proteinuria had normal levels of C1s-C1 inhibitor complex, whereas nonpregnant patients with equivalent proteinuria and hypocomplementemia had high levels, as did pregnant patients with hypocomplementemia who did not have SLE. Pregnant and nonpregnant hypocomplementemic patients with proteinuria had similar levels of C3 and C4. In pregnant patients with SLE, C1s-C1 inhibitor complex was independent of CH50; in nonpregnant patients a linear relationship between C1s-C1 inhibitor complex and CH50 was seen.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Antinuclear↗
The lupus anticoagulant. A disease marker in antinuclear antibody negative lupus that is cross-reactive with autoantibodies to double-stranded DNA.
The lupus anticoagulant (LA) was demonstrated in 37% of 52 consecutive systemic lupus erythematosus (SLE) patients and patients with lupus-like syndromes, who were referred to our unit. The LA was found to be associated with a biologic false-positive VDRL (P less than 0.005), and the apparently paradoxical association of LA with vascular thromboses was confirmed (P less than 0.05). The presence of LA and a biologic false-positive VDRL defined a group of 7 antinuclear antibody negative patients with characteristic features of recurrent thromboses, spontaneous abortions, neurologic involvement, and renal disease. Further studies on 6 selected sera demonstrated LA in all 6 IgM fractions and in 3 of 6 IgG fractions. Inhibition of LA was shown in 6 of 9 Ig fractions after absorption with double-stranded DNA (dsDNA). Anticardiolipin antibody was shown by immunodiffusion in 3 LA positive IgG fractions from VDRL negative sera. Cardiolipin micelles partially inhibited anti-dsDNA binding of 4 IgG fractions, 3 of which were LA negative. In this report we discuss the overlapping specificities due to cross-reactivity between LA, anticardiolipin, and anti-dsDNA antibodies in human SLE, and we suggest that LA be considered equivalent to the biologic false-positive VDRL as a criterion for the diagnosis of SLE.
Antibodies, Antinuclear↗
Immunohistochemical localization of La nuclear antigen in brain. Selective concentration of the La protein in neuronal nucleoli.
We analyzed the subcellular distribution of La antigen in brain tissue using 5 La antisera regarded as monospecific on the basis of immunofluorescence, counterimmunoelectrophoresis and Western blot studies. Staining patterns of these sera on human, rat and guinea pig brain were assessed by indirect immunofluorescence and immunoperoxidase methods. There was an intense staining of the nucleolus in most neurons exposed to the La antisera in guinea pig brain. Human cerebral cortical neurons as well as a subset of neurons of rat brain also showed nucleolar staining with the La antisera. This staining could be inhibited by preincubation of La antisera with immunopurified La protein. One- and two-dimensional analysis of the La antigen from soluble and nuclear extracts suggested that the soluble and nucleolar La antigens are the same protein. The concentration of La in the nucleolus may be related to the proposed processing activity of this protein for the RNA polymerase III transcript, 5S RNA.
Animals↗
Common IgA and IgM rheumatoid factor idiotypes in autoimmune diseases.
Anti-idiotypic antibodies to four monoclonal IgM, one polyclonal IgM, and one polyclonal IgA rheumatoid factors were produced in rabbits and tested for cross reactivity with rheumatoid factors from nine patients with essential mixed cryoglobulinaemia, three patients with rheumatoid arthritis, four patients with systemic sicca syndrome, and one patient with systemic lupus erythematosus. Variable (3/16 to 10/16) cross reactivity for each anti-idiotypic antibody was observed which was not restricted by disease category, clonality, or isotype of the rheumatoid factor. These findings suggest that rheumatoid factor genes are highly conserved from the germ-line antibody repertoire.
Arthritis, Rheumatoid↗
Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting.
Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.
Animals↗
The splenic extraction ratio of antibody-coated erythrocytes and its response to plasma exchange and pulse methylprednisolone.
Splenic blood flow was measured in a series of normal subjects and patients with connective tissue diseases by measuring the rate of equilibration and the partition of 111In-labelled autologous platelets between blood and spleen. These data were used to quantify the role of splenic blood flow in determining the splenic clearance of IgG coated erythrocytes (IgG-RBC) from the circulation. Previous studies have interpreted the clearance rates of IgG-RBC only in terms of splenic reticuloendothelial function. Splenic blood flow was increased in seven of eight patients with systemic lupus erythematosus (SLE), six of 10 patients with rheumatoid arthritis (RA) and in all five patients with essential mixed cryoglobulinaemia (EMC) compared with a series of thirteen normal subjects. Expressing the rate constant of clearance of IgG-RBC as a fraction of splenic blood flow gave a value for the 'extraction ratio' of IgG-RBC (a specific measurement of reticuloendothelial function, corrected for splenic blood flow). Normal splenic extraction ratio of IgG-RBC was calculated to be 32%. All the patients with SLE and with EMC had reduced extraction ratios (in seven out of 13 patients less than 10%). In RA the extraction ratio tended to be normal (average 27.3%) but variable (9-59%). Following plasma exchange in nine patients, a significant increase in IgG-RBC extraction ratio (average of 39% with respect to pre-exchange values, P less than 0.05) was found. In contrast there was no significant change in extraction ratio following pulse methylprednisolone therapy in a further nine patients. Although the rate constant of clearance of IgG-RBC decreased by an average of 33% (P less than 0.01) in the latter group, it was matched by an equal decrease of splenic blood flow (average 37%, P less than 0.01) and so extraction ratio showed no change. These data indicate that quantification of splenic reticuloendothelial function requires measurement of both IgG-RBC clearance and of splenic blood flow.
Adolescent↗
Counterimmunoblotting: detection of non-denatured or denatured antigens in antibody-containing agarose gels following polyacrylamide gel electrophoresis.
Utilizing the principles of counterimmunoelectrophoresis, a technique was devised to immunologically identify non-denatured protein antigens resolved by polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred from polyacrylamide gel to antibody-containing agarose gel in a commercially available blotting apparatus. Detection of the antigen/antibody complex by Coomassie blue staining (in the ng range for unlabeled antigen) or autoradiography (in the pg range for radiolabeled antigen) established the sensitivity and specificity of this method. Similar sensitivity could be obtained following conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis after partial removal of the detergent and possible renaturation of proteins. Placement of a nitrocellulose sheet on the anodal side of the agarose gel produced a 'negative replica' (proteins/polypeptides not reacting with specific antibodies in the agarose gel). Counterimmunoblotting provides an additional method for molecular weight determination of protein antigens with sensitivity and specificity comparable to established nitrocellulose blotting (unlabeled proteins) or immunoprecipitation (labeled proteins) procedures.
Antigen-Antibody Complex↗
Isoelectric focusing of human IgA and secretory proteins using thin layer agarose gels and nitrocellulose capillary blotting.
A simple, rapid and economical method for focusing human immunoglobulin A in low electroendosmosis agarose is described. IgA preparations isolated from serum and secretions were focused with high resolution in thin layer gels. Serum and milk proteins were transferred to nitrocellulose sheets by capillary blotting and the spectrotype of total IgA determined with radiolabeled anti-human IgA antibodies. These studies indicated that the spectrotype of human IgA extends between the pH range 4.3-6.85. The spectrotypes of free secretory component and alpha-1 antitrypsin released from an IgA myeloma protein were determined by a similar technique. These methods should prove useful in determining the diversity of IgA antibodies whether or not the starting material is pure.
Collodion↗
Blotting intact immunoglobulins and other high-molecular-weight proteins after composite agarose-polyacrylamide gel electrophoresis.
A composite agarose-polyacrylamide gel containing urea and sodium dodecyl sulfate reliably resolved unreduced human immunoglobulins according to their molecular weight. Intact immunoglobulins and a number of other macromolecules were readily transferred to nitrocellulose paper by either capillary or electrophoretic blotting, although the latter technique was more effective. Conventional antigen probing as well as immobilized antibody studies can be performed on the nitrocellulose transfers.
Autoradiography↗
The comparative blood clearance kinetics of modified radiolabelled erythrocytes.
The rate of clearance from blood of 111In-labelled heat damaged autologous erythrocytes (HD-RBC) has been compared with that of simultaneously injected autologous 99mTc-labelled erythrocytes (IgG-RBC) coated with a Rhesus anti-D antibody. In 17 studies, the number of antibody molecules coating the erythrocytes was 9000 (high coating) and in nine studies the number was 5000 (low coating). On gamma camera imaging, IgG-RBC uptake, at both levels of coating, could be visualized only in the spleen. HD-RBC were predominantly taken up by the spleen, although slight 111In activity was visible in the liver. The blood clearance of IgG-RBC was monoexponential, whereas that of HD-RBC was biexponential. The reciprocal of the t1/2 (the time taken for the 3 min value to fall by 50%) of the HD-RBC clearance correlated rather poorly with the rate constant of the simultaneous IgG-RBC clearance (r = 0.47, P greater than 0.05 at high coating; r = 0.75, P less than 0.05 at low coating). The rate constant of the second exponential of the HD-RBC clearance showed a correlation with the rate constant of IgG-RBC clearance that was closer than the reciprocal of the t1/2 of HD-RBC clearance (r = 0.89, P less than 0.001 at high coating; r = 0.76, P less than 0.05 at low coating) but significantly closer only at high coating. Splenic blood flow, measured using indium labelled platelets in ten subjects, correlated closely with the initial slope of HD-RBC clearance (r = 0.93, P less than 0.001).
Erythrocytes↗
Detection and immunochemical characterization of a primate type C retrovirus-related p30 protein in normal human placentas.
We find that 12 of 14 specimens of normal human term placentas analyzed by one- or two-dimensional electrophoresis and immunoblotting contain a protein or polypeptide of approximately equal to 30,000 daltons that is antigenically cross-reactive with p30 core protein of the simian sarcoma-associated virus/gibbon ape leukemia virus primate retrovirus group and is physicochemically similar to reference murine and primate type C retrovirus p30s. This finding may lead to an understanding of endogenous type C retrovirus gene expression in humans.
Antibodies, Viral↗
Systemic vasculitis with asthma and eosinophilia: a clinical approach to the Churg-Strauss syndrome.
Drawing on our experience of 16 cases and a review of the English literature, we propose that CSS is under-diagnosed because of exclusive emphasis upon pathologic recognition of the disorder. The classical histological picture comprises a necrotizing vasculitis, eosinophilic tissue infiltration and extravascular granulomas, but it is only found in a minority of cases, and is not pathognomonic of the condition (69, 108). On the other hand, the clinical pattern of the disorder is most distinctive, and CSS can be readily identified on clinical grounds. Typically, it begins with allergic rhinitis, which is often complicated by nasal polyposis and sinusitis. Asthma and peripheral blood eosinophilia are essential features, often accompanied by pulmonary infiltrates. The systemic vasculitis of CSS resembles that of PAN, but severe renal disease is uncommon (the typical renal lesion is a focal segmental glomerulonephritis), and cardiac involvement accounts for 50% of deaths. Diagnostic difficulties arise from the close relationship of CSS to other granulomatous, vasculitic and eosinophilic disorders. CSS is usefully regarded as a point of overlap between these three disease spectrums (Fig. 5). Individual components of each spectrum can occur in the course of CSS; hence cases may be reported as PAN developing as a complication of Löffler syndrome or eosinophilic gastroenteritis (37, 57, 66). The hypereosinophilia of CSS tends to be less severe and more steroid-responsive than in HES, and evidence of eosinophil degranulation was not found in the patients we studied. Complement abnormalities are not a prominent feature of the disorder, and circulating immune complexes were detected in only two cases; both contained IgM. This may be of pathogenetic significance as IgM deposition was a dominant feature in four of the five cases with positive renal immunofluorescence. IgE levels were elevated in all patients studied during the vasculitic phase, and skin-prick tests were positive in 8 of 10 patients tested. CSS responds well to treatment with steroids, although some patients benefit from the addition of immunosuppressive agents. The vasculitic illness is usually of limited duration, but relapses can occur, and should be detected and treated early. Major problems in the post-vasculitic phase stem from hypertension and persisting peripheral nerve damage. Allergic upper and lower respiratory tract disease is an important cause of morbidity in the pre- and post-vasculitic periods.
Adult↗
Autoantibodies in the sicca syndrome (primary Sjögren's syndrome).
IgA and IgM rheumatoid factors as well as antibodies to Ro and La were measured in 37 patients with the sicca syndrome and 9 patients with Sjögren's syndrome secondary to rheumatoid arthritis. Positive results were found in 84% (IgA rheumatoid factor), 76% (IgM rheumatoid factor), 62% (Ro), and 50% (La) of patients with the sicca syndrome. There was no significant difference in the frequency of positive results in patients with glandular versus extraglandular disease. Antibodies to La invariably occurred in patients with antibodies to Ro. In addition, patients who were anti-Ro positive also had significantly higher IgA (p less than 0.05) and IgM (p less than 0.01) rheumatoid factor activity. Since 81% of the sicca patients were concordant for 3 of the 4 antibodies tested, production of these autoantibodies appears to be related.
Antigens↗
Charge heterogeneity of human secretory component: immunoglobulin and lectin binding studies.
Free secretory component purified from human milk showed considerable charge heterogeneity in the pH range 4.7-6.5 when analysed by isoelectric focusing in thin layer agarose gels. Unlike rabbit secretory component, allotypic variation could not be identified by comparing samples from different individuals. Treatment with neuraminidase resulted in a basic shift of secretory component charge isomers. The charge heterogeneity appeared to be unrelated to the immunoglobulin binding property of secretory component since all charge isomers bound 125I-labelled IgM. 125I-labelled secretory component bound more strongly to purified IgM compared with polymeric IgA, and predominantly to the IgM-containing region of focused normal human serum. Lectin-secretory component interaction was demonstrated by concanavalin A and wheat germ agglutinin binding in a dot-blot nitrocellulose assay and precipitation with concanavalin A by Ouchterlony immunodiffusion. Despite the relatively high carbohydrate composition of both secretory component and the polymeric immunoglobulins, no evidence for lectin-like binding was obtained by sugar inhibition and sugar desorption studies. Similarly, desialation of secretory component did not prevent secretory component-IgM binding. These observations suggest that the charge heterogeneity and sugar composition of secretory component are unrelated to immunoglobulin binding in vitro.
Autoradiography↗
Partial immunochemical characterization of the Ro and La proteins using antibodies from patients with the sicca syndrome and lupus erythematosus.
Ro and La are intracellular ribonucleoproteins that are frequent targets for autoantibodies in the sicca syndrome and in lupus erythematosus. We analyzed the m.w. of the protein (antigen) moieties of Ro and La in saline cell extracts of human spleen by SDS-PAGE and immunoblotting, gel filtration, and sucrose density ultracentrifugation with radioimmunoassay. pI values for Ro and La proteins were established by isoelectric focusing on thin layer agarose gels and immunoblotting on nitrocellulose transfers. The La protein had an m.w. of approximately 43 kd and was heterogeneous in charge, with pI values from 4.2 to 4.8. Composite two-dimensional maps developed by immunoblotting revealed a characteristic set of seven dots of m.w. 43 kd. Ro determinants were identified on polypeptides of 50 and/or 57 kd. Antigenic activity was also detected in the void volume of spleen extract fractionated by Sephadex G-200 and in 8 to 9S and greater than 19S regions of sucrose gradients, suggesting either aggregation of the Ro protein or participation in protein-protein complexes. pI values of 4.3 to 5.5 were obtained for the Ro antigen, and two-dimensional maps revealed that the 57 kd polypeptide had a similar charge heterogeneity to the La protein, whereas the 50 kd polypeptide had a different fingerprint. Immunoblotting of extracts from bovine, rabbit, and dog extracts showed that antibodies to Ro and La reacted with a limited number of polypeptides (m.w. 50 and/or 57 kd for anti-Ro and 43 or 50 kd for anti-La). These studies support the physical independence of the isolated Ro and La polypeptides, although a precursor product or functional relationship in vivo is possible. These studies also suggest that, in addition to Western blotting, techniques involving immunodeletion, isoelectric focusing with capillary immunoblotting, and 2D immunoblotting provide useful approaches to characterize saline-soluble cellular antigens.
Antigens↗