The network theory in autoimmunity: In vitro modulation of DNA-binding cells by antiidiotypic antibody present in inactive lupus sera

Lupus or normal controls' sera, depleted of anti-DNA antibody and of DNA, were tested for their capacity to suppress the binding of³H-DNA to DNA-binding cells. Sera from lupus patients with inactive disease were effective in suppressing the binding (P<0.01) and, in the presence of complement, were more efficient in suppression (P<0.001). The same sera were capable of suppressing the in vitro secretion of DNA antibodies but not polyclonal IgG by autologous cells. The suppressor factor in the inactive lupus serum was shown to reside in the F(ab)'₂ fragments of IgG and has a specificity for the F(ab')₂ fragments but not the Fc fragments of anti-DNA antibody. Sera from active lupus patients and human serum albumin were incapable of suppression. Normal sera from donors who had contact with lupus blood components were efficient in suppressing DNA-binding cells (P<0.01).     

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab)₂ fragments. Family members' sera had higher binding to anti-DNA F(ab)₂ and to normal IgG F(ab)₂ fragments (P<0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab)₂ than to normal IgG F(ab)₂ fragments (P<0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The anti-idiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.     

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.     

Family distribution of anti-F(ab'')2 antibodies in relatives of patients with systemic lupus erythematosus.

Recently we reported an inverse relationship between the levels of anti-F(ab')2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on anti-F(ab')2 antibodies in unaffected relatives of SLE patients. Sixty sera from first degree family members from 11 SLE families and 49 sera from 8 control families were studied. Percentage of SLE family members with anti-DNA antibodies (15%) was higher than than control family sera (8%, P less than 0.05). Anti-F(ab')2 antibodies were measured using ELISA assays. The SLE family sera had higher amounts of anti-F(ab')2 antibodies than the normal control family group (P = 0.0051). In an effort to determine if anti-F(ab')2 antibodies found in high titres in the sera of some SLE family members had specificity for the F(ab')2 fragment of anti-DNA antibodies of the SLE relative patients, DNA-anti-DNA inhibition experiments were performed using anti-F(ab')2 prepared from the relative in parallel with anti-F(ab')2 prepared from normal controls with equivalent high titres of serum anti-F(ab')2. Inhibition exhibited by anti-F(ab')2 of first degree relatives was higher than that obtained from control normal donors (P less than 0.02). Such differences in inhibition were not recorded using a control tetanus toxoid-anti-tetanus toxoid assay. In direct binding ELISA experiments, peroxidase-conjugated anti-F(ab')2 antibodies from the same first degree relative showed high relative specificity against purified anti-DNA antibodies of his SLE proband when compared to those obtained against different anti-DNA antibodies isolated from unrelated SLE patients (P less than 0.001). Such a substantial difference was not observed in parallel experiments using peroxidase conjugated anti-F(ab')2 antibodies from normal controls unrelated to SLE subjects.     

Endothelial cell binding by systemic lupus antibodies: functional properties and relationship with anti-DNA activity

Anti-DNA antibodies and anti-endothelial cell antibodies (AECA) are often detected in systemic lupus erythematosus (SLE). Anti-DNA antibodies can also bind the membrane of human umbilical vein endothelial cells (HUVEC), but little is known about the presence of AECA in the population of immunoglobulins from SLE sera that do not bind DNA. The aim of this study is to analyse the ability of anti-DNA and non-anti-DNA antibodies from SLE sera to bind endothelial cell antigens and to investigate their pathogenic potential. Both anti-DNA and non-anti-DNA antibodies display AECA activity by immunoprecipitation and flow cytometry and in some patients recognize antigens of identical molecular weight. Complement-dependent cytotoxicity on HUVEC was not detected with either anti-DNA or non-anti-DNA antibodies. Similarly, apoptosis was not induced in HUVEC and HL60 incubated with anti-DNA or non-anti-DNA antibodies, as shown by the DNA hypodiploid content. These data indicate that AECA are highly heterogeneous, as they recognize a wide variety of surface molecules on HUVEC and equally present in anti-DNA and non-anti-DNA antibodies from SLE patients.     

Pathogenic anti-DNA idiotype-reactive IgG in intravenous immunoglobulin preparations.

This study was addressed to explore the reactivity of natural anti-idiotypes from commercial lots of immunoglobulins to several idiotypes (Ids), usually expressed by anti-DNA molecules in lupus nephritis. Eleven intravenous immunoglobulin (IVIG) preparations and nine (three polyvalent and six hyper-immune) intramuscular IgG were investigated for specific content of anti-DNA, anti-F(ab')2 and antibodies reacting with several anti-DNA IgG Ids. Two samples (nos 6 and 11) showed high reactivity with allogeneic F(ab')2 and with F(ab')2 of myeloma proteins bearing the anti-DNA Id 3I+ and the 8.12+. Since both 3I and 8.12 Id markers are known to characterize pathogenic anti-DNA IgG in systemic lupus erythematosus (SLE), anti-Id antibodies to these markers were obtained by absorbing the IVIG samples nos 6 and 11 to Sepharose columns coupled with pooled F(ab')2 fragments of 3I(+)-F4(+)-8.12(+)-myeloma proteins. Inhibition experiments showed that anti-8.12 Id-eluted IgG induced a selective suppression of the DNA-reactive antibodies derived from patients with active lupus nephritis to their substrate, suggesting the involvement of 8.12+ molecules in the SLE glomerular damage. Since 8.12+ anti-DNA are nephritogenic antibodies, the occurrence of anti-8.12+ Id in commercial IVIG may be of potential therapeutic relevance in modulating the pathogenic SLE Id network. Previous variable results of IVIG treatment in SLE, such as resolution of proteinuria or worsening nephritis, could be related to variable enrichment of different lots of IVIG in suppressive anti-pathogenic Id antibodies.     

Antinuclear and related autoantibodies in sera of healthy subjects with IgA deficiency

The sera of 49 healthy IgA-deficient (SIgAD) subjects were evaluated for the presence of autoantibodies directed against 10 different nuclear and cytoskeletal antigens, as well as for the presence of the common lupus anti-DNA idiotype (16/6 Id). Twenty-nine sera were from IgG subclass-deficient subjects (4 = IgG2, 25 = IgG3), and 25 from normal healthy subjects, used as controls. The incidence of antinuclear but not anti-cytoskeletal antibodies were found to be significantly greater in the SIgAD group, as compared to the IgG-deficient subjects and the normal controls. Overall, 39% of SIgAD sera demonstrated polyreactivity, namely reactivity against more than one nuclear antigen. The incidence of specific antibody detection ranged from 37% against cardiolipin to 12% against RNP in the IgA-deficient group, albeit not with statistical significance in all cases when compared to the control group. Isotype evaluation of the antinuclear and related antibodies in the SIgAD group showed a greater tendency towards IgG. This increased incidence of autoantibody production in SIgAD may preceed the development of an overt autoimmune disease in the future.     

Isolation of human anti-idiotypes broadly cross reactive with anti-dsDNA antibodies from patients with Systemic lupus erythematosus

Antibodies to double stranded (ds)DNA play a central role in clinical diagnosis and disease expression in Systemic lupus erythematosus (SLE). This paper describes the isolation of anti-idiotype reagents (anti/antidsDNA) from four SLE sera and the demonstration of broad and quantitatively similar cross reactivity to both polyclonal and monoclonal anti-dsDNA antibodies isolated from SLE patients. Seven affinity-purified polyclonal and three monoclonal human anti-dsDNA preparations reacted preferentially with anti-idiotype F(ab')(2) coated plates compared to normal immunoglobulin (Ig)G F(ab')(2) coated plates in ELISA. In contrast, autoantibodies of other specificities (anti-Ro/SSA, anti-La/SSB, and anti-U(1)RNP) reacted equally with anti/anti-dsDNA F(ab')(2) and normal IgG F(ab')(2) coated plates. Such anti-idiotypic antibodies could play a significant role in the regulation of anti-dsDNA antibody levels in SLE.     

`Nuclear' antigens and antinuclear antibodies in mink sera

Aleutian disease of mink is transferrable by cell-free extracts and is characterized by hepatitis, vasculitis, nephritis, and hypergammaglobulinaemia. Because of increasing evidence incriminating antigen—antibody complexes in vasculitis disorders, the presence of nuclear antigens and antinuclear antibodies in mink sera was investigated.

Serum pools as well as individual serum specimens were obtained from uninfected mink, mink with experimentally induced Aleutian disease, mink with spontaneous Aleutian Disease, all of genotype Aa as well as from `normal' mink of similar age from colonies without Aleutian disease.

The serum pool from mink before and after experimentally induced Aleutian disease appeared to contain `nuclear' antigens detectable by rabbit anti-DNA antibodies in complement fixation and precipitin tests. The protein-free extracts of these serum pools gave strong reactions for deoxypentose in the diphenylamine tests. These serum pools also were shown to contain antinuclear antibodies by the immunofluorescence tests on human leucocyte nuclei and in precipitin tests against single strand calf thymus DNA. Sera from individual mink were similarly shown to contain `nuclear' antigens and antinuclear antibodies. The incidence and quantity of antigens and antibody detected were much greater in sera from mink after experimentally induced disease than in sera taken from mink before inoculation. The presence of `nuclear' antigens and antinuclear antibodies did not correlate with the degree of hypergammaglobulinaemia. Sera from `normal' mink in colonies without overt disease had neither antigens nor antibodies detectable in precipitin tests. Sera from mink with spontaneously acquired Aleutian disease had a high incidence of `nuclear' antigens and anti-DNA antibodies detectable in precipitin tests.

The `nuclear' antigens were detectable in Ouchterlony precipitin tests by specific rabbit anti-DNA antibodies. The precipitins formed lines of partial identity with those between the rabbit anti-DNA antibodies and single strand calf thymus DNA. However, the antigens in mink sera were not destroyed by prior incubation with DNAse which had been the case with DNA antigens detected in some human and mouse sera.

The antinuclear antibodies were detected in immunofluorescence tests using specific antibodies to mink γ-globulins, were shown to fix complement with single strand calf thymus DNA, but not with DNA that had been digested with DNAse, and formed precipitins with single strand calf thymus DNA which showed complete identity with precipitins formed by rabbit anti-DNA antibodies. Evidence for the simultaneous presence of `nuclear' antigens with antinuclear antibodies in the serum from mink with Aleutian disease was frequently evident. This observation is consistent with the hypothesis for the pathogenetic role of antigen-antibody complexes.

Aleutian disease of mink has certain clinical pathological and serological similarities with disease in New Zealand Black mice and in man with systemic lupus erythematosus. Since Aleutian disease of mink and disease of New Zealand black mice may both be examples of `slow virus' infections, a similar aetiology should be considered for certain autoimmune diseases of man, e.g. systemic lupus erythematosus.

     

Shift of private and not of cross-reactive anti-DNA idiotypes in systemic lupus erythematosus.

The shift of private idiotype (Id) and cross-reactive Id (CRI) on anti-DNA antibodies in a lupus patient KE was investigated during a 7-year period. Anti-private Id and anti-CRI activities were separated by affinity chromatography from rabbit (R)-anti-Ids raised against KE anti-DNA antibodies during active (1/84) and inactive (4/90) stages of the disease. Anti-CRI isolated from the 84 R-anti-Id appeared to recognize binding site-related Ids that are shared with KE non-anti-DNA antibodies, unrelated lupus patients' sera, and certain normal sera. Id expression on serial serum samples of KE using these fractionated R-anti-Ids as probes showed that the 1/84 private Id expression declined while the 4/90 private Id expression gradually increased. Expression of the CRI showed a relatively stable pattern. These results suggest that anti-DNA populations detected by anti-private Id can shift, while populations expressing CRI may stay stable.     

Method for avoiding false-positive results occurring in immunoglobulin M enzyme-linked immunosorbent assays due to presence of both rheumatoid factor and antinuclear antibodies

In a double-sandwich immunoglobulin M (IgM) enzyme-linked immunosorbent assay recently developed for the detection of IgM antibodies to Toxoplasma, the presence of either rheumatoid factor or antinuclear antibodies did not cause false-positive results. We recently noted, however, that in certain sera containing both rheumatoid factor and antinuclear antibodies, false-positive results do occur. In experiments to define the nature of the cross-reaction, these false-positive results were not found to be a consequence of interactions of the sera with Toxoplasma antigens, but rather were due to interactions of rheumatoid factor-antinuclear antibodies with the Fc portion of IgG antibodies used for the enzyme conjugate. This was avoided when the F(ab')2 fragment of IgG was used for the conjugate. The use of such F(ab')2 conjugates did not affect the sensitivity and, thereby, the usefulness of the double-sandwich IgM enzyme-linked immunosorbent assay for the diagnosis of acute acquired or congenital Toxoplasma infections. We concluded that F(ab')2 fragments of IgG antibodies can be used as enzyme conjugates to avoid false-positive results in sera positive for both rheumatoid factor and antinuclear antibodies in either the conventional test or in our double-sandwich IgM enzyme-linked immunosorbent assay.     

Cross-reactivity of anti-DNA antibodies with proteoglycans.

Ten sera of patients with systemic lupus erythematosus (SLE) were tested in an enzyme linked immunosorbent assay for their ability to react with glycosaminoglycans, constituents of proteoglycans, in relation to their anti-DNA reactivity. The SLE sera reacted with hyaluronic acid and chondroitin sulphate and this reactivity correlated with the anti-DNA activity of these sera. By contrast, sera obtained from patients with other autoimmune diseases or normal sera lacked any of these reactivities. Anti-DNA antibodies purified by affinity chromatography with either oligo dT cellulose or Cibracon blue F3Ga Sepharose reacted with DNA as well as with hyaluronic acid. The cross-reactivity of anti-DNA antibodies could be confirmed by the reaction of a mouse monoclonal anti-DNA antibody with DNA, hyaluronic acid, and chondroitin sulphate. This pattern of cross-reactivities of anti-DNA antibodies suggests that several compounds can function as antigenic targets for these antibodies provided that their structures contain repeating negatively charged groups.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin.

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Certain Polyclonal Antinuclear Antibodies Cross-React with the Surface Membrane of Human Lymphocytes and Granulocytes

Five out of 24 human sera with antinuclear antibody (ANA) titers of 1250 or more contained ANA that bound in vitro to normal viable human mononuclear blood cells and granulocytes, but not to erythrocytes. The antibodies can be eluted off from the cell membranes and shown to possess ANA activity. Antinative DNA antibodies and lupus erythematosus factor were not recovered in eluates, indicating that they did not react with the cells. The cells absorbed 75%-87% of the ANA activity from three sera. ANA reacted with both T-lymphocyte-depleted and -enriched mononuclear cells. No or minimal amounts of ANA bound to mouse spleen cells in suspension; in contrast, the ANA eluted from human cells reacted with nuclei of smeared mouse spleen cells. The cross-reacting antibodies were predominantly IgG that bound well at 37 degrees C, and F(ab')2 fragments carried both activities. The ANA-binding plasma membrane antigen was resistant to trypsin and RNAse but was completely inactivated by glutaraldehyde. The data indicate that human leukocyte plasma membranes and cell nuclei from many species contain a cross-specific antigen. Alternatively, the antigen may be produced in the nucleus and somehow attach to the plasma membrane.     

Evaluation of fluorescent antinuclear antibody assays, Crithidia luciliae substrate, and single-stranded DNA-binding capacity in diagnosis of four rheumatic diseases.

Sera from groups of patient with systemic lupus erythematosus, mixed connective tissue disease, rheumatoid arthritis, and progressive systemic sclerosis and normal controls were compared, using different antinuclear antibody assays. Hep-II cells, used as a substrate for the detection of antinuclear antibodies, appeared to be more sensitive than rat liver substrate. In addition, the fluorescent patterns were easier to identify on Hep-II cells. All systemic lupus erythematosus sera with antibodies reactive with kinetoplasts of Crithidia luciliae had binding greater than 43% for single-stranded DNA. Based on the high sensitivity of the Hep-II substrate and the relative specificity of high (greater than 43%) binding for single stranded DNA by sera from patients with systemic lupus erythematosus, it appears that these two tests are most useful in differential diagnosis and for the detection of systemic lupus erythematosus.     

Composition of immune deposits present in glomeruli of NZB/W F1 mice

This paper describes the results of experiments designed to investigate the composition of immune complexes present, in the form of immune deposits, in glomeruli of NZB/NZW F1 mice. Granular deposits of mouse IgG were present along the glomerular capillary walls of 6- to 12-month-old mice. Disappearance of mouse IgG from glomerular deposits, indicating a dissociation of immune complexes, was observed following incubation of kidney sections with an excess of mouse IgG, mouse Fc fragments, rat IgG, and rat Fc fragments, but not with human and rabbit Cohn fraction-II (FII), DNA, nucleohistone, and PBS. Antinuclear antibody activity in mouse sera or in glomerular eluates was removed by absorption with mouse IgG or mouse Fc fragments, rat IgG or rat Fc fragments, DNA, and nucleo-histone, but not by absorption with human or rabbit FII. These results suggest that the IgG antinuclear antibodies present in the sera and in glomerular deposits possess rheumatoid factor (RF) activity. In other experiments, kidney sections were incubated with various concentrations of pepsin, which digests the Fc portion of the IgG. After digestion, the sections were washed and stained for mouse IgG, IgG F(ab')2, and IgG Fc. At concentration of 10 micrograms/ml, pepsin completely removed IgG and IgG Fc, whereas faint IgG F(ab')2 deposits persisted in glomerular deposits. At the concentration of 1 microgram/ml, deposits of mouse IgG, F(ab')2, and Fc persisted, while F(ab')2 was observed bound to nuclei of glomerular cells. At the pepsin concentration of 0.1 microgram/ml or 0.01 microgram/ml, IgG F(ab')2 was bound to the nuclei of glomerular and tubular cells, indicating that the digestion of the Fc portion of IgG had released F(ab')2 with nuclear reactivity from glomerular deposits. The solubilization of mouse IgG from glomerular immune deposits with mouse IgG and the demonstration that pepsin digestion releases mouse F(ab')2 with nuclear reactivity are consistent with the interpretation that the immune deposits present in glomeruli of NZB/NZW F1 mice contain complexes formed by antinuclear IgG and IgG RF. These two antibodies probably cross-react and form multilayer aggregates which contribute to the formation of immune deposits.     

Demonstration of anti-idiotypic antibodies directed against IgM rheumatoid factor in the serum of rheumatoid arthritis patients.

We have identified the presence of anti-idiotypic activity against IgMRF in the sera of RA patients. Only patients seropositive for IgMRF had significant levels of anti-idiotypic activity, while seronegative patients and normal volunteers did not. When this anti-idiotypic activity was affinity-purified from a single RA patient, two separate binding activities were identified. IgG antibodies were pepsin-digested to F(ab')2 fragments before affinity-purification to remove the Fc portion capable of binding to IgMRF. Anti-idiotypic F(ab')2 fragments of IgG were eluted from an IgMRF-Sepharose 4B column. These F(ab')2 bound preferentially to IgMRF bearing an idiotype recognized by the anti-idiotypic murine monoclonal 17.109. A second anti-idiotypic F(ab')2 was affinity purified using rabbit anti-human Fc antibody bound to Sepharose 4B. These eluted antibodies behaved as the internal image of IgG, binding five out of seven IgMRF's tested. The binding of both anti-idiotypic F(ab')2 was inhibited with human IgG. The presence of both IgMRF and anti-idiotypic antibodies directed against it in the sera of RA patients suggests that anti-idiotypic antibodies alone are not capable of inhibiting the production of rheumatoid factor.     

Induction and immunochemical properties of a novel anti-antibody.

This paper describes results which characterize an induced antibody in normal outbred rabbits which we have, for convenience, called parareactant (PR). PR resulting from autoimmunization of rabbits with either keyhole limpet hemocyanin-anti-tetanus toxoid F(ab')2 or with tetanus toxoid-anti-tetanus toxoid F(ab')2 complexes was studied. PR activity was directed solely to autologous, homologous or heterologous F(ab')2 fragments regardless of their specificity. PR failed to react with intact antibodies or with antigen-antibody complexes consisting of homologous antibody bound to specific antigen. Radioimmunoassay and ELISA inhibition assays showed that reactivity between PR and autologous anti-tetanus toxoid F(ab')2 or homologous anti-bovine serum albumin F(ab')2 fragments was specifically inhibited with antigen. Anti-allotypic antibodies specific for a2 and b6 markers strongly inhibited binding of 125I-anti-micrococcal carbohydrate F(ab')2 (a2, b6) with PR (a3, b4, b5). PR specificity thus appears to be directed against non-idiotypic determinants present in Fv regions. Affinity immunoblotting was used to analyze clonality of PR in the sera collected from individual rabbits during the course of an active immune response. PR-positive sera displayed clonally restricted spectrotype patterns. PR molecules were predominantly IgG with isoelectric points of 5.9-6.8. These results strongly suggest that these PR molecules are coded by a small number of V region genes.     

Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies

Monoclonal anti-DNA antibodies produced by hybridomas derived from MRL-lpr/lpr mice and human lupus patients were found to bind to the cytoskeleton of mink lung cells. When tested by indirect immunofluorescence, 17/29 human monoclonal anti-DNA antibodies reacted with the cytoskeleton; 4 of the 29 also produce antinuclear reactions with epithelial cells. The cytoskeletal staining was not inhibited by prior treatment of the cells with DNase, but it was completely blocked by prior incubation of the monoclonal antibodies with DNA and other nucleic acids. The ability of the polynucleotides to inhibit the cytoskeletal staining corresponded to their ability to bind to the antibodies in competitive immunoassays. An (Fab')2 preparation of a monoclonal antibody bound to the cytoskeleton as well as the whole immunoglobulin. The effect of colcemid on the staining pattern, the blocking effect of a monoclonal antivimentin antibody, and results with nitrocellulose blots of cellular proteins indicated that the cytoskeletal protein to which the antibodies bound was vimentin.     

Binding of SLE autoantibodies to native poly(I), ROS-poly(I) and native DNA: a comparative study.

Reactive oxygen species (ROS) are implicated in a variety of human diseases. The formation of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE) has been extensively investigated. ROS-modified DNA has been found to be a better antigen for anti-DNA antibodies found in SLE sera. A comparative binding of SLE autoantibodies with native poly(I), ROS-poly(I) and nDNA has been studied. Affinity-purified SLE IgG exhibited a high degree of specificity towards the ROS-modified poly(I) in comparison to native DNA and native poly(I), reiterated visually by gel retardation assay. The data suggested that hydroxyl radical-modified nucleic acids like RNA and DNA might be agent for the induction of circulating SLE anti-DNA autoantibodies.