单克隆抗DNA自身抗体及其抗血清独特型交叉反应

由严重存在SLE,并无需外源免疫能自发产生抗DNA自身抗体的小鼠MRL-Lpr/Lpr脾细胞与SP2/0-Ag-14骨髓瘤细胞杂交,获得一系列杂交瘤细胞株,它们分别分泌单克隆抗DNA自身抗体。我们用其中的细胞株H241的单抗免疫家兔,从免疫血清中收获到抗H241独特型扰体。通过放射免疫分析法(RIA),对H241进行了敏感度分析,并研究了H241与抗H211血清反应中,H241与另24个抗DNA单抗独特型交叉反应。结果表明,H241是高敏感度的单抗,6ng非标记H241能使~(125)I-H241与抗H241独特型抗体的结合达50%抑制。实验还证明,24个抗DNA单抗中有15个与H241不同程度地存在独特型交叉反应。实验提示,虽然抗DNA自身抗体独特型往往结构分离,从而表现抗体多样性,但有些抗体由于独特型结构类似,多样性受到限制;全身性红斑狠疮小鼠控制免疫球蛋白合成的胚系基因含有某些抗DNA抗体的相关位点。     

Cross-reactions of nucleic acids with monoclonal antibodies to phosphatidylinositol phosphate and cholesterol

Four monoclonal IgM antibodies to phosphatidylinositol phosphate (PIP), four antibodies to cholesterol and one antibody to liposomes containing phosphatidylcholine, cholesterol and dicetyl phosphate were tested for reactivity with denatured DNA. Three of four antibodies to PIP cross-reacted strongly with denatured DNA. The other antibodies did react with denatured DNA but only very weakly. The binding to DNA was competed by synthetic polynucleotides. In competitive assays, one of the anti-PIP antibodies was particularly reactive with poly(dT) and another with poly(I) and poly(dG). Binding of an anti-cholesterol antibody to ssDNA was also inhibited by poly(I) and poly(dG). Two of the anti-PIP antibodies were also reactive with mononucleotides, and all four bound inositol hexaphosphate. High concns of nucleosides did not compete for binding, indicating that phosphate is involved in the binding site. Phospholipids, particularly those containing inositol phosphate, also competed for binding to DNA, but to varying extents, indicating a variable overlap in the antibody binding site for DNA and phospholipid determinants. These antibodies, induced by immunization with liposomes, showed cross-reactivity characteristics often found with certain types of autoantibodies, but they did not bear the H130 idiotype, which was identified on IgM anti-DNA autoantibodies from MRL-lpr/lpr mice.     

Monoclonal anti-tuberculosis antibodies react with DNA, and monoclonal anti-DNA autoantibodies react with Mycobacterium tuberculosis.

Classical models of experimental autoimmune diseases, such as adjuvant arthritis entail the use of mycobacteria. Furthermore, BCG immunotherapy may be followed by arthritic symptoms. To test the infection-autoimmunity relationship of mycobacteria, we used monoclonal antibodies raised against M. tuberculosis and against DNA. Murine monoclonal anti-TB antibodies were found to react with ssDNA, dsDNA and other polynucleotides. Monoclonal anti-DNA autoantibodies derived from patients and mice with SLE bound to three glycolipids shared among all mycobacteria and derived from mycobacterial cell wall. Prior incubation of the antibodies with ssDNA and other polynucleotides or with glycolipid antigens inhibited binding. These results indicate that infecting mycobacteria share antigens with human tissue, thus accounting in part for the production of autoantibodies in mycobacterial infections.     

Human monoclonal anti-DNA antibodies react as lymphocytotoxic antibodies

Two out of 25 monoclonal anti-DNA autoantibodies that were produced by human-human hybridoma were found to have lymphocytotoxic activity. The antibodies reacted with normal B and T lymphocytes at cold (4 degrees C) as well as at warm (37 degrees C) temperatures. The lymphocytotoxic activity of the monoclonal anti-DNA antibodies could be inhibited by prior incubation of the antibodies with either polynucleotides, e.g. poly(I), poly(dT) or anti-idiotypic antibodies, that had been raised against a dominant anti-DNA antibody. The cross-reactivity between nuclear material and lymphocyte membrane raises the question whether these apparently diverse materials have a shared epitope. The cross-reactivity between anti-DNA antibodies and lymphocyte membrane may account in part for the lymphopenia observed in systemic lupus erythematosus patients.     

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.     

Murine lupus anti-DNA antibodies cross-react with the hapten (4-hydroxy-5-iodo-3-nitrophenyl)acetyl, but immunization-induced anti-DNA antibodies do not

The antigen-binding selectivity of 2 sets of anti-DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity-purified anti-DNA auto-antibodies from MRL-lpr/lpr mice were examined for binding to the haptens azobenzenearsonate, phosphorylcholine, (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP-protein conjugates. In contrast, none of 12 monoclonal antibodies to single-stranded DNA or left-handed Z-DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens. In a reciprocal test of the relationship between anti-DNA and anti-NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.     

Cross-reactivity distinguishes serum and nephritogenic anti-DNA antibodies in human lupus from their natural counterparts in normal serum

To distinguish the properties of anti-DNA antibodies in patients with lupus from those in normal individuals, we compared the ligand binding, idiotypic and charge properties of serum anti-DNA antibodies derived from: patients with active lupus; normal individuals; and among Ig eluted from the kidneys of two patients with active lupus nephritis (one with mesangial proliferation and the other with membranous nephropathy). The kidney eluate anti-DNA antibodies were the most cross-reactive; they cross-reacted with ssDNA, poly(GdC), poly(dT), poly(dG), poly(dC), ZDNA, SmRNP and the phospholipids cardiolipin and phosphatidyl serine. Lupus serum anti-DNA antibody cross-reacted with polynucleotides but not with phospholipids, whereas anti-DNA antibodies derived from normal serum reacted only with poly(dT). An anti-idiotype (anti-IdD; produced against serum anti-DNA antibodies from one patient) reacted with: anti-DNA antibodies in 8/9 lupus sera; antibodies in both kidney eluates; and anti-DNA antibodies from 5/7 normal sera. Anti-IdD did not react with Ig that did not bind to DNA. Isoelectric focusing of Ig showed that the charge of anti-DNA antibodies from lupus serum and normal serum were similar and unrestricted (pI 5.4-9.0); Ig in kidney eluates varied: membranous lupus pI 4.5-8.6; mesangial lupus pI 8.1-9.1. We conclude that idiotypically related anti-DNA antibodies in tissue lesions, lupus serum and normal serum from different individuals can be distinguished on the basis of their cross-reactive antigen-binding properties. Furthermore the cross-reactive properties of lupus auto-antibodies may influence their capacity to form glomerular immune deposits.     

A polyspecific monoclonal anti-DNA autoantibody also binds to cell-surface protein(s)

A monoclonal anti-DNA autoantibody (EM85) produced in an autoimmune MRL/lpr/lpr mouse was studied. Its antigenic specificity was demonstrated to be directed against single-stranded DNA, double-stranded DNA, and also a variety of polynucleotides. We have recently reported that a murine monoclonal anti-DNA autoantibody produced in autoimmune B/W mice, with specificity for double-stranded DNA, also binds to cell-surface protein(s). We show here that EM85, which recognizes a variety of polynucleotides, also binds to protein(s) on the surface of Raji cells. These data indicate that the antigenic determinant, recognized by this monoclonal anti-DNA antibody, is common to a variety of polynucleotides and cell-surface protein(s).     

Polynucleotide specificities of murine monoclonal anti-DNA antibodies

Three IgM monoclonal anti-DNA antibodies were produced by hybridoma techniques from an MRL-lpr/lpr mouse using denatured DNA (dDNA) as the selection antigen. All three antibodies also bound poly(dT), poly(rA), and the single-stranded random copolymer poly(dI,dT), and each antibody displayed a unique preference for a limited array of other ribo- and deoxyribopolynucleotides based on direct binding as well as inhibition studies. Inability to identify a common primary structure in the polynucleotides reactive with each antibody suggested that higher ordered structures may be important. This notion was supported by the finding that oligomers of thymidine of 25-30 nucleotides or less were ineffective in blocking antibody binding to dDNA or poly(dT). However, deliberate destabilization of putative secondary structures by decreasing counterion concentration and increasing temperature had little effect on antibody binding to poly(dT). Since the antigenic polynucleotides in general contain little known secondary structure and considerable flexibility, antibody binding may be accompanied by local conformational changes in the polynucleotide that result in a better fit to the antibody combining site.     

Expression of a highly conserved anti-DNA idiotype in normal and autoimmune mice

The specificity and idiotypic relationships of a monoclonal anti-DNA antibody were investigated to evaluate genetic control in this autoantibody response. 6/0 is an IgG2a monoclonal anti-DNA derived by the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse and the cell line NS1. By an enzyme-linked immunosorbent assay (ELISA) for anti-DNA, 6/0 demonstrated preference for single-stranded DNA and bound deoxyribo- and ribohomopolymers of dissimilar base composition. The control of 6/0 expression was evaluated by idiotypic analysis using a rabbit anti-6/0 antiserum made specific by absorption with the BALB/c myelomas UPC 10 (IgG2a) and MOPC 21 (IgG1). The resulting preparation was fractionated by BALB/c IgG affinity columns to provide antibodies to idiotypic determinants essentially unique to 6/0 and those commonly expressed in sera. The commonly expressed 6/0 idiotype was found in sera of ten inbred strains of mice and was not exclusive to the autoimmune strains. MRL-lpr/lpr and A/J strain mice displayed idiotype levels almost fivefold greater than other strains, with 6/0 idiotype-bearing antibodies having serum concentrations as high as 1 mg/ml. Levels of the 6/0 idiotype, however, did not correlate with anti-DNA levels among the various strains. In addition to mice, the majority of individuals of three inbred rat strains showed detectable 6/0 idiotype in their sera. These results suggest that the 6/0 idiotype, although identified using a monoclonal anti-DNA antibody, represents a framework determinant that is phylogenetically conserved. The mechanisms for the expression of this determinant may differ among the normal and autoimmune strains.     

The role of anti-idiotypic antibodies in the induction of experimental systemic lupus erythematosus in mice

We have recently reported the induction of experimental systemic lupus erythematosus (SLE) in mice by a human anti-DNA monoclonal antibody (mAb) that bears a common idiotype, the 16/6 Id. In the present report we investigated the role of the idiotypic network in the induction of experimental SLE by using a murine anti-idiotypic mAb specific for the 16/6 Id. This anti-idiotypic mAb induced experimental SLE similarly to the 16/6 Id. Thus, following immunization, in addition to 16/6 Id+ antibodies, the mice produced antibodies to various nuclear antigens: single-stranded DNA, double-stranded DNA, poly(I), poly(G), Ro, La, Sm and ribonucleoproteins. Similarly to the 16/6 Id-immunized mice, the mice injected with the anti-16/6 Id mAb exhibited elevated erythrocyte sedimentation rate and leukopenia. The murine anti-16/6 Id mAb was found to be more effective than the 16/6 Id, in causing earlier onset of proteinuria and renal damage. These results suggest that the idiotypic network and particularly anti-idiotypic antibodies specific for anti-DNA common idiotypes found in SLE, play an important role in the induction of SLE in mice.     

Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera.     

Monoclonal human anti-DNA antibodies from EB virus-transformed lymphocytes of systemic lupus erythematosus (SLE) patients

Sixteen monoclonal human anti-DNA antibodies were obtained from Epstein-Barr virus-transformed lymphoblastoid cells of patients with systemic lupus erythematosus (SLE) and were studied in terms of antigenic specificity. All of the antibodies showed polyspecificity to polynucleotides. Among them, some antibodies had a specificity to single-stranded (ss) DNA. Especially, O-8 antibodies showed a preference for polynucleotides with pyrimidine bases. The binding specificity of the antibody was also studied using different sizes of dT oligomers in order to assess the size of the epitope. It was revealed that oligonucleotides with a size of more than 25–30 nucleotides are required for inhibition of the antibody to ss-DNA. Other studies also demonstrated that anti-ss-DNA (O-8) antibody and anti-double-stranded (ds) DNA (NE-28) antibody bound to different combining sites in the same polynucleotides, poly(dT). These results suggest that some anti-ss-DNA antibodies are directed to the conformational structure related to the base sequence and that nucleic acids, therefore, might be responsible for the possible immunogenic stimulus causing the anti-DNA immune response. We also indicate that this type of antibody would be popular among serum anti-DNA antibodies in SLE.     

Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.     

Suppression of anti-DNA antibody production in MRL mice by treatment with anti-idiotypic antibodies.

Antibodies against idiotypic determinants carried by a monoclonal polyspecific natural autoantibody were raised in rabbits and in syngeneic BALB/c mice. These anti-idiotypic antibodies were administered to newborn and to pregnant BALB/c mice and to MRL-lpr/lpr mice. Serial measurements of the idiotypes, naturally occurring autoantibodies, and antibodies obtained after antigenic stimulation were performed in the sera of the injected mice and in the offspring of pregnant mice. No idiotypic suppression was noted in newborn injected mice. Transient suppression of idiotypes recognized by the syngeneic anti-idiotypic antibody was noted in the offspring of pregnant mice injected with the rabbit polyclonal anti-idiotypic antiserum. No changes in naturally occurring autoantibodies or in antibodies appearing after antigenic stimulation were noted in BALB/c mice. In contrast, a significant decrease of spontaneously occurring anti-DNA antibodies was found in MRL-lpr/lpr mice treated with rabbit polyclonal anti-idiotypic antiserum. Furthermore in these mice a slight decrease of anti-TNP antibodies was also observed. These results suggest that anti-idiotypic antibodies directed against natural autoantibodies may play a regulatory role in the immune system; this role is more easily appreciated in mice suffering from immune dysregulation.     

A human fetal monoclonal DNA-binding antibody shares idiotypes with fetal and adult murine monoclonal DNA-binding antibodies.

A human DNA-binding monoclonal antibody was produced by fusing the hepatocytes from a 12-week-old human fetus with the lymphoblastoid cell line GM 4672 using polyethylene glycol. This antibody, designated BEG 2, binds to single-stranded (ss) DNA but also binds to double-stranded (ds) DNA, poly(dT), polyI and poly(ADP-ribose), but not to RNA, cardiolipin or K-30. The binding of BEG 2 to these polynucleotides can be inhibited by incubation with polynucleotides in the fluid phase. A rabbit polyclonal anti-idiotype was raised, and using this reagent it was shown that the BEG 2 idiotype is present in normal human serum (7%), systemic lupus erythematosus (SLE) sera (8%) and rheumatoid arthritis sera (23%). The extent of idiotypic sharing between BEG 2 and murine monoclonal DNA-binding antibodies, in particular monoclonal antibody (mAb) 423 (derived from a 15-day-old fetal MRL/Mp-lpr/lpr mouse) and mAb 402 (derived from an adult MRL/lpr mouse), was also investigated. Using a competition ELISA, it was shown that preincubation of BEG 2 with rabbit anti-423 and rabbit anti-402 inhibits the binding of BEG 2 to DNA, and the binding of 402 to DNA by anti-BEG 2 and anti-423. These data suggest that mAb BEG 2, 423 and 402 share common idiotypes, that autoreactivity is present in early fetal life, and that autoantibodies may be encoded for by germline genes, which have been conserved through evolution.     

Monoclonal antibody to a DNA-binding domain of p53 mimics charge structure of DNA: anti-idiotypes to the anti-p53 antibody are anti-DNA

Antibodies to DNA are important markers of various autoimmune diseases and can be pathogenic; however, their generation is not understood. We previously reported that anti-DNA antibodies could be induced in mice by idiotypic immunization to PAb-421, an antibody to a DNA-binding domain of p53. We now report that two monoclonal antibodies of moderate affinity (K(D) asymptotically equal to 10(-7)), raised from PAb-421-immunized mice, specifically recognized both PAb-421 and DNA. These antibodies feature multiple arginine residues in the antigen-binding site, a unique characteristic of disease-associated anti-DNA antibodies; nevertheless, these anti-DNA antibodies show specific complementarity to PAb-421 by competing with p53 for PAb-421 binding and recognize defined oligonucleotides with a specificity similar to that of p53. To study the structural basis for the cross-recognition of PAb-421 and DNA by the anti-DNA antibodies, we constructed computer models (fine-tuned by protein-protein docking) of PAb-421 and one of the monoclonal anti-DNA antibodies. The modeled structures manifested structural complementarity. Most notably, the modeled structure of PAb-421 resembled the structure of DNA by the positions of negatively charged groups and aromatic side chains. Thus, a protein molecule may mimic the structure of DNA and the elusive generation of anti-DNA antibodies could be explained by idiotypic immunity to a DNA-binding protein, like p53.     

Nucleic acid-binding specificity and idiotypic expression of canine anti-DNA antibodies

Serum samples collected from eleven lupus dogs during an active phase of the disease all bound native and denatured DNA, poly(dT), poly(I) and poly(G). Nine bound poly(C); 10 bound poly(U); and 3 bound poly(A). Sera from 22 normal dogs were negative with all of these antigens. The canine sera were also probed for the presence of three idiotypic markers, one related to human lupus anti-DNA antibodies and two related to murine lupus antibodies. One canine lupus serum expressed idiotopes related to murine anti-DNA idiotype (Id) termed H130: (a) the canine serum bound to anti-H130 anti-Id; (b) it inhibited the binding of anti-H130 Id to its homologous Id; and (c) the anti-H130 Id inhibited the binding of the canine serum to DNA. These findings suggest that anti-DNA variable regions exhibit interspecies similarities, probably reflecting the conservation of the encoding gene segments through evolution.     

Specific binding to methylated polynucleotides in monoclonal anti-poly(dT) antibodies from autoimmune mice

Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.     

Anti-DNA and Antiphospholipid Antibodies in IVIG Preparations: In Vivo Study in Naive Mice

Intravenous immunoglobulins (IVIG) are therapeutic preparations of pooled normal polyspecific immunoglobulin G. We investigated the presence and the in vivo pathogenic potential of autoantibodies against phospholipids and DNA in several commercial IVIG preparations. The presence of autoantibodies and their antiidiotypic antibodies in the IVIG preparations was detected by ELISA. Naive mice were actively immunized with either IVIG preparations or pathogenic monoclonal antibodies (mAbs) against cardiolipin (CL) or DNA, in an attempt to induce autoimmune conditions. The mice were tested for the presence of mouse autoantibodies (auto-Abs) and for clinical parameters of autoimmune diseases. We found high levels of auto-Abs against a panel of phospholipids and DNA, as well as their antiidiotypic Abs, in all the IVIGs. Affinity studies pointed to a lower affinity of auto-Abs of IVIG origin to their respective antigens compared to pathogenic mAbs. Mice immunized with pathogenic anti-CL mAb had high levels of antiphospholipid auto-Abs, accompanied by thrombocytopenia, prolonged aPTT, and an increased fetal resorption rate. Mice immunized with pathogenic anti-DNA mAb had elevated anti-DNA and anti-CL auto-Abs, along with a high erythrocyte sedimentation rate, leukopenia, and significant proteinuria. Following immunization with IgGs from IVIG batches, mice developed high levels of auto-Abs against phospholipids and DNA, similar to mice immunized with pathogenic anti-DNA or anti-CL mAbs, but none of the mice expressed the clinical manifestations compatible with the presence of these autoantibodies. We conclude that commercial IVIG preparations contain high levels of antiphospholipid and anti-DNA auto-Abs, as well as their antiidiotypic Abs. Although these Abs induced the generation of mouse auto-Abs upon active immunization, following idiotypic manipulation they did not prove to be pathogenic in vivo.