Expression of idiotype on the surface of human B cells producing anti-DNA antibody

We analyzed the idiotype (Id) expression on the surface of human anti-DNA antibody-producing cells. Murine monoclonal anti-Id antibodies with a specificity for determinants associated with the antigen-binding sites of human monoclonal anti-DNA autoantibodies were prepared. One anti-Id antibody reacted only with surface Id on anti-ssDNA-producing cells, but not with those on anti-dsDNA-producing B cell clones. Another anti-Id antibody did bind the surface Id on anti-dsDNA clones, but not those on anti-ssDNA clones. The interaction between anti-Id and surface Id was inhibited by pretreatment of the clones with DNA or appropriate polynucleotide antigens, or by preabsorption of anti-Id antibodies with free anti-DNA antibodies. Surface IgM and IgD expressed the same Id as the antibody secreted from the clones. The treatment of Id-positive clones by anti-Id antibody induced the redistribution of surface Id on the cells, indicating that these cells serve as targets for the regulatory action of anti-Id antibody.     

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model

B/W mice spontaneously develop IgG antibodies to DNA that cause lethal immune nephritis. T and B cell interactions in the in vitro anti-DNA antibody response of B/W mice were investigated, and two distinct families of helper T cells that drive these responses were defined. First, the anti-DNA antibody-forming cell (AFC) response was found to be increased in B/W mice with nephritis and was inhibited with the monoclonal antibody anti-L3T4, suggesting a major role for helper T cells. Purified splenic T cells from mice with nephritis were able to augment both the IgG and the IgM anti-DNA AFC response of young B/W B cells. T helper cells were cloned from spleens of NZB/W F female mice with high titer anti-DNA antibodies and nephritis. The cloned T cells augmented both IgG and IgM anti-DNA AFC responses of young B/W B cells. Four clones--27.9, 30.7, 30.8, and 30.10--were selected for further study. These cells proliferated, in the context of syngeneic (H2d/z) antigen-presenting cells (APC) but not to allogeneic APC. Analysis of the mechanism of T helper cell clone-mediated augmentation of anti-DNA AFC revealed two populations: "cognate" T helper cells, which specifically augment anti-DNA AFC (30.7 and 30.10), and non-antigen-specific T helper cells (27.9 and 30.8), which augment the response of B cells of differing specificity by a bystander mechanism, probably through increased release of B cell growth and differentiation factors.     

Primary structures and chain dominance of anti-DNA antibodies

Using several anti-DNA autoantibodies, we analyzed the relative involvement of heavy and light chains in their interactions with DNA. We previously obtained eight hybridomas producing monoclonal anti-DNA autoantibodies by fusing spleen cells from an MRL-lpr/lpr mouse with myeloma cells. The chain dominance was analyzed by UV cross-linking experiments, in which the antibodies were covalently cross-linked with radioisotope-labeled oligonucleotides by short-wavelength UV-light, and the cross-linked H and L chains were analyzed by SDS-PAGE and densitometric scanning. Among these, three were found to be heavy chain dominant antibodies in which heavy chains are dominantly involved in DNA binding. The other five were co-dominant antibodies in which both heavy and light chains are involved in DNA binding. To determine the factor(s) that can explain the chain dominance in DNA binding, we determined the amino acid sequences of the variable regions of both heavy (VH) and light (VL) chains of all eight monoclonal antibodies. By analyzing the data, we were able to draw the following conclusions: (1) The arginine residues are found in the CDR3 regions of both VH and VL of the co-dominant antibodies; whereas, the same residues are found only in the CDR3s of VH, but not in VL, of the heavy chain dominant antibodies. (2) The net charges of the V regions affect the chain dominance. From the results of this study it is suggested that the presence of arginine residue in CDR3 is a critical factor in determining chain-dominance, as well as DNA binding of anti-DNA antibodies in general.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Monoclonal antibodies to DNA and RNA from NZB/NZW F1 mice: antigenic specificities and NH2 terminal amino acid sequences

Two anti-DNA hybridoma autoantibodies ( A52 , D42 ) were prepared by fusing spleen cells from unimmunized NZB/NZW F1 female mice with BALB/c myeloma cells. The monoclonal antibodies were purified to homogeneity and were analyzed for their antigen-binding specificities. The two anti-DNA antibodies bound single-stranded, double-stranded, and supercoiled DNA, with a marked preference for the single-stranded conformation. Competition experiments performed with synthetic polynucleotides, as well as chain reconstitution experiments, indicated that both the sugar-phosphate backbone and the heterocyclic bases of the nucleic acid are essential for antibody recognition. Amino terminal sequence analysis of A52 and two RNA-binding hybridoma proteins revealed that the heavy chains from all three were members of the VHII subgroup and that the A52 light chain was homologous to the VK8 subgroup. The D42 heavy chain was found to be similar to a phosphocholine-binding hybridoma of the VHIII subgroup.     

Structural Patterns in Anti-DNA Autoantibodies: A Molecular Modeling Study

Anti-DNA autoantibodies are the hallmark of the autoimmune disease systemic lupus erythematosus. Although these antibodies are both diagnostic and pathogenic, little is known about their structures and the manner in which they recognize DNA antigens. To address the first of these points we have predicted the three-dimensional structures of 40 monoclonal anti-DNA F(ab) fragments derived from lupus-prone mice. These antibodies were chosen to encompass several different autoimmune strains along with the known variable region gene families that encode anti-DNA. We find that the structures of the antigen binding regions of these antibodies fall into three main classes, irrespective of both the mouse strain and genetic origins of the antibody. Specifically, high-affinity anti-ssDNA appear to possess a narrow channel that is presumably used for ligand recognition, whereas the binding site on anti-dsDNA is an open surface that is large enough to accommodate a DNA duplex. These findings provide structural data to support the hypothesis that anti-DNA arise by DNA-driven B cell activation and clonal expansion.     

Cellular basis of in vitro anti-DNA antibody production: evidence for T cell dependence of IgG-class anti-DNA antibody synthesis in the (NZB X NZW)F1 hybrid

An in vitro system was designed to measure anti-DNA antibody synthesis, and the cellular basis of this autoantibody production in NZB X NZW (B/W)F1 (B/W F1) mice was analyzed. The spleen cells from old B/W F1 mice contained a number of B cells that spontaneously produced anti-DNA antibodies of both IgM and IgG classes in the absence of stimulants, thereby demonstrating that these B cells had been activated in vivo. These activated B cells could be removed by Sephadex G-10 column (G-10) filtration. Such G-10-passed, homogeneously small B cells were activated by the stimulant lipopolysaccharide (LPS) and produced both IgM and IgG class anti-DNA antibodies. The G-10-passed cells contained both B and T cells, and the cytotoxic treatment of the cells with monoclonal antibodies to T cells, anti-Thy-1 and anti-L3T4, abolished the LPS-induced IgG class, but not IgM class, anti-DNA antibody syntheses. Thus, the LPS-induced production of IgG class anti-DNA antibodies in B/W F1 mice is regulated by T cells. Reconstitution experiments revealed the requirement of T-B cell contact but not of the proliferative response of T cells. Moreover, there was no apparent adherent cell requirement. Such IgG class anti-DNA antibodies were produced only by spleen cells from old B/W F1 mice, but not from young B/W F1, NZB, NZW, and C57BL/6 mice. Like IgM class anti-DNA antibodies, LPS-induced synthesis of polyclonal IgM was T cell-independent. Only a slight reduction in the polyclonal IgG synthesis was observed after the G-10-passed cells had been treated with anti-Thy-1 antibody plus complement. This study should facilitate investigation of cell to cell interactions in the formation of autoantibodies and their correlations to immunologic abnormalities in autoimmune disease.     

Heat Shock Proteins in the Human Periodontal Disease Process

The production of HSP by periodontopathic Gram-negative bacteria was examined by SDS-PAGE, two dimensional gel electrophoresis, and Western blotting using monoclonal antibodies against HSPs. Strains of Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Treponema socranskii species produced HSP which reacted with anti-Yersinia enterocolitica HSP 60 and/or mycobacterial 65-kDa HSP monoclonal antibodies. It was found that gingival homogenate samples from patients with adult periodontitis reacted with anti-human HSP 60 and bovine brain HSP 70 monoclonal antibodies. Antibodies which reacted with bacterial HSP were also found in a serum sample from a periodontitis patient. The present study suggests that HSPs are implicated in the human periodontal disease process.     

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.     

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.     

Polyspecific binding of Escherichia coli beta-galactosidase by murine antibodies to DNA

To characterize further polyspecific interactions of antibodies to DNA, the binding of sera from autoimmune MRL-lpr/lpr mice to Escherichia coli beta-galactosidase (beta-gal) was analyzed. This protein was selected for study because of preliminary observations that sera from autoimmune mice bound unexpectedly to cloned fusion protein constructions containing beta-gal. Using ELISA assays, sera from MRL-lpr/lpr mice demonstrated high levels of antibodies to both DNA and beta-gal, in titers significantly greater than those of BALB/c controls. Affinity chromatography using beta-gal-Sepharose demonstrated that antibodies enriched for anti-beta-gal activity bound both DNA as well as beta-gal, indicating the presence of a population of cross-reactive anti-DNA antibodies. Furthermore, anti-DNA mAb of MRL-lpr/lpr strain origin also bound beta-gal by ELISA, although these levels were lower than those to DNA. Together, these results extend the range of polyspecific binding of murine anti-DNA antibodies to bacterial proteins. They further suggest caution in the interpretation of immunoassays using fusion protein constructions containing beta-gal, especially with sera from autoimmune mice.     

A public idiotypic determinant is present on spontaneous cationic IgG antibodies to DNA from mice of unrelated lupus-prone strains

A monoclonal anti-idiotypic antibody (anti-Id) to a public idiotype (Id) present on spontaneous IgG antibodies to DNA from NZB/NZW F1 mice recognized similar determinants on polyclonal and monoclonal IgG anti-DNA antibodies from mice of the unrelated MRL/lpr and BXSB strains. Incubation of the anti-Id with four of five monoclonal Id in solid phase inhibited their ability to bind DNA; however, different Id+ antibodies recognized different epitopes within the DNA molecule. Therefore, the public Id was located close to the antigen-binding regions but did not comprise all of those regions. Analysis of multiple polyclonal and monoclonal antibodies to DNA showed the Id on all subclasses of IgG. However, antibodies bearing the Id carried a neutral or cationic charge (10 of 10 monoclonals with pI greater than 7 were Id+); the presence of the Id on anionic IgG (pI less than or equal to 7) was infrequent (one of 21 serums, one of eight monoclonal antibodies). Therefore, IgG autoantibodies to DNA are constructed from closely related public idiotypes in several mouse strains that spontaneously develop lupus, and that Id is restricted to antibodies with a pI of 7 or greater.     

Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells]

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.