Anti‐DNA autoantibodies are the hallmark of human and murine systemic lupus erythematosus (SLE), an autoimmune rheumatic disease of unknown etiology. Some of these antibodies are believed to be pathogenic for kidney tissue and to initiate immune glomerulonephritis. However, the mechanisms by which anti‐DNA antibodies participate in tissue injury remain controversial. We have studied the in vivo pathogenicity of anti‐DNA monoclonal antibodies in immune deficient mice, using a panel of murine B cell hybridomas. No consistent genetic or immunochemical differences were found between pathogenic and non‐pathogenic anti‐DNA antibodies. However, the two antibody populations differed in their cross‐reaction with the acidic actin‐binding protein, α‐actinin, that is known to play a major role in the structural integrity of glomerular filtration components. These results suggest that kidney dysfunction in SLE may be facilitated by protein‐nucleic acid antigenic mimicry.

We have previously constructed knock-in (C57BL/6×BALB/c) F1 mice, each expressing an anti-DNA heavy (H) chain (D42), combined with one of three different light (L) chains, namely V ‹ 1-J ‹ 1, V ‹ 4-J ‹ 4 or V ‹ 8-J ‹ 5. All of these H/L chain combinations bind DNA with similar affinity and fine specificity. However, while mice carrying V ‹ 1-J ‹ 1-transgenic L chain were tolerized almost exclusively by L chain receptor editing, the mice expressing V ‹ 8-J ‹ 5 L chains utilized clonal anergy as their principal mechanism of B cell tolerance. V ‹ 4-J ‹ 4 targeted mice exhibited an intermediate phenotype. In the present study, these three H/L chain combinations were backcrossed onto the autoimmune NZB/NZW F1 mice. We find that the mechanism of clonal anergy is abrogated in these mice, but that receptor editing is maintained. Moreover, diseased NZB/NZW mice utilize L chain secondary rearrangements for the generation of high-affinity, anti-dsDNA-producing B cells from low-affinity precursors. The edited B cell clones are not deleted or anergized in the autoimmune animal; rather they are selected for activation, class-switching and affinity maturation by somatic mutation. These results suggest that B cell receptor editing plays an important role not only in tolerance induction, but also in generating high-affinity autoreactive B cells in autoimmune diseases.

Autoantibodies to SSA/Ro and SSB/La antigens may have a pathogenic role in photosensitive skin disease and congenital complete heart block. Since salivary glands are the major target organ in Sjogren's syndrome (SS) we wondered whether these autoantibodies are present in saliva and may be involved in the sicca syndrome. Whole saliva and serum were collected from 15 patients with SS. Elisa analysis disclosed that 8 of the patients had anti-SSA/Ro antibodies, while 6 of them also had anti-SSB/La antibodies. Studies of immunoglobulin classes showed that the sera contained mainly IgG and IgM anti-SSA/Ro or SSB/La antibodies. One serum also contained IgA antibodies. Analysis of the saliva showed that in all positive samples IgG and IgA classes were present, while none of them contained IgM. Elisa and immunoblot analysis of sera and saliva from SLE patients without the sicca syndrome disclosed that both fluids contained anti-Sm antibodies. These findings suggest that the presence of anti-SSA/Ro and anti-SSB/La antibodies in saliva is not a unique phenomenon, characterizing the sicca syndrome. Therefore, their role in the pathogenicity of the Sjogren's syndrome has to be elucidated.

We have identified two RNA-specific hybridoma autoantibodies in fusions of spleen cells from unimmunized NZB/ NZW female mice with BALB/c myeloma cells. The two fusion experiments were carried out 2 years apart with different myeloma partners. Specificity analyses showed that the two monoclonal antibodies and the total RNA-binding IgG in NZB/NZW serum recognize a G,C-rich sequence of ribonucleotides. The isolated heavy and light chains of the two antibodies and their papain Fab fragments could be distinguished by NaDodSO4/polyacrylamide gel electrophoresis. Rabbit anti-idiotypic antibodies prepared against the two monoclonal proteins showed unique specificities for the antigen-binding sites of their cognate autoantibodies. Moreover, the anti-idiotypic antisera had little effect on the RNA-binding capacity of the total IgG from NZB/NZW serum. These results suggest that a wide range of different idiotypes is involved in the autoimmune response to a similar antigenic determinant.Antibodies to native nucleic acids are found in the sera of patients with systemic lupus erythematosus (SLE), an autoimmune disease of unknown etiology (1). They also are found in several strains of mice (NZB/NZW F1, MRL, BXSB), which serve as animal models for this disease (2).The mechanisms leading to the production of nucleic acidreactive antibodies remain obscure. Both antigenic stimulation (possibly by a virus) and polyclonal activation have been implicated. A particularly interesting question is concerned with the remarkable heterogeneity ofSLE autoantibodies (1, 3). This heterogeneity could arise from multiple antigenic specificities of antibodies directed against a multideterminant macromolecule; alternatively, it could reflect the production of a large number of antibodies that bind to the same antigenic determinant with different affinities. In either case, the autoantibodies could be idiotypically related (i.e., sharing variable region structures) or unrelated. The answer to these questions would have a direct bearing on the development ofcertain therapeutic approaches to SLE. Shared idiotypic determinants would favor the use of suppressive anti-idiotypic antibodies (4, 5), whereas a limited number of antigenic specificities would promote the use of suitable tolerogens (6).We have shown (3) that the frequency of autoantibodies reactive with native, single-stranded RNA in SLE sera is similar to that ofanti-DNA antibodies, and their serum levels correlate well with disease activity. More recently, we have reported (7) the production of an RNA-binding monoclonal antibody (D4) from an unimmunized female NZB/NZW F1 mouse by the cellfusion technique (8). The antibody was shown to be directed against a G,C-rich sequence of ribonucleotides (9). We now describe a second monoclonal antibody (D44) that is derived from a different individual of the same mouse strain and possesses a very similar nucleotide specificity but different idiotypic structures. MATERIALS AND METHODS Production and Purification of Hybridoma Autoantibodies.Fus...

Hyperglycosylated human chorionic gonadotropin (HhCG) is a glycoprotein hormone secreted during embryonic implantation and trophoblast invasion of the uterine wall and is an early marker of pregnancy (1). Relative to hCG, HhCG has a higher molecular mass (38.5-40 kDa, depending on the amount of carbohydrate) and a higher number of asparagine (N)-linked triantennary carbohydrates and serine (O)-linked tetrasaccharide core structures in the-subunit (2). Although both are secreted from the placenta and choriocarcinoma, HhCG is produced by mononucleated cytotrophoblasts, and hCG is produced by syncytiotrophoblast cells (3-6). Because the cytotrophoblasts are primitive and invasive in nature, HhCG is also called invasive trophoblast antigen (ITA) (5). Birken et al. (7) described a monoclonal antibody (B152) specific for the-subunit C-terminal peptide and the O-linked oligosaccharide of HhCG. Although the epitope for this antibody does not require sialic acid, the presence of the O-linked tetrasaccharide core structure is essential (1). Using IRMAs and ELISAs, investigators showed that (a) HhCG rapidly increases in early pregnancy, attaining substantially higher concentrations and decreasing earlier than hCG (1, 8); (b) HhCG is increased in Down syndrome affected pregnancies in both the first and second trimesters (9-11); and (c) the HhCG:hCG ratio appears to be higher in those with invasive vs noninvasive tropho-blastic disease (12). The above HhCG assays were performed manually using large sample volumes (200 L) and long incubation times (turnaround time, 1-2 days). We therefore developed an automated immunochemiluminometric assay (ICMA) that uses two monoclonal antibodies: the HhCG-specific B152 antibody described above and a hCG-sub-unit-specific antibody (B207). Both antibodies were purified from cell lines provided by Dr. O'Connor (Columbia University, New York, NY). B152 was biotinylated with long-chain NHS-biotin (13), and B207 was conjugated with acridinium ester (14). The Nichols Institute Diagnostics Advantage ® instrument automatically pipetted 15 L of sample into a cuvette, followed by 25 L of streptavidin-coated magnetic particles (4 g/L Dynal M-270), 70 L of capture antibody (6 mg/L B152), and 260 L of buffer [0.1 mol/L phosphate-buffered saline (PBS), pH 8.2, containing 50 g/L bovine serum albumin (BSA)]. During a 30-min incubation at 37 °C, HhCG in the sample bound to the B152 capture antibody, which in turn bound to the magnetic particles. The magnetic particles were automatically washed three times to remove unbound materials. Detection antibody [300 L of 1 mg/L B207 in 0.5 mol/L PBS (pH 7.4) with 5 g/L protease-free BSA, 60 mL/L normal mouse serum, and 1 g/L mouse-globulin] was then added to the washed magnetic particles. During this 10-min incubation at 37 °C, the B207 antibody bound to a hCG-shared epitope on the HhCG molecule, forming a sandwich complex. After another three washes, the magnetic particle-containing wells were transferred to the on-board luminometer. Hydrogen peroxide (3.25 mL/L)-...