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In the sera of (NZB x NZW)F1 (B/W) mice that develop a lupus-like syndrome, increased levels of IgG antibodies (Ab) reacting with TNP have been detected before the appearance of IgG anti-DNA Ab and clinical symptoms. A single injection of trinitrophenyl-bovine serum albumin (TNP/BSA) in physiological saline into a young B/W mouse (3 months old), followed by fusion of its splenocytes 3 days later, gave rise to hybridomas simultaneously secreting IgM and IgG Ab with anti-TNP reactivity. Both mu and gamma chains were detected in culture supernatants by ELISA, and double isotype-producing cells were labeled by immunofluorescence. Molecular analysis of two of these double isotype-producing hybridomas showed the presence of mRNA coding for both mu and gamma chains of Ig, and this gamma mRNA could be translated in vitro into a gamma heavy (H) chain. Comparison of the H chain variable-region sequences of IgM and IgG revealed 100% homology between mu and gamma V(H) genes in one clone, while mu and gamma V(H) genes showed only 80% homology in the other clone. Both clones produced a single kappa light (L) chain. These two hybridomas, isolated from a B/W mouse, thus represent two different mechanisms of double isotype expression: the first one corresponds to an IgM to IgG switch, while the second one reflects a lack of allelic exclusion.

NZB is a mouse strain that spontaneously develops autoimmune haemolytic anaemia at 10-12 months of age. We analysed the autoantibodies present throughout their life and compared them to natural autoantibodies found in the normal mouse. Sera and Coombs' antibodies eluted from red blood cells (RBC) were tested for their activities against RBC and a panel of antigens: actin, myoglobin, myosin, tubulin, spectrin, DNA and trinitrophenyl bovine serum albumin (TNP-BSA), F(ab')2 and Fc fragments of IgG by using enzyme immunoassays (EIA) and Western blotting analysis of RBC membrane extracts. In NZB mouse sera, activities of IgM and IgG against the whole panel, compared to those of sera from age-matched BALB/c mice, increased progressively throughout life with oscillating values in parallel with the anti-RBC activity. Two periods of autoantibody production seem to exist: the first is characterized by a fluctuating high level of IgM and stable level of IgG natural autoantibodies, and the second by a rise of IgG natural autoantibodies in parallel with IgG anti-RBC antibodies. The presence of idiotype D23 (IdD23), which is characteristic of natural polyspecific autoantibodies, was high on serum IgM and low on IgG autoantibodies throughout life. To further analyse autoantibody level oscillations, we tested IgM and IgG fractions after their separation from whole serum and observed highly enhanced autoantibody activities of both IgM and IgG. These autoreactivities markedly diminished when the separated IgM and IgG fractions were recombined, suggesting humoral control of the autoreactivity as we had already noted for IgG in normal animals. During the first period of autoantibody production, IgM and IgG antibodies eluted from RBC (Combs' antibodies) and those eluted from serum using an RBC-immunoadsorbent (circulating antibodies) reacted with all RBC membrane components, with all antigens of the panel and with F(ab')2 and Fc. Some of these reactivities were comparable to those exhibited by a monoclonal antibody recognizing bromelain-treated RBC. In the second period, both IgM and IgG Coombs' antibodies reacted more strongly with spectrin, and exhibited new specificities, for example against the band 3 polypeptide. IdD23 was abundant on Combs' IgG antibodies in the second period. Taken together, these data suggest that IgM and IgG natural autoantibodies, able to recognize not only RBC antigens but also other antigens, particularly F(ab')2 and Fc fragment of IgG, predominate in Coomb's antibody population.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoantibodies arising in (NZB x NZW)F1 (B/W) mice during the lupus-like syndrome were studied and compared to natural antibodies present in normal mice. The antibody activities were tested in sera, circulating immune complexes (CIC) and kidney eluates, using an enzyme immunoassay against a panel of self and non-self antigens: actin, myosin, tubulin, DNA, myoglobin, spectrin and trinitrophenylated bovine serum albumin (TNP/BSA). In the B/M mouse sera, IgM antibodies reacting with all the panel of antigens (PAg) and comparable to those of normal mice, increased moderately from 5 to 9 months and markedly during the last stage preceding death (10 months), when particularly high levels of anti-DNA, anti-tubulin and anti-myoglobin antibodies were noted. Polyreactive IgM antibodies present in CIC were moderately increased while those present in complexes deposited in kidneys were strongly enhanced after the 8th month. IgG antibodies showed an early increase (2 months) in B/W sera for anti-TNP activity, which remained more or less constant until death, while a later (5-6 months) and greater increase of activity, mainly directed against DNA but also against the other antigens of the panel, was observed. In CIC, IgG, mainly anti-DNA but also anti-TNP, were enhanced at the end of the disease while at the same time IgG reacting with all the PAg were found in kidney deposits. Isolation of antibodies from sera on a DNA-immunoadsorbent demonstrated that eluted IgM reacted with all the PAg but mainly with DNA, while IgG reactivity was more restricted to DNA and to a lesser degree to TNP. The D23 idiotype, characteristics of natural polyspecific antibodies, was expressed on IgM and IgG autoantibodies from B/W mice and was enhanced, particularly in kidneys, at the end of the disease. These results demonstrate that natural antibodies are a part of the population of increased autoantibodies in this disease and could participate with IgG anti-DNA antibodies in lupus.