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Autoantibodies in the sera of patients with systemic lupus erythematosus were examined with respect to their specificity for proteolytic fragments of histone H1 that retain, or do not retain, DNA-binding domains. 16 of 31 sera contained IgG and IgM antibodies to histone H1. IgM antibodies to H1 in 8 sera (50%) were directed at 18 kD and 20 kD alpha-chymotrypic H1 fragments that bore binding sites for DNA, as identified by staining immunoblots containing the fragments with ssDNA plus 6/0, a mouse monoclonal antibody against ssDNA, IgM with this type of histone H1 specificity did not react with comparably-sized V8 protease fragments of H1. IgM antibodies to H1 in the other patients were directed against entirely different epitopes which were preserved in V8 protease digests of H1. In serial studies of three patients during different phase of their SLE, the level of antibodies against the 18 kD and 20 kD histone H1 fragments varied in parallel with the level of anti-ssDNA antibodies in one and varied inversely in the other two. The data suggest that a significant proportion of autoantibodies to histone H1 are directed at a limited number of epitopes localized to H1 fragments containing DNA-binding sites.

Utilizing nonionic detergent lysates of human lymphoid and non-lymphoid cells as substrate, IgM and/or IgG antibodies to a 110-kDa/isoelectric point 5.4 phosphoprotein (110K) was demonstrated in serum from patients with SLE or certain other systemic autoimmune disorders by immunoblotting and immunoprecipitation. Ig of this specificity was not demonstrable in serum from normal individuals, but, in a limited survey, was detected in serum from patients with acute hepatitis A or infectious mononucleosis. 110K shares a number of properties with nucleolin, i.e., identical Mr and isoelectric point, localization in both the nucleus and the cytosol, increased expression in rapidly dividing cells, and shown to be distinct from already defined autoantigens of similar size, i.e., topoisomerase I, PM-Scl, and RNA polymerase I. Because 110K could bind denatured DNA, as demonstrated by its specific absorption by DNA-cellulose and by its reactivity with monoclonal anti-ssDNA antibody in the presence of denatured DNA, special efforts were made to distinguish reactivity of pre-formed DNA/anti-DNA complexes in SLE serum from that due to specific anti-110K autoantibodies. Although binding to 110K could be mediated by DNA and anti-DNA in some SLE sera, the accumulated evidence supports the existence of a major new autoantibody system in SLE, other autoimmune diseases, and certain virus infections.

Artificial cryoprecipitates have been prepared from SLE sera by incubation of anti-double stranded DNA with 3H-PM2 DNA. IgM rheumatoid factor, and complement to study further the relationships between natural cryoprecipitation and the DNA/anti-DNA system in this disorder. Addition of rheumatoid factor and/or complement to a mixture of SLE serum and 3H-DNA results in increased precipitation of the 3H-DNA following incubation at 4 degrees C. Precipitation formed with added complement or complement plus rheumatoid factor could be rapidly resolubilized or reprecipitated by warming to 37 degrees C or recooling to 4 degrees C, i.e., exhibited the behavior of natural cryoprecipitates. The resolubilized cryoprecipitates contained excess antibody, because they bound significant quantities of additional 3H-DNA in the Farr assay. Of special interest was the observation that DNA binding activity frequently could be recovered from solubilized cryoprecipitates even though 3H-DNA was not added during the original cryoprecipitate preparation. With certain cryoprecipitations prepared in this way, the DNA binding activity was further increased by deoxyribonuclease treatment, raising the possibility that endogenous DNA may be constituent of such cryoprecipitates. The implications of these findings with respect to the nature of antibody-DNA immune complexes in SLE are discussed.

Sera from patients with active systemic lupus erythematosus caused the retention of the radioactively labeled synthetic polynucleotide 14C-dAT on cellulose nitrate filters. The amount of 14C-dAT bound correlated with the presence of hypocomplementemia and active renal disease. Sera containing only anti-single-stranded DNA antibodies did not bind 14C-dAT but did bind 125I DNA from E coli that had been prefiltered to remove ssDNA contamination. Inhibition experiments also indicated the presence of antigenic differences between 14C-dAT and prefiltered 125I-E coli DNA. The data suggest that dAT carries one of the major antigenic specificities for antinative DNA antibodies in SLE, and that cellulose nitrate filtration fails to remove a significant proportion of contaminating single-stranded DNA determinants in non-synthetic DNA.

Although the association of cryoglobulinemia with hypocomplementemia and tissue injury in systemic lupus erythematosus is well recognized, composition of cryoprecipitates in terms of circulating antigens and antibodies in this disease is less clear. To clarify this question, cryoprecipitates from patients with SLE were examined with sensitive assay techniques for certain antipolynucleotide antibodies and DNA antigen. DNA antibodies were highly enriched relative to serum levels in the majority of cryoprecipitates. DNA antigen was also demonstrable. Antibody to ribonucleoprotein, although less frequently present, was similarly enriched in certain cryoprecipitates. In contrast, anti-double strand RNA, which was commonly detectable in relatively high titer in serum, was only minimally concentrated in a minority of cryoprecipitates. Absorption experiments using red blood cells heavily coated with polynucleotide antigen indicated that a major proportion of the IgG in certain cryoprecipitates was specific antibody. The data strongly suggest that the cryoprecipitates in systemic lupus erythematosus represent circulating immune complexes that are soluble at 37 degrees C and come out of solution in the cold. The marked concentration of immune complexes in the cryoglobulin offers a simple and direct method for determination of the nature of the complexes. The accumulated evidence obtained in the present study indicates that these complexes closely reflect, in their composition, the circulating immune complexes which are most significant pathogenetically in renal tissue injury.