Clearance and organ localization of small DNA anti-DNA immune complexes in mice

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.     

SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

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.     

The kinetics of [3H]-dsDNA/anti-DNA immune complex formation, binding by red blood cells, and release into serum: effect of DNA molecular weight and conditions of antibody excess

[3H]dsDNA/anti-DNA immune complexes (IC) formed, fixed complement, and bound rapidly to red blood cells (RBC) in whole blood (less than 5 min), but were released from the cells more slowly. The rate of release was dependent on both the antibody:DNA ratio and the m.w. of the DNA in the complex. For example, complexes formed with high m.w. DNA (6 X 10(6) daltons) were released more slowly (t1/2 = 60 min) than complexes formed with lower m.w. DNA (2 to 6 X 10(5) daltons, t1/2 = 15 to 20 min). The [3H]dsDNA/anti-DNA complexes, which were released from the cells as intact antigen/antibody/complement complexes, did not rebind to RBC, but did bind to Raji cells and could be precipitated by monoclonal antibody to C3d. When these released IC (RIC) containing high m.w. DNA were incubated with additional anti-DNA antibody and fresh complement, they rebound to RBC. However, RIC containing lower m.w. DNA (5 X 10(5) daltons) did not rebind to RBC under the same conditions. These data suggest that IC containing high m.w. DNA bind to and remain bound to RBC more effectively than IC containing lower m.w. DNA, and thus may be more easily cleared from the circulation by the RBC IC clearance mechanism. Thus, the size of the DNA in the IC may be a significant factor in the pathogenicity of DNA/anti-DNA complexes in SLE.     

Effect of antibody excess on the size, stoichiometry, and DNAse resistance of DNA anti-DNA immune complexes

The binding of antibodies to DNA was examined under conditions of increasing antibody excess. DNA anti-DNA immune complexes (IC) formed at increasing antibody to DNA ratios were digested with excess DNAse I, and the DNAse-resistant (protected) IC were analyzed. With increasing antibody excess, the size of the IC that were resistant to DNAse digestion increased, and the size of the protected DNA within the IC also increased. This suggested that IgG molecules could bind in close proximity along the DNA molecule, preventing access of DNAse to the DNA between adjacent IgG. To further define the binding of adjacent IgG, DNAse digested IC containing one or two IgG were isolated, and the DNA contained within these IC was analyzed on DNA sequencing gels. Binding of a single IgG to DNA resulted in the protection of a DNA fragment 35 to 45 base pairs (bp) long, corresponding to the distance between binding sites of a single IgG molecule. Binding of two IgG to DNA protected a DNA fragment 50 to 60 bp long, 1 1/2 times the size of the fragment protected by one IgG. These data suggest that in conditions of Ab excess, IgG molecules can interdigitate along the DNA molecule, resulting in small, stable, DNAse-resistant IC of high antibody density.     

Class-specific regulation of anti-DNA antibody synthesis and the age-associated changes in (NZB x NZW)F1 hybrid mice

Investigations of regulatory helper and suppressor T cells in the in vitro anti-DNA antibody synthesis in NZB x NZW (B/W) F1 hybrid mice were initiated by the development of an in vitro system in which G10-passed B cells from B/W F1 mice were cocultured with mitomycin C-treated T cells in the presence of Con A and either in the presence or in the absence of LPS. It was revealed that each IgG and IgM anti-DNA antibody synthesis was under the regulation of separate L3T4+ helper and Ly-2+ suppressor T cells. The function of these class-specific regulatory T cells was age-dependent. Although the helper effect of L3T4+ T cells on IgG antibody synthesis increased, the effect of L3T4+ T cells on IgM antibody production decreased in B/W F1 mice with aging. The IgG anti-DNA antibody production in the cocultures of L3T4+ T cells and B cells was suppressed by addition of Ly-2+ T cells from young but not aged B/W F1 mice, whereas the production of IgM anti-DNA antibodies was suppressed by Ly-2+ T cells from aged but not young B/W F1 mice. We also found that although IgM anti-DNA antibody-producing B cells were already present in 2-mo-old mice, B cells producing IgG antibodies under the influence of L3T4+ T cells appeared in mice at 7 mo of age. These data clearly indicate that separate class-specific regulatory T cells are involved in the production of IgM and IgG anti-DNA antibodies and that the total serum level of the antibodies is reflected by both their age-associated changes and the generation of antibody-forming B cells in B/W F1 mice.     

Clearance of lymphocytic choriomeningitis virus in antibody- and B-cell-deprived mice.

The role of antibody in immune recovery from infection with lymphocytic choriomeningitis virus (LCMV) strain WE was evaluated in B-cell-depleted mice. Mice were treated from birth with either affinity-purified rabbit anti-mouse immunoglobulin M (IgM), normal rabbit immunoglobulin, or, alternatively, an affinity-purified monoclonal rat anti-mouse IgM antibody (LO-MM-9); untreated mice served as controls. B-cell depletion was considered complete in specifically treated mice according to the following criteria: absence of a significant response to the B-cell mitogen lipopolysaccharide, absence of B cells expressing immunoglobulin on their surfaces, absence of detectable IgM or IgG in serum, and presence in the serum of free anti-IgM antibodies. In organs of mu-suppressed BALB/c mice, LCMV-WE replicated, dependent upon organ, at the same rate or more rapidly and, in general, to higher titers than in normal rabbit immunoglobulin-treated mice; untreated mice eliminated the virus most rapidly and showed lower virus titers. In addition, LCMV-primed control mice cleared a second LCMV challenge very rapidly and contained no virus by day 3, whereas mu-suppressed mice had virus in their blood and organs (except the spleen) up to days 3 to 6. The observed effects of anti-mu treatment may reflect the action of neutralizing antibodies (which so far have been difficult to demonstrate in vivo) or other antibody-dependent antiviral mechanisms which, together with T cells, efficiently control LCMV clearance.     

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.     

Effects of neonatal anti-delta antibody treatment on the murine immune system. II. Functional capacity of a stable sIgM+sIa+sIgD- B cell population

Mice were injected from birth with rabbit anti-mouse IgD (RaM delta). Studies in the accompanying paper indicated that the B cells from these mice have a stable sIgM+sIa+sIgD- B cell population. In the studies presented herein the in vivo and in vitro antibody responses of these mice were examined as well as their responsiveness to various B cell mitogens. The results indicate that splenic B cells from RaM delta-suppressed mice differ from normal adult murine splenic B cells by failure to express increased sIa antigen after in vitro stimulation with soluble anti-mu antibodies and failure to proliferate in response to in vitro stimulation with either soluble or Sepharose-bound anti-mu antibody. Nevertheless, these mice generate relatively normal in vivo IgM and IgG antibody responses to TI-2 and to both high and low epitope density forms of TD antigens as well as secondary IgG antibody responses to a TD antigen. In addition, B cells from RaM delta-treated mice generate relatively normal primary in vitro IgM antibody responses to TI-1, TI-2, and TD antigens. These data suggest that sIgD- B cells can produce antibody responses to the majority of antigenic signals even though they appear to lack one or more differentiative pathways.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Non-specific polyclonal antibody response induced by Mycoplasma pneumoniae

The ability of heat-killed Mycoplasma pneumoniae (MP) organisms to induce polyclonal antibody production in cultures of blood lymphocytes of healthy subjects was studied. MP induced both IgM and IgG production, with a predominance of IgM. Supernatants of MP-stimulated lymphocyte cultures were tested by an enzyme-linked immunosorbent assay for antibodies to measles, rubella, and herpes simplex virus. MP as well as pokeweed mitogen induced production of viral antibodies of IgG class in lymphocytes of donors who had serum antibodies to the corresponding viral antigens. The MP-induced non-specific antibody response was T-cell-dependent. Lymphocytes from four patients with MP pneumonia, collected nine to 13 days after onset of illness, were tested for in vitro Ig production in the absence of MP. These lymphocytes spontaneously produced increased amounts of IgM and/or IgG. Lymphocytes from three of these four patients spontaneously produced viral IgG antibodies to measles and/or varicella antigens, indicating that MP had induced non-specific activation of memory B cells in vivo. Spontaneous viral antibody production was not found in lymphocyte cultures of healthy donors. The non-specific activation of blood B cells in vitro is probably induced by non-specific helper factors from MP-activated T cells. It is possible that in vivo MP also may have a direct activating effect on B cells.     

IgG anti-DNA autoantibodies within an individual autoimmune mouse are the products of clonal selection

Although mice from almost all inbred strains produce IgM anti-DNA antibody in response to B cell mitogens, only (NZB x NZW)F1 mice and mice from other strains that are genetically predisposed to autoimmunity spontaneously produce anti-DNA antibody of the IgG isotype. Because (NZB x NZW)F1 mice display marked B cell hyperactivity, anti-DNA antibody production in these mice has been thought to result from spontaneous, polyclonal B cell activation. Although this may be true for IgM anti-DNA antibodies, our results demonstrate that IgG anti-DNA antibodies are not polyclonal. Rather, IgG anti-DNA autoantibodies within an individual autoimmune mouse are oligoclonal and somatically mutated. These results demonstrate that IgG anti-DNA autoantibodies are the products of clonally selective B cell stimulation and exhibit the same characteristics as secondary immune antibodies to conventional immunogens: they are IgG, they are clonally restricted, and they are somatically mutated.     

Failed self-tolerance and autoimmunity in IgG anti-DNA transgenic mice.

Transgenic mice were generated that express both the H and L chain genes derived from a hybridoma secreting an IgG2a mAb specific for ds- and ssDNA. This hybridoma is derived from a lupus mouse and can accelerate nephritis in young NZB x NZW F1 female mice and induce clinical nephritis in BALB/c mice. Some transgenic B cells did not exhibit allelic exclusion; they expressed both transgene-derived IgG and endogenous IgM intracellularly. Most of the B cells in transgenic mice expressed endogenous IgM, some of them expressed low levels of IgG on cell membranes. The transgenic mice, created in a strain not prone to SLE, expressed elevated serum IgG anti-DNA, and some developed clinical nephritis. The affinity of the spontaneously secreted IgG antibodies for dsDNA were similar in nephritic NZB x NZW F1 and transgenic mice. In contrast to the nontransgenic littermates, immunization of transgenic mice with murine DNA further enhanced serum levels of IgG anti-DNA in transgenic mice. Therefore, expression of transgene-encoded IgG anti-DNA mainly in the secreted form does not provide the signals necessary for allelic exclusion or self-tolerance. Expression of this Ig is sufficient to induce a mild form of autoimmune disease.     

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.     

Mechanism for induction of anti-DNA antibodies by bacterial lipopolysaccharides in mice; II. Correlation between anti-DNA induction and polyclonal antibody formation by various polyclonal B lymphocyte activators.

The capacity of various polyclonal B lymphocyte activators (PBA) to induce, in mice, the formation of anti-DNA antibodies was compared with their ability to mediate the release of DNA in circulating blood and to stimulate polyclonal antibody synthesis in vivo. Anti-DNA antibodies or polyclonal antibody synthesis were induced in mice after the injection of at least 10 microgram lipopolysaccaride (LPS) from Salmonella typhimurium, 1 mg dextran sulfate (DS), or 2 mg purified protein derivative of tubercle bacteria RT32 (PPD). Smaller quantities of LPS (0.1 microgram) or DS (500 microgram) were sufficient to cause the release of DNA in circulating blood, whereas PPD was not able to provoke such a release at any concentration used. The association of anti-DNA antibodies with polyclonal antibody synthesis in mice injected with various PBA contrasts with the lack of correlation between the formation of anti-DNA antibodies and the release of measurable amounts of DNA in circulating blood. These results strongly suggest that the induction of anti-DNA antibodies by PBA is a consequence of the polyclonal B lymphocyte activation.     

Suppression of B cell development and antibody responses in mice with polyclonal rabbit and monoclonal rat anti-IgM antibodies. I. Characterization of the suppressed state

Mice treated from birth with polyclonal, crude or affinity purified rabbit or monoclonal rat anti-mouse IgM antibodies [b-7-6 and C-2-23: Eur. J. Immunol. 14: 753-757, 1984] were found to be heavily suppressed with respect to B-cell activities. Crude or affinity purified rabbit or monoclonal rat anti-mouse IgM gave comparable results as follows: serum IgM was below detectable levels; serum IgG was reduced to about 1-3% of normal levels; free anti-IgM was always detectable; IgM and/or kappa-light-chain positive cells as well as IgM-secreting cells were absent in various lymphoid organs; the B-cell mitogen lipopolysaccharide was unable to induce proliferative responses; primary antibody responses could not be induced against sheep red blood cells and phosphorylcholine; lymphoid organs were reduced in size and B-cell areas were not populated with lymphocytes; besides a 40% reduction in absolute lymphocyte numbers in the blood, we found increased platelet counts and a 10% eosinophilia in anti-IgM-treated mice.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

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.     

IgG antinuclear antibodies with cross-reactive rheumatoid factor activity

To investigate whether IgG antinuclear antibodies have cross-reactive rheumatoid factor activity, monoclonal IgG antibodies to DNA and Sm from autoimmune MRL-lpr/lpr mice were assayed by ELISA for binding to IgG antigens. Of the nine anti-DNA and anti-Sm monoclonals tested, six showed significant binding to affinity-purified rabbit IgG (RIgG) and human IgG (HIgG). To confirm that cross-reactivities were due to a single antibody, immunoabsorption of a representative polyspecific monoclonal termed C11 (anti-DNA, anti-Sm) on either Sepharose-DNA or Sepharose-RIgG resulted in marked loss of activity to the three antigens DNA, Sm and RIgG compared with immunoabsorption on Sepharose-bovine serum albumin. The monomolecular nature of the cross-reacting antibody was also suggested by inhibition analysis of C11; DNA inhibited C11 binding to RIgG 64%, whereas Sm inhibited binding to RIgG 33%. Aggregated RIgG and HIgG, however, did not inhibit binding of C11 to DNA, Sm, or solid-phase RIgG, probably reflecting the low affinity of this antibody for fluid phase Ig. Together, these findings suggest that antinuclear autoantibodies of the IgG, as well as the IgM, class have polyspecific IgG binding activity and suggest that IgG antinuclear antibodies may emerge from rheumatoid factor responses.