Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.     

Comparison of natural antibodies to autoantibodies arising during lupus in (NZB x NZW)F1 mice

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.     

The VH gene sequences of anti-DNA antibodies in two different strains of lupus-prone mice are highly related

The heavy and light chain V region sequences of an IgG anti-DNA autoantibody (PME77), derived from a lupus-prone (NZB x NZW)F1 mouse have been determined by mRNA sequencing. The V kappa gene segment belongs to the V kappa 1A gene sub-group and is found in several (NZB x NZW)F1 and MRL lpr/lpr anti-DNA antibodies, as well as in other antibodies of unrelated specificities. The VH gene segment appears to represent a unique gene or a subfamily of the large J558 VH gene family of the mouse, and is highly related to a germ-line sequence of a major anti-DNA idiotype (H130, IgM) of MRL mice. This anti-DNA-related VH segment has not been found, so far, to be expressed in antibodies with specificities for external or synthetic antigens; therefore, expression of such specificities may be regulated by powerful mechanisms of self tolerance in the healthy animal. In addition, both the heavy and light chain of the PME77 IgG antibody were found to contain somatic point mutations with a high ratio of replacement to silent mutations in complementarity determining regions. This IgM to IgG sequence relationship suggests an affinity maturation process, which is driven by the autoantigen.     

Genesis of antinuclear antibody in NZB/W mice: role of genetic factors and of viral infections

Antinuclear antibodies could be induced in young NZB/W mice long before the natural occurrence of such antibodies by immunization with heat denatured DNA coupled to methylated bovine serum albumin (DNA m BSA). While induction of antinuclear antibodies was possible in several strains of mice (NZB/W, A/J, DBA/2, CBA and AKR), NZB/W mice had by far the highest titre of antibody. A genetic determination of this immune hyperreactivity to DNA was suggested by study of the parental strains. The NZW mice which have a low incidence of spontaneously occurring antinuclear antibody made as much antinuclear antibody upon immunization with DNA m BSA as did the NZB/W mice, while NZB mice which develop naturally a moderate incidence of antinuclear antibody responded relatively poorly to immunization.

It seemed possible that while the native hyperreactivity of NZB/W mice to DNA might be inherited from the NZW parent, the antigenic stimulus calling forth the anti-DNA response might be transmitted from the NZB parent. A possible source of this stimulus might be a viral infection such as that caused by the leukemia virus found in NZB mice which might liberate unusual amounts or kinds of host nuclear antigens or provide viral nucleic acid antigens. In support of this, induced neonatal infection of NZB/W mice with Polyoma virus markedly enhanced their antinuclear antibody response. Also, neonatal thymectomy of NZW mice which predisposes to reduced resistance to infection also enhanced antinuclear antibody formation. And finally, Statolon treatment designed to increase and maintain interferon levels in both normal and thymectomized NZW mice reduced antinuclear antibody formation.

     

Acceleration of autoimmunity in NZB/NZW F1 mice by graft-versus-host disease.

Chronic graft-versus-host (GVH) disease was induced in NZB/NZW F1 (B/W) hybrid female mice by the weekly injection of parental NZB spleen cells. Control mice received injections of syngeneic spleen cells only. The mice were assayed for antibodies to [3H]DNA and [3H]polyadenylic-polyuridylic acid by a cellulose ester filter radioimmunoassay, and for antibody to thymocytes by a cytotoxicity method. GVH disease accelerated the development of all three antibodies in B/W mice. In addition, sucrose density gradient ultracentrifugation of pooled sera suggested that an accelerated switch from 19S to 7S anti-DNA production may be an early effect of GVH. The mechanism of acceleration is discussed in terms of immunological and viral factors generated by the GVH reaction.     

Age-dependent responsiveness to interleukin-6 in B lymphocytes from a systemic lupus erythematosus-prone (NZB x NZW)F1 hybrid.

The cellular mechanisms for the production of IgG anti-DNA antibodies were studied. Culture of T and B cells from old (NZB x NZW)F1 mice led to the production of IgG anti-DNA antibodies. We found that direct cell contact was partly necessary for the production of IgG anti-DNA antibodies. Fixation of the T cells showed that lymphokines were largely responsible for the antibody synthesis. Antibodies to mouse interleukin-6 (IL-6) inhibited the production of these antibodies in the T-B cell coculture. Human IL-6 could induce small "resting" B cells from the old (NZB x NZW)F1 mice to produce IgG anti-DNA antibodies in a dose-dependent fashion. The response was inhibited by an anti-human IL-6 monoclonal antibody. Large or small B cells from young (B/W)F1 mice or Balb/c mice were not induced by IL-6 to antibody production. Therefore, the capacity of the B/W mice to produce the IgG anti-DNA antibodies correlated with the ability of the B cells to respond to IL-6 and with the age at which the mice begin to have signs of the disease.     

A central anti-DNA idiotype in human and murine systemic lupus erythematosus

The monoclonal anti-DNA autoantibody A52 (IgG2b) was obtained from a (NZB X NZW)F1 (B/W) hybridoma. Two rabbits were immunized with the pure monoclonal antibody and produced anti-idiotypic (Id) antibodies. The purified anti-Id reacted with three different B/W monoclonal anti-DNA antibodies at or close to their DNA binding sites. Moreover, the rabbit antibodies had a profound inhibitory effect on the polyclonal anti-DNA activity in the majority of sera derived from B/W mice and human systemic lupus erythematosus (SLE) patients. The A52 IgG must, therefore, represent a major cross-reactive Id of anti-DNA immunoglobulins. In addition, the rabbit anti-Id antibodies may act as the "internal image" of antigen and should prove useful in modulation of the autoimmune response to DNA in SLE.     

Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera.     

Autoantibodies to anti-DNA with anti-allotypic and anti-idiotypic specificities in (NZB X NZW)F1 mice

The monoclonal A52 (IgG2b, kappa) anti-DNA autoantibody represents a major cross-reactive idiotype in the murine and human autoimmune response to DNA. Examination of sera and purified IgG derived from (NZB X NZW)F1 mice showed that these mice develop an age-dependent binding reactivity with the pure anti-DNA IgG. Three monoclonal antibodies possessing this reactivity were prepared from unprimed female (NZB X NZW)F1 mice. One of these monoclonal antibodies appeared to be directed against allotypic determinants present in the NZB IgG2b; the other two antibodies exhibited a marked preference for idiotypic determinants of the A52 IgG. The IgG anti-allotype and anti-idiotype activities in (NZB X NZW)F1 mice may, therefore, represent the products of a deregulated immune system and/or constitute the normal elements of a functional immune regulation system.     

Effects of major histocompatibility complex on autoimmune disease of H-2-congenic New Zealand mice

Autoimmune-prone NZB mice mainly produce IgM-class anti-DNA antibodies and mild SLE develops later in life. The F1 hybrid of NZB and non-autoimmune NZW mice (NZB/W F1 mice) develop a more fulminant SLE, associated with decreases in IgM class, and, in turn, increases in IgG class anti-DNA antibodies. To elucidate the role of the H-2 complex in this mode of anti-DNA antibody production, we established and studied H-2-congenic New Zealand mice, i.e. NZB, NZW, and NZB/W F1 mice with either the homozygous H-2z/H-2z or H-2d/H-2d haplotype or the heterozygous H-2d/H-2z haplotype. The data showed that: (i) although the non-H-2-linked NZB gene(s) seems to determine the IgM anti-DNA antibody production in NZB mice, the effect of this gene is fully expressed only in the case of the H-2d/H-2d homozygous state. (ii) The production of IgG anti-DNA antibodies observed in NZB/W F1 hybrid mice is restricted to the H-2d/H-2z heterozygosity. (iii) Because both NZB and NZW mice with the H-2d/H-2z haplotype produce a lower titer of IgG anti-DNA antibodies than do the NZB/W F1 mice, other complementary non-H-2-linked genetic elements from both NZB and NZW parents are required. The development of lupus nephritis correlated well with that of anti-DNA antibodies. Thus, H-2d/H-2z heterozygosity is a necessary but not sufficient condition for the development of autoimmunity in NZB/W F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific increases in urinary excretion of anti-DNA antibodies in lupus mice induced by lysozyme administration: further evidence for DNA-anti-DNA immune complexes in the pathogenesis of nephritis.

We previously reported that lysozyme electrostatically inhibits the fibronectin-mediated DNA binding to the glomerular basement membrane (GBM) and reduces in situ DNA-anti-DNA complex formation in the GBM in NZB/W F1 mice [1]. In this study, we further noticed significant increases in urinary excretion of anti-DNA antibodies and immune complexes (IC) in lysozyme-treated NZB/W F1 mice. Their clearance ratios of IgG anti-DNA antibody to whole IgG were markedly high compared with those of saline-treated animals. A large number of IgG and C3 positive granules were observed in the tubular cells of NZB/W F1 mice treated with lysozyme. On the contrary, nil or only small amounts of anti-DNA antibodies were detected in the urine of NZB/W F1 mice without lysozyme administration despite a large amount of proteinuria, suggesting entrapment of the antibodies in lupus glomeruli. Lysozyme neither inhibited the binding of anti-DNA antibodies to DNA or heparan sulphate nor did it displace anti-DNA antibodies and IC from the kidney homogenates of lupus mice. It thus appears that the inhibition of DNA binding to the GBM due to lysozyme reduced the entrapment of anti-DNA antibodies in the GBM, resulting in urinary excretion of the antibodies.     

Specificity of mouse hybridoma antibodies to DNA. II. Phospholipid reactivity and biological false positive serological test for syphilis

Using mouse hybridoma monoclonal antibodies to DNA from MRL/lpr and NZB X NZW (B/W F1) mice, the reactivity of anti-DNA antibodies to several phospholipids was analysed. The anti-DNA antibody which reacted with the common antigenic determinants on the phosphate-sugar backbone of nucleic acid could bind to the cardiolipin, but failed to bind to other phospholipids, including VDRL antigen. We tentatively conclude that the anti-cardiolipin antibody is identical with the anti-DNA antibody, but differs from the BFP reactor.     

A peptide derived from a polyreactive monoclonal anti-DNA natural antibody can modulate lupus development in (NZBxNZW)F1 mice

In lupus-prone (NZBxNZW)F1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti-DNA antibodies. While these D23 Id+ antibodies were able to regulate anti-DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti-DNA antibodies and furthermore could participate in the lupus-like syndrome. To explore further the role of the D23 Id+ antibodies, we injected young B/W mice with a peptide corresponding to the VH CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA-reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti-DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.     

Distinct surface phenotypes of B cells responsible for spontaneous production of IgM and IgG anti-DNA antibodies in autoimmune-prone NZB x NZW F1 mice

Autoimmune-prone NZB x NZW F1 (B/W F1) mice produce a high titer of anti-DNA antibodies, In vivo and in vitro studies showed that in the early life of these mice, the immunoglobulin isotype of these antibodies almost exclusively belongs to IgM class, however, IgG anti-DNA antibodies begin to develop when the mice are about 5-6 months old and the titer exceeds that of IgM antibodies from age 7 months on. We asked whether or not the B cell population responsible for IgM and IgG antibody production belongs to the same lineage. The surface phenotypes of B cell populations responsible for the spontaneous production of either IgM or IgG anti-DNA antibodies were examined using panning and sorting methods with several monoclonal antibodies to B cells, including CD5 (Ly-1) and Lp-3; the latter defines a unique B cell differentiation antigen. We obtained evidence that surface phenotypes of B cells secreting IgM anti-DNA antibodies belong to CD5+ Lp-3- and those of B cells secreting IgG anti-DNA antibodies which occur only in old B/W F1 mice belong to CD5- Lp-3+ subpopulations. The majority of peritoneal B cells were CD5+ Lp-3+ throughout the life span of the mice and anti-DNA antibody production was never evidenced. These findings were discussed in relation to age-associated changes of B cell populations in the spleen of this strain of mice.     

Expression of a highly conserved anti-DNA idiotype in normal and autoimmune mice

The specificity and idiotypic relationships of a monoclonal anti-DNA antibody were investigated to evaluate genetic control in this autoantibody response. 6/0 is an IgG2a monoclonal anti-DNA derived by the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse and the cell line NS1. By an enzyme-linked immunosorbent assay (ELISA) for anti-DNA, 6/0 demonstrated preference for single-stranded DNA and bound deoxyribo- and ribohomopolymers of dissimilar base composition. The control of 6/0 expression was evaluated by idiotypic analysis using a rabbit anti-6/0 antiserum made specific by absorption with the BALB/c myelomas UPC 10 (IgG2a) and MOPC 21 (IgG1). The resulting preparation was fractionated by BALB/c IgG affinity columns to provide antibodies to idiotypic determinants essentially unique to 6/0 and those commonly expressed in sera. The commonly expressed 6/0 idiotype was found in sera of ten inbred strains of mice and was not exclusive to the autoimmune strains. MRL-lpr/lpr and A/J strain mice displayed idiotype levels almost fivefold greater than other strains, with 6/0 idiotype-bearing antibodies having serum concentrations as high as 1 mg/ml. Levels of the 6/0 idiotype, however, did not correlate with anti-DNA levels among the various strains. In addition to mice, the majority of individuals of three inbred rat strains showed detectable 6/0 idiotype in their sera. These results suggest that the 6/0 idiotype, although identified using a monoclonal anti-DNA antibody, represents a framework determinant that is phylogenetically conserved. The mechanisms for the expression of this determinant may differ among the normal and autoimmune strains.     

Specific proliferative responses following the induction of experimental SLE in mice

We have previously reported the induction of experimental systematic lupus erythematosus (SLE) in mice by immunization with a human monoclonal antibody that expresses a common anti-DNA idiotype (16/6 Id). Following immunization, antibodies directed against various nuclear autoantigens could be detected in the sera of the mice. In the present study, we investigated the proliferative responses of lymph node cells to one particular autoantigen (DNA) following the induction of experimental SLE. Cells reactive with ssDNA could be detected following immunization of BALB/c mice with the 16/6 Id. The appearance of these DNA-reactive cells succeeded the appearance of 16/6 Id-specific cells. The activation of this subset of autoreactive cells could be achieved only by the immunization of the mice with the 16/6 Id, but not by their immunization with DNA, thus suggesting that the induction of experimental SLE is associated with the alteration of the low responsive potential of the mice to DNA.     

In vivo clearance and tissue uptake of an anti-DNA monoclonal antibody and its complexes with DNA.

In vivo clearance and tissue localization of a purified mouse anti-DNA monoclonal antibody (MoAb) (A52 IgG2b) and its complexes with DNA were studied in normal BALB/c and autoimmune NZB/NZW mice. The plasma half-life of the autoantibody in both mouse strains was significantly shorter (T 1/2 = 10-15 min), compared with that of purified NZB myeloma proteins (T 1/2 greater than or equal to 180 min). DNA antigen and DNA-A52 IgG complexes in antibody excess were cleared very rapidly (T 1/2 = 4-8 min), while complexes formed in antigen excess persisted in the circulation much longer (T 1/2 = 60 min). Organ studies showed that the anti-DNA MoAb was transiently retained by the liver and the spleen but demonstrated a particular affinity for the kidney tissue. We suggest that tissue damage in SLE glomerulonephritis may be facilitated by direct interaction of anti-DNA antibodies with glomerular components.     

Induction of experimental anti-phospholipid syndrome associated with SLE following immunization with human monoclonal pathogenic anti-DNA idiotype.

MIV-7 is a human monoclonal antibody that binds to DNA and carries a pathogenic anti-DNA idiotype 16/6. The antibody was generated by fusing peripheral blood lymphocytes of a healthy donor which were stimulated with an anti-idiotypic antibody to B11 (a human mAb anti-mouse mammary tumor virus-MMTV). The MIV-7, in addition to being an anti-DNA antibody, also binds to MMTV glycoproteins. Following immunization into the footpad of naive BALB/c mice with MIV-7, the mice developed anti-phospholipid syndrome (APLS) and SLE. The APLS was characterized by thrombocytopenia, the presence of anticardiolipin antibodies, lupus anticoagulant (prolonged APTT), high resorption rate of fetuses and lower mean weights of the placentae and fetuses. The SLE was characterized by serological markers (e.g. anti-DNA), laboratory (increased sedimentation rate and proteinuria) and histological findings (deposition of immune complexes in the glomeruli). Active immunization of mice with mouse monoclonal anti-cardiolipin antibodies led to the induction of primary APLS without SLE. The results add to our previous passive transfer model in which mouse monoclonal anti-cardiolipin antibody generated from immunized mice (CAM) was infused into the tail vein and also resulted in induction of pure APLS [11]. Our results demonstrate the ability to induce secondary APLS to SLE following immunization with a pathogenic idiotype of anti-DNA antibodies and to induce primary APLS with anti-cardiolipin mAb. The existence of these experimental models may permit controlled studies of novel therapeutic models.     

Possible role of IL-6 in pathogenesis of immune complex-mediated glomerulonephritis in NZB/W F1 mice: induction of IgG class anti-DNA autoantibody production

This study demonstrates that interleukin-6 (IL-6) increases the autoantibody production by B cells from NZB/W F1 mice. Splenic B cells were cultured for 5 days in the presence or absence of human IL-6, and then the anti-DNA antibody and immunoglobulin contents in the culture supernatants were measured by ELISA. Adding IL-6 increased IgG anti-DNA antibody production by B cells from old mice (30 weeks), but not from young ones (17 weeks). B cells obtained from both young and old mice produced IgM anti-DNA antibody, which increased when IL-6 was added. The increased anti-DNA antibody production was suppressed by anti-recombinant human IL-6 antibody to the background level, i.e. antibody contents in the absence of IL-6. In contrast, murine IL-5 did not increase IgG anti-DNA antibody production, although it promoted the production of IgM anti-DNA antibody. Furthermore, when IL-5 was added in combination with IL-6, there was an additive increase in IgM, but not in IgG anti-DNA antibody production. Similar results were obtained in the measurement of the immunoglobulin contents. These results suggest the possible role of IL-6 in the pathogenesis of autoimmune disease in NZB/W F1 mice.     

The genetic regulation of the induction of experimental SLE.

We have recently reported the induction of systemic lupus erythematosus (SLE) in C3H.SW female mice by their immunization with a human monoclonal anti-DNA antibody that bears a common idiotype termed 16/6 Id. In the present study, the ability to induce experimental SLE in seven inbred mouse strains by immunization with the 16/6 Id was examined. Two out of the seven strains failed to develop the disease. These two strains did not produce antibodies specific to the 16/6 Id, while the other five strains produced high titres of anti-16/6 Id antibodies. The anti-16/6 Id antibody response, followed by the induction of the disease, was not found to be MHC or Ig heavy chain allotype linked. F1 hybrids between a resistant strain and two of the susceptible strains were found to be resistant to the induction of the disease, indicating that susceptibility is inherited as a recessive trait. In the autoimmune NZB/W F1 female mice, immunization with the 16/6 Id resulted in an early onset of the SLE-like disease. The results of the present study indicate the role of the anti-16/6 Id antibodies in the induction of experimental SLE, and provide direct evidence for the importance of the genetic background in determining susceptibility to SLE.