Intrinsic B cell defects in NZB and NZW mice contribute to systemic lupus erythematosus in (NZB x NZW)F1 mice

We have previously shown that long-term in vitro proliferating fetal liver pre-B cell lines derived from autoimmune-prone (NZB x NZW)F1 (BW) mice, but not normal (B6 x DBA2)F1 mice, can differentiate in severe combined immunodeficient (SCID) mice to produce elevated levels of serum immunoglobulin (Ig) M and IgG, and high titers of antinuclear antibodies The contribution of parental NZB and NZW strains to B cell abnormalities of BW hybrid mice was investigated here by preparing pre- B cells and transferring them into immunodeficient SCID- and RAG-2- targeted mice. We show that transfer of NZB pre-B cells led to a marked IgM hypergammaglobulinemia and to the production of limited amounts of IgG2a. On the other hand, the transfer of NZW pre-B cell lines led to moderately elevated IgM levels and marked hypergammaglobulinemia of IgG2a. High IgM and low IgG anti-DNA titers are found in the recipients of NZB pre-B cells, whereas those receiving NZW pre-B cells contained lower levels of IgM and high titers of IgG anti-DNA. In marked contrast, essentially identical titers of antibodies directed against a non-self-antigen, DNP, are found in all group of pre-B cell recipients. Thus, B-lineage cells of both NZB and NZW parental strains manifest abnormalities associated with the development of this lupus-like disease. Therefore, the present study strongly suggests a complex inheritance of B cell abnormalities in autoimmune-prone (NZB x NZW)F1 mice and emphasizes the critical importance of intrinsic B cell defects in the development of murine systemic lupus erythematosus.     

Interleukin 6 promotes murine lupus in NZB/NZW F1 mice.

To investigate the role of IL-6 in systemic lupus erythematosus (SLE), we selectively inhibited IL-6 in lupus-prone NZB/NZW F1(B/W) mice by chronic administration of a rat mAb to mouse IL-6. Anti-IL-6 alone elicited an anti-rat response that blocked its biologic effects. To circumvent this problem, we rendered B/W mice tolerant to the rat mAb by administration of anti-CD4 concurrent with the first dose of anti-IL-6. Thereafter, the mice received weekly injections of anti-IL-6 alone. There were two control groups: one group received the tolerizing regimen of anti-CD4 along with a control rat IgG1 mAb (GL113) instead of anti-IL-6; the other control group received PBS. Mice that received anti-CD4 were tolerant to the rat mAb for 6 mo. Throughout this period, treatment with anti-IL-6 prevented production of anti-dsDNA, significantly reduced proteinuria, and prolonged life. Mice that received anti-IL-6 without anti-CD4 developed an immune response to the rat mAb and then developed anti-dsDNA antibodies, proteinuria, and mortality comparable with control mice. These findings establish that IL-6 promotes autoimmunity in B/W mice. They further indicate that, although mAb to IL-6 can suppress murine lupus, the development of host immunity to the mAb abrogates its beneficial effects. Finally, this is the first study to demonstrate that a brief course of anti-CD4 can induce tolerance to another therapeutic mAb, in this case an anti-cytokine mAb.     

MONOCLONAL MACROGLOBULINEMIA IN NZB/NZW F1 MICE

Serum monoclonal macroglobulins were detected in over 30% of NZB/NZW F₁ mice greater than 11 mo of age. The monoclonal nature of the IgM was shown by restricted electrophoretic mobility, characteristic appearance on immunoelectrophoresis, restriction to a single light chain type, and ability to induce anti-idiotypic antisera. The monoclonal macroglobulins were separated from antibodies to DNA and RNA that migrated in the 7S region of sucrose gradients. Enlarged lymph nodes were often present in mice with monoclonal IgM, and a transplantable IgM-producing lymphoid tumor was established from the spleen of one animal.     

Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4

Autoimmune NZB/NZW mice were treated with weekly injections of monoclonal antibody (mAb) to L3T4, an antigen expressed on a distinct subpopulation of T cells that respond to class II major histocompatibility antigens. Treatment with anti-L3T4 depleted circulating target cells, reduced autoantibody production, retarded renal disease, and prolonged life relative to control mice treated either with saline or with purified nonimmune rat IgG. These findings establish that autoimmune disease in NZB/NZW mice is regulated by T cells. In contrast to mice treated with nonimmune rat IgG, mice treated with rat anti-L3T4 mAb developed little or no antibody to rat Ig. Thus, the benefits of treatment with anti-L3T4 were achieved while minimizing the risks associated with a host immune response to therapy. This study raises the possibility that treatment with mAb against Leu-3/T4, the human homologue for L3T4 might be effective in the treatment of certain autoimmune diseases in people.     

The contribution of NZW genes to lupus-like disease in (NZB x NZW)F1 mice

Unlike parental NZB or NZW mice, (NZB X NZW)F1 mice exhibit a lupus-like disease characterized by high serum levels of IgG antinuclear antibodies and a fatal immune-complex glomerulonephritis. At least three unlinked gene loci can be distinguished in NZW mice that conceivably contribute to a T cell-dependent autoimmune disease, including the MHC (H-2z) and the T cell receptor alpha and beta chain gene complexes. We undertook an (NZB X NZW)F1 X NZB backcross to determine the relative contribution of these NZW genes to lupus-like renal disease and autoantibody production in F1 mice. The incidence of severe renal disease and elevated levels of IgG antibodies to dsDNA and histone in the backcross mice was approximately half of that observed in (NZB X NZW)F1 mice. Furthermore, there was a strong correlation between the presence of the NZW H-2z haplotype and lupus-like disease in backcross mice. Approximately 90% of backcross mice with disease carried the NZW H-2z locus compared with 16% of mice without disease; nearly 90% of H-2d/z mice expressed severe autoimmune disease. In contrast, no association was apparent between the presence of the NZW T cell receptor alpha chain gene complex or beta chain gene complex and severe renal disease or autoantibody production. Thus, the NZW MHC or gene(s) linked to this locus appear to be the only dominant NZW genetic contribution to F1 disease.     

RELATIVE INABILITY TO INDUCE TOLERANCE IN ADULT NZB AND NZB/NZW F1 MICE

Immunologic tolerance to ultracentrifuged bovine gamma globulin could not be induced in 6-wk old NZB or B/W mice, but developed readily in C3H, NZW, and C57Bl mice. NZB and B/W mice, as well as Balb/c mice, failed to become tolerant to ultracentrifuged human gamma globulin. The NZB and B/W mice also showed higher antibody titers to a standard antigenic challenge. This immunologic hyperreactivity and lack of experimental tolerance may be related to the lack of self-tolerance, autoimmunity, and lymphomas that develop in these mice at a later age.     

Mixed lymphocyte reaction in interferon-gamma treated (NZB x NZW)F1 mice

Mouse interferon-gamma was administered to (NZB x NZW)F1 mice, which develop a disease like human systemic lupus erythematosus. Class II antigens on adherent spleen cells and their activity to stimulate allogeneic T lymphocyte in mixed lymphocyte reaction were observed as well as the changes of the clinical findings of the mice. In interferon-gamma treated mice, as compared to control mice, the positive rate for class II antigen on adherent spleen cells was increased, and the mixed lymphocyte reaction using the cells of interferon-gamma treated mice as stimulator cells was enhanced. These findings suggest that the enhancement of antigen presenting activity of adherent spleen cells to T lymphocytes might be associated in the exacerbation of the disease in interferon-gamma treated (NZB x NZW)F1 mice.     

Erythrocyte deformability changes in autoimmune hemolytic anemia during development of NZB mice and their (NZB/NZW)F1 hybrid

NZB and B/W hybrid mice develop compensated hemolytic anemia during the first year of their life. By the age of 3-5 months, their erythrocytes show evidence of spherocytosis, increased osmotic fragility and decreased whole cell deformability, as measured by ektacytometry, a laser diffraction technique. The presence of spherocytes with decreased surface area/volume ratio was confirmed by scanning electron microscopy and osmotic gradient ektacytometry. Whereas these abnormalities persisted and worsened in the NZB mice with further growth, they gradually improved and reverted to normal by the age of 12 months in B/W mice. This spontaneous improvement seems to be due to the accumulation of red cell membrane lipids reflecting the lipemia of immune complex nephritis in B/W mice. The implications of these findings in the modulation of autoimmune hemolytic anemia are discussed.     

CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB x NZW F1 mice

Systemic lupus erythematosus (SLE) is a CD4(+) T cell-dependent, immune complex-mediated, autoimmune disease that primarily affects women of childbearing age. Generation of high-titer affinity-matured IgG autoantibodies, specific for double-stranded DNA and other nuclear antigens, coincides with disease progression. Current forms of treatment of SLE including glucocorticosteroids are often inadequate and induce severe side effects. Immunological approaches for treating SLE in mice using anti-CD4 mAb's or CTLA4-Ig and anti-CD154 mAb's have proven to be effective. However, like steroid treatment, these regimens induce global immunosuppression, and their withdrawal allows for disease progression. In this report we show that lupus-prone NZB x NZW F(1) mice given three injections of anti-CD137 (4-1BB) mAb's between 26 and 35 weeks of age reversed acute disease, blocked chronic disease, and extended the mice's lifespan from 10 months to more than 2 years. Autoantibody production in recipients was rapidly suppressed without inducing immunosuppression. Successful treatment could be traced to the fact that NZB x NZW F(1) mice, regardless of their age or disease status, could not maintain pathogenic IgG autoantibody production in the absence of continuous CD4(+) T cell help. Our data support the hypothesis that CD137-mediated signaling anergized CD4(+) T cells during priming at the DC interface.     

Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZB x NZW)F1 mice

Disease activity in systemic lupus erythematosus is closely associated with the appearance of immunoglobulin (Ig)G antibody to native DNA in both humans and mice. Like normal antibody responses, the anti-DNA autoantibody first appears as IgM and then switches to IgG. Structural studies of IgG anti-DNA suggest that these antibodies are the products of clonally selected, specifically stimulated B cells. The origins of the IgM anti-DNA have been less clear. To determine whether the earlier appearing IgM anti-DNA antibody in autoimmune mice also derives from clonally selected, specifically stimulated B cells or B cells activated by nonselective, polyclonal stimuli, we have analyzed the molecular and serological characteristics of a large number of monoclonal IgM anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. We have also analyzed IgM and IgG anti-DNA hybridomas obtained from the same individual mice to determine how the later-appearing IgG autoantibody may be related to the earlier-appearing IgM autoantibody within an individual mouse. The results demonstrate that: (a) IgM anti-DNA, like IgG, has the characteristics of a specifically stimulated antibody; (b) IgM and IgG anti-DNA antibodies have similar variable region structures and within individual mice may be produced by B cells derived from the same clonal precursors; (c) recurrent germline and somatically derived VH and VL structures may influence the specificity of anti-DNA monoclonal antibody for denatured vs. native DNA; and (d) the results provide a structural explanation for the selective development of IgG antibody to native DNA as autoimmunity to DNA progresses in (NZB x NZW)F1 mice.     

Defective B cell tolerance in adult (NZB X MZW)F1 mice

Hapten-specific tolerance was induced in vitro by trinitrophenyl-human gamma globulin (TNP32HGG) to a comparable degree in B cells from adult autoimmune (NZB X NZW)F1 (B/W) mice and normal BDF1, CBA/J, and DBA/1J mice. When a lower epitope density tolerogen (TNP7HGG) was used, B/W mice were significantly less sensitive than normal mice to the induction of B cell tolerance. This finding of defective B cell tolerance in adult B/W mice is consistent with previous reports that document other B cell abnormalities that may relate to the expression of autoimmune disease.     

Defective dendritic cell accessory function in NZB/W F1 hybrid mice

The proliferative response of NZB/W F1 hybrid mice when activated by sodium periodate (NaIO4) was found to be markedly defective as compared to the response in control NZW mice. This defect was observed in both 4 month old female mice and in 8-10 month old male mice. To determine whether the defect was an intrinsic T cell defect or an accessory cell defect, splenic dendritic cells (DC) were purified and their ability to activate enriched T cells after NaIO4 stimulation was assessed. In mixing experiments it was observed that normal NZW dendritic cells could restore the response of NZB/W F1 hybrid T cells whereas addition of NZB/W F1 dendritic cells to NZW T cells resulted in defective 3H-thymidine incorporation after NaIO4 stimulation. These results indicate that accessory DC of NZB/W F1 mice are defective and unable to support T cell responses to the mitogen NaIO4.     

Loss of suppressor T cells in adult NZB/NZW mice

We have investigated suppressor T-cell activity in female NZB/NZW F1 mice using PWM-driven IgM biosynthesis in vitro as an indicator system. In initial we studied we observed that spleen cells from normal mice (BALB/c, C57BL/6), as well as from young (4 wk) and adult (18 wk) NZB/NZW mice, cultured in the presence of PWM synthesize 860 +/- 120 ng IgM/10(6) cells/7 days. However, when Con A (at 2 mug/ml) was added directly to the cultures (along with PWM), cells obtained from adult normal mice and young NZB/NZW mice showed a 94% suppression of IgM synthesis, whereas cells obtained from adult NZB/NZW mice were suppressed significantly less. To analyze these findings we studied the effect of Con A-induced suppressor cells (cells cultured with Con A for 24 h and washed free of Con A) on PWM-driven IgM biosynthesis. Spleen cells obtained from normal mice cultured in the presence of Con A-pulsed cells obtained from normal mice and young NZB/NZW mice showed an 83-88% suppression of PWM-driven IgM synthesis. Similarly, supernates obtained from Con A-pulsed cells of normal mice or of young NZB/NZW mice suppressed PWM-driven IgM synthesis. This suppression by Con A-pulsed cells and their supernates required T cells since T-cell fractions but not B-cell fractions eluted from anti-Fab Sephadex columns mediated suppression of co-cultured normal cells; in addition, Con A-pulsed cells treated with anti-theta and complement do not mediate suppression. These studies of Con A-induced suppressor cell activity in normal mice and young NZB/NZW mice contrast with studies of Con A-induced suppressor cell activity in adult NZB/NZW mice. We found that adult NZB/NZW Con A-pulsed cells and supernates obtained from the Con A-pulse cells had vastly decreased suppressor potential; in this case the Con A-pulse cells and supernatant fluids derived from such cells did not suppress PWM-driven IgM synthesis by normal cells. Finally, whereas spleen cells from young and adult NZB/NZW mice differ in their suppressor cell potential, cells from both sources could respond equally to suppressor signals in that Con A-pulsed normal cells or supernates derived from such cells caused equivalent suppression of PWM-driven IgM synthesis by young and adult NZB/NZW cells. These observations allow us to conclude that NZB/NZW mice lose suppressor T-cell activity as they age.     

The effects of ciclosporin on the renal histopathological aspects in (NZB x NZW)F1 mice

Ciclosporin A (CYA) is a newly developed immunosuppressant and has a sparing effect on suppressor cell regulatory mechanism. However, accumulation of experimental animal studies has not been enough for the administration of CYA to human autoimmune diseases such as lupus nephritis. In this study, the histopathological aspects of the kidney in (NZB x NZW)F1 mice after administration of CYA were analysed. A dose of 25 mg/kg/day of CYA was injected intraperitoneally so that serum level of CYA was controlled from 250 to 300 ng/ml with this dosage. Typical histopathological changes of lupus nephritis including wire-loop lesions were observed in the kidney of the non-treated control group. In contrast, only few lesions were demonstrated in the CYA-treated group. These results suggest that CYA prevented the renal deterioration in lupus mice.     

The acute-phase response in (NZB X NZW)F1 and MRL/l MICE

The acute-phase plasma protein response to disease activity in murine models of autoimmune lupus-like disease was investigated by measurement of the concentration of serum amyloid P component (SAP) in NZB X W and MRL/l mice. The levels of SAP, which is a major acute-phase protein in mice, did not rise at all in response to progression of disease in NZB X W mice between the ages of 1 and 9 mo. This resembles the behavior of acute-phase proteins such as C-reactive protein and serum amyloid A protein in human systemic lupus erythematosus, and just as in human lupus, where the occurrence of intercurrent microbial infection can stimulate an acute-phase response, so injection of bacterial lipopolysaccharide or casein into the NZB X W mice stimulated "normal" acute-phase SAP production. In marked contrast, MRL/l mice developed greatly increased levels of SAP, which correlated closely with progression of their pathology as they aged. The disease profile of the MRL/l strain includes rheumatoid factors and spontaneous polyarthritis and their SAP response resembles the behavior of acute phase proteins in human rheumatoid arthritis. Different patterns of acute-phase response in different autoimmune disorders may thus be a reflection of the genetic predisposition to particular diseases and/or contribute to their pathogenesis. The existence of animal counterparts for the various clinical patterns of human acute-phase protein production will assist in experimental investigation of the underlying mechanisms and of the biological role of the acute-phase response.     

Enhancing effect of H-2-linked NZW gene(s) on the autoimmune traits of (NZB X NZW)F1 mice

To investigate the possible enhancing effect of the H-2z haplotype of the New Zealand White (NZW) strain on the production of autoantibodies and renal disease observed in B/W F1 mice, we developed the ZWD/8 strain, a NZW congenic line carrying the H-2d haplotype, produced (NZB X ZWD/8)F1 (B/WD8 F1) mice, and examined the difference in several immunological abnormalities between the B/W F1 (H-2d/H-2z) and the B/WD8 F1 (H-2d/H-2d) mice. In comparison with B/W F1 mice, the B/WD8 F1 mice showed markedly lower serum levels of the anti-DNA antibodies and the gp70 ICs, and a later onset and a lower incidence of proteinuria with a lower mortality. In contrast, there was no significant difference in the incidences and the amounts of both natural thymocytotoxic autoantibody and anti-erythrocyte autoantibody between these two hybrid strains. Further, the serum levels of IgG and IgM in B/WD8 F1 mice were as high as those in B/W F1 mice. These findings indicate that the gene(s) that is within or closely linked to the H-2 complex of NZW strain specifically acts to intensify the levels of anti-DNA antibodies and gp70 ICs, and to promote the severity of renal disease in B/W F1 mice. This gene may play a role in the class conversion of anti-dsDNA antibodies from IgM to IgG.     

Characterization of somatically mutated S107 VH11-encoded anti-DNA autoantibodies derived from autoimmune (NZB x NZW)F1 mice

We have studied 19 S107 heavy chain variable region gene (VH11)-encoded monoclonal antibodies from NZBWF1 mice. These studies show that a single VH gene can encode both antibodies to foreign antigens (anti-phosphorylcholine) and to self antigens (anti-double-stranded DNA) in the same animal. All of the anti-DNA antibodies contain many somatic mutations compared with the relevant germline genes. Since the anti-DNA antibodies were extensively somatically mutated and had undergone isotype switching, the response seems to be T cell dependent. While some of the antibodies appear to be the products of an antigen-driven and antigen-selected response, a number of characteristics of the antibodies suggest that forces other than antigen are contributing to the stimulation and selection of this response.     

Treatment of (NZB x NZW)F1 disease with anti-I-A monoclonal antibodies

(NZB x NZW)F1 mice spontaneously develop an autoimmune syndrome characterized by a fatal immune complex glomerulonephritis. Administration of monoclonal antibodies specific for an I region gene product (I-Az) of the H-2 haplotype associated with susceptibility to glomerulonephritis in these animals produced a remission in female mice with established renal disease. The results demonstrated that anti-I-A therapy stabilized the level of proteinuria and increased the 1-yr survival rate from 10% to greater than 90% in treated animals relative to control mice. These findings may ultimately have therapeutic potential for the treatment of systemic lupus erythematosus.     

Treatment of lupus nephritis in adult (NZB + NZW)F1 mice by cortisone-facilitated tolerance to nucleic acid antigens.

Adult female (NZB + NZW)F1 mice were treated with cortisone, cortisone with tolerogen (isologous NZB IgG-nucleosides conjugates) or cortisone with isologous IgG free of nucleosides. Other treatments also included tolerogen or isologous IgG alone, and cortisone together with denatured DNA. All untreated mice died by 10 mo of age. Cortisone prolonged the survival rate. This effect was further improved by combined treatment of cortisone and tolerogen. Prolonged survival was accompanied by a decrease in proteinuria. Other treatments failed to influence either survival or proteinuria. Although cortisone did not prevent the appearance of antibody to denatured DNA, cortisone and tolerogen suppressed them in most of the animals. Preexisting antibody to denatured DNA was reduced by cortisone and cortisone and tolerogen, but not by cortisone and IgG. In contrast, antibody to native DNA bore no relationship to therapy. Animals living beyond 1 yr of age, regardless of the treatment, fall into three histopathological categories: (a) severe nephritis, as in untreated animals, (b) moderate nephritis (with absence of severe alteration of the glomerular basement membrane, i.e. the histological counterpart of prolonged survival), (c) minimal nephritis. In a small number of animals treated with cortisone or cortisone and IgG and in 6/20 animals treated with cortisone and tolerogen, minimal lesions as judged by light, fluorescent, and electron microscopy were found. These last mice were in good health at 15-16 mo of age, twice the life-span of untreated mice. In conclusion, these data suggest that tolerance to nucleic acid antigens facilitated by cortisone offers a promising new approach to treat established murine lupus nephritis.     

Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid prevents proteinuria and prolongs survival in NZB x NZW F1 mice.

Prostaglandins and related compounds are active mediators of inflammation, but data concerning their role in the pathogenesis of the glomerulonephritis of New Zealand Black x New Zealand White (NZB x NZW) F1 mice are conflicting. Dietary eicosapentaenoic acid (EPA, C20:5), a fatty acid analogue of arachidonic acid (C20:4), has been shown to impair platelet aggregation in humans, apparently through inhibition of the synthesis of prostaglandins and thromboxanes from arachidonic acid. We report here the effects of a diet high in EPA on the development of renal disease and survival in female NZB x NZW F1 mice. Animals from 4--5 wk of age were fed diets containing 25% lipid, supplied either as beef tallow or menhaden oil, with fatty acid analysis of less than 0.05 and 14.4% EPA, respectively. In the first experiment, by 13.5 mo of age, mice on the beef tallow diet had all (9/9) developed proteinuria and the majority (6/9) had died, with renal histologic examination revealing severe glomerulonephritis. In contrast, none of 10 menhaden oil-fed animals had developed proteinuria, and all were alive at this time (P less than 0.005 for both proteinuria and survival). In a second experiment using 50 mice in each dietary group, 56% of the beef tallow group vs. none of the menhaden oil group had developed proteinuria at 9 mo of age (P less than 0.005). Native DNA binding at 6 mo of age was 23.9 +/- 14.7 vs. 10.1 +/- 9.7% in the beef and menhaden oil groups, respectively (P less than 0.01). Weights were similar in all groups, and there was no evidence of essential fatty acid deficiency in any group. These results demonstrate that a diet high in EPA protects NZB x NZW F1 mice from the development of glomerulonephritis.