Genetic regulation of erythrocyte autoantibody production in New Zealand black mice

The incidences of positive anti-erythrocyte autoantibodies (AEA) in New Zealand Black (NZB), C57BL/6, their F₁, F₂ hybrid, and the F₁ × NZB backcross mice were 100, 0, 0, 17, and 51%, respectively. This finding is in keeping with the idea that a combined effect of one to three dominant predisposing NZB gene(s) and a single dominant modifying C57BL/6 gene regulates the AEA production. Studies suggested that the modifying locusAem-1 is loosely linked toMup-1 locus on chromosome 4, and the gene order isAem-1: Mup-1: Gpd-1. We analyzed the effects of theAem-1 locus on other autoimmune traits and found that the gene action ofAem-1 is unrelated to the spontaneous productions of dsDNA-specific antibodies, the retroviral gp70-anti-gp70 immune complexes and natural thymocytotoxic autoantibod ies and to the serum level of retroviral gp70. A significant association was observed between the negative AEA and the low (normal) serum IgM level in (C57BL/6 × NZB)F₁ × NZB backcross mice. It remains to be determined whether theAem-1 locus also controls the serum IgM level.Abbreviations used in this paper AEA anti-erythrocyte autoantibody - NTA natural thymocytotoxic autoantibody - gp70 major glycoprotein constituent of the murine C type retrovirus envelope - Mup-1 major urinary protein complex-1 - Gpd-1 glucose-6-phosphate dehydrogenase-1 - Akp-1 alkaline phosphatase-1 - Es-1 esterase-1 - Igh-1 immunoglobulin (IgG2a) heavy chain-1     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

A peripheral and central T cell antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice

Naturally occurring thymocytotoxic autoantibodies (NTA) have been suggested to be the cause of thymic atrophy and T cell disorders in human and murine lupus. Definitive studies on NTA's role in the induction of SLE, however, have been lacking due to the lack of a pure source of NTA. Although it is clear that NTA are a heterogeneous group of antibodies, the nature of their antigens has remained obscure. We report the characteristics of a monoclonal NTA, designated SAG-3, which appears more reflective of the activities previously reported of serum NTA than other NTA-secreting clones. SAG-3 is an IgM autoantibody cytotoxic for 80 to 90% of thymocytes, 20 to 25% of splenic lymphocytes, 25 to 30% of lymph node cells, and less than 3% cortisol-resistant thymocytes, bone marrow, and fetal liver cells. SAG-3 is murine-specific without reactivity towards rat, hamster, or guinea pig, and appears very early in thymic development, on day 17 fetal thymocytes. SAG-3 is equally cytotoxic against several strains of mice, including both Thy-1.1 and Thy-1.2 allotypes, and the cytotoxicity is absorbed by brain but not liver cells. Reactive thymocytes occurred throughout the cortical regions of the thymus, indicating preferential affinity towards immature thymocytes. Although the serologic activities of SAG-3 suggest that Thy-1 alloantigen is its target, SAG-3 antigen is found to be distinct from Thy-1 and also from Lyt-1, Lyt-2, or L3T4 antigens. The binding of SAG-3 to thymocytes could be competitively inhibited by NTA-positive NZB sera.     

Proteins associated with B lymphocyte hyperactivity in New Zealand black mice

New Zealand Black (NZB) mice exhibit polyclonal B cell activation and elevated immunoglobulin production, an abnormality associated with the spontaneous autoimmune disease that affects this strain. To further our understanding of this abnormality of B cell differentiation and maturation, we have employed two-dimensional polyacrylamide gel electrophoresis to analyze the proteins synthesized by lymphocytes of several strains. Two proteins were produced by lymphocytes from NZB mice but not those from normal strains. One was a 16 kd protein with a pI of 5.1, and the other was a 27.5 kd protein with a pI of 4.5. The presence of the xid gene on the NZB background suppressed production of both proteins. They were synthesized by spleen cells but not by bone marrow or lymph node cells, and production was restricted to enlarged B lymphocytes. p16 was synthesized by normal mouse strain B cells upon stimulation with LPS. The 27.5 kd protein was shown to be secreted. On the basis of partial amino acid sequence determination of proteins eluted from gels, p27.5 was identified as J chain and p16 as the C terminal fragment of mu-chain. The synthesis of two other proteins, 13 kd and 18 kd in size, was elevated in NZB spleen lymphocytes. The 18 kd protein was identified as translation initiation factor eIf-4D. The increased level of this protein may be related to the upregulation of immunoglobulin synthesis.     

In vitro regulation of the pathogenic autoantibody response of New Zealand black mice. I. Loss with age of suppressive activity in T cell populations

An investigation of the regulation of specific anti-self responses was initiated with the development of an in vitro system in which spleen cells from NZB mice were stimulated by syngeneic mouse erythrocytes (MRBC) to produce MRBC-specific autoantibody-secreting cells. The response was measured by a modification of the focus-forming cell (FFC) assay, which enumerates cells secreting IgG, which specifically bind MRBC. Spleen cells from 9- to 12-mo-old NZB mice developed MRBC-specific FFC after 3 to 5 days in culture with MRBC. Few FFC were detected in the absence of MRBC in culture. Spleen cells from young (1- to 4-mo-old) NZB mice developed few if any FFC. Spleen cell populations containing T cells from young NZB mice suppressed this anti-MRBC response, whereas B cell populations from these young mice did not. In contrast, spleen cells, including T cell-enriched populations from old, Coombs'-positive mice were not capable under the same conditions of producing equivalent suppression of this in vitro autoimmune response. These data suggest that a population of suppressor T cells that may control the autoimmune anti-MRBC response in young NZB mice is lost, or else its activity is masked in old NZB mice that are actively producing anti-MRBC antibody.     

Superoxide and hydrogen peroxide production by macrophages of New Zealand black mice

Resident peritoneal macrophages from New Zealand Black (NZB) mice release O2- and H2O2 after adherence to a plastic surface without any chemical or particulate stimulant. This phenomenon is age dependent and more pronounced in animals with sever autoimmune disease. Significant differences were observed between the high and low breakage NZB sublines (HB and LB), which were previously developed by selective matings on the basis of chromosome breakage rates. The LB subline differs significantly from the HB subline with respect to autoimmune hemolytic anemia and tumor incidence. When the macrophages were stimulated with the tumor promoter TPA, the number of "responders" was higher in the HB than in the LB subline and correlated with the degree of splenomegaly, that is, with the severity of the disease. A negative response to agonist stimulation and very low spontaneous production of active oxygen species was observed in NZW and Swiss mice, which is the normal finding for resident macrophages according to data from the literature. The increased superoxide and hydrogen peroxide production by macrophages of NZB mice is discussed with respect to autoimmune disease and cancer.     

Defective B-cell tolerance in New Zealand black mice. Fc receptor-independence of resistance to low-epitope-density tolerogens

Trinitrophenyl (TNP) human gamma-globulin with low-epitope-density tolerizes B cells from normal BDF1 mice in a Fc gamma receptor-dependent manner but does not tolerize B cells from preautoimmune NZB mice. In order to investigate the relationships between tolerance induction and epitope density independently of Fc gamma receptor function in these two strains, TNP conjugates of two additional thymic-independent tolerogenic carriers, D-glutamic acid-D-lysine (D-GL) and carboxymethyl cellulose (CMC), were tested. A brief pulse with low-epitope-density conjugates such as TNP4.4-D-GL rendered unfractionated or T-cell-depleted spleen cells from BDF1 but not NZB mice tolerant in a hapten-specific manner. Spleen cells from NZB mice, however, were susceptible to tolerization with TNP13.5-D-GL. NZB mice were also resistant to tolerance induction in vivo with TNP5.5-D-GL, TNP3-CMC, and TNP6-CMC, all of which tolerize BDF1 mice in vivo. Both strains were tolerized with TNP13.5-D-GL and TNP13-CMC in vivo. NZB mice were also significantly less susceptible to tolerance induction with TNP3-CMC when TNP-Ficoll was substituted for TNP Brucella abortus as the challenge antigen. These findings militate against the possibility that an Fc gamma receptor defect is the principal mechanism of resistance of NZB B cells to tolerance induction with-low-epitope density conjugates.     

Atorvastatin inhibits autoreactive B cell activation and delays lupus development in New Zealand black/white F1 mice

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F(1) mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.     

Colocalization of expansion of the splenic marginal zone population with abnormal B cell activation and autoantibody production in B6 mice with an introgressed New Zealand Black chromosome 13 interval

Polyclonal B cell activation is a prominent feature of the lupus-prone New Zealand Black (NZB) mouse strain. We have previously demonstrated linkage between a region on NZB chromosome 13 and increased costimulatory molecule expression on B cells. In this study we have produced C57BL/6 congenic mice with an introgressed homozygous NZB interval extending from approximately 24 to 73 cM on chromosome 13 (denoted B6.NZBc13). We show that B6.NZBc13 female mice not only have enhanced B cell activation but also share many other B cell phenotypic characteristics with NZB mice, including expansion of marginal zone and CD5+ B cell populations, increased numbers of IgM ELISPOTs, and increased serum levels of total IgM and IgM autoantibodies. In addition these mice have increased T cell activation, increased numbers of germinal centers, mild glomerulonephritis, and produce high-titer IgM and IgG anti-chromatin Abs. Male B6.NZBc13 mice have a less pronounced cellular phenotype, lacking expansion of the marginal zone B cell population and IgG anti-chromatin Ab production, indicating the presence of gender dimorphism for this locus. Thus, we have identified a genetic locus that recapitulates with fidelity the B cell phenotypic abnormalities in NZB mice, and we demonstrate that this locus is sufficient to induce an autoimmune phenotype. The data provide further support to the contention that immune abnormalities leading to altered B cell activation and selection contribute to the development of autoimmunity in NZB mice.     

Age-dependent macrophage functions in New Zealand black mice.

Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of β-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ¹²⁵I-labeled Shigella flexneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALB/c mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALB/c mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALB/c mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.     

Tissue localization and biochemical characteristics of a new thymic antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice

Naturally occurring thymocytotoxic autoantibodies (NTA) have been described in both humans and mice with SLE. To define further the role of anti-thymic autoantibodies in murine lupus, we studied the cellular and molecular specificity of a spontaneous monoclonal NTA, designated TC-17, derived from a 4-mo-old New Zealand Black mouse. TC-17, an IgM autoantibody, has been shown previously to be unreactive with Lyt-1, Lyt-2, and L3T4 (T helper) antigens. We have shown further that it is also unreactive with Thy-1. TC-17 recognizes a new thymic antigen that appears to mark a distinct subpopulation of cortisol-sensitive cortical thymocytes. The antigen consists of a single glycoprotein chain with an apparent m.w. of 88,000. TC-17 shows reduced binding to thymocytes treated with tunicamycin, indicating either that glycosylation of TC-17 antigen is necessary for TC-17 to bind to it or that glycosylation is required for expression of the antigen on the cell surface. TC-17 uniquely reacts with two of 17 murine lymphoid tumor cell lines of intermediate cellular maturity. The thymocytotoxic activity of TC-17 is absorbed by single cell suspensions of murine stomach, small intestine, large intestine, kidney, and thymus. Moreover, the specific binding of TC-17 to gut tissue of normal and germfree mice can be demonstrated by indirect immunofluorescence, suggesting antigenic cross-reactions between thymic and gut tissue. TC-17 reacts with rat thymocytes as well as it does with murine cells, indicating moderate evolutionary conservation of the TC-17 antigen. The expression of this glycoprotein by a discrete thymocyte subset may prove to be a valuable probe for the study of murine T cell differentiation.     

Effects of gamma-interferon and FK506 on resting B cell proliferation of New Zealand black/white F1 mice.

We examined the effects of gamma-interferon (gamma-IFN) and the new immunosuppressant FK506 on resting B cell proliferation of New Zealand black/white F1 hybrid (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE). gamma-IFN and FK506 inhibited in a dose-dependent manner both B cell proliferation and autoantibody production of resting B cells respectively. There was a synergistic interaction between gamma-IFN and FK506 in their inhibition and they did not exhibit cell cytotoxicity. This in vitro synergism of gamma-IFN and FK506 may have clinical application in that low doses of gamma-IFN and FK506 combinations may be effective to correct polyclonal B cell activation of patients with SLE.     

Epistatic suppression of fatal autoimmunity in New Zealand black bicongenic mice

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non-lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ-producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21(+)CD1d(+) B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.     

Functional interplay between intrinsic B and T cell defects leads to amplification of autoimmune disease in New Zealand black chromosome 1 congenic mice

Genetic loci on New Zealand Black (NZB) chromosome 1 play an important role in the development of lupus-like autoimmune disease. We have shown previously that C57BL/6 mice with an introgressed NZB chromosome 1 interval extending from approximately 35 to 106 cM have significantly more severe autoimmunity than mice with a shorter interval extending from approximately 82 to 106 cM. Comparison of the cellular phenotype in these mice revealed that both mouse strains had evidence of increased T cell activation; however, activation was more pronounced in mice with the longer interval. Mice with the longer interval also had increased B cell activation, leading us to hypothesize that there were at least two independent lupus susceptibility loci on chromosome 1. In this study, we have used mixed hemopoietic radiation chimeras to demonstrate that autoimmunity in these mice arises from intrinsic B and T cell functional defects. We further show that a T cell defect, localized to the shorter interval, leads to spontaneous activation of T cells specific for nucleosome histone components. Despite activation of self-reactive T cells in mixed chimeric mice, only chromosome 1 congenic B cells produce anti-nuclear Abs and undergo class switching, indicating impaired B cell tolerance mechanisms. In mice with the longer chromosome 1 interval, an additional susceptibility locus exacerbates autoimmune disease by producing a positive feedback loop between T and B cell activation. Thus, T and B cell defects act in concert to produce and amplify the autoimmune phenotype.     

Dissociation of the genetic loci leading to b1a and NKT cell expansions from autoantibody production and renal disease in B6 mice with an introgressed New Zealand Black chromosome 4 interval

Previous mapping studies have linked New Zealand Black (NZB) chromosome 4 to several lupus traits, including autoantibody production, splenomegaly, and glomerulonephritis. To confirm the presence of these traits, our laboratory introgressed homozygous NZB chromosome 4 intervals extending from either 114 to 149 Mb or 32 to 149 Mb onto the lupus-resistant C57BL/6 background (denoted B6.NZBc4S and B6.NZBc4L, respectively). Characterization of aged cohorts revealed that B6.NZBc4L mice exhibited a striking increase in splenic B1a and NKT cells in the absence of high titer autoantibody production and significant renal disease. Tissue-specific expansion of these subsets was also seen in the peritoneum and liver for B1a cells and in the bone marrow for NKT cells. Staining with CD1d tetramers loaded with an alpha-galactosylceramide analog (PBS57) demonstrated that the expanded NKT cell population was mainly CD1d-dependent NKT cells. The lack of both cellular phenotypes in B6.NZBc4S mice demonstrates that the genetic polymorphism(s) that result in these phenotypes are on the proximal region of NZB chromosome 4. This study confirms the presence of a locus that promotes the expansion of B1a cells and newly identifies a region that promotes CD1d-restricted NKT cell expansion on NZB chromosome 4. Taken together, the data indicate that neither an expansion of B1a cells and/nor NKT cells is sufficient to promote autoantibody production and ultimately, renal disease.     

Preferential autoantibody reactivity of the preimmune B cell repertoire in normal mice

Naturally occurring autoantibodies are frequently found in the sera of healthy individuals. They usually exhibit low binding affinities for autoantigens and often react with multiple antigenic determinants. To determine whether their frequencies have been overestimated by sensitive testing procedures, the germ-line B cell repertoire of strain A mice was examined for reactivity with a panel of auto- and foreign Ag. If the high frequencies of autoantibodies result from testing procedures, equally high frequencies would be expected for foreign Ag specificities detected in the same manner. The presence of specific autoantibodies was confirmed in this study by the disparate frequencies observed for antibodies reactive with individual Ag. The frequencies were highest for autoantigens associated with SLE, indicating a bias toward autoreactivity in the preimmune repertoire. Analysis of VH gene usage did not indicate any selection in V gene expression with autoreactivity.