A genetic analysis of lupus.

Systemic lupus erythematosus (SLE) is a complex multigenic inherited disease with susceptibility determined by a combination of genetic, environmental and stochastic factors. Although not yet defined, recent technical advances have provided the means to dissect the component genetic contributions of polygenic traits. We have applied such approaches to mouse models of spontaneous SLE and, in this report, summarize our genome wide mapping studies that identified loci predisposing to several major lupus-related traits. Through the generation and study of interval congenic lines, precise mapping, and screening of candidate genes, identification of the specific genes and mechanisms associated with some of the major loci is currently being pursued.

Role of cyclin kinase inhibitor p21 in systemic autoimmunity.

The cyclin kinase inhibitor protein p21 affects multiple processes relevant to the immune system, including cell cycle progression, replicative senescence, hemopoietic stem cell quiescence, and apoptosis. Therefore, malfunction of this protein may be a contributor to the pathogenesis of systemic autoimmunity. Here, we report that mixed background p21-deficient 129/Sv x C57BL/6 mice showed increased in vitro and in vivo T cell cycling and activation, moderate hypergammaglobulinemia and, at low penetrance, anti-chromatin autoantibodies. Homeostatic anti-self MHC/peptide ligand-induced proliferation of p21-deficient T cells was also enhanced. However, lymphoid organ enlargement was very mild, presumably due to increased apoptosis of the rapidly dividing cells. Moreover, the older p21-deficient mice had kidney pathology representing a similar, but slightly more advanced, state than that seen in the control mice. The timing and severity of the above serologic, cellular, and histologic manifestations in p21-deficient mice were unaffected by gender. Thus, p21 deficiency significantly enhances T cell activation and homeostatic proliferation, and can induce mild autoimmune manifestations at a low incidence without gender bias, but does not in itself generate the full spectrum of lupus-like disease.

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.

The role of alpha beta+ T cells and homeostatic T cell proliferation in Y-chromosome-associated murine lupus.

Male BXSB mice develop an early life, severe lupus-like disease largely attributed to an undefined Y-chromosome-associated autoimmunity accelerator, termed YAA: Although the exact disease pathogenesis is uncertain, indirect evidence suggests that T cells play an important role in the male BXSB disease. We have developed TCR alpha-chain gene-deleted BXSB mice to directly examine the role of alphabeta+ T cells and the mode by which Yaa promotes disease in this strain. All disease parameters, including hypergammaglobulinemia, autoantibody production, glomerulonephritis, and the unique monocytosis of BXSB males, were severely reduced or absent in the alphabeta+ T cell-deficient mice. Adoptively transferred CD4+ T cells of either male or female BXSB origin showed equal homeostatic proliferation in alphabeta+ T cell-deficient male recipients. Moreover, deficient male mice eventually developed equally severe lupus-like disease after adoptive transfer and homeostatic expansion of T cells from wild-type BXSB males or females. The results directly demonstrate that the Yaa-mediated disease requires alphabeta+ T cells that are not, in themselves, abnormal in either composition or properties, but are engaged by a Yaa-encoded abnormality in a non-T cell component. In addition, homeostatic anti-self proliferation of mature T cells derived from a small number of precursors can induce systemic autoimmunity in an appropriate background.

Resistance to xenobiotic-induced autoimmunity maps to chromosome 1.

Although evidence indicates that environmental factors play a major role in precipitating systemic autoimmunity in genetically susceptible individuals, little is known about the mechanisms involved. Certain heavy metals, such as mercury, are potent environmental immunostimulants that produce a number of immunopathologic sequelae, including lymphoproliferation, hypergammaglobulinemia, and overt systemic autoimmunity. Predisposition to such metal-induced immunopathology has been shown to be influenced by both MHC and non-MHC genes, as well as susceptibility to spontaneous lupus, in mice and other experimental animals. Among the various mouse strains examined to date, the DBA/2 appears to uniquely lack susceptibility to mercury-induced autoimmunity (HgIA), despite expressing a susceptible H-2 haplotype (H-2d). To define the genetic basis for this trait, two genome-wide scans were conducted using F2 intercrosses of the DBA/2 strain with either the SJL or NZB strains, both of which are highly susceptible to HgIA. A single major quantitative trait locus on chromosome 1, designated Hmr1, was shown to be common to both crosses and encompassed a region containing several lupus susceptibility loci. Hmr1 was linked to glomerular immune complex deposits and not autoantibody production, suggesting that DBA/2 resistance to HgIA may primarily involve the later stages of disease pathogenesis. Identification and characterization of susceptibility/resistance genes and mechanisms relevant to the immunopathogenesis of mercury-induced autoimmunity should provide important insights into the pathogenesis of autoimmunity and may reveal novel targets for intervention.

The role of IFN-gamma in systemic lupus erythematosus: a challenge to the Th1/Th2 paradigm in autoimmunity.

The classification of T helper cells into type 1 (Th1) and type 2 (Th2) led to the hypothesis that Th1 cells and their cytokines (interleukin [IL]-2, interferon [IFN]-gamma) are involved in cell-mediated autoimmune diseases, and that Th2 cells and their cytokines (IL-4, IL-5, IL-10, IL-13) are involved in autoantibody(humoral)-mediated autoimmune diseases. However, this paradigm has been refuted by recent studies in several induced and spontaneous mouse models of systemic lupus erythematosus, which showed that IFN-gamma is a major effector molecule in this disease. These and additional findings, reviewed here, suggest that these two cross-talking classes of cytokines can exert autoimmune disease-promoting or disease-inhibiting effects without predictability or strict adherence to the Th1-versus-Th2 dualism.

Xenobiotic acceleration of idiopathic systemic autoimmunity in lupus-prone bxsb mice.

The diverse genetic backgrounds of lupus-prone murine models, which produce both quantitative and qualitative differences in disease expression, may be a valuable resource for studying the influence of environmental exposure on autoimmune disease in sensitive populations. We tested this premise by exposing autoimmune-prone BXSB and the nonautoimmune C57BL/6 mice to the heavy metal mercury. Although both strains express a nonsusceptible H-2 haplotype, exposure to mercury accelerated systemic autoimmunity in both male and female BXSB mice, whereas the C57BL/6 mice were resistant. The subclasses of antichromatin antibodies elicited in BXSB mice by mercury exposure were more consistent with the predominant Th1-type response of idiopathic disease than with the Th2-type response found in mercury-induced autoimmunity (HgIA). The appearance and magnitude of both humoral and cellular features of systemic autoimmunity correlated with the mercury dose. Furthermore, environmentally relevant tissue levels of mercury were associated with exacerbated systemic autoimmunity. These studies demonstrate that xenobiotic exposure can accelerate spontaneous systemic autoimmunity, and they support the possibility that low-level xenobiotic exposure enhances susceptibility to systemic autoimmunity in genetically susceptible individuals.

Suggestive evidence for association of human chromosome 18q12-q21 and its orthologue on rat and mouse chromosome 18 with several autoimmune diseases.

Some immune system disorders, such as type 1 diabetes, multiple sclerosis (MS), and rheumatoid arthritis (RA), share common features: the presence of autoantibodies and self-reactive T-cells, and a genetic association with the major histocompatibility complex. We have previously published evidence, from 1,708 families, for linkage and association of a haplotype of three markers in the D18S487 region of chromosome 18q21 with type 1 diabetes. Here, the three markers were typed in an independent set of 627 families and, although there was evidence for linkage (maximum logarithm of odds score [MLS] = 1.2; P = 0.02), no association was detected. Further linkage analysis revealed suggestive evidence for linkage of chromosome 18q21 to type 1 diabetes in 882 multiplex families (MLS = 2.2; lambdas = 1.2; P = 0.001), and by meta-analysis the orthologous region (also on chromosome 18) is linked to diabetes in rodents (P = 9 x 10(-4)). By meta-analysis, both human chromosome 18q12-q21 and the rodent orthologous region show positive evidence for linkage to an autoimmune phenotype (P = 0.004 and 2 x 10(-8), respectively, empirical P = 0.01 and 2 x 10(-4), respectively). In the diabetes-linked region of chromosome 18q12-q21, a candidate gene, deleted in colorectal carcinoma (DCC), was tested for association with human autoimmunity in 3,380 families with type 1 diabetes, MS, and RA. A haplotype ("2-10") of two newly characterized microsatellite markers within DCC showed evidence for association with autoimmunity (P = 5 x 10(-6)). Collectively, these data suggest that a locus (or loci) exists on human chromosome 18q12-q21 that influences multiple autoimmune diseases and that this association might be conserved between species.

Genetics of systemic autoimmunity in mouse models of lupus.

Systemic lupus erythematosus (SLE) is inherited as a complex polygenic trait, involving genetic, environmental and stochastic factors. Although definition of these etiologic processes has been elusive, solid progress has been made toward elucidating the genetic basis for susceptibility. Herein, we summarize our genome wide mapping effort that has defined loci for component phenotypes for lupus-prone NZB, NZW, MRL-Fas(lpr) and BXSB strains. With this framework in place, identification of the specific genetic alterations and mechanisms is now proceeding through the generation of interval congenic lines, precise mapping and screening of candidate genes. In addition to this approach, transgenic and gene knockout studies have begun to identify genes that can induce or modify autoimmunity in nonautoimmune and lupus-prone background mice, including studies by us and others on Th1 and Th2 cytokine genes in lupus. It is apparent that a diversity of genes and mechanisms can independently or in combination promote systemic autoimmunity in mice. This complexity, which is also observed in human lupus, emphasizes the importance of using experimental and less complex mouse models to define these processes, a tactic that has already yielded new insights. With current technologies and the anticipated definition of mammalian genomes, identification of genes predisposing to lupus and elucidation of processes critical for disease pathogenesis appear within grasp.

The pathogenesis of autoimmunity in New Zealand mice.

OBJECTIVE: New Zealand mice were the first spontaneous animal model of human systemic lupus erythematosus (SLE). Since their initial discovery in 1959, studies of these mice have provided insights into the immunopathogenesis and genetics of lupus and have had a substantial impact on our understanding of autoimmunity. METHODS: We extensively reviewed published data for the past 40 years, including work in cellular immunology and molecular biology, to provide new information on the role of lymphoid subpopulations, cytokines, costimulatory molecules, apoptosis, and genetic susceptibility in the natural history of immunopathology in murine lupus. RESULTS: Genetic factors constitute the most important contribution to autoimmunity in New Zealand mice, and specific major susceptibility loci have been described. In addition, there is evidence for a pluripotent stem cell defect, which has implications for developmental and functional defects of T and B cells. The end result of these defects is a breakdown of self-tolerance and production of autoantibodies. Further studies will undoubtedly shape our understanding of this murine model and provide the basis for novel diagnostic and therapeutic approaches in humans. CONCLUSIONS: The advent of molecular biology, including the use of monoclonal antibody therapy in New Zealand mice, has been instrumental in our understanding of the loss of self-tolerance in SLE. Finally, identification of genetic susceptibility loci in the murine system has also led to important comparable studies in humans with SLE. RELEVANCE: The observations in New Zealand mice are of particular importance to systemic lupus erythematosus (SLE).

Treatment of murine lupus with cDNA encoding IFN-gammaR/Fc.

IFN-gamma, a pleiotropic cytokine, is a key effector molecule in the pathogenesis of several autoimmune diseases, including lupus. Importantly, deletion of IFN-gamma or IFN-gammaR in several lupus-predisposed mouse strains resulted in significant disease reduction, suggesting the potential for therapeutic intervention. We evaluated whether intramuscular injections of plasmids with cDNA encoding IFN-gammaR/Fc can retard lupus development and progression in MRL-Fas(lpr) mice. Therapy significantly reduced serum levels of IFN-gamma, as well as disease manifestations (autoantibodies, lymphoid hyperplasia, glomerulonephritis, mortality), when treatment was initiated at the predisease stage, particularly when IFN-gammaR/Fc expression was enhanced by electroporation at the injection site. Remarkably, disease was arrested and even ameliorated when this treatment was initiated at an advanced stage. This therapy represents a rare example of disease reversal and makes application of this nonviral gene therapy in humans with lupus (and perhaps other autoimmune/inflammatory conditions) highly promising.

Genetic studies in systemic autoimmunity and aging.

The etiopathogenesis of systemic lupus erythematosus remains an enigma that will probably not be solved until the genetic basis for susceptibility is defined. Through genomewide searches, we have provided a foundation for this by identifying and characterizing loci predisposing to specific disease traits in four major lupus-susceptible mouse strains. Further ongoing work that includes the study of interval-specific congenic lines and precise mapping of loci should lead to identification of the corresponding genes and elucidation of processes critical for disease pathogenesis. Another important area of investigation is the study of cell-cycle and apoptosis genes in systemic autoimmunity and aging. Based on earlier work, we proposed that the characteristic overexpansion of memory phenotype cells in these conditions may be owing to replicative senescence. Understanding the molecular mechanisms that regulate the generation of these cells may permit selective manipulations to control this process. Other areas of investigation that we are actively engaged in are the role of T cell receptor repertoire in disease and the definition of cellular genes affected by infection with human immunodeficiency virus.

Development of lupus in BXSB mice is independent of IL-4.

Although systemic lupus erythematosus appears to be a humorally mediated disease, both Th1 and Th2 type responses have been implicated in its pathogenesis. The Th1 response, as exemplified by IFN-gamma production, has been uniformly shown in mouse lupus models to be critical for disease induction. The role of Th2 type responses, however, is more complicated, with some studies showing detrimental and others beneficial effects of IL-4 in these models. To further address this issue, we generated and analyzed IL-4 gene-deficient BXSB mice. Mice homozygous for this deletion had significantly lower serum levels of total IgG1 compared with wild-type BXSB, consistent with the lack of IL-4. However, no significant differences were observed in mortality, spleen weight, severity of glomerulonephritis, levels of anti-chromatin and anti-ssDNA Abs, or frequency of activated (CD44high) CD4+ T cells. The anti-chromatin Ab isotype response was virtually all Th1 type in both the knockout and wild-type BXSB. These findings directly demonstrate that IL-4 and, by inference, Th2 cells are not obligatory participants in the induction and maintenance of lupus in this strain.

Lupus-prone mice as models to study xenobiotic-induced acceleration of systemic autoimmunity.

The linkage between xenobiotic exposures and autoimmune diseases remains to be clearly defined. However, recent studies have raised the possibility that both genetic and environmental factors act synergistically at several stages or checkpoints to influence disease pathogenesis in susceptible populations. These observations predict that individuals susceptible to spontaneous autoimmunity should be more susceptible following xenobiotic exposure by virtue of the presence of predisposing background genes. To test this possibility, mouse strains with differing genetic susceptibility to murine lupus were examined for acceleration of autoimmune features characteristic of spontaneous systemic autoimmune disease following exposure to the immunostimulatory metals nickel and mercury. Although NiCl(2) exposure did not exacerbate autoimmunity, HgCl(2) significantly accelerated systemic disease in a strain-dependent manner. Mercury-exposed (NZB X NZW)F1 mice had accelerated lymphoid hyperplasia, hypergammaglobulinemia, autoantibodies, and immune complex deposits. Mercury also exacerbated immunopathologic manifestations in MRL+/+ and MR -lpr mice. However, there was less disease acceleration in lpr mice compared with MRL+/+ mice, likely due to the fact that environmental factors are less critical for disease induction when there is strong genetic susceptibility. Non-major histocompatibility complex genes also contributed to mercury-exacerbated disease, as the nonautoimmune AKR mice, which are H-2 identical with the MRL, showed less immunopathology than either the MRL/lpr or MRL+/+ strains. This study demonstrates that genetic susceptibility to spontaneous systemic autoimmunity can be a predisposing factor for HgCl(2)-induced exacerbation of autoimmunity. Such genetic predisposition may have to be considered when assessing the immunotoxicity of xenobiotics. Additional comparative studies using autoimmune-prone and nonautoimmune mice strains with different genetic backgrounds will help determine the contribution that xenobiotic exposure makes in rendering sensitive populations susceptible to autoimmune diseases.

The genes of systemic autoimmunity.

Autoimmune diseases include a wide spectrum of disorders, which have been divided into systemic and organ-specific disorders. Lupus, the prototypic systemic autoimmune disease, is characterized by female predominance, multiorgan pathology, and autoantibodies, primarily directed against nuclear antigens. The disease is heterogeneous, with variable organ involvement, serology, and clinical course. Susceptibility to lupus is inherited as a polygenic trait with added contributions from environmental and stochastic variance. Concerted efforts have recently been made by several laboratories to define the genetic basis of this disease in predisposed mice and humans. The identification of the Fas/FasL defects in lpr and gld lupus mice was the first example of spontaneous mutations of apoptosis-promoting genes being associated with systemic autoimmunity. This research was instrumental in clarifying the roles of these genes in tolerance and immunoregulation, and in extrapolating these results to other autoimmune diseases, as well as cancer and transplantation. To these findings have been added those from transgenic and gene knockout mouse studies that have helped to define the systemic autoimmunity-inducing or -modifying effects of specific genes in normal background and lupus-congenic mice. In addition, the findings from genome-wide searches have begun to identify predisposing loci (and ultimately genes) for the spontaneous lupus-like diseases in various mouse strains and in humans. The emerging picture is that multiple genetic contributions can independently lead to systemic autoimmunity in mice, which reinforces the view that human lupus may be similarly composed of diverse genotypes. This complexity underscores the importance of defining the predisposing alleles and mechanisms of action, an undertaking that is certainly feasible given current technologies and future advances in the definition of mammalian genomes.

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection.

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.