We previously generated MRL/lpr mice deficient in the activation-induced deaminase (AID) who lack isotype switching and immunoglobulin hypermutation. These mice have high levels of unmutated (germline) autoreactive IgM yet experienced an increase in survival and an improvement in lupus nephritis that exceeded that of MRL/lpr mice lacking IgG. Herein, we test the hypothesis that high levels of germline autoreactive IgM in these mice confer protection against lupus nephritis.
Autoreactive IgM antibodies of various specificities including against dsDNA from AID-deficient-MRL/lpr mice were given to asymptomatic MRL/lpr mice and the levels of cytokines, proteinuria, immune complex deposition in the kidneys, and glomerulonephritis were examined. Novel AID-deficient MRL/lpr mice that lack any antibodies were generated to compare to AID-deficient-MRL/lpr mice that secrete only IgM.
Anti-dsDNA IgM treatment resulted in a dramatic improvement in lupus nephritis. Other autoreactive IgM’s such as anti-phospholipid and anti-Smith antigen did not alter pathology. Secretion of pro-inflammatory cytokines by macrophages, and the levels of inflammatory cells and apoptotic debris in the kidneys were lower in mice receiving anti-dsDNA IgM. Protective IgM derived from AID-deficient-MRL/lpr mice, displayed a distinct B cell repertoire, with a bias towards members of the Vh7183 family.
Anti-dsDNA IgM protected MRL/lpr mice from lupus nephritis likely by stopping the inflammatory cascade leading to kidney damage. A distinct repertoire of Vh usage in anti-dsDNA IgM hybridomas from AID-deficient mice, suggests enrichment in these mice of a dedicated B cell population that secretes unmutated protective IgM.
MRL-lpr mice develop aggressive autoimmune kidney disease associated with increased or de novo renal expression of major histocompatibility complex (MHC) class II molecules and a massive systemic expansion of CD4-CD- double negative (DN) T cells. Whereas non-MHC linked genes can have a profound effect on the development of nephritis, lymphadenopathy, and anti-DNA antibody production in MRL-lpr mice, the role of MHC molecules has not been unequivocally established. To study the role of MHC class II in this murine model of systemic lupus erythematosis, class II-deficient MRL-lpr mice (MRL-lpr -/-) were created. MRL-lpr -/- mice developed lymphadenopathy but not autoimmune renal disease or autoantibodies. This study demonstrates that class II expression is critical for the development of autoaggressive CD4+ T cells involved in autoimmune nephritis and clearly dissociates DN T cell expansion from autoimmune disease initiation.
The effects of biweekly intravenous injections of Staphylococcus Enterotoxin B (SEB) into autoimmune MRL-lpr/lpr (MRL/lpr) mice were investigated. Rather than causing the expansion of V beta 8+ T cells, SEB administration resulted in the reduction V beta 8+, CD4-CD8- "double-negative" (DN) T cells. This was shown by FACS analysis as this putative pathogenic population was diminished in both spleen and lymph node. The symptoms of systemic lupus erythematosus (SLE) in MRL/lpr, which include high titers of anti-DNA antibodies and circulating immune complexes and proteinuria, were reduced in SEB-treated mice in a dose- dependent manner. The clinical parameters of SLE in MRL/lpr, which include lymph node hyperplasia and necrotic vasculitis, were suppressed in 50-micrograms SEB-treated mice. T cells bearing V beta 6 T cell receptor, which does not interact with SEB, were not reduced with SEB administration. Thus, disease suppression was associated with a specific reduction in the number of V beta 8+, DN T cells. These results implicate a possible therapeutic role of superantigen-based immunotherapy in V beta-restricted, T cell-dominated clinical syndromes.
In the present work, we investigate the role of interleukin (IL)27/IL27 receptor α (Rα) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans.
We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties.
In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and IgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)γ and IL4 mRNA by CD4+ T cells from Tg mice decreased in a dose-dependent fashion. CD4+ splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4+ T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells.
WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE.
Anti-DNA antibodies, specifically those that stain nuclei in a homogenous nuclear (HN) fashion, are diagnostic of systemic lupus erythematosus (SLE) and the MRL-lpr/lpr SLE murine model. We have used a heavy chain transgene that increases the frequency of anti-HN antibodies to address whether their production in SLE is the consequence of a defect in B cell tolerance. Anti-HN B cells were undetectable in nonautoimmune-prone transgenic mice, but in MRL-lpr/lpr transgenic mice their Ig was evident in the sera and they were readily retrievable as hybridomas. We conclude that nonautoimmune animals actively delete anti-HN-specific B cells, and that MRL-lpr/lpr mice are defective in this process possibly because of the lpr defect in the fas gene.
A hallmark of systemic lupus erythematosus and the MRL murine model for lupus is the presence of anti–double-stranded (ds)DNA antibodies (Abs). To identify the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune (MRL+/+ and lpr/lpr) mice with that in nonautoimmune (BALB/c) mice. Anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by their developmental arrest and accumulation at the T–B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle. Coincident with the altered localization of anti-dsDNA B cells is a follicular infiltration of CD4 T cells. Together, these data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle.
tolerance; Fas; autoimmunity; antinuclear antibody; splenic architecture
We generated MRL/lpr mice deficient in the Activation Induced Deaminase (AID). Because AID is required for immunoglobulin hypermutation and class switch recombination, these mice lack hypermutated IgG antibodies. Unlike their AID wild-type littermates, AID-deficient MRL/lpr mice not only lacked autoreactive IgG antibodies, but also experienced a dramatic increase in the levels of autoreactive IgM. This phenotype in AID-deficient mice translated into a dramatic reduction in glomerulonephritis, minimal mononuclear cell infiltration in the kidney, and a dramatic increase in survival to levels comparable to previously reported for MRL/lpr mice completely lacking B cells and levels well below those of mice lacking secreted antibodies. Therefore, this study, wherein littermates with either high levels of autoreactive IgM or autorective IgG are directly examined, proves that autoreactive IgM antibodies alone are not sufficient to promote kidney disease in MRL/lpr mice. In addition, the substantial decrease in mortality combined with a dramatic increase in autoreactive IgM antibodies in AID-deficient MRL/lpr mice, suggest that autoreactive IgM antibodies might not only fail to promote nephritis, but may also provide a protective role in MRL/lpr mice. This novel mouse model containing high levels of autoreactive, unmutated IgM antibodies will help delineate the contribution of autoreactive IgM to autoimmunity.
autoimmunity; B cells; Systemic Lupus Erythematosus; Autoantibodies
Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2−/− mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.
anti-DNA antibody; B cell deletion; B cell anergy; recombination-activating gene deficiency; apoptosis
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti–double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti–single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T–B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.
Congenic MRL-lpr mice homozygous and heterozygous for the IFN-gamma gene disruption were created to assess the role of this pleotropic cytokine on the lymphoaccumulation and lupus-like disease of Fas-defective mice. Early death was prevented, and glomerulonephritis severely reduced in IFN-gamma-/- mice. Hypergammaglobulinemia was maintained with a switch from IgG2a to IgG1 predominance, but the dramatic decrease in levels of the dominant IgG2a anti-dsDNA autoantibodies was not associated with a compensatory increase in TH2-associated IgG subclasses. Remarkably, early death and glomerulonephritis were also prevented in IFN-gamma+/- mice, although autoantibody levels and glomerular immune deposits were equivalent to IFN-gamma+/+ lpr mice, indicating the importance of additional locally-exerted disease-promoting effects of IFN-gamma. IFN-gamma-/- mice exhibited reduced lymphadenopathy concomitant to a decrease in DN B220(+) T cells. In vivo BrdU labeling showed reduced proliferation of DN B220(+) cells in IFN-gamma-/- vs. IFN-gamma+/+ lpr mice, while enhanced proliferation of all other T cell subsets was unaffected. Macrophages of IFN-gamma-/-lpr mice expressed markedly decreased levels of MHC class I and II molecules compared with controls. Moreover, the heightened expression of MHC class II molecules on proximal tubules of IFN-gamma+/+ lpr mice was significantly reduced in both IFN-gamma-/- and IFN-gamma+/- mice. The data indicate that IFN-gamma hyperproduction is required for lupus development, presumably by increasing MHC expression and autoantigen presentation to otherwise quiescent nontolerant anti-self T cells, and also by promoting local immune and inflammatory processes.
Adiponectin is an adipocyte-derived cytokine with anti-inflammatory properties. Paradoxically, circulating adiponectin levels are increased in a number of inflammatory diseases. Thus, we sought to define the role of adiponectin deficiency in mouse models of autoimmunity. Adiponectin-deficient mice on a C57BL/6 background do not develop an autoimmune phenotype. Autoimmunity was also not observed in adiponectin-deficient mice generated on the permissive MRL background. However, adiponectin deficiency exacerbated the autoimmune phenotype of MRL-lpr mice. Compared with MRL-lpr mice, MRL-lpr.apn−/− mice displayed greater lymphadenopathy and splenomegaly, as well as increased anti-nuclear antibody and anti-dsDNA production. In addition, evaluation of the kidney revealed larger glomerular tuft size, crescent formation, increased IgG and C3 deposits, and mesangial expansion in the MRL-lpr.apn−/− mice. The effects of adiponectin deficiency on the autoimmune phenotypes were more pronounced in female versus male mice. These data show that, while adiponectin deficiency is not sufficient to confer autoimmunity, adiponectin acts as a negative modulator of the autoimmune phenotype in a murine model of lupus.
Adiponectin; lupus; mouse model; inflammation; autoimmunity
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/faslpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/faslpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/faslpr/lpr B cells was significantly altered, e.g., increased dG/dC transitions, and increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/faslpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer’s patches. In MRL/faslpr/lpr mice, the significant upregulation of SHM and CSR was associated with significantly increased expression of AID, which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone mice, SHM and CSR are dysregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.
activation-induced cytidine deaminase (AID); antibody; autoantibody; B cell; class switch DNA recombination (CSR); DNA deletion; DNA insertion; lupus; somatic hypermutation (SHM)
Clonotypes of IgG anti-DNA antibodies were studied by isoelectric focusing in various autoimmune mice with or without lethal lupus nephritis. MRL/MpJ-lpr/lpr mice exhibited the most heterogeneous spectrotypes of anti-DNA antibodies in the pH range from 6.5 to 8.5, with marked variation in individual mice. Female (NZB X NZW)F1 mice expressed rather uniform DNA-binding bands composed of at least five to six distinct subgroups, having isoelectric points from 6.5 to 8.0. Male BXSB mice showed major characteristic bands confined to alkaline pH range from 7.8 to 8.5, similar to C57BL/6J-lpr/lpr mice, which showed markedly restricted bands in this region. Both AKR/J-lpr/lpr and C3H/HeJ-lpr/lpr mice expressed DNA-binding bands mostly focused between pH 6.5 and 8.2. The aging study indicated that three autoimmune mice (MRL/MpJ-lpr/lpr, [NZB X NZW]F1, and male BXSB) that developed fatal glomerulonephritis showed clonal expansion of anti-DNA antibodies throughout their life. In contrast, such age-dependent expansion of anti-DNA clonotypes was not evident in three lpr cogenic mice (C57BL/6J-lpr/lpr, AKR/J-lpr/lpr, and C3H/HeJ-lpr/lpr) that developed only mild glomerulonephritis; rather, their expression of anti-DNA spectrotypes diminished as they aged. Anti-DNA activities in renal eluates from nephritic autoimmune mice were mostly distributed in the pH range from 6.5 to 8.0, without significant concentrations in the high alkaline range of more than pH 8.0. These results suggest that there exist distinct anti-DNA clonotypes in each mouse strain and that the development of lupus nephritis does not appear to be associated with particular spectrotypes of anti-DNA antibodies. Rather, the age-dependent expansion of anti-DNA clonotypes may be a feature more characteristic of mice developing lethal lupus nephritis.
The systemic autoimmune syndrome of MRL/Mp-lpr/lpr (MRL/lpr) mice consists of severe pan-isotype hypergammaglobulinemia, autoantibody production, lymphadenopathy, and immune complex-associated end-organ disease. Its pathogenesis has been largely attributed to helper alphabeta T cells that may require critical cytokines to propagate pathogenic autoantibody production. To investigate the roles of prototypical Th1 and Th2 cytokines in the pathogenesis of murine lupus, IFN-gamma -/- and IL-4 -/- lupus-prone mice were generated by backcrossing cytokine knockout animals against MRL/lpr breeders. IFN-gamma -/- animals produced significantly reduced titers of IgG2a and IgG2b serum immunoglobulins as well as autoantibodies, but maintained comparable levels of IgG1 and IgE in comparison to cytokine-intact controls; in contrast, IL-4 -/- animals produced significantly less IgG1 and IgE serum immunoglobulins, but maintained comparable levels of IgG2a and IgG2b as well as autoantibodies in comparison to controls. Both IFN-gamma -/- and IL-4 -/- mice, however, developed significantly reduced lymphadenopathy and end-organ disease. These results suggest that IFN-gamma and IL-4 play opposing but dispensable roles in the development of lupus-associated hypergammaglobulinemia and autoantibody production; however, they both play prominent roles in the pathogenesis of murine lupus-associated tissue injury, as well as in lpr-induced lymphadenopathy.
Antibodies directed against IgG and DNA are found in the sera of autoimmune MRL/Mp lpr/lpr mice. Little is known of the molecular mechanisms underlying expression of such autoantibodies. We have investigated the binding diversity and pattern of VH gene expression in a panel of murine anti-IgG antibodies. We constructed eight hybridoma clones secreting IgM antibodies that bound to mouse IgG by using spleen cells from MRL/Mp lpr/lpr mice varying in age from 4 to 15 wk; one clone was derived from a 32-wk-old MRL +/+ mouse. The monoclonal IgM products exhibited varying binding specificities for intact mouse IgG, fragments of mouse IgG [Fc, Fab, (Fab')2], and heterologous IgG. Two of these antibodies crossreacted with B and/or Z DNA. Probes from seven of eight identified mouse VH gene families (7183, S107, Q52, J558, J606, 36-60, and 3609) were hybridized under high-stringency conditions with cytoplasmic RNA blots from each clone. Six clones hybridized only with the probe from the five-member 36-60 family. The remaining three clones crosshybridized with the 36-60 probe and the probe from the 60 member J558 family, perhaps reflecting somatic mutation from the original germline VH gene resulting in recognition by a probe from another family, in addition to the probe from the original germline family. Our results indicate that spontaneous MRL lpr/lpr anti-IgG antibodies are encoded predominantly by the 36-60 VH gene family and imply a nonrandom selection of this VH gene family in the production of these antibodies.
Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp-lpr/lpr (MRL/lpr) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo.
anti-DNA; B cell tolerance; receptor editing; systemic lupus erythematosus; VH replacement
MRL-lpr/lpr mice spontaneously develop an autoimmune disease resembling systemic lupus erythematosus and rheumatoid arthritis. One of the unique serological abnormalities in this strain is remarkably high concentrations of cryoglobulins. Analysis of immunoglobulin components in their cryoglobulins has shown selective enrichment of a particular IgG subclass, IgG3. As IgG3 enrichment is also found in two other cryoglobulins, which are induced after injection with bacterial lipopolysaccharides or infection with malaria, IgG3 apparently represents a major source of murine cryoglobulins. Studies on murine IgG3 monoclonal antibodies (mAbs) have clearly shown that murine IgG3 have the unique physiochemical property to self associate through non-specific IgG3 Fc-Fc interaction, and that most of them can generate monoclonal cryoglobulins. Most strikingly, IgG3 monoclonal cryoglobulins with rheumatoid factor (RF) activity induce extensive pathological manifestations: skin vascular purpura and glomerulonephritis with 'wire loop' lesions. Although the cryoglobulin activity of IgG3 RF mAb is solely responsible for the generation of glomerular lesions (both RF and cryoglobulin activities are necessary for skin vascular lesions), the absence of nephritogenic activity by some IgG3 cryoglobulins supports the idea that qualitative features of cryoglobulins are critical to determine their pathogenic activity. The demonstration of a positive correlation between the production of IgG3 cryoglobulins and the development of lupus nephritis in MRL-lpr/lpr mice further substantiates the pathological importance of cryogenic autoantibodies. On the other hand, it should be emphasised that non-cryogenerating IgG3 autoantibodies may not be harmful, but even protective, as a result of their interaction with pathogenic IgG3 cryoglobulins. Finally, the development of an experimental model of cryoglobulinaemia associated with vascular and glomerular disease certainly represents an invaluable opportunity to study the molecular mechanisms responsible for the generation of cryoglobulins and their associated tissue lesions, and also to assess various therapeutic approaches. Our demonstration that anti-idiotypic mAb can prevent the pathogenic effects of the cryoprecipitable IgG3 RF mAb suggests strongly that such a therapeutic approach might be successful in similar diseases in man.
To investigate the respective roles of Th1 and Th2 cells in the pathogenesis of lupus-like autoimmune disease, we have analyzed the spontaneous and antigen-induced productions of IgG1 vs IgG2a and IgG3 subclasses in relation to the mRNA expression of INF-gamma (Th1 cytokine promoting IgG2a and IgG3 production), IL-4 (Th2 cytokine promoting IgG1 production), and IL-10 (Th2 cytokine) in CD4+ T cells from lupus-prone MRL mice. For this purpose, two paired sets of MRL mice were chosen for the comparison of these parameters: (a) MRL-lpr/lpr (lpr for lymphoproliferation) and its recently described substrain with a prolonged survival, termed MRL-lpr/lpr.ll (ll for long lived) and (b) MRL male mice bearing the Yaa (Y-linked autoimmune acceleration) gene (MRL.Yaa) with an accelerated disease and their male counterparts lacking the Yaa gene. We demonstrate herein that the accelerated development of lupus-like autoimmune disease in MRL-lpr/lpr and MRL.Yaa mice, as compared with MRL-lpr/lpr.ll and MRL-+/+ mice, respectively, was correlated with an enhanced expression of IFN-gamma vs IL-4 and IL-10 mRNA in CD4+ T cells, which paralleled with an increase of spontaneous and foreign T cell-dependent antigen-induced productions of IgG2a and IgG3 vs IgG1 antibodies. These data suggest that an imbalance towards Th1 predominance may play a significant role in the acceleration of lupus-like autoimmune disease in MRL mice.
Young MRL/MPJ-lpr (lpr) mice 8-12 wk old challenged with alloantigen had significantly lower specific cytolytic T lymphocyte (CTL) responses than control MRL/MPJ +/+ mice. Serum from lpr mice compared with serum from ++ or normal C3H mice powerfully suppressed CTL responses in mixed lymphocyte cultures (MLC); absorbing lpr serum on protein G, adding antibody against transforming growth factor beta (TGF-beta) to cultures or dissociating immunoglobulin G (IgG) and TGF-beta before additions to cultures prevented suppression. Apparently autoantibody, similar to IgG produced by normal mice in response to immunization, carries TGF-beta which suppresses CTL responses in vivo and in vitro.
MRL/MpJ-Faslpr (MRL/lpr) mice are an accepted animal model to study human systemic lupus erythematosus. We tested if a commonly used analgesic (buprenorphine hydrochloride) would reduce pain and distress in these mice without impacting the progression of autoimmune disease. Female MRL/lpr mice were randomly separated into four groups. Experimental groups received cyclophosphamide (25 mg/kg i.p. weekly), buprenorphine (0.09 mg/kg/mouse/day via drinking water), or cyclophosphamide + buprenorphine from 11 - 21 weeks of age. Controls received no treatments. Mice were monitored daily by a licensed veterinarian (blinded observer) and assigned a score weekly on parameters associated with pain and distress as well as progression of disease. Proteinuria was measured weekly, and serum anti-dsDNA antibody levels were determined at 11, 15, and 18 weeks of age. At 21 weeks of age, the animals were euthanized and the kidneys and spleens were removed for evaluation. Regardless of the parameter observed, buprenorphine did not significantly decrease distress when compared to the controls. Buprenorphine did not alter the progression of autoimmune disease, based on characteristics of splenic architecture and splenocyte cell profiles, development of lymphadenopathy, or kidney histology as compared to controls. This study indicates that buprenorphine at this dose and route of administration was ineffective in reducing distress associated with disease progression in the MRL/lpr strain. More studies are needed to determine if, at a different dose or route, buprenorphine would be useful as adjunctive therapy in reducing distress in MRL/lpr mice.
Buprenorphine; MRL/lpr; distress; systemic lupus erythematosus; pruritus
Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.
The systemic autoimmune disease lupus erythematosus (SLE) is frequently accompanied by neuropsychiatric manifestations and brain lesions of unknown etiology. The MRL-lpr mice show behavioral dysfunction concurrent with progression of a lupus-like disease, thus providing a valuable model in understanding the pathogenesis of autoimmunity-induced CNS damage. Profound neurodegeneration in the limbic system of MRL-lpr mice is associated with cytotoxicity of their cerebrospinal fluid (CSF) to mature and immature neurons. We have recently shown that IgG-rich CSF fraction largely accounts for this effect. The present study examines IgG levels in serum and CSF, as well as the permeability of the blood–brain barrier in mice that differ in immune status, age, and brain morphology. In comparison to young MRL-lpr mice and age-matched congenic controls, a significant elevation of IgG and albumin levels were detected in the CSF of aged autoimmune MRL-lpr mice. Two-dimensional gel electrophoresis and MALDI-TOF MS confirmed elevation in IgG heavy and Ig light chain isoforms in the CSF. Increased permeability of the blood–brain barrier correlated with neurodegeneration (as revealed by Fluoro Jade B staining) in periventricular areas. Although the source and specificity of neuropathogenic antibodies remain to be determined, these results support the hypothesis that a breached blood–brain barrier and IgG molecules are involved in the etiology of CNS damage during SLE-like disease.
Autoimmunity; Autoantibodies; Lupus; Blood—brain barrier; Cerebrospinal fluid; Immunoglobulin; Albumin; Fluoro Jade B; Western blotting; Mass spectrometry; MRL mice
In young adulthood, MRL/Mp-lpr/lpr mice develop severe systemic lupus erythematosus (SLE)-like syndrome associated with massive T cell proliferation. The congenic MRL/Mp- mice lack the lpr gene and develop chronic SLE late in life. We have exchanged thymic transplants between these substrains so as to determine the role of the thymus in the development of early, severe SLE and of lymphoproliferation. The median survival times of unmanipulated lpr/lpr and mice were 160 and 510 d, respectively. The lpr/lpr and mice thymectomized when newborn and transplanted at 1 mo with the opposite type of thymus retained the diseases phenotype of their unmanipulated counterparts with 50% mortality at 186 and 498 d, respectively. In contrast, lpr/lpr mice thymectomized when newborn but not transplanted with thymus did not develop lymphoid hyperplasia and glomerulonephritis, and 100% of them were alive at 390 d. Serologically, the thymectomized but untransplanted lpr/lpr mice had significantly reduced levels of autoantibodies, whereas thymectomized and transplanted mice of either substrain were similar to unmanipulated controls. The results indicate that: (a) a thymus is essential for expression of lymphoproliferation and early SLE-like disease in the lpr/lpr phenotype; (b) the lpr/lpr disease is not a result of a unique hormonal or microenvironmental defect(s) of the thymus of this substrain because the genotype of the thymus is irrelevant for the development of T cell proliferation and early SLE; (c) differentiation of stem cells under the hormonal or microenvironmental influences of a thymus that possesses the lpr genotype does not lead to abnormal T cell differentiation or early autoimmunity; and (d) the lpr/lpr disease cannot be caused exclusively by an intrinsic B cell defect or environmental stimuli that cause B cell polyclonal activation.
Immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) play important roles in the generation of autoantibodies in systemic lupus erythematosus. Systemic lupus is characterized by the production of an array of pathogenic high-affinity mutated and class-switched, mainly IgG, antibodies to a variety of self-antigens, including nuclear components, such as dsDNA, histones and chromatin. We previously found that MRL/Faslpr/lpr mice, which develop a systemic autoimmune syndrome sharing many features with human lupus, display greatly upregulated CSR, particularly to IgG2a, in B cells of the spleen, lymph nodes and Peyer’s patches. In MRL/Faslpr/lpr mice, the significant upregulation of CSR is associated with increased expression of activation-induced cytidine deaminase (AID), which is critical for CSR and SHM. We also found that HoxC4 directly activates the promoter of the AID gene to induce AID expression, CSR and SHM. Here, we show that in both lupus patients and lupus-prone MRL/Faslpr/lpr mice, the expression of HoxC4 and AID is significantly upregulated. To further analyze the role of HoxC4 in lupus, we generated HoxC4−/− MRL/Faslpr/lpr mice. In these mice, HoxC4-deficiency resulted in reduced AID expression, impaired CSR and decreased serum anti-dsDNA IgG, particularly IgG2a, autoantibodies, which were associated with a reduction in IgG deposition in kidney glomeruli. In addition, consistent with our previous findings that in MRL/Faslpr/lpr mice, upregulated AID expression is associated with extensive DNA lesions, comprising deletions and insertions in the IgH locus, we found c-Myc to IgH(c-Myc/IgH) translocations to occur frequently in B cells of MRL/Faslpr/lpr mice. The frequency of such translocations was significantly reduced in HoxC4−/− MRL/Faslpr/lpr mice. These findings suggest that in lupus B cells, upregulation of HoxC4 plays a major role in dysregulation of AID expression, thereby increasing CSR and autoantibody production, and promoting c-Myc/IgH translocations.
Activation-induced cytidine deaminase (AID); B cell lymphoma; cancer; class switch DNA recombination (CSR); c-Myc/IgH translocation; HoxC4; MRL/Faslpr/lpr mice; systemic lupus erythematosus (SLE)
CD59 is a glycosylphosphatidylinositol (GPI)-anchored membrane regulator of complement expressed on blood cells as well as peripheral tissues. It protects host cells from complement injury by inhibiting formation of the membrane attack complex. Recent studies in mice have suggested also a role of CD59 in T cell immune response that was mechanistically independent of complement. In the present study, we investigated the function of CD59 in the MRL/lpr model of murine lupus. We backcrossed the Cd59a knockout (Cd59a−/−) mouse onto the MRL/lpr background and compared Cd59a+/+-MRL/lpr and Cd59a−/−-MRL/lpr littermates for the development of systemic autoimmunity. We found that CD59a deficiency significantly exacerbated the skin disease and lymphoproliferation characteristic of MRL/lpr mice. It also increased autoantibody titers and caused a higher level of proteinuria in male MRL/lpr mice. Bone marrow transfer experiments indicated that CD59a expression on both BM-derived cells and peripheral tissues played a role in lymphoproliferation, whereas the skin disease phenotype is determined mainly by local CD59a expression. Importantly, C3 gene deletion or C5 neutralization with a blocking mAb in Cd59a−/−-MRL/lpr mice did not rescue the pro-autoimmune phenotype associated with CD59a deficiency. These results together suggest that CD59a inhibits systemic autoimmunity in MRL/lpr mice through a complement-independent mechanism.