B-lineage cell deficits in bone marrow of lpr/lpr mice

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygousfor the lpr mutation (BS.Ipr) disclosed low numbers of pre-Band B cells, as compared with age-matched control B6 mice. BMdepletion in B6.lpr mice was selective for B-lineage cells,appeared in young adults, and developed markedly with age anddisease progression, contrasting with the peripheral lymphocytehypercellularity. Normalization of pre-B and B cellularity inBM of B6.lprmice was observed after administration of polyclonalIg, that also markedly improved the clinical condition. Isolatedpre-B (B220⁺ IgM^–) cells from B6 or B6.lpr mice, however,showed essentially the same rates of IL-7-dependent proliferationand differentiation to B (lgM⁺) cells in culture, indicatingthat the BM B-lineage deficit is not the result of an intrinsicdefect inB cell generation.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

Heat shock protein 90 inhibition by 17-DMAG lessens disease in the MRL/lpr mouse model of systemic lupus erythematosus

Elevated expression of heat shock protein 90 (HSP90) has been found in kidneys and serum of systemic lupus erythematosus (SLE) patients and MRL/Mp-Fas^lpr/Fas^lpr (MRL/lpr) autoimmune mice. We investigated if inhibition of HSP90 would reduce disease in MRL/lpr mice. In vitro, pretreatment of mesangial cells with HSP90 inhibitor Geldanamycin prior to immune-stimulation showed reduced expression of IL-6, IL-12 and NO. In vivo, we found HSP90 expression was elevated in MRL/lpr kidneys when compared to C57BL/6 mice and MRL/lpr mice treated with HSP90 inhibitor 17-DMAG. MRL/lpr mice treated with 17-DMAG showed decreased proteinuria and reduced serum anti-dsDNA antibody production. Glomerulonephritis and glomerular IgG and C3 were not significantly affected by administration of 17-DMAG in MRL/lpr. 17-DMAG increased CD8⁺ T cells, reduced double-negative T cells, decreased the CD4/CD8 ratio and reduced follicular B cells. These studies suggest that HSP90 may play a role in regulating T-cell differentiation and activation and that HSP90 inhibition may reduce inflammation in lupus.     

Effect of cyclophosphamide on lymphokine production in MRL/lpr.Yaa mice

The Y chromosome (Yaa gene) from autoimmune BXSB mice has been shown to be responsible for the acceleration of autoimmune symptoms when transferred to MRL/lpr mice. We examined the pathological, serological and immunological characteristics of MRL/lpr.Yaa mice and the suppressive effect of cyclophosphamide (CP) on the mice. MRL/lpr.Yaa mice spontaneously developed a massive lymphadenopathy characterized by hypergammaglobulinemia, the presence of several autoantibodies, and autoimmune disease. In MRL/lpr.Yaa mice, IL-2, IL-4 and IL-5 production in concanavalin A (Con A)-stimulated splenocytes were about 10-fold lower than in BALB/c mice at 5 weeks of age.The concentrations of these lymphokines remained low until the mice were 16 weeks of age. The production of IFN- and IL-6 in 16 week old MRL/lpr.Yaa mice was about 4- and 2-fold lower, respectively, though these levels were similar in both strains at 8 weeks of age. It was found that this dysregulation of T cell function was almost identical to that in MRL/lpr mice. Administration of CP to MRL/lpr.Yaa mice ameliorated nephritis, and suppressed production of autoantibodies and the accumulation of abnormal T cells. CP also significantly elevated the production of lymphokines. These findings suggest that an abnormality of T cell function may contribute to the autoimmune pathogenesis of MRL/lpr.Yaa mice and that CP probably ameliorates autoimmune disease by improving the T cell functions.     

Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab−/−) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab−/− mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab−/− mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.     

Spontaneous and antibody directed cytotoxicity of double-negative T cells from autoimmune mice

MRL/Mp-lpr/lpr (MRL/lpr) mice develop a syndrome similar tosystemic lupus erythematosus in humans. This strain of miceis characterized by the progressive accumulation of CD4^–CD8^–(double-negative; DN) T cells which express increased levelsof cell adhesion molecules such as CD44 and heat stable antigen(HSA). The DN T cells exhibited a higher level of spontaneouscytolytic activity and contained a higher level of serine esteraseas compared with T cells of MRL/Mp-+/+ (MRL/+) mice. We alsofound that mAbs against CD44, Mei-14, CD45R, and HSA could augmentthe cytolytic activity of DN T cells of MRL/lpr mice. Antibody-mediatedaugmentation of cytolytic activity of DN T cells was due toconjugate formation in which the Fc portion of mAb bound tothe Fc receptor on target cells and the Fab portion of mAb boundto corresponding cell surface antigens on DN T cells. The antibody-mediatedaugmentation of cytolytic activity was not detected in T cellsof MRL/+ mice and lymphokine activated killer (LAK) cells ofC57BL/6 mice. In contrast, anti-CD3 mAbs could augment the cytolyticactivity of DN T cells, T cells as well as LAK cells. mAbs againstLFA-1 and VLA-4 failed to augment the cytolytic activity ofthree different effector cells. It should be noted that antl-CD3mAb-mediated cytolytic activity of DN T cells was substantiallyreduced by anti-LFA-1 mAb. However, CD44, Mel-14, CD45R as wellas HSA-mediated cytolytic activity of DN T cells was not inhibitedby anti-LFA-1 mAb. The cell-cell and cell-matrix interactionsthrough cell adhesion molecules might augment the antigen non-specificcytolytic activity of DN T cells in vivo.     

Treatment effect of a traditional Chinese medicine,Ren-shen-yang-rong-tang (Japanese name: Ninjin-Youeito), on autoimmune MRL/MP-lpr/lpr mice

MRL/Mp-lpr/lpr(MRL/lpr) mice were treated with a traditional Chinese herbal medicine, Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT) intraperitoneally (i.p.) every 3 days or per os (p.o.) 6 times/week from before the onset of autoimmune disease (6 weeks of age). Fifty percent survival time was found in placebo-controlled male and female mice of 28 and 22 weeks of age, respectively. NYT-treatment markedly prolonged the survival time of MRL/lpr mice. That is, 50% survival time was 43 weeks in the i.p.-treated male mice and 30 weeks of age in the p.o.-treated female mice. Further, NYT-treatment significantly reduced occurence of thymic atrophy and prevented the anomalous accumulation of B220⁺ T-cells in lymphnode and spleen, both of which are characteristic in MRL/lpr mice. Moreover, grades of proteinuria were significantly reduced in both the i.p.- and p.o.-treated groups compared with the control groups. Such clinical benefit and increased survival time were interestingly not associated with the decrease in the level of autoantibodies.     

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions,comprising deletions and insertions,in the immunoglobulin locus: Concurrent upregulation of somatic hypermutation and class switch DNA recombination

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/fas^lpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas^lpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas^lpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas^lpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas^lpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (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 MRL/fas^lpr/lpr mice, SHM and CSR are upregulated, 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.     

Bcl-3 inhibits lupus-like phenotypes in BL6/lpr mice

Bcl-3 is an atypical member of the IκB family that modulates NF-κB activity in nuclei. lpr mice carry the lpr mutation in Fas, resulting in functional loss of this death receptor; they serve as models for lupus erythematosus and autoimmune lymphoproliferation syndrome (ALPS). To explore the biologic roles of Bcl-3 in this disease model, we generated BL6/lpr mice lacking Bcl-3. Unlike lpr mice on an MRL background, BL6/lpr mice present with very mild lupus- or ALPS-like phenotypes. Bcl-3 KO BL6/lpr mice, however, developed severe splenomegaly, dramatically increased numbers of double negative T cells — a hallmark of human lupus, ALPS, and MRL/lpr mice — and exhibited inflammation in multiple organs, despite low levels of autoantibodies, similar to those in BL6/lpr mice. Loss of Bcl-3 specifically in T cells exacerbated select lupus-like phenotypes, specifically organ infiltration. Mechanistically, elevated levels of Tnfα in Bcl-3 KO BL6/lpr mice may promote lupus-like phenotypes, since loss of Tnfα in these mice reversed the pathology due to loss of Bcl-3. Contrary to the inhibitory functions of Bcl-3 revealed here, this regulator has also been shown to promote inflammation in different settings. Our findings highlight the profound, yet highly context-dependent roles of Bcl-3 in the development of inflammation-associated pathology.     

lpr T cells vaccinate against lupus in MRL/lpr mice.

MRL/MP-lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8-) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease-accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp-lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This "T cell vaccination" approach resulted in a selective depletion of cells expressing products of the V beta 8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

Effects of the lpr/lpr mutation on T and B cell populations in the lamina propria of the small intestine, a mucosal effector site.

MRL mice, which develop a lymphoproliferative disease characterized by increased numbers of alpha/beta T cell receptor+ (TCR+) B220/6B2+CD4-CD8- T cells [lymphoproliferation (lpr) T cells], were studied for the effect of the lpr/lpr mutation on the mucosal immune system in the gastrointestinal (GI) tract. We analyzed the effect of the lpr gene mutation on T and B cell populations in the Peyer's patches (PP) and the lamina propria lymphocytes (LPLs), as examples of major IgA inductive and effector tissues in the GI tract respectively. Normal mouse PP contain B cells committed to IgA (surface IgA+) but only low numbers of B cells producing IgA. However, enhanced spontaneous IgA and IgG synthesis occurs in the PP of MRL mice. Further, we have now shown that PP of MRL mice are populated by lpr T cells. Interestingly, lpr T cells were not present in significant numbers in LPLs of MRL mice, even in older animals. Of interest was the finding that the ratio of CD4+ to CD8+ T cells in the lamina propria was lower in MRL when compared with control mice, and the CD8+ T cell subset actually predominates in LPLs of autoimmune mice. In addition, the number of gamma/delta TCR+ T cells in LPL of MRL lpr/lpr mice was significantly increased, especially in MRL lpr/lpr mice at 6 and 12 weeks of age. When the isotype distribution of B cells in LPLs was analyzed, no changes were noted in MRL lpr/lpr mice in comparison with MRL +/+ or normal control mice, and the pattern was IgA much greater than IgM greater than IgG. These results show that although increased numbers of CD8+ T cells and gamma/delta TCR+ cells occur in the LPLs of MRL mice, a normal distribution of plasma cell isotypes (IgA much greater than IgM greater than IgG) is found in this mucosal compartment. Further, Ipr T cells do not develop in the lamina propria compartment of the GI tract.     

TCF1 deficiency ameliorates autoimmune lymphoproliferative syndrome (ALPS)‐like phenotypes of lpr/lpr mice

Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease, which is characterized by non‐malignant autoimmune lymphoproliferation. TCF1 is a key effector in the canonical Wnt/β‐catenin pathway, regulating the development, activation and function of T cells. In this study, we aimed to explore the potential role of TCF1 in the development of ALPS‐like phenotypes of lpr/lpr mice. We acquired TCF1^−/−lpr/lpr double mutant mice by crossing TCF1 deficiency mice with lpr/lpr mice. Splenocyte compositions, serum cytokines levels, antidsDNA antibody production and kidney pathology were examined in the TCF1^−/−lpr/lpr mice. With these examinations, we revealed that TCF1 deficiency relieved most manifestations of ALPS‐like phenotype, which were caused by Fas mutation in TCF1^−/−lpr/lpr mice. Splenocyte total numbers and compositions were downregulated to the similar levels with wildtype mice. T_E and T_EM cells were decreased in TCF1^−/−lpr/lpr compared with lpr/lpr mice. The levels of autoantibodies and proinflammatory factors in serum, and the histopathology changes and the relative mRNA levels of proinflammatory factors in kidney all displayed parallel tendency in TCF1^−/−lpr/lpr mice. Our study demonstrated that TCF1 deficiency ameliorated the ALPS‐like phenotypes of TCF1^−/−lpr/lpr mice, which might indicate a potential therapeutic direction for ALPS.     

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice

An MRL strain of mice bearing a Fas-deletion mutant gene, lpr, MRL/MpJ-lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ-lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr) F1 intercross, and MRL/lpr × (MRL/lpr × C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi-squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.     

Enhanced natural but diminished antibody-mediated cytotoxicity in the lungs of MRLlpr/lpr mice

Human systemic lupus erythematosus (SLE) patients, as well as MRLlpr/lpr mice which develop a SLE-like disease, have decreased numbers and functional activity of systemic natural killer (NK) cells. In contrast, it has been found that among lymphocytes recovered from the bronchoalveolar lavage fluid of SLE patients, NK cells were increased in number, correlating with the severity of the lung engagement. The present study was undertaken to assay the capacity for natural killing in the lung compartment of MRLlpr/lpr mice compared with healthy congenic MRL +/+ and heterozygous MRL +/lpr mice. ⁵¹Cr-labelled YAC-1 cells were injected intravenously to settle in the lungs where they were targeted for lysis by NK cells. YAC-1 cell killing inversely correlated with radioactivity remaining in the lungs after the assay, and was inhibited by antibody to the asialo-GM1 antigen expressed on NK cells. To analyse the capacity in the lung for cytolysis of non-NK cell-sensitive target cells, a similar in vivo⁵¹Cr-release assay was set up for antibody-mediated allospecific cytotoxicity. We demonstrate that MRLlpr/lpr mice throughout their lifespan display significantly increased natural cytotoxic activity in the lungs compared with MRL +/+ and MRL +/lpr mice, as demonstrated by more efficient killing of YAC-1 cells. In contrast, antibody-mediated allospecific cytotoxicity in the lungs was significantly less effective in the MRLlpr/lpr strain.     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.