Effect of an exogenous trigger on the pathogenesis of lupus in (NZB × NZW)F1 mice

Objective

This study examined the interactions between exogenous and endogenous factors shaping the phenotype of lupus in autoimmune (NZB × NZW)F₁ mice exposed to pristane, a model environmental trigger.

Methods

Frequencies of various autoantibodies in untreated NZB/NZW mice were determined by various means (immunoprecipitation, enzyme‐linked immunosorbent assay [ELISA], Crithidia luciliae kinetoplast staining). Pristane or saline was administered intraperitoneally to 9–12‐week‐old NZB/NZW mice, followed by serial studies of autoantibodies, total Ig levels (ELISA), and proteinuria (dipstick).

Results

Besides antichromatin/DNA responses, NZB/NZW mice spontaneously produced novel autoantibodies against the double‐stranded RNA binding protein RNA helicase A (RHA). In contrast, NZB/NZW mice (n = 70) did not produce autoantibodies against the nuclear RNP (nRNP), Sm, Ro, or La antigens. Pristane exposure synergistically activated the production of antichromatin/DNA antibodies and dramatically accelerated renal disease. Production of anti‐nRNP/Sm and Su autoantibodies also was induced, indicating that the unresponsiveness of NZB/NZW mice to these antigens can be overcome. Curiously, pristane treatment did not enhance the production of anti‐RHA, suggesting that these autoantibodies are regulated differently than anti‐DNA/chromatin and Sm. In contrast to previous reports that suggest a critical role of deficient interleukin‐12 (IL‐12) production in the pathogenesis of lupus, there was overproduction of IL‐12 in the peritoneal cavity of pristane‐treated NZB/NZW mice, and their spleen cells also produced large amounts of IL‐12.

Conclusion

These data lead us to propose that environmental influences exacerbate autoimmune manifestations in genetically lupus‐susceptible mice through their stimulatory effects on proinflammatory cytokines, such as IL‐12.

     

Delayed lupus onset in (NZB x NZW)F1 mice expressing a human C-reactive protein transgene

OBJECTIVE: Human C-reactive protein (CRP) binds apoptotic cells and alters blood clearance of injected chromatin in mice. To test whether CRP participates in the pathogenesis of systemic lupus erythematosus (SLE), we examined disease development in lupus-prone (NZB x NZW)F(1) (NZB/NZW) mice expressing a human CRP transgene (hCRPtg/BW). METHODS: Mortality was monitored, proteinuria was determined by dipstick, and serum levels of human CRP and anti-double-stranded DNA (anti-dsDNA) were determined by enzyme-linked immunosorbent assay in NZB/NZW and hCRPtg/BW mice. Thin sections of kidneys were analyzed by immunofluorescence microscopy to compare deposition of IgG, IgM, C3, and human CRP, and electron microscopy was used to reveal differences in ultrastructure. In situ hybridization was performed to detect human CRP messenger RNA expression. RESULTS: The hCRPtg/BW mice had less proteinuria and longer survival than NZB/NZW mice. They also had lower IgM and higher IgG anti-dsDNA titers than NZB/NZW mice, although the differences were transient and small. In hCRPtg/BW mice, accumulation of IgM and IgG in the renal glomeruli was delayed, reduced, and more mesangial than in NZB/NZW mice, while end-stage accumulation of IgG, IgM, and C3 in the renal cortex was prevented. There was less glomerular podocyte fusion, basement membrane thickening, mesangial cell proliferation, and occlusion of capillary lumens in hCRPtg/BW mice, but dense deposits in the mesangium were increased. With disease progression in hCRPtg/BW mice, there was little rise in the plasma CRP level, but CRP in the kidneys became increasingly apparent due to local, disease-independent, age-related expression of the transgene. CONCLUSION: In hCRPtg/BW mice, CRP protects against SLE by increasing blood and mesangial clearance of immune complexes and by preventing their accumulation in the renal cortex.     

Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice.

Lupus-like autoimmunity in (NZB x NZW)F1 mice is frequently marked by the development of a severe and fatal renal disease. Genes from both NZB and NZW parents are required for the full expression of disease. We applied a mapping technique based on polymorphism in simple sequence repeats to the analysis of (NZB x NZW)F1 x NZW backcross mice to determine the NZB genetic contribution to disease. The results show that a single NZB locus or tightly linked group of loci on the distal part of chromosome 4 provides the strongest association with renal disease and death. This locus, designated here as nba-1 (New Zealand Black autoimmunity), lies distal to the locus elp-1, 60-70 centimorgans from the centromere. It is of interest that a gene encoding a receptor for tumor necrosis factor maps to the vicinity of this disease-associated gene.     

Antioxidants suppress mortality in the female NZB x NZW F1 mouse model of systemic lupus erythematosus (SLE)

Inflammation produces reactive oxygen intermediates (ROI) that cause vascular damage and activate T lymphocytes. Conversely, antioxidants not only protect tissue from oxidative damage but also suppress immune reactivity. The objective of this study was to examine immunomodulatory effects of the non-enzymatic antioxidants, N-acetylcysteine (NAC) and cysteamine (CYST), on autoimmune disease, glomerulonephritis, and mortality in the female B/W mouse model of human systemic lupus erythematosus (SLE). The development of murine lupus was assessed during the lifespan of female B/W mice given NAC or CYST. Morbidity and mortality were assessed daily. At 6 week intervals mice were examined for weight change, albuminuria, serum BUN, antibodies to DNA, and IgG immunoglobulin levels. Serum prolactin, estrogen and progesterone were measured at 18 weeks of age. In a parallel study, NAC- and CYST-treated and control B/W mice were examined at 24 weeks of age for interval renal histopathology, lymphocyte adhesion molecule expression, and antibody titers and in vitro cytokine production in response to immunization with DNP-KLH. CYST significantly suppressed development of albuminuria and azotemia at 36 and 42 weeks of age compared to control and NAC-treated mice. NAC significantly suppressed anti-DNA antibody levels at 24 weeks. In contrast CYST significantly increased anti-DNA antibody levels at 18 weeks of age (P < 0.001 CYST vs control and NAC-treated mice). Kidneys of CYST-treated mice also had accelerated inflammatory histologic changes despite their lower incidence of albuminuria and azotemia. Mean (+/- s.e.m.) survival of control mice was 33 +/- 2 weeks compared to 38 +/- 2 weeks in NAC-treated mice (P < 0.05 vs control), and 48 +/- 2 weeks in the CYST-treated group (P < 0.01 vs control mice). The antioxidants, NAC and CYST, significantly improved mortality in the female B/W mouse model of SLE. NAC suppressed autoantibody formation and modestly prolonged survival. CYST, despite its augmentation of anti-DNA levels and renal inflammatory changes, inhibited the development of renal insufficiency and markedly improved survival. These findings suggest that ROIs play a role in the pathogenesis of lupus nephritis and that antioxidants reduce the damage causing renal insufficiency. Antioxidants may be a beneficial adjunctive therapy in the treatment of human SLE.     

Inhibition of lupus disease by anti-double-stranded DNA antibodies of the IgM isotype in the (NZB x NZW)F1 mouse

OBJECTIVE: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing pathogenic IgG anti-double-stranded DNA (anti-dsDNA) autoantibodies are deposited in renal capillaries and initiate glomerulonephritis (GN) by the activation of complement and effector cells. In contrast, it has been demonstrated that the presence of IgM anti-dsDNA antibodies correlates negatively with the development of GN in SLE. The aim of this study was to determine whether anti-dsDNA antibodies of the IgM isotype protect against IC-mediated organ damage in SLE. METHODS: Lupus-prone (NZB x NZW)F(1) mice (females) were treated with murine monoclonal IgM anti-dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 mug/week starting at 16 weeks of age (prophylactic) or at 24 weeks of age (therapeutic). RESULTS: Mice treated with IgM anti-dsDNA exhibited a delayed onset of proteinuria and a reduced degree of renal pathology, which resulted in significantly improved survival as compared with control mice. Serum concentrations of IgG anti-dsDNA antibodies were not significantly modified. However, glomerular deposition of ICs was markedly reduced in both treatment protocol groups. In contrast, higher amounts of IgG and IgM and increased expression of Fcgamma receptor were demonstrated in liver sections from the treated mice compared with the untreated mice, suggesting an enhanced clearance of soluble ICs from phagocytic cells of the reticuloendothelial system. CONCLUSION: These data demonstrate the efficacy of IgM anti-dsDNA treatment in inhibiting the pathologic changes of lupus in (NZB x NZW)F(1) mice. Lower glomerular IC deposition is associated with a reduced inflammatory response and impaired organ damage. The reduced frequency of GN in SLE patients who have IgM anti-dsDNA antibodies may therefore reflect a disease-modifying effect of this class of autoantibodies that has potential therapeutic implications. Our findings should encourage the development of new therapeutic modalities using IgM anti-dsDNA antibodies in humans with SLE.     

Galectin-1-induced down-regulation of T lymphocyte activation protects (NZB x NZW) F1 mice from lupus-like disease

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a hyperactive immune system, including activation of autoreactive T and B cells. These studies demonstrate that administration of recombinant galectin-1, a β-galactose binding protein, to SLE-prone (NZB?×?NZW) F1 mice reduced lymphocyte activation, inhibited serum anti-double-stranded DNA(dsDNA) IgG antibody production, decreased the incidence of proteinuria, and increased survival rate. In addition, recombinant galectin-1'-treated mice had a higher frequency of Foxp3 expression, which suggested an increase in the percentage of peripheral regulatory T cells. Consistent with the finding that there were fewer activated T lymphocytes, ex vivo T cells from mice treated with recombinant galectin-1 exhibited less proliferation in response to TCR stimulation. Furthermore, these cells were less efficient at lipid raft clustering in response to TCR/CD28 engagement, consistent with published reports that galectin-1 can reorganize the synaptic contact to interfere with TCR signaling and activation to prevent T cell activation. Aged galectin-1-deficient mice had higher serum levels of antibodies against dsDNA, elucidating a role for endogenous galectin-1 in decreasing susceptibility to autoimmunity. Together, the findings highlight galectin-1 as a novel potential therapeutic immune modulator for treatment of lupus-like disease.     

β2微球蛋白缺乏对鼠狼疮的影响

目的观察β2微球蛋白(β2-microglobulin,β2m)基因敲除(knockout)对狼疮鼠(NZB×NZW)F1(β2m-/- BWF1)的影响,探讨β2m在鼠狼疮发病机制中的作用.方法制备β2m-/- BWF1.酶联免疫吸附(ELISA)法检测血清中IgG型抗磷脂抗体、IgG型抗dsDNA抗体、类风湿因子(RF)、IgG和IgG3.比较β2m-/- BWF1和野生型(β2m+/+ BWF1)及杂合型(β2m+/- BWF1)在生存率、自身抗体、IgG和IgG3的差异.结果①BWF1雄性鼠生存率高于雌性鼠,β2m-/-对狼疮鼠的生存率无明显影响.②β2m-/- BWF1鼠4个月时抗磷脂抗体低,β2m-/-对抗dsDNA抗体无明显影响,β2m-/- BWF1鼠RF在4个月时低于其他两组,到8个月时则高于其他两组.③在4个月和6个月时β2m-/- BWF1鼠IgG显著降低,8个月时差异不显著可能与低蛋白血症有关.β2m-/- BWF1鼠IgG3仅在6个月时显著降低.结论β2m-/-对BWF1鼠的影响是多方面的,但并未影响系统性红斑狼疮(SLE)的生存率,对三种自身抗体的影响并不一致,以抗磷脂抗体和RF的改变明显,由于IgG吸收减少、分解加速,IgG明显降低.     

Effect of low dietary lipid on the development of Sj?gren's syndrome and haematological abnormalities in (NZB x NZW)F1 mice.

A diet low in fat was found to retard the development of autoimmune disease in (NZB x NZW)F1 mice, whereas diets high in fat content were associated with more severe disease. The ability of a reduced lipid intake to ameliorate the progression of autoimmune disease was indicated by preserved lacrimal gland secretion (measured by a modified Schirmer test), decreased infiltration of inflammatory cells into the exocrine tissue, and decreased severity of immunohaemolytic anaemia as indicated by near-normal packed cell volume and reticulocyte values. These results suggest that nutritional intervention may be of some help in reducing the severity of pathological abnormalities associated with human systemic lupus erythematosus and Sjögren''s syndrome.     

Delayed lupus onset in (NZB × NZW)F1 mice expressing a human C‐reactive protein transgene

Objective

Human C‐reactive protein (CRP) binds apoptotic cells and alters blood clearance of injected chromatin in mice. To test whether CRP participates in the pathogenesis of systemic lupus erythematosus (SLE), we examined disease development in lupus‐prone (NZB × NZW)F₁ (NZB/NZW) mice expressing a human CRP transgene (hCRPtg/BW).

Methods

Mortality was monitored, proteinuria was determined by dipstick, and serum levels of human CRP and anti–double‐stranded DNA (anti‐dsDNA) were determined by enzyme‐linked immunosorbent assay in NZB/NZW and hCRPtg/BW mice. Thin sections of kidneys were analyzed by immunofluorescence microscopy to compare deposition of IgG, IgM, C3, and human CRP, and electron microscopy was used to reveal differences in ultrastructure. In situ hybridization was performed to detect human CRP messenger RNA expression.

Results

The hCRPtg/BW mice had less proteinuria and longer survival than NZB/NZW mice. They also had lower IgM and higher IgG anti‐dsDNA titers than NZB/NZW mice, although the differences were transient and small. In hCRPtg/BW mice, accumulation of IgM and IgG in the renal glomeruli was delayed, reduced, and more mesangial than in NZB/NZW mice, while end‐stage accumulation of IgG, IgM, and C3 in the renal cortex was prevented. There was less glomerular podocyte fusion, basement membrane thickening, mesangial cell proliferation, and occlusion of capillary lumens in hCRPtg/BW mice, but dense deposits in the mesangium were increased. With disease progression in hCRPtg/BW mice, there was little rise in the plasma CRP level, but CRP in the kidneys became increasingly apparent due to local, disease‐independent, age‐related expression of the transgene.

Conclusion

In hCRPtg/BW mice, CRP protects against SLE by increasing blood and mesangial clearance of immune complexes and by preventing their accumulation in the renal cortex.

     

雌二醇调控新西兰黑鼠×白鼠子一代雌鼠的骨髓树突状细胞作用研究

目的 通过比较雌二醇(E2)对发病前后系统性红斑狼疮(SLE)模型鼠--新西兰黑鼠×新西兰白鼠子一代(NZB/w F1)雌鼠骨髓来源树突状细胞(BMDC)的作用,探索雌激素参与SLE的作用机制.方法 骨髓单个核细胞在重组小鼠粒细胞-巨核细胞集落刺激因子(rmGM-CSF)、重组白细胞介素-4(rmlL-4)、E2和雌激素受体(ER)调节剂--他莫昔芬(TAM)的作用下培养7 d诱导分化为未成熟DC,脂多糖(LPS)刺激24 h诱导为成熟DC,流式细胞仪检测BMDC表面共刺激分子CD40及胞内IL-6、IL-10、IL-12和肿瘤坏死因子(TNF)-α的产生,混合淋巴细胞反应检测BMDC对脾脏T淋巴细胞的刺激功能.结果 E2升高未成熟DC,但降低成熟DC CD40的表达;E2增强未成熟DC,但降低成熟DC的淋巴细胞刺激功能;E2降低幼龄鼠,但升高高龄鼠BMDC细胞因子的产生.TAM拮抗E2的作用.结论 E2通过ER调控狼疮鼠的BMDC,此调控作用随狼疮的进展及细胞的成熟阶段不同而有所不同.     

Therapeutic Studies in NZB/NZW F1 Mice

Cyclophosphamide (Cy) has been demonstrated to be effective in treating autoimmune disease in NZB/NZW F₁ mice. This study was designed to compare the efficacy of chlorambucil (Chlor) with that of a known effective drug (Cy) in the treatment of murine lupus. NZB/W female mice were treated with Cy, Chlor, or nothing on a once-a-month dosage schedule. The age of onset of proteinuria, the severity of glomerular lesions, and the median survival were compared among the three treatment groups. Cy was found to be superior to Chlor and controls in all measures.     

Protection against renal disease in (NZB x NZW)F(1) lupus-prone mice after somatic B cell gene vaccination with anti-DNA immunoglobulin consensus peptide

OBJECTIVE: Ig molecules contain epitopes that can induce T cell-mediated immune responses. B cells can process and present such epitopes and activate T cells. The purpose of the present study was to test our hypothesis that T cells that recognize an Ig consensus sequence presented by B cells will modulate lupus-like disease in mice. METHODS: (NZB x NZW)F(1) (NZB/NZW) lupus mice received somatic B cell gene transfer of a DNA plasmid encoding a consensus sequence of T cell determinants of murine anti-DNA IgG or control plasmids. Treated animals were monitored for the production of antibody, the development of renal disease, and the phenotype, number, and function of T cells. RESULTS: Treatment of mice with Ig consensus plasmid induced transforming growth factor beta-producing CD8+,CD28- T cells that suppressed the antigen-specific stimulation of CD4+ T cells in a cell-contact-independent manner, reduced antibody production, retarded the development of nephritis, and improved survival. Significantly, adoptive transfer of CD8+,CD28- T cells from protected mice into hypergammaglobulinemic NZB/NZW mice effectively protected the transferred mice from the development of renal disease. CONCLUSION: Gene expression of anti-DNA Ig consensus sequence induces immunoregulatory T cells that delay the development of lupus nephritis by suppressing hypergammaglobulinemia and renal disease.     

Effective therapy for nephritis in (NZB x NZW)F1 mice with triptolide and tripdiolide, the principal active components of the Chinese herbal remedy Tripterygium wilfordii Hook F

OBJECTIVE: Triptolide and tripdiolide are thought to be active components of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F, which has been shown to be effective in treating murine lupus nephritis. This study was undertaken to examine the therapeutic effect of triptolide and tripdiolide on established lupus nephritis in (NZB x NZW)F1 mice. METHODS: (NZB x NZW)F1 mice were treated with vehicle, triptolide, or tripdiolide for 15 weeks beginning at the age of 29 weeks (after the development of lupus nephritis). Body weight, proteinuria, and anti-double-stranded DNA (anti-dsDNA) antibodies were monitored, and the kidney and spleen were assessed histologically. Culture supernatants of spleen mononuclear cells were assayed for cytokines. RESULTS: By 28 weeks, most (NZB x NZW)F1 mice had developed lupus nephritis. Vehicle-treated mice exhibited progressive proteinuria, hypoalbuminemia, elevated blood urea nitrogen (BUN) levels, and evidence of severe nephritis. In contrast, proteinuria and BUN levels were significantly reduced in mice treated with either triptolide or tripdiolide as compared with those treated with vehicle. There was no hypoalbuminemia or apparent evidence of lupus nephritis in mice treated with either of the 2 diterpenoids. At 44 weeks of age, the survival rate in mice treated with vehicle (35.7%) was markedly lower than that in mice treated with either triptolide (87.5%) or tripdiolide (88.2%). The mean level of anti-dsDNA antibody in mice treated with tripdiolide was lower than that in the vehicle-treated mice upon completion of the treatment course. Production of tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein 1 by spleen cells was also decreased after diterpenoid therapy. CONCLUSION: Therapy with triptolide or tripdiolide significantly ameliorated lupus nephritis in (NZB x NZW)F1 mice, reduced cytokine and chemokine production, and prolonged survival.     

Dietary fish oil reduces progression of established renal disease in (NZB x NZW)F1 mice and delays renal disease in BXSB and MRL/1 strains

Previous studies have shown that dietary marine lipids containing large quantities of n-3 polyunsaturated fatty acids, administered to (New Zealand black X New Zealand white)F1 and MRL-lpr/lpr mice before the development of renal disease, reduce the severity of glomerulonephritis in mice of these strains. The present study demonstrated that delayed administration of a marine lipid diet, 25% menhaden oil (MO) by weight, until after the onset of overt renal disease, also resulted in significant improvement in rates of mortality, proteinuria, and histologic evidence of glomerular injury, compared with control animals fed a diet that contained mostly saturated fatty acids, 25% beef tallow. The MO diet also reduced the histologic severity of renal disease in male BXSB/MpJ and male MRL-lpr/lpr mice. In contrast, necrotizing vasculitis was more frequent in small and medium-sized renal arteries of the MRL-lpr/lpr mice fed MO than in those fed beef tallow (33.4% versus 7.6%, respectively).     

Prostaglandin E1 treatment of NZB/NZW mice

NZB/NZW F₁ hybrid mice were treated with pharmacologic doses of prostaglandin E₁ (PGE₁) (200 μg subcutaneously either once or twice daily) from 6 through 52 weeks of age. PGE₁-treated mice were protected against development of anemia, clinical nephritis, and death. At 52 weeks, 18 of 19 treated mice were alive, whereas only 2 of 19 untreated control mice were alive. None of the 10 mice treated with PGE₁ twice daily exhibited significant (>2+) proteinuria at 1 year of age. PGE₁ treatment did not prevent development of antibodies to nuclear antigens. The data also suggest that survival of NZB/NZW mice is prolonged when treatment with PGE₁ is begun at 24 weeks, an age at which mice already show evidence of nephritis. Thus all 6 mice treated with PGE₁ (200 μg sc twice daily) from 24 weeks were alive at 52 weeks, whereas only 2 of 6 untreated control mice were alive. The mechanisms whereby PGE₁ treatment influences the course of disease in NZB/NZW mice are not known.     

Calories versus protein in onset of renal disease in NZB x NZW mice.

Autoimmunity-prone (NZB x NZW)F1 (B/W) female mice are used as a model of human lupus erythematosus. When full-fed, these mice die of glomerulonephritis between 7 and 11 (average 9) months of age. When food intake is restricted to 60% of calories, the onset of this disease is delayed and the mice live greatly prolonged lives free of disease. Since high protein intake is commonly associated with acceleration of kidney damage in humans and experimental animals, the current experiments were designed to employ diets in which protein concentration was as high as possible. The observations demonstrate clearly that with this model of autoimmune disease, total calorie intake (from whatever source) exerts an overriding influence on life span. A higher calorie intake leads to early death and restricted-calorie intake leads to an increased life span. When B/W mice are full-fed, with respect to calories, feeding diets of greatly differing protein composition did not influence life span significantly. By contrast, calorie restriction of diets, even of very high protein content or of lower protein content, greatly prolonged life of B/W mice. Even with exceedingly high protein intake (greater than 83% of the calories) it is not protein per se but the total calorie intake that exerts the greatest influence that determines length of life in mice of this autoimmunity- and glomerulonephritis-prone strain.