Characterization of β2-glycoprotein I-dependent and -independent “antiphospholipid” antibodies from lupus-prone NZW/BXSB F1 hybrid male mice

Male (NZW × BXSB)F1 (W/BF1) mice develop a systemic lupus-like syndrome characterized by thrombocytopenia, coronary vascular disease, nephritis, and anticardiolipin antibodies. Three stable hybridoma cell lines secreting monoclonal anticardiolipin antibodies were developed from these mice by fusing their splenic lymphocytes with nonsecreting myeloma cell line, NS-1. Monoclonal antibody A1.17 reacted with cardiolipin in a β₂-Glycoprotein I-dependent manner. The epitope for this antibody consisted of β₂-glycoprotein I bound to cardiolipin or immobilized on plastic plates. Other anionic phospholipid-binding proteins, such as prothrombin or annexin V, had no significant effect in the reactivity of these antibodies. The specificity is similar to the autoimmune anticardiolipin antibodies described in patients with systemic lupus erythematosus and other infectious diseases. In contrast, monoclonal antibodies A1.72 and A1.84 reacted with cardiolipin in the absence of β₂-glycoprotein I. β₂-glycoprotein I, either in the fluid phase or bound to cardiolipin, inhibited the binding of these antibodies. The specificity of the latter two antibodies was similar to that described in patients with syphilis and allied disorders. Both types of antibodies had lupus anticoagulant properties. Thus lupus-prone male (NZW × BXSB)F1 (W/BF1) mice develop both β₂-glycoprotein I-dependent and β₂-glycoprotein I-independent anticardiolipin antibodies. Am. J. Hematol. 56:86–92, 1997. © 1997 Wiley-Liss, Inc.     

Dexamethasone prodrug treatment prevents nephritis in lupus‐prone (NZB × NZW)F1 mice without causing systemic side effects

Objective

To evaluate the potentially improved therapeutic efficacy and safety of nephrotropic macromolecular prodrugs of glucocorticoids (GCs) for the treatment of lupus nephritis.

Methods

Lupus‐prone female (NZB × NZW)F1 mice received monthly injections of N‐(2‐hydroxypropyl) methacrylamide copolymer–based dexamethasone prodrug (P‐Dex) or daily injections of dexamethasone phosphate sodium (Dex; overall dose equivalent to that of P‐Dex) for 2 months. During treatment, the mice were monitored for albuminuria, mean arterial pressure, and serum autoantibody levels. Nephritis, renal immune complex levels, and macrophage infiltration were evaluated histologically. Bone quality was analyzed using peripheral dual x‐ray absorptiometry and micro–computed tomography. The in vivo distribution of P‐Dex was investigated using optical imaging, immunohistochemistry, and fluorescence‐activated cell sorting (FACS). The antiinflammatory effect of P‐Dex was validated using lipopolysaccharide‐activated human proximal tubule epithelial (HK‐2) cells.

Results

Monthly P‐Dex injections completely abolished albuminuria in the (NZB × NZW)F1 mice; this approach was significantly more efficacious than daily Dex treatment. P‐Dex treatment did not reduce serum levels of anti–double‐stranded DNA antibodies or renal immune complexes but did decrease macrophage infiltration, which is a marker of chronic inflammation. Immunohistochemical and FACS analyses revealed that P‐Dex was primarily sequestered by proximal tubule epithelial cells, and that it could attenuate the inflammatory response in HK‐2 cell culture. In contrast to Dex treatment, P‐Dex treatment did not lead to any significant deterioration of bone quality or reduction in the level of total serum IgG.

Conclusion

Macromolecularization of GCs renders them nephrotropic. Protracted retention, subcellular processing, and activation of GC prodrugs by kidney cells would potentiate nephritis resolution, with a reduced risk of systemic toxicities.

     

Protection against renal disease in (NZB × NZW)F1 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 × NZW)F₁ (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 β–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.

     

Autoimmune polyendocrine syndrome type 1 (APS‐1) as a model for understanding autoimmune polyendocrine syndrome type 2 (APS‐2)

Autoimmune polyendocrine syndromes type 1 and 2 (APS‐1 and APS‐2) are diverse in regards to their component diseases and immunologic features of pathogenesis. Animal models and human studies highlight the importance of alleles of HLA (human leukocyte antigen)‐like molecules determining tissue specific targeting that with the loss of tolerance leads to organ specific autoimmunity. Knowledge of the syndromes and component diseases allows clinicians to recognize and prevent illness prior to morbidity. With the current understanding of the syndromes, a paradigm for diagnosis, screening and treatment can be established. Once genetically susceptible individuals are identified screening for autoantibodies can be performed. Amongst autoantibody positive individuals, monitoring for physiologic decompensation, with a goal of treating prior to morbidity and in some cases mortality, follows. With continued basic and clinical research, therapies aimed at treating the underlying autoimmunity and disease prevention should become possible.     

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.     

雌二醇调节树突状细胞功能参与狼疮鼠的发病机制

目的 比较雌二醇(E2)对SLE模型鼠-(NZB×NZW)F1(NZB/w F1)雌鼠发病前后脾脏树突状细胞(DC)的影响,探索雌激素对DC的调节是否和系统性红斑狼疮(SLE)的进展相关.方法 抗CD11c单抗标记的免疫磁珠纯化DC,流式细胞仪检测DC表面分子和细胞因子,混合淋巴细胞反应检测DC对脾脏T细胞的刺激功能,半定量反转录一聚合酶链反应(RT-PCR)法检测DC内雌激素受体(ER)的表达.结果 E2影响狼疮鼠脾脏DC的CD40、白细胞介素(IL)-6、IL-10、IL-12、肿瘤坏死因子(TNF)-α和ERα的表达及刺激活性;枸橼酸他莫西芬(TAM)拈抗E2的作用;E2对不同周龄鼠DC的作用不同.结论 E2通过与DC的ERα结合而调控DC来参与SLE的病理机制,E2对DC的作用和病程进展有关.     

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.

     

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.

     

Interferon‐α treatment of female (NZW × BXSB)F1 mice mimics some but not all features associated with the Yaa mutation

Objective

Male (NZW × BXSB)F₁ mice develop antiphospholipid syndrome (APS) and proliferative glomerulonephritis that is markedly accelerated by the Yaa locus encoding an extra copy of Tlr7. Female (NZW × BXSB)F₁ mice with only 1 active copy of Tlr7 develop late‐onset glomerulonephritis but not APS. Because a major function of Toll‐like receptor 7 is to induce type I interferons (IFNs), our goal was to determine whether IFNα can induce or accelerate the manifestations of systemic lupus erythematosus (SLE) in female (NZW × BXSB)F₁ mice.

Methods

Eight‐week‐old female (NZW × BXSB)F₁ mice were injected with a single dose of adenovirus expressing IFNα. Mice were monitored for the development of thrombocytopenia and proteinuria. Sera were tested for anticardiolipin and anti‐Sm/RNP antibodies. Mice were killed at 17 or 22 weeks of age, and their kidneys and hearts were examined histologically and by immunohistochemistry. Spleen cells were phenotyped, and enzyme‐linked immunospot assays for autoantibody‐producing B cells were performed.

Results

IFNα markedly accelerated nephritis and death in female (NZW × BXSB)F₁ mice. A significant increase in spleen cell numbers associated with a striking increase in the number of activated B and T cells was observed. Marginal‐zone B cells were retained. IFNα‐induced increased titers of autoantibodies were observed, but thrombocytopenia was not observed. Cardiac damage was milder than that in male mice.

Conclusion

IFNα accelerates the development of renal inflammatory disease in female (NZW × BXSB)F₁ mice but induces only mild APS and does not induce thrombocytopenia. The effect of IFNα on SLE disease manifestations is strain dependent. These findings are relevant to our understanding of the physiologic significance of the IFN signature.

     

Inhibition of lupus disease by anti–double‐stranded DNA antibodies of the IgM isotype in the (NZB × 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 × NZW)F₁ mice (females) were treated with murine monoclonal IgM anti‐dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 μg/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 Fcγ 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 × NZW)F₁ 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.

     

慢性乙型肝炎、乙肝肝硬化、乙肝肝癌患者Th1/Th2型细胞因子水平变化研究

目的分析慢性乙型肝炎(CHB)、乙肝肝硬化(LC)、乙肝肝癌(HCC)患者血清中Th1/Th2型细胞因子水平变化,为慢性乙型肝炎至肝癌的发生发展过程中的免疫变化研究提供线索,并为患者的临床治疗研究提供免疫学指标。方法选取2010年2月-2011年11月于首都医科大学附属北京佑安医院就诊的40例CHB患者、40例LC患者、53例HCC患者,用Luminex技术检测血清中Th1类细胞因子[白细胞介素(interleukin,IL)-12、干扰素(interferon,IFN)-γ和肿瘤坏死因子(tumor necrosis factor,TNF)-α]水平及Th2类细胞因子(IL-4、IL-6和IL-10)水平,并将25例健康志愿者作为正常对照组进行比较。结果除TNF-α外,CHB组、LC组和HCC组Th1类IL-12、IFN-γ和Th2类IL-4、IL-6、IL-10细胞因子水平均低于正常对照组;CHB组中大部分Th1类和Th2类细胞因子都高于LC组和HCC组;HCC组中TNF-α细胞因子水平要高于CHB组、LC组。结论 CHB、LC和HCC患者体内Th1/Th2细胞因子分泌水平受到抑制,Th1/Th2平衡发生漂移,对HBV病毒的清除作用受到抑制。TNF-α在肝癌发生过程中发挥着重要作用。     

Role of F4/80+Mac‐1high adherent non‐parenchymal liver cells in concanavalin A‐induced hepatic injury in mice

Aim: Non‐parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80⁺Mac‐1^high+ cells were involved in the regulative feedback‐modulated regulation of inflammatory responses during concanavalin A (Con A)‐induced hepatitis. Methods: Hepatitis was induced in BALB/c mice by the intravenous injection of Con A. Liver injury was assessed using serum aminotransferase and pathology. The function of NPLC was assessed by FACS analysis. Accessory cell function of adherent Con A NPLC was performed with an ovalbumin specific T‐helper 1 (Th1) clone proliferation assay. The culture supernatant nitric oxide (NO) content was quantified by the Griess reaction. Inducible NO synthase (iNOS) expression was demonstrated by immunohistochemistry and Western blot analysis. Results: The number of hepatic F4/80⁺Mac‐1^high+cells increased in a time‐dependent manner after Con A administration, which consequently suppressed Th1 cell proliferation by a mechanism likely to involve NO. The iNOS expression of NPLC was elevated at 24 h post‐Con A injection. In nude mice, F4/80⁺Mac‐1^high+cells did not increase in the Con A‐treated liver; the NPLC did not suppress Th1 clone proliferation. Conclusion: These findings suggest that the in vivo activation of F4/80⁺Mac‐1^high+cells by Con A administration suppresses Th1 cell proliferation by increasing NO, and subsequently reducing liver injury.     

Anti-Thy-1 treated and irradiated spleen cells from (BALB/cXC57Bl/6) F1 mice infected with Plasmodium chabaudi chabaudi can transfer protection into irradiated hosts

The transfer of spleen cells from (BALB/cXC57Bl/6) F1 mice recovered from a Plasmodium chabaudi chabaudi AS infection into irradiated syngeneic recipients conferred protection. Neither elimination of Thy-1⁺ cells nor in vitro irradiation of immune cells before transfer affected protection while both anti-Thy-1 treatment and irradiation abolished the appearance of anti-P. c. chabaudi antibodies in the recipients. Superinfection of immune spleen cell donors did not improve their capability to transfer protection which was also unaffected by anti-Thy-1 treatment. The serum of mice after one infection was only marginally protective when transferred into irradiated recipients and a second infection improved the protective activity of serum which was not further improved by six infections. The cotransfer of immune serum and immune cells did not result in any synergistic effect. On the other hand, when P. c. chabaudi AS (BALB/cXC57B1/6)F1 infected mice were challenged with a high dose of Plasmodium yoelii 17XL at crisis, the mice were unable to control the heterologous parasite. When mice were challenged with P. yoelii 17XL several weeks after infection with P.c. chabaudi AS, a good degree of cross-protection was observed.     

The influence of the site of parasite inoculation on the development of Th1 and Th2 type immune responses in (BALB/c × C57BL/6) F1 mice infected with Leishmania major

Although inbred strains of mice are classified as genetically resistant or susceptible to Leishmania major based upon their ability to control infection, other factors such as the strain, dose, and site of parasite inoculation can also affect the outcome of the disease. Here we used the Fl progeny of BALB/c (susceptible) and C57BL/6 (resistant) mice (designated CB6F1) to investigate whether mice or intermediate susceptibility to infection differed from the parental strains in their ability to control infections at different cutaneous sites. CB6F1 mice developed progressive disease when inoculated in the dorsal skin, but healed infections in the footpad. Consistent with these observations, mice inoculated in the footpad ultimately developed Th1 responses, known to be required for healing, while Th2 responses developed in mice inoculated in the dorsal skin. However, IL-4 and IFN-γ production during the first few weeks of infection was similar in CB6F1 mice inoculated at either site, suggesting that factors in addition to the relative levels of these cytokines produced early in infection may influence the nature of the antileishmanial immune response, and the eventual disease outcome. Infection in CB6F1 mice provides a model for the study of immunity to L. major in genetically identical animals, in which a prolonged mixed Thl/Th2 cytokine pattern initially develops, but ultimately diverges into more defined Th1 and Thl type responses.