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.

     

An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus

OBJECTIVE: To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)-dependent signaling, could modulate disease in lupus-prone (NZB x NZW)F1 (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. METHODS: R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24-34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. RESULTS: When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. CONCLUSION: We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.     

A peptide derived from an autoantibody can stimulate t cells in the (nzb × nzw)f1 mouse model of systemic lupus erythematosus

Objective. To assess the ability of peptides derived from anti-DNA to stimulate syngeneic T lymphocytes and influence lupus in (NZB × NZW)F₁ (NZB/W) mice. Methods. We synthesized (by Geysen pin method) overlapping 12-mer peptides recapitulating the amino acid sequence of the V_H region of a nephritogenic monoclonal IgG2a anti-DNA antibody (A6.1) from an NZB/W mouse. Splenic T cells were cultured with the peptides; multiple experiments assayed 12-mer and 16-mer peptides which contained a triple-base motif (KFKGK). We immunized 20-week-old NZB/W mice with the 12-mer and evaluated them for evidence of nephritis and for quantities of antibodies in plasma and glomeruli. Results. Three clusters of peptides caused proliferation of spleen cells from young, nonimmunized mice. Both the 12-mer FYNQKEKGKATL and the 16-mer GDTFYNQKEKGKATLT peptides stimulated purified T cells. The KXKXK motif occurs in 15% of murine IgV_H (NBRF protein database), compared with 100% (6 of 6) of NZB/W anti-DNA monoclonal antibody. Immunization with the 12-mer peptide increased plasma levels of IgG, anti-DNA, and immune complexes, and levels of anti-DNA in glomeruli; nephritis was accelerated. Conclusion. NZB/W anti-DNA contain peptide sequences in their V_H regions that stimulate self—T cells. At least one motif is frequent in NZB/W anti-DNA. If some activated T cells provide help, this mechanism may contribute to sustained up-regulation of autoantibodies in murine lupus.     

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

OBJECTIVE: This study examined the interactions between exogenous and endogenous factors shaping the phenotype of lupus in autoimmune (NZB x NZW)F(1) 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.     

Prevention of renal damage in murine lupus nephritis by CTLA-4Ig and cyclophosphamide

OBJECTIVE: To compare the effects of combined administration of cyclophosphamide (CYC) and CTLA-4Ig with the effects of these agents alone on the immunopathology and progression of renal damage in (NZB x NZW)F(1) (B/W) lupus-prone mice, and to explore the clinical implications of this combination by evaluating the ability of CTLA-4Ig to sustain the benefit of CYC in patients with lupus nephritis. METHODS: We carried out a detailed, prospective pathologic and immunohistochemical analysis of the effects of CYC and CTLA-4Ig, alone and in combination, in kidney tissue from B/W mice. The acute effects of these agents on immune cells in the kidney were evaluated by fluorescence-activated cell sorting. We also compared the effect of brief CYC plus sustained CTLA-4Ig administration with the effect of sustained administration of both agents on the progression of renal disease in B/W mice. RESULTS: As a single agent, CTLA-4Ig was generally as effective, and in some cases more effective, than CYC in slowing progression of renal disease. Combined therapy with these two agents very effectively arrested the progression of renal damage and, in some respects, reversed renal pathology. Induction therapy with both CTLA-4Ig and CYC precluded the need for continuous administration of CYC. CONCLUSION: Our results indicate that the combination of CTLA-4Ig and CYC very effectively arrests the progression of murine lupus nephritis. These findings have direct implications for the treatment of lupus nephritis.     

Prevention and reversal of lupus in NZB/NZW mice by costimulatory blockade with adeno-associated virus-mediated gene transfer

OBJECTIVE: To investigate the potency of costimulatory blockade with adeno-associated virus (AAV)-mediated gene transfer in the prevention and reversal of lupus in a murine model. METHODS: AAV vectors expressing CTLA-4Ig or CD40Ig were injected into NZB/NZW mice. Serum levels of transgene expression and autoantibody titers were determined by enzyme-linked immunosorbent assay. The therapeutic effects on proteinuria, renal pathologic features, and survival rate were evaluated. Splenic T cell phenotypes were analyzed by flow cytometry. The humoral immune response to a foreign antigen was also examined in treated mice. RESULTS: A single injection of AAV serotype 8 (AAV8)-CTLA-4Ig in neonatal NZB/NZW mice before the onset of lupus effectively delayed and inhibited autoantibody production, proteinuria, and kidney damage and prolonged their lifespan. In addition, coinjection of AAV8-CTLA-4Ig and AAV8-CD40Ig vectors into neonatal mice achieved a synergistic effect and the best efficacy. The preventive effects were attributed to suppression of CD4+ T cell activation and the transition from naive to memory T cells. Moreover, coinjection of these 2 vectors in adult mice reversed the existing autoantibody levels, suppressed the development of proteinuria, and prolonged their lifespan. The therapeutic effects were found to be dependent on the vector dose. In addition, AAV-mediated long-term gene expression did not severely suppress the host humoral response to foreign antigen. CONCLUSION: Our findings show that delivery of costimulatory inhibitor transgenes by AAV vectors could prevent and reverse lupus in this murine model, suggesting the potential of AAV-mediated gene transfer as an alternative treatment for lupus.     

Enhanced susceptibility to end-organ disease in the lupus-facilitating NZW mouse strain

OBJECTIVE: Although the NZW mouse strain is phenotypically normal, fulminant lupus glomerulonephritis (GN) develops when NZW mice are bred to several other strains, such as NZB, BXSB, B6.Sle1, and B6.Yaa. Based on the observation that aging NZW mice exhibit histologic evidence of GN, we sought to test our hypothesis that NZW mice may be more susceptible to immune-mediated renal damage. METHODS: NZW mice, as well as C57BL/6 (B6) and BALB/c control mice, were challenged with rabbit anti-glomerular basement membrane nephrotoxic sera (NTS), to induce renal disease. The different mouse strains were monitored for the degree of clinical disease, renal pathology, chemokine profiles, and cellular infiltrates. RESULTS: Although the NZW and control strains showed similar glomerular deposits of rabbit Ig and exhibited similar levels of anti-rabbit xenogeneic immune response, the NZW mice had significantly worse pathologic changes and disease. Compared with the control strains, the NTS-injected NZW mice demonstrated significantly increased proteinuria, elevated blood urea nitrogen levels, more severe histologic GN and tubulointerstitial nephritis, increased glomerular crescent formation with macrophage and neutrophil infiltrates, elevated expression of CC and CXC chemokines (monocyte chemoattractant protein 1, RANTES, KC), and significantly accelerated mortality. Importantly, these changes occurred within a few days after NTS administration. Finally, (B6 x NZW)F(1) mice were as susceptible as the NZW parents, which indicates dominant NZW contributions. CONCLUSION: Collectively, these findings support the notion that a lupus-facilitating genome may contribute to disease susceptibility by modulating the degree of immune-mediated end-organ damage. The availability of B6-based congenic strains bearing individual NZW-derived lupus susceptibility loci will permit future genetic dissection of end-organ susceptibility in murine lupus.     

Examining the role of CD1d and natural killer T cells in the development of nephritis in a genetically susceptible lupus model

OBJECTIVE: CD1d-reactive invariant natural killer T (iNKT) cells secrete multiple cytokines upon T cell receptor (TCR) engagement and modulate many immune-mediated conditions. The purpose of this study was to examine the role of these cells in the development of autoimmune disease in genetically lupus-prone (NZBxNZW)F1 (BWF1) mice. METHODS: The CD1d1-null genotype was crossed onto the NZB and NZW backgrounds to establish CD1d1-knockout (CD1d0) BWF1 mice. CD1d0 mice and their wild-type littermates were monitored for the development of nephritis and assessed for cytokine responses to CD1d-restricted glycolipid alpha-galactosylceramide (alphaGalCer), anti-CD3 antibody, and concanavalin A (Con A). Thymus and spleen cells were stained with CD1d tetramers that had been loaded with alphaGalCer or its analog PBS-57 to detect iNKT cells, and the cells were compared between BWF1 mice and class II major histocompatibility complex-matched nonautoimmune strains, including BALB/c, (BALB/cxNZW)F1 (CWF1), and NZW. RESULTS: CD1d0 BWF1 mice had more severe nephritis than did their wild-type littermates. Although iNKT cells and iNKT cell responses were absent in CD1d0 BWF1 mice, the CD1d0 mice continued to have significant numbers of interferon-gamma-producing NKT-like (CD1d-independent TCRbeta+,NK1.1+ and/or DX5+) cells. CD1d deficiency also influenced cytokine responses by conventional T cells: upon in vitro stimulation of splenocytes with Con A or anti-CD3, type 2 cytokine levels were reduced, whereas type 1 cytokine levels were increased or unchanged in CD1d0 mice as compared with their wild-type littermates. Additionally, numbers of thymic iNKT cells were lower in young wild-type BWF1 mice than in nonautoimmune strains. CONCLUSION: Germline deletion of CD1d exacerbates lupus in BWF1 mice. This finding, together with reduced thymic iNKT cells in young BWF1 mice as compared with nonautoimmune strains, implies a regulatory role of CD1d and iNKT cells during the development of lupus.     

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.     

Strong acceleration of murine lupus by injection of the SmD1(83-119) peptide

OBJECTIVE: The mechanisms of IgG anti-double-stranded DNA (anti-dsDNA) antibody induction are incompletely understood. We recently demonstrated a high prevalence of autoantibodies to the C-terminus of SmD1 in patients with systemic lupus erythematosus (SLE) that was closely associated with anti-dsDNA reactivity. The aim of the present study was to analyze the influence of the SmD1 C-terminus on the generation of pathogenic anti-dsDNA antibodies in a murine model of SLE. METHODS: Female lupus-prone prenephritic (NZB x NZW)F1 mice (NZB/NZW mice) as well as female control BALB/c, NZW, and (BALB/c x NZW)F, mice (CWF1 mice) were subcutaneously injected with keyhole limpet hemocyanin (KLH)-coupled SmD1(83-119). Controls received injections of recombinant SmD1 (rSmD1), KLH-rSmD1, KLH-coupled randomized peptide of SmD1(83-119), ovalbumin, or saline. Animals were monitored for survival and proteinuria and for levels of plasma creatinine, urea, and autoantibodies. In addition, histologic examinations were performed and T cell responses against SmD1(83-119) peptide and rSmD1 protein were determined in SmD1(83-119)-treated and -untreated NZB/NZW mice. RESULTS: Immunization with KLH-SmD1(83-119), but not with control peptide, significantly accelerated the natural course of lupus in NZB/NZW mice, with premature renal failure and increased development of anti-dsDNA antibodies. Control strains of mice remained healthy, with no relevant anti-SmD1(83-119) antibodies detectable even after immunization. In contrast to findings in control mice, a T cell response against SmD1(83-119) was already present in unmanipulated NZB/NZW mice, and this response was further amplified after immunization. CONCLUSION: The SmD1(83-119) peptide can influence the pathogenic anti-dsDNA response in the NZB/NZW murine lupus model. The data suggest that an SmD1(83-119)-specific T cell response is critical. Therefore, modulation of these autoantigen-specific T cells by tolerance induction may provide a therapeutic approach to specific immunosuppression in lupus.     

CD19 hyperexpression augments Sle1-induced humoral autoimmunity but not clinical nephritis

OBJECTIVE: B cell hyperactivity is a common denominator in murine and human systemic lupus erythematosus. Some susceptibility genes in lupus are associated with B cell hyperactivity, but others are clearly not. While the Sle1 lupus susceptibility locus of NZM2410/NZW origin leads to chromatin-focused autoimmunity, genetically engineered overexpression of CD19 leads to "generalized" B cell hyperactivity. We undertook this study to determine the degree to which generalized B cell hyperactivity can amplify lupus pathogenesis. METHODS: To elucidate the impact of generalized B cell hyperactivity on Sle1-triggered autoimmunity, B6 mice bearing the human CD19 transgene were rendered congenic for the Sle1(z) genetic locus and phenotyped for serologic, cellular, and pathologic evidence of lupus. RESULTS: As expected, B6.Sle1.hCD19(Tg/Tg) mice, homozygous at Sle1 and bearing the hCD19 transgene, exhibited high levels of IgM and IgG anti-DNA/antiglomerular autoantibodies, skewed B cell subsets, and profoundly activated B and T cells. Despite exhibiting glomerular IgM, IgG, and complement deposits, these mice did not exhibit accelerated mortality or any clinical evidence of renal dysfunction. CONCLUSION: Generalized B cell hyperactivity may augment humoral autoimmunity, but this may not suffice to engender end-organ disease in lupus. These findings allude to the presence of an additional distal checkpoint that dissociates pathogenic autoantibody formation and renal immunoglobulin deposition from the progression to clinical nephritis in lupus.     

Disparate effects of depletion of CD1d-reactive T cells during early versus late stages of disease in a genetically susceptible model of lupus

Some T cells react with lipid antigens bound to antigen-presenting molecule CD1d. Numbers and functions of a subset of such lipid-reactive T cells are reduced in patients with systemic lupus erythematosus (SLE) and their relatives, as well as in genetically susceptible and chemically induced animal models of lupus-like disease. We have reported that the germline deletion of CD1d exacerbates lupus, suggesting a protective role of these cells in the development of lupus. The use of a knockout mouse model in this study, however, did not allow examination of the role of these cells at different stages of disease. Here, we describe an approach to deplete CD1d-dependent T cells, which allowed us to investigate the role of these cells at different stages of disease in genetically lupus-prone NZB/NZW F1 (BWF1) mice. Repeated intravenous injections of large numbers of CD1d-transfected cells resulted in ～50-75% reduction in these cells, as defined by the expression of CD4, NK1.1 and CD122, and lack of expression of CD62 ligand. TCR γδ (+)NK1.1(+) cells were also reduced in the recipients of CD1d-transfected cells as compared with control recipients. Such depletion of CD1d-reactive T cells in preclinical BWF1 mice resulted in disease acceleration with a significant increase in proteinuria and mortality. In older BWF1 mice having advanced nephritis, however, such depletion of CD1d-reactive T cells resulted in some disease improvement. Taken together, these data as well as our published studies suggest that CD1d-reactive T cells protect against the development of lupus in animal models. However, these cells appear to be unable to suppress established lupus nephritis in these animals, and might even play a disease aggravating role in late stages of disease.     

新西兰黑鼠肾脏H2区域与蛋白尿强度的相关性探讨

目的探讨狼疮性肾炎的发生机制。方法取新西兰黑鼠，建立NZB（雌鼠）×NZW（雄鼠）F1×NZW回交鼠（以下简称回交鼠）模型，以尿蛋白强度为肾脏损伤标记，进行基于微卫星遗传标记的数量性状位点（QTL）分析，寻找可能与NZB第17号染色体有关的狼疮性肾炎易感区域。结果QTL分析发现NZB第17号染色体H2区域的D17Mit22与蛋白尿的发生存在连锁关系（LOD〉3．3）；携有该位点的回交鼠的尿蛋白强度最高（P〈0．005）。结论NZB第17号染色体的H2区域可能是诱导回交鼠狼疮性肾炎的易感基因位点。     

Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F₁ mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly, in vitro stimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fas^high and highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Fas^low and apoptosis-resistant. The Fas^low cells were included in the CD5⁺ B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F₁ mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fas in vivo and were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.     

Prevention of renal damage in murine lupus nephritis by CTLA‐4Ig and cyclophosphamide

Objective

To compare the effects of combined administration of cyclophosphamide (CYC) and CTLA‐4Ig with the effects of these agents alone on the immunopathology and progression of renal damage in (NZB × NZW)F₁ (B/W) lupus‐prone mice, and to explore the clinical implications of this combination by evaluating the ability of CTLA‐4Ig to sustain the benefit of CYC in patients with lupus nephritis.

Methods

We carried out a detailed, prospective pathologic and immunohistochemical analysis of the effects of CYC and CTLA‐4Ig, alone and in combination, in kidney tissue from B/W mice. The acute effects of these agents on immune cells in the kidney were evaluated by fluorescence‐activated cell sorting. We also compared the effect of brief CYC plus sustained CTLA‐4Ig administration with the effect of sustained administration of both agents on the progression of renal disease in B/W mice.

Results

As a single agent, CTLA‐4Ig was generally as effective, and in some cases more effective, than CYC in slowing progression of renal disease. Combined therapy with these two agents very effectively arrested the progression of renal damage and, in some respects, reversed renal pathology. Induction therapy with both CTLA‐4Ig and CYC precluded the need for continuous administration of CYC.

Conclusion

Our results indicate that the combination of CTLA‐4Ig and CYC very effectively arrests the progression of murine lupus nephritis. These findings have direct implications for the treatment of lupus nephritis.

     

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.     

Anti-DNA Ig peptides promote Treg cell activity in systemic lupus erythematosus patients

OBJECTIVE: Treg cells oppose autoreactive responses in several autoimmune diseases, and their frequency is reduced in systemic lupus erythematosus (SLE). In murine lupus models, treatment with anti-DNA Ig-based peptides can expand the number of Treg cells in vivo. This study was undertaken to test the possibility that functional human Treg cells can be induced by exposure to anti-DNA Ig-based peptides. METHODS: Peripheral blood mononuclear cells were isolated from 36 lupus patients and 32 healthy individuals matched for ethnicity, sex, and age. Short-term culture experiments in the presence of several independent stimuli including anti-DNA Ig peptides were followed by flow cytometric analysis for identification of CD4+,CD25(high) T cells, cell sorting for in vitro suppression assays, and analysis of correlations between the expression of forkhead box P3 (FoxP3) and serologic and clinical characteristics of the SLE patients. RESULTS: The number of in vitro CD4+,CD25(high) T cells increased after culture with anti-DNA Ig peptides in the SLE patients, but not in the controls. The expanded CD4+,CD25(high) T cells required FoxP3 for cell contact-mediated suppression of proliferation and interferon-gamma production in target CD4+,CD25- T cells. The induction of FoxP3 in SLE Treg cells occurred only in seropositive patients, and was correlated with anti-DNA and IgG serum titers. CONCLUSION: These results suggest a new modality to reverse the functional deficit of Treg cells in SLE patients with positive autoimmune serology, and identify a new strategy to enhance immunoregulatory T cell activity in human SLE.     

Alteration of lymphocyte function in NZB/NZW mice. IV. Response to levamisole.

NZB/NZW F1 female mice received levamisole in an attempt to increase suppressor T cell function. Responses varied with dosage of drug and age of mice. When treatment with 25 microgram/gm doses (intermittent or daily) was begun at 12 weeks of age, nephritis was accelerated in spite of transient reduction of anti-DNA antibodies. When treatment was begun at 4 weeks, or when doses of 250 microgram/gm were given, no clinical effects were observed. In the mice with accelerated disease, delayed hypersensitivity and antibody responses to sheep erythrocytes increased; antibody responses to pneumococcal polysaccharide were unaffected. It is possible that some increased immune responses associated with levamisole are undesirable in murine lupus.     

Cyclin‐dependent kinase inhibition limits glomerulonephritis and extends lifespan of mice with systemic lupus

Objective

To examine whether the cyclin‐dependent kinase (CDK) inhibitor seliciclib ameliorates autoimmune nephritis in (NZB × NZW)F₁ mice.

Methods

In experiment 1, NZB × NZW mice received seliciclib (100 mg/kg or 200 mg/kg) or vehicle by gavage, beginning at age 2 months and ending at 8 months of age. In experiment 2, seliciclib (200 mg/kg) was administered alone or combined with low‐dose methylprednisolone, starting at age 5 months, when immune complex deposition in the kidney had already occurred. Animals were followed up until all vehicle‐treated mice died. In 2 additional groups of NZB × NZW mice treated with seliciclib or vehicle from 2 months of age until 5 months of age, splenocytes were isolated and tested ex vivo for T cell and B cell activity.

Results

Seliciclib, given at an early phase of disease, prolonged survival, delayed the onset of proteinuria and renal function impairment, and protected the kidney against glomerular hypercellularity, tubulointerstitial damage, and inflammation. Combining seliciclib with low‐dose methylprednisolone in mice with established disease extended the lifespan and limited proteinuria and renal damage more than treatment with either agent alone. Seliciclib limited immunologic signs of disease, reducing glomerular IgG and C3 deposits and levels of serum anti‐DNA antibodies. Moreover, it inhibited ex vivo T cell and B cell proliferative responses to polyclonal stimuli. T cell production of interferon‐γ and interleukin‐10 and B cell release of IgG2a were reduced by treatment with seliciclib.

Conclusion

These findings suggest that CDK activity may be a useful target in the treatment of systemic lupus erythematosus. A direct immunomodulatory action of seliciclib on T cells and B cells may be one of the mechanisms underlying the beneficial effects.