Control MRL Research Articles

Benzo[a]pyrene exposure reduces cell-type diversity in lupus-prone mice

Abstract Studies indicate that genetic factors only account for approximately thirty percent of all autoimmune diseases, while the rest of autoimmune pathogenesis is attributed to environmental factors including toxic chemicals, dietary components, and infections. To understand if and how toxic chemicals trigger autoimmunity, we investigated the effect of benzo[a]pyrene (BaP) exposure on the development of autoimmune phenotypes in the lupus-prone MRL/MpJ strain. Since BaP is a polycyclic aromatic hydrocarbon commonly found in byproducts of industrial incineration, motor vehicle emissions and cigarette smoke, we subjected mice to skin plus lung exposure to BaP over the course of 8 weeks before lupus onset. BaP treatment resulted in total body weight loss in male mice, while marginal changes in anti-dsDNA levels occurred. Multi-organ analyses of BaP-treated and control MRL mice suggested that kidney is a major organ directly affected by the metabolism of benzene-containing compounds, with increased expression of BaP-target genes including Cyp4b1 and Hao2. Intriguingly, spatial transcriptomic data showed that BaP caused drastic reduction in cell type diversity in both kidney and spleen of MRL mice. In addition, BaP exposure triggered C3 deposit in a more severe manner in the kidney of male MRL mice compared to female. Collectively, our results show that BaP exposure reduces cell type diversity in lupus-prone mice before disease onset, and stimulates sex-biased molecular pathways which may help explain the increased likelihood of end organ damage in males with lupus.

Benzo[a]pyrene Exposure Reduces Cell-Type Diversity and Stimulates Sex-Biased Damage Pathways in End Organs of Lupus-Prone Mice.

Studies indicate that genetic factors only account for approximately thirty percent of all autoimmune diseases, while the rest of autoimmune pathogenesis is attributed to environmental factors including toxic chemicals. To understand if and how environmental pollutants trigger autoimmunity, we investigated the effect of benzo[a]pyrene (BaP) exposure on the development of autoimmune phenotypes in the lupus-prone MRL strain. The exposure of MRL mice to BaP over the course of 8 weeks before lupus onset resulted in total body weight loss in males, while marginal changes in anti-dsDNA levels occurred. Multi-organ analyses of BaP-treated and control MRL mice suggested that the kidney is a major organ directly affected by the metabolism of benzene-containing compounds, with increased expression of BaP-target genes including Cyp4b1 and Hao2. Intriguingly, spatial transcriptomic data showed that BaP caused a drastic reduction in cell-type diversity in both the kidneys and spleen of MRL mice. Further analysis of the molecular pathways affected suggested a sex-biased effect of BaP treatment, with the upregulated expression of angiogenesis genes in the lungs and an increased deposition of C3 in the kidneys of male mice. While SLE is more common in women, the disease is more severe in male patients, with an increased risk of disease progression to renal failure and lung cancer. Our results reveal sex-biased molecular pathways stimulated by BaP which may help explain the increased likelihood of end organ damage in males with lupus.

Iron Status Alters Murine Systemic Lupus Erythematosus

The effect of Fe status on murine systemic lupus erythematosus was investigated. Weanling female MRL/MPJ-lpr/lpr mice (systemic lupus erythematosus strain) were fed diets with the following levels (mg Fe/kg diet): 3 (severely deficient), 10 (moderately deficient), 35 (control) and 250 (supplemented). A fifth group was pair fed the control diet in the amounts consumed by the severely deficient group. C3H/Hej mice fed the same diets were used as nonlupus controls. Anemia was more severe in severely deficient mice than in all other MRL groups and C3H severely deficient mice. Incidence of skin lesions was highest in MRL severely and moderately deficient mice compared with pair-fed, control and supplemented mice. By 22 wk of age, mortality was higher in supplemented and severely deficient mice than in moderately deficient, pair-fed and control MRL mice. Anti-dsDNA activity in serum was not altered by Fe. In a second experiment, kidney function was examined in mice fed severely deficient, control, supplemented and pair-fed diets. Urine protein concentration was highest in supplemented mice at 14 wk of age. Serum urea nitrogen was significantly higher in MRL severely deficient mice than in pair-fed and control mice at 18 wk of age. Glomerular filtration rate, measured by creatinine clearance, was significantly lower in MRL severely deficient mice than in pair-fed and Fe supplemented mice at 16 wk of age and pair-fed and control mice at 18 wk of age. Renal histopathology was more severe in Fe supplemented mice than in pair-fed and control mice, and more severe in severely deficient and pair-fed mice than in control mice. Fluorescent staining of kidneys with anti-Ig G and anti-C3 fluorescein-conjugated antibodies was most intense in severely deficient mice, and the concentration of circulating immune complexes in serum was significantly higher in severely deficient mice than in all other groups. These data demonstrate that systemic lupus erythematosus in MRL/MPJ-lpr/lpr mice is altered by dietary iron.

Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Faslpr/lpr Mice.

Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFNγ), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2+ lymphocytes (miR-182−/−B6/lpr and miR-183C−/-B6/lpr). The miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/−B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183Cfl/flB6/lpr controls. In vitro activated splenocytes from miR-182−/−B6/lpr mice and miR-183C−/−B6/lpr mice showed reduced ability to produce lupus-associated IFNγ. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4+ cells of miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182−/−B6/lpr and miR-183C−/-B6/lpr mice significantly increased IFNγ expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFNγ in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4+, CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells in the miR-183C−/−B6/lpr and miR-182−/−B6/lpr mice and their respective littermate controls, miR-183Cfl/flB6/lpr and miR-182fl/flB6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFNγ in in vitro activated splenocytes from B6/lpr mice.

EGR2 is elevated and positively regulates inflammatory IFN\u03b3 production in lupus CD4+ T cells

BackgroundRecent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly.ResultsWe performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells.ConclusionsEGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.

Similar dysregulation of lupus-associated miRNAs in peripheral blood mononuclear cells and splenic lymphocytes in MRL/lpr mice

ObjectiveMicroRNAs (miRNAs) play an important role in the pathogenesis of various autoimmune diseases including systemic lupus erythematosus (SLE; lupus). We have previously reported a common pattern of miRNA dysregulation in...

Sustained Immunosuppression Alters Olfactory Function in the MRL Model of CNS Lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is frequently accompanied by diverse neuropsychiatric manifestations. An increased frequency of olfactory deficits has been recently reported as another marker of CNS involvement in SLE patients. Similarly, we observed that spontaneous development of lupus-like disease in MRL/lpr mice is accompanied by altered olfaction-related behaviors. However, it remained unclear whether the behavioral deficits are due to systemic autoimmunity, or the distinct genetic make-up. To address this question, we presently examine whether prolonged treatment with the immunosuppressive drug cyclophosphamide (CY) restores odor-guided behaviors in MRL/lpr mice. Over 12 weekends, MRL/lpr and control MRL +/+ males were given ad lib access to a sweetened CY solution or a vehicle. Their responsiveness to different scents was assessed at ages corresponding to mild, modest, and severe disease. Odor-guided exploratory behavior was further examined in the novel object test at 21weeks of age, shortly before terminal assessment of immunopathology. In comparison to control groups, MRL/lpr mice exposed to CY exhibited normal spleen size and antibody levels, as well as increased responsiveness to an attractant and a novel object. However, CY treatment also exacerbated their aberrant response to a repellent, suggesting a dual mode of action on brain olfactory systems. The present results reveal that generalized immunosuppression modulates odor-guided behaviors in lupus-prone animals. Although key pathogenic mechanisms are not clear, the findings strengthen the construct validity of the MRL model by supporting the hypothesis that onset of systemic autoimmunity alters the activity of olfactory circuits.

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation.

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4+ T, CD19+ B, and splenic CD4-CD19- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19+ and CD4-CD19- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.

Neuropsychiatric lupus occurs independently of systemic autoimmunity (BA6P.120)

Abstract Neuropsychiatric disease is a common manifestation of systemic lupus erythematosus (SLE). Potential mechanisms underlying neuropsychiatric SLE (NPSLE) include thrombosis, complement deposition, brain-reactive autoantibodies, and cytokine mediated inflammation. However, it is not clear whether the neuropsychiatric manifestations of SLE are secondary to systemic disease, or a primary pathogenic process in the brain. In order to distinguish between central nervous system (CNS) and hematopoietic contributions, we generated bone marrow chimeras between lupus-prone MRL/lpr and congenic control MRL/+ mice. We monitored systemic disease progression, performed extensive behavioral testing, and analyzed brain tissue for evidence of inflammation. MRL/lpr (donor) → MRL/lpr (recipient) chimeric mice showed significantly elevated autoantibodies and marked proteinuria while MRL/+→MRL/+ and MRL/+→MRL/lpr did not, indicating abrogation of the MRL/lpr systemic disease phenotype. Surprisingly, MRL/+→MRL/lpr mice displayed a behavioral phenotype consistent with MRL/lpr→MRL/lpr mice, including depression-like behavior and increased spatial memory deficits. In contrast, MRL/+→MRL/+ mice behaved normally. Additionally, there was elevated RANTES expression, cellular infiltration, and neurodegeneration in MRL/lpr→MRL/lpr and MRL/+→MRL/lpr chimeric mice. The data presented herein indicate that the MRL/lpr CNS is sufficient to mediate NPSLE development absent significant hematopoietic contribution.

Effects of prolonged treatment with memantine in the MRL model of central nervous system lupus

Neuropsychiatric manifestations and brain atrophy of unknown etiology are common and severe complications of systemic lupus erythematosus (SLE). An autoantibody that binds to N-methyl-D-aspartate (NMDA) receptor NR2 has been proposed as a key factor in the etiology of central nervous system (CNS) SLE. This hypothesis was supported by evidence suggesting memantine (MEM), an uncompetitive NMDA receptor antagonist, prevents behavioral dysfunction and brain pathology in healthy mice immunized with a peptide similar to an epitope on the NR2 receptor. Given that SLE is a chronic condition, we presently examine the effects of MEM in MRL/lpr mice, which develop behavioral deficits alongside SLE-like disease. A broad behavioral battery and 7-Tesla MRI were used to examine whether prolonged treatment with MEM (~25 mg/kg b.w. in drinking water) prevents CNS involvement in this spontaneous model of SLE. Although MEM increased novel object exploration in MRL/lpr mice, it did not show other beneficial, substrain-specific effects. Conversely, MEM was detrimental to spontaneous activity in control MRL +/+ mice and had a negative effect on body mass gain. Similarly, MRI revealed comparable increases in the volume of periventricular structures in MEM-treated groups. Sustained exposure to MEM affects body growth, brain morphology, and behavior primarily by pharmacological, and not autoimmunity-dependant mechanisms. Substrain-specific improvement in exploratory behavior of MEM-treated MRL/lpr mice may indicate that the NMDA system is merely a constituent of a complex pathogenenic cascade. However, it was evident that chronic administration of MEM is unable to completely prevent the development of a CNS SLE-like syndrome.

Potential roles for soluble MD-1 in disease progression of autoimmune prone MRLlpr/lpr mice (171.2)

Abstract MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) on B cells, macrophages and dendritic cells. This complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRLlpr/lpr mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor 4. We have now established a flow cytometry-based assay to detect endogenous soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRLlpr/lpr mice had higher levels of serum sMD-1 than control MRL+/+ mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRLlpr/lpr mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRLlpr/lpr kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model mouse. Also, sMD-1 may be feasible to monitor autoimmune disease severity.

Serum soluble MD-1 levels increase with disease progression in autoimmune prone MRLlpr/lpr mice

MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) and this complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRLlpr/lpr mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor (TLR) 4. We have now established a flow cytometry-based assay to detect the soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRLlpr/lpr mice had higher levels of sMD-1 than control MRL+/+ mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRLlpr/lpr mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRLlpr/lpr kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model.

Lupus nephritis reoccurs following transplantation in the lupus prone mouse

The incidence and pathomechanism of recurrent lupus nephritis (RLN) after transplantation is not clearly understood. Burning out of the autoimmune process or local immunoregulatory mechanisms in the kidney may be responsible for the low incidence of recurrence. These mechanisms cannot be investigated in human subjects, due to post-transplant immunosuppression. To investigate the pathomechanisms of RLN, male and female kidneys were transplanted from FAS deficient lupus prone (LPR) or control (FAS intact) MRL mice into either LPR or MRL recipients. Urinary protein and blood urea were assessed. Double negative (DN) lymphocyte proliferation was determined by flow cytometry. Two months after transplantation inflammatory infiltration of the glomerular, vascular and interstitial compartments were determined. Renal function as demonstrated by blood urea levels was normal in MRL recipients, but elevated in LPR recipients, independent of the donor strain. Paralleling functional results, inflammatory infiltration was mild or absent in MRL recipients of MRL grafts, and mild to moderate in MRL recipients of LPR grafts, suggesting that kidney removal from the autoimmune (LPR) environment significantly reduced inflammation. Graft infiltration was most severe in LPR recipients: grafts were similarly inflamed independent of the donor. All LPR recipients had significantly less CD4+ Th cells versus MRL mice. Transplantation of LPR grafts into MRL recipients reduced CD4+ Th cell percentage, accompanied by a slight induction of lupus autoantibody production. Our results demonstrate that lupus nephritis is not kidney specific in the LPR model with recurrence after transplantation in the absence of immunosuppression.

Modulation of intra-pulmonary TGF-b expression by mycophenolate mofetil in lupus prone MRL/lpr mice

We investigated the expression profile of inflammatory cytokines in the lung of lupus-prone MRL/lpr mice and evaluated the therapeutic potential of mycophenolate mofetil (MMF) in reducing pulmonary cytokines in active lupus. Eight-week old female MRL/lpr mice (n = 20) were treated with MMF in vehicle by oral gavage. Disease control MRL/lpr mice (n = 30) or normal control MRL mice (n = 20) received vehicle alone. The mice were sacrificed after eight or 12 weeks of treatment. Gene expression and protein synthesis of IL-1beta, MCP-1 and TGF-beta1 in lung tissues were determined. We found an increase in the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues of untreated MRL/lpr mice compared with MRL mice at either 16 weeks or 20 weeks of age. MMF treatment significantly prolonged the survival of MRL/lpr mice, down-regulated the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues at the end of eight or 12 weeks of treatment. Protein synthesis of TGF-beta1 was decreased following eight weeks of MMF treatment. We conclude that MMF treatment can reduce the TGF-beta1 gene expression and protein synthesis in lung tissues of lupus-prone mice. Our findings provide experimental data suggesting a beneficial potential of MMF therapy in pulmonary involvement of lupus.

C5a promotes development of experimental lupus nephritis which can be blocked with a specific receptor antagonist

The MRL/lpr murine SLE model has widespread complement activation and deposition of complement fragments in affected tissues. The potent anaphylatoxin C5a has the potential to play a key role in the pathogenesis of lupus nephritis. We found that renal expression of C5aR mRNA and protein was significantly increased in MRL/lpr mice compared to control MRL/+ mice. To examine the role of C5a signaling through C5aR, a specific small molecule antagonist (a) of C5aR was administered continuously to MRL/lpr mice from 13 to 19 wks of age. Littermate controls were given vehicle alone. The progressive impairment in renal function exhibited in the control group was prevented by C5aRa treatment. Infiltration of neutrophils and macrophages into kidneys was significantly reduced in animals treated with C5aRa compared to controls. Furthermore, renal expression of IL-1beta and MIP-2 mRNA as well as the extent of apoptosis were significantly decreased with blockade of C5aR, indicating their dependence upon signals delivered through C5aR. Thus, pharmacological blockade of C5aR reduces disease manifestations in experimental lupus nephritis. These data support an important role for the C5a anaphylatoxin in lupus nephritis, and that blockade of C5aR represents a potentially viable treatment for human lupus nephritis.

Activation of the lectin pathway in murine lupus nephritis

In systemic lupus erythematosus (SLE), hypocomplementaemia and complement deposition have been described both in man and in experimental models. A major involvement of the classical pathway of complement activation has been demonstrated in this disease, however relatively little is known about the involvement of the lectin pathway. Therefore in the present study we have analyzed the activity of all three pathways of complement activation in murine models of SLE. In the mouse, MBL is expressed in two forms, namely MBL-A and MBL-C. In the present study young and old MRL-lpr and control MRL+/+ mice were compared for the levels of complement activity with specific attention for the lectin pathway. It was found that upon aging of both MRL-lpr and MRL+/+ mice, a marked decrease in the activity of the classical pathway (CP) occurs. Levels of alternative pathway (AP) and lectin pathway (LP) activity remain unchanged. Key-molecules of these pathways, C1q, C3, MBL-A and MBL-C were analyzed and were all found to be decreased in aged mice of both strains. The levels of MBL-A and MBL-C showed a high degree of correlation and decreased equally. In aged MRL-lpr mice in which autoimmunity is most pronounced, we observed high autoantibody titers and strong deposition of glomerular immune complexes in association with deposition of C1q, C3, MBL-A and MBL-C. In conclusion, these data suggest that in addition to the classical pathway and the alternative pathway also the lectin pathway of complement activation is involved in murine lupus nephritis.

Anti-C1q autoantibodies in murine lupus nephritis.

Autoantibodies against C1q can be found in the circulation of patients with several autoimmune diseases including systemic lupus erythematosus (SLE). In SLE there is an association between the occurrence of these antibodies and renal involvement. How anti-C1q autoantibodies contribute to renal disease is currently unknown. Cohorts of MRL-lpr mice, which are known to develop age-dependent SLE-like disease, were used to study the relationship between levels of anti-C1q autoantibodies and renal disease. We collected serum, urine and renal tissue and analysed autoantibodies, complement levels and renal deposition as well as renal function. At 2 months of age all mice already had elevated levels of anti-C1q autoantibodies, and elution of kidneys revealed the presence of these antibodies in renal immune deposits in MRL-lpr mice and not in control MRL+/+ mice. In conclusion, anti-C1q antibodies are already present in serum and immune deposits of the kidney early in life and therefore can play a role in nephritis during experimental SLE-like disease in mice.

MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte adhesion and transendothelial migration in response to TNF-alpha.

Endothelial activation and dysfunctional leucocyte-endothelial interactions are thought to play key roles in the pathogenesis of systemic lupus erythematosus (SLE). The object of this study was to investigate directly the effect of increased endothelial adhesion molecule expression on leucocyte-endothelial cell interactions, using the MRL/lpr mouse model. Leucocyte rolling, arrest and transendothelial migration were quantified in the cremaster muscle microcirculation of 20-week-old MRL/lpr mice, using intravital microscopy. Endothelial adhesion molecule expression was quantified using intravenously injected radiolabelled monoclonal antibodies. Basal expression of intercellular adhesion molecule 1 (ICAM-1) by cremaster endothelium was 2-fold greater in MRL/lpr than in MRL/++ mice (P<0.05). There was a 1.6-fold increase in expression of vascular adhesion molecule 1 (VCAM-1), but no increase in E-selectin or P-selectin expression. Following intrascrotal injection of saline, no difference was detected in leucocyte-endothelial interactions between MRL/lpr and control MRL/++ mice. In contrast, intrascrotal injection of tumour necrosis factor alpha (TNF-alpha) (2 h test period) led to significantly increased numbers of adherent and extravasated leucocytes in MRL/lpr (5.98+/-0.71 and 5.45+/-0.34 leucocytes per 100 micro m vessel segment respectively) compared with MRL/++ mice (3.63+/-0.26 and 2.97+/-0.24 respectively, each P<0.05). Treatment of TNF-alpha-stimulated mice with anti-ICAM-1 F(ab')2 (YN1) abolished the difference between MRL/lpr and MRL/++ mice, whereas a negative control anti-DNP F(ab')2 had no effect. MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte-endothelial interactions in response to TNF-alpha. Increased leucocyte-endothelial interactions due to endothelial priming could contribute to the clinical link between infection and flares of lupus disease activity.

Effects of dietary ω3 and ω6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ- lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1α (MIP-1α), and Macrophage Inflammatory Protein-1β (MIP-1β) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (ω3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (ω-6 and ω-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/ lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months ω-6 and ω-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37°C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1α) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/ lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets ( p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1α as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1α compared to the group of mice fed the diets containing 269 IU of vitamin E ( p < 0.0001). The data obtained from this study in MRL/ lpr and MRL/++ mice suggest that FO diets containing ω-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/ lpr mouse model.

Elevated Plus Maze Behavior, Auditory Startle Response, and Shock Sensitivity in Predisease and in Early Stage Autoimmune Disease MRL/lpr Mice

Behaviors indicative of anxiety have been suggested to emerge with the onset of autoimmune disease in MRL/MpJ-Faslpr (MRL/lpr) mice. This study extends the behavioral characterization of MRL/lpr and congenic MRL/MpJ+/+ (MRL/+) mice using the elevated plus maze (EPM), acoustic startle response, and foot-shock sensitivity tasks. In the elevated plus maze, predisease MRL/lpr mice exhibited less anxiety while MRL/lpr mice in the early stage of autoimmunity did not differ from age-matched control MRL/+ mice. MRL/lpr mice exhibited lower startle responses compared to MRL/+ mice. Similarly, predisease MRL/lpr mice were less reactive to various foot-shock levels than MRL/+ mice. Both the MRL/lpr and the MRL/+ strains exhibited startle habituation deficits, implicating the background MRL strain in the impairment in this process. These data do not support the hypothesis that increased anxiety is apparent with the emergence of autoimmune disease in MRL/lpr mice; however, anxiety may appear as the disease advances.