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The accelerated development of atherosclerosis with increased risk of cardiovascular disease in systemic lupus erythematosus (SLE) patients is not well understood. An appropriate mouse model would greatly help to understand the mechanisms of this association. We have therefore combined the ApoE-/- model of atherosclerosis with three different murine models of SLE. We found that induction of cGVH in B6.ApoE-/- mice, breeding a Fas null gene onto these B6/lpr.ApoE-/- mice, and breeding the ApoE-/- defect onto MRL/lpr mice all caused a modest increase of atherosclerosis at 24 weeks of age compared to B6.ApoE-/- controls. B cells in B6.ApoE-/- mice had certain phenotypic differences compared to congenic C57BL/6 mice, as indicated by high expression of MHC II, Fas, CD86, and by increased number of cells bearing marginal-zone phenotype. Furthermore, B6ApoE-/- mice had significant titers of anti-oxLDL and anti-cardiolipin autoantibodies compared to their B6 counterparts. Our studies also indicate that, following induction of cGVH, marginal zone B cells in B6.ApoE-/- are depleted, and there is considerable increase in anti-oxLDL and anti-cardiolipin abs along with secretion of lupus-specific autoantibodies, such as anti-dsDNA and anti-chromatin abs. Histological sections showed that cGVH and/or Fas deficiency could exacerbate atherosclerosis. The production of anti-oxLDL and anti-cardiolipin in ApoE-/- mice was also increased. These observations define a connection between induction of lupus-like symptoms and development of severe atherosclerosis in apoE deficient lupus mouse models.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic lupus erythematosus (SLE)-like disease. The natural history of the pulmonary involvement and the underlying mechanism of leukocyte infiltration into the lungs of MRL/lpr mice and SLE patients remains elusive. We aimed to investigate the expression profiles of chemokines and chemokine receptors in the lung of the SLE-prone mouse. We examined the correlation between lung inflammation and expression of IP-10 (interferon-γ-inducible protein 10), a CXC chemokine, and TARC (thymus- and activation-regulated chemokine), a CC chemokine, in MRL/lpr mice, MRL/Mp-+/+ (MRL/+) mice, and C57BL/6 (B6) control mice. The extent of cell infiltration in the lung was assessed histopathologically. Reverse transcriptase PCR showed up-regulation of IP-10 mRNA expression in the lungs (P < 0.05) of MRL/lpr mice, in comparison with MRL/+ or B6 mice. The increase paralleled increased expression of a specific IP-10 receptor, CXCR3, and correlated with the degree of infiltration of mononuclear lymphocytes. In contrast, lung expression of TARC and its specific receptor, CCR4, were suppressed in MRL/lpr mice. Immunohistology showed that macrophage-like cells were the likely source of IP-10. Flow cytometric analyses revealed that the CXCR3-expressing cells were mainly infiltrating CD4 T cells and macrophages, which correlated with the degree of mononuclear lymphocyte infiltration. Recent data suggest that Th1 cells and Th1-derived cytokines play an important role in the development of SLE-like disease in MRL/lpr mice. Our results suggest that IP-10 expression in the lung is involved, through CXCR3, in the pathogenesis of pulmonary inflammation associated with migration of Th1 cells.

Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems. It is characterized by the presence of autoantibodies reactive against various self-antigens. Susceptibility to SLE is found to be associated with many major histocompatibility complex (MHC) and non-MHC genes, one of which is APO-1/Fas gene, which is present on chromosome 10 in humans. The APO-1/Fas promoter contains consensus sequences for binding of several transcription factors that affect the intensity of Fas expression in cells. The mutations in the APO-1/Fas promoter are associated with risk and severity in various autoimmune diseases and other malignancies. The APO-1/Fas receptor is expressed by many cell types. Two forms of APO-1/Fas protein that are involved in regulation of apoptosis have been identified. Fas receptor-mediated apoptosis plays a physiological and pathological role in killing of infected cell targets. In this review, we have focused on APO-1/Fas gene structure, promoter variants and its association with SLE and other autoimmune diseases. Functional aspects of Fas receptor in apoptosis are also discussed.

apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.

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Background

Prolactin is secreted from the pituitary gland and other organs, as well as by cells such as lymphocytes. Prolactin has an immunostimulatory effect and is associated with autoimmune diseases that are characterised by abnormal B cell activation, such as systemic lupus erythematosus (SLE). Our aim was to determine if different splenic B cell subsets express the prolactin receptor and if the presence of prolactin influences these B cell subsets and correlates with development of lupus.

Results

Using real-time PCR and flow cytometry, we found that different subsets of immature (transitional) and mature (follicular, marginal zone) B cells express different levels of the prolactin receptor and are differentially affected by hyperprolactinaemia. We found that transitional B cells express the prolactin receptor at higher levels compared to mature B cells in C57BL/6 mice and the lupus-prone MRL/lpr and MRL mouse strains. Transitional-1 (T1) B cells showed a higher level of prolactin receptor expression in both MRL/lpr and MRL mice compared to C57BL/6 mice. Hyperprolactinaemia was induced using metoclopramide, which resulted in the development of early symptoms of SLE. We found that T1 B cells are the main targets of prolactin and that prolactin augments the absolute number of T1 B cells, which reflects the finding that this B cell subpopulation expresses the highest level of the prolactin receptor.

Conclusions

We found that all B cell subsets express the prolactin receptor but that transitional B cells showed the highest prolactin receptor expression levels. Hyperprolactinaemia in mice susceptible to lupus accelerated the disease and increased the absolute numbers of T1 and T3 B cells but not of mature B cells, suggesting a primary effect of prolactin on the early stages of B cell maturation in the spleen and a role of prolactin in B cell differentiation, contributing to SLE onset.

Three independent mutations involving the apoptosis-1 (APO-1)/Fas receptor or its putative ligand have led to lupuslike diseases associated with lymphadenopathy in different strains of mice. To determine whether humans with SLE also have a defect in this apotosis pathway, we analyzed the expression of APO-1 on freshly isolated blood mononuclear cells and on lymphocytes activated in vitro using flow cytometry and the monoclonal antibody anti-APO-1. Significantly higher level of APO-1 expression were detected on freshly isolated peripheral B cells and both CD4+ and CD8+ T lymphocyte populations obtained from lupus patients when compared with normal controls (P < 0.001). Almost 90% of the cells that stained positive for APO-1 also expressed the CD29 antigen, suggesting that APO-1 was upregulated after lymphocyte activation in vivo. No defect in APO-1 regulation was detected after activation of SLE T (with anti-CD3) or B (with Staphylococcus aureus Cowan 1) lymphocytes in the presence of IL-2 in vitro. Similarly, the anti-APO-1 antibody induced apoptosis in 74 +/- 5% of activated SLE T cells in vitro compared with 79 +/- 6% of the normal controls (P > 0.05). These results reveal that, while APO-1/Fas may play an important role in the regulation of lymphocyte survival in SLE, no consistent defect in the expression or function of the receptor could be detected in these studies.

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Anti-DNA antibodies, specifically those that stain nuclei in a homogenous nuclear (HN) fashion, are diagnostic of systemic lupus erythematosus (SLE) and the MRL-lpr/lpr SLE murine model. We have used a heavy chain transgene that increases the frequency of anti-HN antibodies to address whether their production in SLE is the consequence of a defect in B cell tolerance. Anti-HN B cells were undetectable in nonautoimmune-prone transgenic mice, but in MRL-lpr/lpr transgenic mice their Ig was evident in the sera and they were readily retrievable as hybridomas. We conclude that nonautoimmune animals actively delete anti-HN-specific B cells, and that MRL-lpr/lpr mice are defective in this process possibly because of the lpr defect in the fas gene.

Background

Monocyte activation and migration into the arterial wall is a key event in atherogenesis associated with hypercholesterolemia. CD11c/CD18, a β2 integrin expressed on human monocytes and a subset of mouse monocytes, has been shown to play a distinct role in human monocyte adhesion on endothelial cells, but the regulation of CD11c in hypercholesterolemia and its role in atherogenesis are unknown.

Methods and Results

Mice genetically deficient in CD11c were generated and crossbred with apoE-/- mice to generate CD11c-/-/apoE-/- mice. Using flow cytometry, we examined CD11c on blood leukocytes in apoE-/- hypercholesterolemic mice and found that compared to wild-type and apoE-/- mice on normal diet, apoE-/- mice on western high-fat diet (HFD) had increased CD11c+ monocytes. Circulating CD11c+ monocytes from HFD-fed apoE-/- mice exhibited cytoplasmic lipid vacuoles and expressed higher levels of CD11b and CD29. Deficiency of CD11c decreased firm arrest of mouse monocytes on vascular cell adhesion molecule-1 and E-selectin in a shear flow assay, reduced monocyte/macrophage accumulation in atherosclerotic lesions, and decreased atherosclerosis development in apoE-/- mice on HFD.

Conclusions

CD11c, which increases on blood monocytes during hypercholesterolemia, plays an important role in monocyte recruitment and atherosclerosis development in an apoE-/- mouse model of hypercholesterolemia.

The development and progression of systemic lupus erythematosus (SLE) is strongly associated with complement activation and deposition. The anaphylatoxin C3a is a product of complement activation with immunomodulatory properties, and the receptor for C3a (C3aR) is not only expressed by granulocytes and antigen presenting cell populations, but it is also strongly up-regulated in lupus prone mice with active nephritis. In order to characterize the role of the C3aR in inflammatory nephritis, we bred C3aR knock-out mice onto the MRL/lpr genetic background (C3aR KO MRL). Compared to control MRL/lpr mice, C3aR KO MRL mice had elevated autoantibody titers and an earlier onset of renal injury. At 8 weeks, renal expression of a wide range of chemokines and chemokine receptors was increased in C3aR KO MRL kidneys compared to controls. Only the expression of MCP-1 was significantly decreased in the C3aR KO MRL mice. The increased chemokine and chemokine receptor expression seen in the C3aR KO MRL mice was associated with a more rapid rise in serum creatinine and the acceleration of renal fibrosis. However, loss of the C3aR had little impact on long-term kidney injury and did not alter survival. These findings suggest that activation of the C3aR plays a protective, not pathologic, role in the early phase of inflammatory nephritis in the MRL/lpr model of SLE.

Liver cells absorb apolipoprotein (Apo) B48-carrying lipoproteins in ApoE’s absence, albeit not as efficiently as the ApoE-mediated process. Our objective was to identify differentially expressed hepatic endosome proteins in mice expressing ApoB48 but lacking ApoE and ApoB100 expression (ApoE−/−/B48/48). We purified early and late endosomes from ApoE−/−/B48/48 and wild-type mouse’s livers. In ApoE−/−/B48/48 mouse’s hepatic endosomes, proteomic analysis revealed elevated protein levels of major urinary protein 6 (MUP), calreticulin, protein disulfide isomerases (PDI) A1, and A3. These proteins are capable of interacting with lipids/lipoproteins and triggering receptor-mediated endocytosis. In addition, hepatic endosomes from ApoE−/− /B48/48 mice exhibited significantly reduced protein levels of haptoglobin, hemopexin, late endosome/lysosome interacting protein, cell division control protein 2 homolog, γ-soluble Nethylmaleimide- sensitive factor attachment protein, vacuolar ATP synthase catalytic subunit A1, dipeptidyl peptidases II, cathepsin B, D, H, and Z. These proteins participate in plasma heme clearance, receptor-mediated signaling, membrane fusion, endosomal/lysosomal acidification, and protein degradation. The significance of increasing endosomal MUP, calreticulin and PDIs in ApoE−/−/B48/48 mouse liver cells is not clear; however, reducing endosomal/ lysosomal membrane proteins and hydrolases might be, at least partially, responsible for the retarded clearance of plasma ApoB-carrying lipoproteins in ApoE−/−/B48/48 mice.

Background

Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis.

Methodology/Principal Findings

Hypomorphic apoE (Apoeh/h) mice expressing wildtype mouse apoE at ∼2–5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoeh/h allele in Apoeh/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoeh/hLysM-Cre and Apoeh/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoeh/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoeh/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7). On HCD, Apoeh/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoeh/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoeh/h mice (167×103±16×103 µm2 versus 259×103±56×103 µm2, n = 7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol.

Conclusions/Significance

Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.

Systemic lupus erythematosus (SLE) is a chroni c autoimmune disease characterized by loss of tolerance to self-antigens and activation of autoreactive T cells. Regulatory T cells (Treg) play a critical role in controlling the activation of autoreactive T cells. Here, we investigated mechanisms of potential Treg defects in SLE using MRL-Faslpr/lpr (MRL/lpr) and MRL-Fas+/+ (MRL+/+) mouse models. We found a significant increase in CD4+CD25+Foxp3+ Treg cells, albeit with an altered phenotype (CD62L–CD69+) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared to non-autoimmune C3H mice. A search for mechanisms underlying the altered Treg phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155 that can target CD62L and may thus confer the altered Treg phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H) Treg cells reduced their CD62L expression, which mimics the altered Treg phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.

Objective

Systemic lupus erythematosus (SLE) T cells display a hyperactive calcineurin-NFAT pathway. The aim of this study is to answer whether this pathway is responsible for the aberrant SLE T cell function and test the effectiveness of the recently recognized calcineurin inhibitor dipyridamole in limiting SLE related pathology.

Methods

T and mononuclear cells were isolated from the peripheral blood of patients with SLE and healthy individuals. Murine cells were isolated from the spleens and lymph nodes of lupus prone MRL/lpr mice and control MRL/MpJ mice. Cells were treated in vitro with tacrolimus, dipyridamole or control. MRL/lpr mice were injected intraperitoneally with dipyridamole 50 mg/kg three times a week for 3 weeks.

Results

MRL/lpr T cells, especially CD3+CD4-CD8- displayed a robust calcium influx upon activation and increased levels of NFATc1. MRL/lpr T cells (both CD4+ and CD3+CD4-CD8- cells) provided help to B cells to produce immunoglobulin in a calcineurin-dependent fashion. Dipyridamole treatment of SLE T cells inhibited significantly the expression of CD154, the production of IFN-γ, IL-17, IL-6, and the T cell dependent B cell immunoglobulin secretion. Treatment of MRL/lpr mice with dipyridamole alleviated lupus nephritis and prevented the appearance of skin ulcers.

Conclusion

NFAT activation is a key step in the activation of SLE T cells and the production of immunoglobulin. Dipyridamole inhibits SLE T cell function and improves disease pathology in lupus-prone mice. We propose that dipyridamole can be used in treatment regimens of SLE patients.

Apolipoprotein (apo) E4 plays a major role in the pathogenesis of Alzheimer’s disease (AD). Brain amyloid-beta (Aβ) accumulation depends on age and apoE isoforms (apoE4 > apoE3) both in humans and in transgenic mouse models. Brain apoE levels are also isoform-dependent, but in the opposite direction (apoE4 < apoE3). Thus, one prevailing hypothesis is to increase brain apoE expression to reduce Aβ levels. To test this hypothesis, we generated mutant human amyloid precursor protein (hAPP) transgenic mice expressing one or two copies of the human apoE3 or apoE4 gene that was knocked-in and flanked by LoxP sites. We report that reducing apoE3 or apoE4 expression by 50% in 6-month-old mice results in efficient Aβ clearance and does not increase Aβ accumulation. However, 12-month-old mice with one copy of the human apoE gene had significantly reduced Aβ levels and plaque loads compared to mice with two copies, regardless of which human apoE isoform was expressed, suggesting a gene dose-dependent effect of apoE on Aβ accumulation in aged mice. Additionally, 12-month-old mice expressing one or two copies of the human apoE4 gene had significantly higher levels of Aβ accumulation and plaque loads than age-matched mice expressing one or two copies of the human apoE3 gene, suggesting an isoform-dependent effect of apoE on Aβ accumulation in aged mice. Moreover, Cre-mediated apoE4 gene excision in hippocampal astrocytes significantly reduced insoluble Aβ in adult mice. Thus, reducing, rather than increasing, apoE expression is an attractive approach to lowering brain Aβ levels.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/faslpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/faslpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/faslpr/lpr B cells was significantly altered, e.g., increased dG/dC transitions, and increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/faslpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer’s patches. In MRL/faslpr/lpr mice, the significant upregulation of SHM and CSR was associated with significantly increased expression of AID, which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone mice, SHM and CSR are dysregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.

Summary

The impact of IFNα secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental B6 congenic partner using an adenovirus expression vector containing a recombinant IFNα gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFNα. Most IFNα-induced phenotypes were similar in B6 and B6.Sle123, however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFNα exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFNα were identical in both strains, severe GN only occurred in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum ANA levels. In summary, the predominant impact of systemic IFNα in this murine model is an exacerbation of mechanisms mediating end organ damage.

Background

Angiotensin II (AII) is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express AII type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an AII-responsive setting induced by uninephrectomy (UNx).

Methods and Results

AT1−/− or AT1+/+ marrow from apolipoprotein E deficient (apoE−/−) mice was transplanted into recipient apoE−/− mice with subsequent UNx or sham operation: apoE−/−/AT1+/+→apoE−/− + Sham; apoE−/−/AT1+/+→apoE−/− + UNx; apoE−/−/AT1−/−→apoE−/− + Sham; apoE−/−/AT1−/−→apoE−/− + UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the two UNx groups. ApoE−/−/AT1+/+→apoE−/− + UNx had significantly more atherosclerosis (16907 ± 21473 vs 116071 ± 8180 μm2, p<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174 ± 9947 vs 75714 ± 11333 μm2, p=NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE−/−/AT1−/−→apoE−/− + UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE−/−/AT1−/−→apoE−/− whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE−/−/AT1+/+→apoE−/− mice. Instead, apoE−/−/AT1−/−→apoE−/− had 5-fold-increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1−/− macrophages vs AT1+/+.

Conclusions

AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury–induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.

A number of macrophage functions were sequentially expressed when the bone marrow precursors of mononuclear phagocytes differentiated in culture in the presence of a specific growth factor, colony-stimulating factor-1. We have defined the expression of apoprotein E (ApoE), a major secreted protein of resident peritoneal macrophages, during maturation of adherent bone marrow-derived mononuclear phagocytes into macrophages. By 5 d the bone marrow macrophages were active secretory cells, but few cells contained intracellular immunoreactive ApoE, and little, if any, ApoE was secreted. ApoE secretion was initiated at 9 d, and this correlated with an increase in the percentage of macrophages containing intracellular ApoE. The onset of ApoE secretion was selective, and little change occurred in the other major secreted proteins detected by [35S]methionine incorporation. In parallel, the high rate of plasminogen activator secretion, which peaked at 7 d, decreased markedly. ApoE secretion was not associated with altered expression of the macrophage surface antigen, Ia, or with secretion of fibronectin. Virtually all cells in independent colonies of bone marrow- derived macrophages eventually expressed ApoE. The proliferating monocyte/macrophage-like cell lines P388D1, J774.2, WEHI-3, RAW 264.1, and MGI.D+ secreted little or no ApoE. These data establish that ApoE secretion is developmentally regulated.

Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp-lpr/lpr (MRL/lpr) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo.

MRL/1 and BXSB male mice have a systemic lupus erythematosus (SLE)-like disease similar to but more acute than that occurring in NZB X W mice. The common elements of lymphoid hyperplasia, B-cell hyperactivity, autoantibodies, circulating immune complex (IC), complement consumption, IC glomerulonephritis with gp70 deposition, and thymic atrophy were found in all three kinds of SLE mice. On the basis of these common elements, SLE seen in these mice can be considered a single disease in the same sense that human SLE is one disease. The differences in the SLE expressed in the different mice are no greater than those found in an unselected series of humans with SLE. However, the significant quantitative and qualitative variations in abnormal immunologic expression suggest that different constellations of factors, genetic and/or pathophysiologic, may operate in the three murine strains and that each constellation is capable of leading, via its particular abnormal immunologic consequences, to the activation of common immunopathologic effector mechanisms that cause quite similar SLE-like syndromes. From an experimental point of view, the availability of several inbred murine strains of commonplace histocompatibility types that express an SLE-like syndrome makes possible innumerable manipulations which should help to elucidate the nature and cause(s) of this disorder.

The aim of this study was to quantify soluble Fas/APO-1 (sFas/APO-1) protein in the serum of patients with Behcet's disease (BD) in active and inactive stages, compared with patients with systemic lupus erythematosus (SLE) and patients with rheumatoid arthritis (RA). Soluble Fas/APO-1 was quantified using a sandwich enzyme-linked immunosorbent assay. Increased serum sFas/APO-1 levels were observed in active BD, compared with inactive BD, RA patients and SLE patients. Increased serum sFas/APO-1 levels were correlated with the presence of neurologic manifestations or pulmonary involvement in active BD. In conclusion, increased levels of sFas/APO-1 occurred frequently and exclusively in active BD patients. Preliminary evidence suggested that elevated levels of sFas/APO-1 are associated with the clinical stage and clinical manifestations in BD.

Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2−/− mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.

CXCL13 is a key B cell chemoattractant and marker of disease activity in patients with SLE; however, the mechanism of its induction has not been identified yet. Here, we have shown that the proteoglycan biglycan triggers CXCL13 expression via TLR2/4 in macrophages and dendritic cells. In vivo, levels of biglycan were markedly elevated in the plasma and kidneys of human SLE patients and lupus-prone (MRL/lpr) mice. Overexpression of soluble biglycan in MRL/lpr mice raised plasma and renal levels of CXCL13 and caused accumulation of B cells with an enhanced B1/B cell ratio in the kidney, worsening of organ damage, and albuminuria. Importantly, biglycan also triggered CXCL13 expression and B cell infiltration in the healthy kidney. Conversely, biglycan deficiency improved systemic and renal outcome in lupus-prone mice, with lower levels of autoantibodies, less enlargement of the spleen and lymph nodes, and reduction in renal damage and albuminuria. This correlated with a marked decline in circulating and renal CXCL13 and a reduction in the number of B cells in the kidney. Collectively, our results describe what we believe to be a novel mechanism for the regulation of CXCL13 by biglycan, a host-derived ligand for TLR2/4. Blocking biglycan-TLR2/4 interactions might be a promising strategy for the management of SLE and other B cell–mediated inflammatory disease entities.

The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE−/− mice before the development of manifest atherosclerotic lesions. Platelet–endothelial cell interaction involved both platelet glycoprotein (GP)Ibα and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE−/− mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.

Adhesive interactions between endothelial cells and leukocytes contribute to atherosclerotic plaque growth. However, mechanism(s) responsible for endothelial priming and deactivation in inflammatory diseases such as atherosclerosis are not clear. Apolipoprotein E deficient mice were generated with deficiency of P-selectin glycoprotein ligand-1 (Psgl-1−/−, ApoE−/−). On both standard chow and Western diet, Psgl-1−/−, ApoE−/− mice were protected against atherosclerosis compared to Psgl-1+/+, ApoE−/− controls. Psgl-1−/−, ApoE−/− mice also showed reduced leukocyte rolling and firm attachment on endothelial cells, however, adoptively transferred Psgl-1+/+, ApoE−/− leukocytes into Psgl-1−/−, ApoE−/− hosts displayed similar reduced rolling as Psgl-1−/−, ApoE−/− leukocytes. Hematopoietic deficiency of Psgl-1 conferred resistance to the effects of interleukin-1β (IL-1β) on leukocyte rolling along with reduced circulating levels of sP-sel and sE-sel. Antibody blockade of Psgl-1 also reduced endothelial activation in response to IL-1β, eliminated leukocyte rolling, and was protective against atherosclerosis in ApoE−/− mice. Monocyte depletion with clodronate restored the endothelial response to IL-1β in Psgl-1−/− mice. This study suggests that Psgl-1 deficiency leads to reduced atherosclerosis and adhesive interactions between endothelial cells and leukocytes by indirectly regulating endothelial responses to cytokine stimulation.