Compensatory phospholipid digestion is required for cholesterol absorption in pancreatic phospholipase A(2)-deficient mice.

BACKGROUND AND AIMS: Numerous studies have suggested phospholipid inhibition of dietary cholesterol absorption through the gastrointestinal tract. This study addressed the importance of luminal phospholipid hydrolysis in this process. METHODS: The effect of phospholipase inhibition on cholesterol transport from intestinal lumen to the lymphatics was evaluated in lymph fistula rats. Cholesterol and phospholipid absorption efficiency in intact animals was evaluated in control and phospholipase A(2) (PLA2) gene-targeted mice. RESULTS: The PLA2 inhibitor FPL 67047XX retarded cholesterol absorption in a lymph fistula rat model. Under basal chow-fed dietary conditions, cholesterol absorption efficiency from a single bolus meal, and plasma lipid levels, were similar among PLA2+/+, PLA2+/-, and PLA2-/- mice. Interestingly, the nonhydrolyzable phospholipid dioleoyl ether phosphatidylcholine suppressed cholesterol absorption by 10% to 18% in mice without regard to their PLA2 genotype. When 1-palmitoyl-2-[(14)C]oleoyl-phosphatidylcholine was used as the substrate, the radiolabeled phospholipid was found to be hydrolyzed and absorbed with equal efficiency in PLA2+/+, PLA2+/-, and PLA2-/- mice. CONCLUSIONS: These results suggested that although phospholipid digestion in the intestinal lumen is a prerequisite for efficient cholesterol absorption, additional enzyme(s) can compensate for pancreatic PLA2 in catalyzing phospholipid digestion and facilitating cholesterol absorption in PLA2 knockout mice.

Apolipoprotein E inhibition of vascular smooth muscle cell proliferation but not the inhibition of migration is mediated through activation of inducible nitric oxide synthase.

Initial experiments revealed that low concentrations of apolipoprotein (apo) E (0.1 to 5 microg/mL) were effective in inhibiting platelet-derived growth factor (PDGF)-directed smooth muscle cell (SMC) migration by 60% to 80%. In contrast, higher concentrations of apoE, at 25 and 50 microg/mL, were necessary to achieve similar inhibition of PDGF-induced SMC proliferation. The potential role of nitric oxide (NO) in mediating the inhibitory effects of apoE was explored. Results showed that, although 0.1 to 5 microg/mL of apoE had no effect on NO production by SMCs, physiological concentrations of apoE (25 to 50 microg/mL) enhanced NO synthesis by 2-fold in a dose-dependent manner (P<0.001). Reverse transcription-polymerase chain reaction amplification of RNA obtained from control and apoE-treated SMCs demonstrated a direct role of apoE in activating inducible nitric oxide synthase (iNOS) gene expression. The apoE-induced nitric oxide production was significantly reduced by coincubation of the cells with aminoguanidine or N(G)-monomethyl-L-arginine (P<0.05) or with antisense iNOS oligodeoxynucleotides (P<0.01). Moreover, the inhibition of iNOS was shown to overcome apoE suppression of PDGF-induced vascular SMC proliferation. However, apoE suppression of PDGF-directed SMC migration was not affected by these treatments. Taken together, these results document that apoE exerts its inhibitory effects on cell proliferation via activation of iNOS. However, apoE inhibition of cell migration is mediated by a mechanism independent of iNOS activation.

Maternal high density lipoproteins affect fetal mass and extra-embryonic fetal tissue sterol metabolism in the mouse.

Previous studies have shown that antibodies to cubulin, a receptor on the yolk sac that binds high density lipoproteins (HDL) and cobalamin, induce fetal abnormalities. Mice with markedly low concentrations of plasma HDL-cholesterol (HDL-C) give birth to healthy pups, however. To establish whether maternal HDL-C has a role in fetal development, sterol metabolism was studied in the fetus and extra-embryonic fetal tissues in wild-type and apolipoprotein A-I-deficient mice (apoAI-/-). Maternal HDL-C content was markedly greater in apoAI+/+ mice prior to pregnancy and at 13 days into gestation. By 17 days into gestation, HDL-C content was similar between both types of mice. Fetuses from apoAI (-/- x -/-) matings were 16;-25% smaller than control mice at 13 and 17 days of gestation and contained less cholesterol. The differences in size and cholesterol content were not due to a lack of cholesterol synthesis or apoA-I in the fetus. In the yolk sac and placenta, sterol synthesis rates were approximately 50% greater in the 13-day-old apoAI-/- mice as compared to the apoAI+/+ mice. Even though synthesis rates were greater, cholesterol concentrations were 22% lower in the yolk sac and similar in the placenta of apoAI-/- mice as compared to tissues of wild-type mice. These data suggest that a difference in maternal HDL-C concentration or composition can affect the size of the fetus and sterol metabolism of the yolk sac and placenta in the mouse.

Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase.

The anti-atherogenic effects of apolipoprotein (apo) E have been attributed to its ability to reduce plasma cholesterol level and to limit foam cell formation. The purpose of this study was to ascertain if apoE also may have cytostatic functions that could attenuate vascular occlusive diseases. Purified apoE inhibited smooth muscle cell migration directed to platelet-derived growth factor (PDGF) or oxidized LDL (oxLDL) (p < 0.0001). The purified apoE also suppressed PDGF- and oxLDL-induced smooth muscle cell proliferation (p < 0.001). These apoE inhibitory effects were not because of suppression of PDGF binding to its receptors on the smooth muscle cells, but was correlated with a significant reduction in agonist-stimulated mitogen-activated protein kinase activity (p < 0.01). ApoE also inhibited PDGF-induced cyclin D1 mRNA expression, suggesting that the apoE effect was mediated by growth arrest at the G0 to G1 phase. Taken together, these results suggest that apoE has cytostatic functions in the vessel wall and may protect against vascular diseases through inhibition of cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation.

Enhancement of renal disease in BXSB lupus prone mice after prior exposure to bacterial lipopolysaccharide.

The mechanism, or mechanisms, responsible for enhancement of renal disease after episodes of infection are poorly understood. We used the BXSB mouse as a lupus model of autoimmune disease and we used bacterial lipopolysaccharide (LPS) as a surrogate infectious agent to gain some insight into the mechanism by which infections promote enhancement of autoimmune disease to chronicity. BXSB mice were exposed to LPS for 5 weeks, LPS was withdrawn and various tests and measurements were performed 6 weeks thereafter. Matched BXSB mice exposed to vehicle injections for 5 weeks served as controls. We verified that previous exposure to LPS enhances polyclonal B cell activation, impairs carrier function of blood cells for immune complexes, increases deposition of immune complexes in the microcirculation and promotes glomerular inflammation and sclerosis. These changes occurred at 6 weeks after withdrawal of LPS in the presence of unimpaired function of mononuclear phagocytes. Some of the effects of LPS are reversible, others are partially so and others are irreversible. Altered immune functions elicited by prior exposure to LPS can result in enhanced involvement of various renal compartments and can result in renal insufficiency.

Long-lasting effects of bacterial lipopolysaccharide promote progression of lupus nephritis in NZB/W mice.

Lupus prone NZB/W mice repeatedly exposed to bacterial lipopolysaccharide (LPS) develop enhanced polyclonal B cell activation and exacerbated nephritis by a mechanism that results in increased deposits of immunoreactants in kidneys without measurable impairment of mononuclear phagocyte function. In this paper, we investigate whether the referenced effects of LPS are reversible after withdrawal of LPS, or whether their persistence could contribute to progression of nephritis to chronicity. In NZB/W mice previously exposed to LPS, features of enhanced polyclonal B cell activation, more severe glomerulonephritis with tubulointerstitial involvement, increased deposits of immunoreactants in glomeruli, and altered protein excretion persisted 6 weeks after LPS was discontinued. Additionally, mononuclear phagocyte function, assessed through liver uptake of radiolabeled immune complexes, was found to be impaired. The results indicate that some of the effects of prior exposure to LPS may be partially reversible; however, they last long after LPS has been discontinued, and additional impairment of immune function develops together with permanent glomerular dysfunction, thereby contributing to progression of nephritis to chronicity.

Pathogenesis of early nephritis in lupus prone mice with a genetic accelerating (lpr) factor.

We investigated the relative roles of B cell activity, circulating immune complexes, complement concentration and kinetics of disappearance and uptake of immune complexes from the circulation in the pathogenesis of early nephritis of MRL/lpr mice. In comparison to data in control (C57BL/6J) mice, B cell activity was enhanced and the concentration of autoantibodies and endogenous immune complexes in plasma were increased, whereas complement (C3) concentration was not significantly different in MRL/lpr mice. The kinetics of disappearance of radiolabeled immune complexes from the circulation were similar in MRL/lpr and control mice, whereas uptake of radiolabeled immune complexes by the liver was decreased in MRL/lpr mice. Features of polyclonal B cell activation and impaired mononuclear phagocyte function are early events that may set the stage for progressive systemic involvement in MRL/lpr mice.

Bacterial lipopolysaccharide causes variable deposits of diverse immunoglobulin isotypes in kidneys of lupus-prone mice.

We investigated the role of immunoglobulin isotypes in the exacerbation of lupus nephritis associated with exposure to bacterial lipopolysaccharide. The data indicate that enhanced polyclonal B-cell activation and exacerbated autoimmune disease evoked by lipopolysaccharide are associated with an increase in the concentration of isotypes in plasma and in renal eluate, that this isotype response is polyclonal and preferential but not restrictive, that all B cells are responsive but all are not equally sensitive to the effects of lipopolysaccharide, and that some expanded isotypes may be more nephritogenic in certain strains of lupus-prone mice.

Autoimmunity, polyclonal B-cell activation and infection.

It is widely believed that autoimmunity is an integral part of the immune system, and that genetic, immunologic, hormonal, environmental and other factors contribute to the pathogenesis of autoimmune disease. Thus, autoimmune disease may represent an abnormal expression of immune functions instead of loss of tolerance to self, and it can be organ specific or systemic in its manifestations. We review the various factors that contribute to the development of autoimmune disease; we also review the mechanisms of polyclonal B-cell activation, with emphasis on the role of infectious agents. We consider systemic lupus erythematosus in humans and in experimental animals as prototypic autoimmune disease, and we summarize data to indicate that polyclonal B-cell activation is central to the pathogenesis of systemic autoimmune disease. The effect of polyclonal B-cell activation, brought about by injections of a B-cell activator-lipopolysaccharide from Gram-negative bacteria-is sufficient to cause autoimmune disease in an immunologically normal host. In fact, autoimmune disease can be arrested if excessive polyclonal B-cell activation is suppressed; alternatively, autoimmune disease can be exacerbated if polyclonal B-cell activation is enhanced. We explore the mechanism of tissue injury when autoimmune disease is induced or exacerbated, and we consider the pathogenic roles of autoantibodies, immune complexes, complement, the blood cell carrier system, and the mononuclear phagocyte system. Although polyclonal B-cell activation may be the mechanism whereby various factors can cause or exacerbate systemic autoimmune disease, polyclonal B-cell activation may cause autoimmune disease on its own.

Bacterial lipopolysaccharide enhances deposition of immune complexes and exacerbates nephritis in BXSB lupus-prone mice.

Systemic autoimmune disease is influenced by genetic, immunological, hormonal, and environmental factors. Although environmental factors are major agents that induce or exacerbate autoimmune diseases, the mechanism(s) and the molecular events by which they operate remain poorly understood. Here we used the lupus-prone BXSB mouse as an animal model of systemic autoimmune disease, and we used a bacterial lipopolysaccharide (LPS) as a surrogate infectious agent to gain some insight into the mechanism(s) by which infectious agents exacerbate autoimmune diseases. Our experimental protocol was designed to address three questions: (i) whether spontaneous polyclonal B cell activation (PBA) that occurs in BXSB mice could be further enhanced by bacterial LPS; (ii) whether repeated exposure to LPS would exacerbate autoimmune disease, as reflected by enhanced deposits of immune complexes (ICs) in kidneys and exacerbated nephritis; and (iii) whether the mechanism by which LPS exacerbates nephritis might involve interference with blood cell carrier function, mononuclear phagocyte function, or both. BXSB mice were injected with LPS (25 micrograms) twice a week for 5 weeks; control autoimmune BXSB mice and immunologically normal (C57BL/6) mice were injected with vehicle only. The three groups of mice were then challenged with soluble ICs to assess the kinetics of their disappearance from the circulation, their uptake by the mononuclear phagocyte system (liver, spleen), their distribution in target organ (kidney), and blood cell carrier function. The results indicate that: (i) spontaneous PBA can be enhanced further by LPS; (ii) exposure to LPS results in increased deposits of endogenous ICs in kidneys and exacerbated nephritis; and (iii) defective handling of ICs by the mononuclear phagocyte system and impaired blood cell carrier function are contributory factors to exacerbated nephritis, but that mechanisms in addition to passive localization of ICs may also be operative.

Bacterial lipopolysaccharide induces long-lasting IgA deficiency concurrently with features of polyclonal B cell activation in normal and in lupus-prone mice.

Polyclonal B cell activation (PBA) and autoimmune disease can be induced in immunologically normal mice, or enhanced in lupus-prone mice, by bacterial lipopolysaccharide (LPS). Because immune defects are common in autoimmune diseases and IgA deficiency is prevalent in patients with systemic lupus erythematosus, we investigated: (i) whether LPS might induce IgA deficiency in normal mice; (ii) whether IgA deficiency might be a feature in lupus-prone mice; (iii) whether, if present in lupus-prone mice, IgA deficiency could be further accentuated by LPS; and (iv) whether the effects of LPS on IgA concentrations of normal and lupus-prone mice might be reversible upon withdrawal of LPS. We injected normal (C57BL/6) and lupus-prone (NZB/W) mice with 50 micrograms of LPS from Salmonella minnesota Re595 twice a week for 5 weeks and then discontinued LPS for 6 weeks. We determined the concentrations of plasma immunoglobulins, DNA antibodies, and circulating immune complexes before, during, and after mice were exposed to LPS. Our results indicate that: (i) LPS induces IgA deficiency in normal mice concurrently with PBA; (ii) IgA deficiency is a feature of lupus-prone mice; (iii) LPS accentuates naturally occurring PBA and IgA deficiency in lupus-prone mice; and (iv) LPS induced, or LPS enhanced, IgA deficiency and PBA in normal and lupus-prone mice persist long after withdrawal of LPS. Thus, LPS triggers or enhances autoimmune disease by a mechanism that involves in part PBA with selective increase (IgG, IgM) and concurrent decrease (IgA) of specific isotypes.

Lipopolysaccharide from gram-negative bacteria enhances polyclonal B cell activation and exacerbates nephritis in MRL/lpr mice.

Depletion of B cells in mice bearing the lymphoproliferation (lpr) gene reduces lymphoproliferation and polyclonal B cell activation (PBA) and attenuates mononuclear cell vasculitis. We sought to verify whether the obverse was true, i.e. whether enhancement of B cell activity might exacerbate the nephritis of MRL/lpr (MRL) mice, a lupus-prone strain. The experimental approach was designed to address three questions: whether naturally occurring PBA in MRL mice could be further enhanced; whether enhanced PBA would exacerbate nephritis; and whether the mechanism of nephritis exacerbation involved interference with mononuclear phagocyte system (MPS) function. To enhance B cell activity, we injected MRL mice with lipopolysaccharide (LPS) from Gram-negative bacteria, a potent B cell activator. To determine whether nephritis was exacerbated, we performed immunopathologic studies and tests of renal function. To verify whether nephritis exacerbation involved impairment of MPS function, we probed the kinetics of immune complex removal from the circulation, their uptake by the liver and spleen, and their localization in kidney tissue. The results indicate that in MRL mice: (i) spontaneous PBA can be enhanced by LPS; (ii) enhancement of PBA by LPS exacerbates nephritis; and (iii) the MPS is already saturated, presumably due to excessive production of endogenous immune complexes. Thus, further increase in immune complex formation due to enhanced PBA by LPS results in increased localization of immune complexes in kidneys and exacerbated nephritis.

Prolonged circulation of immune complexes due to various altered immune functions contributes to nephritis in MRL/lpr mice.

To gain some insight into the pathogenesis of proliferative lupus nephritis in MRL/lpr mice we investigated the kinetics of removal of immune complexes from the circulation, the carrier state of blood cells, the uptake of complexes by the mononuclear phagocyte system, and the localization of complexes in kidneys. In nephritic MRL/lpr mice challenged with a subsaturating dose of radiolabelled complexes (2.5 mg bovine serum albumin-anti-bovine serum albumin) liver uptake was profoundly decreased, removal of circulating complexes was delayed, and 12-h kidney localization of complexes was enhanced 7.3-fold, in comparison to control mice. The findings were not encumbered by differences in complement concentration and most likely are attributable to various altered immune functions: spontaneous polyclonal activation of B cells, enhanced production of endogenous immune complexes, delayed removal of complexes from the circulation, and decreased uptake of complexes by the mononuclear phagocyte system. In concert, such altered functions contribute to prolonged circulation of complexes to result in their enhanced deposition in the microcirculation.

Bacterial lipopolysaccharide transforms mesangial into proliferative lupus nephritis without interfering with processing of pathogenic immune complexes in NZB/W mice.

Systemic lupus erythematosus is a multifactorial systemic disease in which genetic, immunologic, hormonal, and environmental factors may contribute to disease pathogenesis. Bacterial products (eg, bacterial lipopolysaccharide [LPS]) induce a lupuslike disease in normal mice and trigger an early and accelerated form of lupus nephritis in NZB/W mice. To investigate whether the mechanism by which LPS accelerates nephritis in the NZB/W mice involves interference with processing of immune complexes (IC), we administered LPS to NZB/W mice for 5 weeks and probed the kinetics of removal, liver uptake, and organ localization of a subsaturating dose of radiolabeled IC (2.5 mg of bovine serum albumin-antibovine serum albumin). Control NZB/W mice received vehicle (saline) alone. In NZB/W exposed to LPS, features of polyclonal B-cell activation (PBA) were enhanced, anti-DNA antibodies were raised, and a proliferative glomerulonephritis developed that was associated with renal insufficiency and substantial proteinuria. This LPS-accelerated nephritis could not be attributed to altered complement concentration, to altered blood cell carrier function, to delayed removal of pathogenic (large-sized) ICs from the circulation, to impaired liver uptake of ICs, or to enhanced localization of ICs in kidney. The findings indicate that transformation of nephritis is probably the result of LPS-induced PBA, that defective processing of pathogenic IC is not a contributory factor to nephritis, and that mechanisms other than passive renal localization of circulating ICs must be operative.

Repeated exposure to bacterial lipopolysaccharide interferes with disposal of pathogenic immune complexes in mice.

Patients with systemic lupus erythematosus (SLE) experience clinical flares in association with superimposed bacterial infection. To investigate whether heightened immune phenomena during the course of bacterial infections were related to abnormal disposal of immune complexes, we administered bacterial lipopolysaccharide (LPS) to C57BL/6 mice for 5 weeks. Control mice received vehicle only. We then challenged the mice with a subsaturating dose of radiolabelled immune complexes intravenously and determined the localization of immune complexes in liver, spleen and kidney. In comparison to control mice, mice exposed to LPS developed features of polyclonal B cell activation, autoimmune phenomena, delayed removal of immune complexes from the circulation, diminished liver uptake of immune complexes, and enhanced localization of immune complexes in the kidneys. The findings could not be attributed to biological processes dependent on complement concentration. Instead, interferences with Fc receptor function, or with endocytosis of immune complexes may represent likely possibilities. Thus, clinical flares in patients with SLE, in the presence of a superimposed infection, may result from enhanced localization of immune complexes in organs due to altered mechanisms of their disposal.

Mechanism of localization of immune complexes in NZB/W mice with early nephritis.

We investigated the pathogenesis of mesangial proliferative lupus nephritis in NZB/W mice under conditions that allowed us to examine removal of immune complexes from the circulation, uptake by the mononuclear phagocyte system, and localization in kidney tissue. These studies were performed at a time when variables such as the quantity of endogenous immune complexes, complement concentration, and carrier state of blood cells (platelets) were controlled. NZB/W mice and C57BL/6 (control) mice showed comparable kinetics for removal of a subsaturating dose of immune complexes (2.5 mg bovine serum albumin-antibovine serum albumin) from the circulation; additionally, the liver uptake and kidney localization of these immune complexes were comparable between NZB/W and control mice. The localization of immune complexes in the glomerular mesangium of NZB/W mice could not be attributed to enhanced production of endogenous immune complexes, to decreased removal of immune complexes from the circulation, to impaired uptake by the liver, or to complement concentration and carrier state of blood cells. It appears, by exclusion, that mesangial deposits of immunoreactants in early lupus nephritis may result from interaction of antibodies with antigens in mesangia.

Defective disposal of immune complexes and polyclonal B cell activation persist long after exposure to bacterial lipopolysaccharide in mice.

Patients with systemic lupus erythematosus experience clinical exacerbation during superimposed bacterial infection. Previous studies in mice indicated that heightened immune phenomena during exposure to bacterial lipopolysaccharide (LPS) appear to be related, in part, to polyclonal B cell activation, to abnormal disposal of immune complexes (IC), and to increased localization of IC in tissues. To investigate whether such effects were reversible, we administered bacterial LPS to C57BL/6 mice for 5 weeks. Control mice received vehicle alone. We then discontinued LPS, and 6 weeks later LPS and control mice were challenged with a subsaturating dose of radiolabeled IC; the removal of IC from the circulation, their localization in the liver, spleen, and kidney were determined. In comparison to values in control mice, in mice previously exposed to LPS, serologic features of polyclonal B cell activation persisted; liver uptake of pathogenic IC (greater than Ag2Ab2) was normal, but removal of small size IC (less than or equal to Ag2Ab2) from the circulation was delayed; localization of IC in the kidneys was enhanced, and pathologic proteinuria developed. The effects of repeated exposure to bacterial LPS are partially reversible, but they last long after LPS is discontinued and may contribute to altered disposal of IC, enhanced organ localization of IC, and organ dysfunction.

Clonality of the spontaneous immune response to DNA in murine lupus.

To probe the mechanism of spontaneous formation of anti-DNA antibodies, we studied the isotype distribution of anti-DNA antibodies in the plasma of NZB/W mice with incipient, chronic, and drug-attenuated nephritis. The concentration of anti-DNA antibodies in plasma did not discriminate between the various groups of mice, and the anti-DNA activity in renal eluates was very low and did not reflect the course of nephritis. Progression of incipient to chronic nephritis was associated with increase, and drug attenuation of nephritis with decrease, in plasma concentration of all isotypes tested. Anti-DNA antibodies were detected in all classes (IgG, IgM) of antibodies studied and the anti-DNA antibodies were found to be unrestricted with respect to IgG isotypes and within a given (IgG2a) isotype. The data indicate that the spontaneous immune response to DNA in NZB/W mice reflects activation of several autoreactive clones and that, overall, anti-DNA activity in the plasma or renal eluate is a poor predictor of extent of renal disease.

Pathogenic role of anti-DNA antibodies in murine lupus nephritis.

We studied the relative role of anti-DNA antibodies in pathogenesis of murine lupus nephritis, and we used, as index of their contribution, the association between attenuation, or progression, of renal disease and decrease or increase in concentration of anti-DNA antibodies in the plasma and renal eluate of NZB/W mice. The concentration of anti-DNA antibodies in plasma did not discriminate mice with incipient or drug attenuated nephritis, who had normal renal function, from mice with progressive nephritis, who developed renal failure. Although the quantity of IgG eluted from kidneys reflected the extent of renal disease (mice with progressive nephritis greater than mice with attenuated nephritis greater than mice with incipient nephritis), the anti-DNA activity of such antibodies was negligible. The low anti-DNA activity could not be attributed to either excess DNA or DNAase in renal eluates. In fact, the eluates contained a factor that inhibited the interaction between anti-DNA antibody and DNA. The results indicate that immune complex systems other than, or in addition to, DNA-anti-DNA are likely to play a role in the pathogenesis of murine lupus nephritis.