IL-33 reduces the development of atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1-like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE(-/-) mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33-treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFNgamma in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG(1), but decreased IgG(2a), which is consistent with a Th1-to-Th2 switch. IL-33-treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE(-/-) mice compared with control IgG-treated mice. Furthermore, coadministration of an anti-IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.     

Renin inhibition reduces hypercholesterolemia-induced atherosclerosis in mice

The role of the renin angiotensin system (RAS) in atherosclerosis is complex because of the involvement of multiple peptides and receptors. Renin is the rate-limiting enzyme in the production of all angiotensin peptides. To determine the effects of renin inhibition on atherosclerosis, we administered the novel renin inhibitor aliskiren over a broad dose range to fat-fed LDL receptor-deficient (Ldlr(-/-)) mice. Renin inhibition resulted in striking reductions of atherosclerotic lesion size in both the aortic arch and the root. Subsequent studies demonstrated that cultured macrophages expressed all components of the RAS. To determine the role of macrophage-derived angiotensin in the development of atherosclerosis, we transplanted renin-deficient bone marrow to irradiated Ldlr(-/-) mice and observed a profound decrease in the size of atherosclerotic lesions. In similar experiments, transplantation of bone marrow deficient for angiotensin II type 1a receptors failed to influence lesion development. We conclude that renin-dependent angiotensin production in macrophages does not act in an autocrine/paracrine manner. Furthermore, in vitro studies demonstrated that coculture with renin-expressing macrophages augmented monocyte adhesion to endothelial cells. Therefore, although previous work suggests that angiotensin peptides have conflicting effects on atherogenesis, we found that renin inhibition profoundly decreased lesion development in mice.     

MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B

The earliest recognizable atherosclerotic lesions are fatty streaks composed of lipid-laden macrophages (foam cells). Circulating monocytes are the precursors of these foam cells, but the molecular mechanisms that govern macrophage trafficking through the vessel wall are poorly understood. Monocyte chemoattractant protein-1 (MCP-1), a member of the chemokine (chemotactic cytokine) family, is a potent monocyte agonist that is upregulated by oxidized lipids. Recent studies in hypercholesterolemic mice lacking apo E or the low-density lipoprotein receptor have suggested a role for MCP-1 in monocyte recruitment to early atherosclerotic lesions. To determine if MCP-1 is critically involved in atherogenesis in the setting of elevated physiological plasma cholesterol levels, we deleted the MCP-1 gene in transgenic mice expressing human apo B. Here we report that the absence of MCP-1 provides dramatic protection from macrophage recruitment and atherosclerotic lesion formation in apo B transgenic mice, without altering lipoprotein metabolism. Taken together with the results of earlier studies, these data provide compelling evidence that MCP-1 plays a critical role in the initiation of atherosclerosis.     

Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice

Human atherosclerotic lesions overexpress the lysosomal cysteine protease cathepsin S (Cat S), one of the most potent mammalian elastases known. In contrast, atheromata have low levels of the endogenous Cat S inhibitor cystatin C compared with normal arteries, suggesting involvement of this protease in atherogenesis. The present study tested this hypothesis directly by crossing Cat S-deficient (CatS(-/-)) mice with LDL receptor-deficient (LDLR(-/-)) mice that develop atherosclerosis on a high-cholesterol diet. Compared with LDLR(-/-) mice, double-knockout mice (CatS(-/-)LDLR(-/-)) developed significantly less atherosclerosis, as indicated by plaque size (plaque area and intimal thickening) and stage of development. These mice also had markedly reduced content of intimal macrophages, lipids, smooth muscle cells, collagen, CD4(+) T lymphocytes, and levels of IFN-gamma. CatS(-/-)LDLR(-/-) monocytes showed impaired subendothelial basement membrane transmigration, and aortas from CatS(-/-)LDLR(-/-) mice had preserved elastic laminae. These findings establish a pivotal role for Cat S in atherogenesis.     

Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice.

Atherosclerosis is associated with immune activation. T cells and macrophages infiltrate atherosclerotic plaques and disease progression is associated with formation of autoantibodies to oxidized lipoproteins. In the apo E knockout mouse, a genetic model of cholesterol-induced atherosclerosis, congenital deficiency of macrophages, lymphocytes, or interferon-gamma receptors result in reduced lesion formation. We have now evaluated whether immune modulation in the adult animal affects disease development. Injections of 7-wk-old male apo E knockout mice with polyclonal immunoglobulin preparations (ivIg) during a 5-d period reduced fatty streak formation over a 2-mo period on cholesterol diet by 35%. Fibrofatty lesions induced by diet treatment for 4 mo were reduced by 50% in mice receiving ivIg after 2 mo on the diet. ivIg treatment also reduced IgM antibodies to oxidized LDL and led to inactivation of spleen and lymph node T cells. These data indicate that ivIg inhibits atherosclerosis, that it is effective both during the fatty streak and plaque phases, and that it may act by modulating T cell activity and/or antibody production. Therefore, immunomodulation may be an effective way to prevent and/or treat atherosclerosis.     

High phosphate diet reduces atherosclerosis formation in apolipoprotein E-deficient mice

Although higher serum phosphate level is a risk factor for cardiovascular diseases in general population as well as chronic kidney disease patients, it has not been clarified whether higher phosphate can affect atherosclerotic plaque formation. In this study, we investigated the effect of prolonged-intake of different concentrations of phosphate on atherosclerosis formation using apolipoprotein E-deficient mice. Apolipoprotein E-deficient mice were fed with high fat diet including 0.6%, 1.2% or 1.8% phosphate. After 20-week treatment, atherosclerotic plaque formation in aorta in 1.8% phosphate diet group was unexpectedly less than that in the other groups. To elucidate mechanisms of suppression of plaque formation by high phosphate diet, we hypothesized that high phosphate diet may modify a profile of monocytes/macrophages suppressing plaque formation. We confirmed that elevated peripheral monocytes (CD11b+, F4/80+ cell numbers) in apolipoprotein E-deficient mice were decreased by feeding with 1.8% P diet. In addition, ex vivo study indicated that high dose of phosphate induced macrophage apoptosis. These observations suggest that excess phosphate intake decreased atherosclerosis formation, at least in part, by changing the profile of peripheral monocytes or inducing apoptosis of macrophages in apolipoprotein E-deficient mice.     

Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice

Current therapies for non-Hodgkin lymphoma commonly include CD20 mAb to deplete tumor cells. However, the response is not durable in a substantial proportion of patients. Herein, we report our studies in mice testing the hypothesis that heterogeneity in endogenous tissue CD20+ B cell depletion influences in vivo lymphoma therapy. Using highly effective CD20 mAbs that efficiently deplete endogenous mature B cells and homologous CD20+ primary lymphoma cells through monocyte- and antibody-dependent mechanisms, we found that lymphoma depletion and survival were reduced when endogenous host B cells were not depleted, particularly a rare IL-10-producing B cell subset (B10 cells) known to regulate inflammation and autoimmunity. Even small numbers of adoptively transferred B10 cells dramatically suppressed CD20 mAb-mediated lymphoma depletion by inhibiting mAb-mediated monocyte activation and effector function through IL-10-dependent mechanisms. However, the activation of innate effector cells using a TLR3 agonist that did not activate B10 cells overcame the negative regulatory effects of endogenous B10 cells and enhanced lymphoma depletion during CD20 immunotherapy in vivo. Thus, we conclude that endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells able to inhibit lymphoma depletion by CD20 mAbs. Consequently, B10 cell removal could provide a way to optimize CD20 mAb-mediated clearance of malignant B cells in patients with non-Hodgkin lymphoma.     

Fibrinogen deficiency is compatible with the development of atherosclerosis in mice.

A critical role of the coagulation system in the development of atherosclerosis has been frequently postulated based on a variety of indirect observations, including the expression of procoagulants and fibrinolytic factors within atherosclerotic vessels, the presence of substantial amounts of fibrin(ogen) and fibrin degradation products within intimal lesions, the cellular infiltration and assimilation of mural thrombi into developing plaques, and the identification of high plasma fibrinogen (Fib) levels as an independent risk factor for the development of ischemic heart disease. To directly examine the role of fibrin(ogen) in atherogenesis, Fib-deficient mice were crossed to atherosclerosis-prone apolipoprotein E (apo E)-deficient mice. Both apo E-/- and apo E-/-/Fib-/- mice developed lesions throughout the entire aortic tree, ranging in appearance from simple fatty streaks to complex fibrous plaques. Furthermore, remarkably little difference in lesion size and complexity was observed within the aortae of age- and gender-matched apo E-/- and apo E-/-/Fib-/- mice. These results indicate that the contribution of fibrin(ogen) to intimal mass and local cell adhesion, migration, and proliferation is not strictly required for the development of advanced atherosclerotic disease in mice with a severe defect in lipid metabolism.     

B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.     

Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice.

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.     

Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice

OBJECTIVE: Erythropoietin is a potent stimulator of erythroid progenitor cells, and its expression is enhanced by hypoxia. In the present study, we investigated the effects of erythropoietin (1000 IU/kg subcutaneously) on the development of nonseptic shock caused by zymosan. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: Male CD mice. INTERVENTIONS: Mice received either intraperitoneally zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) or vehicle (0.25 mL/mouse saline). Erythropoietin was administered at the dose of 1000 IU/kg subcutaneously, 1 and 6 hrs after zymosan administration. Organ failure and systemic inflammation in mice was assessed 18 hrs after administration of zymosan and/or erythropoietin. MEASUREMENTS AND MAIN RESULTS: Treatment of mice with erythropoietin (1000 IU/kg subcutaneously, 1 and 6 hrs after zymosan administration) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. Erythropoietin also attenuated the lung, liver, and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase activity caused by zymosan in the lung and intestine. Immunohistochemical analysis for nitrotyrosine and poly(ADP-ribose) revealed positive staining in lung and intestine tissues obtained from zymosan-treated mice. The degree of staining for nitrotyrosine and poly(ADP-ribose) was markedly reduced in tissue sections obtained from zymosan-treated mice, which received erythropoietin. In addition, administration of zymosan caused severe illness in the mice characterized by a systemic toxicity, significant loss of body weight, and a 70% mortality rate at the end of observation period (7 days). Treatment with erythropoietin significantly reduced the development of systemic toxicity, the loss in body weight, and the mortality caused by zymosan. CONCLUSIONS: This study provides evidence, for the first time, that erythropoietin attenuates the degree of zymosan-induced nonseptic shock in mice.     

Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor- deficient mice.

Previous studies of osteopetrotic (op) mice lacking macrophage colony-stimulating factor (M-CSF) have revealed an inhibition of atherosclerosis development in the apolipoprotein E (apo E)-deficient model and in a diet-induced model. Using LDL receptor-deficient mice, we now show that atheroma development depends on M-CSF concentration, as not only did homozygous osteopetrotic (op/op) mice have dramatically reduced lesions (approximately 0.3% of control lesion size) but heterozygous (op/+) mice had lesions < 1% of controls. Mice heterozygous for the op mutation (op/+) had plasma levels of M-CSF about half those in controls (+/+). The finding that an approximately 2-fold reduction in M-CSF expression reduced lesion size approximately 100-fold suggests the requirement for a threshold level of M-CSF. The effect of M-CSF on atherosclerosis did not appear to be mediated either by changes in plasma lipoprotein levels or alterations in the number of circulating monocytes, since both op/op and op/+ mice exhibited higher levels of atherogenic lipoprotein particles and (op/+) mice showed a near normal number of circulating monocytes. LDL receptor-null littermates of genotypes from op/op, op/+, to +/+ showed monocyte differentials of approximately 4.5, 8, and 10%, respectively. Taken together, these results suggest that the effects of M-CSF on atherogenesis may not be mediated by expression of M-CSF systemically or by modulation of the number of circulating monocytes. These studies support the conclusion that M-CSF participates critically in fatty streak formation and progression to a complex fibrous lesion.     

Chromium depletion in the pathogenesis of diabetes and atherosclerosis.

Dietary Cr deficiency may cause the aging-related Cr depletion observed in humans. Such depletion, when it occurs with excessive consumption of sugar and other carbohydrates, may result in glucose intolerance, glycosuria, hyperinsulinemia, and hyperlipidemia. These metabolic disorders could explain why athero-sclerotic diseases are endemic to most Western industrial societies. This review detailed significant current research and findings on the manifestations of Cr deficiency in humans and animal models.     

Modification of apolipoprotein(a) lysine binding site reduces atherosclerosis in transgenic mice.

Lipoprotein(a) contributes to the development of atherosclerosis through the binding of its plasminogen-like apolipoprotein(a) component to fibrin and other plasminogen substrates. Apolipoprotein(a) contains a major lysine binding site in one of its kringle domains. Destruction of this site by mutagenesis greatly reduces the binding of apolipoprotein(a) to lysine and fibrin. Transgenic mice expressing this mutant form of apolipoprotein(a) as well as mice expressing wild-type apolipoprotein(a) have been created in an inbred mouse strain. The wild-type apolipoprotein(a) transgenic mice have a fivefold increase in the development of lipid lesions, as well as a large increase in the focal deposition of apolipoprotein(a) in the aorta, compared with the lysine binding site mutant strain and to nontransgenic littermates. The results demonstrate the key role of this lysine binding site in the pathogenic activity of apolipoprotein(a) in a murine model system.     

Polyclonal B cell activation in lupus-prone mice precedes and predicts the development of autoimmune disease.

Polyclonal B cell activation is an early feature of autoimmune disease in humans and mice with systemic lupus erythematosus. The contribution of polyclonal activation to the progression of autoimmunity is unclear, however, since it precedes the development of end-organ damage by months or years. To examine this issue, 109 autoimmune-prone (NZB X NZW)F1 X NZB backcross mice were hemi-splenectomized at 10 wk and the number and antigenic specificity of their Ig-secreting B cells quantitated by ELISA spot assay. Of the 61 mice that had polyclonally increased numbers of Ig-secreting cells/spleen, 31 died by 6 mo. In contrast, 0/48 backcross mice with normal numbers of Ig-secreting B cells at 10 wk died over the same period (P less than 0.001). Polyclonally activated mice also developed proteinuria earlier and more frequently than littermates with normal numbers of Ig-secreting cells (P less than 0.001). As adults, backcross mice with proteinuria expressed repertoires skewed towards the production of anti-DNA antibodies. At 10 wk these same mice expressed repertoires marked by polyclonal activation rather than preferential anti-DNA production. These findings indicate that autoimmune disease in SLE is accompanied by the autoantigen-driven production of autoantibodies but is preceded and predicted by polyclonal B cell activation.