Joseph J. Boyle

Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans

Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.

Coronary Intraplaque Hemorrhage Evokes a Novel Atheroprotective Macrophage Phenotype

Intraplaque hemorrhage accelerates atherosclerosis via oxidant stress and contributes to lesion development and destabilization. Normally, macrophages scavenge hemoglobin-haptoglobin (HbHp) complexes via CD163, and this process provokes the secretion of the anti-inflammatory atheroprotective cytokine interleukin (IL)-10. We therefore tested the hypothesis that HbHp complexes may drive monocyte differentiation to an atheroprotective phenotype. Examination of the macrophage phenotype in hemorrhaged atherosclerotic plaques revealed a novel hemorrhage-associated macrophage population (HA-mac), defined by high levels of CD163, but low levels of human leukocyte antigen-DR. HA-mac contained more iron, a pro-oxidant catalyst, but paradoxically had less oxidative injury, measured by 8-oxo-guanosine content. Differentiating monocytes with HbHp complexes reproduced the CD163(high) human leukocyte antigen-DR(low) HA-mac phenotype in vitro. These in vitro HA-mac cells cleared Hb more quickly, and consistently showed less hydrogen peroxide release, highly reactive oxygen species and oxidant stress, and increased survival. Differentiation to HA-mac was prevented by neutralizing IL-10 antibodies, indicating that IL-10 mediates an autocrine feedback mechanism in this system. Nonlinear dynamic modeling showed that an IL-10/CD163-positive feedback loop drove a discrete HA-mac lineage. Simulations further indicated an all-or-none switch to HA-mac at threshold levels of HbHp, and this conversion was experimentally verified. These data demonstrate the creation of a novel atheroprotective (HA-mac) macrophage subpopulation in response to intraplaque hemorrhage and raise the possibility that therapeutically reproducing this macrophage phenotype may be cardio-protective in cases of atherosclerosis.

Immunoglobulin M Is Required for Protection Against Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Background— Immunoglobulin M (IgM) natural antibodies bind oxidatively-modified low-density lipoprotein (LDL) and apoptotic cells and have been implicated as being important for protection against atherosclerosis. We have directly investigated the requirement for IgM by studying the effects of IgM deficiency in LDL receptor–deficient (Ldlr−/−) mice. Methods and Results— Mice deficient in serum IgM (sIgM) or complement C1q were crossed with Ldlr−/− mice and studied on both low-fat and high-fat semisynthetic diets. On both diets, en face and aortic root atherosclerotic lesions in sIgM.Ldlr−/− mice were substantially larger and more complex, with accelerated cholesterol crystal formation and increased smooth muscle cell content in aortic root lesions. Combined C1q and IgM deficiency had the same effect as IgM deficiency alone. Increased apoptosis was observed in aortic root lesions of both sIgM.Ldlr−/− and C1qa.Ldlr−/− mice. Because lesions were significantly larger in IgM-deficient mice than in the absence of C1q, IgM protective mechanisms appear to be partially independent of classical pathway activation and apoptotic cell clearance. Levels of IgG antibodies against copper-oxidized LDL were lower in sIgM.Ldlr−/− mice fed a high-fat diet, suggesting compensatory consumption of IgG in the absence of IgM. Conclusions— This study provides direct evidence that IgM antibodies play a central role in protection against atherosclerosis. The mechanism appears to be at least partly independent of classical pathway complement activation by C1q.

Cooperative Interactions Between RB and p53 Regulate Cell Proliferation, Cell Senescence, and Apoptosis in Human Vascular Smooth Muscle Cells From Atherosclerotic Plaques

Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105RB (retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.

Specific C-Terminal Cleavage and Inactivation of Interleukin-8 by Invasive Disease Isolates of Streptococcus pyogenes

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.

Tumor Necrosis Factor-α Promotes Macrophage-Induced Vascular Smooth Muscle Cell Apoptosis by Direct and Autocrine Mechanisms

Objective—We have previously shown that human macrophages induce human plaque vascular smooth muscle cell (VSMC) apoptosis by cell-cell proximity, Fas-L, and nitric oxide (NO), thereby predisposing to plaque rupture. This study sought to analyze whether tumor necrosis factor-&agr; (TNF-&agr;) contributes additionally to macrophage-induced VSMC apoptosis. Methods and Results—Macrophage-induced VSMC apoptosis was examined in direct coculture. Antagonistic antibodies to TNF-receptor (R1) inhibited VSMC apoptosis, and preincubation of monocytes and VSMCs indicated that TNF-R1 on both cell types contributed to macrophage-induced VSMC apoptosis. Correspondingly, both monocytes and VSMCs expressed TNF-R1, and macrophages expressed cell surface TNF-&agr;. Two NO donors upregulated VSMC surface TNF-R1, and exogenous TNF-&agr; induced VSMC apoptosis synergistically with the NO donor diethylenetriamine/NO, indicating that NO sensitizes VSMCs to TNF-&agr;. Neutralizing anti–TNF-R1 antibodies inhibited macrophage activation assessed by Fas-L expression and NO secretion. Conclusions—TNF-&agr; promotes macrophage-induced VSMC apoptosis by autocrine and direct pathways.

Activating Transcription Factor 1 Directs Mhem Atheroprotective Macrophages Through Coordinated Iron Handling and Foam Cell Protection

Rationale: Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH. Objective: This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. Methods and Results: To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-&bgr;). Heme-induced HO-1 and LXR-&bgr; were suppressed by knockdown of ATF-1, and HO-1 and LXR-&bgr; were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-&bgr; in turn led to induction of other genes central to cholesterol efflux, such as LXR-&agr; and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. Conclusions: These results show that ATF-1 mediates HO-1 induction by heme and drives macrophage adaptation to intraplaque hemorrhage. Our definition of an ATF-1–mediated pathway for linked protection from foam cell formation and oxidant stress may have therapeutic potential.

Apoptosis of vascular smooth muscle cells in atherosclerosis

Apoptosis (programmed cell death) of vascular smooth muscle cells and macrophages has recently been demonstrated in the following: (a) human atherosclerotic plaques; (b) physiological remodelling of the vessel; and (c) a variety of disease states. Apoptosis is a highly regulated mechanism of death, controlled by the interactions between the following: (i) cellular receptors; (ii) cytoplasmic and nuclear gene products; and (iii) the local cytokine and cellular environment within the plaque. The knowledge of the key regulators of apoptosis does, however, offer novel therapeutic targets in both the prevention and treatment of atherosclerosis.

Mycobacterium tuberculosis Up-Regulates Matrix Metalloproteinase-1 Secretion from Human Airway Epithelial Cells via a p38 MAPK Switch

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung’s tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-α and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-α synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.

Human Macrophage-Induced Vascular Smooth Muscle Cell Apoptosis Requires NO Enhancement of Fas/Fas-L Interactions

Objective—We have previously shown that macrophages induce vascular smooth muscle cell (VSMC) apoptosis in vitro by cell-cell proximity and Fas-L/Fas interactions. Because NO is a short-range mediator, we tested whether NO mediates macrophage-induced VSMC apoptosis. Methods and Results—NO synthase (NOS) inhibitors markedly inhibited macrophage-induced apoptosis of carotid plaque VSMCs (apoptotic indices, 81±2.9% for control and 28.2±3.9% for NG-nitro-l-arginine methyl ester [L-NAME] treatment) and coronary medial VSMCs (apoptotic indices, 76±5.5% for control and 3.5±0.8% for L-NAME treatment). Inactive enantiomers were without effect (P >0.05). Cultured macrophages, but not VSMCs, expressed inducible NOS (but not neuronal NOS or endothelial NOS) concomitant with activation and secreted 1.51±0.3 fmol nitrite per cell, which was blocked by L-NAME (100 &mgr;mol/L). Diethylene triamine nitric oxide (DETA/NO) and sodium nitroprusside (NO donors) induced VSMC cell-surface Fas and enhanced plaque VSMC apoptosis induced by agonistic anti-Fas antibody (apoptotic indices, 6.6±1.8% for control, 6.3±1.5% for DETA/NO, 26±1.8% for Fas, and 44±6.9% for Fas+DETA/NO). In isolated macrophages, NOS inhibitors reduced and NO donors increased surface Fas-L, indicating an NO-dependent autocrine enhancement of macrophage surface Fas-L. Conclusions—Together, these data indicate that macrophage-derived NO is required for macrophage-induced VSMC apoptosis and that it acts by enhancing Fas-L/Fas interactions.