Macrophages, Oxidative Stress, and Atherosclerosis - Springer Link

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Macrophages, Oxidative Stress, and Atherosclerosis Anna V. Mathew and Subramaniam Pennathur Department of Medicine, Division of Nephrology, University of Michigan, Ann Arbor, MI, USA

Synonyms Atherosclerosis and macrophages

Definition Vascular inflammation and oxidative stress mediated by macrophages play a key role in the development and progression of atherosclerotic disease. Both of these processes accentuate a proinflammatory milieu which propagates atherosclerosis.

Introduction Atherosclerotic vascular disease is the leading cause of cardiovascular disease (CVD) and stroke, the two major causes of death in the United States. In response to physiologic stimuli, the vasculature responds dynamically to regulate arterial vascular tone and maintains endothelial integrity and dynamics by producing vasodilators and vasoconstrictors. Risk factors such as

diabetes, smoking, hypercholesterolemia, autoimmune disease, and hypertension interfere with this response, promoting macrophage activation, endothelial dysfunction, and atherosclerosis. Recent evidence suggests a central role for vascular inflammation mediated by macrophages and resultant oxidative stress in atherosclerosis and endothelial dysfunction. These studies support the hypothesis that unique oxidants generated in microvasculature and inflammatory cells promote atherosclerosis by lipoprotein oxidation and impairment of macrophage function. Therapies interrupting these oxidative pathways in vascular tissue might help prevent cardiovascular disease. According to the recent report by the National Vital Statistics, in 2008 over 616,000 people died of heart disease, which translates to roughly 1 in 4 Americans (Minino et al. 2011). CVD remains the leading cause of death for both men and women. In 2008, 405,309 people died from coronary heart disease (CHD). The costs due to health-care services, medications, and loss of productivity for CHD alone have ballooned to an estimated $108.9 billion in 2010 and expected to grow in the future (Heidenreich et al. 2011). Additional manifestations of atherosclerosis include peripheral vascular disease and cerebrovascular events which further increase morbidity, mortality, and economic impact of this process. Therefore, understanding molecular mechanisms of initiation and progression of atherosclerosis has been a prime focus of research of several investigators.

I.R. Mackay, N.R. Rose (eds.), Encyclopedia of Medical Immunology – Autoimmune Diseases, DOI 10.1007/978-0-387-84828-0, # Springer Science+Business Media New York 2014

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Macrophages, Oxidative Stress, and Atherosclerosis

Atherosclerosis is a chronic inflammatory disease characterized by infiltration of lipids and inflammatory cells, such as monocytederived macrophages and T-lymphocytes into the artery wall (Ross 1999). Macrophages were the first inflammatory cells to be associated with atherosclerosis. Macrophages produce proinflammatory cytokines, participate in lipid retention and vascular cell remodeling, and express pattern-recognition receptors (PRR), including scavenger receptors (SR) and tolllike receptors (TLRs) that connect the innate and adaptive immune response during atherosclerosis. Macrophages are linked to all stages of atherosclerosis including lesion initiation, propagation of atheroma, and rupture and acute myocardial events (Moore and Tabas 2011). Macrophages can produce reactive oxidants that can damage proteins and lipids and accentuate atherosclerosis (Fig. 1).

levels of interleukin-10 (IL-10) and secrete proinflammatory cytokines. The alternatively activated protective M2 macrophages are driven by Th2 cytokines. M1 and M2 macrophages play complimentary but opposing roles during inflammation restraining the process. Indeed, both are present in atherosclerotic lesions, and the balance between the two types determines the degree of vascular inflammation. Flow cytometric analysis of the aortic lesions of low-density lipoprotein (LDL) receptor-deficient mice fed an atherogenic diet for 30 weeks revealed that 39 % of the aortic macrophages express M1 marker CD86, 21 % expressed M2 marker CD206, 45 % expressed Mox marker heme oxygenase 2, and 10 % co-expressed CD86 (Kadl et al. 2010). Upon entry in to the subendothelial space, the macrophages express high levels of scavenger receptor A (SR-A), lipoxygenase 1 (LOX1), chemokine (C-X-C motif) ligand 16 (CXCL16), and CD36. Endoplasmic reticulum (ER) stress is a key regulator to the macrophage differentiation and cholesterol deposition. When macrophages from diabetics were exposed to oxidized LDL (OxLDL) after either alternative activation into M2 or classic activation into M1, the M2 macrophages formed more foam cells and expressed the scavenger receptors CD36 and SR-A1. ER stress was necessary to generate M2 macrophages through c-Jun N-terminal kinases (JNK) activation and increased peroxisome proliferatoractivated receptor (PPAR g) expression. Absence of SR signaling decreased ER stress and prevented M2 macrophage formation. Furthermore, suppression of ER stress decreased M2 formation and foam cell formation by increasing the high-density lipoprotein (HDL) and Apo lipoprotein A1 (Apo A1)-mediated efflux (Oh et al. 2012).

Macrophage Polarization in Atheroma Mac-1+ cells are the resident macrophages in the arterial wall and express CD11b, CD68, and F4/80. Classically activated M1 macrophages, driven by Th1 cytokines (interferon gamma (IFN-g) and tumor necrosis factor (TNF)) or by lipopolysaccharide (LPS), produce high levels of interleukins 12 and 23 (IL-12 and IL-23) and low

Oxidized LDL and Atherosclerosis Although it is well known that elevated levels of LDL greatly increase the risk for atherosclerosis, in vitro studies suggest that LDL by itself is not atherogenic but needs to be modified to initiate atherosclerotic disease. This conclusion led to the “oxidation hypothesis,” which proposed that LDL must be oxidatively modified to become

Some conditions like obesity, diabetes, autoimmune diseases, and kidney disease magnify the burden of atherosclerotic disease by accentuating oxidant stress. In this review, the pivotal role of macrophages and oxidative stress in atherosclerosis and the link between the two processes will be discussed. The potential relationship of lipoproteins and reactive carbonyls to macrophage-derived oxidant-generating pathways and the rationale for therapies aimed at decreasing oxidative stress are emphasized. Identifying specific pathways of reactive oxidant generation in vascular tissue will ultimately lead to design of drugs to interrupt this process and prevent atherosclerotic complications.

Role of Macrophages in Atherosclerosis


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Inflammatory Lipid Mediators Lipoxygenase

−NO2

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MPO

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Tyr O2

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NO2•

HOCl

Tyrosyl Radical

ATHEROSCLEROSIS

NOX

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Macrophage Polarization Inflammatory cytokines

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Macrophages, Oxidative Stress, and Atherosclerosis, Fig. 1 Macrophages-derived oxidants and inflammatory stimuli promote atherosclerosis. Pathologic conditions result in increased superoxide (O2•) by macrophages through NADPH oxidase (NOX). O2• can dismutate to yield hydrogen peroxide (H2O2). H2O2 can react with redox-active metal ions (Mn+) to form hydroxyl radical or

with phagocyte-derived myeloperoxidase (MPO) to nitrate (nitrogen dioxide; NO2•), chlorinate (hypochlorous acid; HOCl), or cross-link (tyrosyl radical) proteins. Macrophages can also promote a proinflammatory milieu by generation of a multitude of cytokines which can overwhelm endogenous anti-inflammatory antioxidant defenses resulting in atherosclerosis

atherogenic. Many lines of evidence support this hypothesis. OxLDL is taken up by SR-A and CD 36 of macrophages, which then become lipidladen foam cells, the pathologic hallmark of early atherosclerotic lesions. OxLDL has been isolated from human and animal atherosclerotic tissue, and immunohistochemical studies have detected oxidized lipids in atherosclerotic lesions. All major cell types involved in atherosclerosis – smooth muscle cells, endothelial cells, and macrophages – produce reactive oxidants that can oxidize LDL in vitro. Moreover, OxLDL attracts mononuclear cells and stimulates the production of monocyte chemoattractant protein-1 (MCP-1) and other inflammatory cytokines such as macrophage colonystimulating factor (M-CSF), leading to the conversion of fatty streaks to more advanced

complex lesions as smooth muscle cells migrate from the media into the subendothelial space. OxLDL may also stimulate smooth cells to synthesize extracellular matrix and activate a signaling cascade by interacting with the lectin-like OxLDL receptor. Finally, several structurally unrelated lipid-soluble antioxidants that inhibit LDL oxidation in vitro also inhibit atherosclerosis in hypercholesterolemic animals (reviewed in Vivekanadan-Giri et al. 2008). There is some controversy about the function of scavenger receptors in atherogenesis. Early studies showed that SR plays a pro-atherogenic role in atherosclerosis. Crossing CD36/ null mice on apolipoprotein E (ApoE)-deficient background showed protection from atherosclerosis (Febbraio et al. 2000). In a separate study, ApoE/ mice lacking SR-A or CD36 showed

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increased aortic sinus lesion areas with abundant foam cells, suggesting alternative lipid uptake mechanisms and a possible athero-protective role for SR-A and CD36 (Moore et al. 2005). ApoE/ mice with a combined deficiency of CD36 and SR-A I/II showed no further reduction of atherosclerosis compared with Cd36/ApoE/ mice (Kuchibhotla et al. 2008). High-Density Lipoproteins and Reverse Cholesterol Transport HDL, a lipid-protein complex, has long been recognized for its inverse association with CVD, largely attributed to its ability to remove excess cholesterol from arterial wall macrophages, a process known as reverse cholesterol transport (RCT; Tall et al. 2008). Once internalized, free cholesterol trafficking to the cell membrane may play a role in lesion regression, and deficiency in this pathway in certain macrophages might be the reason for lesion progression. Once at the membrane, the ABCA1 (ATP-binding cassette protein A1)- and ABCG1 (ATP-binding cassette G1)-mediated transfer to Apo A1 and HDL respectively occurs (Tall et al. 2008). Passive diffusion occurs as well. Along with its important role in mediating RCT, HDL has multiple endothelial actions that also afford cardiovascular protection, including antioxidative, anti-inflammatory, antiapoptotic, and antithrombotic activities. Further, HDL activates endothelial nitric oxide (NO) synthase (Besler et al. 2011), enhances endothelial progenitor cell-mediated endothelium repair, and stimulates endothelial cell proliferation and migration. Additionally, increasing the levels of HDL by drugs, gene therapy, or direct infusion has been shown to improve outcomes in animal models and in patients with CVD. Role of Macrophages in Thinning of Fibrous Cap/Plaque Rupture The older and mature plaques rupture and ulcerate at the shoulder regions enriched with macrophages and macrophage-derived foam cells. Macrophages trigger smooth muscle cell


apoptosis by activating apoptotic pathways and by secreting TNF-a and NO (Boyle et al. 2003). Macrophages also decrease transforming growth factor (TGF) b production causing decrease in collagen production (Fadok et al. 1998). With prolonged ER stress, the unfolded protein response (UPR) effector C/EBP homologous protein (CHOP) can trigger UPR. Macrophages secrete metalloproteinases (MMP) which can lead to thinning of the fibrous cap due to their protease activity and may predispose to the unstable plaque and rupture (Tabas 2010). Macrophages produce tissue plasminogen activator (TPA) and plasminogen activator inhibitor type 1(PAI-1) for control of local fibrinolysis. PAI-1 and alpha 2-antiplasmin are elevated in atherosclerotic plaques while TPA is low, which may favor thrombosis and predispose to acute MI (Schwartz et al. 1988). Diabetes Promotes Inflammatory Macrophage Phenotype Recent evidence points out to direct effects of diabetes on atherosclerotic lesion cells, such as macrophages, which play an additional important role in accounting for increased atherosclerotic risk in diabetes. Thus, the increased macrophage expression of inflammatory mediators associated with diabetes can be mimicked by elevated glucose concentrations in vitro (Wen et al. 2006). In addition, fatty acids exert inflammatory effects in macrophages, which could contribute to inflammation in the setting of diabetesaccelerated atherosclerosis and possibly other complications (Hummasti and Hotamisligil 2010). After entering the cell, fatty acids are thio-esterified into their acyl-CoA derivatives catalyzed by long-chain acyl-CoA synthetases (ACSLs). Kanter et al. (2012) demonstrated that monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protected monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic


mice without affecting lesions in nondiabetic mice (Kanter et al. 2012). These observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with diabetes. Macrophage Sterol Regulation and Atherosclerosis Sterol-regulated transcriptional factors liver X receptors (LXR a and b) appear to be antiatherogenic as mice lacking LXR’s have advanced atherosclerosis and LXR agonists decrease atherosclerosis. Transplanting aortic arches from atherosclerotic ApoE-deficient mice with LXR a and LXR b deficiency into wild-type mice resulted in marked regression of atherosclerotic lesions (Feig et al. 2010). The salutary effects of LXR’s in part are thought to be due to upregulation of RCT due to increased ABCA1 and ABCG1 activity in macrophages. Activation of PPAR g can inhibit foam cell formation with and without help from the ABCA1 pathways. PPAR g activation reduces cholesterol esterification and induces ABCG1 expression. Becker et al. showed that the peritoneal macrophages from chow- versus high-fat-fed LDL receptor-deficient mice differentially expressed cytoskeleton, lipid trafficking, and lipid-binding proteins utilizing mass spectrometry (MS)-based proteomic techniques (Becker et al. 2010). Their analysis revealed a sterol-responsive network that is highly enriched in proteins with known physical interactions and established roles in vesicular transport associated with atherosclerotic phenotypes in mice. Pharmacologic intervention with a statin or rosiglitazone and use of mice deficient in LDL receptor or ApoE implicated the network in atherosclerosis. Biochemical fractionation revealed that most of the sterol-responsive proteins resided in microvesicles, providing a physical basis for the network’s functional and biochemical properties. These observations identify a highly integrated network of macrophage proteins whose expression is influenced by environmental, genetic, and pharmacological factors implicated in atherogenesis.

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Role of Oxidative Stress in Atherosclerosis Although oxidative stress has a well-established role in atherosclerosis, its origins and magnitude remain poorly understood. Moreover, it is not known whether oxidative stress is a primary event that occurs early in the disease or whether it represents a secondary phenomenon that merely reflects end-stage tissue damage. This distinction has important clinical relevance. If oxidative stress simply reflects tissue damage, interventions that reduce it may fail to affect the disease process. If oxidative stress promotes tissue injury, therapies that interrupt oxidative pathways early in the disease may prevent complications, and those that act later may slow disease progression. Pathways for Generating Oxidants Many pathways oxidize proteins in vitro. However, the specific pathways that promote oxidative stress in atherosclerosis have not been conclusively identified. One reason is that oxidizing intermediates are difficult to detect in vivo because they are short lived and generated at low levels. Macrophage-Derived Oxidants

(a) The NADPH pathway. The major pathway through which macrophages and other phagocytic cells of the innate immune system generate oxidants begins with the cell membrane-bound NADPH oxidase (NOX), which produces superoxide. Superoxide dismutates into hydrogen peroxide which can be used as a substrate for propagating oxidative reactions by free metal ions (hydroxyl radical) or used as substrate by myeloperoxidase (MPO). Superoxide can also react with NO to form the potent oxidant peroxynitrite. Several NOX isoforms are present in the endothelium, and smooth muscle cells are selectively upregulated by pathologic stimuli, which can augment macrophage-derived oxidants. Potential factors

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include angiotensin II, endothelin-1, hypercholesterolemia, shear stress, nonesterified fatty acids, hyperglycemia, and growth factors. Angiotensin II may represent a pathophysiologically relevant pathway for stimulating the production of reactive intermediates by artery wall cells because inhibitors of this pathway lower the risk for cardiovascular events. In humans, NOX activity correlates inversely with endothelial function, even after other major risk factors for atherosclerosis, including diabetes and hypercholesterolemia, are taken into account (Guzik et al. 2006). (b) The MPO pathway. The peroxide generated by NOX can be used by another phagocyte enzyme, MPO, to convert chloride ion to hypochlorous acid (HOCl). Oxidation of NO with oxygen yields nitrite (NO2), which MPO converts to nitrogen dioxide radical (NO2•), a potent nitrating intermediate (Gaut et al. 2002). Reactive nitrogen species, including peroxynitrite and NO2•, might contribute to the inflammatory process by nitrating lipoproteins and other biomolecules. Hyperglycemia can activate protein kinase C (PKC) (Ishii et al. 1998), which leads to phagocyte activation, secretion of MPO, and oxidant generation. Nonesterified fatty acids (NEFA) that commonly are overabundant in diabetes can also activate phagocytes in vitro. These changes might enhance the production of superoxide and hydrogen peroxide, which myeloperoxidase converts into more potent cytotoxic oxidants, such as HOCl and NO2•. MPO is a major source of reactive oxidants in the human vasculature and has been widely implicated in the development of human atherosclerotic lesions. In large epidemiological studies, the level of plasma MPO is a strong independent predictor of CVD (Brennan et al. 2003). Further, MPO has been localized to atherosclerotic plaques, and oxidants produced by MPO activate protease cascades and plaque rupture (Fu et al. 2001). Lipoproteins that have been modified by MPO and HOCl have also been


detected in human atherosclerotic lesions (Vivekanadan-Giri et al. 2008). For example, MPO has been linked to LDL oxidation, which triggers the formation of lipid-laden foam cells. Oxidants produced by MPO also activate protease cascades that weaken atherosclerotic plaques, increasing the likelihood of rupture (Fu et al. 2001). Moreover by consuming the NO that relaxes blood vessel walls, MPO may cause vasoconstriction and endothelial dysfunction and reduce antiinflammatory function (Baldus et al. 2006). MPO is also a source of reactive nitrogen species because it generates nitrotyrosine in vitro and in vivo (Gaut et al. 2002). Expression of human MPO in macrophages accelerated atherosclerosis in mice (McMillen et al. 2005), strongly supporting the hypothesis that the enzyme’s ability to produce reactive intermediates promotes vascular disease. Consistent with this potential pro-atherogenic role, certain MPO polymorphisms associate with cardiovascular risk. Vita and colleagues (Vita et al. 2004) examined the relationship between serum MPO levels and endothelial function (measured with flow-mediated and nitroglycerin-mediated dilation of the brachial artery) in 298 subjects with or without CVD. Correlates of vasodilator function included established CVD risk factors, such as age and HDL level, as well as serum MPO level. When the data were stratified into quartiles, MPO levels predicted endothelial dysfunction even after multivariate adjustment. MPO binds to glycosaminoglycans in vessel walls, where it has been proposed to oxidize endothelium-derived NO and impair endothelial function. Baldus et al. (2006) demonstrated that heparin mobilizes MPO from vascular compartments in humans with and without CVD. Thus, administration of heparin increased plasma levels of MPO. Heparin treatment also improved endothelial NO bioavailability, as monitored by flowmediated dilation and acetylcholine-induced changes in forearm blood flow. Improved endothelial function correlated with the


extent of heparin-induced MPO release. These observations suggest that MPO represents a mechanism for heparin’s antiinflammatory effects and that it affects vascular NO bioavailability. (c) The reactive nitrogen pathway. Another pathway for generating oxidants involves nitric oxide (NO). NO is produced during inflammation by macrophages, which are early components of atherosclerotic lesions from inducible nitric oxide synthase (iNOS). NO reacts with superoxide (O2•) to generate peroxynitrite, a potent oxidant that converts tyrosine residues to 3-nitrotyrosine. Thus, 3-nitrotyrosine is a marker for the reactive nitrogen pathway. It has been detected in low-density and high-density lipoproteins (LDL and HDL) isolated from human diabetic atherosclerotic lesions (Pennathur et al. 2004), and plasma nitrotyrosine levels are elevated in patients with CHD (Shishehbor et al. 2003). Because acute hyperglycemia promotes vasodilation in humans, glucose might directly or indirectly enhance NO release and oxidant generation. Other Oxidant Pathways That Promote Oxidative Stress in the Vascular Wall

There are several sources of non-macrophagederived oxidants in the artery wall that can propagate oxidative stress in the vascular wall. These include the glycoxidation pathway, which involves glucose-derived oxidants which generate advanced glycosylation end products (AGEs), the mitochondrial electron transport chain, uncoupled endothelial nitric oxide synthase, xanthine oxidase, and non-phagocytic NOX enzymes. These pathways can accentuate oxidative damage in a pro-atherogenic milieu. Experimental Studies Identifying Pathways of Oxidation in Atherosclerosis Mass spectrometry (MS) is a powerful approach for detecting biomarkers of oxidative stress in vivo. Antibody-based assays and dihydroethidium fluorescence have been extensively used to study oxidation-specific epitopes and oxidant production in atherosclerosis.

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These techniques are highly sensitive, and the ability of immunochemical studies to provide anatomical data can localize oxidative events. However, they are nonspecific and, at best, only semiquantitative. In contrast, MS offers a powerful set of analytical tools for quantifying and identifying biomolecules. Isotope dilution gas or liquid chromatography (GC and LC)/MS is a highly sensitive and specific method that is used to quantify oxidation of specific amino acid markers (reviewed in Vivekanandan-Giri et al. 2011). Biomolecules such as oxidized amino acids derived from plasma or tissue are separated by GC or LC and ionized. The mass-to-charge ratios of ions derived by fragmenting the ionized, parent compound are determined by MS. Such a spectrum can unequivocally identify a target biomolecule because each compound has a unique fragmentation pattern. The analyte is quantified by adding a stable, isotopically labeled internal standard, which is identical to the target analyte except for the heavy isotope. With certain ionization processes, such as electron capture negative-ion chemical ionization, it is possible to detect and quantify sub-femtomole levels of biomolecules. Oxidized Amino Acids Serve as Molecular Fingerprints for Specific Oxidation Pathways To understand the molecular mechanisms that promote oxidative stress in vivo, a chemical approach to define the patterns of oxidation products that are formed by well- characterized oxidant-generating systems in vitro has been employed. Similar patterns in tissue and plasma were identified. Focus was on proteins because aromatic amino acids constituting proteins retain the initial footprint of the reactive intermediate that initiates oxidative damage. In contrast, lipid peroxidation products readily undergo subsequent chain-propagating reactions, which mask the products formed early in the pathway. Moreover, many different oxidizing intermediates give the same spectrum of oxidized lipids, making it difficult to identify specific pathways that trigger lipid oxidation. Oxidizing intermediates are difficult to detect in vivo because they are short lived and generated

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at low levels. To sidestep this problem, acidstable products of protein oxidation were identified and monitored, both in vitro and in vivo (Bhattacharjee et al. 2001; Pennathur et al. 2001, 2004, 2005). The overall approach is to use isotope dilution MS to accurately identify oxidized amino acids isolated from tissue proteins. These markers, which include ortho-tyrosine, meta-tyrosine, dityrosine, 3-nitrotyrosine, and 3-chlorotyrosine indicate which biochemical pathway has damaged a protein (reviewed in Vivekanadan-Giri et al. 2008, 2011). MS Quantitation of Oxidized Amino Acids in Aortic Proteins of Diabetic Cynomolgus Monkeys Reveals Localized Oxidative Damage by Hydroxyl Radical in the Artery Wall To investigate the potential role of localized oxidative stress in diabetic macrovascular disease, cynomolgus monkeys that had been hyperglycemic for 6 months due to streptozotocin-induced diabetes were used (Pennathur et al. 2001). Samples from seven controls and eight diabetic cynomolgus monkeys were analyzed, sampling three different areas from the thoracic aorta of each animal. Compared with the control samples, the aortic wall proteins from the diabetic animals showed a significant (40 %) increase in ortho-tyrosine and a similar (60 %) increase in meta-tyrosine. This pattern of oxidized amino acids suggests that a hydroxyl radical-like oxidant promotes aortic damage in this animal model of diabetic vascular disease. To determine whether glucose promotes protein oxidation in vivo, we assessed the relationship between level of glycemic control (measured as serum glycated hemoglobin) and levels of amino acid oxidation products in aortic tissue in control and diabetic cynomolgus monkeys. Linear regression analysis demonstrated a strong correlation between levels of both ortho-tyrosine and meta-tyrosine and glycated hemoglobin (r2 ¼ 0.9 and 0.8, respectively; both p < 0.001). Levels of o,o0 -dityrosine and glycated hemoglobin correlated less strongly (r2 ¼ 0.3; p ¼ 0.07).


In contrast, there was no correlation between levels of 3-nitrotyrosine and glycated hemoglobin. These observations support the hypothesis that glucose promotes the formation of orthotyrosine and meta-tyrosine in the artery wall and suggest that both glucose and other pathways contribute to the generation of o,o0 -dityrosine (Pennathur et al. 2001). Plasma and Urinary Levels of Oxidized Amino Acids Are Potential Markers for Assessing Oxidative Stress In Vivo There is increasing evidence that oxidized amino acids in plasma and urine can serve as markers for noninvasive assessment of oxidative stress in vivo. Plasma and urinary levels of these markers are likely to be proportional to the rate of generation and thus can serve as indices of chronic oxidative stress in vivo (Bhattacharjee et al. 2001; Bergt et al. 2004). These observations may be relevant to human pathophysiology. For example, a case–control study demonstrated that systemic levels of protein-bound nitrotyrosine were significantly higher in patients with CVD than in controls with healthy arteries. Moreover, statin therapy lowered levels of oxidation markers in plasma, raising the possibility that these drugs can potentially be antioxidants. Therefore, these markers might serve not only to assess oxidative stress but also to monitor the efficacy of therapy.

HDL Oxidation and Altered HDL Proteome in Atherosclerosis Oxidized amino acids in plasma lipoproteins are markers of lipoprotein oxidation which are implicated in atherogenesis. Preliminary studies demonstrated that HDL, but not LDL, isolated from plasma of subjects with established CVD contained high levels of 3-chlorotyrosine, a highly specific marker for the myeloperoxidase pathway. The level of 3-chlorotyrosine was 13-fold higher in HDL isolated from plasma of subjects with established CVD than in HDL from plasma of healthy subjects (Bergt et al. 2004). Levels of 3-nitrotyrosine were twice as high in


HDL from plasma of patients with established CVD (Pennathur et al. 2004). Circulating HDL from patients with known CVD has impaired reverse cholesterol transport (Bergt et al. 2004; Pennathur et al. 2004; Shao et al. 2012). HDL isolated from atherosclerotic plaques is bound to MPO and enriched in MPO-derived oxidation products, supporting an important role of MPO in the development of dysfunctional HDL. Proteomic approaches were applied to the composition of HDL isolated from healthy subjects and subjects with CVD. Multiple complement regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity were identified. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. MS and biochemical analyses demonstrated that HDL3 from subjects with CVD was selectively enriched in ApoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant CVD. HDL protein cargo plays a role in regulating the complement system and protecting tissue from proteolysis and contributes to its anti-inflammatory and anti-atherogenic properties (Vaisar et al. 2007). These observations raise the exciting possibility that oxidized HDL and HDL proteome might be a novel marker for clinically significant CVD. Furthermore, it highlights the importance of MPO as a mechanistic cause of atherosclerosis.

Lipoxidation and Atherosclerosis Lipoxygenases The LOX enzymes are present in macrophages and are implicated in human atherosclerosis. They are named for the numbered carbon where they oxygenate their polyunsaturated fatty acid (PUFA) substrates (e.g., 12-LOX and 5-LOX). The human LOX enzymes include 5-LOX (which produces leucotrienes), 12-LOX (with platelet-type and leukocyte-type forms), and 15-LOX (which is further separated into the reticulocyte or leukocyte-type, 15-LOX-1).

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The human leukocyte-type 12-LOX and the human reticulocyte-type 15-LOX-1 can form similar products from common substrates and are often referred to in the literature as 12/15-LOXs (Dobrian et al. 2011). In humans, 12/15-LOXs act upon arachidonic acid to form a number of important lipid mediators including 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), 12- and 15-hydroxyeicosatetraenoic acids (HETEs), and hydroxyoctadecadienoic acids (HODEs) from linoleic acid. These lipid products in addition to oxidizing LDL can have a myriad of proinflammatory functions that can propagate atherosclerosis. While the pro-atherogenic role of the 5-LOX pathway is generally better established in animal models and human studies, the role of the 12- and 15-LOX pathways is not yet clear. Indeed, some 15-LOX metabolites are known to be anti-inflammatory and promote a more anti-inflammatory macrophage phenotype in the vessel wall. Hyperlipidemia in Concert with Hyperglycemia Stimulates the Proliferation of Macrophages in Atherosclerotic Lesions: Potential Role of Glucose-Oxidized LDL Macrophage proliferation has been implicated in the progression of atherosclerosis. Recent studies have investigated the effects of hyperglycemia and hyperlipidemia on macrophage proliferation in murine atherosclerotic lesions and isolated primary macrophages (Lamharzi et al. 2004). Glucose promoted lipid and protein oxidation of LDL in vitro (Pennathur et al. 2005). Oxidation of LDL with glucose resulted in a selective increase in protein-bound ortho-tyrosine and meta-tyrosine. Moreover, glucose-oxidized LDL – but not elevated levels of glucose alone – stimulated proliferation of isolated macrophages. These observations may be pertinent to diabetic vascular disease because macrophage proliferation in atherosclerotic lesions was observed in LDL receptor-deficient mice that were both hypercholesterolemic and hyperglycemic but not in mice that were only hyperglycemic (Lamharzi et al. 2004).

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Conclusion and Perspectives Many lines of evidence implicate macrophages and oxidative stress in atherosclerosis. Macrophages play a critical determinant in initiation and progression of atherosclerosis, and its effect, is in part mediated by oxidative stress. Measuring levels of specific oxidized biomolecules by MS show promise for evaluating the role of oxidative stress in the pathogenesis of vascular disease and determine therapeutic efficacy of agents that promote diminished vascular inflammation. Our observations also suggest that chlorinated and/or nitrated HDL might serve as a marker – and perhaps a mechanism – of active cardiovascular disease in humans. If oxidation of HDL by MPO converts the cardioprotective lipoprotein into a dysfunctional form, the enzyme might be a suitable therapeutic target for preventing vascular disease. Additionally agents that improve HDL function and ability to perform RCT would potentially have a major therapeutic role. These include LXR agonists, creating oxidant resistant forms of HDL, peptide, or small molecule HDL mimetics. Additionally, agents that would alter macrophage phenotype to anti-inflammatory state would also be beneficial in preventing CVD.

Cross-References ▶ Dendritic Cells in Atherosclerosis

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Macrophages, Oxidative Stress, and Atherosclerosis Bhattacharjee S, Pennathur S, et al. NADPH oxidase of neutrophils elevates o,o0 -dityrosine cross-links in proteins and urine during inflammation. Arch Biochem Biophys. 2001;395(1):69–77. Boyle JJ, Weissberg PL, et al. Tumor necrosis factor-alpha promotes macrophage-induced vascular smooth muscle cell apoptosis by direct and autocrine mechanisms. Arterioscler Thromb Vasc Biol. 2003;23(9):1553–8. Brennan ML, Penn MS, et al. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med. 2003;349(17):1595–604. Dobrian AD, Lieb DC, et al. Functional and pathological roles of the 12- and 15-lipoxygenases. Prog Lipid Res. 2011;50(1):115–31. Fadok VA, Bratton DL, et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest. 1998;101(4):890–8. Febbraio M, Podrez EA, et al. Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest. 2000;105(8):1049–56. Feig JE, Pineda-Torra I, et al. LXR promotes the maximal egress of monocyte-derived cells from mouse aortic plaques during atherosclerosis regression. J Clin Invest. 2010;120(12):4415–24. Fu X, Kassim SY, et al. Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem. 2001;276(44):41279–87. Gaut JP, Byun J, et al. Myeloperoxidase produces nitrating oxidants in vivo. J Clin Invest. 2002; 109(10):1311–9. Guzik TJ, Sadowski J, et al. Coronary artery superoxide production and NOX isoform expression in human coronary artery disease. Arterioscler Thromb Vasc Biol. 2006;26(2):333–9. Heidenreich PA, Trogdon JG, et al. Forecasting the future of cardiovascular disease in the United States: a policy statement from the American Heart Association. Circulation. 2011;123(8):933–44. Hummasti S, Hotamisligil GS. Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circ Res. 2010;107(5):579–91. Ishii H, Daisuke K, et al. Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J Mol Med. 1998;76:21–31. Kadl A, Meher AK, et al. Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2. Circ Res. 2010;107(6):737–46. Kanter JE, Kramer F, et al. Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1. Proc Natl Acad Sci USA. 2012;109(12):4353–4. Kuchibhotla S, Vanegas D, et al. Absence of CD36 protects against atherosclerosis in ApoE knock-out mice with no

Mammalian Target of Rapamycin (mTOR) additional protection provided by absence of scavenger receptor A I/II. Cardiovasc Res. 2008;78(1):185–96. Lamharzi N, Renard CB, et al. Hyperlipidemia in concert with hyperglycemia stimulates the proliferation of macrophages in atherosclerotic lesions: potential role of glucose-oxidized LDL. Diabetes. 2004;53 (12):3217–25. McMillen TS, Heinecke JW, et al. Expression of human myeloperoxidase by macrophages promotes atherosclerosis in mice. Circulation. 2005;111(21):2798–804. Minino AM, Murphy SL, et al. Deaths: final data for 2008. Natl Vital Stat Rep. 2011;59(10). Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145(3):341–55. Moore KJ, Kunjathoor VV, et al. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice. J Clin Invest. 2005;115(8):2192–201. Oh J, Riek AE, et al. Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J Biol Chem. 2012;287(15): 11629–41. Pennathur S, Wagner JD, et al. A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J Clin Invest. 2001;107(7):853–60. Pennathur S, Bergt C, et al. Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species. J Biol Chem. 2004;279(41):42977–83. Pennathur S, Ido Y, et al. Reactive carbonyls and polyunsaturated fatty acids produce a hydroxyl radical-like species: a potential pathway for oxidative damage of retinal proteins in diabetes. J Biol Chem. 2005;280(24):22706–14. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999;340(2):115–26. Schwartz CJ, Valente AJ, et al. Thrombosis and the development of atherosclerosis: Rokitansky revisited. Semin Thromb Hemost. 1988;14(2):189–95. Shao B, Pennathur S, et al. Myeloperoxidase targets apolipoprotein A-I, the major high density lipoprotein protein, for site-specific oxidation in human atherosclerotic lesions. J Biol Chem. 2012;287(9):6375–86. Shishehbor MH, Brennan ML, et al. Statins promote potent systemic antioxidant effects through specific inflammatory pathways. Circulation. 2003;108(4): 426–31. Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ Res. 2010;107(7): 839–50. Tall AR, Yvan-Charvet L, et al. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis. Cell Metab. 2008;7(5):365–75. Vaisar T, Pennathur S, et al. Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest. 2007;117(3):746–56.

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Vita JA, Brennan ML, et al. Serum myeloperoxidase levels independently predict endothelial dysfunction in humans. Circulation. 2004;110(9):1134–9. Vivekanadan-Giri A, Wang JH, et al. Mass spectrometric quantification of amino acid oxidation products identifies oxidative mechanisms of diabetic end-organ damage. Rev Endocr Metab Disord. 2008;9(4):275–87. Vivekanandan-Giri A, Byun J, et al. Quantitative analysis of amino acid oxidation markers by tandem mass spectrometry. Methods Enzymol. 2011;491:73–89. Wen Y, Gu J, et al. Elevated glucose and diabetes promote interleukin-12 cytokine gene expression in mouse macrophages. Endocrinology. 2006;147(5):2518–25.

Mammalian Target of Rapamycin (mTOR) Christopher J. Gamper and Jonathan D. Powell Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA

Synonyms

M FK506 binding protein 12-rapamycin associated protein 1 (FRAP1); Mammalian target of rapamycin (mTOR); Rapamycin and FK506 binding protein 12 targets 1 (RAFT1)

Definition mTOR is a 289-kD evolutionarily conserved protein with homology to phosphoinositide 3 (PI3)-kinase. Unlike PI3-kinase, mTOR possesses serine-threonine kinase activity sensitive to inhibition by the macrolide antibiotic rapamycin isolated from Streptomyces hygroscopicus collected on Rapa nui (Easter Island) (reviewed in (Dennis et al. 1999)). The rapamycin-dependent inhibition of kinase activity was used to identify both yeast and mammalian TOR proteins. Rapamycin was quickly noted to have profound immunosuppressive effects, but an understanding of the mechanisms by which mTOR inhibition mediates immunologic tolerance has only recently been more clearly understood.

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Yeast have two separate genes, TOR1 which regulates protein translation and TOR2 which regulates cytoskeletal reorganization, while mammalian cells utilize the mTOR protein encoded by the single Frap1 gene to form two distinct multi-protein complexes termed mTORC1 and mTORC2. Each complex has different inputs for activation and unique downstream substrates and functions (reviewed in (Laplante and Sabatini 2009)). A schematic view of mTOR signaling is depicted in Fig. 1. mTORC1 consists of mTOR, the regulatoryassociated protein of mTOR (raptor), the mammalian lethal with Sec13 protein 8 (mLST8), the proline-rich Akt substrate 40 kDa (PRAS40), and the DEP-domain-containing mTOR-interacting protein (Deptor). mTORC2 consists of mTOR, the raptor-independent companion of TOR (rictor), mLST8, the mitogen-activated protein kinase-associated protein 1 (MAPKAP1 also called mSIN1), and the protein observed with rictor (Protor). Inputs that activate mTORC1 include growth factor signaling such as insulin, the presence of adequate cellular energy stores via inhibition of the AMP-activated protein kinase (AMPK) pathway, the presence of sufficient amino acids, the presence of normoxia, and in T cells, activation of CD28 costimulatory molecules and signaling via the interleukin-2 receptor (IL-2R) (reviewed in (Thomson et al. 2009)). Pathways linking several of these inputs to mTORC1 activation include protein kinase B (also called AKT)-dependent phosphorylation of the tuberous sclerosis 1 and 2 protein complex (TSC1/TSC2). The TSC1/2 complex functions as a GTPase-activating protein (GAP) for the GTPase Ras-homolog enriched in brain (Rheb). Phosphorylation of TSC1/2 inhibits GAP activity, increasing Rheb-GTP levels resulting in mTORC1 activation (Laplante and Sabatini 2009). Inputs that activate mTORC2 are less well understood, but like mTORC1, mTORC2 is strongly activated in T cells in the presence of costimulation and cytokine exposure (reviewed in (Cantrell 2002)). mTORC1 is exquisitely sensitive to inhibition by the complex of rapamycin with FKBP12, resulting in loss of phosphorylation of the eukaryotic initiation

Mammalian Target of Rapamycin (mTOR)

factor 4E-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase 1 (S6K1) and subsequent inhibition of protein translation (Laplante and Sabatini 2009). Measurement of 4E-BP1 and S6K1 phosphorylation is utilized to monitor the activation status of mTORC1. mTORC2 activity is insensitive to low doses of rapamycin in vitro; however, upon incubation of cells with higher rapamycin doses or following prolonged exposure to rapamycin, loss of mTORC2-dependent AKT S473 phosphorylation is detected (Delgoffe et al. 2011). More recently, small molecules that directly inhibit the kinase activity of mTOR have been developed. These interfere with both mTORC1- and mTORC2-dependent phosphorylation events (Thoreen et al. 2009). Consequences of pharmacologic and genetic blockade of mTOR signaling in various immune cell types are explored below and summarized in Table 1.

mTOR Function in T Cells Normal T cell activation is dependent on simultaneous recognition of antigen in the form of peptide bound to major histocompatibility complex (MHC) on the antigen-presenting cell (APC) surface by cognate TCR (Signal 1) with concurrent activation of T cell costimulatory molecules including CD28 by APC-expressed B7-1 or B7-2 surface proteins (Signal 2). Such activation leads to T cell interleukin (IL)-2 secretion, autocrine IL-2 stimulation, and proliferation resulting in clonal expansion of the peptide-specific T cell. Biochemically, signals resulting from costimulation, cytokine receptors, and the local nutrient supply are integrated via mTOR signaling in T cells. mTOR activation results in increased expression of metabolic enzymes to permit cell growth and division, as well as acquisition of effector functions such as cytokine expression. Following TCR stimulation in the absence of costimulation (Signal 1 alone), mTOR is not activated, and a T cell is rendered unresponsive to subsequent full stimulation (Signal 1 + 2) in vitro, a condition termed anergy (reviewed in (Powell 2006)). Full stimulation (Signal 1 + 2) in the presence of 2-deoxyglucose


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mTOR Signaling in Immune Cells TCR

CD28

TLR

Growth Factor Receptor

IL2 Receptor S1P(1) Receptor

PIP2

PIP3 PDK1

= Kinase = Adapter

PI3 Kinase

= GTPase

ATP/AMP

Hypoxia

= Other

P-T308 AKT

TSC1

= Small Molecule

AMPK

TSC2

Amino Acids Rheb

Rheb

GDP

GTP RagA/RagB

Protor

Deptor

RagC/RagD

mTOR (mTORC1)

P

mLST8

4EBP1 P

Raptor PRAS40

mTOR Kinase Inhibitor

Deptor mTOR (mTORC2)

mLST8 P mSIN1

Rictor

SGK1 P-T308

S6K1

AKT P-S473

Rapamycin FKBP12 mTORC1 Dependent Events

mTORC2 Dependent Events

Increased ProteinTranslation, Glycolysis, Proliferation

Cytoskeletal Reorganization, Survival

CD4 Th1 and Th17 Differention

CD4 Th2 Differentiation

Inhibition of CD4 Foxp3 Expression

Inhibition of CD4 Foxp3 Expression

CD8 Effector Differentiation

B cell maturation

BM-DC IL-12 and B7-1/2 Expression Plasmacytoid DC Cytokine Expression Monocyte IL-10 Expression

Mammalian Target of Rapamycin (mTOR), Fig. 1 mTOR activation by environmental stimuli: an overview of the mTOR signaling cascade

(2DG) or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimicking energy depletion, N-acetyl-leucine (NALA) mimicking amino acid deprivation, and rapamycin all inhibit

normal mTOR activation and result in anergy upon restimulation of the T cell in the absence of the inhibitory drug (Zheng et al. 2009). Importantly, induction of anergy depends on

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Mammalian Target of Rapamycin (mTOR), Table 1 Summary of mTOR-dependent signaling in immune cells Cell type CD4 T cell

Naı¨ve CD4 T cell

Naı¨ve CD4 T cell

Naı¨ve CD4 T cell Naı¨ve CD4 T cell

Naı¨ve CD8 T cell Naı¨ve CD8 T cell Bone marrowderived dendritic cell Plasmacytoid dendritic cell Monocyte, macrophage, mature dendritic cell B cell

B cell B cell

Experimental manipulation of mTOR Proximal blockade of mTOR activation (costimulatory blockade, 2-DG, AICAR, NALA)a, rapamycin, or mTOR kinase inhibitor during T cell activation Initial T cell activation in the presence of rapamycin or after genetic inactivation of mTOR

Effect Decreased proliferation and cytokine secretion upon restimulation (anergy)

Enhanced expression of FoxP3 and generation of Treg; inhibition of HIF-1a-dependent Th17 differentiation by rapamycin Thymopoiesis of T cells overexpressing S1P(1) Inhibition of FoxP3 expression, enhanced Th1 with hyperactivation of mTOR after TCR/CD28 differentiation, spontaneous autoimmunity signaling Initial T cell activation after genetic inactivation Inhibition of Th1 and Th17 differentiation with of mTORC1 (via deletion of Rheb) preservation of Th2 differentiation Initial T cell activation after genetic inactivation Inhibition of Th2 differentiation with of mTORC2 (via deletion of rictor) preservation of Th17 and preservation or decrease of Th1 and differentiation Initial T cell activation after genetic inactivation Decreased CD8 effector gene expression of mTORC1 (via deletion of Rheb) Initial T cell activation after blockade of mTOR Enhanced frequency of antigen-specific memory with rapamycin CD8 T cells LPS and IL-4 stimulation during blockade of Inhibition of costimulatory molecule surface mTOR with rapamycin expression and IL-12 secretion (generation of “tolerogenic” DC) CpG oligonucleotide stimulation during Inhibition of IFN-a, IFN-b, TNF-a, and IL-6 blockade of mTOR with rapamycin secretion LPS stimulation during blockade of mTOR with Inhibition of IL-10 and enhancement of IL-12 rapamycin secretion

Partial blockade of mTOR in hypomorphic knock-in mouse

Partial block in B cell maturation in the bone marrow and reduced numbers of transitional, marginal zone, and follicular B cells in the spleen; decreased T-independent antibody production Selective disruption of mTORC2 by deletion of Reduction of mature IgM + B cells mSin1 Constitutive activation of mTOR via conditional Decreased B cell maturation and a reduction in deletion of TSC1 marginal zone B cells in the spleen; decreased T-dependent and loss of T-independent antibody production

a

Abbreviations: 2-DG 2-deoxyglucose, AICAR 5-aminoimidazole-4-carboxamide ribonucleoside, NALA N-acetyl leucine

TCR-mediated, calcium-dependent activation of nuclear factor of activated T cells (NF-AT), as demonstrated by the fact that the immunosuppressive drugs cyclosporine or FK506 that block calcium-dependent NF-AT activation actually prevent the induction of anergy by Signal 1 alone or by Signal 1 + 2 in the presence of rapamycin (Powell 2006). Clinical implications of this difference in mechanism of immune

modulation between rapamycin and calcineurin inhibitors are discussed below. Following activation, naı¨ve CD4 T cells acquire specialized effector function in response to the particular cytokine and costimulatory milieu present at the time of activation. Different effector fates are associated with induction of specific master transcriptional regulators and restricted patterns of cytokine expression,


for example, Th1 cells express T-box expressed in T cells (Tbet) and secrete interferon (IFN)-g, Th2 cells express GATA3 and secrete IL-4, Th17 cells express retinoic acid receptor-related orphan receptor g (RORgT) and express IL-17A/F, and a subset of CD4 T cells termed Tregs acquires suppressive regulatory function dependent on Forkhead-box-P-3 (Foxp3) transcription factor expression (reviewed in (Weaver et al. 2006)). mTOR plays a critical role in CD4 effector differentiation. Activation of T cells in the presence of rapamycin in vitro increases the frequency of cells expressing Foxp3 that have suppressor function (Battaglia et al. 2005). Genetic blockade of mTOR signaling by conditional deletion of mTOR in T cells using CD4-Cre transgenic mice does not interfere with generation of naı¨ve T cells. However, following activation, mTOR-deficient CD4 T cells upregulate FoxP3 expression, fail to express effector cytokines, and become functional suppressor cells (Delgoffe et al. 2009). Reciprocally, excessive activation of mTOR in mice bearing T cells transgenic for the sphingosine 1-phosphate receptor S1P(1) suppressed generation of normal FoxP3+ Tregs and promoted Th1 effector cytokine expression resulting in spontaneous autoimmunity (Liu et al. 2009). Inhibition of FoxP3 expression downstream of mTOR signaling has been demonstrated to partly result from the induction of hypoxia-induced factor-1 alpha (HIF-1a) expression which enhances Th17 cell generation by cooperating with RORgT to promote IL-17 expression and simultaneously direct proteolytic degradation of FoxP3 protein (Dang et al. 2011). Somewhat unexpectedly, the enhanced FoxP3 expression in the absence of mTOR is dependent on complete loss of both mTORC1 and mTORC2 signals, as selective deletion of mTORC1 alone via T cell conditional deletion of Rheb results in selective inhibition of Th1 and Th17 cell generation but permits Th2 cells (Delgoffe et al. 2011). Conversely, selective deletion of mTORC2 alone via T cell conditional deletion of rictor results in inhibition of Th2 cells but permits Th17 effectors and has variable effects on Th1 cells in two reports (Delgoffe et al. 2011; Lee et al. 2010).

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The complete mechanism by which loss of either mTORC1 or mTORC2 selectively impairs cytokine responses of naı¨ve T cells remains under investigation, but mTORC1 null T cells demonstrate decreased Signal Transducers and Activators of Transcription(STAT)4 phosphorylation after stimulation with IL-12 and STAT3 phosphorylation after stimulation with IL-6 (Delgoffe et al. 2011). Variable effects of mTORC2 deletion on cytokine signaling in T cells have been reported, with impaired STAT6 activation after stimulation with IL-4 in rictor-floxed mice on a CD4-Cre background (Delgoffe et al. 2011) but normal IL-4 induced STAT6 phosphorylation with abnormal TCR-induced proximal AKT and distal PKC-y activation in rictor-floxed mice on the dLck-iCre background (Lee et al. 2010). In summary, while selective loss of mTORC1 or mTORC2 impairs a subset of CD4 effector fates, complete loss of mTOR signaling results in generation of Tregs instead of effector cells. mTOR has also been shown to participate in generation of effector CD8 T cells. TCR stimulation, IFN-g signaling, and IL-12 signaling in naı¨ve CD8 cells lead to strong mTORC1 activation and resulting upregulation of Tbet, promoting proliferation and expression of effector genes such as perforin and granzyme B (Rao et al. 2010). CD8 T cells lacking mTORC1 due to deletion of Rheb or with constitutively activated mTORC1 due to deletion of TSC2 have impaired and enhanced CD8 effector function, respectively, consistent with necessity of mTORC1 in activation of the CD8 effector program (Pollizi, K.P. and Powel, J.D., manuscript in preparation). Following control of an acute infection, the number of antigen-specific CD8 T cells declines, and the cells downregulate expression of Tbet and increase expression of a homologous transcription factor eomesodermin (Eomes) along with the IL-7 receptor, CD127, and the IL-2/ IL-15 receptor beta chain, CD122, in order to persist as a smaller population of long-lived memory CD8 cells. Somewhat unexpectedly, blockade of mTOR with low-dose rapamycin during acute infection of mice with lymphocyte choriomeningitis virus (LCMV) does not impair the antiviral response; rather, it enhances the

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frequency and persistence of virus-specific CD8 T cells (Araki et al. 2009). Furthermore, mTOR inhibition during vaccination has also been shown to enhance antitumor immune responses (Rao et al. 2010). Mechanistically, this appears to be due to effects of rapamycin on promoting Eomes expression at the expense of Tbet, thereby increasing the frequency of memory CD8 cells.

mTOR Function in Antigen-Presenting Cells Antigen-presenting cells (APCs) are critical bridges between innate immune recognition of infectious pathogens and the adaptive immune response. The cytokines produced by APCs following activation with pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide, viral ribonucleic acid, and fungal cell wall components initiate the inflammatory response and recruit adaptive immune cells to fight infection. Following activation, APCs upregulate cell-surface expression of costimulatory molecules including B7-1 and B7-2, resulting in full activation of T cells that recognize antigenic peptides taken up and presented by the APC. Effector differentiation of the activated T cells is then influenced by the local cytokines secreted from the APC. mTOR signaling within the APC regulates many of these critical steps in a manner that is cell type- and activation state-specific. Resting bone marrow-derived dendritic cells (DCs) are poor stimulators of T cells but acquire potent immune-stimulatory capacity when “matured” following LPS stimulation and culture with IL-4 or CD40 cross-linking as a consequence of IL-12 secretion and induction of surface B7-1/B7-2 expression. Maturation in the presence of rapamycin inhibits both IL-12 and costimulatory molecule expression, rendering the rapamycin-treated DC tolerogenic rather than stimulatory in a manner at least partly dependent on an enhanced ability to induce CD4 T cells to become FoxP3+ Treg (Turnquist et al. 2007). In plasmacytoid dendritic cells, CpG oligonucleotides which mimic viral infection elicit robust


secretion of IFN-a, IFN-b, tumor necrosis factor (TNF)-a, and IL-6 in a manner sensitive to inhibition by rapamycin (Cao et al. 2008). In contrast, in monocytes in peripheral blood, macrophages, and mature DCs, mTOR signaling appears to limit secretion of Th1-promoting IL-12 by enhancing secretion of the immunosuppressive cytokine IL-10. Thus, stimulation of these cell types with LPS in the presence of rapamycin has the paradoxical effect of inhibiting IL-10 with resulting enhanced IL-12 secretion and stronger Th1 immune responses in some systems (Weichhart et al. 2008). Which of these two opposing effects of mTOR inhibition on APC function are dominant in vivo appears to depend on the type of inflammatory stimulus and the dose and timing of mTOR inhibition used. For example, rapamycin treatment protects susceptible Balb/C mice from Listeria infection and augments LCMV immunity but impairs immunity to yellow fever vaccine (Araki et al. 2009; Cao et al. 2008; Weichhart et al. 2008).

mTOR Function in B Cells The PI3-kinase/mTOR axis is activated in B cells following surface immunoglobulin (Ig) crosslinking, LPS stimulation, and CD40 ligation and resulting proliferation and antibody secretion that is inhibited by rapamycin (reviewed in (Thomson et al. 2009)). Several genetic models have very recently been utilized to examine the mechanism for these effects. An mTOR hypomorphic mouse generated by insertion of a neo-cassette within the Frap1 gene was found to have lymphopenia and reduced numbers of both T and B cells, with a partial block in B cell maturation in the bone marrow and reduced numbers of transitional, marginal zone, and follicular B cells in the spleen. These mice make poor responses to the T-independent antigen nitrophenol-LPS (Zhang et al. 2011). Selective disruption of mTORC2 by deletion of mSin1 resulted in a reduction of mature IgM + B cells in irradiated mice reconstituted with mSin1-deficient fetal liver cells. This was due to a partial arrest at the pro-B cell stage associated with persistent Rag1


gene expression and enhanced IL-7 receptor expression. Mechanistically, this was found to be due to enhanced nuclear translocation of the Forkhead family transcription factor FoxO1 as a consequence of reduced mTORC2-dependent FoxO1 phosphorylation that retains FoxO1 in the cytoplasm (Lazorchak et al. 2010). Unlike in T cells, constitutive activation of mTOR via conditional deletion of TSC1 using CD19-Cre did not produce a reciprocal phenotype to the mTOR hypomorph or mTORC2 null mice reported above. Instead, mice with constitutively active mTOR in B cells also have decreased B cell maturation and a reduction in marginal zone B cells in the spleen. This effect was partially rescued by rapamycin treatment. Such mice make poor responses to T-dependent antigen and fail to make a response to a T-independent antigen (Benhamron and Tirosh 2011). While more details regarding the targets of mTOR-dependent signaling in B cells remain to be identified, these early data are consistent with mTOR-dependent signaling being necessary for normal generation of naı¨ve B cells and for regulation of mature B cell proliferation/activation. They also suggest that physiologic limitation of the duration or intensity of mTOR signaling during B cell terminal differentiation may be necessary for to permit normal antibody production during an immune response.

mTOR Inhibition as Clinical Immunosuppressant Given the pleiotropic effects of mTOR signaling on cells involved in both innate and adaptive immunity, it is not surprising that rapamycin and related compounds have found clinical utility in the fields of solid organ and hematopoietic stem cell transplantation. Numerous examples of both animal models and human systems are reported, but due to space limitations, only a few are highlighted that help to illustrate important differences between tolerogenic mTOR inhibition and conventional calcineurin-based immunosuppression that may interfere with generation of long-term tolerance.

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In a murine model of cardiac allograft rejection, DCs were matured in vitro in the presence of rapamycin and infused into cardiac allograft recipients who received a short course of systemic rapamycin. This resulted in long-term allograft survival off immunosuppression, increased allograft infiltration by Foxp3+ Tregs, and the ability to adoptively transfer tolerance to secondary recipients by infusion of CD4 T cells isolated from tolerant graft recipients (Turnquist et al. 2007). Several groups have isolated human Tregs for expansion in vitro, relying on culture with rapamycin to promote sustained Foxp3 expression, with the goal of applying these to treat autoimmune disease or prevent graft versus host disease (GVHD). This technology has been applied in a phase I clinical trial of Treg infusion following unrelated umbilical cord blood transplantation and demonstrated safety and lower rates of GVHD compared to historical controls from the same institution (Brunstein et al. 2011). Systemic treatment with rapamycin is also used to promote tolerance. The longer clinical experience with calcineurin-based GVHD prophylaxis in hematopoietic stem cell transplant has resulted in most trials adding rapamycin to regimens containing cyclosporine or tacrolimus. Despite the mechanistic antagonism, there is encouraging evidence of a favorable incidence of acute GVHD, as low as 20.5 % Grade II–IV acute GVHD following myeloablative transplant with tacrolimus- and sirolimus-based GVHD prophylaxis (Cutler et al. 2007). However, based on the potential inhibition of rapamycininduced tolerance by calcineurin inhibitors and preclinical work demonstrating that treatment with low-dose total body irradiation (TBI) and rapamycin alone permitted stable mixed donor chimerism in a murine model of non-myeloablative haploidentical bone marrow transplant, a human clinical trial was opened for the treatment of severe sickle cell anemia with matched sibling donor peripheral blood stem cell transplant using a regimen of alemtuzumab, low-dose TBI, and post-transplant rapamycin. This study achieved stable mixed donor chimerism in 9 of 10 treated patients, no patients experienced acute or chronic GVHD, all had

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resolution of sickle cell-related symptoms, and two patients who discontinued rapamycin have remained stable mixed chimeras off of immunosuppression (Hsieh et al. 2009). In summary, mTOR signaling plays an essential role in integration and transmission of signals in multiple immune cell types leading to cellular activation, proliferation, and effector differentiation. Thoughtful blockade of mTOR signaling appears to offer significant promise as a means to achieve immunologic tolerance in the setting of organ and hematopoietic stem cell transplantation while preserving the ability to make protective immune responses against viruses, bacteria, and tumors. mTORC1 consists of mTOR, raptor, mLST8, PRAS40, and Deptor. mTORC2 consists of mTOR, rictor, mLST8, Deptor, MAPKAP1 (also called mSIN1), and Protor. In the figure, straight arrows indicate activating signals, and lines ending in a bar indicate inhibitory signals. Curved arrows indicate chemical reactions catalyzed by the indicated enzymes. “P” indicates select sites of protein phosphorylation. Inputs that activate mTORC1 include growth factor signaling such as insulin, the presence of adequate cellular energy stores via activation of the AMP-activated protein kinase (AMPK) pathway, the presence of sufficient amino acids via the Rag (RagA/B/C/D) family of GTPases that promote Rheb colocalization with mTORC1, the presence of normoxia via regulated in development and DNA damage responses 1 (REDD1), and in T cells activation of TCR, CD28 and other costimulatory molecules, sphingosine 1-phosphate receptor 1 (S1P(1) receptor), and signaling via the interleukin-2 receptor (IL-2R). Pathways linking several of these inputs to mTORC1 activation include protein kinase B (also called AKT)-dependent phosphorylation of the tuberous sclerosis 1 and 2 protein complex (TSC1/TSC2). The TSC1/2 complex functions as a GTPase-activating protein (GAP) for the GTPase Ras-homolog enriched in brain (Rheb). Phosphorylation of TSC1/2 inhibits GAP activity, increasing Rheb-GTP levels resulting in mTORC1 activation. Inputs that activate mTORC2 are less well understood, but like


mTORC1, mTORC2 is activated in T cells by costimulation and cytokine exposure. mTORC1 is inhibited by the complex of rapamycin with FKBP12, resulting in loss of phosphorylation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase 1 (S6K1) and subsequent inhibition of protein translation. Measurement of 4E-BP1 and S6K1 phosphorylation is utilized to monitor the activation status of mTORC1. mTORC2 activity is insensitive to low doses of rapamycin in vitro; however, upon incubation of cells with higher rapamycin doses or following prolonged exposure to rapamycin, loss of mTORC2-dependent AKT S473 phosphorylation is detected. Phosphorylation of both mTORC1 and mTORC2 substrates is blocked by small molecule inhibitors of mTOR kinase activity. Boxes at the bottom catalogue cellular responses known to be downstream of mTORC1 or mTORC2. Global functions ascribed to each pathway are listed at the top in shaded areas, and the growing list of functions defined in immune cells is listed at the bottom.

Cross-References ▶ B7 and CD28 Families ▶ Cytotoxic T Lymphocytes ▶ PI3K

References Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, Bachmann MF, Larsen CP, Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460:108–12. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4 + CD25 + FoxP3+ regulatory T cells. Blood. 2005;105:4743–8. Benhamron S, Tirosh B. Direct activation of mTOR in B lymphocytes confers impairment in B-cell maturation and loss of marginal zone B cells. Eur J Immunol. 2011;41:2390–6. Brunstein CG, Miller JS, Cao Q, McKenna DH, Hippen KL, Curtsinger J, Defor T, Levine BL, June CH, Rubinstein P, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Blood. 2011;117:1061–70. Cantrell D. Protein kinase B (Akt) regulation and function in T lymphocytes. Semin Immunol. 2002;14:19–26.

Marginal Zone B Cells Cao W, Manicassamy S, Tang H, Kasturi SP, Pirani A, Murthy N, Pulendran B. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3) K-mTOR-p70S6K pathway. Nat Immunol. 2008;9: 1157–64. Cutler C, Li S, Ho VT, Koreth J, Alyea E, Soiffer RJ, Antin JH. Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood. 2007;109:3108–14. Dang EV, Barbi J, Yang HY, Jinasena D, Yu H, Zheng Y, Bordman Z, Fu J, Kim Y, Yen HR, et al. Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1. Cell. 2011;146:772–84. Delgoffe GM, Kole TP, Zheng Y, Zarek PE, Matthews KL, Xiao B, Worley PF, Kozma SC, Powell JD. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity. 2009;30:832–44. Delgoffe GM, Pollizzi KN, Waickman AT, Heikamp E, Meyers DJ, Horton MR, Xiao B, Worley PF, Powell JD. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat Immunol. 2011;12:295–303. Dennis PB, Fumagalli S, Thomas G. Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation. Curr Opin Genet Dev. 1999;9:49–54. Hsieh MM, Kang EM, Fitzhugh CD, Link MB, Bolan CD, Kurlander R, Childs RW, Rodgers GP, Powell JD, Tisdale JF. Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. N Engl J Med. 2009;361:2309–17. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci. 2009;122:3589–94. Lazorchak AS, Liu D, Facchinetti V, Di Lorenzo A, Sessa WC, Schatz DG, Su B. Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells. Mol Cell. 2010;39:433–43. Lee K, Gudapati P, Dragovic S, Spencer C, Joyce S, Killeen N, Magnuson MA, Boothby M. Mammalian target of rapamycin protein complex 2 regulates differentiation of Th1 and Th2 cell subsets via distinct signaling pathways. Immunity. 2010;32:743–53. Liu G, Burns S, Huang G, Boyd K, Proia RL, Flavell RA, Chi H. The receptor S1P1 overrides regulatory T cell-mediated immune suppression through AktmTOR. Nat Immunol. 2009;10:769–77. Powell JD. The induction and maintenance of T cell anergy. Clin Immunol. 2006;120:239–46. Rao RR, Li Q, Odunsi K, Shrikant PA. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin. Immunity. 2010;32:67–78. Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol. 2009;9:324–37.

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Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, Reichling LJ, Sim T, Sabatini DM, Gray NS. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 2009;284:8023–32. Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang Z, Thomson AW. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol. 2007;178:7018–31. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity. 2006;24:677–88. Weichhart T, Costantino G, Poglitsch M, Rosner M, Zeyda M, Stuhlmeier KM, Kolbe T, Stulnig TM, Horl WH, Hengstschlager M, et al. The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity. 2008;29:565–77. Zhang S, Readinger JA, DuBois W, Janka-Junttila M, Robinson R, Pruitt M, Bliskovsky V, Wu JZ, Sakakibara K, Patel J, et al. Constitutive reductions in mTOR alter cell size, immune cell development, and antibody production. Blood. 2011;117:1228–38. Zheng Y, Delgoffe GM, Meyer CF, Chan W, Powell JD. Anergic T cells are metabolically anergic. J Immunol. 2009;183:6095–101.

Marginal Zone B Cells Raul M. Torres and Lindsey Pujanauski Integrated Department of Immunology, University of Colorado School of Medicine and National Jewish Health, Denver, CO, USA

Synonyms CD27+ IgM+ IgD+ B cells; IgM memory B cells

Definition Marginal zone (MZ) B cells are a subpopulation of B lymphocytes that localize at the border of the red and white pulp in the spleen where they are positioned to sample blood-borne antigens. Accordingly, MZ B cells mount rapid and T cell–independent antibody responses to bloodborne pathogens that display repetitive epitopes such as the polysaccharides found on

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encapsulated bacteria and viral coat proteins. They are derived as a separate lineage from naı¨ve follicular B cells and express a unique phenotype. The antibodies produced by MZ B cells bear little-to-no mutation but often display polyreactive and auto-reactive specificities.

Introduction Antibodies are a critical component of adaptive immunity and their production is a cooperative effort between different B cell subpopulations (Swanson et al. 2013). MZ B cells contribute to humoral immunity upon pathogen infection by rapidly producing pathogen-specific antibodies that limit and contain infection and during the time needed for the generation of somatically mutated high affinity antibodies of appropriate subclass that are produced by naı¨ve follicular B cells (Cerutti et al. 2013; Martin and Kearney 2002; Pillai et al. 2005; Weill et al. 2009). MZ B cell antibody responses are typically comprised of IgM and limited IgG subclass (IgG2 in humans and IgG3 in mice) antibodies that harbor little-tono mutations and thus are of relatively weak affinity. However, these pathogen-specific antibodies are effective due to the avidity provided by the pentameric IgM and because the MZ B cell–derived antibodies often display polyreactive specificities that include determinants common to many microbial pathogens. Much of our understanding about the development and function of MZ B cells comes from rodent studies performed over the last several decades (Martin and Kearney 2002; Pillai et al. 2005). However, human and murine MZ B cells are analogous populations that share many similar features that include phenotype, localization in lymphoid organs and ability to respond to pathogenic encapsulated bacteria in a T cell–independent manner (Cerutti et al. 2013; Weill et al. 2009).

Phenotype The MZ B cells in both humans and mice express a very similar pattern and level of receptors

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defined as IgMhi IgDlo CD21hi CD1hi CD23lo, presumably reflecting a similar function in both species. Human MZ B cells additionally express CD27, a surface antigen previously considered to be a marker of memory B cells (Klein et al. 1998). In contrast to murine MZ B cells, whose antibodies are germline encoded, human MZ B cells harbor (relatively few) somatic mutations in their immunoglobulin (Ig) genes (Cerutti et al. 2013; Weill et al. 2009). These features led to their initial consideration as “IgM memory” B cells (Klein et al. 1998). However, introduction of somatic mutations and production of memory B cells occurs in germinal centers, and mutated IgM+ IgD+ CD27+ human B cells are nevertheless present in individuals with mutations that preclude germinal center formation (Weill et al. 2009). Furthermore, IgM+ IgD+ CD27+ human B cells function analogously to murine MZ B cells (Kruetzmann et al. 2003; Weller et al. 2004). Thus, CD27+ human MZ B cells are functionally analogous to murine MZ B cells and are not germinal-center experienced conventional memory B cells. Human and mouse MZ B cells are larger cells compared to naı¨ve follicular B cells. In the mouse, these large MZ B cells are considered “pre-activated” as they display elevated levels of activation antigens such as CD86 and have a lower threshold of activation by either specific antigen or toll-like receptor ligands (Capolunghi et al. 2008; Snapper et al. 1993). The antibody repertoire expressed by both mouse and human MZ B cells also shares a number of features and differs from the major follicular B cell population. Notably, the murine MZ B cell antibody repertoire is known to harbor polyreactive specificities that include weak reactivity to autoantigens and that are important in promoting the development of the MZ B cell population (Martin and Kearney 2002; Pillai et al. 2005). Consistent with this, MZ B cell antibodies from humans and mice also harbor relatively short complementary determining region 3 (CDR3) Ig heavy chain sequences (Briney et al. 2012; Schelonka et al. 2007) that are also a common feature of antibodies with polyreactive specificities (Chen et al. 1991). Finally, human MZ


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B cells recirculate and, thus, are present in blood and other lymphoid organs where they reside in microenvironments among macrophages similar to those present in the marginal zone of the spleen. Murine MZ B cells do not recirculate and are restricted to the spleen. However, they are not restricted to the marginal zone as they continuously shuttle antigen into the B cell follicle to deliver it to follicular dendritic cells (Cyster and Schwab 2012).

Finally, the development and maintenance of both mouse and human MZ B cells is dependent on the spleen. B cells with a mature MZ B cell phenotype are not present in the spleen of children until approximately 2 years of age or mice until approximately 3–4 weeks of age (Cerutti et al. 2013; Weill et al. 2009). This neonatal lack of MZ B cells is thought to account for the poor antibody responses to encapsulated bacteria by children and neonatal mice.

Development

Localization

MZ B cells in the mouse develop from bone marrow–derived transitional B cells as a separate lineage from the major follicular B cell population in a process that is influenced by both antigen specificity and signaling ability of the B cell antigen receptor (BCR) (Martin and Kearney 2002; Pillai et al. 2005). The precise signals that dictate development of immature B cells into FO or MZ B cells are unknown, but certain specificities provided by Ig transgenes promote or discourage MZ B cell development in mouse models. In addition, a number of studies have suggested that a weak BCR signaling leads to MZ B cell preferential development, as shown in mice with deficient BCR signaling components that include the Btk tyrosine kinase and surface CD19 antigen (Martin and Kearney 2002; Pillai et al. 2005). In addition, both Notch- and Pyk2deficient mice have suggested that both these signaling molecules are important for MZ B cell development. Whether human MZ B cells also develop as a separate lineage from immature bone marrow B cells similar to their mouse counterparts is not established but their development appears to depend on TLR signaling (Weller et al. 2012). Furthermore, in contrast to mouse MZ B cells, human MZ B cells harbor somatic mutations within their Ig genes that are present even in the absence of germinal center formation. It has been postulated that human MZ B cells introduce somatic mutations during their development independent of antigen and in a mechanism that diversifies the MZ B cell antibody repertoire similar to other species (Weill et al. 2009).

In both humans and rodents, MZ B cells reside within the marginal zone that lies at the border between the red and white pulp. In mice, the marginal zone is separated from the white pulp by a marginal sinus into which arterial blood empties, promoting MZ B cell sampling of blood-borne antigens (Mebius and Kraal 2005). In humans, the marginal sinus within the marginal zone appears to be replaced by a plexus of capillaries that terminate in the perifollicular region surrounding the marginal zone (Steiniger et al. 2011). However, the juxtaposition of the marginal zone to this region presumably allows human MZ B cells to similarly sample blood antigens. Importantly, as the marginal zone is juxtaposed to the site where arterial blood empties into the spleen, the marginal zone also harbors post-germinal center memory B cells. Studies in the mouse have demonstrated that MZ B cell localization in the marginal zone is a result of a signaling balance between chemokine and sphingosine-1-phosphate G-protein coupled receptors (GPCR) and that the aLb2 and a4b1 integrins mediate MZ B cell adhesion to their respective ICAM and VCAM ligands expressed by cells within the marginal zone (Cyster and Schwab 2012).

Participation in the Immune Response MZ B cells are best characterized to mount rapid antibody responses independent of T cells and directed against repetitive surface epitopes on

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blood-borne pathogens referred to as T cell– independent type 2 (TI-2) antigens. Physiological TI-2 antigens to which MZ B cells are known to respond include the polysaccharide capsules of pathogenic bacteria such as Streptococcus pneumoniae, Hemophilus influenzae, and Neisseria meningitides (Kruetzmann et al. 2003; Martin and Kearney 2002) and the viral coat proteins of polyoma virus, vesicular stomatitis virus, and foot and mouth disease virus (Gatto and Bachmann 2005). The ability of MZ B cells to rapidly respond to these pathogens is, in large part, due to their location in the spleen within the marginal zone, an area close to the interface between the red and white pulp and where arterial blood is emptied and where in collaboration with specific macrophages, its contents are efficiently sampled. In contrast to germinal center–derived antibody responses, which take time to develop but are somatically mutated and highly specific, MZ B cell antibody responses are rapid and display relatively weak antigen affinity and can be polyreactive. A notable feature of human MZ B cells is the presence of somatic mutations in Ig genes, and these mutations are introduced during development and independent of germinal centers (Weill et al. 2009). Accordingly, the anti-polysaccharide antibodies produced by human MZ B cells are typically mutated (Zhou et al. 2002). In contrast, abundant evidence from mouse studies suggests that the MZ B cell antibody response to TI-2 antigens is devoid of mutations and, consequently, it is presumed that MZ B cells do not initiate germinal centers. However, it is now clear that the model antigens used to define the MZ B cell antibody response in rodents (typically haptencoupled polysaccharides) would not be able to engage CD4+ T cells nor facilitate germinal center formation. Thus, the ability of MZ B cells to mount a mutated antibody response to bona fide TI-2 antigens, such as those associated with microbial pathogens, may be underappreciated. Indeed, when directly tested mouse MZ B cells are able to contribute to the antibody response against T cell–dependent (TD) protein antigens by seeding germinal centers and undergoing somatic hypermutation (Song and Cerny 2003).


CD21 is expressed at relatively high levels by both human and mouse MZ B cells, and serves not only as a costimulatory molecule but also as a receptor for complement components, thus allowing for the capture of complement-bound antigen. MZ B cells also express nonclassical major histocompatibility complexes (MHC) such as CD1d in rodents and CD1c in humans that allow these cells to interact with a wider range of T cells, including iNKT cells. Finally, MZ B cells can affect more long-term responses by shuttling antigen into the follicle for delivery to follicular dendritic cells (FDCs), which mediate selection during germinal center reactions (Cyster and Schwab 2012).

Conclusion MZ B cells fill an important niche in humoral immunity by their ability to rapidly produce antibodies against blood-borne pathogen and independent of T lymphocytes. This antibody response contributes to limiting infection during the time needed for highly specific antibodies of appropriate subclass to be generated in germinal centers. A number of features contribute to the expedited MZ B cell antibody response including their localization in the marginal zone of the spleen that facilitates blood sampling, their expressed antibody repertoire that often is capable of recognizing common microbial determinants, and the decreased threshold necessary to be activated.

Cross-References ▶ BCR Signaling ▶ Regulatory B cells

References Briney BS, Willis JR, McKinney BA, Crowe Jr JE. High-throughput antibody sequencing reveals genetic evidence of global regulation of the naive and memory repertoires that extends across individuals. Genes Immun. 2012;13(6):469–73. doi:10.1038/ gene.2012.20.

Mechanisms of Endothelial Activation Capolunghi F, Cascioli S, Giorda E, Rosado MM, Plebani A, Auriti C, et al. CpG drives human transitional B cells to terminal differentiation and production of natural antibodies. J Immunol. 2008;180(2): 800–8. Cerutti A, Cols M, Puga I. Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes. Nat Rev Immunol. 2013;13(2):118–32. doi:10.1038/ nri3383. Chen C, Stenzel-Poore MP, Rittenberg MB. Natural autoand polyreactive antibodies differing from antigeninduced antibodies in the H chain CDR3. J Immunol. 1991;147(7):2359–67. Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu Rev Immunol. 2012;30:69–94. doi:10.1146/annurevimmunol-020711-075011. Gatto D, Bachmann MF. Function of marginal zone B cells in antiviral B-cell responses. Crit Rev Immunol. 2005;25(4):331–42. Klein U, Rajewsky K, Kuppers R. Human immunoglobulin (Ig)M + IgD + peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med. 1998;188(9):1679–89. Kruetzmann S, Rosado MM, Weber H, Germing U, Tournilhac O, Peter HH, et al. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J Exp Med. 2003;197(7):939–45. doi:10.1084/ jem.20022020. Martin F, Kearney JF. Marginal-zone B cells. Nat Rev Immunol. 2002;2(5):323–35. doi:10.1038/nri799. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol. 2005;5(8):606–16. doi:10.1038/ nri1669. Pillai S, Cariappa A, Moran ST. Marginal zone B cells. Annu Rev Immunol. 2005;23:161–96. doi:10.1146/ annurev.immunol.23.021704.115728. Schelonka RL, Tanner J, Zhuang Y, Gartland GL, Zemlin M, Schroeder Jr HW. Categorical selection of the antibody repertoire in splenic B cells. Eur J Immunol. 2007;37(4):1010–21. doi:10.1002/ eji.200636569. Snapper CM, Yamada H, Smoot D, Sneed R, Lees A, Mond JJ. Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells. J Immunol. 1993;150(7):2737–45. Song H, Cerny J. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J Exp Med. 2003;198(12):1923–35. doi:10.1084/ jem.20031498. Steiniger B, Bette M, Schwarzbach H. The open microcirculation in human spleens: a three-dimensional

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approach. J Histochem Cytochem. 2011;59(6): 639–48. doi:10.1369/0022155411408315. Swanson CL, Pelanda R, Torres RM. Division of labor during primary humoral immunity. Immunol Res. 2013;55((1–3)):277–86. doi:10.1007/s12026012-8372-9. Weill JC, Weller S, Reynaud CA. Human marginal zone B cells. Annu Rev Immunol (Rev). 2009; 27:267–85. doi:10.1146/annurev.immunol.021908. 132607. Weller S, Braun MC, Tan BK, Rosenwald A, Cordier C, Conley ME, et al. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood. 2004;104(12):3647–54. doi:10.1182/blood-2004-010346. Weller S, Bonnet M, Delagreverie H, Israel L, Chrabieh M, Marodi L, et al. IgM + IgD + CD27+ B cells are markedly reduced in IRAK-4-, MyD88-, and TIRAPbut not UNC-93B-deficient patients. Blood. 2012;120(25):4992–5001. doi:10.1182/blood-201207-440776. Zhou J, Lottenbach KR, Barenkamp SJ, Lucas AH, Reason DC. Recurrent variable region gene usage and somatic mutation in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23 F. Infect Immun. 2002;70(8): 4083–91.

M Mechanisms of Endothelial Activation Matthew S. Waitkus1,2, Daniel P. Harris1,3 and Paul E. DiCorleto1,2,3 1 Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic, Lerner College of Medicine, Cleveland, OH, USA 2 Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH, USA 3 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA

Synonyms Endothelial cell activation; Endothelial dysfunction; Vascular endothelium

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Mechanisms of Endothelial Activation

Definition

to fluctuate between antagonistic functions of vascular homeostasis. At different times or locations, the endothelium can be nonadhesive or hyperadhesive to leukocytes, can be procoagulatory or anticoagulatory, and act as a vasoconstrictor or vasodilator (DiCorleto and Fox 2005). In this way, the endothelium displays a remarkable phenotypic plasticity to adapt to environmental changes. These phenotypic changes are usually normal, adaptive responses to the changing vascular environment, and are an essential contribution to vascular homeostasis. Localized, nonadaptive changes to endothelial physiology can also alter its phenotype to induce changes in vessel tone, leukocyte adhesiveness, coagulation, and the production of autocrine and paracrine factors, including vasorelaxants and vasospastic substances. These aberrant alterations are collectively referred to as endothelial dysfunction, and are involved in the initiation and progression of numerous cardiovascular diseases. Endothelial activation, a type of endothelial dysfunction, is a distinct term that describes the process by which blood-borne and environmental stimuli cause endothelial cells to undergo dramatic functional changes and acquire new physiological properties (De Caterina et al. 2007). Proper endothelial function is critical for maintaining vascular health, and endothelial dysfunction may cause or contribute to numerous vascular diseases. This entry will provide a brief overview of normal endothelial physiology that will serve as a foundation for an in-depth explanation of the molecular mechanisms underlying endothelial cell activation as well as the contribution of endothelial cell activation to the genesis and progression of vascular diseases.

The vascular endothelium is a multifunctional organ that actively participates in the maintenance of vascular homeostasis. Proper endothelial function is critical for vascular health, and endothelial dysfunction causes or contributes to numerous diseases. Endothelial activation is a type of endothelial dysfunction that describes the process by which blood-borne and environmental stimuli cause endothelial cells to undergo dramatic functional changes.

Introduction The vascular endothelium is a monolayer of cells that lines the entire luminal surface of the vasculature and forms a regulatory interface between circulating blood components and underlying tissue compartments. The vascular endothelium covers a network of blood vessels that exceeds 100,000 km in aggregate length, with a surface area of approximately 5,000 m2 (De Caterina et al. 2007). Its massive size and distribution into all organs and tissues gives the endothelium the capacity to monitor physiological perturbations throughout the entire body. By virtue of its juxtaposition with circulating blood components, the endothelium is critically situated to sense and respond to blood-borne stimuli on a systemic scale, making it the body’s largest homeostatic organ (DiCorleto and Fox 2005). Although it was originally considered a passive barrier, modern research has uncovered numerous mechanisms by which the endothelium actively maintains vascular homeostasis. Under physiological conditions, the endothelium prevents vasospasm, resists leukocyte adhesion, inhibits smooth muscle cell proliferation, and acts as a non-thrombogenic surface for the entire circulatory system. In addition, the endothelium acts as a selective physical barrier by actively regulating the passage of macromolecules and leukocytes into and out of the bloodstream (Pober and Sessa 2007). These physiological roles are influenced by environmental stimuli that can cause the endothelial phenotype

Normal Vascular Endothelium Morphology and Barrier Function As a continuous monolayer of cells, the vascular endothelium forms an extensive regulatory interface between the bloodstream and underlying tissues with cells that are elongated in the direction of blood flow (Chiu and Chien 2011).


Abundant intercellular junctions including tight junctions, gap junctions, and adherens junctions allow the endothelium to act as an active physical barrier. The relative abundance of these junctions varies between anatomic locations and confers site-specific specialization of endothelial permeability (Pober and Sessa 2007). Arteries and blood vessels of the brain contain more tight junctions, which restrict macromolecular flux between intravascular and extravascular compartments. Gap junctions, which consist of connexin proteins, link the cytoplasm of neighboring endothelial cells and likely play roles in intercellular communication. Adherens junctions, which consist of cadherin proteins, are important for endothelial cell organization, growth, and migration (De Caterina et al. 2007). The modulation of intercellular junctions via posttranslational modifications of junction proteins plays a role in the regulation of vascular permeability. The mechanisms vary according to stimuli, but permeability is usually increased by phosphorylation of junction proteins, actin-myosin-dependent morphological changes, and an increased number of intercellular gaps in the endothelial monolayer (Pober and Sessa 2007). Under physiological conditions, these changes are controlled and reversible, representing adaptive control of macromolecular flux between the bloodstream and tissues. Regulation of Vascular Tone The endothelium acts as a regulator of vascular tone by synthesizing and secreting vasodilatory or vasoconstrictive substances that act on underlying smooth muscle cells. The most important endothelial-derived vasorelaxing factor is nitric oxide (NO), which is synthesized by endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). NO exerts a local vasorelaxing effect by diffusing into the smooth muscle cell and activating guanylate cyclase, thereby increasing levels of cyclic guanosine monophosphate (cGMP). Higher levels of cGMP inhibit calcium entry into the smooth muscle cell, resulting in decreased vasoconstriction (Kinlay and Ganz 2007; L€ uscher et al. 2005). Endothelial cells are

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also capable of synthesizing vasoactive metabolites of arachidonic acid called eicosanoids. The most important eicosanoid for maintaining physiological vessel tone is the vasorelaxant prostacyclin. Endothelial cells express the enzymes cyclooxygenase-1,2 (COX-1, COX-2) which synthesize prostaglandin H2 (PGH2) from arachidonic acid (L€uscher et al. 2005). PGH2 can then be converted to a number of vasoactive metabolites, but endothelial cells express significant levels of the enzyme prostacyclin synthase, thus favoring the formation of prostacyclin. Prostacyclin is secreted by endothelial cells and is a potent vasodilatory substance which acts on smooth muscle cells to decrease vascular tone (DiCorleto and Fox 2005). NO and prostacyclin antagonize a number of endothelial-derived vasoconstrictive substances. These substances, which include angiotensin II, endothelin-1 (ET-1), and platelet-derived growth factor (PDGF), act as agonists of smooth muscle cell contraction (DiCorleto and Fox 2005; Kinlay and Ganz 2007). All three of these molecules are expressed as propeptides by normal endothelial cells until appropriate stimuli trigger their activation by proteolytic cleavage. So, whereas the expression of vasorelaxing substances NO and prostacyclin is normally constitutive, the activation of endothelial-derived vasoconstrictive substances is rapidly inducible, allowing the endothelium to quickly and reversibly regulate vascular tone (De Caterina et al. 2007). Regulation of Hemostasis Under normal conditions, the endothelium actively participates in the prevention of blood clot formation. This non-thrombogenic property is maintained by the production of several endothelial-derived factors that act on platelets and enzymes of the coagulation cascade. Endothelial cells synthesize thrombomodulin which binds to thrombin and inactivates thrombin’s procoagulant activity (DiCorleto and Fox 2005). Additionally, endothelial cells synthesize the arachidonic acid metabolite prostacyclin, which was previously discussed in terms of its role as a vasodilator. Prostacyclin is a particularly potent inhibitor of platelet aggregation and

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platelet adherence to the endothelium. Under physiological conditions, endothelial-derived prostacyclin activates the prostacyclin receptor IP1 on platelets to inhibit aggregation and adhesion (DiCorleto and Fox 2005). Endothelial production of prostacyclin, thrombomodulin, and other anticoagulant factors appears to be constitutive under normal conditions, necessitating the rapid production of the procoagulant factors when clotting is required. The endothelium synthesizes numerous thrombotic factors including tissue factor (TF), platelet-activating factor (PAF), and von Willebrand factor (vWF) (DiCorleto and Fox 2005). TF is particularly important for the transformation of the vessel wall into a thrombogenic surface because it promotes the activation of factors X and IX of the coagulation cascade (De Caterina et al. 2007). Healthy vessels maintain a strict balance between thrombotic and antithrombotic factors, favoring the expression of antithrombotic factors under physiological conditions and rapidly producing TF and other coagulant factors following exposure to thrombotic stimuli. Transduction of Biomechanical Forces The endothelium is situated in direct contact with circulating blood. As a result, endothelial cells are constantly exposed to a variety of hemodynamic forces including shear stress, hydrostatic pressure, and cyclic strain. Shear stress is particularly significant because it is transduced by endothelial cell-surface proteins to activate signaling pathways that regulate changes in cell morphology, growth, and gene expression (Hahn and Schwartz 2009). The endothelial response to shear stress depends on the type and rate of flow. For example, exposure of endothelial cells to steady laminar flow may induce cell-cycle arrest in G0 or G1, whereas exposure to disturbed blood flow exhibits pro-proliferative effects (Chiu and Chien 2011). Additionally, laminar flow causes endothelial cells to become aligned and elongated in the direction of flow, while disturbed flow causes endothelial cells to adopt a more polygonal shape without a clear orientation (Chiu and Chien 2011). Rearrangement of intercellular junctions and cytoskeletal proteins


underlies the morphological changes of endothelial cells in response to both types of flow. Fluid shear stress can also modulate endothelial gene expression. Shear stress response elements (SSRE) have been discovered in the promoters of many genes and act as critical cis-acting elements for shear stress-regulated gene expression (DiCorleto and Fox 2005). Mechanotransducers at the endothelial cell surface respond to shear forces to activate signaling pathways and transcription factors that regulate gene induction. The exact mechanisms by which endothelial cell-surface proteins transduce mechanical forces are not known, but cytoskeletal proteins, receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and ion channels all play a role (Hahn and Schwartz 2009). The ability of endothelial cells to react to specific types of biomechanical forces emphasizes its role as a type of organism-wide sensory organ, capable of detecting and responding to a variety of hemodynamic forces depending on both the type and magnitude of the force.

Mechanisms of Endothelial Activation The original models of the endothelium’s role in disease progression were largely based on the response-to-injury hypothesis. This hypothesis stated that the endothelium was a protective, passive barrier whose absence following injury led to abnormal contact between circulating platelets and thrombogenic basement membrane. However, after researchers observed that the endothelium could interact with circulating monocytes, oxidize low-density lipoprotein, and synthesize growth factors and cytokines, it was suggested that the endothelium may play an active role in the development of atherosclerosis and other vascular diseases (DiCorleto and Chisolm 1986). After decades of research, it is well accepted that endothelial activation is associated with a number of vascular diseases including atherosclerosis. Inflammatory and pro-thrombotic substances transition the endothelium to an activated state


characterized by increased intimal permeability, expression of leukocyte adhesion factors, and shifts in protein secretion. Alterations in the secretion profile affect the balance of proand antithrombotic factors, pro- and antiinflammatory cytokines, growth factors and inhibitors, and vasodilatory and constrictive substances. Endothelial reactions to stimulation are largely coordinated by a central signaling pathway, NF-kB. Stimulation of this pathway results in the induction of hundreds of genes associated with the activated phenotype. The results of endothelial activation are manifested as altered interactions between the endothelium, other cells of the vascular wall, and components of the blood. NF-kB as a Central Regulator of Endothelial Cell Activation The endothelium responds to inflammatory agonists primarily by inducing genes not transcribed in the resting state. Signals for a variety of activating agonists, such as TNF, IL-1, LPS, or oxidized LDL, converge on the NF-kB signaling pathway to activate transcription of target genes (De Caterina et al. 2007). The NF-kB family includes five transcription factors, RelA (p65), RelB, c-Rel, NF-kB1 (p50), and NF-kB2 (p52), and their inhibitors, the IkB subunits, all of which are ubiquitously expressed in mammalian cells. Characteristic of NF-kB family members is a common domain essential for dimerization and DNA binding, the Rel homology domain. At rest, the transcription factors are sequestered by their inhibitory partners in the cytosol. These inhibitors are phosphorylated, usually by the IkB kinase complex (IKK), ubiquitinated, and targeted for proteasomal degradation upon stimulation. Newly liberated NF-kB factors dimerize and translocate to the nucleus, where they bind the kB sequence in the promoter of target genes. The transcriptional activity of NF-kB members is further regulated through associations with coactivators and through a variety of posttranslational modifications (Oeckinghaus et al. 2011). The NF-kB binding site is a remarkably loose, decameric sequence written as GGGRNNTYCC (R ¼ G or A, Y ¼ C or T, N ¼ any nucleotide)

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(Baltimore 2011). Genes important in the inflammatory program often are induced under the control of NF-kB. These include the adhesion molecules E-selectin, VCAM-1 and ICAM-1, as well as IL-1, IL-6, IL-8, MCP-1, TF, PAI-1, COX-2, and iNOS (De Caterina et al. 2007). Expression of Leukocyte Adhesion Molecules, Cytokines, and Chemokines Leukocytes do not interact with healthy, unstimulated endothelial cells, but following exposure to an activating agent, adhesion molecules are expressed on the luminal surface and chemokines are secreted into the blood to interact with and recruit leukocytes (Fig. 1, bottom). Initial interactions between endothelial cells and leukocytes involve the immune cell rolling along the endothelium, followed by arrest, focal adhesion, spreading, and emigration of the leukocyte from the lumen across the endothelial barrier. Leukocyte rolling on the endothelial surface is a relatively weak interaction governed by the selectin family of proteins, P-, E-, and L-selectin. P-selectin is present in platelet secretory granules, as well as the Weibel-Palade bodies of endothelial cells, and is therefore rapidly displayed on the endothelial cell surface within minutes of agonist exposure (De Caterina et al. 2007; Pober and Sessa 2007). L-selectin is expressed on leukocyte populations and has affinity for ligands expressed on endothelial cells and other leukocytes. E-selectin is only expressed in activated endothelial cells. Stronger interactions mediated by the immunoglobulin superfamily tether the rolling leukocyte to the endothelium. These adhesion molecules include ICAM-1, ICAM-2, and ICAM-3, VCAM-1, and PECAM-1. While low levels of ICAM-1 are constitutively expressed by endothelial cells, this gene and other members of the immunoglobulin family are induced upon activation. Migration of the tethered leukocyte through the intima is promoted by cell-type-specific chemoattractants secreted by endothelial cells, smooth muscle cells, and leukocytes (DiCorleto and Fox 2005). Chemokines also play a role in sustaining inflammation and are often expressed under the control of NF-kB. Chemokines of particular importance

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Mechanisms of Endothelial Activation, Fig. 1 Mechanisms of endothelial activation. Clockwise from the left: Blood vessels are lined by a single-cell-thick layer of endothelial cells surrounded by extracellular matrix proteins and concentric layers of smooth muscle cells. Endothelial cells synthesize and secrete a variety of growth factors and vasoactive substances (top) to regulate smooth muscle cell growth, motility, and vessel tone. Endothelial cells also respond to growth factors to create angiogenic sprouts to form new blood vessels (top right). Endothelial cells actively regulate the hemostatic/thrombotic balance of circulating blood by modulating production of procoagulant and anticoagulant factors (right).

Inflammatory activation of endothelial cells (bottom) is caused by a wide variety of factors and is implicated in numerous disease states, including atherosclerosis. Inflammatory factors such as TNF induce expression of leukocyte adhesion molecules and chemokines which recruit leukocytes to sites of inflammation, permit leukocyte rolling and firm adhesion to endothelial cells, and facilitate the extravasation of leukocytes into the subendothelial space. The accumulation of monocytes in the subendothelial space, and the activation of monocytes by oxidized lipoproteins and endothelial-derived factors, are critical events in the initiation of atherosclerosis

in atherosclerosis include IL-6, IL-8, and monocyte chemotactic protein 1 (MCP-1) (De Caterina et al. 2007). Recent research has discovered that NF-kB signaling also regulates the formation and release of endothelial-derived microparticles. Microparticles are membrane vesicles less than 1 mm in

diameter that are released from the cell in response to various stimuli. These microparticles can be generated by the same agents that induce adhesion molecule and cytokine expression (e.g., TNF and thrombin) and are functionally heterogenous (Rabelink et al. 2010). Depending on their lipid and protein compositions,


these NF-kB-regulated microparticles can be procoagulant and chemotactic or promote leukocyte adhesion to the endothelium (Rautou et al. 2011). Circulating levels of endothelial-derived microparticles increase at early stages in atherosclerosis and may contribute to disease progression, in part, by reducing eNOS phosphorylation and the bioavailability of NO (Rautou et al. 2011). The overall contribution of microparticles to vascular disease progression remains uncertain. However, as a functionally heterogenous group of bioactive substances, microparticles may represent an additional target of pathophysiologically relevant factors regulated by NF-kB transcription factors. Altered Permeability of the Activated Endothelium LDL, in a pathway common to many cells, normally enters endothelial cells through an LDL receptor-mediated endocytic route, leading to the hydrolysis of cholesteryl ester and reesterification of free cholesterol. This pathway is usually downregulated in hypercholesterolemic animals. Experiments have shown enhanced endothelial permeability to plasma lipoproteins in hypercholesterolemic animals, leading to plasma-derived buildup of LDL and VLDL particles in the subendothelial space (De Caterina et al. 2007). This accumulation of relatively large lipoproteins and lipids results from changes in vessel wall permeability in response to bloodborne inflammatory molecules in the local environment. Agonists such as histamine transiently open intercellular junctional proteins, allowing plasma-derived lipids and lipoproteins to become trapped in the subendothelial space (DiCorleto and Fox 2005). Once there, these lipoproteins can become oxidized by free radicals released by endothelial cells and leukocytes located within the lesion. Conversion of LDL to oxidized forms allows the particles to be recognized and taken up by various scavenger receptors, including CD36. Further, other products of lipid and lipoprotein oxidation function to induce changes in endothelial behavior. Cell culture experiments show that atherogenic concentrations of both oxidized LDL and unmodified

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LDL trigger the binding of monocytes to endothelial cells by activating transcription of the adhesion molecules VCAM-1 and ICAM-1 (DiCorleto and Fox 2005). Other functional changes, such as alterations in motility, and the expression of growth factors and cytokines have also been reported. Thrombogenicity and Vasospasm The healthy endothelium actively inhibits coagulation. Endothelial synthesis of prostacyclin and other antithrombotic factors inhibit platelet aggregation and adherence to the endothelium. During endothelial dysfunction, however, localized alterations in the balance of hemostatic factors may render the endothelial surface thrombogenic (Fig. 1, right). Endothelial expression of the pro-thrombotic tissue factor can be induced by a variety of agonists including thrombin, TNF, shear stress, and oxidized lipoproteins (Chiu and Chien 2011; DiCorleto and Fox 2005). Additionally, changes in the relative expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) contribute to increased thrombogenicity of the activated endothelium. Under normal conditions, the expression of these enzymes is tightly regulated to maintain a hemostatic/thrombotic balance. Activated endothelial cells tend to express higher levels of PAI-1, which can be induced by oxidized lipoproteins and shear stress, and lower levels of tPA (DiCorleto and Fox 2005). Together, these changes reduce the rate of fibrinolysis at localized sites of endothelial cell activation, creating a more thrombogenic environment. The vasorelaxants NO and prostacyclin are key mediators of vascular tone. Availability of NO is curtailed in a number of vascular pathologies, including atherosclerosis and coronary artery disease, and is considered characteristic of vascular disease (De Caterina et al. 2007). Reductions in NO availability occur though multiple mechanisms, including decreased NO production, stabilization, or inactivation by ROS. A number of endothelial-derived vasoconstrictive substances antagonize the actions of NO and prostacyclin to induce smooth muscle cell

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contraction (Fig. 1, top). ET-1 is the most potent and perhaps the most pathophysiologically important vasoconstrictor reported to date. It is expressed as a propeptide by endothelial cells and is activated by proteolytic cleavage mediated by ET-converting enzyme. Endothelial cells secrete most of the protein basolaterally, where the active form interacts with endothelin receptors on smooth muscle cells to induce vasoconstriction. Multiple atherogenic factors induce ET-1, including lipoproteins, interferon-g, IL-1b, thrombin, and shear stress. Aberrant expression of endothelial-derived ET-1 may function in a number of vascular diseases, including atherosclerosis, where it is expressed in atherosclerotic vessels and contributes to pathophysiological vasoconstriction (L€uscher et al. 2005; De Caterina et al. 2007). Biomechanical Forces and Endothelial Activation As previously discussed, the location of the endothelium exposes it to a variety of biomechanical forces, which regulate cell shape, growth, and gene expression. Mechanotransduction of laminar flow and high shear stress is normally adaptive and atheroprotective, but low-level shear stress or disturbed flow is atherogenic. Atherosclerotic lesions form at sites of disturbed blood flow in arterial vessels, suggesting that changes in shear stress may alter patterns of atheroprotective or atherogenic gene expression (Chiu and Chien 2011). While the mechanisms by which disturbed blood flow contributes to lesion formation are not completely understood, some differences in the activation of signaling pathways between laminar and disturbed flow have been observed. Both types of flow activate intracellular signaling kinases and transcription factors including NF-k B, early growth response 1 (EGR1), activator protein 1 (AP1), and JUN N-terminal kinase (JNK) (Hahn and Schwartz 2009). Under laminar flow, the activation of these molecules is transient, increasing over several hours and then returning to basal levels. In contrast, endothelial cells exposed to disturbed flow show sustained activation of kinases and transcription factors,


including NF-kB and JNK, which coincides with increased expression of inflammatory genes, such as ICAM-1, VCAM-1, E-selectin, MCP-1, ET-1, and PDGF (Chiu and Chien 2011; Hahn and Schwartz 2009). These differences in the kinetics of pathway activation and gene expression between laminar and disturbed flow may represent a potential basis for the nonrandom formation of atherosclerotic lesions at vessel bifurcations where blood flow is disturbed or slowed. Growth Factor Activation of the Endothelium and Angiogenesis Normally quiescent under physiological conditions, endothelial cells can be activated by growth factors to acquire proliferative, migratory, and invasive properties. Important stimulators of endothelial cell proliferation include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), PDGF, and epidermal growth factor (EGF) (Manka et al. 2005). These substances also induce angiogenesis, the formation of new blood vessels from the preexisting vasculature. The primary event governing the initiation of angiogenesis is the formation of an endothelial cell sprout from a preexisting endothelial cell monolayer (Potente et al. 2011). Environmental cues, primarily in the form of growth factors such as VEGF, cause normally quiescent endothelial cells to become activated and acquire a migratory and invasive phenotype (Herbert and Stainier 2011; Potente et al. 2011). This phenotypic change coincides with a reduction in cell-to-cell contacts and a corresponding increase in vascular permeability (Herbert and Stainier 2011). Based on relative amounts of VEGF receptor-2 (VEGFR-2), a “tip cell” (TC, high VEGFR-2 expression) is selected to lead an endothelial cell sprout to form a new vessel. The TC leads migration by following chemoattractant guidance cues and degrading extracellular matrix (ECM) proteins. Other activated endothelial cells, termed “stalk cells,” (SC) follow behind TCs, maintain a proliferative phenotype, and form a rudimentary new vessel lumen (Fig. 1, top right). Together, TCs and SCs constitute the


endothelial cell sprouts that continue to migrate until an adjacent sprout is reached. Sprouts then fuse together in a process called anastomosis. Following anastomosis, the motile and proliferative phenotypes of TCs and SCs are gradually lost, and cell-to-cell junctions are reestablished to form a proper lumen and allow for blood flow. The endothelium plays an additional role in forming a mature neovasculature by synthesizing and depositing ECM proteins as well as synthesizing growth factors such as PDGF which recruit pericytes to support the new vessel (Potente et al. 2011). Clinical Definitions of Endothelial Dysfunction The results from many studies using in vitro systems and animal models have led to the currently accepted definition of endothelial dysfunction. An unresolved challenge in vascular research is to create a reliable method for the clinical diagnosis of endothelial dysfunction, and various plasma biomarkers have been proposed and investigated for this purpose. In particular, plasma levels of von Willebrand factor (vWF) may be useful as a prognostic factor for cardiovascular diseases. Other potential biomarkers include ET-1, soluble adhesion molecules, angiotensin II, PDGF, asymmetric dimethylarginine ADMA, and NO (Freestone et al. 2010). Importantly, some studies have shown that NO bioavailability is reduced in early atherosclerotic lesions (Chiu and Chien 2011). Additionally, it has long been known that ADMA is an endogenous inhibitor of NO synthesis, and ADMA levels are elevated in patients with various vascular diseases including coronary artery disease, hypertension, hypercholesterolemia, and atherosclerosis (Freestone et al. 2010; Leone et al. 1992). It has therefore been suggested that ADMA may be a plasma marker for early-stage endothelial dysfunction. More recently, endothelial-derived microparticles in the plasma have been proposed as a diagnostic indicator of endothelial dysfunction. As previously mentioned, endothelial microparticles are known to be generated by a variety of atherogenic cytokines. Additionally, increased

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levels of microparticles generated from the endothelium have been observed in a variety of disease states including atherosclerosis, coronary artery disease, pulmonary hypertension, and end-stage renal failure (Rautou et al. 2011). As the body’s largest homeostatic organ, the vascular endothelium forms a marvelous nonthrombogenic surface lining every vessel in the body. Its function is critical for maintaining vascular health, and its dysfunction is associated with the genesis and progression of various vascular diseases. As a dynamic biological interface, the endothelium has the capacity to sense environmental changes, synthesize vasoactive substances, interact with other cell types, regulate vascular permeability, and form collateral vessels from the preexisting vasculature. Dysregulation of these processes is present in many vascular diseases, and endothelial dysfunction often precedes atherosclerosis and associated conditions, suggesting that endothelial activation may play a causative role in serious vascular illnesses. However, while the endothelium is known to participate in the genesis and progression of vascular diseases, the specific triggering events of endothelial activation, which distinguish pathophysiological dysfunction from physiological adaptation, are still incompletely characterized. Therefore, a more thorough understanding of endothelial dysfunction may lead to descriptions of causative events underlying endothelial dysfunction for distinct disease states. Such knowledge may allow for better risk stratification of patients before major adverse events as well as the identification of novel opportunities for therapeutic intervention to prevent or treat vascular diseases.

Cross-References ▶ Atherosclerosis and Cytokines ▶ Cell Adhesion Molecules ▶ Chemokines ▶ Dendritic Cells in Atherosclerosis ▶ Macrophages, Oxidative Stress, and Atherosclerosis ▶ NF-kB ▶ Nitric Oxide

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References

Micro-RNA in Autoimmunity Baltimore D. NF-kB is 25. Nat Immunol. 2011;12:683–5. De Caterina R, Massaro M, Libby P. Endothelial Functions and Dysfunctions, in Endothelial Dysfunctions in Vascular Disease (De Caterina R, Libby P, editors). Blackwell Publishing, Oxford, UK; 2007. p. 3–25. doi: 10.1002/9780470988473. Chiu J-J, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327–87. DiCorleto PE, Chisolm GM. Participation of the endothelium in the development of the atherosclerotic plaque. Prog Lipid Res. 1986;25:365–74. DiCorleto PE, Fox PL. Vascular endothelium. In: Fuster V, Topol EJ, Nabel EG, editors. Atherothrombosis and coronary artery disease. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 391–9. Freestone B, Krishnamoorthy S, Lip GYH. Assessment of endothelial dysfunction. Expert Rev Cardiovasc Ther. 2010;8:557–71. Hahn C, Schwartz MA. Mechanotransduction in vascular physiology and atherogenesis. Nat Rev Mol Cell Biol. 2009;10:53–62. Herbert SP, Stainier DYR. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol. 2011;12:551–64. Kinlay S, Ganz P. Endothelial vasodilatory dysfunction: basic concepts and practical implementation. In: Caterina RD, Libby P, editors. Endothelial dysfunctions and vascular disease. Malden: Blackwell; 2007. p. 179–88. Leone A, Moncada S, Vallance P, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet. 1992;339:572–5. L€uscher TF, Noll G, Spieker L, et al. Coronary spasm and atherothrombosis. In: Fuster V, Topol EJ, Nabel EG, editors. Atherothrombosis and coronary artery disease. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 599–622. Manka D, Compernolle V, Carmeliet P. Molecular basis of vasculogenesis, angiogenesis, and arteriogenesis. In: Fuster V, Topol EJ, Nabel EG, editors. Atherothrombosis and coronary artery disease. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 333–46. Oeckinghaus A, Hayden MS, Ghosh S. Crosstalk in NF-kB signaling pathways. Nat Immunol. 2011;12:695–708. Pober JS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev Immunol. 2007;7:803–15. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146:873–87. Rabelink TJ, de Boer HC, van Zonneveld AJ. Endothelial activation and circulating markers of endothelial activation in kidney disease. Nat Rev Nephrol. 2010;6:404–14. Rautou P-E, Vion A-C, Amabile N, et al. Microparticles, vascular function, and atherothrombosis. Circ Res. 2011;109:593–606.

To-Ha Thai Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Synonyms Micro-ribonucleoprotein (miRNP); Micro-RNA (miRNA, miR); RNA polymerase II (pol II); RNA-induced silencing complex (RISC); Systemic lupus erythematosus (SLE)

Definition Micro-RNAs are small, 21- to 22-nucleotide (nt) long, noncoding regulatory RNAs that contribute to the regulation of coding gene expression.

Introduction In vivo gain- and loss-of-function studies in mouse models demonstrate without a doubt that micro-RNAs (miRNAs), alongside with coding genes, control the mammalian immune system. The regulation of miRNA expression is tightly controlled, and often the same rules and regulations that govern coding gene expression apply also to miRNAs. Similar to coding genes, altering the levels and the temporal expression of a specific miRNA clearly affects the proper development and function of the tissue where it is expressed. Therefore, it is reasonable to argue that the dysregulated control of miRNA expression would affect immune functions and development as has been well established for coding genes. Along the same line of reasoning, there is an impetus to use miRNAs as diagnostic biomarkers and to develop therapeutic agents to target miRNAs for the treatment of various diseases such as autoimmune diseases.


Micro-RNAs are small, 21 to 22 nucleotide (nt) long, noncoding regulatory RNAs, first discovered in Caenorhabditis elegans (C. elegans) for its role in regulating the expression of coding genes. Most mammalian miRNAs are transcribed by RNA polymerase II (pol II), the same polymerase that directs the transcription of coding genes. MiRNA genes are encoded in intergenic regions, in sense or antisense orientation within introns of specific genes, or by noncoding transcripts (Pillai et al. 2007). In the nucleus, miRNAs are derived from larger precursors called primary (pri)-miRNAs that are processed into 75-nt-long precursor (pre)-miRNA hairpins by the RNAse III enzyme Drosha. The hairpins are exported into the cytoplasm by exportin 5. In the cytoplasm, Dicer, an RNAse III-like enzyme, processes the pre-miRNA hairpins to generate small RNA duplexes. One and sometimes both of the strands will be incorporated into the RNA-induced silencing complex (RISC) or micro-ribonucleoprotein (miRNP) complex containing Argonaute 2 (Ago 2), among other proteins. The RISC/miRNP complex executes miRNA functions, and miRNAs perform their functions by forming a duplex with the target gene(s) in the 30 untranslated (UTR) region of its messenger RNA (mRNA). Depending on the degree of complementarity between the miRNA: mRNA duplex, this interaction usually leads to the downregulation of protein expression by translational repression, mRNA cleavage, or promotion of mRNA decay (Fabian et al. 2010). Interestingly, the interaction between mRNAs and miRNAs not only affects the translation of the target mRNA, but also the miRNA stability, and this reciprocal relationship depends on the extent of base pairing between the two molecules (Ameres and Fukunaga 2010). To date, hundreds of miRNAs have been identified in species including viruses, plants, nematodes, mice, and humans, and the number is still increasing (miRBase, http://www.mirbase. org/). Most human miRNAs are conserved in the mouse, and about one-third of C. elegans miRNAs have vertebrate homologues, suggesting that a large fraction of miRNAs play evolutionary conserved developmental and/or functional roles.

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Effects of Global Reduction of miRNA Expression The first hint that miRNAs play an important role in the immune system came from mouse studies in which the dicer-1 or Ago2 gene is conditionally deleted. The ablation of dicer leads to a global reduction of miRNAs and other small RNAs, and it is clear that inactivation of Dicer activity, thus the expression of most if not all miRNAs, impacts the development and differentiation of T and B lymphocytes (Thai et al. 2010). Additionally, B-cell-specific deletion of dicer in female mice leads to the development of autoimmunity characterized by the presence of high titers serum autoreactive B-cells. It is noteworthy that not all miRNAs are equally ablated. It is also evident that not all T-cell lineage commitment programs are subjected to Dicer control. Irrespective of the cell type examined, Dicer activity is required for cell survival and proliferation, suggesting that the defects in lymphoid development may be partially due to the reduction of a group of miRNAs controlling cell survival and proliferation. Could all these phenotypes be ascribed to miRNAs, if so, which subsets of miRNAs are involved in each cell type and at which stage of lineage commitment? The answer awaits further studies. Although Dicer and Ago2 are among the major factors involved in miRNA biogenesis and function, they are also involved in small interfering RNA (siRNA) and other small RNA biology. Therefore, the defects observed in the above mutant mice may not be solely due to the loss of miRNAs.

Lymphocyte Development Through several seminal genetic studies, it is apparent by 2004 that individual miRNAs exert control on the immune system. First, mice reconstituted with bone marrow cells overexpressing the thymus-enriched miRNA, miR181, whose expression is dynamically regulated during T-cell development, have a higher and lower percentage of B- and T-cells in the

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periphery, respectively. MiR-181a is also overrepresented in tolerized and re-tolerized CD8+ T-cells (Schietinger et al. 2012). The generation and analysis of miR-181 loss-of-function mice would be valuable to provide a more physiological flavor to these observations. Subsequently, work from two different groups demonstrates the important role of miRNAs in B-cell development (Xiao et al. 2007; Zhou et al. 2007). Loss- and gain-of-function studies show that miR-150, a mature lymphocyte-specific miRNA, regulates B-cell differentiation by controlling the transcription factor c-myb in a dose-dependent manner (Xiao et al. 2007). Therefore, miR-150 may act as a dimmer rather than an on/off switch in the modulation of B-cell development, and it achieves this delicate balance by fine-tuning the expression levels of its targets, such as c-myb, in B-cells. Although c-myb plays an important role in both T- and B-cell development, ectopic expression or deletion of miR-150 has a stronger effect on B- than T-cell development. Thus, other miR-150 targets may be more crucial to T-cell development. The developmental programs in B- and T-cells are controlled by many transcription factors, among which some are lineage- and stagespecifically expressed and some are more ubiquitously expressed. It is not surprising that it is the case with miRNAs such as the miR-1792 cluster, and mature miRNAs from this cluster are broadly but differentially expressed in various mouse tissues. Mice overexpressing the miR-17-92 cluster suffer from lymphoproliferative and autoimmune manifestations with no obvious defects in B-cell development (Xiao et al. 2008). By contrast, miR-17-92/ mice have a profound impairment of both fetal and adult B-cell development while their peripheral B-cell effector function appears to remain intact (Ventura et al. 2008). These results suggest that miR-17-92 overexpression and miR-17-92 deficiency affect overlapping but not identical targets. It would be interesting to determine whether miR-17-92 deficiency would alleviate autoimmunity.


Adaptive Immunity Many miRNAs seem to participate in both hematopoiesis and immune responses, and the same mechanisms may be employed in both processes. In addition to the dysregulated B-cell development observed, miR-150/ mice (discussed above) also displayed augmented steady state levels of serum immunoglobulin (Ig) A, IgG1, IgG2b, and IgM, as well as an enhanced T-dependent immune response, perhaps due to the increased c-myb levels. It has been shown that c-myb promotes lymphocyte survival by controlling the pro-survival factor Bcl2 (Taylor et al. 1996). The data suggests that miR-150 also regulates the immune response; doing so whether through c-myb or other targets remains to be determined. One miRNA that participates in both innate and adaptive immunity is miR-155. It does not regulate lymphocyte development. MiR-155 expression is transiently induced upon activation. Dysregulated miR-155 expression results in impaired immune responses due in part to defects in germinal center (GC) formation (Rodriguez et al. 2007; Thai et al. 2007). In addition, results from many groups clearly showed that miR-155 plays an essential role in both T- and B-cell effector function (Thai et al. 2010). One might wonder how does miR-155 regulate B- and T-cell effector functions? Does it do so by controlling the expression of a yet to be identified master regulator or by modulating different targets? Another plausible scenario would be that miR-155 controls a pathway(s) downstream of receptor activation, which might modulate genetic/epigenetic modifications in lymphoid effector genes.

Innate Immunity The importance of miRNAs in innate immunity is also underscored by work demonstrating that miR-223, a myeloid-specific miRNA encoded in the X chromosome, negatively regulates granulocyte lineage specification and inflammatory


response (Johnnidis et al. 2008). In the absence of miR-223 (miR-223/y), granulocytes are hypermature, hypersensitive to stimuli and they display enhanced fungicidal activity. As a result, miR-223/y mice develop spontaneous inflammatory lung pathology and exhibit an exaggerated tissue destruction following endotoxin challenge. The mutant mice display an expanded granulocytic compartment as a result of progenitor hyperproliferation. Ablation of Mef2c, a transcription factor that promotes progenitor proliferation and a miR-223 target predicted with the highest confidence in miR-223/y mice, does not rectify the functional defects, while the progenitor proliferation and granulocyte differentiation defects are rescued. These results suggest that miR-223 is a negative regulator of progenitor proliferation, and granulocyte differentiation and activation. In addition, the data imply that miR-223 may regulate distinct targets during granulocyte development and function. Similar to miR-155, miR-146a is induced in human monocytes after in vitro activation by LPS, suggesting that this miRNA may be involved in mammalian microbial infection. In a recent publication, the same group demonstrates that miR-146a null mice manifest an exaggerated inflammatory response to endotoxin challenge (Boldin et al. 2011). In addition, old miR-146a/ mice develop spontaneous autoimmune symptoms characterized by splenomegaly, lymphadenopathy, and multi-organ inflammation that ultimately leads to premature death. MiR-146a also regulates immunological tolerance by controlling the IFN-g signaling pathway in Treg cells (Lu et al. 2010). Thus, miR-155 and miR-146a appear to govern both the innate and adaptive immune responses to foreign and endogenous antigens. The dysregulated expression of either miRNA will lead to the breakdown of immune regulation and tolerance in mice. Because of their inducible nature, miR-155 and miR-146a would be excellent targets for the treatment of inflammatory and autoimmune diseases without interfering with normal immune responses.

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Autoimmunity Data from animal studies highlighted above clearly demonstrate that the dysregulated expression of a subset of miRNAs involved in normal lymphoid development and functions leads to some form of autoimmunity. MiR-17-92 cluster controls targets that may contribute to autoimmunity. By contrast, miR-146 and miR-223 targets appear to suppress autoimmunity. Although overexpression of miR155 in mice does not appear to contribute to autoimmunity, its deficiency clearly alleviates inflammatory and autoimmune manifestations. It is suggested that miR-101 might be involved in the pathogenesis of lupus-like diseases in the sanroque mouse; however, there is no direct genetic evidence to support this observation (Yu et al. 2007). While the observations are intriguing, the final proof implicating miR-101 in lupus pathogenesis awaits the generation of a miR-101 mutant mouse models. The paucity of available miRNA mouse models for the study of autoimmune diseases such as systemic lupus erythematosus (SLE) is not due to a lack of interest in linking miRNAs to autoimmune diseases. Indeed, many groups have identified miRNAs that are differentially expressed in lupus patients by miRNA array assays using mostly peripheral blood mononuclear cells (PBMCs) (Thai et al. 2010). The type of miRNAs, and their putative targets, identified in these studies ranges from those associated with innate immunity to inflammation and DNA methylation. Suggestions from the data are tantalizing, yet again the final proof requires the generation and in-depth analysis of animal models for each of these miRNAs.

Micro-RNAs as Potential Drug Targets Due to their small size and potential roles in disease processes, miRNAs have been intensively exploited as potential drug targets by both academic institutions and biopharmaceutical companies. Several studies have demonstrated that the introduction of a small inhibitory nucleic acid

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molecule, either RNA or DNA, into animal models of disease abrogates molecular pathology and, in some cases, the disease itself. To date, only three miRNAs have been effectively targeted in vivo, miR-122, miR-155, and miR-21. The injection of an anti-miRNA modified oligonucleotide (locked nucleic acids, LNA) against the liver-specific miR-122 into mice and non-human primates selectively and effectively lowers the level of this miRNA in the liver, reduces serum cholesterol, and resolves Hepatitis C infection, and the effects are long-lasting (Lanford et al. 2010). The US Food and Drug Administration (FDA) approves a Clinical Phase 2a trial to assess the efficacy and tolerability of the anti-miR-122 LNA (miravirsenSPC3649) in treatment-naı¨ve patients with chronic Hepatitis C infection. This represents the first miRNA-targeted drug to enter clinical trials. At this point, no data is available to indicate if miravirsen-SPC3649 is efficacious in humans. As discussed above, the dysregulated expression of miR-155 and miR-146a has been implicated in inflammation and cancer in mice. However, to date, no targeted drugs for these miRNAs have moved into in vivo animal models or clinical trials. One group though demonstrated that it is possible to inhibit miR-155 in vivo (Zhang et al. 2012). Recently, miR-21 was effectively targeted in vivo in the lupus-prone mouse model B6sle123 with miR-21 LNA (Garchow et al. 2011). Since miR-21 is ubiquitously and constitutively expressed, it is not clear how targeting miR-21 would ameliorate lupus-like symptoms in their study. In addition, there is no mouse or human genetic data to support the results. In vivo miRNA replacement therapy has not been fully explored. However, one group is successful in inhibiting cancer cell proliferation, inducing tumor-specific apoptosis and providing protection from disease progression by systemically administering miR-26a into a hepatocellular carcinoma (HCC) mouse model (Kota et al. 2009). The replacement protocol described here requires the use of adeno-associated virus to express miR-26a in vivo. It might be more advantageous to develop means to introduce miRNA mimics,


thus avoiding unwanted adjuvant effects inherent in adenovirus vector. Although results in animal models proved promising, many hurdles must be overcome before anti-miRNAs or miRNA replacement therapy could be routinely used as therapeutic agents in humans: first, the mode of delivery to achieve specificity in vivo; second, the bioavailability of theses reagents; third, the potential offtarget effects of these RNA molecules; and finally the cost-effectiveness of developing large-scale RNA agents.

Perspective One can conclude without any doubt that miRNAs participate, along with coding genes, in the regulation of complex biological processes in mammalian hematopoietic/immune system. However, there is a dearth of genetic studies in human to implicate the dysregulation of miRNA expression in the pathogenesis of human diseases. To date only one study in which targeted deletion of a miRNA region faithfully recapitulates a disease state: deletion of the LEU2/miR-15a/16-1 region results in the onset of a form of CLL manifested by a subset of patients. What accounts for the paucity of human genetic evidence to implicate miRNAs in human diseases? Is it because most miRNAs do not act as master regulators of gene expression, but rather as modifiers of gene levels? Or most miRNAs reside in more stable genomic regions that are protected from agents that induce genetic instability? Or perhaps the direction of research carried out by miRNA biologists precludes the identification of genetic evidence implicating miRNAs in human diseases? The answer is probably all of the above. While the first two issues are beyond scientists’ ability to address since the nature of biology could not be modified, the miRNA biology community could focus its creativity and resources on in-depth genetic and in vivo functional studies such as the one described for the DLEU2/ miR15a/16-1 in CLL, to definitively draw a connection between miRNA expression/ function and autoimmunity.

Microscopic Polyangiitis

Cross-References ▶ Cytotoxic T Lymphocytes ▶ Resolution of Inflammation ▶ Systemic Lupus Erythematosus, Animal Models ▶ Systemic Lupus Erythematosus, Autoantibodies ▶ Therapeutic Considerations in Kidney Diseases Due to Glomerulonephritis

References Ameres SL, Fukunaga R. Riding in silence: a little snowboarding, a lot of small RNAs. Silence. 2010;1(1):8. Boldin MP, Taganov KD, Rao DS, Yang L, Zhao JL, Kalwani M, et al. miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med. 2011;208(6):1189–201. Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem. 2010;79:351–79. Garchow BG, Encinas OB, Leung YT, Tsao PY, Eisenberg RA, Caricchio R, et al. Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice. EMBO Mol Med. 2011;3(10):605–15. Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, et al. Regulation of progenitor cell proliferation and granulocyte function by microRNA223. Nature. 2008;451(7182):1125–9. Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell. 2009;137(6): 1005–17. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science. 2010;327(5962): 198–201. Lu LF, Boldin MP, Chaudhry A, Lin LL, Taganov KD, Hanada T, et al. Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses. Cell. 2010;142(6):914–29. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 2007;17(3):118–26. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, et al. Requirement of bic/microRNA-155 for normal immune function. Science. 2007; 316(5824):608–11. Schietinger A, Delrow JJ, Basom RS, Blattman JN, Greenberg PD. Rescued tolerant CD8 T cells are preprogrammed to reestablish the tolerant state. Science. 2012;335(6069):723–7.

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Taylor D, Badiani P, Weston K. A dominant interfering Myb mutant causes apoptosis in T cells. Genes Dev. 1996;10(21):2732–44. Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y, et al. Regulation of the germinal center response by microRNA-155. Science. 2007; 316(5824):604–8. Thai TH, Christiansen PA, Tsokos GC. Is there a link between dysregulated miRNA expression and disease? Discov Med. 2010;10(52):184–94. Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008;132(5):875–86. Xiao C, Calado DP, Galler G, Thai TH, Patterson HC, Wang J, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell. 2007;131(1):146–59. Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol. 2008;9(4): 405–14. Yu D, Tan AH, Hu X, Athanasopoulos V, Simpson N, Silva DG, et al. Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature. 2007;450(7167):299–303. Zhang Y, Roccaro AM, Rombaoa C, Flores L, Obad S, Fernandes SM, et al. LNA-mediated anti-miR-155 silencing in low-grade B-cell lymphomas. Blood. 2012;120(8):1678–86. Zhou B, Wang S, Mayr C, Bartel DP, Lodish HF. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci USA. 2007;104(17):7080–5.

Microscopic Polyangiitis Atul Khasnis and Leonard H. Calabrese Department of Rheumatic and Immunologic Diseases, Section of Clinical Immunology, Cleveland Clinic Foundation, Cleveland, OH, USA

Definition A systemic inflammatory disease characterized by necrotizing small and medium vessel vasculitis.

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History Microscopic polyangiitis (MPA) was first described as a microscopic variant of polyarteritis nodosa (Wohlwill. 1923). It was classified as a small and medium vessel vasculitis under the Chapel Hill Consensus Conference (Jennette et al. 1994). In 1950, Wainwright and Davson used the term “microscopic polyarteritis” to describe this illness (Wainwright and Davson 1950).


multiple pathways that include apoptosis, necrosis, and neutrophil extracellular traps. Although this has been well documented in some preclinical models, it has been inconsistently demonstrated in clinical studies. Although there appears to be an important antigen-antibody interaction in the pathogenesis of this illness, immune complexes are not observed in tissues on biopsy. Abnormal host immune responses undoubtedly participate in the onset and maintenance of vascular injury.

Epidemiology Clinical Features The annual incidence of MPA has been reported to be 2–17 per million in various populations (Gibelin et al. 2011). There is no gender or age predilection for MPA, but the incidence has been reported to peak in the sixth to seventh decade.

Etiology and Pathogenesis The etiology of MPA is unknown. The disease is characterized by necrotizing inflammation of arterioles and venules with infiltration of neutrophils and lymphocytes, often with leukocytoclasis and fibrinoid necrosis. The pathogenesis of MPA is incompletely understood but has been proposed to involve the formation of antineutrophil cytoplasmic antibodies (ANCAs) (Jennette et al. 2011). The mechanisms that lead to ANCA production are unclear. ANCAs are, however, neither absolutely necessary nor sufficient to cause histologic or clinical manifestations of MPA. The initial event in MPA may involve the priming of neutrophils with cytokines such as tumor necrosis factor alpha (TNF-a) which could be triggered by environmental factors, such as infection. Neutrophil priming results in translocation of normally intracellular neutrophil granule contents to its surface. One of the proteins translocated in this way is myeloperoxidase (MPO), an enzyme that forms the antigenic target for the perinuclear ANCA (pANCA). The binding of ANCA to MPO results in neutrophil activation, degranulation, and demise by

MPA tends to involve predominantly small and medium sized blood vessels in multiple visceral organs. In one study of patients with MPA (Guillevin et al. 1999), the mean age was 56.8 years and major manifestations included renal disease (78.8 %), weight loss (72.9 %), rash (62.4 %), fever (55.3 %), mononeuritis multiplex (57.6 %), arthralgias (50.6 %), myalgias (48.2 %), hypertension (34.1 %), lung involvement (24.7 %), alveolar hemorrhage (11.8 %), and cardiac failure (17.6 %). Patients with MPA tend not to have ear, nose, and throat (ENT) involvement or lung nodules and they lack granulomatous inflammation. These features, along with the ANCA pattern and specificity (see below), help to distinguish it from granulomatous polyangiitis (Wegener’s) (GPA).

Diagnosis The diagnosis of MPA is based on a compatible clinical presentation accompanied by laboratory test results (including a positive ANCA serology) and/or imaging studies. The clinical presentation of systemic inflammation is often accompanied by concordant laboratory tests. These include a normochromic normocytic anemia, leukocytosis, thrombocytosis, low serum albumin, abnormal urinalysis (microscopic hematuria, proteinuria, and/or red blood cell casts), and elevated acute phase reactants such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP).


Positive ANCAs are present in 75–80 % patients with a predominant pANCA pattern on immunofluorescence and antibodies directed at myeloperoxidase (MPO) by Enzyme Linked ImmunoSorbent Assay (ELISA) testing. Chest imaging with CT scan without radiographic contrast can be used to evaluate pulmonary involvement in the form of capillaritis/alveolitis/diffuse alveolar hemorrhage which typically shows a “ground glass appearance,” In patients with multiple episodes of previous diffuse alveolar hemorrhage or capillaritis, pulmonary fibrosis in the form of “honeycombing” may be seen. Renal biopsy in indicated cases classically demonstrates a pauciimmune (relative lack of immune complexes on immunofluorescence and electron microscopy) crescentic glomerulonephritis.

Treatment The treatment of MPA is guided by the severity of disease. The treatment paradigm for MPA has been extrapolated from treatment of related small vessel vasculitides such as GPA. The treatment is based on the classification of MPA as severe (organ-threatening or life-threatening) or non-severe. With either type, glucocorticoids form the cornerstone of treatment. The choice of a second immunosuppressive medication is driven by the severity of disease. In 1996, the French Vasculitis Study Group devised a five-factor score (FFS) (Guillevin et al. 1996) based on patients with polyarteritis nodosa, MPA, and Churg Strauss syndrome (CSS) which included proteinuria >1 g/dL, renal insufficiency (stabilized peak creatinine >1.4 mg/dL), cardiomyopathy, severe gastrointestinal manifestations, and CNS involvement. The revised FFS (Guillevin et al. 2011) based on patients with PAN, MPA, CSS, and GPA found that age >65 years, cardiac symptoms, gastrointestinal involvement, and renal insufficiency (stabilized peak creatinine >1.69 mg/dL) were associated with a high 5-year mortality. The presence of each was assigned 1 point. ENT symptoms (most commonly affecting patients with GPA and CSS) were

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associated with a lower risk of death, and therefore, their absence was also assigned 1 point. For patients with non-severe disease (FFS ¼ 0), glucocorticoids alone may be sufficient as they have been shown to result in survival rates comparable to those of patients who received glucocorticoids and cyclophosphamide. For severe disease (FFS 1), the typical treatment regimen involves high-dose glucocorticoids (often intravenous followed by oral) and either oral cyclophosphamide (2 mg/kg/day) or rituximab. In a study that compared cyclophosphamide to rituximab for remission induction in patients with new onset or relapsing GPA or MPA, rituximab was found to be non-inferior to cyclophosphamide (Stone et al. 2010). At 3–6 months, upon achievement of remission, the cyclophosphamide may be replaced with a maintenance immunosuppression medication such as methotrexate, azathioprine, or mycophenolate mofetil (Pagnoux et al. 2008) with continued taper of glucocorticoids. The choice of the maintenance immunosuppressant should be individualized based on patient preference, comorbidities, and other factors such as thiopurine methyltransferase (TPMT) genotype status for patients being considered for azathioprine. In patients who receive rituximab as induction therapy, whether to add a maintenance immunosuppression medication or continue the glucocorticoid taper alone is controversial. In patients with nonsevere disease, methotrexate along with high-dose glucocorticoids can be used to induce remission provided renal insufficiency does not contraindicate the use of methotrexate. Informed decision making by the patient in terms of risk and benefit is imperative in the selection of immunosuppression and close clinical and laboratory monitoring is warranted. Plasma exchange has been shown to be effective as adjunctive therapy to conventional immunosuppression in patients with advanced renal failure in the setting of MPA (Jayne et al. 2007).

Prognosis The FFS was also used to derive prognostic information in patients with MPA based on the

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severity of illness. Based on the original FFS of 0, 1, and > ¼2 were associated with 5-year mortality rates of 12 %, 26 %, and 46 %, respectively. According to the revised FFS, 5-year mortality rates for scores of 0, 1, and > ¼2 were 9 %, 21 %, and 40 %, respectively. For 13 patients with MPA who died during the first year, older age, renal involvement, central nervous system involvement, and possibly cardiomyopathy seemed to confer the greatest risk of death. Treatment confers a good prognosis in patients with MPA. Even in patients with a FFS ¼ 0, with a good overall 5-year survival, glucocorticoids achieved and maintained remission in only about half of the patients (40 % patients required additional immunosuppressive therapy) (Ribi et al. 2010). ANCA-associated vasculitides (GPA, MPA, CSS, and renal limited vasculitis) can result in end-stage renal disease and account for 1–2 % of patients on renal replacement therapy (Stegeman CA. 2012); however, separate data on the contribution of MPA is unavailable.

Conclusion MPA is a systemic small and medium vessel vasculitis that merits prompt recognition and treatment. The choice of immunosuppression is guided by the severity of illness and mandates close monitoring. The introduction of newer therapeutic agents such as rituximab and the controlled duration of use of agents such as oral cyclophosphamide with change to maintenance immunosuppression after securing disease remission have made the treatment of this illness safer and more effective.

Cross-References ▶ Polyarteritis Nodosa ▶ Therapeutic Considerations in Kidney Diseases Due to Glomerulonephritis ▶ Vasculitis: Granulomatosis with Polyangiitis (Wegener’s)


References Gibelin A, Maldini C, Mahr A. Epidemiology and etiology of Wegener granulomatosis, microscopic polyangiitis, churg-strauss syndrome and goodpasture syndrome: vasculitides with frequent lung involvement. Semin Respir Crit Care Med. 2011; 32(3):264–73. Guillevin L, Lhote F, Gayraud M, et al. Prognostic factors in polyarteritis nodosa and Churg-Strauss syndrome. A prospective study in 342 patients. Medicine (Baltimore). 1996;75(1):17–28. Guillevin L, Durand-Gasselin B, Cevallos R, Gayraud M, Lhote F, Callard P, et al. Microscopic polyangiitis: clinical and laboratory findings in eighty-five patients. Arthritis Rheum. 1999;42(3): 421–30. Guillevin L, Pagnoux C, Seror R, et al. The Five-Factor Score revisited: assessment of prognoses of systemic necrotizing vasculitides based on the French Vasculitis Study Group (FVSG) cohort. Medicine (Baltimore). 2011;90(1):19–27. Jayne DR, Gaskin G, Rasmussen N, Abramowicz D, Ferrario F, Guillevin L, et al. Randomized trial of plasma exchange or high-dosage methylprednisolone as adjunctive therapy for severe renal vasculitis. J Am Soc Nephrol. 2007;18(7):2180–8. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum. 1994;37: 187–92. Jennette JC, Falk RJ, Gasim AH. Pathogenesis of antineutrophil cytoplasmic autoantibody vasculitis. Curr Opin Nephrol Hypertens. 2011;20(3):263–70. Pagnoux C, Mahr A, Hamidou MA, Boffa JJ, Ruivard M, Ducroix JP, et al. Azathioprine or methotrexate maintenance for ANCA-associated vasculitis. N Engl J Med. 2008;359(26):2790–803. Ribi C, Cohen P, Pagnoux C, et al. Treatment of polyarteritis nodosa and microscopic polyangiitis without poor-prognosis factors: A prospective randomized study of one hundred twenty-four patients. Arthritis Rheum. 2010;62(4):1186–97. Stegeman CA. Microscopic polyangiitis. In: Hoffman GS, Weyand CM, Langford CA, Goronzy JJ, editors. Inflammatory diseases of blood vessels. Chichester: Wiley Blackwell; 2012. p. 227–37. Stone JH, Merkel PA, Spiera R, Seo P, Langford CA, Hoffman GS, et al. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010;363(3):221–32. Wainwright J, Davson J. The renal appearances in the microscopic form of periarteritis nodosa. J Pathol Bacteriol. 1950;62(2):189–96. € Wohlwill F. Uber die nur mikroskopisch erkennbare Form der Periarteritis nodosa. Virchows Arch Pathol Anat Physiol. 1923;246:36.

Mixed Connective Tissue Disease (MCTD)

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Mixed Connective Tissue Disease (MCTD), Table 1 The Alarcon-Segovia classification criteria for MCTD

Mixed Connective Tissue Disease (MCTD) Eric L. Greidinger Division of Rheumatology, Miami VAMC and University of Miami Miller School of Medicine, Miami, FL, USA

1. High-titer RNP+ 2. Swollen hands 3. Synovitis 4. Myositis 5. Raynaud’s 6. Acrosclerosis To be classified with MCTD, patients must have criterion 1, plus at least 3 of the remaining 5 criteria

Synonyms Mixed connective syndrome

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Definition MCTD is an autoimmune syndrome in which high-titer autoantibodies to the U1 small nuclear ribonucleoprotein (anti-RNP) are present along with overlapping clinical manifestations of lupus, scleroderma, and/or inflammatory myositis. Several classification criteria for MCTD have been published that perform similarly in identifying patients with MCTD (Alarcoń-Segovia and Cardiel 1989). The criteria set of Alarcon-Segovia and colleagues is particularly concise and easy to apply in clinical settings (Table 1). However, it may be less likely to identify patients with primarily pulmonary manifestations as having MCTD compared to more complex schemes (Gunnarsson et al. 2012). Clinical overlap syndromes in the absence of anti-RNP antibodies exist and are distinct from MCTD. Patients with other well-defined rheumatic diseases (especially lupus and scleroderma) may have anti-RNP antibodies without having MCTD. Among lupus patients, anti-RNP is a marker of more severe disease including an increased risk of nephritis (Kirou et al. 2005). In contrast, MCTD often follows a mild and benign clinical course, though a significant risk of lung diseases emerges in some MCTD patients later in the course of their disease (Burdt et al. 1999).

Historical Background MCTD was first described by Gordon Sharp and colleagues, working initially at Stanford and then at the University of Missouri-Columbia in the late 1960s and early 1970s (Sharp 2002). It was the first rheumatic syndrome to be defined in part by the presence of a specific set of autoantibodies (anti-RNP). Controversy has surrounded MCTD regarding the extent to which it was or was not distinct from lupus and other rheumatic diseases. Reports showing that, compared to lupus, MCTD has different Class II MHC disease associations, different autoantibody epitope targeting, and can be clinically distinguished by machine learning approaches have muted these concerns to some extent (Hoffman and Greidinger 2012). Concerns remain that the name “Mixed Connective Tissue Disease” may connote too general a sense of an overlap syndrome despite its acceptance as a clearly defined clinical entity (Swanton and Isenberg 2005).

Clinical Features The most common manifestations of MCTD include Raynaud’s Phenomenon, swollen hands, arthritis, sicca complaints, and esophageal disease (Table 2). Other typical manifestations include lupus-like and/or scleroderma-like skin changes, myositis, serositis, and the potential for lung disease. The manifestations of MCTD are

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Mixed Connective Tissue Disease (MCTD), Table 2 Clinical manifestations present in >50 % of MCTD patients in multiple cohorts Raynaud’s Phenomenon Hand swelling Synovitis Dry eyes and/or mouth Gastroesophageal reflux

>85 % >75 % >75 % >56 % >51 %

generally consistent across geographical and ethnic boundaries (Maldonado et al. 2008). A nationwide retrospective study in Norway estimated the point prevalence of MCTD in adults to be 3.8/100,000, with an incidence of 2.1/1,000,000, and a 3.3:1 female predominance (Gunnarsson et al. 2011). These values are similar to those observed elsewhere. Since the initial reports by Sharp and others, MCTD has been recognized to be a generally more mild form of rheumatic disease that tends not to be associated with the most serious complications of lupus, scleroderma, or myositis. Diffuse proliferative glomerulonephritis, scleroderma renal crisis, and acute respiratory failure due to profound muscle weakness are all rare in MCTD. There is, however one exception: at least one third of MCTD patients develop serious lung disease manifestations including pulmonary hypertension, interstitial pneumonitis, or pulmonary fibrosis. Lung disease is the predominant mechanism of death attributable to MCTD, and it tends to emerge later in the evolution of the condition. Although MCTD may be less likely to lead to major organ complications than SLE, disease activity can be substantial and impact the ability of patients to perform their activities of daily living. Most MCTD patients typically require some level of ongoing treatment. Among patients who escape the major organ complications of MCTD, some may ultimately lose their anti-RNP antibodies and enter sustained clinical remission. Given the potential for lung involvement in MCTD, active surveillance for evidence of lung disease with pulmonary function testing and echocardiography to assess right heart pressures

is commonly performed, though these tests may miss some patients with evolving disease. Right heart catheterization to assess for pulmonary hypertension and/or high-resolution chest CT to assess for interstitial lung disease can be helpful when clinical suspicion is high. Investigators are seeking safer, less invasive tests with higher diagnostic yield to improve future disease management.

Treatment In contrast to scleroderma lung disease in which immunosuppressive therapy has yielded minimal clinical benefit, lung disease in MCTD may be highly responsive to aggressive immunosuppression, including approximately 50 % of cases with pulmonary hypertension (Jais et al. 2008). No validated prediction rule has yet emerged to distinguish MCTD lung disease patients who will respond to immunosuppression from those who will not. The relative effectiveness of other immunosuppressive therapies (such as mycophenolate or calcineurin inhibitors) for MCTD lung disease compared to cyclophosphamide remains to be established. There are scant treatment trials published regarding MCTD patients. The accrued evidence and anecdotal reports supports the use antimalarials, corticosteroids, disease modifyingantirheumatic drugs (DMARDs), and immunosuppressives for the treatment of MCTD activity. Treatments for organ-specific manifestations of MCTD, such as vasodilators for Raynaud’s Phenomenon, proton pump inhibitors for gastroesophageal reflux disease, and topical steroids for inflammatory rashes, appear to be similarly helpful as in other rheumatic diseases. Biologic antirheumatic therapies have not been systematically tested in MCTD. Progress in understanding the pathogenesis of MCTD may offer more opportunities to develop biologic drugs for use in this disease. Potential targets may include T cell response modifiers, anti-B cell therapies, Toll-like receptor antagonists, Type I Interferon antagonists, and IL-17 axis antagonists. Some promise exists that


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Other Targets Autoantibodies Mixed Connective Tissue Disease (MCTD), Fig. 1 Putative pathogenesis of MCTD: Dead cell debris functions as a source of autoantigenic and autoinflammatory stimuli. Taken up by myeloid dendritic cells (mDC) or other antigen presenting cells (APC), the U1-RNA and other pro-inflammatory components of this cellular debris activate TLR3 and potentially TLR7 and other innate immune sensors to induce pro-inflammatory responses including elaboration of type I interferons and presentation of autoantigens

to autoreactive T cells. A subset of TLR3-expressing mDC traffic to the lungs and induce the influx of autoreactivce T cells to the lungs leading to interstitial lung disease and pulmonary hypertension. Under the influence of other APC, T cells traffic to other target organs and support the development of antigen-driven autoantibody responses that also induce end organ pathology in target tissues. Target tissue injury induces additional cell debris that can cause perpetuation and amplification of the autoimmune process

antigen-targeted tolerogenic therapies may also be effective in anti-RNP autoimmunity, based on animal models and studies in SLE (Page et al. 2009; Trivedi et al. 2010).

to have relevance to autoimmune disease pathogenesis, including metal-catalyzed oxidation, granzyme B cleavage, phosphorylation, and RNA conjugation (Hof et al. 2005). The RNA backbone of the U1 snRNP has been found to be a potent agonist of Toll-like receptors 3 and 7 (Greidinger et al. 2007). A murine model of MCTD induced by immunization with an RNP peptide and U1-RNA has supported the model of MCTD as an antigen-driven immune process dependent on TLR activation (Greidinger et al. 2006). Oligoclonal anti-RNP T cell responses are seen in the mice that are homologous to those seen in human patients (Greidinger et al. 2008). Adoptive transfer studies have shown the importance of myeloid dendritic cells for the development of MCTD-like lung disease (Greidinger et al. 2009). A figure integrating the established data regarding MCTD pathogenesis (Fig. 1) must be regarded as largely speculative.

Pathogenesis Autoantigen-specific T cells and immunoglobulin have been identified in MCTD patients (Greidinger and Hoffman 2005). Patterns of epitope spreading in MCTD patients over time implicate immune responses against U1 snRNP macromolecules. Early recognition of apoptosis-specific RNP epitopes in some patients suggests that, as has been hypothesized in lupus, apoptotic cell debris may constitute a significant source of antigenic stimulus in MCTD. RNP antigens targeted in MCTD are also susceptible to other forms of posttranslational modification that have been hypothesized

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Cross-References ▶ Complement in Rheumatic Diseases ▶ Discoid Lupus ▶ Juvenile Diseases: SLE in Children ▶ Raynaud’s Phenomenon ▶ Scleroderma (Systemic Sclerosis): Pathogenesis and Clinical Manifestations ▶ Scleroderma-Like Conditions of the Skin ▶ Skin in Systemic Lupus Erythematosus ▶ Systemic Lupus Erythematosus, Animal Models ▶ Systemic Lupus Erythematosus, Autoantibodies ▶ Systemic Lupus Erythematosus, Clinical Features and Diagnosis ▶ Systemic Lupus Erythematosus, Pathogenesis ▶ Systemic Lupus Erythematosus, Treatment ▶ Therapeutic Considerations in Kidney Diseases Due to Glomerulonephritis

References Alarcoń-Segovia D, Cardiel MH. Comparison between 3 diagnostic criteria for mixed connective tissue disease. Study of 593 patients. J Rheumatol. 1989;16(3):328–34. Burdt MA, Hoffman RW, Deutscher SL, Wang GS, Johnson JC, Sharp GC. Long-term outcome in mixed connective tissue disease: longitudinal clinical and serologic findings. Arthritis Rheum. 1999;42(5): 899–909. Greidinger EL, Hoffman RW. Autoantibodies in the pathogenesis of mixed connective tissue disease. Rheum Dis Clin North Am. 2005;31(3):437–50. vi. Greidinger EL, Zang Y, Jaimes K, Hogenmiller S, Nassiri M, Bejarano P, Barber GN, Hoffman RW. A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen. Arthritis Rheum. 2006;54(2):661–9. Greidinger EL, Zang Y, Martinez L, Jaimes K, Nassiri M, Bejarano P, Barber GN, Hoffman RW. Differential tissue targeting of autoimmunity manifestations by autoantigen-associated Y RNAs. Arthritis Rheum. 2007;56(5):1589–97. Greidinger EL, Zang YJ, Jaimes K, Martinez L, Nassiri M, Hoffman RW. CD4+ T cells target epitopes residing within the RNA-binding domain of the U1-70-kDa small nuclear ribonucleoprotein autoantigen and have restricted TCR diversity in an HLA-DR4-transgenic murine model of mixed connective tissue disease. J Immunol. 2008;180(12):8444–54.

Mixed Connective Tissue Disease (MCTD) Greidinger EL, Hoffman RW. Mixed connective tissue disease. In: Wallace DJ, Hahn BH,editors. 9th ed. Dubois’ Lupus Erythematosus and Related Syndromes; Philadelphia: Elsevier, 2013. Greidinger EL, Zang Y, Fernandez I, Berho M, Nassiri M, Martinez L, Hoffman RW. Tissue targeting of antiRNP autoimmunity: effects of T cells and myeloid dendritic cells in a murine model. Arthritis Rheum. 2009;60(2):534–42. Gunnarsson R, Molberg O, Gilboe IM, Gran JT. PAHNOR1 study group. The prevalence and incidence of mixed connective tissue disease: a national multicentre survey of Norwegian patients. Ann Rheum Dis. 2011;70(6):1047–51. Gunnarsson R, Aaløkken TM, Molberg O, Lund MB, Mynarek GK, Lexberg AS, Time K, Dhainaut AS, Bertelsen LT, Palm O, Irgens K, Becker-Merok A, Nordeide JL, Johnsen V, Pedersen S, Prøven A, Garabet LS, Gran JT. Prevalence and severity of interstitial lung disease in mixed connective tissue disease: a nationwide, cross-sectional study. Ann Rheum Dis. 2012;71:1966–72. May 1. [Epub ahead of print]. Hof D, Raats JM, Pruijn GJ. Apoptotic modifications affect the autoreactivity of the U1 snRNP autoantigen. Autoimmun Rev. 2005;4(6):380–8. Jais X, Launay D, Yaici A, Le Pavec J, Tche´rakian C, Sitbon O, Simonneau G, Humbert M. Immunosuppressive therapy in lupus- and mixed connective tissue disease-associated pulmonary arterial hypertension: a retrospective analysis of twenty-three cases. Arthritis Rheum. 2008;58(2):521–31. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum. 2005;52(5):1491–503. Maldonado ME, Perez M, Pignac-Kobinger J, Marx ET, Tozman EM, Greidinger EL, Hoffman RW. Clinical and immunologic manifestations of mixed connective tissue disease in a Miami population compared to a Midwestern US Caucasian population. J Rheumatol. 2008;35(3):429–37. Page N, Schall N, Strub JM, Quinternet M, Chaloin O, Dećossas M, Cung MT, Van Dorsselaer A, Briand JP, Muller S. The spliceosomal phosphopeptide P140 controls the lupus disease by interacting with the HSC70 protein and via a mechanism mediated by gammadelta T cells. PLoS One. 2009;4(4):e5273. Epub 2009 Apr 23. Sharp GC. MCTD: a concept which stood the test of time. Lupus. 2002;11(6):333–9. Swanton J, Isenberg D. Mixed connective tissue disease: still crazy after all these years. Rheum Dis Clin North Am. 2005;31(3):421–36. v. Trivedi S, Zang Y, Culpepper S, Rosenbaum E, Fernandez I, Martinez L, Hoffman RW, Greidinger EL. T cell vaccination therapy in an induced model of anti-RNP autoimmune glomerulonephritis. Clin Immunol. 2010;137(2):281–7.

Mucous Membrane Pemphigoid

Mucous Membrane Pemphigoid Bryan D. Sofen and Nicholas A. Soter The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY, USA

Synonym Cicatricial pemphigoid

Definition Mucous membrane pemphigoid (MMP), previously known as “cicatricial pemphigoid,” (Chan et al. 2002) is a chronic, autoimmune, subepithelial blistering disorder characterized by involvement of predominantly mucosal surfaces and a tendency for scarring. HLA type DQB1*0301(DQ7) is common amongst patients with both MMP and classic bullous pemphigoid.

Clinical A chronic and progressive disease, MMP may be associated with substantive morbidity due to the tendency for scarring and fibrosis. When present on the conjunctivae, these effects may lead to blindness. The histopathologic presentation of MMP is similar to that observed in classic bullous pemphigoid but with fewer eosinophils and more plasma cells being present in the lymphohistiocytic infiltrate that surrounds the subepidermal blisters. Mast cells and other mononuclear leukocytes are also found. Later stages also show granulation tissue in the submucosa and the characteristic fibrosis in the upper dermis.

Pathophysiology The heterogenous nature of this condition is reflected in the variety of antigenic targets that

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are associated with mucous membrane pemphigoid. The principal antigens for pathogenic autoantibodies, which are all in the basement membrane zone, are the hemidesmosomal bullous pemphigoid antigen 180 (BP180, BPAG2, or type XVII collagen) and laminin 5 (laminin 332), which is a component of the anchoring filaments outside the hemidesmosomal plaque. Several other pathogenic target antigens have been suggested, which include the a6 (predominant in oral pemphigoid) and b4 (predominant in ocular pemphigoid) subunits of the transmembrane and hemidesmosomal a6b4 integrins (Bhol et al. 2000, 2001; Rashid et al. 2006), the major ligand for which is laminin 5. Collagen VII and BP230 have also been implicated in some cases (Chan et al. 2002). Autoantibodies are mostly IgG1 and IgG4 isotypes, with IgA1 antibodies present in a minority of cases (Bernard et al. 1991). Despite the similarity of target antigens with bullous pemphigoid and other bullous diseases in many cases of MMP, the specific mechanisms and reasons for the mucosal predilection, chronicity, and tendency for scarring are not well understood. For BP180, the carboxy terminal is the immunodominant epitope in oral pemphigoid, with the shed ectodomain (LABD97) autoreactive in other forms of MMP (Calabresi et al. 2007; Kromminga et al. 2002; Leverkus et al. 2001; Schmidt et al. 2001; Schumann et al. 2000). The a3 subunit of laminin 5 is the principal autoreactive epitope in patients with laryngeal involvement; less frequently the b3 and/or g2 subunits are involved (Chan et al. 2002; Seo et al. 2001). NC16A domain-specific T cells are present in a minority of patients, which contributes to blister formation along with released TNF-a and IFN-g. Complement is not required (Lazarova et al. 2000). Increased expression of TGF-b1 and collagen-binding heat shock protein 47 likely contributes to conjunctival scarring in ocular disease (Black et al. 2004). Perilesional biopsy specimens for direct IF are often difficult to obtain owing to the mucosal site, but if obtained, most patients display a fine, linear deposition along the epithelial

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BMZ of IgG and/or C3. Less commonly, IgA and/or IgM may be observed (Bean et al. 1972). Circulating antibodies are observed by indirect IF in only a few cases.

Seo SH, et al. Antiepiligrin cicatricial pemphigoid with autoantibodies to the beta subunit of laminin 5 and associated with severe laryngeal involvement necessitating tracheostomy. Dermatology. 2001;202(1):63–6.

References


Bean SF, et al. Cicatricial pemphigoid. Immunofluorescent studies. Arch Dermatol. 1972;106(2):195–9. Bernard P, et al. The subclass distribution of IgG autoantibodies in cicatricial pemphigoid and epidermolysis bullosa acquisita. J Invest Dermatol. 1991;97(2): 259–63. Bhol KC, et al. The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4. J Immunol. 2000;165(5):2824–9. Bhol KC, et al. Autoantibodies to human alpha6 integrin in patients with oral pemphigoid. J Dent Res. 2001;80(8):1711–5. Black AP, et al. Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid. Br J Dermatol. 2004;151(6):1160–4. Calabresi V, et al. Oral pemphigoid autoantibodies preferentially target BP180 ectodomain. Clin Immunol. 2007;122(2):207–13. Chan LS, et al. The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators. Arch Dermatol. 2002;138(3):370–9. Kromminga A, et al. Cicatricial pemphigoid differs from bullous pemphigoid and pemphigoid gestationis regarding the fine specificity of autoantibodies to the BP180 NC16A domain. J Dermatol Sci. 2002;28(1):68–75. Lazarova Z, et al. Fab fragments directed against laminin 5 induce subepidermal blisters in neonatal mice. Clin Immunol. 2000;95(1 Pt 1):26–32. Leverkus M, et al. Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230. Br J Dermatol. 2001;145(6):998–1004. Rashid KA, Gurcan HM, Ahmed AR. Antigen specificity in subsets of mucous membrane pemphigoid. J Invest Dermatol. 2006;126(12):2631–6. Schmidt E, et al. Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180. Br J Dermatol. 2001;145(5):778–83. Schumann H, et al. The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am J Pathol. 2000;156(2):685–95.

Marinos C. Dalakas Department of Pathophysiology, Neuroimmunology Unit, National University of Athens Medical School, Athens, Greece Thomas Jefferson University, Philadelphia, PA, USA

Synonyms Dermatomyositis; Inclusion body myositis; Inflammatory myopathies; Necrotizing myositis; Polymyositis

Definition Myositis refers to a group of inflammatory myopathies (IM) that constitute a heterogeneous group of subacute, chronic, or sometimes acute acquired muscle diseases, which have in common the presence of muscle weakness and inflammation on muscle biopsy. Based on distinct clinical, histological, and immunopathological criteria, as well as different degrees of response to therapies, the most common types of myositis seen in practice can be separated into four distinct subsets: polymyositis (PM), dermatomyositis (DM), necrotizing autoimmune myositis (NAM), and inclusion body myositis (IBM) (Dalakas 1991, 2010a, 2011; Dalakas and Hohlfeld 2003). PM was first recorded by Wagner in 1863 but became a recognized clinical entity 75 years later when Walton and Adams published a remarkable monograph entitled Polymyositis. The first definitive description of dermatomyositis was reported by Unverricht in 1891. Inclusion body myositis (IBM) was first recognized as a separate entity between 1967 and 1971 based on distinct


microtubular filamentous inclusions by electron microscopy in the muscle biopsies of some patients. The term “inclusion body myositis” was coined by Yunis and Samaha in 1971 to stress the uniqueness of the disease and to separate it from polymyositis. NAM has been known for some time but it has been always included within the group of PM, until the last 10 years when it has been increasingly recognized as a distinct entity attracting considerable attention for diagnosis and therapy (Dalakas 2010a, 2011). Main Clinical Features All forms have in common a myopathy characterized by muscle weakness which develops subacutely (weeks to months, as in PM and DM); acutely, even in days, as in some cases of NAM; or insidiously over years, as in IBM, simulating the tempo of a limb-girdle muscular dystrophy leading eventually to wheelchair confinement (Dalakas 1991, 2010a, 2011; Dalakas and Hohlfeld 2003). DM affects both children and adults, and females more often than males, whereas PM is seen after the second decade of life and very rarely in childhood. IBM is more frequent in men than women and more likely to affect persons over the age of 50 years comprising the most commonly acquired myopathy in this age group (Dalakas 1991, 2010a, 2011; Dalakas and Hohlfeld 2003; Mastaglia et al. 2003; Engel et al. 2008); in contrast, PM is the least common while DM is the most frequent inflammatory myopathy in children. Patients with these inflammatory myopathies have difficulty performing tasks requiring the use of proximal muscles, such as getting up from a chair, climbing steps, or lifting objects; IBM patients however may experience early difficulties with distal muscles such as inability to hold certain objects, turning keys, shaking hands, or buttoning a shirt. Falling is common among IBM patients because of early involvement of the quadriceps muscle that causes buckling of the knees and weakness of foot extensors (Dalakas 1991). All forms of myositis can be seen with increased frequency in patients with other

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systemic autoimmune, viral, or connective tissue diseases. Up to 10 % of patients have interstitial lung disease and anti-Jo-1 antibodies, directed against histidyl-transfer RNA synthetase; the presence of anti-Jo-1 antibodies denotes a high probability of having or developing interstitial lung disease (Dalakas 1991, 2011; Dalakas and Hohlfeld 2003; Mastaglia et al. 2003). Polymyositis has no unique clinical features, and its diagnosis is one of exclusion, best defined as an inflammatory myopathy of subacute onset (weeks to months) and steady progression occurring in adults that do not have the following: rash; involvement of eye and facial muscles; family history of a neuromuscular disease; endocrinopathy; history of exposure to myotoxic drugs or toxins; any neurogenic, dystrophic, or metabolic myopathy; and no signs of inclusion body myositis (Dalakas 1991, 2010a; Dalakas and Hohlfeld 2003). Unlike dermatomyositis, in which the rash secures early recognition, the actual onset of polymyositis cannot be easily determined, and the disease may exist for several months before the patient seeks medical advice. Dermatomyositis is a distinct clinical entity identified by a rash, which accompanies or, more often, precedes the muscle weakness. The typical skin changes include a heliotrope (blue-purple discoloration) on the upper eyelids with edema; a flat red rash on the face and upper trunk, anterior chest (often in a V sign), or shoulders (shawl sign); and erythema of the knuckles with a raised violaceous scaly eruption (Gottron rash) (Dalakas 1991, 2010a, 2011; Dalakas and Hohlfeld 2003; Mastaglia et al. 2003; Engel et al. 2008). Dermatomyositis usually occurs alone, but it may overlap with systemic sclerosis and mixed connective tissue disease. An increased incidence of malignancies is seen in patients with DM (particularly over 50 years of age), but not in PM or IBM, requiring frequent malignancy work-up and vigilance at least for the first 3 years (Dalakas 1991, 2011; Dalakas and Hohlfeld 2003). Ovarian cancer is most frequent, followed by breast, lung, and liver cancer. In Asian populations, nasopharyngeal cancer is more common.

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Necrotizing autoimmune myositis (NAM) has been an overlooked entity, misdiagnosed as polymyositis for many years. NAM may in fact be more common than polymyositis based on the number of cases increasingly recognized in large clinics. The patients present with very high CK, in the thousands, and moderate to severe muscle weakness of acute or subacute onset. The cause of NAM is multifactorial. Some patients have cancer or an active viral infection (i.e., HIV); others have been exposed to statins, which can induce both a toxic and an autoimmune necrotizing myositis that responds to immunotherapy; others may have a smoldering underlying autoimmune process; and still others have no other disease or apparent exposure to exogenous agents (Dalakas 2010a, 2011). The muscle biopsy shows only necrotic fibers and macrophages necessitating the absolute need to exclude dystrophic or toxic processes (Dalakas 2009), especially when the disease has subacute onset. Inclusion body myositis has a distinct presentation with the earliest and most frequent symptom falling and tripping due to weakness and atrophy of the quadriceps muscle and foot extensors (Dalakas 1991, 2010a; Mastaglia et al. 2003). In other patients, the disease may begin with weakness of the distal muscles of the hands leading to a weak grip. Inclusion body myositis is, therefore, a proximal and distal myopathy. Weakness is asymmetrical in one third of patients (Engel et al. 2008), progresses slowly over years, and is always associated with worsening atrophy. Dysphagia and facial muscle weakness are also common. Diagnosis The diagnosis of DM is relatively easy when the typical skin changes are apparent. The diagnosis of PM or NAM is one of exclusion and based on finding a steadily progressive myopathy of acute or subacute onset in adults who do not have a family history of a neuromuscular disease, history of exposure to myotoxic drugs or toxins, endocrinopathy, neurogenic disease, dystrophy, or IBM (Dalakas 1991). The diagnosis of IBM should be considered in an adult who has


slow-onset disease that involves distal muscles, especially foot extensors and deep finger flexors, and should be always suspected when a patient with presumed polymyositis did not respond to therapy (Dalakas 1991, 2010a; Dalakas and Hohlfeld 2003). The diagnosis is established or confirmed by elevated levels of serum muscle enzymes, electromyographic findings of an active myopathy, and, definitively, by the muscle biopsy. The serum CK, in the presence of active disease, can be elevated by as much as 50 times above normal and usually parallels disease activity; it can be however normal in active DM and IBM. In NAM, the CK is very high, usually around 8,000–15,000 IU/L. The muscle biopsy shows distinct changes in each subset. In DM the inflammation is perivascular or at the periphery of the fascicle and is often associated with perifascicular atrophy (Dalakas 1991; Engel et al. 2008). The presence of perifascicular atrophy even in the absence of inflammation should raise the suspicion of DM. In NAM, there are necrotic fibers invaded by macrophages; T cells are characteristically absent and MHC-I is not upregulated (except in necrotic fibers). In PM and IBM, the inflammation is in multiple foci within the endomysial parenchyma and consists predominantly of CD8+ T cells that invade healthy muscle fibers expressing the MHC-I antigen which is ubiquitously upregulated on the surface of most fibers. The MHC/DC8 complex is characteristic of PM and IBM, as discussed later. In IBM, in addition to inflammation, there are rimmed vacuoles and tiny amyloid deposits in a variable number of fibers, usually in or near the vacuoles, identified with Congo-red or crystal violet stains or with Texas-red fluorescent optics. The vacuoles contain 12–16 nm filamentous masses, reported to be identical to the paired helical filaments found in the brains of Alzheimer’s disease. Ragged-red fibers with mitochondrial excess as well as cytochrome oxidase-negative fibers are frequent (Engel et al. 2008; Dalakas 2011). A number of degeneration-associated and stressor molecules accumulate within the myofibers of IBM muscles, including beta-amyloid and a series of amyloid-related proteins including phosphorylated


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Myositis, Pathogenesis, Fig. 1 Immunopathogenesis of dermatomyositis. Activation of complement, possibly by autoantibodies (Y), against endothelial cells and formation of C3 via the classic or alternative pathway. Activated C3 leads to formation of C3b, C3bNEO, and membrane attack complexes (MAC), which are deposited in and around the endothelial cell wall of the endomysial capillaries. Deposition of MAC leads to destruction of capillaries, ischemia, or microinfarcts most prominent in the periphery of the fascicles and perifascicular atrophy.

B cells, CD4 T cells, and macrophages traffic from the circulation to the muscle. Endothelial expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) is induced by cytokines released by the mononuclear cells. Integrins, specifically very late activation antigen (VLA)-4 and leukocyte function-associated antigen (LFA)-1, bind VCAM and ICAM and promote T cell and macrophage infiltration of muscle through the endothelial cell wall

tau, best detected with antibodies to transporter protein p62/SQSTMI, ubiquitin, and others (Dalakas 2010a, 2011). Currently, there is no unique molecule that serves as a specific IBM biomarker of diagnostic value.

dilatation of the remaining capillaries in an effort to compensate for impaired perfusion (Dalakas 1991; Engel et al. 2008). Larger intramuscular blood vessels are also affected in the same pattern, leading to muscle fiber destruction (often resembling microinfarcts) and inflammation. The perifascicular atrophy often seen in more chronic stages is a reflection of the endofascicular hypoperfusion that is prominent distally (Dalakas 2011). Membranolytic attack complex and the early complement components C3b and C4b can be detected in the serum, correlate with disease activity, and are deposited on the capillaries before inflammatory or structural changes are seen in the muscle (Engel et al. 2008; Dalakas 2010a) (Fig. 1). The endomysial infiltrates, consisting of CD4+ cells, macrophages, B cells, CD8+ cells, and plasmacytoid dendritic cells, are prominent in the perimysial, perivascular, and perifascicular regions.

Immunopathogenesis Dermatomyositis The primary antigenic target in DM is the vascular endothelium of the endomysial blood vessels (Dalakas 1991; Hohlfeld and Dornmair 2007; Engel et al. 2008). The disease begins when putative antibodies directed against endothelial cells activate complement C3 that subsequently forms C3b and C4b fragments and leads to the formation of C5b-9 (MAC), the lytic component of the complement pathway that leads to osmotic lysis of the endothelial cells and necrosis of the capillaries. This results in marked reduction in the number of capillaries per muscle fiber and

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The release of cytokines and chemokines related to complement activation upregulates VCAM-I and ICAM-I on the endothelial cells and facilitates the exit of lymphoid cells through the blood vessel wall to the perimysial and endomysial spaces (Fig. 1). After successful therapy, these abnormalities are no longer prominent in the repeat biopsies (Dalakas 2010a, 2011). The perifascicular regions of DM contain many regenerating or degenerating fibers and they are in a stage of continuous remodeling. As a result, they stain with alkaline phosphatase, desmin, and NCAM, with the autoantibody against chromatin remodeler Mi-2 and with a variety of antibodies against immune or stressor molecules, including TGF-b, MHC-I, aB-crystallin, cathepsins, amyloid precursor protein, STAT-1 (triggered by interferon-g), or myxovirus-resistance MxA protein (triggered by a/b-interferon) (Dalakas 2010a, 2011). The theory that the perifascicular myofibers may be primarily injured by chronic overproduction of a/b-interferon-inducible proteins because they stain with MxA lacks specificity for DM and does not explain the aforementioned sequence of events.

Immunopathology of Polymyositis and IBM In PM and IBM, there is evidence of a T cellmediated and MHC-1-restricted cytotoxicity process directed against heretofore unidentified muscle antigens (Engel and Arahata 1986; Emslie-Smith et al. 1989; Dalakas 1991, 2010a, 2011; Dalakas and Hohlfeld 2003; Mastaglia et al. 2003; Engel et al. 2008). The immune components associated with this process are identical in both PM and IBM, in spite of poor response to immunotherapies of the latter, and include the following (Fig. 2). Activated, Cytotoxic CD8+ T Cells that Invade Healthy-Appearing Muscle Fibers Overexpressing MHC-I Antigen In PM and IBM, the CD8+ T cells invading muscle fibers are activated and cytotoxic. They express ICAM-I, MHC-I, CD45RO, and


inducible co-stimulator (ICOS) on their surface; send spikelike processes into nonnecrotic muscle fibers, which traverse the basal lamina and focally displace the fibers; contain perforin and granzyme granules, which are vectorially directed towards the surface of the muscle fiber inducing necrosis upon release; and are cytotoxic in vitro when exposed to autologous myotubes. An early event in PM and IBM is the widespread overexpression of MHC-I, even in areas remote from the inflammation (Engel and Arahata 1986; Emslie-Smith et al. 1989), probably induced by cytokines and chemokines such as IFN-g or TNF-a which are found increased in the muscle fibers. The presence of perforin-positive CD8 + T cells that surround and invade MHC-I-classexpressing, nonnecrotic muscle fibers (the CD8/ MHC-I lesion) is characteristic of IBM and PM and helpful to distinguish them from nonimmune myopathies (Dalakas and Hohlfeld 2003; Dalakas 2010a, 2011). In other chronic myopathies including the inflammatory dystrophies, the muscle fibers do not express the MHC-I antigen in a ubiquitous and consistent pattern as in PM and IBM, and the few T cells found in the proximity to the muscle fibers do not release cytotoxic granules. Rearrangement of the TCR Genes of the Endomysial T Cells and Formation of Immunological Synapses Between the MHC-I-Expressing Muscle Fibers and Autoinvasive CD8+ T Cells T cells recognize an antigen via the CDR3 region of the T cell receptors (TCR), a heterodimer of two a- and b-chains, encoded by specific genes. In patients with PM and IBM, but not in those with DM or dystrophies, only certain T cells of specific TCRa and TCRb families are recruited to the muscle from the circulation. Cloning and sequencing of the amplified endomysial or autoinvasive TCR gene families has demonstrated a restricted use of certain gene families with conserved amino acid sequence in the CDR3 region, indicating that these cells are specifically selected and clonally expanded in situ probably driven by local antigen(s). This has been further confirmed by combining spectratyping with


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Antigen MHC class I

Cytokines

VCAM-1

CD8+ T

CD8+ T

ICAM-1 CTLA4 CD28

VLA-4 CD8+ T

VCAM-1

CD40 ICOS ICOSL

Perforin

CD40L

ICAM-1

ICAM-1 BB1 ICOS-L Chemokine receptors

LFA-1

CD80

Necrosis

MCH-1

IL1, TGF-β, TNF-α Chemokines, MMPs

Myositis, Pathogenesis, Fig. 2 Cell-mediated mechanisms of muscle damage in polymyositis (PM) and inclusion body myositis (IBM). Antigen-specific CD8 cells are expanded in the periphery, cross the endothelial barrier, and bind directly to muscle fibers via T cell receptor (TCR) molecules that recognize aberrantly expressed MHC-I. Engagement of co-stimulatory molecules (BB1 and ICOS-L) with their ligands (CD28, CTLA-4, and ICOS) along with ICAM-1/LFA-1 stabilizes the CD8–muscle fiber interaction between MHC-I and TCR

(immunological synapse). Muscle fiber necrosis occurs via perforin granules released by the autoaggressive T cells. The stimulated muscle fiber also secrets myocytotoxic cytokines such as interferon (IFN)-g, interleukin (IL)-1, or tumor necrosis factor (TNF)-a, which stimulate further the production of cytokines in an autoamplificatory mechanism that may perpetuates disease chronicity. Death of the muscle fiber is mediated by necrosis

molecular laser-assisted microdissection and by demonstrating that the clonally expanded TCR families persist over time even in different muscles (Engel and Arahata 1986; Hofbauer et al. 2003; Salajegheh et al. 2007; Engel et al. 2008). In an important case of PM, a single clone of g/d T cells was the primary cytotoxic effectors that recognized genuine muscle antigens. Myeloid dendritic cells (DC), potent cells in antigen presentation, as well as large numbers of plasma cells and clonally expanded B cells are also found within the endomysial infiltrates of all inflammatory myopathies including PM, DM, and IBM (Bradshaw et al. 2007). The presence

of these cells, frequently noted in the targeted tissues in several autoimmune disorders, denotes that different effector mechanisms may concurrently play an active role in the autoimmune process and contribute to muscle fiber injury. Cytokines, Chemokines, and Adhesion Molecules The muscle fibers and their autoinvasive CD8+ T cells coexpress co-stimulatory molecules (B7-1, B7-2, BB1, CD40, or ICOS-L) and the respective counter-receptors CD28, CTLA-4 (cytotoxic T lymphocyte antigen 4), CD40L, or ICOS (Dalakas 2011). Cytokines, chemokines,

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and various adhesion and extracellular matrix molecules, such as VCAM, ICAM, thrombospondins, and metalloproteinases MMP-9 and MMP-2, are also upregulated in the tissues of patients with PM and IBM and may enhance T cell adhesion, transmigration, and cytotoxicity. Of interest, the muscle has the potential to secrete proinflammatory cytokines upon cytokine stimulation, such as INF-g, in an auto-amplificatory mechanism that may facilitate the self-sustaining nature of endomysial inflammation and disease chronicity (Fig. 2).

Immunopathology of NAM Although poorly studied, emerging data indicate that in NAM the main effector cell associated with muscle fiber necrosis is the macrophage. Histologically, there are a large number of necrotic fibers invaded by macrophages, but no T cell infiltrates or MHC-I expression as seen in polymyositis and inclusion body myositis (Dalakas 2010a, 2011). In a number of patients, there is deposition of complement on blood vessels (Christofer-Stine et al. 2010). It is likely that NAM is an antibody-mediated disease, as suggested by the presence of antibodies against signal recognition particles (SRP) or against a 100-kd protein corresponding to 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR) (Christofer-Stine et al. 2010; Mammen et al. 2011). The recruitment of macrophages is probably consistent with an antibody-dependent cell-mediated cytotoxicity (ADCC) process (Dalakas 2010a, 2011). Because statins upregulate the expression of HMGCR in cultured cells, the major target of autoantibodies in statin-associated NAM is probably the HMGCR (Mammen et al. 2011). These autoantibodies were found in 6 % of patients with myopathies but in 92.3 % of those taking statins and aged 50 years and older. Autoantibodies Various autoantibodies against nuclear antigens (antinuclear antibodies) or cytoplasmic antigens are found in as many as 20 % of patients with inflammatory myopathies. These antibodies are


directed against ribonucleoproteins involved in translation and protein synthesis and include various synthetases and translation factors, including Jo-1, PL-7, PL-12, OJ, EJ, PMS1, and PMS2. Of those, the antibody directed against the histidyltransfer RNA synthetase, called anti-Jo-1, accounts for 75 % of all the antisynthetases and is clinically useful because up to 80 % of patients with Jo-1 antibodies develop interstitial lung disease. In general, the role of these antibodies in the pathogenesis of inflammatory myopathies is unclear because they are directed against ubiquitous cytoplasmic antigens with undefined function. Further, they are present in less than 15 % of the patients, and with the exception of Jo-1, they do not define an immunopathologically distinct myositis subset. Among the autoantibodies, the ones of pathogenic significance and of diagnostic value are those against SRP and HMGCR which are being evolved as specific markers of necrotizing myositis, as mentioned above.

Viral Triggers Although several viruses have been implicated in chronic and acute myositis, sensitive PCR studies have repeatedly failed to confirm the presence of such viruses in patients’ muscle biopsies. The best evidence of a viral connection is with the retroviruses. Monkeys infected with the simian immunodeficiency virus and humans infected with HIV and human T cell lymphotropic virus (HTLV-1) develop PM or IBM with typical clinical and immunopathological features as the HIV-negative myositis (Dalakas and Hohlfeld 2003; Dalakas 2010a, 2011). Viral antigens are not, however, detected within the muscle fibers but only in occasional endomysial macrophages. Molecular immunology studies using tetramers have shown that among the autoinvasive T cells, there are retrovirally specific CD8+ cells that clonally expand in situ (Dalakas et al. 2007). It is likely that the chronic infection triggers a persistent, in situ inflammatory response, which, via infected macrophages and viral-specific T cells, changes the local milieu leading to myositis.


Degenerative Pathomechanisms in IBM and Interrelationship with Inflammation IBM is a complex disorder because in addition to the immunopathogenic events described above, there is an equally strong degenerative process as evidenced by the presence of rimmed vacuoles (almost always in fibers not invaded by T cells) and intracellular deposition of congophilic amyloid and degeneration-associated or stressor molecules, including beta-amyloid and its precursor protein, alpha-chymotrypsin, phosphorylated tau, ubiquitin, apolipoprotein E, prion protein, and others (Dalakas 2010a, 2011). One line of evidence suggests that the proteasome machinery is malfunctioning or overloaded with aberrant proteins, which may explain why these proteins accumulate in the cytoplasm of muscle fibers. The b-secretase, a major enzyme relevant for processing of amyloid precursor protein (APP), has been overexpressed in sIBM muscle fibers and may explain why processing of APP may be shifted towards the generation of b-amyloid. The aforementioned accumulations, although extensively studied in sIBM, do not seem to be unique to this disease, because they are also observed in other vacuolar myopathies. What appears unique to IBM, however, compared to other chronic vacuolar myopathies, is the concomitant accumulation of these molecules with a strong primary inflammatory response and the overexpression of proinflammatory mediators and MHC-I on all fibers, vacuolated or not. The T cell invasion appears to occur early and in higher frequency than the Congo-red-positive fibers, suggesting that inflammation precedes the accumulation of amyloid and stressor molecules. Regardless of whether the primary event in IBM is an inflammatory or protein dysregulation process, the unique coexistence of the two processes and co-localization of APP with cytokines has provided evidence that there is an interrelationship between inflammation and degeneration and that inflammatory mediators affect or increase the accumulation of degenerative molecules (Dalakas 2008, 2010a, 2011; Schmidt et al. 2008).

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Therapeutic Strategies As the specific target antigens in DM, PM, and IBM are unknown, current therapies are not selectively targeting either the autoreactive T cells or the complement-mediated process on intramuscular blood vessels. Instead, they induce nonspecific immunosuppression or immunomodulation. Furthermore, many of these therapies are empirical. Based on experience, but not controlled studies, the majority of patients with PM and DM respond to corticosteroids to some degree and for a period of time (Dalakas 2006, 2010b). Intravenous immunoglobulin (IVIg), as tested in a controlled study, is effective in DM as a second, and at times, first-line therapy (Dalakas et al. 1993). IVIg also appears to be effective in PM and NAM. Immunosuppressants are used as steroid-sparing agents but their efficacy remains unclear. New agents in the form of monoclonal antibodies or fusion proteins that target cytokines, adhesion molecules, T cell transduction or transmigration molecules, and B cells or their activation factors are emerging as promising immunotherapeutic drugs. Among them, rituximab, a B cell-depleting agent, has been helpful in some cases of DM and NAM but a controlled study – although with a problematic design – did not show efficacy (Oddis et al. 2013). In contrast to PM and DM, there is currently no effective treatment for IBM. Prednisone, cyclosporine, azathioprine, methotrexate, total body irradiation, and IFN-b have generally failed justifying the contention that the condition could be more of a degenerative disease rather than an autoimmune condition. A number of patients with IBM however may respond to common immunotherapeutic agents early on to some degree and for a period of time; up to 25 % of patients in a controlled study have also responded transiently to IVIg. These benefits are arguably limited and short-lived; IBM remains a steadily progressive disease that, over time, is uniformly resistant to all therapies. Although transient therapeutic responses are also seen in other autoimmune diseases with poor response to immunotherapies, such as

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primary progressive multiple sclerosis, the lack of treatment efficacy remains of concern and a reason for tilting the pathogenetic theories towards a primary degenerative process rather than autoimmune. It should be noted, however, that in IBM, therapy is typically initiated late when the degenerative cascade has already begun or is too advanced, due to insidious onset and very slow disease progression. This is supported by the observations that IBM patients, even with minimal clinical weakness, already exhibit significant muscle atrophy and extensive histopathologic changes.

Cross-References ▶ Dermatomyositis, Skin ▶ Juvenile Dermatomyositis ▶ Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies ▶ Therapeutic Considerations in Kidney Diseases Due to Glomerulonephritis

References Bradshaw EM, Orihuela A, McArdel SL, Salajegheh M, Amato AA, Hafler DA, Greenberg SA, O’Connor KC. A local antigen-driven humoral response is present in the inflammatory myopathies. J Immunol. 2007;178(1):547–56. Christofer-Stine L, Casciola-Rosen LA, Hong G, Chung T, Corse AM, Mammen AL. A novel autoantibody recognizing 200 and 100 kDa proteins is associated with an immune-mediated necrotizing myopathy. Arthritis Rheum. 2010;62:2757–66. Dalakas MC. Polymyositis, dermatomyositis, and inclusion-body myositis. N Engl J Med. 1991;325:1487–98. Dalakas MC. Treatment of polymyositis and dermatomyositis. Signaling pathways and immunobiology of inflammatory myopathies. Nat Clin Prac Rheumatol. 2006;2:219–27. Dalakas MC. Interplay between inflammation and degeneration: using inclusion body myositis to study neuroinflammation. Ann Neurol. 2008;64(1):1–3. Dalakas MC. Toxic and drug-induced myopathies. J Neurol Neurosurg Psychiatry. 2009;80(8):832–8. Dalakas MC. Inflammatory muscle diseases: a critical review on pathogenesis and therapies. Curr Opin Pharmacol. 2010a;10(3):346–52.

Myositis, Pathogenesis Dalakas MC. Immunotherapy of myositis: issues, concerns and future prospects. Nat Rev Rheumatol. 2010b;6(3):129–37. Dalakas MC. An update on inflammatory and autoimmune myopathies. Neuropathol Appl Neurobiol. 2011;37(3): 226–42. Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis. Lancet. 2003;362:1762–3. Dalakas MC, Illa I, Dambrosia JM, et al. A controlled trial of high-dose intravenous immunoglobulin infusions as treatment for dermatomyositis. N Engl J Med. 1993;329:1993–2000. Dalakas MC, Rakocevic G, Shatunov A, Goldfarb L, Salajegheh M. Inclusion body myositis with human immunodeficiency virus infection: four cases with clonal expansion of viral-specific T cells. Ann Neurol. 2007;61(5):466–75. Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility complex class-I antigen expression, immunolocalization of interferon subtypes and T-cell mediated cytotoxicity in myopathies. Hum Pathol. 1989;20:224–31. Engel AG, Arahata K. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigen-specific cell-mediated cytotoxicity in some diseases, and implications for the pathogenesis of the different inflammatory myopathies. Hum Pathol. 1986;17:704–21. Engel AG, Hohlfeld R, Banker BQ. The polymyositis and dermatomyositis syndrome. In: Engel AG, Franzini-Armstrong C, editors. Myology. New York: McGraw-Hill; 2008. p. 1335–83. Hofbauer M, Wiesener S, Babbe H, Roers A, Wekerle H, Dornmair K, Hohlfeld R, Goebels N. Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3spectratype analysis. Proc Natl Acad Sci USA. 2003;100(7):4090–5. Hohlfeld R, Dornmair K. Revisiting the immunopathogenesis of the inflammatory myopathies. Neurology. 2007;69:1966–7. Mammen AL, Chung T, Christopher-Stine L, Rosen P, Rosen A, Doering KR, Casciola-Rosen LA. Autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme a reductase in patients with statin-associated autoimmune myopathy. Arthritis Rheum. 2011;63(3):713–21. Mastaglia FL, Garlepp MJ, Phillips BA, Zilko PJ. Inflammatory myopathies: clinical, diagnostic and therapeutic aspects. Muscle Nerve. 2003;27:407–25. Oddis CV, Reed AM, Aggarwal R, Rider LG, Ascherman DP, Levesque MC, Barohn RJ, Feldman BM, HarrisLove MO, Koontz DC, Fertig N, Kelley SS, Pryber SL, Miller FW, Rockette HE. RIM study group. Rituximab in the treatment of refractory adult and juvenile dermatomyositis and adult polymyositis: a randomized, placebo-phase trial. Arthritis Rheum. 2013;65(2):314–24. Salajegheh M, Rakocevic G, Raju R, Shatunov A, Goldfarb LG, Dalakas MC. T cell receptor profiling

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies in muscle and blood lymphocytes in sporadic inclusion body myositis. Neurology. 2007;69(17):1672–9. Schmidt J, Barthel K, Wrede A, Salajegheh M, Bahr M, Dalakas MC. Interrelation of inflammation and APP in sIBM: IL-1 beta induces accumulation of beta-amyloid in skeletal muscle. Brain. 2008;131(Pt 5):1228–40.

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies Ritu Valiyil Division of Rheumatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Synonyms DM (dermatomyositis); IBM (inclusion body myositis); Idiopathic inflammatory myopathies (IIM); Inflammatory myopathies; Myositis; PM (polymyositis)

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developed myalgias with muscle weakness and atrophy. Unverricht then coined the term “dermatomyositis” to describe her condition. Inclusion body myositis was not named until more recently in 1967 when the pathology of inclusion bodies was described (Levine 2003). Criteria for the diagnosis of polymyositis and dermatomyositis were later created by Bohan and Peter in 1975 and these are still used today.

Epidemiology The reported incidence of idiopathic inflammatory myopathy ranges from 2 to 10 new cases per million persons at risk per year (Oddis and Medsger 2004). Dermatomyositis has a bimodal incidence with a peak incidence at childhood and then again between the ages of 50 and 70. Polymyositis, in contrast, rarely occurs in childhood. The overall female to male incidence is 2.5:1, although this ratio is lower (1:1) in childhood disease and with malignancies and is much higher (10:1) when myositis occurs with a coexisting connective tissue disease. Disease onset is more frequent in the winter and spring months, especially in childhood cases of inflammatory myositis (Becezny 1935).

Definition The idiopathic inflammatory myopathies are a group of connective tissue diseases manifested by chronic inflammation of the striated muscle, characteristic cutaneous features (dermatomyositis), and a variety of systemic complications.

History In 1886, Wagner first coined the term “polymyositis” to describe a woman presenting with muscle weakness, diffuse muscle and joint pain, swelling of the extremities, and erythema of the forearms (Oddis and Medsger 2004). In 1891, Unverricht described a 39-year-old pregnant woman with facial erythema and swollen, erythematous legs and thighs who subsequently

Classification Criteria of PM and DM Many classification systems have been proposed for polymyositis and dermatomyositis, but the 1975 Bohan & Peter criteria are still most frequently used for defining patient groups for clinical research and to aid in diagnosing individual patients. These criteria are shown in Fig. 1. The essential elements of the criteria include proximal muscle weakness on physical examination, increased serum muscle enzymes, abnormal electromyogram (EMG), muscle biopsy consistent with myositis, and the characteristic rash of dermatomyositis. Inclusion body myositis (IBM) and amyopathic dermatomyositis (in which patients have the rash of dermatomyositis but no overt muscle weakness) are not included in the

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Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies, Fig. 1 Bohan & Peter criteria for diagnosis of polymyositis and dermatomyositis. The Bohan and Peter classification criteria are shown

1. Symmetrical proximal muscle weakness 2. Muscle biopsy evidence of myositis (degeneration/regeneration, perifascicular atrophy, necrosis, phagocytosis, fiber size variation, mononuclear inflammatory infiltrate) 3. Increase in serum skeletal muscle enzymes 4. Characteristic electromyographic pattern (polyphasic, short small motor unit potentials; fibrillations; positive sharp waves; insertional irritability; high frequency repetitive discharges) 5. Typical rash of dermatomyositis (Gottron’s sign, Gottron’s papules, heliotrope rash) Diagnostic Criteria: Polymyositis: Definite: all of 1–4 Probable: any 3 of 1–4 Possible: any 2 of 1– 4 Dermatomyositis: Definite: 5 plus any 3 of 1–4 Probable: 5 plus any 2 of 1–4 Possible: 5 plus any 1 of 1–4

Bohan and Peter classification criteria. Most experts have agreed that the Bohan and Peter criteria should be updated and such efforts are currently underway. Myositis-specific serum autoantibodies and magnetic resonance imaging (MRI) have been proposed to be added to the new criteria.

Clinical Features of PM and DM Skeletal Muscle Weakness The most frequent presenting symptom is insidious, progressive, painless symmetrical proximal muscle weakness that is subacute in onset occurring over weeks to months. In polymyositis and dermatomyositis, the hip flexors or pelvic girdle is often affected initially and patients often complain of difficulty walking up steps or arising from a chair. Upper extremity or shoulder girdle symptoms usually follow with patients experiencing difficulty raising their arms overhead or difficulty washing and combing their hair. Dysphagia can occur with nasal regurgitation of liquids or difficulty swallowing, indicating pharyngeal striated muscle involvement. Ocular or facial weakness is very uncommon in PM and DM and should prompt consideration of other neuromuscular diagnoses.

Skin Manifestations Dermatomyositis has characteristic skin manifestations which may precede, develop simultaneously with, or follow muscle symptoms. Pathognomonic cutaneous features of dermatomyositis include Gottron’s papules and the heliotrope rash. Gottron’s papules are scaly, erythematous, or violaceous papules located over the dorsal aspect of the metacarpophalangeal and proximal and distal interphalangeal joints of the hands. Gottron’s sign is a macular erythematous rash that occurs over extensor areas such as elbows, knees, and ankles (Sontheimer and Provost 2004). Sixty to eighty percent of dermatomyositis patients demonstrate either Gottron’s papules or sign. The heliotrope rash is a characteristic erythematous or violaceous rash seen over the eyelids and periorbital area and is seen in fewer than 50 % of patients with dermatomyositis. A “V” sign with erythema over the anterior chest can be seen as well as the “shawl” sign with a macular erythema over the nape of the neck, upper back, and across both shoulders. The facial rash of dermatomyositis can appear similar to the malar rash of lupus but can involve the nasolabial folds and forehead which is atypical for lupus, and pruritus is a common and underrecognized complaint in dermatomyositis especially in the scalp (Shirani 2004).


Another characteristic rash is “mechanics hands” which is hyperkeratosis, scaling, and cracking/fissuring of the palms and lateral fingers, and is most frequently seen in patients with anti-synthetase myositis autoantibodies. Other common skin findings include cuticular hypertrophy, periungual erythema, nailfold telangiectasias, infarcts, and capillary dilation. These findings can be seen in both dermatomyositis and myositis in overlap with other connective tissue diseases. Calcinosis Soft tissue calcifications can occur in dermatomyositis, and is more common in the juvenile form of dermatomyositis (30–70 %) than in adult-onset disease (10 %) (Dalakas and Holhfeld 2003). Typically, calcinosis is associated with increased disease activity and duration. Calcinosis may be intracutaneous, subcutaneous, fascial, or intramuscular in location and is typically located at sites of compression. Joints Arthralgias and arthritis can occur early in the disease course. Typically, symptoms are mild; however, the arthropathy associated with antiJo1 antibody can be erosive and deforming. Lung The lung is the most common extramuscular organ affected in polymyositis and dermatomyositis and is a major cause of morbidity and mortality. Pulmonary complications of PM and DM can be due to hypoventilation from respiratory muscle weakness, aspiration pneumonias, and interstitial lung disease. Interstitial lung disease (ILD) has been reported to occur in 26–64 %, of patients with PM or DM, and can occur before, concomitantly, or after the onset of skin and muscle disease (Khan and Christopher-Stine 2011). The strongest predictors of poor outcomes in a large retrospective cohort study of PM/DM and ILD included older age, symptomatic ILD, low diffusion capacity (DLCO) and decreased forced vital capacity (FVC) at diagnosis, and usual interstitial pattern on chest computed

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tomography (CT) scan (Marie et al. 2011). Myositis-specific anti-synthetase autoantibodies such as anti-Jo1 were the strongest predictive marker for ILD. Heart Cardiac manifestations are quite common in PM and DM but are usually asymptomatic and thus under-recognized. In a long-term follow-up study of PM and DM patients, cardiovascular involvement was the most common cause of death in patients with myositis (Danko et al. 2004). Cardiac abnormalities can include arrhythmias, conduction abnormalities, cardiac arrest, congestive heart failure, myocarditis, pericarditis, angina, and fibrosis. Gastrointestinal Because pharyngeal muscle is striated and can also become inflamed in myositis, patients can exhibit dysphonia and swallowing dysfunction with difficulty initiating swallow and nasal regurgitation of liquids. Cricopharyngeal muscle dysfunction can also occur, resulting in dysphagia with a “blocking” or “sticking” sensation with swallowing. Peripheral Vascular System Raynaud’s phenomenon can occur in all idiopathic inflammatory myopathies. Systemic vasculitis can occur commonly in childhood dermatomyositis, but it does not occur frequently in adults. The vascular lesions of dermatomyositis can include dermal or subcutaneous nodules, periungual infarcts, and digital ulcerations (Oddis and Medsger 2004).

Differential Diagnosis The differential diagnosis of adult polymyositis and dermatomyositis is broad and can include numerous conditions affecting skeletal muscle. Inherited or genetic causes of muscle disease include the muscular dystrophies, metabolic myopathies, glycogen storage disorders, and mitochondrial myopathies. These can be

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differentiated from acquired myopathies, because they are more slowly progressive and can involve a family history of myopathy. While the idiopathic inflammatory myopathies are considered acquired myopathies, other acquired myopathies can also present with skeletal muscle weakness including toxic (or drug-induced) myopathies, endocrinopathy (especially hypothyroidism), or myopathies triggered by infections such as viruses. History, physical examination, differences in laboratory tests, and muscle biopsies are essential in differentiating among these conditions.

involves testing a patient’s proximal and distal muscle groups against resistance and grading the strength on a 0–5 scale. Serum Muscle Enzymes Enzymes that leak from injured skeletal muscle into the serum are valuable in detecting muscle injury. The serum creatine kinase (CK) can be quite elevated in myositis (over 1,000 to several thousands) and is the most sensitive marker of ongoing muscle injury. Other enzymes that can also be elevated in decreasing order of sensitivity include aldolase, aspartate and alanine aminotransferases (AST and ALT), and lactate dehydrogenase (LDH) (Oddis and Medsger 2004).

Association with Malignancy Epidemiologic studies have shown an association between dermatomyositis and polymyositis and a higher risk of cancer. National registries and population-based cohort studies of biopsy proven cases have found that the risk of malignancy is highest in patients with dermatomyositis. In a pooled analysis of all cases of dermatomyositis and polymyositis in Sweden, Denmark, and Finland, 618 cases of dermatomyositis were identified (Hill et al. 2001). Thirty-two percent of the DM cases had cancer, suggesting that patients with DM had a three-fold increased risk of developing cancer. The same study identified a lower but still statistically significant increased risk of cancer associated with polymyositis with a standardized incidence ratio (SIR) of 1.3. The overall risk of cancer is greatest in the first 3 years after the diagnosis of myositis. In dermatomyositis, the strongest associations are with ovarian, lung, pancreatic, breast, and colorectal cancers. Lung, bladder cancer, and non-Hodgkin’s lymphoma are more strongly associated with polymyositis.

Diagnosis of PM and DM Physical Examination The examining physician should assess for objective signs of weakness and impaired muscle function through manual muscle strength testing which

Electromyography Electromyography (or EMG) evaluates the electrical activity of muscle fibers and is a sensitive but nonspecific method of evaluating muscle inflammation. The electrical activity is recorded via surface electrodes during needle insertion, rest, and voluntary muscle contractions. Typical EMG findings in inflammatory myopathies include irritability of myofibrils on needle insertion and at rest (fibrillation potentials, complex repetitive discharges, positive sharp waves) and short duration, low amplitude complex (polyphasic) potentials on contraction. More than 90 % of patients with active myositis will have an abnormal EMG. Muscle Biopsy Muscle biopsy is the gold standard for confirmation of the diagnosis of inflammatory myopathy, and open surgical biopsy tends to provide more definitive pathology compared with percutaneous needle muscle biopsy. Degeneration and regeneration of myofibrils occurs in 90 % of cases. Chronic inflammatory cells in the perivascular and interstitial areas surrounding myofibrils are present in 80 % of cases and lymphocytic invasion of non-necrotic fibers is considered pathognomonic of polymyositis (Lundberg 2004). In dermatomyositis, B cells, CD4+ T cells, and the late component of complement (C5-C9, membrane attack complex) predominate in the perivascular area. Perifascicular myofibril


atrophy and endothelial cell hyperplasia of blood vessels are also noted. In contrast, polymyositis features cytotoxic T cell (mostly CD8+) invasion of myofibrils with sparing of the vasculature. Muscle biopsies in inclusion body myositis can also demonstrate inflammatory infiltrate primarily of cytotoxic T cells, although inflammation is usually mild. The notable finding on muscle biopsy in inclusion body myositis is rimmed vacuoles representing abnormal filaments. MRI Magnetic resonance imaging (MRI) can be a useful, noninvasive tool for evaluating myositis (Scott and Kingsley 2004). The T1-weighted images provide anatomic detail, and normal tissue appears homogenously dark with a low signal, whereas fat appears bright. Inflammation is bright on both T1 and T2 images, but adding fat suppression to the T2 technique, in the form of short tau inversion recovery (STIR) sequences, improves the detection of muscle inflammation by enhancing the bright signal of inflammation and decreasing the fat signal (dark). Thus, T1-MRI demonstrates damage and chronicity of disease, while STIR-MRI demonstrates inflammatory disease activity.

Pathophysiology of PM and DM The true pathogenesis of the inflammatory myopathies is unknown. The presence of T lymphocytes in muscle tissue in a majority of patients with myositis as well as serum autoantibodies suggests that immune mechanisms are involved in the pathogenesis, and both T and B cells may play a pathogenic role in these diseases. Mechanisms that have been proposed for the development of muscle inflammation include possible direct cytotoxic effects of infiltrating leukocytes such as T cells and macrophages on muscle cells, indirect effects of pro-inflammatory cytokines on muscle function, or microvessel involvement and disturbed circulation which could lead to reduced muscle function (Lundberg 2004).

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Immunohistochemistry analysis of muscle tissue from myositis patients has revealed that lymphocytes such as CD8+ T lymphocytes in polymyositis and CD4+ T lymphocytes in dermatomyositis appear to invade muscle fibers, and different lymphocytes tend to aggregate in different areas of the muscle (Engel and Arahata 1986). Inflamed muscle also expresses MHC class I and II molecules in muscle fibers, which normally do not express these antigens. Although MHC class I molecules are expressed on most nucleated cells in the body, mature muscle fibers lose class I MHC expression during their differentiation from immature fibers. In myositis patients, MHC class I antigen expression is not only found in a majority of patients’ muscle but can also be found distant from sites of inflammation (Karpati et al. 1988). The exact role of induced MHC expression on muscle fibers is not known. In terms of other possible cytotoxic mechanisms, perforins and granzymes have also been suggested as possible mechanisms of disease. Perforin-containing granules within CD8+ T cells have been found oriented toward adjacent muscle fibers (Goebels et al. 1996). It has been suggested that perforin may mediate the transfer of granzymes into the target cell, which may then activate caspases, leading to cell lysis and nuclear damage. Many of the myositis autoantigens are cleaved by granzyme B, and this process may play a role in creating proteins that are immunogenic and resulting in autoimmunity. Several cytokines and chemokines have been detected in muscle tissue from patients with myositis, and it has been hypothesized that these cytokines may cause direct toxicity to muscle fibers or indirectly affect muscle fiber metabolism and contractility. The most frequently reported cytokines are pro-inflammatory cytokines IL-1a, IL-1b, and TNF-a (Lundberg 2004). With respect to dermatomyositis, perifascicular atrophy is a unique feature of this subset of myositis, and some have speculated that this may provide a clue into the pathogenesis of DM. In muscle fibers of DM patients, capillary density tends to be highest in the middle of the fiber and lowest in the periphery of the fiber,

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or the perifascicular region. Histopathologic analysis has also demonstrated that in early DM patients, there seems to be localization of the late components of complement (C5-C9) in the areas with fewer capillaries. There is also complement deposition seen on endothelial cells and dropout of endothelial cells in the perifascicular region. It is unclear if this complement deposition and possible injury to the vasculature precedes the infiltration of inflammatory cells, leading to muscle fiber damage. It has also been hypothesized that perifascicular atrophy may be the result of microvessel involvement and metabolic disturbance, leading to localized hypoxia in the perifascicular region. A combination of immune-mediated mechanisms and different molecular pathways may ultimately contribute to the muscle pathology seen in polymyositis and dermatomyositis. The key pathways, however, are still unclear and require further research.

Myositis Autoantibodies Serum autoantibodies can be found in up to 50 % of patients with polymyositis and dermatomyositis and can be useful in defining subsets of patients with certain distinguishing clinical features and unique phenotypes. The role of autoantibodies is unclear as it is not well understood whether they are involved in pathogenesis of myositis or just an epiphenomenon. The autoantibodies are typically classified into myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs). Myositis-specific autoantibodies are specific for autoimmune disease as they are not associated with other neuromuscular diseases characterized by inflammation. The myositis-specific autoantibodies are classified into anti-synthetase antibodies directed against aminoacyl tRNA synthetase antigens and non-synthetase antibodies. The most frequent myositis-specific antibodies are those directed against aminoacyl-tRNA synthetase. There are 20 different aminoacyl-tRNA synthetases, and seven of them have been found to be autoantigens in myositis. Anti-synthetase antibodies are generally

found in 16–26 % of patients with inflammatory myopathies. There are several well-known anti-synthetase antibodies (Fig. 2). Anti-synthetase Myositis-Specific Antibodies Anti-Jo1 antibodies are directed against histidyltRNA synthetase and are the most common MSAs since they are found in 20 % of patients with myositis (Khan and Christopher-Stine 2011). The clinical associations of the anti-synthetase antibodies are similar and have been collectively described as the “anti-synthetase syndrome.” Classically, patients with anti-Jo1 autoantibodies and other anti-synthetase antibodies experience symptoms of myositis with recurrent fevers, nonerosive arthritis, mechanic’s hands, Raynaud’s phenomenon, and interstitial lung disease. The myositis and interstitial lung disease in this syndrome can be quite severe with multiple exacerbations, often requiring combination therapy with corticosteroids and immunosuppressive agents. It is not clear whether anti-Jo1 has a pathogenic role in myositis, but anti-Jo1 titers have also been correlated with disease activity. Longitudinal analysis of 11 patients showed modest correlation of anti-Jo1 titers and CK levels, myositis, lung, and joint disease activity (Stone et al. 2007). In three of these patients, anti-Jo1 antibody became negative when their disease was inactive. Non-synthetase Myositis-Specific Antibodies Non-synthetase MSAs are also associated with unique clinical phenotypes (Fig. 3). Anti-MI2 autoantibodies are directed against MI-2, a multiunit protein complex participating in transcription regulation at the chromosome level. Anti-MI2 antibodies are found in 10–30 % of adult dermatomyositis patients and are strongly associated with dermatomyositis. Anti-MI2 myopathy is characterized by severe skin manifestations of DM but has excellent response to immunosuppressive therapy. Anti-p155 is an autoantibody directed against a 155/140-kd doublet and has been found to be significantly associated with cancer in adult dermatomyositis. A more recent meta-analysis of six cohort studies involving 312 DM patients found


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Antibody

Antigen

Frequency

Clinical Manifestations

Anti-Jo1

Histidyl-tRNA synthetase

18–20%

PM, DM + ILD

Anti-PL7

Threonyl-tRNA synthetase

≤3%

DM, PM + ILD

Anti-PL12

Alanyl-tRNA synthetase

≤3%

ILD > PM, DM

Anti-EJ

Glycyl-tRNA synthetase

≤2%

PM>DM + ILD

Anti-OJ

Isoleucine-tRNA synthetase

≤1%

ILD + DM, PM

Anti-KS

Asparaginyl-tRNA synthetase

≤1%

ILD > DM, PM

Anti-Zo

Phenylalanyl-tRNA synthetase

≤1%

ILD + DM, PM

Anti-Ha

Tyrosyl-tRNA synthetase

≤1%

ILD + DM, PM

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies, Fig. 2 Myositis-specific autoantibodies: anti-synthetase antibodies. The myositis-specific anti-synthetase

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autoantibodies are shown. PM polymyositis, DM dermatomyositis, ILD interstitial lung disease, less than or equal to, > more often than (Adapted with permission from Khan and Christopher-Stine (2011))

Antibody

Antigen

Clinical Manifestations

Anti-SRP

Signal recognition particle

Severe, resistant necrotizing myopathy

Anti-MI2

Chromatin remodeling enzyme

DM with rash > muscle symptoms

Anti-SAE

Small ubiquitin-like modifier activating enzyme

DM with severe skin symptoms and rapidly progressive ILD

Anti-HMGCR

HMG-CoA Reductase

Necrotizing myopathy associated with statin

Anti-MDA5

MDA5

DM with rapidly progressive ILD, Cancer associated myositis

Anti-155/140

Transcriptional intermediary factor 1Y

Cancer associated myositis

Anti-140

Nuclear matrix protein (NXP-2)

Juvenile DM

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies, Fig. 3 Myositis-specific autoantibodies: non-synthetase antibodies. The myositis-specific non-synthetase autoantibodies are shown. DM dermatomyositis, ILD

interstitial lung disease, HMGCR HMG-CoA reductase, MDA5 melanoma differentiation-associated gene 5, SAE small ubiquitin-like modifier-activating enzyme, > more often than (Adapted with permission from Khan and Christopher-Stine (2011))

pooled sensitivity of anti-P155 for cancerassociated myositis of 78 % and specificity of 89 % (Trallero-Araguas et al. 2012). This study found that patients with anti-P155 antibodies have 27-fold higher odds of having cancerassociated myositis than patients without the autoantibody. Anti-SRP is an autoantibody directed against the signal recognition particle (SRP), a ribonucleoprotein involved in protein translocation across the endoplasmic reticulum. The prevalence of anti-SRP antibodies is estimated to be 5 % of myositis cases. Patients with anti-SRP myopathy present with a unique clinical syndrome with markedly elevated CK levels and rapidly progressive severe proximal muscle

weakness that leads to significant disability. Patients often demonstrate early muscle atrophy and dysphagia is quite common in the later stages of the disease. Muscle biopsy specimens in antiSRP myopathy are often distinct because they demonstrate necrosis with often scant or no inflammatory infiltrate. Staining also shows vigorous regeneration and deposition of the terminal components of the complement (C5b-9 or membrane attack complex) in the endomysial capillaries and the sarcolemma. The myopathy is also difficult to treat as it is poorly responsive to corticosteroid monotherapy. An autoantibody directed against 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCoA reductase) has also been described and is

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Antibody

Antigen

Clinical Manifestation

PM-Scl

Unidentified

PM/SSc overlap

U1RNP

U1 small RNP

MCTD, overlap syndrome

Non-U1 snRNPs

U2, U4/6, U5, U3 snRNPs

PM or DM/SSc or SSc overlap

Ku

DNA-binding proteins

Myositis/SSc/SLE overlap

Ro (SS-A) includes Ro60 and Ro52

RNA protein

Myositis often with SS or SLE, Ro52 with ILD

56 kDa

RNP particle

Myositis, often with Jo-1

KJ

Unidentified translation factor

PM, ILD, RP

Fer

Elongation factor 1α

Myositis

Mas

tRNAser-related antigen

Myositis, rhabdomyolysis, chronic hepatitis

MJ

Unidentified nuclear pore

Juvenile DM

hPMS1

Protein related to DNA repair

Myositis

Myositis: Polymyositis, Dermatomyositis, Inclusion Body Myositis, and Myositis Autoantibodies, Fig. 4 Myositis-associated autoantibodies. The myositis-associated autoantibodies are shown. RNP ribonucleoprotein, snRNP small nuclear ribonucleoprotein,

RP Raynaud’s phenomenon, SLE systemic lupus erythematosus, SS Sjogren’s syndrome, SSc systemic sclerosis or scleroderma, > more often than (Adapted with permission from Khan and Christopher-Stine (2011))

associated with an autoimmune necrotizing myopathy and statin medication use. The initial study described 16 patients with necrotizing myopathy on muscle biopsy with serum that precipitated a 200/100 kd antibody (Christopher-Stine et al. 2010). In 12 of these patients over the age of 50, 83 % had been exposed to statin medications prior to the onset of weakness which was higher than age-matched DM and PM controls. Discontinuation of the statin medication did not lead to clinical improvement in these patients, and the myopathy responded well to immunosuppression. A subsequent study demonstrated that specifically regenerating muscle fibers from patients with the anti-HMGCR myopathy expressed high levels of HMG-CoA reductase (Mammen et al. 2011).

patients with “mixed connective tissue disease” and can have clinical findings of Raynaud’s disease, systemic lupus erythematosus, polymyositis, dermatomyositis, or systemic sclerosis, or a combination of these diseases.

Myositis-Associated Autoantibodies There are multiple myositis-associated autoantibodies (MAAs) which are found in patients with myositis but can also be found in other connective tissue diseases. Although these antibodies may not occur exclusively in patients with inflammatory myopathies, they can also have unique clinical phenotypes (Fig. 4). Anti-PM Scl is an antinucleolar antibody that identifies a subset of patients with myositis and features of systemic sclerosis. Anti-U1 RNP antibodies can occur in

Inclusion Body Myositis Inclusion body myositis (IBM) is included among the idiopathic inflammatory myopathies, but there is still debate whether IBM should be regarded as an inflammatory myopathy or as a degenerative myopathy with secondary inflammation. Symptoms of muscle weakness begin insidiously and progress slowly. IBM primarily affects individuals over the age of 50, and is more common in males. Although patients can manifest proximal muscle weakness, distal weakness is common and is often asymmetric.

Treatment of the Inflammatory Myopathies Because of the rarity of the inflammatory myopathies, randomized controlled studies are lacking and treatment of these diseases remains empiric


based on case series. The mainstay of treatment is with daily oral corticosteroids initially dosed 1–2 mg/kg/day. Combination therapy with cytotoxic drugs is commonly prescribed to reduce corticosteroid side effects and to treat more aggressive therapy. A variety of cytotoxic drugs have been recommended including methotrexate, azathioprine, cyclosporine, mycophenylate mofetil, intravenous immunoglobulin (IVIg), rituximab, and cyclophosphamide. The response to treatment of IBM has been considered so poor that most have recommended not treating the disease with immunosuppressive medications. Although several controlled trials have investigated the use of IVIg (Dalakas et al. 1997), these have shown variable responses. In many cases, weakness appears to progress despite immunosuppressive treatment.

Cross-References ▶ Cancer and Dermatomyositis ▶ Dermatomyositis, Skin ▶ Juvenile Dermatomyositis ▶ Mixed Connective Tissue Disease (MCTD) ▶ Myositis, Pathogenesis ▶ Raynaud’s Phenomenon

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Engel AG, Arahata K. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigen-specific cell-mediated cytotoxicity in some disease and implications for the pathogenesis of the different inflammatory myopathies. Hum Pathol. 1986;17:704–21. Goebels N, et al. Differential expression of perforin in muscle-infiltrating t cells in polymyositis and dermatomyositis. J Clin Invest. 1996;97:2905–10. Hill CL, et al. Frequency of specific cancer types in dermatomyositis and polymyositis: a populationbased study. Lancet. 2001;357:96–100. Karpati G, et al. Expression of immunoreactive major histocompatibility complex products in human skeletal muscles. Ann Neurol. 1988;23:64–72. Khan S, Christopher-Stine L. Polymyositis, dermatomyositis, and autoimmune necrotizing myopathy: clinical features. Rheum Dis Clin N Am. 2011;37:143–58. Levine TD. History of dermatomyositis. Arch Neurol. 2003;60:780–2. Lundberg IE. Etiology and pathogenesis of inflammatory muscle disease. In: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH, editors. Rheumatology, Chapter 137. 4th ed. Philadelphia: Elsevier; 2004. p. 1433–48. Mammen et al. Autoantibodies against 3-hydroxy-3methylglutaryl-coenzyme A reductase in patients with statin-associated autoimmune myopathy. Arthritis Rheum. 2011;63(3)713–21. Marie I, et al. Short-term and long-term outcomes of interstitial lung disease in polymyositis and dermatomyositis: a series of 107 patients. Arthritis Rheum. 2011;63:3439–47. Oddis CV, Medsger TA. Clinical features, classification and epidemiology of inflammatory muscle disease. In: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH, editors. Rheumatology. 4th ed, Chapter 137. New York: Mosby; 2004. p. 1433–48. Scott DL, Kingsley GH. Use of imaging to assess patients with muscle disease. Curr Opin Rheumatol. 2004;16:678–83. Shirani Z, et al. Pruritus in adult dermatomyositis. Clin Exp Derm. 2004;29:273–6. Sontheimer RD, Provost TT. Cutaneous manifestations of rheumatic diseases. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2004. Stone K, et al. Anti-Jo1 antibody levels correlate with disease activity in idiopathic inflammatory myopathy. Arthritis Rheum. 2007;56(9):3125–31. Trallero-Araguas E, et al. Usefulness of anti-p155 autoantibody for diagnosing cancer-associated dermatomyositis: a systematic review and metal-analysis. Arthritis Rheum. 2012;64(2):523–32.

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