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Ontogeny and Development
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Eosinophils develop in the bone marrow from multipotential, stem cell-derived CD34+ myeloid progenitor cells in response to eosinophilopoietic cytokines and growth factors (see Fig. 31-1). They are released into the circulation as mature cells.1–3 Important stimulatory cytokines and growth factors for eosinophils include interleukin (IL)-3, granulocyte macrophage colony stimulating factor (GM-CSF), and IL-5. Activated T cells likely are the principal sources of IL-3, GM-CSF, and IL-5 that induce eosinophil differentiation in bone marrow. However, depending on pathogenic stimuli, eosinophilopoietic cytokines may be released by other cell types, including mast cells, macrophages, natural killer cells, endothelial cells, epithelial cells, fibroblasts, and even eosinophils, themselves.4 IL-3 and GM-CSF are pluripotent cytokines that have effects on other hematopoietic lineages. IL-5 is the most selective eosinophil-active cytokine, but it is relatively late acting. Although it is both necessary and sufficient for eosinophil differentiation, IL-5 demonstrates maximum activity on the IL-5 receptor (IL-5R)-positive eosinophil progenitor pool that first is expanded by earlier acting pluripotent cytokines such as IL-3 and GM-CSF4; expression of the high affinity IL-5R is a prerequisite for eosinophil development. Exodus from the bone marrow also is regulated by IL-5. IL-3, GM-CSF, along with IL-5, promote survival, activation, and chemotaxis of eosinophils through binding to receptors that have a common β chain (CD131) with IL-5R, and unique α chains. See ch31etb0.1 for designations of many factors involved in eosinophil biology.
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Eosinophil/Basophil Colony Forming Unit
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Eosinophils and basophils share the CD34+ progenitor, also referred to as the “eosinophil/basophil colony forming unit.”
5 This hybrid cell is characterized by granule contents that have features of both cell types and by the expression of common receptors, the high-affinity IL-5R and the CC chemokine receptor 3 (CCR3), which binds most eosinophil-specific chemokines.
6 This hybrid cell has been identified in the circulation of patients with atopic dermatitis and in inflamed tissues of patients with allergic diseases.
7 Cells within these tissues produce eosinophilopoietins; therefore, extramedullary eosinophilopoiesis may occur at sites of allergic inflammation.
8 Eosinophil/basophil progenitors also are altered in cord blood of infants at risk of atopy and asthma, suggesting that hematopoietic processes underlying the allergic phenotype may begin in the prenatal period.
8
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Corroborating Mouse Studies on Cytokines
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Studies in mice corroborate the primary importance of the cytokine, IL-5, and the CC chemokine, CCL11 (eotaxin-1).
9 For example, IL-5 transgenic mice develop peripheral blood and tissue eosinophilia.
10,11 Furthermore, administration of neutralizing IL-5 antibodies in wild-type mice leads to a significant reduction in baseline levels of circulating eosinophils and diminished tissue eosinophilia in response to infection with various parasites or allergen challenge. It is important to note that despite the massive eosinophilia observed in IL-5 transgenic mice, these mice do not develop the tissue damage observed in eosinophilic diseases. This is likely because eosinophils also must be activated to degranulate and/or release their inflammatory mediators. Mice genetically deficient in IL-5 have little or no blood eosinophilia, but have eosinophils in their bone marrow, albeit at reduced levels. A mouse strain deficient in the common β chain, CD131, shared by the IL-3, GM-CSF, and IL-5 receptors have reduced lung eosinophilia in asthma models,
12 whereas mice deficient in both CCL11 and IL-5 have even greater reduction in tissue eosinophils in asthma models.
13 The highly eosinophil-specific expression of eosinophil peroxidase (EPO) has been exploited for the development of an eosinophil-deficient mouse (so-called PHIL mice, “eosinoPHIL” minus “eosino” mice) in which expression of a toxin is molecularly linked to EPO expression, resulting in eosinophil death before they leave the bone marrow.
14 These eosinophil-less mice have subsequently been employed in various disease models, including asthma. Importantly, the mouse models with reduced (IL-5 knockout) or no eosinophils (PHIL) have normal life spans, reproduce normally, do not have increased infections, increased malignancies, or other apparent health defects (personal communications with Paul S. Foster, Ph.D. University of Newcastle, Newcastle, Australia, and James J. Lee, Ph.D., Mayo Clinic, Scottsdale, Arizona). A human correlate to the IL-5 transgenic mouse is the recently recognized
lymphocytic variant of hypereosinophilic syndromes (see
Chapter 36), in which a clonal population of T cells producing IL-5 causes persistent and profound eosinophilia and which often is responsive to anti-IL-5 (
mepolizumab) therapy.
15–17 The ability to propagate eosinophils from bone marrow and umbilical cord stem cells is dependent on IL-5 and further substantiates its role as the most critical cytokine for eosinophil proliferation, maturation, terminal differentiation, and survival.
18,19 In summary, allergen challenge, inflammatory eosinophilic disorders, and parasitic infections induce the production and release of eosinophil precursors from the bone marrow in response to endocrinological actions of cell-derived IL-5, with a contribution by CCL11.
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Interactions of Eosinophilic Factors and Cytokines and Intracellular Signaling
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The interactions of eosinophilopoietic factors with their receptors stimulate a cascade of complex biochemical events through signal transduction. Signaling events progress in four steps: (1) juxtamembranous signaling in which membrane-anchored tyrosine kinases and lipid kinases are activated; (2) signal interfacing which serves to transduce juxtamembranous signals to cytosolic signals; (3) mobile signaling in which cytosolic signaling molecules translocate from the receptor site to other cellular compartments including the nucleus, mitochondria, and cytoskeleton; and (4) transcription activation resulting from nuclear translocation and initiation of gene transcription. Studies have shown the pivotal role of IL-5 in immune responses involving eosinophils through receptor-driven signaling.20 IL-5 binds to the α chain of the IL-5R and induces recruitment of the common β (βc) chain to IL-5R. Janus kinase (JAK)2 tyrosine kinase is constitutively associated with IL-5Rα, and JAK1 tyrosine kinase with IL-5Rβc; both are activated with IL-5 binding as part of the juxtamembranous step. Lyn and Fes are other tyrosine kinases involved in the first step; these tyrosine kinases also are activated by IL-3 and GM-CSF. Adaptor proteins, src homologues and collagen (Shc), SH2-containing phosphatase-2 (SHP-2), growth factor receptor-bound protein 2 (Grb2), Vav, and lipid kinases, phosphatidylinositol 3-kinase (PI-3K), function in the interfacing step. The activation of JAK2 and signal transducer and activator of transcription (STAT) 5 is essential for IL-5 dependent signal transduction. The Ras GTPase-extracellular signal-regulated kinase (Ras-ERK) and also known as Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, in addition to the JAK2-STAT5 pathway, is important in IL-5 signaling in the mobile step. The JAK-STAT and Ras-MAPK pathways converge at various levels in IL-5 signaling of eosinophils. IL-5 induces the expression of cytokine-inducible SH2 protein (CIS) and JAK-binding protein (JAB), which is one of the negative feedback loops in the regulation of IL-5 signaling. Multiple other interactive signal transduction pathways induce and regulate gene expression for eosinophil growth, development, activation, and survival.21,22 Much of the discovery in these pathways has been in murine systems with presumed general applicability to humans. However, at least part of the immune response to IL-5 in mice is NOT part of the biological effect in humans, i.e., in mice, IL-5 enhances several functions of B cells, including immunoglobulin production, growth, and differentiation, whereas human B-cells are influenced by IL-5 only in the presence of specific cytokines and under certain conditions.20 However, human IL-5 does act on T cells by increasing the expression of IL2Rα and augmenting cytotoxic T cell generation.23
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Role of Transcription Factors
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Eosinophil development occurs as a result of functional interactions among various transcription factors.
24 The key transcription factors involved in eosinophil lineage commitment and terminal differentiation are GATA-1, FOG-1 (friend of GATA-1), C/EBPα (CCAAT enhancer-binding protein α), and the ets (E-twenty six) family transcription factor, PU.1.
4 GATA-1 functions primarily to facilitate the differentiation of granulocyte–macrophage progenitors into eosinophils. As a result, GATA-1-deficient mice do not develop eosinophils, and deletion of a specific GATA binding site of the mouse GATA-1 promoter (ΔdblGATA mice) results in strains of mice in which terminal differentiation of eosinophils is prevented.
25,26 FOG-1 functions to antagonize GATA-1 activity and must be downregulated for eosinophil development to occur.
19,27 Mice deficient in C/EBPα. are devoid of all granulocytes,
28 and mice congenitally deficient in PU.1 are unable to generate terminally differentiated eosinophils.
4 As may be predicted, many of these transcription factors are important in generating eosinophil lineage-specific granule proteins, such as major basic protein (MBP)-1, along with CCR3 and the α subunit of IL-5R.
29 For progenitor cells to become committed to eosinophil development requires concomitant expression of C/EBPα, PU.1, and low-to-moderate GATA-1, with no expression of FOG-1. Another member of the C/EBP transcription factor family, C/EBPϵ, is required for terminal differentiation and functional maturation of eosinophils and neutrophils. C/EBPϵ-null (knockout) mice lack functionally mature granulocytes.
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In humans, a novel loss-of-function mutation in the C/EBPϵ transcription factor results in failure of terminal differentiation of both eosinophils and neutrophils along with failed expression of secondary/specific granule protein genes in both granulocytes.
30 These patients are severely immunocompromised and develop frequent bacterial infections. As knowledge of the intricate interactions among transcription factors that direct eosinophil commitment and differentiation continues to unfold, new understanding of eosinophil regulation will emerge, including potential therapies for eosinophil-associated diseases.
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Eosinophil Ultrastructure and Granule Content
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Mature eosinophils are 12–17 μm in diameter and, therefore, slightly larger than neutrophils. They typically have a bilobed nucleus with highly condensed peripheral chromatin. Eosinophils have distinctive cytoplasmic granules, demonstrated by their staining properties with acidic dyes such as eosin and by their unique electron microscopic appearance. These specific or secondary granules are composed of an electron-dense core and a less electron dense matrix, the core being a crystalline lattice by electron microscopy. In cross section, the eosinophil contains approximately 30 of these membrane-bound, core-containing, secondary granules.1 Five highly basic proteins are found within the granules: (1) major basic protein (MBP)-1, (2) MBP-2, (3) eosinophil-derived neurotoxin (EDN) also known as ribonuclease (RNase)2, (4) eosinophil cationic protein (ECP) also known as RNase3, and (5) eosinophil peroxidase (EPO). Several other types of proteins are found in secondary granules and include enzymes, cytokines, growth factors, and chemokines. Eosinophils contain three other types of cytoplasmic granules, referred to as (1) primary granules, (2) small granules, and (3) secretory vesicles. Primary granules are of variable size, round, uniformly dense, present in 1–3 per electron microscopic cross section, and more common in immature eosinophilic promyelocytes. These granules may contain Charcot–Leyden crystal protein (also known as galectin-10), which can be found in neutrophils as well31; Charcot–Leyden crystals (CLCs) are characteristically found in asthmatic sputum and in feces from patients with helminth infections or eosinophilic gastroenteritis. Small granules contain acid phosphatase and arylsulfatase and are present at 2–8 per electron microscopic cross section. Secretory vesicles, also referred to as tubulovesicular structures or microgranules, are characterized by their small, dumbbell-shaped appearance and their albumin content. They are the most abundant granules in number, with approximately 160 per electron microscopic cross section. Normal eosinophils contain varying numbers of nonmembrane-bound lipid bodies, which are the principal stores of arachidonic acid. Lipid bodies also contain the enzymes, cyclooxygenase, 5- and 15-lipoxygenase, which are required to synthesize prostaglandins, leukotrienes (LTs), and eoxins (vide infra), and are increased in activated eosinophils.1
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Non-Mammalian Studies
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Eosinophils likely evolved from an ancestral phagocytic cell that existed in primitive invertebrates. When immune functions emerged separately from digestive functions, cells resembling granulocytes, including eosinophilic cells, evolved. Eosinophils have been described in numerous species of fish and are also present in frogs. They are present in reptiles although they may not contain a crystalloid internus.
32 Reptiles are pivotal on the evolutionary scale because they are progenitors of both avian and mammalian classes. Eosinophils are found in numerous mammals other than humans. Controversy exists as to the evolutionary pressures that directed the development of these cells and their current biological functions. Eosinophils are likely an important part of the innate and adaptive immune response. The effector response of eosinophils may also contribute to the physiological and pathological reactions associated with disease.
33 Through elaboration of remodeling and fibrogenic growth factors, eosinophils may have important roles in the maintenance of tissue homeostasis, wound healing, and repair.
34,35 Eosinophils regulate mast cell functions through release of granule proteins and cytokines, and, in a reciprocal manner, mast cells also activate eosinophils.
36,37
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In mammals, such as the mouse and humans, the eosinophil is released as a mature cell into the circulation from the bone marrow, but is present in the blood only transiently, ranging from 8–18 hours. Eosinophils comprise a small portion, normally 6% or less, of circulating leukocytes. They are primarily tissue dwelling cells, but only in certain tissues in humans, with an average tissue life span of 2–5 days. This may be prolonged by cytokines that increase eosinophil survival for up to 14 days. Under normal circumstances, a balance exists between bone marrow production and release of eosinophils, their time in circulation, and their entrance into tissues. Changes in any one of the compartments causes an increase or decrease in circulating and tissue eosinophils. Eosinophilia in blood or tissue or both is associated with helminthiasis, allergic hypersensitivity, and other pathological conditions. In humans, bone marrow, spleen, lymph node, thymus, and gastrointestinal tract from the stomach through the colon, sparing the esophagus, are the only tissues in which eosinophils normally reside.38 Furthermore, the gastrointestinal tract is the only organ other than bone marrow in which extracellular eosinophil granule protein deposition is observed even under homeostatic conditions. Eosinophils and their granule proteins are found in the lamina propria in normal gastrointestinal tract and are not found in Peyer's patches or epithelium. Eosinophils may be important for thymocyte deletion based on the localization of eosinophils within the thymus and the timing of their migration during the neonatal period.39 Murine observations indicate that eosinophils are also important for postnatal mammary gland and uterine development, and their recruitment into the uterus heralds estrus. Although eosinophils, themselves, are not known to participate in human reproduction, eosinophil proMBP-1 is expressed in the uterus by placental X and giant cells during pregnancy, and its production peaks 2–3 weeks before parturition.40,41 The recruitment of eosinophils to the gastrointestinal, thymic, uterine, and mammary tissues is under the control of the CC chemokine, CCL11.42,43
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Once eosinophils enter tissues, most do not recirculate. Several possible mechanisms exist for removal of tissue eosinophils. These include shedding of the cells across mucosal surfaces into the lumen of the intestinal or respiratory tract, engulfment of apoptotic eosinophils by macrophages, and lysis or degranulation with cellular degeneration. In various inflammatory conditions, including those affecting the skin (Chapter 36), striking numbers of free granules and/or eosinophil granule protein deposition are present in the absence of intact eosinophils.1 Studies recently have revealed that isolated eosinophil granules express extracellular domains for interferon (IFN)-γ receptor and CCR3 and, upon stimulation, respond independently as organelles by releasing ECP.44
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Role of Eosinophils in Immune Function
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Shortly after their discovery by Paul Ehrlich in 1879, eosinophils were observed in association with helminth infections. Theories have been promulgated that eosinophils are important for host defense against parasites spawning numerous studies.45 For example, in vitro studies demonstrated that eosinophils are cytotoxic to large nonphagocytosable organisms, such as multicellular helminthic parasites. Eosinophils bind to host-derived immunoglobulins and complement components on the surface of their targets (so-called antibody- or complement-) dependent cytotoxicity. They also bind to carbohydrate ligands expressed on parasites, such as the Lewisx-related molecules, and cell adhesion molecules similar to selectins. Eosinophils are activated to release their granule products with deposition of these biologically active proteins in and around the parasites causing disruption of the parasite's integument and, ultimately, death of the organism. The granule proteins have different effects. ECP produces fragmentation and disruption whereas MBP-1 produces a distinctive ballooning detachment of the tegumental membrane, and EDN is active only at high concentrations causing crinkling of the tegumental membrane.46 However, in murine models in which blood, marrow, and tissue eosinophilia is largely abolished by neutralizing IL-5 activity, the intensities of primary or secondary parasitic infection are unchanged indicating that eosinophils have little or no role in parasitic host defense in these models.1 The results must be interpreted cautiously because mouse and human eosinophils have functional differences, and mice are not natural hosts of many of the parasites tested experimentally.
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Eosinophils also release cytotoxic granule proteins onto the surface of fungal organisms and into the extracellular milieu in fungal infections. Eosinophils kill fungi in a contact-dependent manner. Eosinophils adhere to the fungal cell wall component, β-glucan, via a β2-integrin surface molecule, CD11b.47 Eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide, and, specifically, do not react with chitin. However, chitin, which is a polymer that confers structural rigidity to fungi, helminths, crustaceans, and insects, induces accumulation of eosinophils in tissues through production of LT B4 in mice.48 Eosinophils also are activated by fungal organisms that release proteases, such as Alternaria, through protease-activated receptors (PARs). For example, fungal aspartate protease activates eosinophils through PAR-2 and, thereby, mediates eosinophils’ innate responses to certain fungi.49
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As a granulocyte, the eosinophil is capable of phagocytosing and killing bacteria and other small microbes in vitro, but eosinophils cannot effectively defend against bacterial infections when neutrophil function is deficient. Nevertheless, recent investigations reveal that eosinophils may have a role in innate immunity against bacteria using a unique mechanism, DNA trap.50 Eosinophils rapidly release mitochondrial DNA when exposed to bacteria, a complement component, C5a, or CCR3 ligands. The traps contain eosinophil granule proteins, ECP and MBP, and have antimicrobial effects. In the extracellular space, the granule proteins and mitochondrial DNA form structures that bind and kill bacteria both in vitro and in vitro. Eosinophils, unlike neutrophils, do not undergo cell death as part of this process. This may be an important innate immune response, particularly in mucosal epithelium.50
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Another protective function that eosinophils may have is in viral infections. Eosinophils and their granule proteins are increased in the respiratory tracts of patients with respiratory syncytial virus, an RNA-viral infection. EDN (RNase2) and ECP (RNase3), eosinophil granule matrix proteins, are ribonucleases (vide infra). Purified eosinophils, as well as EDN and ECP individually, reduced viral titers when added to respiratory syncytial viral suspensions. In mice, at least 11 eosinophil-associated ribonucleases degrade single stranded RNA containing viruses.51 Despite divergence of the coding regions, conserved eosinophil ribonuclease activity across species suggests a strong evolutionary pressure to preserve this critical enzymatic activity.51 In other studies, pretreatment of parainfluenza-infected guinea pigs with anti-IL-5, to reduce numbers of eosinophils, strikingly increased viral load in the airways. Viruses, including parainfluenza virus, respiratory syncytial virus, and rhinovirus, induce the release of another eosinophil granule protein, EPO, when coincubated with antigen-presenting cells and T cells.52 Paradoxically, eosinophils may be a potential reservoir for the human immunodeficiency virus (HIV)-1.53
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Eosinophils may have other roles in immune responses as well. Through MHC class II expression and IL-1α production, they can function as antigen-presenting cells for a variety of viral, parasitic, and microbial antigens, including staphylococcal superantigens, and allergens.54,55 Eosinophils are recruited to secondary lymphoid structures to promote the proliferation of effector T cells although they are unable to affect naïve T cells.56 Eosinophils, as sources of cytokines, influence T cell-dependent responses.1 In keeping with the prominence of eosinophils in allergic disorders, eosinophils are involved in T cell polarization favoring Th2 by promoting Th1 apoptosis in addition to their influence via cytokine expression.54,57–60
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Role of Eosinophils in Disease
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The activities of eosinophil-derived products include direct cytotoxic effects on structural cells and microbes, increased vascular permeability, procoagulant effects, innate immune responses to some parasites, viruses, fungi and tumor cells, enhancement of leukocyte migration, amplification of effector T-cell responses and, possibly even mammary gland development. Collectively, these varied biologic actions provide the pathophysiological basis for the signs and symptoms observed in eosinophil-associated diseases.
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Eosinophils in lymph nodes and spleen are especially increased after allergen exposures or microbial insults.61,62 Eosinophils have been found in several cancers, particularly in lymphomas, leukemias, and colon cancer. Clinical studies indicate that certain tumors associated with tissue and/or peripheral eosinophilia have a more favorable prognosis,63 whereas in other tumors, they are thought to confer a poor prognosis, such as nodular sclerosing Hodgkin disease, Sézary syndrome, and gastric carcinomas. In Sézary syndrome (see Chapter 145), the tumor cells produce IL-5 and, therefore, are responsible for the eosinophilia, which is a reflection of tumor burden.64 Where eosinophilia is a good prognostic factor, eosinophils are considered to be part of an effective host response to the tumor.65,66
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Antitumor Activity, Gastrointestinal Diseases and Asthma
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The specific nature of eosinophil-dependent antitumor activity has not been sufficiently explored but may include, in addition to direct cytotoxic effects on tumor cells, tumor antigen presentation, vascular compromise via microthrombi formation, or synergism with the antitumor response of other leukocytes. There are many gastrointestinal tract disorders in which eosinophil numbers are substantially increased such as drug reactions, helminthic infections, inflammatory bowel disease, gastroesophageal reflux, and the primary eosinophilic gastrointestinal disorders (EGID) including eosinophilic esophagitis, eosinophilic gastritis, and eosinophilic gastroenteritis. The skin and respiratory tract, in contrast to lymphatic and gastrointestinal tissues, normally are devoid of eosinophils. Both intact and degranulated eosinophils are a significant component of the inflammatory infiltrate in many cutaneous diseases as described in
Chapter 36. Eosinophils were identified in anaphylaxis and immunoglobulin (Ig) E-mediated hypersensitivity where they were originally hypothesized to play a protective role. They were subsequently defined as a component of Th2 immunity. Asthma is the prototypic disease in which eosinophil involvement has been most extensively characterized.
67–69
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Along with parasitic infections, asthma was recognized soon after eosinophil discovery as a disease in which eosinophils were prominent. The findings of two disparate disorders, parasitism and asthma, linked by eosinophils prompted the more recent concept that the mechanisms by which eosinophils participate in host defense may have deleterious effects to the host. Investigations followed and revealed that eosinophils are unique among circulating leukocytes in their amazing abilities to wage chemical warfare. They are endowed with highly toxic granule proteins and an arsenal of enzymes that are released upon activation or with cell lysis that inflict damage on biological targets.
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Eosinophil Constituents and Their Activities
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The eosinophil contains and produces a myriad of factors that implicate its role in inflammation and tissue destruction and remodeling (see Fig. 31-2).70 Products released by eosinophils include chemoattractants, colony-stimulating factors, and endothelial-activating cytokines. In addition to toxic cationic proteins from specific granules and oxidative products released into tissues following activation, these factors include arachidonic acid-derived lipids, hydrolytic enzymes, neuropeptides, colony-stimulating factors, and cytokines/chemokines that facilitate further leukocyte recruitment to sites of inflammation (see Fig. 31-2). Surface molecule expression is important in all aspects of eosinophil biology from promoting growth and differentiation to eosinophil trafficking into tissue to activation and/or priming of the cells to senescence. Numerous membrane factors are expressed on eosinophils that further direct eosinophil biological effects. (See ch31etb0.1 for designations of various eosinophil factors.)
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Eosinophil Granule Proteins
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Among the products of eosinophils that are most damaging to the host are the specific granule's cationic proteins. Furthermore, the granule proteins are markers of eosinophil activity because the eosinophil often loses its characteristic morphology through cytolysis in tissues.71 Knowledge of their biological actions provides insight into their functions in human disease. Once deposited, the granule proteins persist in tissues for extended times—EPO for 1 week, ECP for 2 weeks, EDN for 2.5 weeks, and MBP-1 for 6 weeks.72 Each of these proteins have been shown to induce direct tissue damage to both host cells, including myocytes, endothelium, neurons, epithelium, and smooth muscle, and to microbes (vide supra). For example, MBP-1, ECP, or EPO application to airway epithelium in primates produces ciliostasis, desquamation, and hyperreactivity of respiratory smooth muscle mimicking the pathology of asthma.73,74 Damage to endothelium in eosinophilic endomyocardial disease is thought to be the initiating factor in the cardiomyopathy observed in the hypereosinophilic syndromes (see Chapter 36).75 All four of the cationic granule proteins [(1) EPO, (2) ECP, (3) EDN, and (4) MBP-1] likely contribute to the edema observed in skin diseases due to their vasodilatory effects with contribution from mast cells and basophil histamine release by MBP-1.76 Eosinophil granule proteins stimulate various cells in addition to mast cells and basophils, including neutrophils and platelets. Nodules, eosinophilia, rheumatism, dermatitis and swelling (NERDS), episodic angioedema with eosinophilia (Gleich syndrome), urticaria, eosinophilic cellulitis (Wells syndrome), and insect bite reactions demonstrate variable degrees of edema that are probably explained, at least in part, by this mechanism (see Chapter 36). Eosinophil granule proteins injected into skin produce lesions including dose-dependent wheal-and-flare reactions by MBP and ulcerations by ECP and EDN.77,78 Wound healing is delayed in the presence of eosinophils and eosinophil granule proteins.79,80 Eosinophils, through activities of granule proteins, have procoagulant activity. Thromboses have developed in the hypereosinophilic syndromes, including several case reports of hepatic vein obstruction (Budd–Chiari syndrome). The thromboses could be the result of direct endothelial damage or due to the ability of MBP and ECP to neutralize heparin. In addition, MBP is a strong platelet agonist, and platelet-activating factor (PAF), which is released by eosinophils, causes platelet aggregation.1
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General Characteristics
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Major basic protein (MBP) comprises the crystalloid core of the specific eosinophil granule. It was so named because it accounts for a major portion, about 55% (in guinea pig), of the eosinophil granule protein, and has a high isoelectric point, calculated at greater than pH 11, so strongly basic that it cannot be measured accurately. It is now known that MBP is expressed as two homologs, MBP-1 and MBP-2, coded by different genes on chromosome 11.
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Detailed Major Basic Protein's Chemistry
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MBP-1 and, presumably, MBP-2 are translated as a preproprotein with a strongly acidic proportion. The combination of the prosection with MBP-1 or MBP-2 yields a molecule with a relatively neutral pI, 6.2. ProMBP-1, and possibly proMBP-2, lacks the cytotoxic properties of MBP-1 and MBP-2; synthesis as a neutral proprotein may function to protect the cell during transport from the Golgi apparatus to the granule. ProMBP-1 is the predominant form of MBP-1 in the blood of pregnant women and circulates as a complex with pregnancy-associated plasma protein-A (PAPP-A), with angiotensin, and with complement component, C3dg. It localizes to placental X cells and functions as a novel enzyme inhibitor of PAPP-A to modify its ability to release active insulin-like growth factor during primate pregnancy.
1 Both MBP-1 and MBP-2 are present in eosinophil granules; MBP-1 also is present in basophils in a much lesser concentration than in eosinophils, but MBP-2 is not in basophils. Mature eosinophils lose the ability to transcribe mRNA encoding MBP-1 indicating that all MBP-1 stored in the crystalloid granule cores is synthesized during early eosinophil development. MBP-2 is approximately 100 times less basic than MBP-1, with calculated pI of 8.7. MBP-1 and MBP-2 have 42 identical amino acids of the approximately 117 in each of these proteins. The crystal structure of MBP-1 indicates that it is a member of the C-type lectin family but does not have all the biochemical activities of lectins. Rather, MBP-1 and MBP-2 have many other activities. Comparative analyses of the biological effects of MBP-1 and MBP-2 demonstrate that they are similar in cytotoxic and cytostimulatory effects, but with reduced potency of MBP-2. Most of the eosinophil granule protein activities have been characterized for MBP-1.
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Tissues Effects of Basic Proteins
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MBP-1 directly damages helminths and also lethally damages mammalian cells and tissues, examples of which are its ability to cause exfoliation of bronchial epithelial cells and to kill tumor cells. MBP-1 exerts its effects by increasing cell membrane permeability through surface charge interactions leading to disruption of the cell surface lipid bilayer.81 MBP-1 and MBP-2, but none of the other eosinophil granule proteins, stimulate histamine and LTC4 release from human basophils. Further, MBP-1 and MBP-2 stimulate neutrophils, inducing release of superoxide, lysozyme, and IL-8. MBP-1 and EPO are potent platelet agonists causing release of 5-hydroxytryptamine and promoting clotting.
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Eosinophil Peroxidase
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Eosinophil peroxidase (EPO) is highly basic, pI 10.8, localized in the matrix of the specific eosinophil granule and is a key participant in generating reactive oxidants and free radical species in activated eosinophils. EPO consists of a heavy chain and a light chain encoded with a prosequence. The EPO gene is on chromosome 17 and maps closely to myeloperoxidase and lactoperoxidase genes, two other members of the mammalian peroxidase family found in neutrophils and mucosal glands, respectively. Although MBP is present in the highest molar concentration in eosinophil granules, EPO, by weight, is the most abundant protein constituting approximately 25% of the specific eosinophil granule's total protein mass. EPO catalyzes the oxidation of halides, pseudohalides, and nitric oxide to form highly reactive oxygen species (hypohalous acids), reactive nitrogen metabolites (nitric dioxide), and other oxidants that then oxidize targets on proteins with oxidative stress and subsequent cell death by apoptosis and necrosis. EPO kills numerous microorganisms in the presence of hydrogen peroxide, generated by eosinophils and other phagocytes, and halide. This combination of products also initiates mast cell secretion. As noted, both EPO and MBP induce noncytolytic, dose-dependent 5-hydroxytryptamine release from platelets.82 Binding of EPO to neutrophils reversibly inhibits EPO peroxidase activity but increases neutrophil aggregation and adhesion to endothelial cells.83 EPO binding to microbes, including Staphylococcus aureus, greatly potentiates their killing by phagocytes. EPO-coated tumor cells are spontaneously lysed by activated macrophages.
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Eosinophil Cationic Protein and Eosinophil-Derived Neurotoxin
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General Characteristics
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Eosinophil cationic protein (ECP or RNase3) and eosinophil-derived neurotoxin (EDN or RNase2) are homologous proteins with sequence identity in 37 of 55 amino acid residues and are encoded on chromosome 14. ECP also has neurotoxic activity. ECP and EDN play a role in viral host defense to RNA viruses.51,88,89 New roles for these proteins continue to be identified.90 EDN induces the migration and maturation of dendritic cells.91 It also is an endogenous ligand of Toll-like receptor 2 (TLR2) and can activate myeloid dendritic cells by triggering the TLR2-myeloid differentiation factor 88 (Myd88) signaling pathway.60 Based on its ability to serve as a chemoattractant and activator of dendritic cells along with enhancing antigen-specific Th2-biased immune responses, EDN functions as an alarmin alerting the adaptive immune system to preferentially enhance antigen-specific Th2 responses.60
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Specific Chemicals Characteristics of ECP and ECN
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They are found in the matrices of specific granules in mature eosinophils. Both proteins also are present in mature neutrophils.84 They are highly basic proteins, the pI of ECP is 10.8 and the pI of EDN is 8.9. It is likely that these two proteins arose as a consequence of gene duplication 25–40 million years ago.85 EDN, in particular, is a rapidly evolving protein, accumulating nonsilent mutations at a rate exceeding those of most other functional coding sequences studied in primates.86 Although the homology in EDN and ECP sequences accounts for certain similarities, their differences are considerable. ECP and EDN are members of the human RNase A family, ECP is a relatively weak ribonuclease, and EDN is a potent ribonuclease. ECP is a potent toxin for parasites through a different mechanism than MBP-1 and is more effective at killing certain helminths than MBP-1. EDN, as its name implies, was discovered as the neurotoxin that accounted for the Gordon phenomenon, described in 1933, in which splenic extracts from patients with the Hodgkin's disease caused severe damage to myelinated neurons in experimental animals.87
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Charcot–Leyden Crystal Protein
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Distinctive hexagonal, bipyramidal crystals were initially described in 1853 in a patient with leukemia and later, in 1872, in the sputa of asthmatic patients. Since then, CLCs have been regarded as a hallmark of eosinophilia. CLC protein is an abundant, characteristic, although not unique, protein of eosinophils. It is also found in lesser amounts in basophils. CLC mRNA and EDN mRNA are among the most highly expressed mRNAs in mature peripheral blood eosinophils suggesting de novo synthesis.1
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Charcot–Leyden Crystals
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Specific Biochemistry
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CLC belongs to a family of galactose-binding lectins and also is called galectin-10.
92 CLC protein is localized to primary eosinophil granules but also is detected in the nuclear matrix and cytoplasm of eosinophils derived from IL-5-treated umbilical cord cell cultures. Moreover, in cells expressing the recombinant molecular form, CLC protein is detected in the nucleus, the cytoplasm and the plasma membrane. The role of CLC protein is unknown. Previous studies suggested that it may have lysophospholipase activity or lysophospholipase inhibitor binding activity, with the theory that this activity could protect the cells from the lytic effects of lysophospholipase at sites of inflammation. However, CLC protein has no sequence similarities to known lysophospholipases, and depletion of CLC protein from eosinophil lysates did not alter their lysophospholipase activity. Further, affinity-purified CLC protein lacked lysophospholipase activity. Therefore, CLC protein and eosinophil lysophospholipases are distinct proteins.
93 Lastly, through its lectin-like domains, CLC protein may bind carbohydrates expressed on microorganisms or biological molecules, such as IgE or laminin.
94
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Other Eosinophil Enzymes
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Many enzymes, in addition to EPO and the RNases (EDN and ECP), have been identified in human eosinophils, granules, and membranes (see
Fig. 31-2).
1 Arylsulfatase B, β-glucuronidase, lysozyme, acid phosphatase, catalase, and histaminase are also present in eosinophils. Eosinophils are a source of matrix metalloproteinase-9 (MMP-9), which is important for their migration through basement membranes. Eosinophil-derived MMP-9 is found in basal cell and squamous cell carcinomas
95,96 and in bullous pemphigoid lesions.
97 MMP-9 cleaves type XVII
collagen (BP 180), a transmembrane molecule of the epidermal hemidesmosome, likely contributing to or causing basement membrane zone separation in bullous pemphigoid.
Collagenase, which degrades type I and type III
collagen, may play a role in tissue remodeling. Eosinophils generate extracellular superoxide anions through activation of a plasma membrane NADPH oxidase, expressed in high levels in human eosinophils. Activated eosinophils form more of the functional NADPH oxidase complex and produce more superoxide than neutrophils or macrophages.
98 Secretory phospholipase A
2 is mainly located in specific granules in the eosinophil and copurifies with ECP in granule fractions. It is known to degrade phospholipids of Gram-negative bacteria and to cause airway inflammation and smooth muscle contraction. Secretory phospholipase A
2 may act together with bactericidal/permeability-increasing protein (BPI), also present in eosinophil granules, in microorganism defense. Cytosolic phospholipase A
2 plays an essential role in mediating hydrolytic cleavage of arachidonic acids in membrane phospholipids and generating various lipid mediators (vide infra).
1
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Reactive Oxygen Metabolites
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Reactive oxygen species are important mediators of the tissue injury caused by eosinophils. The reactive oxygen intermediates produced by NADPH oxidase during the respiratory burst initiated on eosinophil activation include superoxide, hydroxyl radicals, and hydrogen peroxide. Hydrogen peroxide can generate hypohalous acids through the action of EPO, which are cytotoxic as well. Reactive oxygen species also can augment the inflammatory response by inducing gene expression and T-cell proliferation.98
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Lipid bodies in eosinophils are storage sites for arachidonic acids. Eosinophils produce several arachidonic acid metabolites, including cysteinyl LTs from the 5-lipoxygenase pathway (LTC4, LTD4, and LTE4) and thromboxanes and prostaglandins from the cyclooxygenase pathway [thromboxane B2, prostaglandin (PG) E2, and PGF1α].99,100
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Detailed Lipid Mediator Biochemistry
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LTC
4 is the only LT produced intracellularly by eosinophils. LTD
4 and LTE
4 are metabolites of LTC
4 produced by extracellular cleavage. LTC
4, LTD
4, and LTE
4 have several biologic effects, including bronchoconstriction, increased airway reactivity, increased vascular permeability, and leukocyte recruitment. Not surprising, these cysteinyl LTs are major mediators of asthma and allergic rhinitis. Whether LTs play a role in eosinophilic disorders of the skin is less clear. Human eosinophils contain abundant amounts of 15-lipoxygenase, coded by a Th2-induced gene that is upregulated in asthma and eosinophilic esophagitis and likely other eosinophil-associated diseases. In addition to 5-lipoxygenase products, 15-lipoxygenase metabolites are produced, including 5,15- and 8,15-dihydroxy-hydroxyeicosatetraenoic acid (HETE) and a novel chemotactic lipid, 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid. Recently, new 15-lipoxygenase compounds were discovered by incubating eosinophils with exogenous arachidonic acid, analogous to the 5-lipoxygenase-derived LTs. To mitigate confusion with LTC
4, LTD
4, and LTE
4, these compounds have been named eoxins (EX). Therefore, EXC
4, EXD
4, and EXE
4 correspond to 14,15-LTC
4, 14,15-LTD
4, and 14,15-LTE
4, respectively.
101 EXC
4 is produced by mast cells and nasal polyps in addition to eosinophils. The fact that eosinophils produced EXC
4 after challenge with the proinflammatory factors, LTC
4, PGD
2 and IL-5, indicates that eosinophils can produce EX from endogenous arachidonic acid. EXs enhance vascular permeability in vitro almost as potently as LTC
4 and LTD
4, and 100 times more potently than histamine.
101 The predominant cyclooxygenase product of eosinophils is thromboxane B
2, but this, and the other mediators produced through this pathway, are minor compared to those produced by the 5- and 15-lipoxygenase pathways. Eosinophils contain high levels of other phospholipids, the stored precursor to platelet-activating factor (PAF). Eosinophils produce at least three molecular species of PAF, most of which remain cell-associated.
102 PAF has important pharmacological activities, including activation of platelets and neutrophils and induction of bronchoconstriction.
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Eosinophils are a considerable source of growth factors and regulatory as well as proinflammatory cytokines and chemokines.1,103 The various growth factors produced by eosinophils include transforming growth factor (TGF)-α, TGF-β, fibroblast growth factor-2, vascular endothelial growth factor, nerve growth factor, and platelet-derived growth factor-β. There is evidence that these growth factors induce stromal fibrosis and basement membrane thickening at sites of chronic eosinophilic inflammation including nasal polyps, asthmatic airways and, likely, in certain skin disorders such as atopic dermatitis.1 The production and release of nerve growth factor by eosinophils promotes neurites in nerve cells.104 Another group of cytokines produced by eosinophils modulates other immune cells and includes tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α (CCL3), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, CXCL8 (IL-8), IL-10, IL-12, IL-13, IL-16, GM-CSF, and IFN-γ.103 Additional chemokines produced by eosinophils are CXCL13 (B lymphocyte chemoattractant factor), CCL5 [regulated on activation, normal T cells expressed and secreted (RANTES)], and CCL11, in addition to CCL3 and CXCL8. CCL5 apparently is stored in at least two intracellular compartments, namely, (1) the matrix of specific eosinophil granules and (2) small secretory vesicles. All of these cytokines are constitutively produced in low levels in resting eosinophils and induced in inflammatory conditions with activation of eosinophils by engagement of receptors (vide infra) with immunoglobulins, complement and cytokines, including those produced by eosinophils, themselves, in an autocrine manner. Notably, eosinophils produce the three principal cytokines involved in their own growth and differentiation—(1) IL-3, (2) IL-5, and (3) GM-CSF, as well as chemokines important in their own chemotaxis, CCL5 and CCL11. It is known that eosinophils synthesize and secrete GM-CSF by a peptidyl-prolyl isomerase (PIN1)-dependent mechanism.105 However, although eosinophils produce quantities of certain cytokines comparable to T cell production,106 the relative contributions of eosinophil-derived cytokines to inflammation remain to be determined. In summary, the eosinophil-derived cytokines may function in both an autocrine and paracrine fashion and likely have pathophysiological relevance.
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Eosinophils express numerous receptors and other factors on their surface membranes through which they communicate with the extracellular environment, but no single surface protein is uniquely expressed on eosinophils. These receptors have been identified either by flow cytometry or by functional assays, and can be grouped as follows: chemotactic factor and complement receptors including chemokine, LT and PAF; immunoglobulin supergene family member receptors including immunoglobulins; cytokine receptors including those discussed above; adhesion molecule receptors; receptors involved in apoptosis; and miscellaneous receptors and surface factors. Eosinophil membrane proteins are promising targets for therapeutic modulation of eosinophil effects (see section “Pharmacological Manipulation”).
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Chemotactic Factor and Complement Receptors
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Chemotactic factors are important in orchestrating cellular trafficking to sites of inflammation as well as physiologic homing (e.g., eosinophils to gastrointestinal tract). The eosinophil has receptors for many chemotactic agents such as LTB4, PAF, bacterial products (N-formyl-methionyl-leucyl-phenylalanine), complement anaphylatoxins, C3a and C5a. Eosinophils express complement receptor (CR)1 (CD35), a receptor for C1q that also binds C4b, C3b, and iC3b, and CR3 (Mac-1, CD11b/CD18) in addition to receptors for C3a and C5a. These are important receptors in eosinophil effector functions. The binding of chemokines to their respective receptors mediates many biological effects, which, in addition to cell shape change and migration, includes cell activation, receptor internalization, induction of the respiratory burst with generation of toxic oxygen metabolites, and transient activation of integrin adhesiveness. PAF activity on eosinophils is mediated through PAF receptors that have been cloned. PAF is one of the most potent chemoattractants for eosinophils and selectively recruits eosinophils over neutrophils. PAF also induces release of granule proteins, reactive oxygen species, and LTC4 from eosinophils. LTB4 through its eosinophil receptor potently stimulates eosinophil chemotaxis and elicits arachidonic acid metabolism and a respiratory burst. It does not induce eosinophil degranulation. Eosinophils also may have LTD4 receptors.1 The chemotaxins listed above have potent effects on eosinophils but are nonselective in that they are active on other leukocytes. Because many eosinophil-associated diseases are characterized by tissue eosinophil infiltration with little or no neutrophil infiltration, the identification of the CCR3 receptor and its ligands was an important breakthrough in discovering eosinophil-selective chemotaxins.107 Specific members of the chemokine family are critical for the cellular trafficking of eosinophils. The major ligands of CCR3, CCL5, CCL11, CCL13 (monocyte chemotactic protein-4), CCL24 (eotaxin-2), CCL26 (eotaxin-3), play a critical role in both the homeostatic and inflammation-induced recruitment of eosinophils to tissue sites.13,108 Not surprisingly, the most highly expressed chemokine receptor on human eosinophils is CCR3 (40,000–400,000 receptors per cell). CCR3 is a seven-transmembrane spanning G-protein coupled receptor that can deliver both powerful positive and negative signals depending on the interacting ligand.109
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Immunoglobulin Supergene Family Member Receptors
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Many of the studies of eosinophil functions, including phagocytosis, antigen-dependent cytotoxicity, oxygen metabolism, LTC4 production, and eosinophil survival, have been performed using IgG-coated targets. Among eosinophil surface receptors for the immunoglobulin family members, the most highly expressed receptor is FcγRII (CD32), which binds aggregated IgG, particularly of the subclasses IgG1 and IgG3. The binding of IgG to this receptor may be important in eosinophil degranulation in parasitic and allergic diseases along with other eosinophil functions.1 Freshly isolated eosinophils express only FcγRII (CD32) of the IgG receptors, but eosinophils can be stimulated by cytokines, particularly IFN-γ, to express FcγRI (CD64) and FcγRIII (CD16) and augment FcγRII (CD32) expression.
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FcαRI (CD89), the IgA receptor, is present on the surface of eosinophils. Eosinophils also possess a binding site for secretory component that may account for the finding that secretory IgA is the most potent immunoglobulin stimulant for eosinophil degranulation. The interaction of eosinophils with IgA is enhanced by Th2 cytokines, IL-4 and IL-5. These observations, coupled with the observation that many eosinophils are found in epithelial tissues such as gastrointestinal tract, suggest that eosinophils and secretory IgA play an important role in mucosal immunity.
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Members of the immunoglobulin superfamily are type I transmembrane molecules that share common structural characteristics of the globular domains found in immunoglobulins. Intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-3 (CD50) are members of this superfamily expressed on eosinophils and are likely important in leukocyte–leukocyte and leukocyte–tissue cell adhesion through leukocyte function-associated antigen (LFA)-1 (αLβ2; CD11a/CD18) as its counterligand (see Fig. 31-1).
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Cytokine receptors are present at low levels on the surfaces of eosinophils. Receptors for IL-3 (CD123), IL-5 (CD125), and GM-CSF (CD116) are readily detected and, as noted previously, all share a common β chain (CD132). Eosinophil activation has been observed by a variety of other cytokines through presumed and/or detected receptors. These include: stem cell factor (c-kit receptor; CD117), IFN-γ (CD119), TNF-α (CD120), IL-4 (CD124), IL-9 (CD129 and CD132), IL-13 (gp65), IL-2 (CD25), IL-31, and TGF-β receptors. Many of these receptors are for cytokines that eosinophils produce providing further evidence that they have autocrine functions.
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Adhesion Molecule Receptors
++
Adhesion molecule receptors are expressed on the eosinophil cell surface to mediate trafficking to and within tissues, and for general cell–cell interactions.112 These receptors fall into three groups: (1) immunoglobulin superfamily (reviewed above), (2) selectins and their glycoprotein counterligands, and (3) integrins. l-selectin (CD62L) and p-selectin glycoprotein ligand-1 (PSGL-1, CD162) are expressed at high levels on eosinophils, whereas e-selectin ligands, as an example, sialyl-Lewis X (CD15s), are expressed at very low levels. p-selectin together with PSGL-1 is the most important selectin pair in eosinophil migration into tissues.
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Eosinophils express a variety of integrins (β1, β2, and β7) on their surface, which facilitate their adhesion to extracellular matrix proteins, vascular cellular adhesion molecule (VCAM)-1 (CD106) on activated endothelium, or ICAM-1 present on resting or activated epithelium and activated endothelium. Integrins are composed of two subunits that exist as noncovalently associated heterodimers, with α and β subunits. The β1 integrins expressed on eosinophils include α4β1 (very late antigen-4 or VLA-4), which binds to VCAM-1 found on activated endothelium and the extracellular matrix protein, fibronectin. Eosinophil adhesion to fibronectin induces the autocrine production of eosinophil-activating survival cytokines, IL-3, IL-5, and GM-CSF. The only other β1 integrin expressed on eosinophils is α6β1, which mediates the binding to another matrix protein, laminin. Four β2 integrins are found on eosinophils: (1) αLβ2 (LFA-1), (2) αMβ2 (CD11b/CD18 or Mac-1), (3) αXβ2, and (4) αDβ2. These integrins bind to ICAM-1, ICAM-2, ICAM-3, VCAM-1, fibrinogen, and the complement fragment, C3bi. Lastly, eosinophils also express α4β7, which is the ligand for the gut mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is likely important in homing of eosinophils to gastrointestinal mucosa; α4β7 also mediates adhesion to fibronectin and VCAM-1.
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Receptors Involved in Apoptosis
++
Eosinophils express several “death receptors,” that are involved in apoptotic pathways, such as Fas receptor (CD95), Siglec-8, CD30, CD45, Campath (CD52), and CD69, along with important intracellular regulators of eosinophil apoptosis such as the members of the B-cell leukemia/lymphoma (Bcl)-2 and inhibitor of apoptosis families.113 Diseases characterized by eosinophilia likely result, in part, from delayed or defective apoptotic pathways allowing accumulation and persistence of eosinophils in blood and/or tissues.
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Miscellaneous Surface Receptors and Other Surface Factor Expression
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Eosinophils express some of the requisite costimulatory molecules characteristic of the T cell-antigen presenting cells synapse, including the IL-2 receptor (CD25), B7 homologs, and HLA-DR [major histocompatibility complex class II (MHC-II)].
55 Eosinophils, similar to many antigen-presenting cells, are activated through Toll-like receptors 7 and 8 suggesting that they participate in the recognition and killing of viruses and bacteria.
114–116 Eosinophils also have PAR-2 on their surfaces indicating their ability to respond to fungal and other organisms.
49,117 CD48 is an activation molecule on eosinophils that, when neutralized, is associated with decreased eosinophil infiltration into lung tissue.
118 CD52 is expressed on eosinophils and lymphocytes, but not neutrophils, and cross-linking CD52 inhibits, in a dose-dependent manner, production of reactive
oxygen species after stimulation with C5a, PAF, and GM-CSF.
119 Other surface receptors and factors including enzymes are expressed on eosinophils.
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Factors Working Together
++
The various products elaborated by eosinophils in response to receptor activation do not necessarily function independently but often act in concert to mediate their biological effects. For example, the release of TGF-α, TGF-β, fibroblast growth factor-2, vascular endothelial growth factor, MMP-9, and inhibitors of MMPs from activated eosinophils collectively induce fibroblast proliferation and extracellular matrix protein production. Eosinophils contribute factors of their own and influence factor production from other cells, for example, eosinophil mediators induce platelet release of TGF-β. After intradermal eosinophil infiltration, there is production of extracellular proteins, including tenascin and procollagen 1, as well as myofibroblast formation.120 Eosinophil-induced fibrosis is observed in the lungs and heart of patients with hypereosinophilic syndrome, in and around organs in other fibrosing/sclerosing disorders and in the skin of patients with eosinophilic fasciitis (Shulman syndrome), eosinophilia–myalgia syndrome, and toxic oil syndrome (see Chapter 36).121 Eosinophil granule proteins, MBP-1 and EDN, along with other neuroactive mediators produced by eosinophils such as nerve growth factor, vasoactive intestinal peptide, and substance P, likely affect nerve physiology. In fact, eosinophils and eosinophil granule proteins are often observed in close proximity to nerve endings.122,123 Eosinophil-induced nerve dysfunction is likely an important part of the gastric dysmotility observed in subjects with food allergies, the dysfunction of vagal muscarinic M2 receptors observed in asthmatics, and may also contribute to itch along with other physiological aberrations in atopic dermatitis and other cutaneous diseases.123,124 Collectively, the eosinophil's response to surface factors determines its role in health and disease.
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The selective recruitment of eosinophils into sites of inflammation results from interactions among eosinophil-activating cytokines, chemokine-inducing cytokines, and endothelial-activating cytokines (see Fig. 31-1). Similar to other leukocytes, selectin, integrin, and immunoglobulin gene superfamily members contribute to the signaling involved in eosinophil trafficking. In particular, eosinophils constitutively express the integrin, VLA-4, which interacts with its ligand, VCAM-1, induced on endothelial cells by cytokines, especially Th2 cytokines (IL-4 and IL-13).125 After movement through vessels, eosinophils adhere to extracellular matrix proteins. Here, surface factors, b2 integrins such as CD11b/CD18 (Mac-1), bind to fibrous proteins such as fibronectin, laminin, and collagen, and, not only determine where eosinophils will reside, but likely prolong their survival.126 In this regard, CD11b/CD18 (Mac-1) integrin is also critical for eosinophil effector functions, including degranulation.127
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Detailed Mechanisms of Tissue Recruitment
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Several lines of investigation indicate that eosinophils are recruited to and activated in tissues by Th2 cytokines and also by Th1 cytokines. Mast-cell cytokines, through generation of IL-5 and GM-CSF, likely contribute to prolonged tissue survival and activation of eosinophils. In addition, human natural killer cells, which respond to some of the same CC chemokines as eosinophils, also produce IL-5. Eosinophils, themselves, after recruitment into tissues, function in an autocrine manner, elaborating important inflammatory and regulatory cytokines as reviewed previously.
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Tissue recruitment of eosinophils from circulation occurs in a sequential process beginning with tethering and slow rolling along endothelial cells, followed by firm adhesion which is accompanied by a shape change that is observed as a spreading of the eosinophil on the endothelial cell surface. Next, eosinophils migrate between endothelial cells, traversing the basement membrane barrier. Finally, eosinophils enter the extravascular tissue. Their fate in tissues depends on their activation state and tissue factors. Intuitively, these processes must progress with stepwise cell activation such that the highly active eosinophil transmigrates the vessel without release of oxidants and granule proteins that would damage the vessel.
+++
Eosinophil-Activating Cytokines
++
Eosinophil-activating cytokines can be produced by many cell types in addition to T cells and mast cells, including keratinocytes, endothelial cells, and monocytes, along with eosinophils, themselves. The eosinophil-activating cytokines, IL-3, IL-5, GM-CSF, and others, enhance chemotactic responses, in addition to multiple other effects on eosinophils such as promoting maturation, cell survival, and LT production.128
+++
Specific Studies of Activating Cytokines
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In cytotoxicity assays, eosinophils are maximally activated by GM-CSF, followed by IL-3, IL-5, TNF-α, and IL-4, in order of potency. The role of IL-4 and IL-13 in eosinophil trafficking is mainly indirect; they induce CCL11 expression in a STAT6-dependent signaling pathway providing an explanation for an integrated mechanism that accounts for eosinophil infiltration in Th2 responses,
129 although, in lungs, eosinophil infiltration may be dissociated from IL-13 signaling.
130 Eosinophils from individuals with atopic dermatitis have increased migratory responses in vitro to several common chemotaxins, such as
N-formyl-methionyl-leucyl-phenylalanine, IL-4, and PAF, in comparison to eosinophils from unaffected individuals, indicating that they have been exposed to an activating cytokine in vitro.
112 In fact, IL-5 treatment of eosinophils from normal donors induced the migratory responses observed in eosinophils from atopic dermatitis subjects.
131 Similarly, the kinetics of eosinophil recruitment in response to intradermal CCL5 challenge are profoundly faster in allergic subjects.
132 Eosinophils from atopic dermatitis donors also have prolonged cell survival ex vivo compared with normal controls providing further evidence that they have been primed by activating cytokines such as IL-5 in vitro.
133 Numerous studies have demonstrated the importance of IL-5 for tissue eosinophilia; nevertheless, tissue eosinophilia does occur independent of IL-5 as observed in anti-IL-5-treated asthmatic patients and in IL-5-deficient mice.
128,134,135 Interestingly, the local reactions observed in response to subcutaneous GM-CSF administration given as an adjunct to chemotherapy demonstrate both intact and degranulated eosinophils, suggesting that eosinophil activation by GM-CSF coupled with cutaneous trauma may be sufficient for tissue recruitment.
136 GM-CSF-stimulated eosinophils also induce T-cell proliferation in response to infectious agents, including staphylococcal superantigens (
Staphylococcus enterotoxins A, B, and E). Eosinophil-activating cytokines are important for migration of eosinophils into tissues and also are important for the effector functions of eosinophils (vide infra).
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Endothelial-Activating Cytokines
++
++
During eosinophil migration, at least three types of endothelial activations occur. The first is the expression of p-selectin, which occurs when Weibel–Palade bodies in endothelial cells are transported to the cell surface rapidly after exposure to histamine, LTs, and a host of other inflammatory mediators. Expression of p-selectin on the endothelial-cell surface initiates leukocyte rolling, via CD162 (PSGL-1), which is the important initial step before firm adhesion and transendothelial migration. A second type of endothelial activation is that induced by nonspecific activators such as IL-1 and TNF-α. These cytokines stimulate endothelial expression of e-selectin, ICAM-1, and VCAM-1, to which eosinophils firmly adhere, or “tether.” They also induce production of chemokines by endothelial cells. The third type of endothelial activation is that induced by IL-4 and IL-13. These cytokines selectively induce VCAM-1, which is centrally involved in the recruitment of VLA-4-positive cells, including eosinophils, basophils, and lymphocytes into sites of allergic inflammation.
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The transition from rolling to firm adherence is substantially increased by CCR3 ligands, the CC chemokines. Induction of the expression of chemokines by activated endothelial cells results in higher levels of chemokines on or near the endothelial surface, which transiently affect β1 and β2 integrin avidity, resulting in firm adhesion of the eosinophil to the endothelial cell. However, chemokines produced by structural cells such as fibroblasts, smooth muscle cells, and epithelium probably are more important in directing migration and activation of eosinophils within tissues than those expressed on endothelial cells.137
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Chemokines as Eosinophil Chemoattractants
++

Several members of the CC chemokine gene superfamily are chemotactic for eosinophils;
138 these chemoattractants include CCL3, CCL5 and the eotaxin family, CCL11, CCL24, and CCL26, along with CCL7 (MCP-3), CCL8 (MCP-2), and CCL13 (MCP-4).
139 Eosinophil-active chemokines signal primarily through the CCR3, which is expressed by eosinophils.
140 CCL24 (eotaxin-2) and CCL26 (eotaxin-3) are only distantly related to CCL11 (eotaxin-1), with approximately 30% sequence homology and encoded in different chromosomal locations; nevertheless, their three-dimensional structure allows them to bind to the same receptor. CCL11, CCL24, and CCL26 (eotaxins 1–3) are specifically chemotactic for eosinophils. CCL5 (RANTES) is chemotactic, not only for eosinophils, but also for monocytes, T lymphocytes, natural killer cells, and basophils, but not for neutrophils. CCL3 (MIP-1α) is a chemoattractant to a variety of cells including monocytes, T cells, B cells, and eosinophils and signals through the CCR1 and CCR5 chemokine receptors. In addition to their chemotactic properties, CCL11, CCL24, CCL26, and CCL5 induce production of reactive
oxygen species by eosinophils, indicating that they have both chemotactic and functional activation effects. As eosinophil chemoattractants, the eotaxins are more potent than CCL5, and CCL11 and CCL24 also are more potent in inducing reactive
oxygen species by eosinophils than CCL26 and CCL5. Eotaxins are produced by resident cells in tissues including respiratory and intestinal epithelial cells and dermal fibroblasts; CCL5 is also produced by dermal fibroblasts and keratinocytes, positioning them well for participation in cutaneous inflammation.
141 Allergen-induced infiltrative cells, macrophages, and eosinophils, additionally produce eotaxins.
142 Moreover, eotaxin expression exhibits a cadence; CCL11 is induced early correlating with eosinophil recruitment at 6 hours and CCL24 and CCL26 expression correlates with eosinophil accumulation at 24 hours.
129
++

Negative signaling is important in homeostatic, inflammatory, and repair processes in leukocytes, but little is known about these processes in eosinophils. An inhibitory receptor of the immunoglobulin superfamily, the paired immunoglobulin-like receptor B (PIR-B), is highly expressed by eosinophils. PIR-B-deficient mice have increased gastrointestinal eosinophils, and there is evidence that PIR-B, in a direct and negative manner, regulates eotaxin-dependent eosinophil chemotaxis.
130
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Tissue chemokine expression forms a gradient signal that guides eosinophils into tissue. CCL11 guides eosinophils into tissue locations in which eosinophils are normally present, thymus, uterus, mammary gland, and gastrointestinal tract.143 In Th2 disorders, Th2 cytokines induce chemokine expression. In lungs affected by asthma and eosinophilic pneumonia, CCL11, CCL24, and CCL26 are increased along with other chemokines,144 such as LTB4 and galectin-9.145,146 In the intestinal tract, CCL26 plays a key role in eosinophilic esophagitis, whereas CCL11 is involved in lower gastrointestinal eosinophil disorders. In skin, IL-4, IL-13, and TNF-α stimulate CCL11, CCL24, and CCL26 production from mast cell and lymphocyte sources as well as from fibroblasts (CCL11 and CCL26) and keratinocytes (CCL26).147 As in eosinophilic esophagitis, CCL26 may be important in atopic dermatitis in which serum CCL26 levels correlate with disease activity.148
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Arachidonic acid metabolites, particularly, the cysteinyl LTs, LTC4, LTD4 and LTE4, and PGD2, are involved in eosinophil trafficking as evidenced by the observations that LT receptor antagonists reduce blood and lung eosinophilia and that mice, depleted of LTB4 receptors, have markedly reduced lung eosinophilia after allergen exposure. Eosinophil, basophil, and Th2 cell recruitment occurs, to some extent, through CRTH2 (CD294), the high affinity PGD2 type 2 receptor. Other factors that contribute to eosinophil trafficking are being identified. For example, eosinophils express high levels of histamine 4 receptors that mediate chemoattraction and activation.149 An extracellular matrix protein, periostin, encoded by an IL-13 inducible gene, likely facilitates eosinophil infiltration into tissues by directly affecting eosinophil adhesion; periostin is overexpressed in eosinophilic esophagitis and correlates with eosinophil numbers in biopsies.150
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Activation of Eosinophils
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As reviewed previously, various inflammatory mediators activate eosinophils. In addition to cytokines, TNF-α, GM-CSF, IL-3, and IL-5, these include complement components, C3a and C5a, lipid mediators, LTC4 and PAF, chemokines, as well as engagement of IgA and IgG Fc receptors. CD11b/CD18 (Mac-1)-dependent cellular adhesion is a critical component for degranulation and superoxide production induced by GM-CSF and PAF eosinophil activation and likely is an in vitro mechanism that results from eosinophil contact with stromal cells and/or proteins.151 Members of the CC chemokine subfamily, CCL5, CCL7 (MCP-3), CCL11, CCL13 (MCP-4), CCL24 that bind to the chemokine receptor, CCR3, also potently activate eosinophils. Activated eosinophils develop a number of phenotypic changes, including a reduction in granules, vacuolization, and an expansion of their cytoplasm, leading to a reduction in cell density and are referred to as hypodense. The number of hypodense cells predicts allergic disease severity. A cell surface marker that distinguishes hypodense from normodense eosinophils has not been identified, but there are several surface markers with enhanced expression on in vitro or in vitro activated or hypodense cells: αM integrin (CD11b), αX integrin (CD11c), FcγR111 (CD16), hyaluronic acid receptor (CD44), ICAM-1 (CD54), CD69, and HLA-DR.152
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Upon recruitment and activation in tissues, eosinophils have various effects as detailed in previous sections. In tissues, eosinophils release granule contents into their extracellular space via three mechanisms: (1) piecemeal degranulation, (2) regulated secretion (also referred to as regulated exocytosis), and (3) cytolytic degranulation. First, in piecemeal degranulation, eosinophils selectively release specific granule components; as an example, IFN-γ activation promotes mobilization of granule-derived CCL5 to the eosinophil's surface without inducing cationic protein release.153–155 Second, in regulated secretion, a docking complex forms consisting of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on the vesicle (vSNAREs) and the target membrane (tSNAREs). Two types of SNAREs have been described based on the presence of a conserved amino acid, arginine (R) or glutamine (Q). Human eosinophils express the R-SNARE, vesicle-associated membrane protein (VAMP)-2 on cytoplasmic secretory vesicles, and the Q-SNAREs, SNAP-23, and syntaxin-4, on the plasma membrane.156 VAMP-7 also plays a critical role in both eosinophil and neutrophil mediator release.157 The current understanding is that receptor-induced eosinophil activation leads to rapid mobilization of cytoplasmic vesicles to the plasma membrane, leading to the formation of a SNARE complex (VAMP-2, -7/SNAP-23/syntaxin-4) and mediator release. Third, cytolytic degranulation occurs in many inflammatory diseases including skin disease, such as atopic dermatitis, eosinophilic esophagitis, and lesions found in affected organs with the hypereosinophilic syndromes. It is characterized by organelle rupture, chromatolysis of nuclei with loss of morphologic integrity and identity of eosinophils, and extensive deposition of eosinophil granules and granule products in tissue.71 Therefore, it seems somewhat paradoxical that eosinophils from atopic dermatitis patients have prolonged eosinophil survival and yet exhibit such marked cytolytic degranulation in skin lesions. Clearly, there is much more to learn about eosinophil biology and its relevance to human diseases.
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Pharmacological Manipulation
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Eosinophil-associated disease is a term that, strictly speaking, refers to diseases in which eosinophil numbers or eosinophil granule protein levels (or other eosinophil products) are associated with disease activity. This term encompasses multiple heterogeneous disorders, including skin diseases (see Chapter 36), in which targeting eosinophils and/or their products is a therapeutic goal. Many available treatments reduce eosinophils numbers, thereby inhibiting eosinophilic inflammation, including glucocorticoids, calcineurin inhibitors, IFN-α, IFN-γ, LT antagonists, myelosuppressive/cytotoxic drugs, and, possibly, even antihistamines. However, none is specific for eosinophils. It is only in recent years that selective and direct reduction of eosinophils has been achieved, and these therapies have provided new insight into disease pathogenesis.9
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Among the nonselective drugs for eosinophil reduction, glucocorticoids generally are very effective. The immediate (within 3 hours) reduction in circulating eosinophils observed after systemic administration of glucocorticoids likely occurs as a consequence of sequestration into extramedullary organs (liver, spleen, and lymph node), as has been shown in rodents. Glucocorticoids affect eosinophil infiltration into tissues by four mechanisms: (1) sequestration into lymphoid tissues, (2) induction of eosinophil apoptosis, (3) reduction of eosinophil production by bone marrow, and (4) alterations in the production of the cytokines/chemokines important in eosinophil trafficking.158–160 Glucocorticoids suppress the production of several cytokines important for the induction of adhesion molecules on endothelial cells, including IL-1, TNF-α, IL-4, and IL-13, and the release of eosinophil-active chemokines, including CCL5, CCL7, and CCL11. Unfortunately, “steroid resistance” develops in some patients, and long-term administration of glucocorticoids is associated with limiting side effects. The mechanism of glucocorticoid resistance is unclear but, in part, may be explained by decreased numbers of glucocorticoid receptors, and polymorphisms and alterations in transcription factor activator protein-1 (AP-1).161 Patients who have or who develop glucocorticoid resistance require other therapies. Interestingly, lidocaine and sulfonylureas (such as glyburide) also inhibit cytokine-induced eosinophil survival with glucocorticomimetic effects and may have antieosinophilic effects clinically.162–164
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Calcineurin antagonists, such as cyclosporine, tacrolimus, and pimecrolimus, broadly inhibit T-cell cytokine release including those that specifically induce eosinophilic inflammation (IL-4, IL-5, and GM-CSF). They also decrease the expression of CCL5, CCL11, and IL-5 with associated decreased tissue eosinophilia as has been shown in atopic dermatitis.165 Mammalian target of rapamycin (mTOR) inhibitors, including rapamycin, have direct effects on eosinophils, decreasing eosinophil granule protein release after IL-5 activation.166 The use of these therapeutic agents are limited by their side effects including immunosuppression, as well as other metabolic effects that may be in part genetically determined.167
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Several myelosuppressive drugs, including hydroxyurea, vincristine sulfate, cyclophosphamide, methotrexate, 6-thioguanine, 2-chlorodeoxyadenosine and cytarabine combination therapy, pulsed chlorambucil, and etoposide, may be beneficial in eosinophil-associated disease alone or as steroid-sparing agents. Hydroxyurea has been particularly effective in decreasing circulating eosinophil numbers.
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In myeloproliferative hypereosinophilic syndrome (chronic eosinophilic leukemia) with the FIP1L1-PDGFRA mutation that codes for a tyrosine kinase, imatinib mesylate, a tyrosine kinase inhibitor, is approved for the treatment of chronic myelogenous leukemia and the hypereosinophilic syndrome and has produced rapid, complete or near complete remissions.168 Patients who have features of myeloproliferative HES but who lack FIP1L1-PDGFRA still may respond to imatinib (see Chapter 36).169
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Alemtuzumab is a monoclonal antibody to CD52 that is used to deplete CD52+ lymphocytes in the treatment of chronic (B-cell) lymphocytic leukemia and T-cell lymphoma. Eosinophils, but not neutrophils, also express CD52, and alemtuzumab has been useful in treating patients with refractory hypereosinophilic syndrome, including those with abnormal T cells,170–172 but has serious limiting side effects from cytopenias, infusion reactions and infections.
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Interferons, both IFN-α and IFN-γ, may be therapeutically beneficial in eosinophil-associated disease by inhibiting eosinophil degranulation and inflammatory mediator release. IFN-α may be better tolerated than IFN-γ and is used as a steroid-sparing agent predominantly in patients with lymphocytic variant hypereosinophilic syndrome, but also may be useful in myeloproliferative variant hypereosinophilic syndrome (see Chapter 36).173,174
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Other Therapeutic Agents
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Other therapeutic agents interfere with products of eosinophil activation, the process of eosinophil activation and/or eosinophil recruitment, and, therefore, alter disease expression. The cysteinyl LTs elaborated by eosinophils are mediators of asthma and allergic rhinitis. LT antagonists, including the 5-lipoxygenase inhibitor,
zileuton, and the LT receptor antagonists,
zafirlukast and
montelukast, decrease tissue eosinophils and their biological effects in allergic individuals and improve clinical symptoms.
175 In eosinophilic pustular folliculitis, nonsteroidal anti-inflammatory drugs, particularly
indomethacin, are often first-line therapy; clinical improvement may be observed within 2 weeks and is associated with a decrease in peripheral blood eosinophil counts.
176–178 A mechanism for this has been proposed based on the observation that
indomethacin not only inhibits cyclooxygenases and subsequent prostaglandin D2 synthesis, but also is associated with reduction in the prostaglandin D2 receptor (chemoattractant receptor homologous molecule expressed on Th2 cells, CRTH2) on eosinophils and lymphocytes.
179 Cromoglycate and
nedocromil inhibit effector functions of eosinophils such as antibody-dependent cytotoxicity.
180 Second-generation H1 blocking agents, such as
cetirizine and
fexofenadine, inhibit eosinophil accumulation in allergen challenge models of the skin or airways.
181–183 These other therapies may augment or synergize the beneficial effects of more toxic medications, such as glucocorticoids, calcineurin inhibitors, or myelosuppressants, to reduce the dose needed for effective management.
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New, more specific eosinophil-directed therapeutic strategies are emerging.
2,3,9 Based on the importance of IL-5 as a cytokine that directly affects eosinophil proliferation, differentiation, and activation, two humanized monoclonal antibodies to IL-5 have been developed, (1)
mepolizumab and (2)
reslizumab. These antibodies decrease peripheral blood and tissue eosinophils with clinical benefit and relative safety in patients with eosinophil-associated diseases, including hypereosinophilic syndromes, Churg–Strauss syndrome, and asthma.
16,184–188 Notably, although eosinophil numbers were strikingly reduced,
mepolizumab did not reduce deposition of eosinophil granule proteins in tissue.
134 Keeping in mind that eosinophil granule proteins are readily deposited with eosinophil activation and persist in tissues for weeks, more robust and long-term suppression of eosinophils may be needed.
72 With awareness that IL-5 critically mediates its effects on eosinophils through the
receptor for IL-5, a humanized anti-IL5Rα monoclonal antibody was developed (MEDI-563) and is in clinical trials.
189 This antibody binds with high affinity to and mediates the lysis of IL5Rα+ cells, including eosinophils and basophils, through antibody-dependent cytotoxicity. A single, well-tolerated intravenous dose of the antibody decreased circulating eosinophil counts to less than detection limits within 2 days for up to 12 weeks. With the key role that CCR3 and its ligand, CCL11, have in eosinophil chemotaxis, agents that interfere with interactions of IL-13, CCL11, and CCR3 have a strong likelihood of being effective therapeutically.
190 IL-13, CCL11, and CCR3-blocking antibodies, along with small molecule inhibitors of CCR3 are in development.
191 Agents that block eosinophil adhesion to endothelium through the interactions of CD18 (β
2 integrin) and ICAM-1 or between VLA-4 and VCAM-1 also may prove useful. Other molecules implicated in tissue eosinophilia identified through studies of deficient mice or with antagonists as potential treatment pursuits are H4 histamine receptor and thymic stromal lymphopoietin (TSLP).
192–194 Fingolimod (FTY720), recently approved for treatment of multiple sclerosis, is a structural analog of sphingosine which, when phosphorylated, occupies one of the five sphingosine-1-phosphate receptors involved in immune-cell trafficking. In mice, it inhibits eosinophil egress from bone marrow with subsequent decreased tissue eosinophils and inhibition of hapten-induced dermatitis, and, although attended with significant side effects, may be useful in human eosinophil-associated diseases.
195,196 Eosinophil death can result from withdrawal or blocking of survival factors, and also by activation of proapoptotic pathways via death factors. Antibody blocking of CD48, an activation molecule on eosinophils, decreased lung eosinophil infiltration.
118,197 Recent observations suggest a role for cell surface death receptors and mitochondria in facilitating eosinophil apoptosis, although the mechanisms that trigger each of these death pathways remain obscure. A recently identified eosinophil surface marker that may prove to be a therapeutic target is the apoptosis factor, Siglec-8. Siglec-8 is a sialic acid binding lectin family member that contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that induce apoptosis when cross-linked.
198 Therefore, the control of eosinophil apoptosis may become a strategy for treatment.
199 Although several of these eosinophil surface molecular targets, such as Siglec-8, CCR3, and CRTH2, are not eosinophil-specific, they are expressed on other cells such as basophils, mast cells, and Th2 cells, so that multiple aspects of allergic reactivity and other eosinophil-associated inflammation could be therapeutically managed.
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The eosinophil is a multifunctional cell, the prototype of which was present as reptiles evolved, and it has been retained and refined through mammalian evolution. It is likely an active player in defense against parasitic infections. Recent evidence suggests that it has a much broader role in immune responses, both innate and acquired, with potent antibacterial and antiviral activities. Along the way, the eosinophil may have evolved to respond to other environmental insults and exposures, leading to the rise of eosinophilic diseases such as atopy.
61 Eosinophils seemingly play an effector role not only in allergic diseases, but in fibrotic disorders and antitumor host responses as well. How and why these functions emerged remains a mystery. Only recently has it been recognized that eosinophils are present under “normal” conditions in lymphoid organs and in major parts of the gastrointestinal tract, suggesting that it has homeostatic functions as well. Understanding the relevant inflammatory versus homeostatic functions of this leukocyte will likely yield therapies for eosinophil-associated diseases with improved risk/benefit ratios.