Redwan Moqbel
Solomon O. (Wole) Odemuyiwa
Paige Lacy
Darryl J. Adamko
The eosinophil was first described for its characteristic intracytoplasmic granules that exhibit a high affinity for the negatively charged dye eosin. Although rare in healthy individuals, the eosinophil is prominent in peripheral blood and tissue in association with various disease conditions including allergy (1,2,3), inflammatory responses against metazoan helminthic parasites (4,5) and certain skin and malignant conditions. The eosinophil has received special attention for its potential pathophysiologic role in the manifestation of allergic diseases such as asthma, rhinitis, eczema, eosinophilic esophagitis, and Crohn disease. Disorders of the respiratory tract, particularly allergic asthma and rhinitis, exhibit a strong correlation with the number as well as activation status of infiltrating tissue eosinophils. Similarly, many disorders of the gastrointestinal system exhibit prominent eosinophilic inflammation in the mucosa. The presence of eosinophils in the airway and gut mucosa has been associated with both allergic (immunoglobulin [IgE] IgE-dependent) and nonallergic (IgE-independent) manifestations of disease. Although clinically these conditions have been characterized as either allergic or nonallergic, it appears that the mechanisms underlying recruitment and activation of eosinophils in both types of disease are similar. Despite some difficulties in defining the exact immunologic role of the eosinophil in disease, there is evidence that the eosinophil remains a major effector cell in many types of allergic and nonallergic inflammation.
Eosinophils are mobile, terminally differentiated granulocytes that arise principally from the bone marrow (6). They are approximately 8 μm in diameter and their nuclei are usually bilobed, although three or more lobes are also often observed. The eosinophil is characterized by large crystalloid granules, also known as secondary or specific granules, as shown in light microscopy by their bright red staining properties with acidic dyes such as eosin (Fig. 10.1). As apparent in electron micrographs, the crystalloid granules contain electron-dense crystalline cores surrounded by an electron-lucent granule matrix (Fig. 10.2). Eosinophils contain up to four other “granule” types: Primary granules, small granules, lipid bodies, and small secretory vesicles. Crystalloid granules are membrane bound and contain a number of highly cationic basic proteins (see “Granule-Derived Proteins,” below). The latter have been implicated in the tissue damage observed in asthma and other similar allergic conditions. Allergen- and parasite-induced eosinophilia have been shown to be T cell dependent, and are mediated by soluble factors (cytokines) released from sensitized lymphocytes (7). Recent advances in human eosinophil research have also indicated that eosinophil infiltration into the tissue in allergic-type responses and asthma is regulated by a series of biologic events that includes a complex interplay between immunologic and inflammatory mechanisms including cytokines and chemokines (8,9).
Eosinophil Differentiation
Peripheral blood and tissue eosinophils are derived by hemopoiesis from CD34+ myelocytic progenitors found in the bone marrow and in inflamed tissues. Eosinophils make up approximately 3% of the bone marrow from healthy individuals, of which 37% are fully differentiated, and the remainder are promyelocytes/ myelocytes and metamyelocytes (6,10). The appearance of newly matured cells in the blood occurs approximately 2.5 days from the time of the last mitotic division (6). The turnover of eosinophils is approximately 2.2 × 108 cells/kg/day, and the bone marrow possesses the largest end-differentiated eosinophil reservoir in the healthy body (9 to 14 × 108 cells/kg) (11). Progenitors differentiate upon exposure to a network of cytokines and chemokines to become committed to the eosinophil/basophil (Eo/B) lineage (12). Eosinophils are more closely related to basophils than neutrophils and monocytes due to lineage differentiation at this stage (13). In addition, eosinophils retain elements of expression of basophil/mast cell–specific high-affinity Fc∊ receptor (α subunit) (14), while basophils continue expression of low concentrations of eosinophil major basic protein (MBP) (15). Cytokines and chemokines are soluble factors generated under appropriate stimulation from T cells in the bone marrow. The three key cytokines that are critical for stimulation of bone marrow production of eosinophils are interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (16,17). These three cytokines are also produced by CD4+ and CD8+ T lymphocytes from peripheral blood as well as inflamed tissues (18). In bone marrow samples, committed eosinophil precursors can be recognized by their expression of the IL-5 receptor (IL-5R) and the C-C chemokine receptor, CCR3, in addition to CD34 (19). It is now well recognized that IL-5 is a key cytokine in terminal differentiation of eosinophils (20), and expression of the IL-5R on the progenitor cell is one of the first signs of commitment to the eosinophil lineage. The expression of IL-5R is almost exclusively limited to eosinophil progenitors and mature peripheral blood eosinophils, with some expression on basophils but not neutrophils or monocytes. This selectivity in receptor distribution indicates that IL-5 acts primarily as an eosinophilopoietic cytokine. This has resulted in several groups proposing that inhibition of IL-5 with anti–IL-5 antibody therapy will result in the complete loss of eosinophils from the body, thus preventing the manifestation of allergic symptoms. The obligatory role of IL-5 in the differentiation of the eosinophil has been confirmed by numerous studies on transgenic mice in which the expression of the gene for IL-5 caused marked eosinophilia and increased numbers of eosinophil precursors in their bone marrow (21,22). Interestingly, eosinophil differentiation in this transgenic model appeared to be completely indepen-dent of IL-3 and GM-CSF, suggesting that IL-5 alone may be sufficient to generate an eosinophilia from stem cell precursors. However, although IL-5 gene-deficient mice exhibit almost no eosinophils in their blood, a small pool of apparently IL-5–independent eosinophils persist in the mucosal tissues of these animals. Tissue eosinophils appear to sustain their survival through autocrine release of GM-CSF. Additional eosinophilopoietic factors may assist in inducing the differentiation of Eo/B progenitors in the bone marrow, including IL-4, IL-6, IL-11, IL-12, stem cell factor (SCF), and others (23). C-C chemokines, named for their adjacent cysteine residues in the C-terminus amino acid sequence as distinct from the CXC chemokines, include eotaxin and RANTES (regulated upon activation, normal T-cell expressed and secreted), which have also been shown to be important in the development of eosinophils (24). Overall, at the level of the bone marrow, the early development of Eo/B progenitors is driven by IL-3 and GM-CSF, among other factors, while at later stages, IL-5 regulates the terminal differentiation of eosinophils. Eotaxin may facilitate the efflux of fully mature eosinophils into the peripheral circulation.
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Figure 10.1. Photomicrograph of a peripheral blood eosinophil stained with May-Grünwald-Giemsa. |
The half-life of eosinophils in the circulation is approximately 18 hours with a mean blood transit time of 26 hours (25), although this is extended in eosinophilic conditions, possibly due to the elevation of systemic eosinophil-activating cytokines that promote eosinophil survival. Based on a study of 740 medical students, the normal range of blood eosinophils was shown to be between 0 and 0.5 × 109/L, with counts ranging from 0.015 to 0.65 × 109/L (26). Circulating eosinophil counts exhibit diurnal variation in humans, in which the lowest and highest levels are seen in the morning and evening, respectively, often exhibiting more than 40% variation within a day (27,28). Mild eosinophilia is generally considered to be 0.5 to 1.5 × 109/L, moderate eosinophilia as 1.5 to 5.0 × 109/L, and marked eosinophilia >5.0 × 109/L. Allergy is commonly associated with eosinophilia in the mild range, whereas parasitic infestation is often characterized by a marked eosinophilia.
Eosinophils are predominantly tissue cells, and their major target organs for homing in the healthy individual is the gastrointestinal tract. In states of disease, eosinophils also home to the lungs, the skin, and the brain (e.g., during strokes). Once they enter target tissues, eosinophils do not return to the blood circulation. Tissue eosinophil numbers can remain high in tissues even when peripheral numbers are low, suggesting that their survival is enhanced upon extravasation. Curiously, pathogen-free laboratory animals have no eosinophils in their blood, while tissue eosinophils are difficult to find, suggesting that the appearance of eosinophils may be disease related (10).
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Figure 10.2. Electron photomicrographs of peripheral blood eosinophils from buffy-coat. (Original magnification 7,655× and 22,000× courtesy of Dr. G.E. Quinonez, Department of Pathology, University of Manitoba.) |
Eosinophil Production and Survival in Peripheral Tissue
Eosinophil development and maturation may also occur in situ in peripheral (extramedullary) sites outside of the bone marrow. In this case, Eo/B precursors are released into the bloodstream directly from the bone marrow to circulate to sites where they specifically transmigrate in response to locally produced cytokines and chemokines. This may provide an alternative mechanism for the persistence or accumulation of tissue eosinophils. Like neutrophils, eosinophils are end-stage cells, which, in culture, rapidly undergo cell death by either apoptosis or necrosis. However, eosinophil-active cytokines, such as IL-3, IL-5, and GM-CSF, as well as interferon-γ (IFN-γ), prolong eosinophil survival in culture for up to 2 weeks (29,30,31). They also enhance receptor expression as well as cell function including cytotoxicity against metazoan targets and mediator release. Activated eosinophils can generate a number of cytokines themselves in vitro. This may lead to autocrine prolongation of eosinophil maturation and survival in tissues (32). Local tissue types such as endothelial cells, fibroblasts, and epithelial cells may also contribute to the production of IL-5 and GM-CSF for in situ eosinophil maturation and differentiation in airway or gut mucosa.
Extracellular matrix proteins have been shown to modulate eosinophil response to physiologic soluble stimuli (33). Eosinophils were shown to adhere specifically to fibronectin (34), an abundant extracellular matrix protein, and VLA-4, a known receptor for fibronectin (35), was involved in mediating eosinophil/fibronectin interactions (34). Similarly, VLA-6 expressed on eosinophils was shown to interact with the connective tissue protein laminin.
IL-5 delays eosinophil apoptosis and promotes eosinophil priming and activation (36). IL-5 production by airway CD4+ T cells may be directly stimulated by eosinophils in a paracrine manner to enhance survival of tissue eosinophils (37). Eosinophil progenitors in nasal explants from atopic patients have been shown to survive and develop into fully mature eosinophils ex vivo using similar mechanisms (38). Allergen challenge of these explants, as well as lung explants of Brown-Norway rats, was shown to evoke a rapid (6-hour) accumulation of MBP-positive cells after allergen challenge (39). This was shown to be depen-dent on IL-5 production within the explant, a key cytokine in eosinophil survival.
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Figure 10.3. Signaling pathway leading from binding of interleukin-5 (IL-5) to its receptor in the membrane to transcriptional activation in the cell nucleus via the Ras-Raf1-MEK-ERK pathway. The β subunit of the receptor is also able to activate the Jak2-STAT1 pathway. Transcriptional activation is proposed to generate antiapoptotic effects in eosinophils. |
The IL-5R consists of two subunits, an α subunit of 60 to 80 kDa, and a common βc subunit of between 120 and 140 kDa, which is shared with IL-3R and GM-CSF receptor. IL-5 interacts with its α subunit specifically but at a lower affinity than the βc subunit (40). IL-5 stimulation through the βc subunit leads to phosphorylation of the tyrosine kinases Jak2, Lyn, and Syk. While Jak2 signals through the nuclear translocation factor STAT1, Lyn and Syk signal through the mitogenic Ras-Raf1-MEK-ERK pathway (Fig. 10.3). Tyrosine phosphorylation enhances the expression of the antiapoptotic protein Bcl-xL in eosinophils, and decreases translocation of the proapoptotic signaling molecule Bax, resulting in decreased activation of apoptotic signaling through the caspase family (41,42). GM-CSF prolongs the survival of eosinophils bound to tissue sites via α4integrin for up to 2 weeks (34), and has also been shown to inhibit eosinophil apoptosis similarly to IL-5. Thus, the growth, maturation, and prolongation of survival of eosinophils in extramedullary tissues may occur in sites other than the bone marrow.
Eosinophil Heterogeneity
Human peripheral blood eosinophils exhibit marked heterogeneity based on their physical, morphologic, and functional properties. There are three different populations of eosinophils that can be characterized based on their intrinsic buoyant density and responsiveness to stimuli. These are the normodense, hypodense, and primed eosinophils, which can be described in both normal and eosinophilic subjects. Each of these populations responds differently to stimuli, which may be related to their stage of maturation. In addition, they may derive from distinct pools of eosinophils that are genetically regulated. The majority of blood eosinophils (>90%) from normal individuals are normodense, which separate out from other leukocytes in the lower interfaces of Percoll or metrizamide discontinuous density gradients. Hypodense eosinophils can be seen in a proportion of eosinophils from individuals with a raised eosinophil count that exhibit lower density than eosinophils from normal subjects, resulting in a spread of eosinophil populations in the gradient, with contaminating mononuclear cell bands (43). Morphologically, hypodense eosinophils appear vacuolated, contain more lipid bodies, express less MBP, and possess smaller-size crystalloid granules that appear to be slightly more lucent and take up less cell volume, although these are of equal numbers to normodense eosinophils (44). They also exhibit a greater cell volume than normodense eosinophils (45). The mechanism for this heterogeneity is not clear. The presence of low density (or hypodense) eosinophils appears to be a nonspecific phenomenon, which occurs in any eosinophilic condition including parasitosis, asthma, allergic rhinitis, idiopathic hypereosinophilic syndrome, and certain malignancies. It was originally thought that the numbers of hypodense cells correlated with the degree of eosinophilia, although this has not been consistently observed (43,46,47,48,49). Thus, the mechanisms governing the production of hypodense eosinophils are likely to be distinct from those that control eosinopoiesis.
Functionally, hypodense eosinophils appear to be more activated, since they exhibit elevated oxygen consumption (43) and increased cytotoxicity toward helminthic targets (46) and release more LTC4after physiologic stimulation (50). Activation of eosinophils in vitro with inflammatory mediators such as platelet-activating factor (PAF), as well as long-term culture with cytokines (e.g., IL-3, IL-5, and GM-CSF), has been associated with a decrease in eosinophil density (29,51). Two possible explanations may account for the enhanced responsiveness of hypodense eosinophils. The first is that hypodense eosinophils frequently comigrate to the same density as neutrophils in metrizamide or Percoll gradients, thus making it difficult to separate these two cell types. Neutrophils could, therefore, enhance the responsiveness of eosinophils through cell–cell interaction. For example, total LTC4 produced by a mixture of eosinophils and neutrophils was found to be greater than the amount produced by either cell type alone (52). However, other studies assessing the possibility of neutrophils enhancing eosinophil responsiveness have been negative (53,54). Secondly, hypodense eosinophils have been shown to express a greater number of receptors for IgG, IgE, CD44, complement, and the p55 subunit of the IL-2 receptor when compared with normodense cells (43,55,56,57), which may explain their enhanced responsiveness to stimuli. However, the surface expression of numerous other receptors does not differ between normodense and hypodense eosinophils, with some populations (e.g., CD18) even showing decreased expression in hypodense cells (58). Furthermore, normodense eosinophils from patients with an eosinophilia have enhanced effector function compared with eosinophils from normal individuals. It is possible, therefore, that the formation of low-density eosinophils results from the migration of normodense eosinophils from the bone marrow to the circulation, whereupon they become activated by elevated systemic factors. Another scenario may be that the association between hypodensity and activation is coincidental, with the less dense cells being immature.
Eosinophil Tissue Accumulation
Eosinophils migrate to the gastrointestinal tract during their normal development (59,60), and possibly in response to environmental factors as part of a role in innate defense against parasites.
The mechanisms involved in the selective tissue recruitment of eosinophils across the vascular endothelium and into tissues in allergic reactions occur sequentially in four well-defined steps: (a) the tethering of the eosinophil to the luminal surface of the vascular endothelium during normal transport through the blood vessel, (b) rolling of the eosinophil along the luminal surface of the activated endothelium in a reversible manner, (c) firm adhesion of the eosinophil to endothelial cells, and (d) transmigration of the eosinophil through the endothelium into target tissues (Fig. 10.4). A further, less understood, step in eosinophil trafficking in the tissues is the in situ differentiation of circulating committed Eo/B precursors. Most migration through endothelium occurs at postcapillary venules. Each of these steps is controlled by a complex network of chemotactic factors and adhesion molecules, which collectively direct the movement of the eosinophil into the tissues. For eosinophils, selectins and α4 integrins are thought to be important in tethering and rolling, while α4 and β2 (CD18) integrins mediate firm adhesion. The transmigration step is primarily regulated by β2 integrins as well as C-C chemokines such as eotaxin. Cytokines and chemokines are elaborated by surrounding tissues to modulate the transmigration of eosinophils into tissues. Many of these mechanisms appear to be controlled at the level of the T-cell response to antigen (allergen)-presenting cells and the subsequent release of cytokines and chemokines, which in turn regulate the activity of eosinophils.
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Figure 10.4. Eosinophil tethering, rolling, adhesion, transmigration, and chemotaxis in response to inflammatory signals in tissues. During chemotaxis, eosinophils may either become activated in response to local inflammation and release mediators, as in asthma and other related conditions, or accumulate in tissues in the apparent absence of mediator release. |
Tethering and Rolling
Our knowledge of the mechanisms involved in eosinophil interactions with the endothelium extends primarily from in vitro assays of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVECs) both in stable and under flow conditions. Antibodies specific for adhesion molecules have been applied in this system and have identified critical regulatory molecules required for adhesion and transmigration of eosinophils. Tethering and rolling of eosinophils on HUVECs under flow conditions is regulated by selectins (L-selectin [CD62L]) expressed on the eosinophil surface interacting with E- and P-selectins (CD62E and CD62P) on endothelial cells (61). Selectins are characterized by a lectin-binding domain, which is involved in the initial anchoring of inflammatory cells to the venular endothelium. This interaction is enhanced after the release of inflammatory mediators from these cells as well as neighboring tissues. Once tethered, eosinophils roll until they become stimulated by a chemoattractant stimulus (indicating local inflammation), which induces activation of α4 integrin receptors on the leukocyte. In addition, rolling appears to facilitate the subsequent adherence and transmigration of eosinophils into tissues. Eosinophils also express PSGL-1 and the integrins α4β1 (VLA-4) and α4β7, which are involved in cell rolling (61). Eosinophil integrins bind to target sites in the endothelium, primarily ICAM-1 (CD54) and VCAM-1 (CD106), through their Mac-1 (a β2 integrin, also known as CR3 or CD11b/CD18) and VLA-4 (α4β1 integrin) receptors, respectively. The constitutive expression of VLA-4 (α4β1 integrin) is limited to a small number of leukocytes, including eosinophils, monocytes, basophils, and T cells, suggesting that regulated expression of its ligand, VCAM-1, on endothelial cells may be important in selective recruitment of these cells (62).
Adhesion
The firm adhesion of eosinophils involves the interaction of α4 and β2 integrins with the endothelial layer. Specifically, eosinophils adhere to tumor necrosis factor-α (TNF-α)-, IL-1β-, and LPS-activated HUVECs through CR3/ICAM-1 and VLA-4/VCAM-1 interactions (62,63,64,65,66). Other adhesion molecules that may contribute to this process are LFA-1, VLA-6 (α4β1), α4β7 integrin, p150,95, and CD11d. Eosinophils exhibit differential binding properties through VCAM-1 and ICAM-1, which are dependent on their activation status. Freshly prepared unstimulated eosinophils preferentially bind to endothelial VCAM-1 via VLA-4 (α4β1) rather than β2 to ICAM-1 (24,67). Once activated, eosinophil preference for VCAM-1 shifts to that of endothelial ICAM-1 via β2 integrins (24,68,69,70). During extravasation (diapedesis) into tissues, the eosinophil becomes progressively more activated upon contact with extracellular matrix proteins and other stimulated cells. Tissue eosinophils from an antigen challenge model express increased CD11, CD69, and ICAM-1 (71). Eosinophil binding in tissues switches to ICAM-1 and the CS-1 region of tissue fibronectin (61,68,72). The change in the activation status is also confirmed by the changes in the expression of cell surface molecules seen as the eosinophil goes through tissue. Eosinophils recovered from bronchoalveolar lavage (BAL) express increased ICAM-1, Mac-1, and CD69 and decreased L-selectin, suggesting an activated state (42).
Cytokines such as IL-4 and IL-13 have been shown to up-regulate eosinophil adhesion, primarily through up-regulation of VCAM-1 on endothelial cells (73,74). The effects of IL-4 and IL-13 are mediated through Jak3 and the nuclear transcription factor STAT-6 (75,76). Interestingly, a decrease in tissue eosinophilia has been observed in allergen-challenged STAT-6-/- mice, in spite of high levels of VCAM-1 expression (76). This difference was thought to be due to decreased expression of CCR3 in eosinophils, which is directly controlled by STAT-6 (77). Results from STAT-6-/- mice would suggest that IL-4 and IL-13 also have a role in the induction of CCR3 on eosinophils and T cells. These findings underline the importance of cytokine and chemokine cross-talk in the generation of blood eosinophilia and tissue diapedesis.
The switch to ICAM-1–mediated adhesion and transmigration may be associated with facilitation of eosinophil entrance into the tissue. Increased β1 expression (VCAM associated) has been shown to slow eosinophil migration compared with ICAM-1/β2 (78). It is important to note that anti–VLA-4 antibodies may not prevent eosinophil migration into tissue if ICAM-1 or P-selectin sites are the first targets for activated eosinophils (61).
IL-5 also up-regulates eosinophil, but not neutrophil, adhesion to unstimulated endothelium, offering a selective pathway of eosinophil adhesion (64). IL-5 has been shown to activate transendothelial migration of eosinophils through ICAM-1 via decreased β1 and increased β2 integrin expression (79). Similarly, stimulation of eosinophil CCR3 with a chemokine such as eotaxin, which can be released from endothelial cells, also increases β2 integrin expression, resulting in preferential binding to ICAM-1 (80). Thus, numerous cytokines and chemokines have been shown to enhance eosinophil adhesion to endothelium.
Complement proteins are also important in eosinophilic trafficking in tissues. Complement-mediated inflammation, as seen with parasite infection, is associated with the release of C3a and C5a. While C3a increases binding of eosinophil to endothelium but does not increase migration, C5a increases both adhesion and migration (81). VCAM-1 and ICAM-1 are involved in complement-mediated binding and migration of eosinophils, as this process is blocked by the application of anti-α4 and β2 antibodies. These findings illustrate the importance of adhesion molecules VCAM-1 and ICAM-1 in the complement-mediated pathway of anaphylaxis and host defense.
Other more general inflammatory cytokines, such as IL-1 and TNF-α, are also released by inflamed tissues and have significant effects on eosinophil migration (61). Messages encoding both IL-1 and TNF-α are increased in the airways of symptomatic as opposed to nonsymptomatic asthmatics (82), and IL-1 is increased in tissues from sites of cutaneous allergy (83). Antibodies to IL-1 have been shown to decrease the expression of VCAM-1 and ICAM-1 in endothelial cells (84). Mice deficient in IL-1 expression (IL-1-/-) have decreased eosinophil rolling, adhesion, and transmigration (85). TNF-α has also been shown to increase expression of endothelial ICAM-1, VCAM-1, P-selectin, and E-selectin, causing increased eosinophil rolling and adhesion (86,87,88). In addition, TNF-/- mice show decreased eosinophil adhesion and migration into tissue, similar to IL-1-/- mice (89). These factors may have important roles in allergic asthma where preferential accumulation of eosinophils is a feature of atopic (IgE-dependent) inflammatory conditions.
Transmigration and Chemotaxis
Once eosinophils adhere to vascular endothelium, they commence diapedesis whereby they emerge out of the capillaries and traverse the adjacent connective tissue en route to the focus of the inflammatory response. Eosinophils move through the endothelium by extending lamellipodia in the form of a uropod, thus leading to lamellar motion (61). For cells to move, there must be increased binding forward via the uropod and release of binding to the rear. Changes in the binding affinity for adhesion molecules and extracellular matrix proteins are thought to contribute to cell movement on a substratum. A gradient in binding affinity of eosinophil VLA-4 to fibronectin has been demonstrated (90), where increased adherence at the leading edge of the cell is followed by deadherence at the rear of the cell, allowing the cell to move on. Cytokines and chemokines also influence the binding of eosinophils to tissue surfaces, such as GM-CSF, which increases the binding affinity of VLA-4 to VCAM-1 or CS-1 (91), and eotaxin, which stimulates the reverse reaction (80). In addition, eotaxin may induce cytoskeletal changes via mitogen-activated protein kinases (MAPKs) (61). Other chemokines or chemotactic factors, such as RANTES, MCP-3, and C5a, may also alter β1 integrin affinity (68,92). The balance of these factors determines the rate of eosinophil migration.
Although cytokines (e.g., IL-3, IL-5, and GM-CSF) are essential for the development and proliferation of eosinophils, they are likely to play an immunomodulatory role in priming eosinophils for better chemotactic responses to target tissue sites. The most potent eosinophil chemoattractants include PAF, LTD4, C5a, IL-2, and C-C chemokines such as eotaxin and RANTES (93,94). C-C chemokines appear to be essential for inducing the specific migration of eosinophils to inflamed sites. Several distinct families of chemokines have been identified, and the CCR3-binding family in particular plays a crucial role in generating tissue eosinophilia due to the nearly exclusive expression of CCR3 in eosinophils (24). This family of chemokines consists of eotaxin (1,2,3); RANTES; monocyte chemoattractant protein (MCP) 2, 3, and 5; and macrophage inhibitory protein (MIP)-1α. Chemokines binding CCR3 may be selective for granulocytes such as eosinophils and basophils, as neutrophils do not express this receptor. Eotaxin is the only chemokine specific to eosinophils, making it a key member of the CCR3 family (93,95). CCR3 chemokines are produced by endothelial cells, epithelial cells, parasympathetic nerves, T cells, macrophages, fibroblasts, and eosinophils, among other tissue sources (32,96,97).
Basal expression of eotaxin in the gut is elevated compared with other tissues in the normal animal (98). During allergen-induced eosinophilia, eotaxin expression is further increased within tissues (99). Some synergism exists between IL-5 and eotaxin, as IL-5 stimulation enhances the eosinophil response to eotaxin both in vitro and in vivo (100,101). In order to define the specific role of eotaxin in inflammation, eotaxin gene knockout (Eo-/-) mice have been deployed (59,102). These mice produce IL-5 normally, and thus continue to develop blood eosinophilia similar to their wild-type heterozygotes. However, Eo-/- mice do not develop tissue eosinophilia. Thus, the primary role of CCR3 appears to be involved in the homing of circulating eosinophils to target tissues expressing eotaxin.
Additional chemokines of the CCR3 family have been shown to exert important effects in situations where eotaxin may not be necessarily essential to the response (61,103). Each chemokine appears to have a unique role in the timing and location of tissue eosinophilia. Peripheral blood levels and cultured mononuclear cells from patients with allergic dermatitis produce increased levels of RANTES, MCP-1, and MIP-1α compared with nonallergic controls (104). Similarly to eotaxin, IL-5–stimulated eosinophils have an increased affinity for RANTES. However, unlike eotaxin, RANTES was specifically associated with exacerbations of eosinophilic bronchitis, thought to be provoked by viral infection. Infections with respiratory syncytial virus (RSV) leading to eosinophilia have been correlated with increased RANTES, MCP-1, and MIP-1α expression (105,106). Children with asthma have large increases in eosinophil-associated MBP, RANTES, and MIP-1α in their nasal secretions during naturally acquired viral infections (107). Therefore, the apparently broader range of effects of RANTES, MIP-1α, and MCP-1 may also increase the range of eosinophil activity in disease, even though all of these bind specifically to CCR3 on eosinophils.
Other factors are also produced in mucosal tissues, which are moderately or strongly chemotactic for eosinophils. These include bacterial products (e.g., endotoxin and the tripeptide f-Met-Leu-Phe [fMLF]), the anaphylatoxin complement factor C5a, opsonized particles (which exert their effect via complement [CR1, CR3] and FcγRII receptors), and other cytokines (IL-4, IL-8, and possibly IL-13). In addition, the lipid-derived mediators, leukotriene B4 (LTB4) and PAF, which are elevated in allergic responses and induce eosinophil respiratory burst and degranulation at higher doses (23,33,108,109), are also eosinophilotactic. Eosinophil cytokines IL-3, IL-5, and GM-CSF are able to enhance the chemotactic ability of each of these factors. Although PAF antagonists are not sufficient at preventing eosinophilic inflammation in allergy, treatment of allergic individuals with leukotriene modifiers has been effective at reducing eosinophil numbers and inhibiting eosinophil activation (110).
Eosinophils also express a range of receptors for immunoglobulins that may contribute to chemotactic and activation responses in tissues. These include receptors for IgA, IgD, IgE, IgG, and IgM, which may possess up to three chains (α, β, and γ). Some controversy has surrounded the existence of the high-affinity receptor for IgE (Fc∊RI) on eosinophils. Studies have shown that the α subunit of Fc∊RI in eosinophils is expressed intracellularly rather than on the cell surface in resting cells, which may be mobilized to the surface and released during activation (111,112). Interestingly, although murine Fc∊RI contains α, β, and γ subunits, the human homolog lacks the β subunit, suggesting that this subunit is redundant in signaling in cells expressing Fc∊RI. Eosinophils express an IgE-binding protein, galectin-3 (Mac-2/∊ binding protein), as well as the low-affinity Fc∊RII (CD23), which may have contributed to apparent high-affinity binding for IgE in earlier studies. Cross-linking of immunoglobulin receptors on eosinophils has been shown to be highly effective at inducing respiratory burst and eosinophil-derived neurotoxin (EDN) degranulation in eosinophils, with a hierarchy of effectiveness in degranulation demonstrated to be in the order of secretory IgA (sIgA) = IgA > IgG ≫ IgE (113). Eosinophil cytokines such as IL-3, IL-5, and GM-CSF were demonstrated to enhance this process (114). IgA, particularly the secretory isoform, is an important mucosal antibody involved in supporting the body’s first line of defense. Thus, the sensitivity of the eosinophil to IgA is in agreement with its proposed role in protection against invasive organisms in mucosal tissues.
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Figure 10.5. Mediators released by activated eosinophils. De novo synthesized lipid mediators and oxidative metabolites are elaborated directly from the cell membrane or lipid bodies following enzyme activation, while granule-derived cationic proteins and cytokines, chemokines, and growth factors are released following granule–plasma membrane fusion during degranulation. GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; MIP, macrophage inhibitory protein; NGF, nerve growth factor; PAF, platelet-activating factor; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; SCF, stem cell factor; TGF, transforming growth factor; TNF, tumor necrosis factor; TXB2, thromboxane B2. |
Eosinophil Mediators
The eosinophil is considered to be both a factory and a store for a large array of mediators that are released upon activation and are thought to be important in various inflammatory reactions associated with this cell (Fig. 10.5).
Membrane-derived Mediators
Eosinophils produce a wide variety of lipid-derived mediators, which have profound biologic activity. The more important products are eicosanoids, which include leukotrienes (especially LTC4), prostaglandins (particularly PGE2), thromboxane, and lipoxins (especially LXA4) as well as PAF. The main substrate for these mediators is arachidonic acid (AA), which is specifically liberated from membrane phospholipids possessing this fatty acid at the sn-2 position by phospholipase A2 (PLA2) during receptor stimulation. Of the nine known families of PLA2, two families are expressed in eosinophils, the type IIA and type IV enzymes, which are commonly known as secretory and cytosolic PLA2, respectively (115,116). These enzymes are distinguished by their distribution, size, and sensitivity to Ca2+, where granule-stored sPLA2 (13 to 15 kDa) requires millimolar amounts of Ca2+ for activity, while cytosolically localized cPLA2 (85 kDa) is catalytically active in the presence of micromolar amounts of Ca2+. Interestingly, eosinophils express 20- to 100-fold higher levels of secretory PLA2 in their granules than other circulating leukocytes, suggesting a functional role in inflammatory processes involving eosinophil degranulation.
Eosinophils are a rich source of LTC4 (5S-hydroxy-6R,S-glutathionyl-7,9,-trans-11,14-cis-eicosatetraenoic acid) (117,118). Stimulation with the calcium ionophore A23187 generates up to 40 ng/106cells of LTC4 from normal-density eosinophils, while light-density eosinophils elaborate 70 ng/106 cells. Eosinophils produce negligible amounts (6 ng/106 cells) of LTB4 (5S-12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid) compared with up to 200 ng/106 cells from neutrophils. LTC4 generation by human eosinophils was also observed after stimulation with both opsonized zymosan and via an Fc∊RII-dependent mechanism using Sepharose beads coated with IgG (50). Release was maximal at 45 minutes, was greater in hypodense eosinophils than normal-density eosinophils, and was enhanced by fMLF. The production of LTC4 is critically dependent upon the activation of 5-lipoxygenase, an enzyme that resides in the euchromatin region of the nucleus, which translocates to the nuclear membrane upon cell activation, where it activates an 18-kDa protein called FLAP (119). The substrate for 5-lipoxygenase is AA, which may be released from membrane phospholipids by PLA2. The first product of this enzyme is an intermediary compound, 5-HPETE, which is transformed into the unstable epoxide LTA4. At this point, human eosinophils predominantly generate LTC4 through the action of LTC4 synthetase (117,118). Eosinophils are particularly rich in LTC4 synthetase, and account for 70% of all LTC4 synthetase-positive cells in the airway mucosa of normal and asthmatic individuals (120). LTC4 is generated intracellularly in human eosinophils stimulated with the calcium ionophore A23187. LTC4 is later exported from the cell in a regulated manner (121).
The production of 15-HETE, a lipid mediator generated via the 15-lipoxygenase pathway, occurs in activated eosinophils. The product 15-HETE has proinflammatory actions and can modulate the chemotactic effects of LTB4 on neutrophils (122). The enzyme 15-lipoxygenase may be distinguished from 5-lipoxygenase in that it can modify a larger pool of fatty acid substrates than the latter enzyme, and will oxygenate fatty acids that are esterified in phospholipids. Substrates include arachidonic acid, linoleic acid, polyenoic acids, and more complex lipids, such as lipoproteins. Eosinophils are the major cellular source of elevated 15-HETE in asthmatic airways, and are capable of generating 100 to 300 times more 15-HETE than neutrophils, endothelial cells, and fibroblasts (123). Eosinophils also account for 85% of cells positive for 15-lipoxygenase in the airway submucosa of normal and asthmatic subjects (124,125).
Eosinophils generate large amounts of PAF after stimulation with either calcium ionophore, opsonized zymosan, or IgG-coated Sepharose beads (126,127,128,129). PAF (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine) is a potent phospholipid mediator, which causes leukocyte activation. For instance, eosinophils elaborated 25 ng/106 cells of PAF after stimulation with calcium ionophore and up to 2 ng/106 cells after IgG stimulation. Much of the PAF remained cell associated, possibly acting as an intracellular messenger, or alternatively binding to PAF receptors on eosinophils, thus acting as an autocrine agent. Interestingly, stimulation of eosinophils with fMLF did not augment PAF release and hypodense eosinophils from patients with a marked eosinophilia released less PAF than normal eosinophils. [3H]PAF added to hypodense eosinophils was more rapidly incorporated into the phospholipid pool than [3H]PAF with normal-density cells (128). This suggested that hypodense eosinophils were metabolizing the exogenous PAF at a greater rate than normodense cells and may explain why stimulation with fMLF did not result in an increased amount of PAF generation. As with leukotriene synthesis, eosinophil-derived release of PAF was maximal at 45 minutes. Regulated PAF production is controlled by the release of biologically inactive lyso-PAF from membrane phospholipids by PLA2, which is later acetylated to form PAF by an acetyltransferase (127).
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Table 10.1 Content of Human Eosinophil Granules and Secretory Vesicles |
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The cyclooxygenase pathway is prominent in eosinophils as well, and eosinophils are capable of producing PGE1 and PGE2, and thromboxane B2from cyclooxygenase acting on free AA. In studies with guinea pig eosinophils, thromboxane B2 and PGE2 were shown to be generated following PAF or A23187 stimulation (130,131).
Many of the enzymes associated with membrane-derived mediator release from eosinophils, including cyclooxygenase and 5-lipoxygenase, are found stored in association with lipid bodies (Table 10.1) (132,133,134).
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Figure 10.6. Structure of the eosinophil crystalloid granule. This membrane-bound organelle is a major site of storage of eosinophil cationic granule proteins as well as a number of cytokines, chemokines, and growth factors. |
Granule-derived Proteins
Eosinophils contain at least five different populations of phospholipid bilayer membrane-bound granules.
1. Crystalloid granules: These specialized and unique granules measure between 0.5 and 0.8 mm in diameter, contain crystalline electron-dense cores (internum) surrounded by an electron-lucent matrix, and can take up acidic dyes avidly due to their cationic nature (2,135). They are mainly present in mature eosinophils, although coreless granules have been observed in immature eosinophils. These granules contain the bulk of highly charged cationic proteins present in eosinophils, including MBP, eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), and EDN. There are approximately 200 crystalloid granules in each cell. The core is predominantly composed of crystallized MBP (Fig. 10.6).
2. Primary granules: These coreless granules are enriched with Charcot-Leyden crystal protein (CLC), and are present in immature as well as mature eosinophils. Some authors refer to immature crystalloid granules as primary granules in eosinophil promyelocytes. These measure between 0.1 and 0.5 mm in diameter and are less abundant than crystalloid granules.
3. Small granules: These granules are also free of cores and contain acid phosphatase, arylsulphatase B, catalase, and cytochrome b558.
4. Lipid bodies: There are around five lipid bodies per mature eosinophil, the number of which increases in certain eosinophilic disorders, especially in idiopathic hypereosinophilia. Lipid bodies are enriched in arachidonic acid esterified into glycerophospholipids.
5. Secretory vesicles: Eosinophils are densely packed with small secretory vesicles in their cytoplasm. These vesicles appear as dumbbell-shaped structures in cross-sections, and contain albumin, suggesting an endocytotic origin. These structures are also known as microgranules or tubulovesicular structures.
Eosinophil MBP (13.8 kDa) is an arginine-rich 117–amino acid protein that constitutes a significant proportion of total cell protein in human eosinophils (5 to 10 pg/cell). MBP was originally named for its abundance in guinea pig eosinophils, which contain as much as 250 pg/cell, making up 50% of the total cellular protein (136). The high isoelectric point of MBP (10.9) cannot be measured accurately due to the extremely basic nature of the protein (137). MBP has been shown to be cytotoxic to airway tissues, including bronchial epithelial cells and pneumocytes. Thus, MBP may be at least partly responsible for tissue damage and neural dysfunction associated with eosinophil infiltration into the bronchial mucosa in asthma (2,138,139). Indeed, airway sections from patients with status asthmaticus exhibit intense MBP-specific immunofluorescence, suggesting that infiltrated eosinophils were fully activated, undergoing extracellular secretion of their contents of MBP (140). Parasympathetic ganglia in the airways of patients dying of asthma exhibit eosinophil infiltration, and MBP is an allosteric antagonist for the M2 muscarinic receptor. Loss of M2 receptor function results in airway hyperreactivity (139). The effects of MBP, in the absence of opsonization, on target cells such as parasites is thought to result from increased membrane permeability through surface charge interactions leading to perturbation of the lipid bilayer (141).
MBP is synthesized during the promyelocytic stage of eosinophil development, characterized by the presence of message encoding this protein, in a neutral prepro-MBP form that is later processed to form pro-MBP that is subsequently transported to the immature crystalloid granule and cleaved to form MBP (142,143). Mature MBP undergoes condensation from the periphery of immature crystalloid granules to the internum, where it develops a crystalline core as its concentration is increased (143,144). Once eosinophils have reached full maturity, MBP is no longer synthesized and messenger RNA encoding MBP disappears from the cell (143,145). MBP acts on other inflammatory cells, including neutrophils and eosinophils, to induce degranulation and lipid mediator release (146,147).
Other eosinophil basic proteins include EPO, ECP, and EDN, which reside in the matrix compartment of the crystalloid granule. EPO is a highly basic (pI of 10.9) heme-containing protein composed of two subunits, a heavy chain of 50 to 57 kDa and a light chain of 11 to 15 kDa. EPO is a haloperoxidase with 68% sequence identity to neutrophil myeloperoxidase, suggesting that a peroxidase multigene family may have developed through gene duplication (137,148). Eosinophils store approximately 15 pg/cell of EPO, which is important in catalyzing the peroxidative oxidation of halides and pseudohalides, leading to the formation of bactericidal hypohalous acids in reaction with hydrogen peroxide generated during respiratory burst (149,150,151).
The molecular mass of ECP is between 16 and 21 kDa, with around 15 pg/cell expressed in human eosinophils. The pI of ECP (10.8) is identical to that of MBP due to a similar arginine-rich sequence. Early studies have demonstrated that ECP, a member of a subfamily of RNase A multigenes and which possesses intrinsic ribonuclease (RNase) activity, is bactericidal, promotes degranulation of mast cells, and is toxic to helminthic parasites on its own (152,153). The mechanism of action of ECP is thought to involve the formation of pores or channels in the target membrane, which is apparently not dependent on its reversible RNase activity (154). ECP is perhaps most well known for its ability to elicit the Gordon phenomenon when it was injected into the cranial ventricles of rabbits, causing the destruction of Purkinje cells and leading to spongiform changes in the cerebellum, pons, and spinal cord (155,156).
EDN, another member of the RNase A multigene family of 18.5 kDa with approximately 100-fold higher RNase activity than ECP, is less basic than MBP or ECP with a pI of 8.9 due to a relatively smaller number of arginine residues in its sequence. ECP and EDN share a remarkable sequence homology of 70% at the amino acid level for the preform of both proteins, suggesting that evolutionarily, these proteins are derived from the same gene (157,158). Eosinophils express approximately 10 pg/cell of EDN, but there is marked variation between individuals. EDN similarly induces the Gordon phenomenon when injected intracranially in laboratory animals (155,156). Messenger RNA encoding EPO, ECP, and EDN has been detected in mature eosinophils, suggesting that eosinophils have the capacity to continue to synthesize these proteins (145).
The gene family expressing ECP and EDN has among the highest rates of mutation in the primate genome, ranking with those of immunoglobulins, T-cell receptors, and major histocompatibility complex (MHC) classes (158). These genes effectively comprise a superfamily of RNases expressed in the mammalian genome. Such an extreme rate of mutation suggests that the evolutionary constraints acting on the ECP/EDN superfamily have promoted the acquisition of a specialized antiviral activity. This may be inferred from the high mutation rates of other genes commonly associated with host protection against viral infection. Whether ECP or EDN possess any antiviral activity has yet to be demonstrated, although some studies have indicated that EDN may be a potent antiviral factor in respiratory infections (159).
The CLC protein (17.4 kDa) is produced in eosinophils at very high levels (accounting for 10% of the total cellular protein), although its functional role is still obscure. CLC is a hydrophobic protein with strong sequence homology to the carbohydrate-binding galectin family of proteins, and has been designated galectin-10 (160). CLC is released in large quantities in the tissues in eosinophilic disorders, resulting in the formation of distinct, needle-shaped structures that are colorless and measure 20 to 40 μm in length and 2 to 4 μm across. CLC crystals are abundant in the sputum and feces of patients with severe respiratory and gastrointestinal eosinophilia, which were first observed by Charcot and Robin in 1853.
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Table 10.2 Cytokines, Chemokines, and Growth Factors Produced by Human Eosinophils |
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A list of these and other granule proteins synthesized and stored in eosinophils is presented in Table 10.1 and published elsewhere (23,161,162).
Eosinophil-derived Cytokines
Human eosinophils have been shown to produce over 25 different cytokines, chemokines, and growth factors (Table 10.2) with the potential to regulate various immune responses. These cytokines have been identified in eosinophils by detecting messenger RNA (mRNA) and/or protein using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunocytochemical staining (163,164,165). In addition, picogram amounts of cytokines, chemokines, and growth factors were measured in supernatants of stimulated eosinophils (164,166). These cytokines are likely to act in an autocrine, paracrine, or juxtacrine manner, thereby regulating local inflammatory events. Studies have demonstrated that the production of eosinophil-activating cytokines (e.g., IL-3 and GM-CSF) by eosinophils may be important in prolonging the survival of these cells by a putative autocrine loop (34,164). For instance, adherence of highly purified eosinophils to the extracellular matrix protein fibronectin resulted in prolongation of survival of these cells in the absence of exogenous cytokines (34). Fibronectin-induced eosinophil survival was inhibitable by antibodies against fibronectin and VLA-4 and up-regulated by picogram amounts of IL-3 and GM-CSF derived from eosinophils (34). Observations on eosinophil cytokine release have been mainly studied in vitro, but a few have been confirmed in vivo (167,168,169,170).
A major distinction in cytokine production between eosinophils and T cells is that the former store their cytokines intracellularly as preformed mediators, while the latter produce and release cytokines only following activation. Although many eosinophil-derived cytokines are elaborated at lower concentrations than other leukocytes, eosinophils possess the ability to release these cytokines immediately (within minutes) following stimulation. Stored cytokines include IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-16, GM-CSF, TNF-α, eotaxin, IL-8, RANTES, nerve growth factor (NGF), and transforming growth factor-α (TGF-α) (32). Studies using immunogold electron microscopic analysis or confocal laser scanning microscopy coupled with double immunofluorescence labeling have indicated that several of these cytokines are found in close association with either the crystalline core or matrix of the crystalloid specific granules of the cell (Table 10.2) (165,171,172,173,174,175,176). For example, RANTES was found to be associated predominantly with the matrix compartment of the crystalloid granule in eosinophils (Fig. 10.7).
Developing eosinophils possess the ability to express cytokine message and protein at early stages of maturation. Eosinophils generated from semi-solid culture of cord blood–derived CD34+ cells in the presence of IL-3 and IL-5 were shown to express IL-5 and GM-CSF mRNA after 10 days of culture (177). Freshly purified CD34+ cells expressed IL-4 and RANTES mRNA, but not IL-4 and RANTES protein. On day 23 of culture, IL-4 and RANTES localized to the matrix of MBP+ crystalloid granules as determined by immunofluorescence (178). In addition, IL-6 protein expression was found in cells after day 16 of culture (144).
Another site of storage of cytokines and chemokines is within the small secretory vesicle. At least two such proteins were shown to be associated with these vesicles, namely RANTES and TGF-α immunolabeling (165,179). These organelles belong to the same group of secretory vesicles identified by electron microscopy analysis as tubulovesicular structures. RANTES-positive vesicles are highly sensitive to stimulation by IFN-γ and are rapidly mobilized (within 10 minutes of stimulation) to secrete RANTES extracellularly (165,180). Crystalloid granules, which also contain RANTES within their matrix compartment, were found to release this chemokine more slowly in response to IFN-γ (1 hour), while the majority of MBP remained associated with the core of these granules. These observations suggest that eosinophils have the ability to “shuttle” RANTES from the crystalloid granules to the cell exterior, and may provide an important in vitro model for eosinophil piecemeal degranulation (see “Degranulation Mechanisms,” below).
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Figure 10.7. Translocation of the chemokine RANTES in human eosinophils activated by interferon-γ (IFN-γ) in vitro. Immunoreactivities for RANTES (green fluorescence) and eosinophil major basic protein (red fluorescence) are shown in control (A) and IFN-γ–stimulated (10 minutes, 500 U/ml) (B) cells. The yellow color in (B) resulted from colocalization of green and red immunofluorescence stains. Note that immunoreactivity for major basic protein (MBP) remained associated with the cores of the crystalloid granules in both cells, while the green label for RANTES translocated toward the cell membrane. RANTES was proposed to be released from eosinophils by piecemeal degranulation. (Experimental conditions described in Lacy P, Mahmudi-Azer S, Bablitz B, et al. Rapid mobilization of intracellularly stored RANTES in response to interferon-g in human eosinophils. Blood 1999;94:23–32.) |
Respiratory Burst
Eosinophils undergo respiratory burst concurrently with the release of other mediators during cell activation. Respiratory burst is defined as the increase in cell metabolism (measured by the elevated activity of the hexose monophosphate shunt) and oxygen consumption, coupled with the release of reactive oxygen species (ROS). Many stimuli are capable of inducing respiratory burst in eosinophils, including LTB4, PAF, fMLF, C5a, opsonized particles, and RANTES (23). The principal product of respiratory burst is superoxide (O2-•), a potent oxidant with a highly reactive electron in its outer valence, possessing relatively weak intrinsic microbicidal activity. The function of O2-• is thought to reside in its ability to dismutate into more reactive ROS, including hydrogen peroxide (H2O2), the hydroxyl radical (OH-•), and formation of hypohalous acids (HOBr) upon reaction with EPO produced following eosinophil degranulation. The formation of ROS subsequent to O2-• generation is dependent upon the presence of a number of catalysts, such as superoxide dismutase (SOD), which accelerates the formation of H2O2, and the ferrous ion, which induces OH-• production from H2O2. O2-• is also able to react with nitric oxide (NO) produced from nitric oxide synthase enzymes (e.g., iNOS, eNOS) to form the highly reactive peroxynitrite (ONOO-), which has been shown to alter cell function.
The regulated burst of O2-• production is largely mediated through the activation of a membrane-associated enzyme complex, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Fig. 10.8). This enzyme complex is crucial for maintenance of host defense as it mediates the destruction of ROS-sensitive organisms. In addition, overactivation of the NADPH oxidase is likely to be cytotoxic to tissues, and has been implicated in the pathogenesis of many eosinophil-related disorders including allergic asthma (181). Interestingly, eosinophils possess the ability to generate up to 10-fold more superoxide than other phagocytes, including neutrophils, in which the mechanisms associated with NADPH oxidase activation have been studied in greater detail (182). The ability of eosinophils to release more O2-• is thought to be the result of higher levels of expression of the protein components that make up the NADPH oxidase complex (183,184,185,186,187). In addition, preferential assembly of NADPH oxidase occurs at the cell membrane in eosinophils, eliciting a predominantly extracellular form of O2-• release (188). This was in contrast to neutrophils, which showed intracellular NADPH oxidase assembly during respiratory burst stimulation and bacterial infection (188,189).
NADPH oxidase is a complex of several proteins, of which two (p22phox and gp91phox) reside in the membrane as part of the cytochrome b558protein, and the remaining proteins (p40phox, p47phox, p67phox, and Rac1 or Rac2) are cytosolic in resting states (182). In resting cells, the phagocytic oxidases p40phox, p47phox, and p67phox are found in a complex in the cytosol, while Rac1 or Rac2 are bound to the cytosolic guanine dissociation inhibitor RhoGDI. Binding between p47phox and p67phox occurs through the C-terminal SH3 domain of p67phox and a proline-rich region (PRR) in p47phox. The p67phox protein also contains a C-terminal Bem1p (PB1) motif that allows a high-affinity interaction with a C-terminal phox and Cdc motif in p40phox (190,191). An SH3 domain also exists in p40phox that is capable of interacting with the PRR domain in p47phox, although in vitro binding studies indicate that the affinity of this interaction is lower than that of p40phox for p67phox(192,193).
During activation by cell surface receptors engaged by opsonized microbes or soluble inflammatory mediators, p47phox is phosphorylated by cellular kinases on multiple serine residues, unmasking tandem SH3 domains to allow binding to p22phox in the membrane. Concurrently, p67phox and p40phox, which are still complexed to p47phox, translocate to the cell membrane, and the “activation domain” in p67phoxpromotes electron transfer between NADPH and flavin adenine dinucleotide (194). Superoxide generation from NADPH oxidase also requires concurrent activation and membrane translocation of Rac1 or Rac2, which binds to tetratricopeptide repeat motifs in the N-terminus of p67phoxand cytochrome b558 to induce additional conformational changes necessary for efficient electron transport to O2 (195). The function of p40phoxis still uncertain, although a recent study suggests that it may have a role in activating superoxide production during IgG-mediated phagocytosis (191).
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Figure 10.8. Assembly and activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex during respiratory burst. This complex is essential for the inducible release of superoxide for microbicidal reactions and is also present in neutrophils. During cell activation, the GTPase Rac, normally bound to guanosine diphosphate (GDP) in the resting cell, is activated by a guanine exchange factor (GEF) to bind to guanosine triphosphate (GTP). This results in translocation of Rac-GTP to the cell membrane, where other cytosolic proteins p67phox and p47phox have also translocated, to bind to the two subunits of cytochrome b558 (gp91phox and p22phox). Following the assembly of the oxidase, electrons are transferred from NADPH in the cytosol via flavin adenine dinucleotide (a cofactor) to oxygen molecules to form the highly reactive oxygen intermediate, superoxide. Assembly of this complex is reversed by GTPase-activating protein (GAP), which hydrolyzes GTP on Rac to GDP, and by the dissociation of the phox subunits. PMA, phorbol myristate acetate. |
Rac1 and Rac2 are monomeric guanine triphosphatases (GTPases), which exhibit 92% homology in their amino acid sequence, and are functionally interchangeable in their ability to activate NADPH oxidase, although they differ in their tissue distribution. Another potential modulator of the oxidase is the monomeric GTPase Rap1a, although its precise role is unknown. Rac1 is ubiquitously expressed throughout the body, while neutrophils, eosinophils, and other blood cells predominantly express Rac2, which is mainly expressed in hemopoietic tissues (188,196,197,198). The five minimal components of the NADPH oxidase complex were determined using cell-free assays (199), although several other proteins, such as p40phox and Rap1a, which also translocate to the oxidase complex during activation, may be involved in “fine-tuning” the activity of the oxidase (200).
The pathway leading from receptor stimulation to activation of the oxidase is still poorly understood. Many studies on NADPH oxidase activation utilize phorbol esters, such as phorbol myristate acetate (PMA), as highly potent artificial stimuli to activate respiratory burst in eosinophils. Phorbol esters are classically known for their ability to directly activate protein kinase C (PKC) (201,202,203). The use of pharmacologic inhibitors of PKC has generated paradoxic results, in that PKC inhibitors only partially inhibit agonist-induced H2O2 release in guinea pig eosinophils (202,204). However, there is a species difference in sensitivity to PMA, as in human eosinophils, where PKC inhibitors actually augment the rate of oxygen consumption in response to opsonized particles (205). These findings suggest that PKC is not critical for agonist-induced respiratory burst in eosinophils, although stimulation of PKC appears to be able to induce superoxide release on its own.
Taken together, eosinophils generate substantial amounts of O2-• as part of their role in host defense, and the mechanisms associated with the release of this toxic mediator are under investigation. The release of O2-• from eosinophils is likely to be a crucial component of the pathophysiologic processes underlying eosinophilic inflammation in mucosal tissues.
Degranulation Mechanisms
Degranulation is defined as the exocytotic fusion of granules with the plasma membrane during receptor-mediated secretion. During exocytosis, the outer leaflet of the lipid bilayer membrane surrounding the granule encounters the inner leaflet of the plasma membrane, a process known as “docking.” The docking step is hypothesized to be regulated by intracellular membrane-associated proteins that act as receptors directing the specificity of granule targeting. After docking, the granule and plasma membrane fuse together and form a reversible structure called the fusion pore, which is also thought to be regulated by similar, or the same, membrane-associated proteins regulating granule docking. Depending on the intensity of the stimulus, the fusion pore may either retreat, leading to reseparation of the granule from the plasma membrane, or expand and allow complete integration of the granule membrane into the plasma membrane as a continuous sheet. The inner leaflet of the granule membrane becomes outwardly exposed, and the granule contents are subsequently expelled to the exterior of the cell (206).
There are four main forms of eosinophil granule release, which have been observed in vitro and in vivo (Fig. 10.9). The first is the classical sequential release of single crystalloid granules, which was the original hypothesis suggested for a predominant route of degranulation in eosinophils. This type of release is typically seen in vitro and can be elegantly demonstrated electrophysiologically using patch-clamp procedures that measure changes in membrane capacitance, which are directly proportional to increases in the surface area of the cell membrane. During the sequential release of individual crystalloid granules, a stepwise increment in capacitance may be observed as their membranes fuse with that of the cell membrane (207,208,209). The second mode of granule release is compound exocytosis, also demonstrated by patch-clamp analysis, in which sudden, very large increments in whole cell capacitance occur resulting from individual granules fusing with the cell membrane (210). Ultrastructural studies of guinea pig eosinophils have also demonstrated evidence for compound exocytosis (211) similar to that observed in rat eosinophils adhering to the outer surface of opsonized parasitic larvae (210,212). Additional evidence for compound exocytosis was suggested in eosinophils stimulated with a cocktail of IL-3, IL-5, and GM-CSF, which were observed to fuse their granules following activation as determined by immunofluorescence for CD63, a marker for crystalloid granules (213). The third manner in which eosinophils degranulate is by piecemeal degranulation (PMD). PMD was first characterized by Dvorak et al. for the appearance of numerous small vesicles in the cytoplasm coupled with the apparent loss of crystalloid granule core and matrix components, creating a “mottled” appearance in electron microscopy analysis (214). This was thought to be due to small vesicles budding off from the larger secondary granules and moving to the plasma membrane for fusion, thereby causing gradual emptying of the crystalloid granules to the outside of the cell. PMD was the most commonly observed pattern of degranulation seen in situ in biopsy samples from the upper airways of allergic individuals (215), and is likely to be physiologically the most important mechanism for eosinophil mediator release in allergic disease. An in vitro model for PMD has been established using IFN-γ–stimulated eosinophils, in which a piecemeal manner of RANTES release was detected by a combination of confocal laser scanning microscopy, immunogold labeling, and subcellular fractionation (165). Airway tissue eosinophils not undergoing PMD appeared necrotic, which is a fourth pattern of granule release, also termed “cytolysis” (216). This type of release has been previously observed to occur following in vitro stimulation of human eosinophils with the calcium ionophore A23187 (217), and appears to be a physiologically relevant granule release event.
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Figure 10.9. Four putative physiologic modes of eosinophil degranulation. The most commonly observed forms of degranulation in allergic disease are piecemeal degranulation and necrosis (cytolysis). Parasitic and fungal diseases typically exhibit eosinophils undergoing compound exocytosis. |
The mechanisms associated with classical exocytosis, compound exocytosis, and PMD, but not cytolysis, are thought to require specific intracellular membrane-associated proteins acting as receptors for granule docking and fusion. These proteins include a family of molecules known as SNAREs (an acronym for SNAP receptors). The paradigm associated with SNARE molecule function predicts that these proteins are essential for exocytosis. SNAREs were originally described in neuronal tissues and were found to group themselves into two distinct locations, the granule-associated SNAREs (the so-called vesicular SNAREs or v-SNAREs) and the plasma membrane-associated SNAREs (target SNAREs or t-SNAREs) (218). In order for a functional SNARE complex to form, allowing the granule to dock with the plasma membrane, one v-SNARE binds to two t-SNARE molecules. In neuronal cells, a commonly observed v-SNARE is vesicle-associated membrane protein (VAMP)-1 or its isoform VAMP-2. In these cells, the t-SNAREs associating with VAMP-2 that were originally described were synaptosome-associated protein of 25 kDa (SNAP-25) and syntaxin-1A. These three molecules form a stable detergent-resistant four-helix coiled-coil bundle, which may be regulated by protein phosphorylation. The precise mechanisms regulating SNARE binding and activation are not yet known.
Nonneuronal cells also express SNAREs, although some isoforms have been identified with high-sequence homology to the neuronal SNAREs. Up to three SNAP-25 isoforms and approximately 16 syntaxin-1 isoforms have been characterized based on detection of homologous SNARE motif messenger RNA sequences. Interestingly, most nonneuronal secretory cells appear to require SNAP-23 and syntaxin-3, syntaxin-4, or syntaxin-6 (219,220,221) for control of exocytosis. Eosinophils have been shown to express the v-SNARE, VAMP-2, in their small secretory vesicles containing RANTES, but not their crystalloid granules (222). Crystalloid granules express mainly VAMP-7 and VAMP-8, the former being important in regulation of crystalloid granule secretion during degranulation responses from permeabilized eosinophils (223). The t-SNARE isoforms syntaxin-4 and SNAP-23 are expressed in the cell membrane of eosinophils, and these have the potential to act as cognate membrane-binding partners for VAMP-2 and VAMP-7 degranulation (224). The SNARE molecules VAMP-2, SNAP-23, and syntaxin-4 identified in eosinophils are proposed to regulate docking and fusion of RANTES-containing small secretory vesicles during piecemeal degranulation (Fig. 10.10).
Mechanisms associated with granule release in eosinophils are critical for effector function of eosinophils. Without degranulation and mediator secretion, the eosinophil is a relatively inert cell, and does not affect the surrounding tissues, as seen in cases of idiopathic pulmonary eosinophilia and eosinophilic pneumonia. In these conditions, eosinophil numbers are increased in the capillaries and tissues of the lung, but no cellular or structural damage is evident, probably because of the lack of eosinophil degranulation.
In contrast, asthmatic patients show profound eosinophilia in the airways combined with significant tissue destruction, suggesting that, in addition to eosinophilic infiltration, their undergoing degranulation may contribute to mucosal damage in the airways and related symptoms of asthma.
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Figure 10.10. Schematic model for molecular regulation of granule–plasma membrane fusion proposed to occur in piecemeal degranulation in eosinophils. In this model, the v-SNARE VAMP-2 is expressed on small secretory vesicles, which store RANTES as a preformed mediator, while the t-SNAREs SNAP-23 and syntaxin-4 reside on the inside of the plasma membrane. Following cell activation, v- and t-SNAREs bind together to form a SNARE complex, resulting in fusion and release of vesicular contents including RANTES. (See Lacy P, Logan MR, Bablitz B, et al. Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-g-induced piecemeal degranulation in human eosinophils from atopic individuals. J Allergy Clin Immunol 2001;107:671–678; and Logan MR, Lacy P, Bablitz B, et al. Expression of eosinophil target SNAREs as potential cognate receptors for vesicle-associated membrane protein-2 in exocytosis. J Allergy Clin Immunol 2002;109:299–306.) |
Eosinophils as Immunoregulatory Cells
The consistent association of eosinophils with a specific pattern of immune response common to helminth infection and atopy suggests that eosinophils may be either a bystander cell or an active component of a complex immune disease. To determine the involvement of eosinophil-derived factors in modulating the immune response, the bioactivity of cytokines released from eosinophils has been explored for their potential physiologic effects in immune regulation. Studies have shown that IL-2, IL-4, IL-12, IL-16, GM-CSF, RANTES, and TGF-β derived from eosinophils are capable of exerting bioactive effects on bystander cells using in vitro assays (32,162). For example, the release of IL-4 from eosinophils is important in driving the initiation of a Th2-type response in Schistosoma mansoni infection in mice (225). IL-2 and IFN-γ from CD28-stimulated eosinophils were shown to stimulate proliferation in an IL-2–dependent cell line and MHC class II expression on Colo 205 cells, respectively (226). These studies have demonstrated that eosinophil-derived cytokines and chemokines have the ability to at least regulate local inflammatory responses.
Generally speaking, eosinophils produce significantly smaller amounts of cytokines than T cells, B cells, and other cells in the immune system. However, in eosinophilic inflammation, eosinophils outnumber T cells in the tissues by as much as 100-fold. As such, the magnitude of the presence of eosinophils may be a determining factor in regulating immune responses at a local level. The release of eosinophil cytokines often takes place within a much shorter period than cytokines released by T cells (which may be several hours), as eosinophil-derived cytokines are stored as preformed mediators in crystalloid granules and may be secreted in response to stimuli in a matter of minutes.
Allergy is often characterized by a significant polarization of the immune response toward enhanced production of Th2 cytokines and a dramatic increase in allergen-specific and total IgE levels. Allergic disease is initiated by the generation of allergen-specific CD4+ cells that produce the Th2 cytokines IL-4, IL-5, IL-9, and IL-13 (Fig. 10.11). These cytokines are crucial for the maturation, proliferation, survival, and activation of mast cells, basophils, and eosinophils, important effector cells. They also regulate IgE synthesis by B cells and mucous production by epithelial cells. In contrast, Th1 cells are characterized by the production of the Th1 cytokines IL-2 and IFN-γ. The immunologic responses of eosinophils are dependent on an array of cytokines and chemokines that may traditionally be associated with Th1 or Th2 responses. Interestingly, eosinophils synthesize many of these cytokines and chemokines to which they can also respond. Based on their ability to synthesize, store, and release both Th1 and Th2 cytokines and chemokines, and significant evidence indicating the bioactivity of their released cytokines, eosinophils have been implicated as active components of allergic disease, rather than as bystander cells.
Classically, the Th1 response has been modeled as an immune response that exerts inhibitory effects on Th2 responses. It is therefore paradoxic that eosinophils, as a cell type marking Th2-type responses to allergic diseases or helminthic infections, synthesize and store both Th1 and Th2 cytokines (Table 10.2). For example, binding of CD28 on human eosinophils induced the release of bioactive IL-2 and IFN-γ (226), both of which are Th1 cytokines, and IL-13, a Th2 cytokine (227). Since Th1 and Th2 responses are mutually inhibitory, it has been very difficult to dissect the specific roles of eosinophil-derived cytokines in the initiation and effector phases of the allergic immune response. The role of eosinophil-derived Th1 or Th2 cytokines may therefore depend on the timing of eosinophil infiltration into sites of allergic inflammation. Indeed, recent studies using a mouse model of Nippostrongylus brasiliensis infection have shown that while eosinophils may be crucial for secondary Th2 polarized immune response against this parasite, the primary response does not require eosinophil-derived IL-4 or IL-13 (228).
Experimental models that induced eosinophilia in the absence of tissue infiltration of eosinophils have exquisitely demonstrated the importance of the timing and location of chemokine release in recruitment of eosinophils. For example, eotaxin gene knockout (Eo-/-) mice showed decreased tissue eosinophils during the early (but not late) phase of allergic inflammation (72). Moreover, Eo-/-mice also showed a significant defect in clearing the larvae of Trichinella spiralis from skeletal muscles compared to wild-type animals in spite of similar levels of blood eosinophilia (229). Thus, the presence of eosinophils in allergic disease results from a carefully orchestrated process that involves cytokines and chemokines controlling the maturation and recruitment of eosinophils to the site of allergen exposure.
The implications of eosinophil cytokine production are extensive, such as in the case of IL-4, where this cytokine may be released from eosinophils to direct Th2 cell differentiation in local lymph nodes. In support of this possibility, eosinophils have been shown to traffic to paratracheal draining lymph nodes (in a mouse model of asthma), where they were demonstrated to function as antigen-presenting cells expressing MHC class II and costimulatory CD80 and CD86 to stimulate CD4+ T cells (230). During intimate cell–cell contact, the production of IL-4 and IL-13 is not required in abundance to effect important immunoregulatory events, such as enhanced switching of T cells to Th2 phenotype and increased IgE synthesis, both of which are hallmarks of allergic disorders (231,232). Under such conditions, antigen-loaded eosinophils acting as antigen-presenting cells were found to preferentially initiate the generation of a Th2 response to ovalbumin in an experimental mouse model (233,234). These studies have confirmed the ability of eosinophils to participate in the generation of allergic immune response through the secretion of immunomodulatory cytokines.
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Figure 10.11. A proposed scheme addressing the multiple roles the eosinophil may potentially play in asthma and related allergic conditions. In addition to other immune cells (antigen-presenting cells, dendritic cells, T-helper cells) as well as inflammatory cells such as the mast cell in the initial stages of exposure to the specific allergen trigger, eosinophils are now proposed to play a role in regulating the type of the immune response predominant in asthma and allergy, namely the Th2-type. Eosinophils have the capacity to exert powerful effector function against the mucosal tissue that may contribute to airway hyperresponsiveness among other changes in the affected site. Additionally, eosinophils have recently been suggested to play a role in tissue remodeling, a main feature of even the most mild forms of asthma. Eosinophils constitutively express indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the metabolism of the amino acid tryptophan (W). IDO, when stimulated intracellularly with interferon-γ (IFN-γ), would lower the level of extracellular W and break it down to a series of other products including kynurenines (KYNs) that we had shown to target Th1 cells for limited proliferation and apoptosis, or while selectively allowing Th2 proliferation. The precise mechanism and receptors involved in this distinct Th1 versus Th2 response remain unknown and under active study. APC, antigen-presenting cell; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LT, leukotriene; MBP, major basic protein; MHC, major histocompatibility complex; PAF, platelet-activating factor; PG, prostaglandin; TCR, T-cell receptor; TGF, transforming growth factor; TNF, tumor necrosis factor; VGEF, vascular endothelial growth factor. |
Apart from cytokines, other eosinophil-derived immunoregulatory factors have been recently recognized. Based on initial data showing that its inhibition resulted in an immune-mediated abortion in mice (235), studies in the last 5 years have shown that indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the oxidative catabolism of tryptophan, may play a central role in immune regulation (236). Subsequent studies have associated tryptophan catabolism with mechanisms of tumor escape from T cells (237); dysfunctional tolerance in autoimmune diabetes mellitus in NOD mice (238); and protective negative regulation of trinitrozobenzene-induced (Th1) model of colitis in the mouse (239). According to this mechanism, a discrete population of dendritic cells (DCs) that express functional IDO in lymphoid tissues is able to inhibit T-cell proliferation and induce T-cell apoptosis (240). The presence of this minority population of inhibitory DCs in lymphoid tissue can override the activating properties of other DCs and, therefore, act as a strong negative feedback mechanism on immune activation (241). Further studies have shown that regulatory T cells (Tregs) that constitutively express cytotoxic T-lymphocyte antigen-4 (CTLA-4), the so-called central Tregs, down-regulate the immune response using this mechanism (242,243).
Human eosinophils constitutively express the enzyme IDO (244), in contrast to DCs where IDO is inducible. Furthermore, coculture of IDO-expressing eosinophils with T lymphocytes selectively inhibited the proliferation of Th1 cells while having no effect on Th2 cells. Immunostaining of histologic sections of human lymphoid tissues and those from ovalbumin-sensitized and -challenged mice also showed the presence of IDO-expressing eosinophils. Similarly, a study investigating the expression of IDO in tumor biopsies from 25 cases of non–small cell lung cancer showed that eosinophils were the only IDO-expressing cells in these tissues, and concluded that eosinophil-derived IDO may play a significant role in the escape of non–small cell lung cancer from immune surveillance (245). In this study, the presence of IDO-expressing tumor-infiltrating eosinophils was strongly correlated with poor survival. When these findings are juxtaposed with previous studies investigating the prognostic value of tumor-associated tissue eosinophilia, it becomes apparent that eosinophil-derived IDO may be a major immunoregulatory mechanism in several eosinophil-associated conditions (246,247).
The recognition of the capacity of the eosinophil to synthesize and release cytokines, and produce other immunomodulatory molecules, has introduced a new paradigm shift toward understanding the potential of the eosinophil as an effector cell in allergic inflammation and other eosinophil-associated conditions. However, the full capacity of the eosinophil to elaborate cytokines, the precise microenvironment requirements for such synthesis, and the intracellular pattern of production and storage, including the timing of its immunomodulatory function during the immune response, remain the subject of intensive investigation.
Eosinophils and Allergic Disease
The association between eosinophils and allergic disease has been known for many years. Eosinophils are a feature of allergic and nonallergic asthma and large numbers of eosinophils (248) and eosinophil granule products are commonly found in and around the bronchi in patients who have died of asthma (140). The gastrointestinal tract is a natural homing site for eosinophils; however, excessive eosinophilia and active secretion in various compartments of the gut result in disease states such as eosinophilic esophagitis, gastroesophageal reflux disease (GERD), or inflammatory bowel disease (IBD) (60). Patients with atopic dermatitis also show strong evidence of eosinophil presence and mediator release (249,250).
Eosinophils and Asthma
A correlation between the degree of bronchial hyperresponsiveness (a cardinal clinical feature of asthma) and peripheral blood eosinophilia has been observed in subjects who exhibited a dual response following allergen challenge (27,251). Eosinophils in the airways may be assessed using bronchial biopsy, bronchoalveolar lavage (BAL), or induced sputum. The use of fiberoptic bronchoscopy is considered the gold standard for acquiring a better appreciation of eosinophil involvement in asthma (252,253). A consistent finding in asthmatic airways is the presence of increased numbers of activated eosinophils or their release products, which correlate broadly with the severity of disease as reflected in symptoms, bronchial hyperreactivity, and lung function (254,255). Segmental allergen challenge of human subjects’ bronchi induces increased eosinophils in the airways (256) that were positive for the early activation marker CD69 (257). Even in mild disease, there may be a significant increase in numbers of eosinophils in the airways of patients, and these are CD69+ (254,258).
Sputum analysis offers a noninvasive technique showing correlation of eosinophil numbers with clinical outcomes (259,260,261). Increases in sputum eosinophils correlate with the degree of airway responsiveness, asthma symptom scores, and asthma exacerbations (254,262,263). Monitoring eosinophilic inflammation in induced sputum samples has been suggested as an important adjunct to the clinical management of asthma beyond relying on lung function and symptoms alone (262). Conversely, a lack of eosinophils in sputum analysis appears to correlate with a lack of response to inhaled corticosteroids (264).
Challenge of the airways with inhaled allergen induces local airway inflammation characterized by influx of both eosinophils and neutrophils. In the late phase of the response, eosinophils are the predominant feature (251,256). A transient peripheral blood eosinophilia may also occur following these challenges (251). Challenges with low-molecular-weight occupational compounds such as toluene diisocyanate have also been associated with eosinophilia (265). Even nonatopic asthma, which appears to lack an IgE-mediated immune response, shows similar increases in airway eosinophils (254,265).
The Eosinophil Controversy
Early studies on eosinophil function in the immune response suggested that eosinophils played an immunoprotective role in allergy. For example, eosinophils produce histaminase, which was thought to act by down-regulating mast cell–mediated early-phase responses to allergen (266). However, reports emerging in the latter part of the 20th century suggested that eosinophils may have a destructive role in allergy and asthma, based on the discovery of intensely stained deposits of eosinophil MBP in the airways of individuals who died from fatal asthma (140). More recent studies have thrown this concept into doubt, including animal models of asthma (IL-5 gene knockouts and anti–IL-5–treated mice) and clinical trials using anti–IL-5, IL-12, and IFN-γ. Treatments or strategies targeting IL-5 in animal models of allergic inflammation were shown to reduce eosinophil numbers but have variable effects on airway hyperresponsiveness (267).
Mouse Models of Airway Hyperresponsiveness
Animal models of asthma have utilized IL-5-/- mice to determine the contribution of eosinophils to the pathogenesis of airway inflammation induced by allergens. One report showed that airway hyperresponsiveness was not affected in allergen-sensitized and -challenged IL-5-/- mice, although blood eosinophil numbers were significantly diminished (268). Studies using anti–IL-5 injections in mice generated similar observations (269,270). In another allergen challenge model, increased blood and tissue IL-5 levels were evident in wild-type mice (271). These levels correlated with both blood and tissue eosinophilia and airway hyperresponsiveness. In the same study, IL-5-/- mice did not mount a blood or tissue eosinophil response after allergen challenge, nor did they develop airway hyperresponsiveness (271). Restoring IL-5 expression in these animals via vaccinia virus encoding IL-5 reconstituted blood and tissue eosinophilia was associated with the development of airway hyperresponsiveness. In an inducible model of T cell–specific transgenic expression of IL-5, mice produced severe skin lesions, gastrointestinal dysfunction, splenic enlargement, and airway hyperresponsiveness similar to symptoms associated with human eosinophilic disorders (272), supporting a crucial role for the eosinophil in tissue damage associated with allergy.
The roles of IL-5 and eosinophilia in these mouse models are still in question. Airway hyperresponsiveness may still persist in animals in spite of treatment with an antibody to IL-5 and depletion of blood eosinophils, depending on the protocol used for sensitization and challenge. Thus, airway hyperresponsiveness may persist during allergen challenge even though blood eosinophilia will be lost. The answer to this dilemma may be in the persistence of tissue eosinophils even during IL-5 depletion. Although IL-5 is important in the differentiation and proliferation of eosinophils in the bone marrow, once they arrive in peripheral mucosal tissues, they may switch to an IL-5–independent mechanism of activation, and possibly recruitment, due to the strongly down-regulatory effects of IL-5 on eosinophil IL-5 receptor expression.
The possibility of persistence of IL-5-dependent tissue eosinophils has been implicated in results from Eo-/- mice. Despite developing blood eosinophilia, Eo-/- mice do not generate tissue eosinophilia (37) with its associated eosinophil-mediated tissue damage following allergen challenge (273). However, in double-knockout mice where IL-5 and eotaxin expression is deficient (IL-5-/-Eo-/-), both blood and tissue eosinophilia were eradicated, and airway hyperresponsiveness was significantly decreased during allergen challenge (P. Foster, unpublished observations). These studies indicate that there are distinct roles for eotaxin and IL-5 in eosinophil maturation, proliferation, and homing to target tissues. Thus, while IL-5 is critical for the maturation and proliferation of eosinophils in the bone marrow, eotaxin may be equally essential for movement and maintenance of eosinophilia in the tissues. Therefore, a key event in eosinophil-mediated inflammation leading to airway hyperresponsiveness may lie in the persistence of activated eosinophils in the tissue.
Recent studies in an eosinophil knockout mouse, generated by linking the diphtheria toxin promoter to the gene expressing eosinophil peroxidase (the so-called “PHIL” mouse), indicate a key role for eosinophils in establishing airway hyperresponsiveness using an acute model of allergic inflammation (274). In contrast, another study showed that GATA-1 promoter disruption, which leads to the ablation of eosinophils, had no effect on airway hyperresponsiveness to methacholine challenge in either acute or chronic models of allergic inflammation, but showed reduced airway remodeling (275). These studies show conflicting data on the role of eosinophils in mediating airway hyperresponsiveness, at least in the mouse model.
Indeed, the appropriateness of the mouse model for investigating airway hyperresponsiveness has been brought into question. A major limitation of mouse models is that murine eosinophils seem to lack the ability to degranulate in vivo or in vitro in response to any known eosinophil-specific agonists. As mentioned earlier, eosinophil degranulation appears to be a vital component of the symptoms associated with allergic airway disease, and the use of mice may be counterproductive in providing clues relating to a better understanding of the role of the eosinophil in airway hyperresponsiveness. In support of this suggestion, knocking out eosinophil MBP or EPO was shown to have little effect on the development of airway hyperresponsiveness following allergen challenge in murine studies (276).
In conclusion, these findings have important implications for the treatment of asthmatic patients with anti–IL-5, such that IL-5 depletion may not be sufficient to clear the airways of a persistent population of IL-5–independent, activated tissue eosinophils. Further studies will be essential for determining the precise role of the eosinophil in contributing to mucosal inflammatory events in allergy.
Antieosinophil Strategies in Asthma
Although eosinophils are closely associated with the clinical pre-sentation of asthma and other atopic diseases, the evidence that this reflects a direct cause-and-effect relationship is still a subject of debate. The discovery of IL-5 in the 1980s as the most crucial cytokine in the regulation of growth and terminal differentiation of the eosinophil (20,277) led to major pharmaceutical investments aimed at antagonizing IL-5 with a view to blocking the eosinophil influx into the tissue and presumably inhibiting its associated inflammatory sequelae. Animal models, particularly simian, pointed optimistically to such a possibility (267). However, clinical trials with a humanized anti–IL-5 monoclonal antibody, mepolizumab, concluded that targeting the eosinophil is far more complex than blocking its differentiation at the level of the bone marrow and blood (278,279,280). As expected, a single intravenous injection of anti–IL-5 induced a profound reduction in peripheral blood eosinophils in patients for up to 16 weeks. However, in spite of the loss of eosinophils from the circulation, bronchial hyperresponsiveness to histamine persisted in these patients for up to 6 weeks after treatment. Based on these findings, the authors questioned the role of the eosinophil in the late-phase asthmatic response and bronchial hyperresponsiveness. This proposal was reinforced by findings from mouse models of asthma, some of which indicated that the eosinophil may be redundant in mechanisms associated with the development of airway hyperresponsiveness.
However, the study design and patient selection criteria may have been deficient in determining the success of mepolizumab in these reports (281). Moreover, the measurement of eosinophils in sputum or airway fluids alone may not reflect the complete contribution of airway tissue eosinophils. Mepolizumab only depleted ∼50% of bronchial tissue eosinophils in spite of its ability to eradicate blood and BAL fluid eosinophils (282). Airway tissue eosinophils may be less dependent on IL-5 for their survival. The development and maturation of eosinophils can occur in situ in peripheral sites of inflammation. Eosinophil progenitors are released into the circulation to reach such tissue sites (16). Eosinophils can release GM-CSF in an autocrine fashion (34,163), a cytokine that is stored in association with eosinophil granules (173), which can enhance tissue eosinophil maturation and prolong their survival in tissues. Autocrine GM-CSF stimulation of eosinophils bound to fibronectin, via α4integrin, promoted eosinophil survival for 2 weeks (34). Additionally, GM-CSF appears to have a strong role in inhibiting eosinophil apoptosis at the tissue level. Other eosinophil-derived and stored cytokines (e.g., IL-4 [174] and IL-13 [227,283]) and chemokines (e.g., RANTES [165,284]) may further amplify the inflammatory milieu. Eosinophils may enhance their own survival by directly stimulating CD4+ T cells within tissue to produce IL-5 (37). Nasal explants from atopic patients were shown to survive ex vivo using similar mechanisms to promote extramedullary eosinophil maturation and survival (36,38). Shen et al. demonstrated that eosinophils, when instilled into the trachea of IL-5 knockout mice, not only survive in the absence of IL-5, but in concert with CD4+ T cells, also migrate back into lung and reconstitute the asthma phenotype of wild-type antigen challenged animals (285). Overall, while IL-5 is essential in the maturation and differentiation of eosinophils in the bone marrow (12), the recruitment to tissues and function within tissues may be IL-5 independent.
The position of eosinophils in the airways may also play a role. Patients with eosinophilic bronchitis have increased sputum and tissue eosinophils, but do not develop asthma (286). From histopathologic samples of patients with asthma, eosinophils can be found clustered around vagal nerve ganglia in the lung (139). As well, positive staining for extracellular eosinophil MBP has been detected in the vicinity of these nerves (139). In guinea pig models of antigen sensitization followed by challenge (287) and antigen sensitization followed by virus infection (138), the release of MBP from eosinophils has been shown to cause M2 receptor dysfunction and hyperreactivity. This may be related to the increased number of eosinophils found in closer proximity to the parasympathetic nerves of sensitized guinea pigs compared to nonsensitized animals (288).
In summary, despite uncertainty surrounding the specific role of eosinophils in asthma, significant correlations between numbers of activated airway eosinophils (and their released products) and disease severity have been provided in a large body of literature (2,3,280,289,290).
Eosinophilic Esophagitis
Eosinophils have been shown to be associated with the pathogenesis of gastrointestinal disorders such as eosinophilic esophagitis (EE). Often patients undergoing biopsy for diagnosis of GERD have increased numbers of intraepithelial eosinophils (291,292), which appear to clear with effective antireflux therapy (293). The condition of EE is described as a distinct clinical entity, although the feature of esophageal dysfunction is shared with GERD. The pathogenesis of EE has an allergic basis, and EE has been called asthma of the esophagus. Patients with EE present with increased gastroesophageal reflux, and also exhibit choking or food impaction. The clinical symptoms of EE respond well to antiasthma therapy such as systemic corticosteroids (294), topical corticosteroids (295,296), and montelukast (297). Similar to asthma, a key stimulus for developing exacerbations of EE symptoms is not always food allergen, but inhaled aeroallergen (298,299). The mechanism of this surprising observation is not known, but may relate back to the shared embryonic origin of the gastrointestinal and respiratory tracts (300).
The Effector Role of the Eosinophil in Worm Infections
There is a strong apparent relationship between parasitic infection and eosinophilia. Infection with helminths is the most common cause of moderate to marked eosinophilia. The relationship between peripheral blood eosinophilia and tissue-invading helminths has been recognized for many years. In particular, studies in the late 1970s demonstrated that eosinophils had the capacity to kill parasitic targets and led to the concept that eosinophils were immunoprotective (181).
As in allergic inflammation, the precise role of eosinophils in the immunopathologic changes associated with helminth infections remains ill-understood and rather controversial. Increases in the number of tissue and peripheral blood eosinophils, together with elevations in the levels of total and parasitic-specific IgE and mastocytosis, have been considered for a long time to be hallmarks of infection with parasitic worms (4), especially during their tissue migratory phases. Much has been published about the inimical role these cells may play in protection against helminths, but there is equally important evidence to suggest that their presence may be a reflection of their participation in the pathology of the disease rather than immunity to the parasitic metazoa (301). The original observation of Basten and Beeson (7) that helminth-associated eosinophilia is T-cell dependent was an important turning point in our current understanding of eosinophil-mediated inflammation in worm infections. The identification and subsequent cloning of GM-CSF, IL-3, and particularly IL-5 helped to explain the T-cell control of eosinophilic response both in terms of eosinophilopoiesis and differentiation as well as priming and activation of the mature cell. The question, however, remains as to why there is a selective increase of eosinophils and what is their function, both locally and systematically, in infected subjects.
In Vitro and Murine Parasitic Helminth Studies
Much has been published on the helminthicidal effects of human, primate, and rodent eosinophils against metazoan targets coated with either IgG, IgA, IgE, and/or complement components. In this context, a number of parasitic targets have been studied including schistosomula of Schistosoma mansoni, newborn larvae of Trichinella spiralis, larvae of Nippostrongylus brasiliensis, Fasciola hepatica, and others (135,302).
Eosinophils adhere readily to appropriately coated larvae and undergo exocytosis, which results in the deposition of the basic and cytotoxic granule-associated proteins. On their own, these preformed products of eosinophils (including MBP, ECP, and EPO) have potent helminthicidal properties at low molar concentrations (135). The exogenous addition of a number of chemotactic agents, such as LTB4, PAF, and fMLF (303,304), and cytokines, such as GM-CSF, IL-3 TNF-α and IL-5 (305), to eosinophil preparations enhances their cytotoxic capacity against parasitic larvae. In addition to killing worm larvae, eosinophils that adhere to schisto-somula via IgG, IgE, or complement generate substantial amounts of membrane phospholipid-derived mediators, especially LTC4 (306). More recent studies have shown that in IL-5-/- mice, skin implants containing parasites failed to eliminate larval forms of the organisms (307). The mechanism underlying larval expulsion was shown to be dependent on eosinophils as well as IgM, and the results suggested that the function of eosinophil granule proteins might be associated with disrupting parasitic larvae to allow processing by antigen-presenting cells, including the eosinophil itself.
Helminthiases in Humans and Nonhuman Primates
The precise regulatory and functional roles of eosinophils in human helminthiases during the well-documented inflammatory reaction require urgent and extensive attention. In general, no clear evidence exists of direct contact between eosinophils and adult worms, although accumulation of eosinophils about helminthic parasites has been described. Eosinophil-rich granulomas surrounding dead fragments of skin-invading larvae of the skin-invading nematode Strongyloides ratti have been described in hyperimmune rats following challenge (308). Eosinophils were also found in close contact with the surface tegument of schisto-somula of Schistosoma haematobium in the cutaneous tissue of immune monkeys, associated with the presence of a large number of dead larvae in eosinophil-rich sites (303). Similar observations were made in other host-parasite systems (135). Using appropriate antibodies, eosinophil-derived toxic proteins such as MBP have been identified on filarial worm targets in vivo (304) and levels of blood ECP is elevated in patients with filariasis, which may suggest the activation and degranulation of eosinophils (305).
Conclusions
The eosinophil is an enigmatic and fascinating cell that has intrigued biomedical scientists for more than a century. The precise function of this cell in allergic inflammation and asthma remains a matter of debate and requires further study in appropriately designed research projects. However, it is important to recognize that no single cell type, whether the eosinophil, T cell, mast cell, neutrophil, or other lung cell, is on its own responsible for all aspects of the immunopathology and clinical sequelae of airway inflammation in asthma and related diseases. In recognition of this fact, the attention currently focused on the eosinophil is warranted and timely. This relates partially to the overwhelming evidence in favor of a potential effector role of the eosinophil in parasitic helminthic and allergic diseases, including asthma. While the mechanisms of eosinophilia in association with allergic disease are not yet fully understood, they seem likely to be controlled at the level of the T-cell response to antigen and the subsequent elaboration of cytokines, which exert both direct and indirect effect on these inflammatory cells. The profile of cytokines generated in allergic reactions, such as the allergen-induced late-phase response in the skin, nose, and lung, appears to conform to a Th2 profile, because mRNA expression of IL-4 and IL-5, but not IFN-γ or IL-2, occurs or is up-regulated during these reactions. The release of IL-5 by Th2-type T cells following stimulation with allergen may, therefore, be responsible for the eosinophilia of allergic disease. Thus, a complex network of T cells, eosinophils, and other inflammatory cells as well as their cytokine products may participate in a cascade of events that leads to specific accumulation of eosinophils in sites of allergic inflammation and asthma. Whether tissue damage, a feature of these disease conditions, is the consequence of the activation and exocytosis of these infiltrating cytotoxic cells and the release of their highly basic protein products is yet to be demonstrated unequivocally.
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