Eosinophil function in allergic inflammation: From bone ... - Springer Link

Eosinophil Function in Allergic Inflammation: From Bone Marrow to Tissue Response Darryl Adamko, MD, FRCPC, Paige Lacy, PhD, and Redwan Moqbel, PhD, FRCPath

Address Department of Medicine, 550A HMRC, University of Alberta, Edmonton, AB T6G 2S2, Canada. E-mail: [email protected] Current Allergy and Asthma Reports 2004, 4:149–158 Current Science Inc. ISSN 1529-7322 Copyright © 2004 by Current Science Inc.

The role of the eosinophil in the pathophysiology of allergy and asthma has been the focus of intense interest during the past two decades. Although the presence of eosinophils in humans with allergy and asthma is well established, the precise role of this cell in human and animal tissue response is still unclear. However, recent developments in research on many organ systems have provided novel insights into the possible underlying role of the eosinophil in both allergic and nonallergic inflammation. In this review, we examine the pathways associated with eosinophil recruitment and activation, and discuss these findings with reference to clinically defined categories.

Introduction The eosinophil is a key effector cell involved in inflammatory disorders of numerous organ systems within the body. In the gastrointestinal system, eosinophilic inflammation on mucosal biopsy has been associated with both allergic (IgE-dependent) and nonallergic (IgEindependent) disorders of the esophagus, stomach, and intestine. Similarly, disorders of the respiratory tract have a strong association with the presence and activation status of eosinophils in states of atopy or otherwise. In addition, the eosinophil has long been recognized to be important in the cellular immune response against helminthic parasite infection. Although clinically these conditions have been characterized as either allergic or nonallergic, from a basic cellular level, the mechanisms underlying recruitment and activation of eosinophils appear to be similar. Therefore, in this article, we review the basic mechanisms at play in eosinophil recruitment and activation that both encompass and transcend these clinical categories.

Eosinophil Recruitment Begins in the Bone Marrow The source of tissue or blood eosinophilia is ultimately from progenitor cells in the bone marrow. Both eosinophils and basophils arise from a common CD34+ myelocytic prog enit or, which is later commit ted to the eosinophil/basophil lineage (Eo/B) [1]. At this level, committed eosinophil progenitors are characterized by positive staining for interleukin (IL)-5 receptor, the chemokine receptor CCR3, and CD34 antigen [2]. Expression of the IL-5 receptor on the progenitor cell is a sign of commitment to the eosinophil lineage [1]. Patients with atopy have higher levels of Eo/B progenitors in the blood and bone marrow compared with nonatopic individuals [3]. Asthma exacerbation or specific experimental allergen challenge of such atopic individuals causes a further increase in Eo/B progenitors, both in the blood and in the bone marrow [4]. Associated with these increases in eosinophil recruitment from the marrow is the development of airway hyperresponsiveness (AHR). Additionally, treatment with inhaled corticosteroids has been shown to prevent such increases in Eo/B progenitors and the related AHR [5]. The control of Eo/B lineage production is mediated via a network of cytokines and chemokines produced locally within the bone marrow and distally in inflamed tissues. The three key eosinophil cytokines that have been shown to stimulate the bone marrow production of eosinophils are IL-3, IL-5, and granulocyte/monocyte-colony stimulating factor (GM-CSF) [6,7]. To further aid in eosinophil de vel op me nt , b ot h IL- 5 an d GM - CSF upr e g ul at e expression of IL-5 receptors on the immature cell [1]. In addition, IL-3, IL-5, and GM-CSF continue to stimulate the eosinophil through to maturity [8,9]. These three eosinophil-activating cytokines are produced by CD4 + and CD8 + T lymphocytes from peripheral blood and inflamed tissues [10]. The switch to Th2 cytokine production is secondary to stimulation by IL-4 and IL-13 [10]. In addition to T cells, IL-3, IL-5, and GM-CSF are produced locally within the bone marrow by the eosinophils themselves [11]. Mice that are deficient in IL-5 and GM-CSF lack eosinophils in both the blood and tissue. These mice

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have continued eosinophil presence in the bone marrow, suggesting that an additional role for IL-5 and GM-CSF might be mobilization of eosinophils from bone marrow to blood [12]. Other potentially eosinophilopoietic signals are also produced by Eo/B progenitors, including IL-4, IL-6, and RANTES (regulated upon activation, normal T-cell expressed and secreted) [13,14]. Therefore, in an autocrine fashion, eosinophils might self-induce their maturation and prolong their survival. Overall, at the level of the bone marrow, the fundamental role of IL-3, IL-5, and GM-CSF is to regulate the production of eosinophils available to the peripheral circulation.

Eosinophil Production and Survival within Tissues In addition to bone marrow sources of eosinophils, the development and maturation of eosinophils might also occur in situ in peripheral sites of inflammation where an increased eosinophil presence exists. As discussed, Eo/B progenitors can be released into the circulation to reach tissue sites. Autocrine stimulation of eosinophil production might again occur with increased local production of IL-3, IL-5, and GM-CSF by eosinophils [15]. Additionally, IL-5 and GM-CSF are produced by local fibroblasts and epithelial cells. Eosinophils might enhance their own survival by directly stimulating CD4+ T cells within tissue to produce IL-5 [16]. Nasal explants from atopic patients have been shown to survive ex vivo using similar mechanisms to promote extramedullary eosinophil maturation and survival [17]. These explants, as well as lung explants of Brown-Norway rats, were shown to exhibit rapid (6 h) accumulation of major basic protein (MBP)-positive cells after allergen challenge of the explants in vitro [18]. The major signaling pathway of these events has been shown to be associated with IL-5 release, the key cytokine in eosinophil survival. IL-5 delays and might inhibit eosinophil apoptosis [19]. Stimulation of the IL-5 receptor (IL-5 R) leads to phosphorylation of JAK-2 and Lyn kinases, and decreases BAX translocation, resulting in decreased activation of the caspase family of enzymes [20,21]. Additionally, although the mechanism is not as well understood, GMCSF appears to have a strong role in inhibiting eosinophil apoptosis at the tissue level. GM-CSF stimulation of eosinophils bound to tissue sites via α4 integrin have led to eosinophil survival for 2 weeks [22]. Therefore, development, maturation, and maintenance of eosinophils at a peripheral level can also be achieved.

Chemokines Regulate Migration of Eosinophils to Peripheral Tissues Although cytokines are important in the generation of eosinophils, chemokines are responsible for the migration of eosinophils to specific tissue sites. The CCR3 family of chemokines appears to play a crucial role in generating

tissue eosinophilia [23]. Because neutrophils express negligible CCR3 receptors, this family of chemokines might be selective for granulocytes such as eosinophils and basophils. The CCR3-specific family of chemokines consists of eotaxin (1, 2, and 3), RANTES, monocyte chemoattractant protein (MCP) 2, 3, and 5, and macrophage inhibitory protein (MIP)-1α. A key member of the CCR3 family is eotaxin, as it is the only chemokine specific to eosinophils (Fig. 1) [24]. In normal humans, eosinophils migrate to the gastrointestinal tract as a part of normal development [12,25], possibly as part of their role in maintaining innate defense against parasites. Gut expression of eotaxin is higher at a basal level in these tissues [26]. During allergeninduced eosinophilia, eotaxin expression is further increased within tissues [27]. IL-5 and eotaxin appear to work synergistically, as IL-5 stimulation enhances the eosinophil response to eotaxin both in vitro and in vivo [28,29]. Stimulation enhances the eosinophil response to eotaxin both in vitro and in vivo [28,29]. Both IL-5 and eotaxin appear to be increased in the airways during asthma exacerbation [30]. Allergen challenge also increases expression of CCR3 on bone marrow eosinophil progenitor cells. To specifically isolate the role of eotaxin, eotaxin gene knockout (KO) mice have been deployed [12,31]. Eotaxin KO mice (Eo-/-) produce IL-5 normally, and therefore, like wild type (WT) mice, continue to develop blood eosinophilia. In contrast, Eo -/- mice do not develop tissue eosinophilia. Therefore, the primary role of the CCR3 receptor appears to be involved with the stimulation of eosinophils and eosinophil progenitors to migrate from the bone marrow and blood to tissue targets expressing eotaxin. While eotaxin has been shown to be a key factor in the development of tissue eosinophilia, the additional chemokines of the CCR3 family play important roles where eotaxin is not necessarily essential [32,33]. In addition to epithelial and endothelial sources, RANTES, MCP-1, and MIP-1α are produced by T cells, macrophages, fibroblasts, and eosinophils [15,34]. Similar to eotaxin, increased expression of these chemokines has been associated with eosinophilmediated AHR [32] and childhood asthma [35]. Also, each chemokine appears to have a unique role in the timing 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 [36]. Similar to eotaxin, eosinophils stimulated with IL-5 have increased affinity to RANTES. However, unlike eotaxin, RANTES has been associated with exacerbations of eosinophilic bronchitis, which often arise from viral infection. Eosinophilia in association with respiratory syncytial virus (RSV) infection has been correlated with increased IL-5, MCP-1, MIP-1α, and RANTES expression [37,38]. During naturally acquired viral infection, children with asthma have large increases in eosinophil-associated MBP, RANTES, and MIP-1α in their nasal secretions [39]. Therefore, although RANTES, MIP-1α,

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Figure 1. Receptors expressed in resting human peripheral blood eosinophils. These receptors, representing a small proportion of the total surface receptors detected in eosinophils, are crucial to eosinophil function and in many cases lead to activation of degranulation in these cells.

and MCP-1 bind specifically to CCR3 receptor on eosinophils, their apparent broader range of effects might also increase the range of eosinophil activity.

Development of Tissue Eosinophilia The process of migration of eosinophils into tissues through the endothelium is under complex regulation. Most migration through endothelium occurs at postcapillary venules. The first step in eosinophil entrance to tissue during transmigration from circulating blood involves adhesion to the vessel endothelium. As for other leukocytes, the initial phase of eosinophil contact with the endothelium is cell rolling. The endothelial cell surface expresses various receptors, including E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule (ICAM)-1 (CD54), and vascular cell adhesion molecule (VCAM)-1 (CD106) [33]. The eosinophil expresses L-selectin (CD62L), P-selectin glycoprotein ligand-1 (PSGL-1), and the integrins α4β1 (VLA-4), α4β7, and β2 (CD18), which are involved in cell rolling [33]. VLA-4 (α4β1) and α4β7 are unique to the eosinophil and allow binding to VCAM-1 [40]. Although all these molecules can interact to play a role in the initial step of eosinophil rolling, the final step of firm binding to endothelium remains solely dependent on either VCAM-1– or ICAM-1–related mechanisms [33]. The expression of these molecules, whether on the eosinophil or endothelium, depends on many factors related to their respective activation state. It appears that IL-4 and IL-13 play a role in the migration of eosinophils into tissues. Again, CD4+ and CD8+ T cells are responsible for the production of these key cytokines. IL-13 KO mice have been shown to develop pulmonary eosinophilia and AHR following allergen

challenge. When these mice were treated with an antibody to IL-4, pulmonary eosinophil numbers were reduced, along with AHR, despite the continued development of blood eosinophilia [41]. Loss of either cytokine alone did not deplete the lungs of eosinophils or prevent the development of AHR. IL-13 acts on the IL-4 receptor α subunit, suggesting redundancy in the actions of these two cytokines in allergic disease [42,43]. The mechanism for generation of tissue eosinophilia appears to be through VCAM-1. Both IL-4 and IL-13 upregulate the expression of VCAM-1 [44] and P-selectin [45] on the endothelium, thus allowing increased eosinophils to leave the circulation via rolling and binding. Both IL-4 and IL-13 mediate many of their activities through the nuclear transcription factor STAT-6 [46,47]. In STAT-6-/- mice, decreased tissue eosinophilia was observed, although VCAM-1 expression remained high after allergen challenge. The difference in tissue eosinophil numbers in STAT-6-/- mice was thought to be due to decreased eosinophil expression of CCR3 receptor, which is directly controlled by STAT-6 [48]. The results from STAT-6-/- mice suggests that IL-4 and IL-13 also have a role in the induction of CCR3 receptor on eosinophils and T cells. Therefore, IL-4 and IL-13 appear to be important in the generation of blood eosinophilia and the entrance of these cells into tissue.

Activation of Eosinophils Increases Binding and Migration The type of eosinophil binding occurring on the endothelium, whether via VCAM or ICAM, depends on the activation status of the eosinophil. From fresh blood samples, unactivated eosinophils prefer binding via VLA-4 (α4β1) to endothelial VCAM-1 rather than β2 to ICAM-1

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binding [23,49]. If activated, the preference for VCAM binding has been shown to decrease, whereas increased binding via ICAM-1 to β2 integrins has been observed [23,50–52]. During entrance into tissues, the eosinophil behaves in a more activated state. Tissue eosinophils from an antigen challenge model express increased CD11, CD69, and ICAM-1 [53]. Eosinophil binding in tissues switches to ICAM-1 and the CS-1 region of tissue fibronectin [33,50,54]. Eotaxin appears to increase CD11/CD18 expression, further promoting ICAM-1–mediated migration [55]. The change in the activation status is also confirmed by the cell surface changes 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 [21]. The switch to an ICAM-1–mediated pathway might be related to a need for faster eosinophil tissue entrance. Increased β1 expression (VCAM-associated) has been shown to slow eosinophil migration compared with ICAM-1/β2 [56]. If one is attempting to block tissue eosinophilia, an important point is that anti-VLA-4 antibodies might not prevent eosinophil migration into tissue if ICAM-1 or P-selectin sites are selected first by these activated eosinophils [33]. Factors in tissue responsible for this activation might be IL-5 and GM-CSF, which have been shown to enhance transendothelial migration [57]. IL-5 has been shown to activate eosinophils such that increased transendothelial migration occurs through ICAM-1 via decreased β1 and increased β2 expression [58]. Similarly, stimulation of the eosinophil CCR3 receptor also increases β2 expression and ICAM-1 binding [59]. Complement-mediated inflammation, as seen with parasite infection, increases release of complement proteins C3a and C5a. Whereas C3a increases binding of eosinophil to endothelium but does not increase migration, C5a increases both adhesion and migration [60]. Both factors are blocked by anti-α4 and -β2 antibodies, suggesting the importance of both VCAM and ICAM in the complementmediated pathway of anaphylaxis and host defense. In addition to these specific eosinophil adherence pathways, general inflammatory cytokines, such as IL-1 and tumor necrosis factor (TNF), are released by inflamed tissue and have significant effects on eosinophil migration [33]. Both IL-1 and TNF messenger RNA (mRNA) levels are increased in the airways of symptomatic versus nonsymptomatic asthmatics [61]. Increased IL-1 is found in the tissue of cutaneous allergy sites [62]. Antibodies to IL-1 decrease the tissue expression of VCAM-1 and ICAM-1 [63]. IL-1 KO mice have decreased eosinophil rolling, adhesion, and transmigration [64]. Similarly, TNF increases expression of endothelial ICAM-1, VCAM, P-selectin, and E-selectin, leading to increased eosinophil rolling and adhesion [65–67]. Finally, like the IL-1 KO mouse, TNF KO mice show decreased eosinophil adhesion and migration into tissue [68].

Factors Influencing Eosinophil Migration through Tissues Eosinophils move in tissue via extensions of their cytoplasm, called lamellae, thus leading to lamellar motion [33]. In theory, to move, there must be increased binding forward via the uropod and release of binding to the rear. The mechanisms for this are not well understood, but changes in binding affinity secondary to various stimuli are likely to be important. A change in binding affinity of VLA-4 to fibronectin has been demonstrated [69], thus stimulating de-adherence, which might allow the release of the cell and enable it to move on. Factors such as GM-CSF have been shown to increase binding affinity of VLA-4 to VCAM or CS-1 [70], whereas eotaxin stimulates the reverse [59]. Eotaxin might also be involved in cytoskeletal changes via mitogen-activated protein kinases (MAPK) [33]. RANTES, MCP-3, and C5a might also alter β1 integrin affinity [50,71]. Inhaled corticosteroids might also have a role in inhibiting intracellular cytoskeletal changes required for eosinophil adhesion and migration [72]. The balance of these factors determine the rate of eosinophil migration.

Tissue Eosinophilia Is More Important than Blood Eosinophilia in Mediating Airway Hyperresponsiveness The mechanisms involved in the development of blood eosinophilia are distinct from those of tissue eosinophilia. Again, work in transgenic mice has confirmed this. In an allergen challenge model, increased blood and tissue IL-5 levels are evident in WT mice [73]. These levels correlate with both blood and tissue eosinophilia and AHR. In the same study, IL-5 KO mice did not mount a blood or tissue eosinophil response after allergen challenge, nor did they develop AHR. Restoring IL-5 expression in these animals via vaccinia virus encoding IL-5 reconstituted blood and tissue eosinophilia with an associated development of AHR. The role of IL-5 in the mouse model is still in question. Depending on the protocol used for sensitization and challenge, AHR might still persist in spite of treatment with an antibody to IL-5 and depletion of blood eosinophils. Therefore, blood eosinophilia will be lost, but AHR might persist during allergen challenge. The answer to this dilemma might be in the persisting presence of eosinophils in tissue sites even during IL-5 depletion, which has been suggested in results from Eo-/- mice. Despite developing blood eosinophilia, Eo -/- mice do not mount the same degree of tissue eosinophil response [16], nor do they develop eosinophil-mediated tissue damage following allergen challenge [74]. However, in double KO mice (IL-5 -/- /Eo -/- ), both the blood and tissue eosinophil numbers are depleted, and AHR is significantly decreased during allergen challenge [75]. These studies indicate distinct roles for eotaxin and IL-5 in eosinophil maturation, proliferation, and homing to target tissues. Whereas


IL-5 is critical for the maturation and proliferation of eosinophils in the bone marrow, it appears that eotaxin is equally essential for movement and maintenance of eosinophilia in the tissues. Shen et al. [76•] recently demonstrated that eosinophils 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, migrate back into lung, and reconstitute the asthma phenotype seen in wild-type antigen-challenged animals. Therefore, although IL-5 might be essential in maturation and differentiation of eosinophils in the bone marrow [1], the recruitment of eosinophils to the tissues and their subsequent function might be IL-5independent. Therefore, a key event in eosinophil-mediated inflammation leading to AHR might be the persistence of activated eosinophils in the tissue.

The Importance of Activation in Eosinophil-mediated Effects Similar to the relation of AHR to eosinophilia, which goes beyond the circulating level of eosinophils, both the presence and activation of tissue eosinophils seem to be important. In the mouse, transgenically induced expression of IL-5 or eotaxin caused increased lung eosinophilia, but not AHR [77]. Only with subsequent allergen challenge did AHR develop [29]. In the same way, in guinea pigs, allergen sensitization alone without challenge increased the number of eosinophils in the lung lavage compared with nonsensitized controls, but airway function remained normal [78]. This is different from many mouse models in which aggressive allergen sensitization plus challenge are required to even mount an increased eosinophil presence in tissue. Perhaps paradoxically, despite having increased tissue eosinophil numbers, allergen-challenged mice frequently have normal airway function. In contrast, if sensitized guinea pigs are allergen-challenged or virus-infected, they develop eosinophil-mediated AHR [78–80]. Similarly, in humans with asymptomatic asthma, increased eosinophils are seen in the airways on lavage and biopsy [81]. It is only during exacerbation [82,83] or experimental challenge [84,85] that evidence of eosinophil-induced AHR is obtained. The manifestation of AHR appears to correlate strongly with evidence of eosinophils actively degranulating in the airways. Murine models of asthma suffer from an apparent deficiency when compared with clinical disease, in that murine eosinophils fail to exhibit any ability to undergo active degranulation either in vivo or in vitro. This might account for the discrepancies in “normal” lung function that is sometimes observed in allergen-challenged mice. In addition, the specific location of the eosinophils within the tissue might also be important. In the lung [86] and in the intestines [25], the close association of eosinophils with nerves has been related to eosinophil-mediated dysfunction [87]. Unlike nonsensitized, control guinea pigs, ovalbumin-sensitized animals appear to develop AHR in response to virus infection through an eosinophil-

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mediated process. The difference appeared to be related to the close positioning of eosinophils to the airway nerves [88•]. Therefore, just as producing blood eosinophilia does not appear to be enough to develop AHR, tissue eosinophilia per se might not be enough to induce symptoms. These findings suggest that, even within tissue eosinophils, there are additional factors controlling the activation of eosinophils to become effector cells through degranulation and mediator release at key sites.

Factors in Eosinophil Degranulation and Mediator Release The morphology of eosinophils as seen by electron microscopy varies from that of a resting state, to partially activated or “primed” cells (stimulated without undergoing active secretion), and finally to that of fully activated degranulating cells (Fig. 2) [89]. Morphologic markers of eosinophil priming include increased size and numbers of lipid bodies, as well as increased numbers of primary granules, small granules, and vesiculotubular structures. Smooth endoplasmic reticulum and crystals of CLC protein, the latter of which are not membrane-bound, might also appear in the cytoplasm. With further activation and degranulation, there is often a marked change in the appearance of crystalloid secretory granules, which might appear translucent, as if emptied of their contents, particularly in tissue eosinophils (Fig. 3A). Degranulation is the end result of full activation of the eosinophil. Eosinophils can release their secretory granule contents by four possible mechanisms: piecemeal degranulation, granule exocytosis, compound exocytosis, and necrosis (cytolysis). The most commonly observed form of eosinophil degranulation in situ in allergic tissues is piecemeal degranulation [90]. In piecemeal degranulation, numerous small vesicles appear in the cytoplasm, whereas the crystalloid granules appear to be gradually losing their matrix components and crystalline cores (creating a “mottled” appearance) [89]. This is 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 secondary granules to the outside of the cell. In granule exocytosis, sometimes referred to as the “classic” form of exocytosis, the crystalloid granules fuse directly with the plasma membrane prior to releasing their contents to the outside of the cell (Fig. 3B). Compound exocytosis might also occur, whereby secondary granules fuse with each other prior to release from the cell through a single fusion pore, which has been demonstrated in tannic acid-arrested degranulating eosinophils [91]. Although granule and compound exocytosis have been shown to occur in eosinophils from patients with inflammatory bowel disease and tissue invasive infections, it is unusual to see morphologic evidence of classic or compound exocytosis in eosinophilic

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Figure 2. Events associated with priming and activation of eosinophils during recruitment to peripheral tissues. In the upper panel, scanning electron microscopy analysis of human peripheral blood eosinophils adhering to fibroblasts in culture is shown. Images indicate: (A) a resting eosinophil, (B) a “primed” eosinophil showing lamellipodia formation, and (C) a fully activated, degranulating eosinophil. Bars indicate 10 ␮m. Drawings below the photographs show upregulation of activity during eosinophil emigration from bone marrow, circulation through the blood, and transmigration into sites of tissue inflammation.

inflammation. In cytolysis, eosinophils at sites of inflammation appear disrupted, with cells exhibiting loss of integrity of plasma membrane and spilling their contents into the surrounding tissues. Release of intact or disrupted granules into the interstitium is likely to have toxic effects on the surrounding cells. A novel in vitro model of piecemeal degranulation was reported in which secretory vesicles containing RANTES were demonstrated to be selectively released following interferon (IFN)-γ stimulation of eosinophils [92]. Using confocal microscopy and subcellular fractionation, eosinophils were shown to mobilize secretory vesicles containing RANTES to the cell membrane immediately following stimulation by IFN-γ (10 min), whereas RANTES stored in the crystalloid granules was selectively released at a later stage of incubation (60 min) [92]. These findings suggested that eosinophils have the ability to selectively remove crystalloid granule contents, possibly via a shuttling mechanism through the small secretory vesicles, and transport these to the outside of the cell during receptor stimulation.

Guanosine Triphosphatases and SNARE Fusion Proteins Are Implicated in Regulation of Eosinophil Exocytosis The two essential effectors known to induce exocytosis in eosinophils are Ca2+ and guanosine triphosphate (GTP) (as determined in studies using the slowly hydrolyzed analog, GTPγ S) [93]. The requirement for GTP and Ca2+ has been shown in permeabilization studies [93] and by patch-clamp analysis of eosinophils [94]. The obligatory requirement of eosinophils for GTP suggests that at least one GTP-binding protein is crucial to the process of degranulation. The evidence gathered from these studies has been used to support the notion that a hypothetical GTP-binding protein, GE, exists to regulate exocytotic release [95]. Studies det ermin in g which proteins migh t be candidates for GE in eosinophils have been lacking in the literature. Several publications have alluded to a ras-related GTP-binding protein, Rab3, as being an important regulatory GTPase required for exocytotic fusion in many cell types [96]. Recent data suggest that Rab3D is expressed in


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Figure 3. Modes of degranulation in tissue eosinophils in allergic disease. A, Earlier studies using electron microscopic analysis have shown that approximately 67% of tissue eosinophils appear to be degranulating in a piecemeal manner in nasal biopsies of allergic individuals, while the remaining eosinophils appeared to undergo cytolysis (necrotic release). B, The process of granule exocytosis involves membrane fusion between the granule and the plasma membrane, which is tightly controlled by signaling molecules in the cytoplasm as well as on membrane surfaces. The mechanisms controlling eosinophil degranulation in granule exocytosis and piecemeal degranulation are believed to be very similar.

mast cells [97,98] and is functionally involved in activating phosphorylation of fusion proteins belonging to a family of SNARE molecules essential for membrane fusion (see later), suggesting that Rab3D might be the regulatory GTP-binding protein required for IgE-mediated degranulation in mast cells [99]. However, a later study utilizing a Rab3D gene deletion model in mice showed that there was no effect of this mutation on the exocytotic release of mediators from mast cells [100]. Furthermore, Rab3 isoforms have not been detected so far in eosinophils [101], indicating that Rab3 might not be important in exocytosis in all secretory cells. In addition to GTP-binding proteins, cells that undergo secretion require a set of intracellular “receptors” that specifically recognize and fuse with membrane-bound compartments during vesicular trafficking. In exocytosis, it has been suggested that intracellular receptors must reside on the surface of the mobilized secretory granule or vesicle in order for it to recognize and bind (“dock”) to another receptors on the inner leaflet of the plasma membrane prior to membrane fusion. Such specific protein-protein interaction is hypothesized to be essential for granule release to occur in a directional and highly regulated manner. Studies on many types of secretory cells, particularly neuronal cells and yeast, have revealed the existence of a highly homologous complex of fusion proteins that might

at least partially fulfill this role. These are known as SNAREs (SNAP receptors) for their ability to bind with the cytosolic fusion complex proteins SNAP (soluble NSF attachment protein) and NSF (N-ethylmaleimide-sensitive factor) (Fig. 3B) [102]. SNAREs have been hypothesized to lend specificity to granule-granule and granule-plasma membrane fusion and have been divided into two groups, vesicular SNAREs (v-SNAREs) deployed on the surface of secretory vesicles, and target SNAREs (t-SNAREs), present on the inner leaflet of the target membrane. The key components of the mammalian neuronal SNARE complex are syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP)-1 [102]. Interestingly, a different set of isoforms of SNAREs (syntaxin-4, SNAP-23, and VAMP-2) have been detected in inflammatory cells. Eosinophils do not express detectable levels of classic SNARE proteins (syntaxin-1, SNAP-25, and VAMP-1) [101]. However, the vSNARE isoform VAMP-2 was recently found to localize to the same population of small secretory vesicles containing RANTES, and translocated in correlation with RANTES during IFN-γ stimulation [103]. In addition, eosinophils express the t-SNAREs, SNAP-23, and syntaxin-4 as potential cognate binding sites for VAMP-2 in degranulation [104•]. These results indicate that non-neuronal SNAREs might play a crucial role in the regulation of granule fusion and mediator release in eosinophils.

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Conclusions Research into the immunobiology of the eosinophil has recently encountered a number of interesting challenges, including whether this inflammatory cell type is relevant in the pathophysiology of the late phase response in asthma. However, the studies that questioned the role of the eosinophil seemed to have been mainly influenced by their design and/or the animal model used. Because allergic inflammation involves a complex spatial and temporal interplay between a complex array of cells and mediators, a precise functional role for any cell type at a given stage of disease will likely remain elusive. Therefore, like many other cell types in the immune system, the eosinophil is an inflammatory cell that exhibits a spectrum of effects spanning both positive and negative consequences. Regardless, we remain convinced that the eosinophil is a key effector cell involved in inflammatory disorders of numerous organ systems. This enigmatic cell has a potential role in regulating immune responses beyond its capacity to secrete proinflammatory mediators and cytokines. Indeed, ongoing studies will further extend our understanding of potential and novel roles for the eosinophil in homeostatic, immune, and inflammatory reactions in human health.

References and Recommended Reading Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1.

2.

3.

4.

5.

6.

7. 8.

9.

Denburg JA, Sehmi R, Saito H, et al.: Systemic aspects of allergic disease: bone marrow responses. J Allergy Clin Immunol 2000, 106:S242–S246. Sutherland DR, Stewart AK, Keating A: CD34 antigen: molecular features and potential clinical applications. Stem Cells 1993, 11:50–57. Sehmi R, Howie K, Sutherland DR, et al.: Increased levels of CD34+ hemopoietic progenitor cells in atopic subjects. Am J Respir Cell Mol Biol 1996, 15:645–655. Gibson PG, Manning PJ, O’Byrne PM, et al.: Allergen-induced asthmatic responses: relationship between increases in airway responsiveness and increases in circulating eosinophils, basophils, and their progenitors. Am Rev Respir Dis 1991, 143:331–335. Gauvreau GM, Wood LJ, Sehmi R, et al.: The effects of inhaled budesonide on circulating eosinophil progenitors and their expression of cytokines after allergen challenge in subjects with atopic asthma. Am J Respir Crit Care Med 2000, 162:2139–2144. Denburg JA: Bone marrow in atopy and asthma: hematopoietic mechanisms in allergic inflammation. Immunol Today 1999, 20:111–113. Foster PS: Allergic networks regulating eosinophilia. Am J Respir Cell Mol Biol 1999, 21:451–454. Yamaguchi Y, Hayashi Y, Sugama Y, et al.: Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor. J Exp Med 1988, 167:1737–1742. Clutterbuck EJ, Hirst EM, Sanderson CJ: Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF. Blood 1989, 73:1504–1512.

10. 11.

12.

13.

14.

15. 16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

28.

29.

30.

Hamelmann E, Gelfand EW: IL-5-induced airway eosinophilia—the key to asthma? Immunol Rev 2001, 179:182–191. Gauvreau GM, O’Byrne PM, Moqbel R, et al.: Enhanced expression of GM-CSF in differentiating eosinophils of atopic and atopic asthmatic subjects. Am J Respir Cell Mol Biol 1998, 19:55–62. Mishra A, Hogan SP, Lee JJ, et al.: Fundamental signals that regulate eosinophil homing to the gastrointestinal tract. J Clin Invest 1999, 103:1719–1727. Mahmudi-Azer S, Velazquez JR, Lacy P, et al.: Immunofluorescence analysis of cytokine and granule protein expression during eosinophil maturation from cord blood-derived CD34 progenitors. J Allergy Clin Immunol 2000, 105:1178–1184. Velazquez JR, Lacy P, Mahmudi-Azer S, et al.: Interleukin-4 and RANTES expression in maturing eosinophils derived from human cord blood CD34+ progenitors. Immunology 2000, 101:419–425. Lacy P, Moqbel R: Eosinophil cytokines. Chem Immunol 2000, 76:134–155. Foster PS, Mould AW, Yang M, et al.: Elemental signals regulating eosinophil accumulation in the lung. Immunol Rev 2001, 179:173–181. Cameron L, Christodoulopoulos P, Lavigne F, et al.: Evidence for local eosinophil differentiation within allergic nasal mucosa: inhibition with soluble IL-5 receptor. J Immunol 2000, 164:1538–1545. Eidelman DH, Minshall E, Dandurand RJ, et al.: Evidence for major basic protein immunoreactivity and interleukin 5 gene activation during the late phase response in explanted airways. Am J Respir Cell Mol Biol 1996, 15:582–589. Simon HU, Yousefi S, Schranz C, et al.: Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia. J Immunol 1997, 158:3902–3908. Pazdrak K, Olszewska-Pazdrak B, Stafford S, et al.: Lyn, Jak2, and Raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only Raf-1 kinase is essential for eosinophil activation and degranulation. J Exp Med 1998, 188:421–429. Wardlaw AJ, Brightling C, Green R, et al.: Eosinophils in asthma and other allergic diseases. Br Med Bull 2000, 56:985–1003. Anwar AR, Moqbel R, Walsh GM, et al.: Adhesion to fibronectin prolongs eosinophil survival. J Exp Med 1993, 177:839–843. Bochner BS, Schleimer RP: Mast cells, basophils, and eosinophils: distinct but overlapping pathways for recruitment. Immunol Rev 2001, 179:5–15. Rothenberg ME: Eotaxin: an essential mediator of eosinophil trafficking into mucosal tissues. Am J Respir Cell Mol Biol 1999, 21:291–295. Rothenberg ME, Mishra A, Brandt EB, Hogan SP: Gastrointestinal eosinophils. Immunol Rev 2001, 179:139–155. Zimmermann N, Hogan SP, Mishra A, et al.: Murine eotaxin-2: a constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression. J Immunol 2000, 165:5839-5846. Rothenberg ME, Luster AD, Lilly CM, et al.: Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung. J Exp Med 1995, 181:1211–1216. Collins PD, Marleau S, Griffiths-Johnson DA, et al.: Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo. J Exp Med 1995, 182:1169–1174. Mould AW, Ramsay AJ, Matthaei KI, et al.: The effect of IL-5 and eotaxin expression in the lung on eosinophil trafficking and degranulation and the induction of bronchial hyperreactivity. J Immunol 2000, 164:2142–2150. Park SW, Kim DJ, Chang HS, et al.: Association of interleukin-5 and eotaxin with acute exacerbation of asthma. Int Arch Allergy Immunol 2003, 131:283–290.


31.

32.

33. 34. 35.

36.

37.

38.

39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49.

50.

Matthews AN, Friend DS, Zimmermann N, et al.: Eotaxin is required for the baseline level of tissue eosinophils. Proc Natl Acad Sci U S A 1998, 95:6273–6278. Gonzalo JA, Lloyd CM, Wen D, et al.: The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J Exp Med 1998, 188:157–167. Broide D, Sriramarao P: Eosinophil trafficking to sites of allergic inflammation. Immunol Rev 2001, 179:163–172. Schall TJ: Biology of the RANTES/SIS cytokine family. Cytokine 1991, 3:165–183. Rojas-Ramos E, Avalos AF, Perez-Fernandez L, et al.: Role of the chemokines RANTES, monocyte chemotactic proteins-3 and -4, and eotaxins-1 and -2 in childhood asthma. Eur Respir J 2003, 22:310–316. Kaburagi Y, Shimada Y, Nagaoka T, et al.: Enhanced production of CC-chemokines (RANTES, MCP-1, MIP-1 beta, MIP-1beta, and eotaxin) in patients with atopic dermatitis. Arch Dermatol Res 2001, 293:350–355. Noah TL, Becker S: Chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus. Clin Immunol 2000, 97:43–49. Kim CK, Kim SW, Park CS, et al.: Bronchoalveolar lavage cytokine profiles in acute asthma and acute bronchiolitis. J Allergy Clin Immunol 2003, 112:64–71. Teran LM, Seminario MC, Shute JK, et al.: RANTES, macrophageinhibitory protein 1alpha, and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children. J Infect Dis 1999, 179:677–681. Bochner BS, Luscinskas FW, Gimbrone MA Jr, et al.: Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. J Exp Med 1991, 173:1553–1557. Webb DC, McKenzie AN, Koskinen AM, et al.: Integrated signals between IL-13, IL-4, and IL-5 regulate airways hyperreactivity. J Immunol 2000, 165:108–113. Grunig G, Warnock M, Wakil AE, et al.: Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998, 282:2261–2263. Wills-Karp M, Luyimbazi J, Xu X, et al.: Interleukin-13: central mediator of allergic asthma. Science 1998, 282:2258–2261. Bochner BS, Klunk DA, Sterbinsky SA, et al.: IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells. J Immunol 1995, 154:799–803. Woltmann G, McNulty CA, Dewson G, et al.: Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow. Blood 2000, 95:3146–3152. Kuperman D, Schofield B, Wills-Karp M, Grusby MJ: Signal transducer and activator of transcription factor 6 (STAT6)deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J Exp Med 1998, 187:939–948. Akimoto T, Numata F, Tamura M, et al.: Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6deficient mice. J Exp Med 1998, 187:1537–1542. Matsukura S, Stellato C, Georas SN, et al.: Interleukin-13 upregulates eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism. Am J Respir Cell Mol Biol 2001, 24:755–761. Matsumoto K, Sterbinsky SA, Bickel CA, et al.: Regulation of alpha4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1. J Allergy Clin Immunol 1997, 99:648–656. Weber C, Katayama J, Springer TA: Differential regulation of beta1 and beta2 integrin avidity by chemoattractants in eosinophils. Proc Natl Acad Sci U S A 1996, 93:10939–10944.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61. 62.

63.

64.

65.

66.

67.

68.

69.

157

Ebisawa M, Bochner BS, Georas SN, Schleimer RP: Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1betainduced transendothelial migration. J Immunol 1992, 149:4021–4028. Shahabuddin S, Ponath P, Schleimer RP: Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines. J Immunol 2000, 164:3847–3854. Georas SN, Liu MC, Newman W, et al.: Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge. Am J Respir Cell Mol Biol 1992, 7:261–269. Rothenberg ME, MacLean JA, Pearlman E, et al.: Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J Exp Med 1997, 185:785–790. Liu L, Hakansson L, Ridefelt P, et al.: Priming of eosinophil migration across lung epithelial cell monolayers and upregulation of CD11b/CD18 are elicited by extracellular Ca2+. Am J Respir Cell Mol Biol 2003, 28:713–721. Kuijpers TW, Mul EP, Blom M, et al.: Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration. J Exp Med 1993, 178:279–284. Ebisawa M, Liu MC, Yamada T, et al.: Eosinophil transendothelial migration induced by cytokines. II. Potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J Immunol 1994, 152:4590–4596. Werfel SJ, Yednock TA, Matsumoto K, et al.: Functional regulation of beta1 integrins on human eosinophils by divalent cations and cytokines. Am J Respir Cell Mol Biol 1996, 14:44–52. Tachimoto H, Burdick MM, Hudson SA, et al.: CCR3-active chemokines promote rapid detachment of eosinophils from VCAM-1 in vitro. J Immunol 2000, 165:2748–2754. DiScipio RG, Daffern PJ, Jagels MA, et al.: A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo. J Immunol 1999, 162:1127–1136. Broide DH, Lotz M, Cuomo AJ, et al.: Cytokines in symptomatic asthma airways. J Allergy Clin Immunol 1992, 89:958–967. Bochner BS, Charlesworth EN, Lichtenstein LM, et al.: Interleukin1 is released at sites of human cutaneous allergic reactions. J Allergy Clin Immunol 1990, 86:830–839. Hirata N, Kohrogi H, Iwagoe H, et al.: Allergen exposure induces the expression of endothelial adhesion molecules in passively sensitized human bronchus: time course and the role of cytokines. Am J Respir Cell Mol Biol 1998, 18:12–20. Broide DH, Campbell K, Gifford T, Sriramarao P: Inhibition of eosinophilic inflammation in allergen-challenged, IL-1 receptor type 1-deficient mice is associated with reduced eosinophil rolling and adhesion on vascular endothelium. Blood 2000, 95:263–269. Walsh LJ, Trinchieri G, Waldorf HA, et al.: Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1. Proc Natl Acad Sci U S A 1991, 88:4220–4224. Mackay F, Loetscher H, Stueber D, et al.: Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J Exp Med 1993, 177:1277–1286. Neumann B, Machleidt T, Lifka A, et al.: Crucial role of 55kilodalton TNF receptor in TNF-induced adhesion molecule expression and leukocyte organ infiltration. J Immunol 1996, 156:1587–1593. Broide DH, Stachnick G, Castaneda D, et al.: Inhibition of eosinophilic inflammation in allergen-challenged TNF receptor p55/p75- and TNF receptor p55-deficient mice. Am J Respir Cell Mol Biol 2001, 24:304–311. Masumoto A, Hemler ME: Multiple activation states of VLA-4. Mechanistic differences between adhesion to CS1/fibronectin and to vascular cell adhesion molecule-1. J Biol Chem 1993, 268:228–234.

158

70.

Asthma

Sung KL, Li Y, Elices M, et al.: Granulocyte-macrophage colonystimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils. J Immunol 1997, 158:919–927. 71. Kitayama J, Mackay CR, Ponath PD, Springer TA: The C-C chemokine receptor CCR3 participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow. J Clin Invest 1998, 101:2017–2024. 72. Delbrouck C, Doyen I, Belot N, et al.: Galectin-1 is overexpressed in nasal polyps under budesonide and inhibits eosinophil migration. Lab Invest 2002, 82:147–158. 73. Hogan SP, Koskinen A, Matthaei KI, et al.: Interleukin-5producing CD4+ T cells play a pivotal role in aeroallergeninduced eosinophilia, bronchial hyperreactivity, and lung damage in mice. Am J Respir Crit Care Med 1998, 157:210–218. 74. Hogan SP, Mishra A, Brandt EB, et al.: A pathological function for eotaxin and eosinophils in eosinophilic gastrointestinal inflammation. Nat Immunol 2001, 2:353–360. 75. Mattes J, Yang M, Mahalingam S, et al.: Intrinsic defect in T cell production of interleukin (IL)-13 in the absence of both IL-5 and eotaxin precludes the development of eosinophilia and airways hyperreactivity in experimental asthma. J Exp Med 2002, 195:1433–1444. 76.• Shen HH, Ochkur SI, McGarry MP, et al.: A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse. J Immunol 2003, 170:3296–3305. This paper describes the ability of eosinophils instilled into the trachea of IL-5 knockout mice to reconstitute the asthma phenotype seen in wild-type, allergen-challenged animals in an IL-5–independent manner, suggesting that eosinophils might not require IL-5 for their persistence and function within tissues. 77. Mould AW, Matthaei KI, Young IG, Foster PS: Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice. J Clin Invest 1997, 99:1064–1071. 78. Adamko DJ, Yost BL, Gleich GJ, et al.: Ovalbumin sensitization changes the inflammatory response to subsequent parainfluenza infection: Eosinophils mediate airway hyperresponsiveness, M2 muscarinic receptor dysfunction, and antiviral effects. J Exp Med 1999, 190:1465–1478. 79. Evans CM, Fryer AD, Jacoby DB, et al.: Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs. J Clin Invest 1997, 100:2254–2262. 80. Costello RW, Evans CM, Yost BL, et al.: Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils. Am J Physiol 1999, 276:L709–L714. 81. Kirby JG, Hargreave FE, Gleich GJ, O’Byrne PM: Bronchoalveolar cell profiles of asthmatic and nonasthmatic subjects. Am Rev Respir Dis 1987, 136:379–383. 82. Bousquet J, Chanez P, Lacoste JY, et al.: Eosinophilic inflammation in asthma. N Engl J Med 1990, 323:1033–1039. 83. Frigas E, Loegering DA, Solley GO, et al.: Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clin Proc 1981, 56:345–353. 84. Wu W, Samoszuk MK, Comhair SA, et al.: Eosinophils generate brominating oxidants in allergen-induced asthma. J Clin Invest 2000, 105:1455–1463. 85. Kelly EA, Busse WW, Jarjour NN: Inhaled budesonide decreases airway inflammatory response to allergen. Am J Respir Crit Care Med 2000, 162:883–890. 86. Costello RW, Schofield BH, Kephart GM, et al.: Localization of eosinophils to airway nerves and effect on neuronal M2 muscarinic receptor function. Am J Physiol 1997, 273:L93–L103. 87. Jacoby DB, Costello RM, Fryer AD: Eosinophil recruitment to the airway nerves. J Allergy Clin Immunol 2001, 107:211–218.

88.• Adamko DJ, Fryer AD, Bochner BS, Jacoby DB: CD8+ T lymphocytes in viral hyperreactivity and M2 muscarinic receptor dysfunction. Am J Respir Crit Care Med 2003, 167:550–556. Eosinophils were shown to position themselves close to airway nerves in this study, and were suggested to be responsible for inducing airway hyperresponsiveness during viral infections by degranulating onto the nerves themselves and causing M2 muscarinic receptor dysfunction. This was suggested to lead to enhanced cholinergic release, an important contributing factor in airway hyperresponsiveness. 89. Dvorak AM, Weller PF: Ultrastructural analysis of human eosinophils. Chem Immunol 2000, 76:1–28. 90. Erjefalt JS, Greiff L, Andersson M, et al.: Allergen-induced eosinophil cytolysis is a primary mechanism for granule protein release in human upper airways. Am J Respir Crit Care Med 1999, 160:304–312. 91. Newman TM, Tian M, Gomperts BD: Ultrastructural characterization of tannic acid-arrested degranulation of permeabilized guinea pig eosinophils stimulated with GTP-gamma-S. Eur J Cell Biol 1996, 70:209–220. 92. Lacy P, Mahmudi-Azer S, Bablitz B, et al.: Rapid mobilization of intracellularly stored RANTES in response to interferonalpha in human eosinophils. Blood 1999, 94:23–32. 93. Cromwell O, Bennett JP, Hide I, et al.: Mechanisms of granule enzyme secretion from permeabilized guinea pig eosinophils: dependence on Ca2+ and guanine nucleotides. J Immunol 1991, 147:1905–1911. 94. Nüsse O, Lindau M, Cromwell O, et al.: Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils. J Exp Med 1990, 171:775–786. 95. Gomperts BD: GE: a GTP-binding protein mediating exocytosis. Annu Rev Physiol 1990, 52:591–606. 96. Simons K, Zerial M: Rab proteins and the road maps for intracellular transport. Neuron 1993, 11:789–799. 97. Roa M, Paumet F, Le Mao J, et al.: Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc-epsilonRI). J Immunol 1997, 159:2815–2823. 98. Tuvim MJ, Adachi R, Chocano JF, et al.: Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis. Am J Respir Cell Mol Biol 1999, 20:79–89. 99. Pombo I, Martin-Verdeaux S, Iannascoli B, et al.: IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes. J Biol Chem 2001, 276:42893–42900. 100. Riedel D, Antonin W, Fernandez-Chacon R, et al.: Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules. Mol Cell Biol 2002, 22:6487–6497. 101. Lacy P, Thompson N, Tian M, et al.: A survey of GTP-binding proteins and other potential key regulators of exocytotic secretion in eosinophils: apparent absence of rab3 and vesicle fusion protein homologues. J Cell Sci 1995, 108:3547–3556. 102. Söllner T, Whiteheart SW, Brunner M, et al.: SNAP receptors implicated in vesicle targeting and fusion. Nature 1993, 362:318–324. 103. Lacy P, Logan MR, Bablitz B, Moqbel R: Fusion protein vesicleassociated membrane protein 2 is implicated in IFN-alphainduced piecemeal degranulation in human eosinophils from atopic individuals. J Allergy Clin Immunol 2001, 107:671–678. 104.• Logan MR, Lacy P, Bablitz B, Moqbel R: Expression of eosinophil target SNAREs as potential cognate receptors for vesicleassociated membrane protein-2 in exocytosis. J Allergy Clin Immunol 2002, 109:299–306. This was the first report of the expression of two target SNARE molecules, SNAP-23 and syntaxin-4, in eosinophils, which were postulated to serve as targets for binding by vesicle-associated membrane-2, and suggested to be critical for exocytosis of eosinophil granules.