Eosinophil granulocytes are pleiotropic multifunctional leukocytes implicated in the pathogenesis of allergic disorders including asthma, rhinitis, atopic dermatitis (AD) and responsible for host defense against selected pathogens (1,2). Eosinophils are also present in diseases without allergy and parasitic infections including infiltrates in cancer and responses to some viral infections (3).
The characteristic morphological features of eosinophils include the bilobed nucleus, and large acidophilic cytoplasmatic granules (Figs 1 and 2). Eosinophils are a rich source of chemokines including eotaxin, IL-8, macrophage inflammatory protein (MIP)-1a, RANTES as well as growth factors such as heparin-binding epidermal growth factor (HB-EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-β, stem cell factor (SCF), transforming growth factor (TGF)-α, TGF-β and interferons including IFN-γ, tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF) (Fig. 2). Once activated, eosinophils release cytokines (IL-1α, IL-2-6, IL-8-10, IL-12, IL-16, IFN-γ, GM-CSF, TGF-α and TGF-β), lipid mediators [platelet-activating factor (PAF), leukotriene (LT)C4, LTD4], prostaglandins, superoxide anions, singlet oxygen and an armoury of potent cytotoxic granule proteins including major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (4,5) (Fig. 2). EDN is cytotoxic, neurotoxic and has RNase activity; MBP is a potent helminthotoxin and cytotoxin; whereas ECP possesses ribonuclease activity, is neuro- and helminthotoxic, correlates with disease severity of AD patients (6), is bactericidal and promotes degranulation of mast cells (7).
Figure 1. Cardinal structure of human peripheral blood eosinophils highly purified by CD16-negative selection (purity >98%, CD16; Microbeads Miltenyi Biotec, Borgisch, Cladbach, Germany). Eosinophils display the typical morphology with bilobed nucleus (a,b), including specific red stained granules (a). Staining with a modified Giemsa preparation [Dade Behring AG, Dudingen, Switzerland (a)]. Original magnifications. (a) ×400, (b) ×18 000. Electron micrograph of an eosinophil with a diameter of approximately 8 μm (b). In the cytoplasm packed with many large, membrane-enclosed, dense crystalloid-containing ovid granules. Eosinophil conventionally fixed (glutaraldehyde).
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Figure 2. Cardinal structure of the eosinophil granulocyte [adapted from Giembyzcz et al. (7)] with the four main granules including the primary granules (1) with charcot-leyden crystals (CLC); the secondary granules (2) with MBP, ECP, EDN, EPO and cytokines/chemokines; the small granules (including arylsulfatase B, acid phosphatase); lipid bodies (including 5′-lipoxygenase (5′-LO), cyclo-oxygenase (COX), leukotrien (LTC)-4 synthase, arachidonic acid (AA); and the core of the secondary granules including MBP, GM-CSF, NGF, IL-2 and IL-4-5.
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Despite their presumed role in tissue destruction, eosinophils also appear to be capable of modulating acute phase and innate inflammatory responses as well as acquired immunity associated with both Th1 and Th2 immune responses as recently reviewed by Jacobsen et al. (3). Eosinophils have been shown to express Toll-like receptors (TLR) including TLR-7 (8), which are part of the innate immune responses that generally lead to Th1-acquired immune responses. With the expression of IL-12 receptor, tissue-infiltrating eosinophils are capable of recognizing and responding to a localized Th1 immune microenvironment. Furthermore, the ubiquitous presence of eosinophils at sites of Th2-mediated inflammation and their demonstrated abilities to express each of the representative Th2 cytokines have led to the speculation that eosinophils are capable of modulating and/or sustaining the Th2 character of the local tissue immune microenvironment (3). Eosinophils also have the capability to link immune suppression that potentially modulates T-regulatory cell functions and thymocyte development (9). In addition, eosinophils play important roles in remodelling and repair processes (10).
Furthermore, eosinophils bidirectionally interact with nerves, highlighting a novel pathophysiological activity of this granulocyte. Eosinophils are not only a source of neuromediators but also respond to and release neuromediators including neurotrophins and neuropeptides. As eosinophils can be found in the close vicinity of nerves (11), there is increasing evidence that eosinophil-associated neural mechanisms are important in modulating organ function in diseases such as asthma.
The goals of this review are to: (i) highlight new aspects of neuroimmune interactions between nerves and eosinophils, (ii) present evidence that eosinophils are tissue-resident cells; and (iii) explain why eosinophils have a role in allergic diseases such as asthma.
The tissue and its target: trafficking eosinophils
Eosinophils comprise 2–10% of peripheral leukocytes. The turnover of eosinophils occurs almost exclusively in the bone marrow, although ancillary sites of production can include the spleen, thymus and lymph nodes (7). In the bone marrow of healthy individuals, the number of eosinophils is estimated to be approximately 3% of which 37%are mature, non-dividing granulocytes while the remainder are promyleocytes/myelocytes (37%) and metamyelocytes (26%) (12). The maturation of progenitors, such as pluripotent CD34+ stem cells, into eosinophils includes effects on the bone marrow, mediated principally by IL-5, which results in a fourfold increase in circulating eosinophils (13). CD34+ IL-5R+ eosinophil progenitor cells have also been recently identified in murine lung tissue (14). For the selective tethering of eosinophils to venular endothelium, the combined effects of P-selectin/P-selectin glycoprotein ligand 1, very late activation antigen-4/vascular cell adhesion molecule-1 and selective chemotaxis under the influence of chemokines (e.g. RANTES and the eotaxins) are needed for eosinophil migration and chemotaxis (15–20). All these events are orchestrated by allergen-specific Th2 lymphocytes through the generation of IL-5, IL-4 and IL-13 (Fig. 3). The time taken from the last mitosis to the appearance of eosinophils as mature cells in the blood is approximately 2.5 days (7). Once in the circulation, eosinophils have a half-life of approximately 18 h and a mean blood transit time of 26 h (21). Eosinophils exhibit diurnal variation with the highest and lowest levels in the evening and in the morning. Eosinophils are considered predominantly tissue-resident cells and do not re-enter the circulation. In humans, there is relatively little data on the kinetics of eosinophil trafficking and it is difficult to make a firm statement about the relative contribution of eosinophilopoiesis, recruitment into tissue and removal either by apoptosis or luminal egress to eosinophil tissue numbers. However, the much more marked effect of anti-IL-5 on blood eosinophils compared with tissue eosinophils in asthma (22), the observation that large numbers of eosinophils can be found in tissues even when the peripheral blood count is low and conversely that the tissue eosinophil count can be normal in conditions, such as benign eosinophilia, where there is a marked blood eosinophilia, suggest that the mechanisms controlling the blood eosinophil count, which are presumably related to IL-5-dependent growth signals and tissue count (related to chemoattractant and adhesion receptor recruitment), can be dissociated. Once in the tissues, eosinophils may persist for several days or weeks, surviving under the influence of locally generated cytokines, which may also partly explain the selective tissue accumulation of eosinophils (13). The principal site for eosinophil accumulation in healthy subjects is thought to be the gastrointestinal tract, whereas the airways and the skin are commonly involved in eosinophilic disease. In AD, a superficial tissue distribution of eosinophilia has been described with <10% of total eosinophil granule protein deposition below a depth of 1.39 mm from the epidermis (23). Still, the tissue distribution of eosinophils in subjects with disease has not been systematically quantified. However, in rats, the number of bone marrow and tissue eosinophils is estimated to be 300 times higher than the circulating count (7).
Figure 3. Selective accumulation of eosinophils is a multi-step process directed by Th2-associated cytokines: Step 1 involves selective eosinophilopoiesis under the influence of IL-5 and egress from the bone marrow promoted by chemoattractants such as eotaxin 1. Step 2 involves selective migration through venular endothelium, which is promoted by α4β1-VCAM-1, PSGL-1-P-selectin and (CCL26) eotaxin 3. After transmigration (step 3), tissue accumulation is controlled by a balance between prolonged survival and retention in tissues as a result of adhesion to matrix proteins such as fibronectin (step 4), local differentiation from precursors within tissue (step 5) and signals for egress into the lumen provided by epithelial-derived chemoattractants such as CCL26 (step 6). All this is orchestrated by IL-4, IL-13 and IL-5 released by Th2 lymphocytes as well as other cells within the airway mucosa.
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A final aspect of eosinophil trafficking deals with the survival and functioning within the peripheral tissue (24). In in vitro studies, we have shown that peripheral blood eosinophils derived from patients with atopic dermatitis and allergic rhinitis have a prolonged viability with delayed programmed cell death (PCD) in comparison with eosinophils of non-atopic donors (25,26). As eosinophils are able to release GM-CSF, IL-3 and IL-5, we speculated that this effect is explained by autocrine-releasing mechanisms. Several other studies have shown in vitro data revealing other eosinophil apoptosis inhibitors including IL-5, eotaxin, TNF-α, etc. (24,27–29). Only one study has been carried out in human tissue performed by Simon et al., which revealed an association of the prolonged survival of eosinophils in explants of nasal polyps by IL-5 (30).
Networking of eosinophils: expression of surface molecules
Our understanding of the repertoire of surface molecules expressed by eosinophils has expanded considerably in recent years. Although there is no single cell surface marker that is uniquely expressed by eosinophil granulocytes, the eosinophil displays a unique pattern of cell surface markers, more closely resembling the basophil than the neutrophil granulocyte (31). This subject has recently been reviewed (32).
We will focus on the receptors for neuromediators including neurotrophins and neuropeptides expressed on eosinophil granulocytes (Table 1). It is important to note that a majority of investigations have been performed on peripheral blood eosinophils. It is not clear whether tissue eosinophils display the same receptor expression as found in the peripheral blood.
Table 1. Human eosinophils derived from subjects with allergic asthma, allergic rhinitis and atopic dermatitis express receptors for neuromediators
|Allergic asthma||PB, BAL||PB, BAL||PB, BAL||PB, BAL||nk||nk|
Noga et al. described the expression of functionally active neurotrophin receptors such as tyrosine kinase (trk) A, B and C on peripheral blood eosinophils of patients with asthma and allergic rhinitis with release of IL-4 and EPX upon neurotrophin stimulation (33).
Trk receptors are receptors to which mature neurotrophins bind with high affinity and specificity, trkA for NGF (34), trkB for brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4 (35) and trkC for NT-3 (36). Nassenstein et al. only found trkA expression on peripheral blood eosinophils. However, they found an increased expression of trkA, -B and -C and the pan-neurotrophin receptor (p75NTR) on bronchoalveolar lavage eosinophils of asthmatic patients after segmental allergen provocation (37). P75NTR belongs to the TNF superfamily to which all mature neurotrophins bind with low affinity and specificity (38,39). We have found that trkB and p75NTR are expressed on peripheral blood eosinophils, with greater expression on eosinophils from patients with AD compared with non-atopic subjects (40) (Table 1). So far, this is the only study describing a higher expression profile of neurotrophin receptors on peripheral blood eosinophils in atopic disease compared with that in healthy subjects. Whether or not the expression of neurotrophin receptors is different on eosinophils of patients with asthma, allergic rhinitis, atopic dermatitis and healthy subjects is currently under investigation.
With regard to neuropeptides, the neurokinin-1 (NK-1) receptor to which the neuropeptide substance P (SP) binds with high affinity and specificity has not been described on peripheral blood or tissue-resident eosinophils. However, SP has been described to induce peripheral blood eosinophil chemotaxis in vitro, which was abolished by the NK-1 receptor antagonist (FK 888), suggesting a possible surface expression of NK-1 (41). NK-2, which is the specific receptor for Neurokinin-A, has been described on tissue-resident eosinophils in the human intestine (42). Receptors for other neuromediators including calcitonine gene-related product (CGRP) and vasoactive intestinal polypeptide (VIP) as well as alpha-melanocyte-stimulating hormone (α-MSH) have not yet been described on peripheral or tissue eosinophils.
Eosinophils and nerves: Interaction via granule proteins
The inflammatory allergic reaction modulates the functional activity of peripheral nerves and induces structural changes in the diseased tissues including the airways and the skin. On the contrary, it has become clear that the inflammatory allergic reaction is also controlled by tissue-resident cells including neurons and eosinophil granulocytes (43). Eosinophils have been described in close vicinity of peripheral nerves in the airways (allergic asthma) and the skin (prurigo nodularis) (11,44). In prurigo nodularis, a skin disease with intense pruritus, eosinophil granule proteins ECP and EDN/EPX have been described in close connection with peripheral nerves compared with healthy subjects or uninvolved skin (44). ECP and EDN have been described for their neurotoxic activity (45). However, the number of peripheral nerves was higher in areas with increased levels of eosinophil granulocytes, ECP and EDN in prurigo nodularis (44). In an in vitro study, eosinophil adhesion to a nerve cell line via ICAM-1 has been shown to result in an adhesion-dependent release of granule proteins. Culture in serum-deprived media induced apoptosis in those nerve cell lines that were dose-dependently abolished by MBP-1 but not by EDN, giving evidence that MBP-1 release from eosinophils at inflammatory sites regulates peripheral nerve apoptosis (46).
Furthermore, MBP is an antagonist of inhibitory M2 muscarinic receptors on nerves. In addition to being cytotoxic, MBP increases smooth muscle reactivity by causing dysfunction of vagal muscarinic M2 receptors, which may contribute to the development of airway hyper-reactivity (46–48). Together, eosinophil granule proteins including EDN/EPX, ECP and MBP have a pivotal role in regulating peripheral nerve plasticity.
Bidirectional interaction of eosinophils and nerves via neurotrophins
Neurotrophins, such as NGF, BDNF, NT-3 and NT-4, have recently gained widespread attention in allergic asthma, allergic rhinitis and AD (37,49–53). Initially, neurotrophins were described for their neurotrophic activity (54). More recently, it has become evident that neurotrophins modulate the functional activity of a variety of immune cells including eosinophils, which are also a source of neurotrophins (40,43,49,55,56).
Normal human eosinophils constitutively express messenger RNA for NGF and NT-3. They synthesize and store these neurotrophins intracellularly and continuously replenish them (57). Recently, it was found that NGF was localized in the central core of stable granules in human peripheral blood eosinophils from patients with AD (Fig. 2) (58). In addition, the levels of NGF were increased in freshly isolated peripheral blood eosinophils of AD compared with healthy control subjects. NGF levels significantly correlated with MBP in eosinophils of AD patients (58). Moreover, NGF was found to increase the release of IL-4 of peripheral blood eosinophils (33).
As discussed above, PCD of eosinophils from patients with AD is significantly delayed compared with non-atopic subjects (26). Interestingly, NGF stimulation led to a further delay of PCD in peripheral blood eosinophils of AD (59). In our own studies, BDNF also had a functional role with an inhibition of PCD and induction of chemotaxis in AD, but not in eosinophils from non-atopic control subjects (40). Additionally, eosinophils are releasers of BDNF suggesting autocrine stimulation mechanisms. Again, this effect was only seen in eosinophils from AD patients (40). Further, BDNF and NT-3 increase the release of EPX of peripheral blood eosinophils, whereas ECP release was not enhanced (33,40).
Regarding direct neuroimmune interactions, Kobayashi et al. revealed a promotion of neurite extension of the PC-12 pheochromocytoma cell line by supernatants derived from human peripheral blood eosinophils stimulated with IgA immune complex and IL-5. Eosinophil supernatants contained increased levels of NGF and neurite outgrowth was abolished by pretreatment of supernatants with anti-NGF-neutralizing antibody. Thus, Kobayashi et al. have provided evidence for a modulation of the functional activity of nerves by eosinophils through the release of neuromediators such as NGF (57).
Eosinophils and the non-adrenergic non-cholinergic nervous system
The sensory non-adrenergic non-cholinergic (NANC) system is divided into an excitatory (e-NANC) and an inhibitory (i-NANC) parts, which contribute to the afferent nervous system, e.g. controlling airway function (60). Neurotransmitters of the e-NANC system are SP, neurokinin A (NKA) and CGRP. Mediators of the i-NANC system represent nitric oxide and VIP (61).
Substance P is a potent vasodilatory neuropeptide, which is released from peripheral nerve endings of sensory neurons. In addition, eosinophils have the capacity to synthesize, store and release large amounts of SP and VIP that may act in an autocrine fashion (62,63). High concentrations of SP effectively degranulate eosinophils with increased release of EPO and ECP (64,65). However, submicromolar concentrations of SP have been reported to upregulate FCε and FCγ on human eosinophils enhancing antibody-dependent eosinophil-mediated cytotoxicity (66). As shown in in vitro studies, SP induces eosinophil infiltration via degranulation of mast cells pointing to indirect capabilities of this neuropeptide (67). In an animal model, SP increased the production of TNF-α and IL-1, providing an additional mechanism for eosinophil recruitment as TNF-α and IL-1 are known to upregulate adhesion molecules on endothelial cells (68). In this regard, SP and NK-A induced the recruitment of eosinophils to alveolar walls in allergic asthma (69). In allergic rhinitis, a single administration of SP in nebulized form together with nasal allergen provocation significantly increased the high number of eosinophil granulocytes in nasal lavage fluid compared with lavage fluid with only nasal allergen provocation. In addition, the SP caused increase in eosinophil numbers correlated with clinical symptoms such as nasal obstruction (70).
Substance P stimulation of eotaxin-primed normal human eosinophils caused an exaggerated EDN release, which was blocked by 7B11 and herbimycin A, the CCR3 antagonist and the tyrosine kinase inhibitor respectively (71). The amino acid fragment 7–11 but not 1–7 of SP caused superoxide production, suggesting receptor-mediated effects of SP (72). Neuropeptides including SP, CGRP and VIP also induce chemotaxis of preactivated eosinophils. Interestingly, this effect was only seen in eosinophils of patients with allergic disease, but not in healthy donors (73,74). Further, SP, VIP and CGRP have been shown to reduce IL-16 release of purified peripheral blood eosinophils in vitro, which led to a reduced chemotaxis of lymphocytes (75).
Eosinophils and sensory nerves most probably influence each other in a bi-directional pattern in the pathogenesis of allergic inflammatory diseases (Fig. 4). Although release of sensory neuropeptides is involved in most conditions of airway hyper-responsiveness, airway obstruction and airway eosinophilia, the role of these nervous system-derived mediators in pulmonary diseases still seems to be underestimated (60,76).
Figure 4. Bidirectional interaction between sensory nerve and eosinophil granulocyte. Eosinophil capable of BDNF, NGF, NT-3 and SP release leading to afferent stimulation via p75NTR and NK-1 expression on sensory nerves. Inhibition of neuronal apoptosis and blockade of neuronal M2 receptor by MBP-1 granule protein. Neurotoxic activity of EDN and ECP. On the contrary, eosinophil expressing p75NTR, trkA, trkB, trkC receptors as a target of NGF, BDNF and NT-3 released by sensory neuron.
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Clinical implications – why eosinophils have a role in asthma
Asthma is a disease characterized by reversible airflow obstruction, and airway hyper-responsiveness to a variety of antigen-specific and non-specific triggers (77). The asthmatic lung is associated with a prominent eosinophil-, macrophage-, and lymphocyte-rich inflammatory response and by a variety of structural alterations including mucus production, smooth muscle hyperplasia, neovascularization and deposition of extracellular matrix components (78,79).
Over the years, the role of eosinophils in asthma has been disputed. This debate has been intensified by the disappointing effects of anti-IL-5 in both allergen challenge and clinical disease. In an allergen challenge study, anti-IL-5 treatment revealed no effect on PC20 or the late response despite a dramatic decline in peripheral blood eosinophil counts (22). However, the number of tissue-resident eosinophils was not taken into account in that study. In a later study, anti-IL-5 treatment only reduced, but did not deplete the number of airway and bone marrow eosinophils and had no effect on airway hyper-responsiveness, FEV1 and peak flow recordings (80). In addition, there is increasing evidence that eosinophils are involved in exacerbations of asthma rather than day-to-day symptoms or changes in lung function (81), and as yet no study has reported on the effect of anti-IL-5 in eosinophilic patients with severe exacerbations as the primary outcome.
The importance of the compartmentalization of eosinophils is supported by our observations using an anti-inflammatory neuropeptide in a mouse model of allergic airway inflammation. Application of α-MSH inhibited allergic airway inflammation with a significant reduction in eosinophils in broncho-alveolar lavage fluid, but with no effect on airway hyper-reactivity. In the lung tissue itself, we found a significant reduction, but not a complete depletion of tissue-resident eosinophils (82). This result suggest that a reduction of tissue-resident eosinophils still allows the neuronal activation leading to airway hyper-reactivity, letting the remaining eosinophils drive the manifestation of the disease. Additionally, it has been shown recently that intercellular adhesion molecule (ICAM)-1 is induced in human parasympathetic nerves by TNF-α (83). This novel finding gives direct evidence for the adhesion of eosinophils to peripheral nerves via ICAM-1.
Furthermore, airway nerves and cultured parasympathetic neurons express eotaxin, recruiting eosinophils to neurons with alterations in neuronal function (3). However, application of CCR3 antagonist did not decrease the number of eosinophils in lung tissues, but prevented the antigen-induced clustering of eosinophils along nerves as assessed histologically (84). Thus, signalling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to the blockade of inhibitory M2Rs by eosinophil MBP.
Eosinophils are a rich source of fibrogenic factors implicated in airway remodelling including IL-11, IL-17, IL-25, TGF-α, matrix metalloproteinase (MMP)-9 and particularly TGF-β1 (10). TGF-β1 stimulates fibroblasts in vitro to promote the synthesis and secretion of many proteins of the extracellular matrix such as collagen I and III, fibronectin, proteoglycans and tenascin (85). Furthermore, TGF-β1 correlates with disease severity and degree of subepithelial fibrosis in patients with asthma (86).
Recently, eosinophils have been shown to induce the expression of several cholinergic genes involved in the synthesis, storage and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase and acetylcholinesterase (87). These eosinophil-induced changes in enzyme content were found to be associated with a reduction in intracellular neural acetylcholine, but an increase in choline content suggesting that eosinophil localization to cholinergic nerves induces neural remodelling, promoting a cholinergic phenotype.
Together with the release of neuronal mediators, such as neurotrophins and neuropeptides, eosinophils can interact with the neuronal network, which may have an important role in orchestrating airway hyper-responsiveness (88).
Another prominent feature of patients with eosinophilic airway inflammation and eosinophilic bronchitis, eosinophilic pneumonia and hypereosinophilic syndrome (HES) as well as asthma is coughing (89,90). However, whether or not coughing is induced by eosinophil neural interactions remains to be elucidated.
Eosinophils: pro-or anti-inflammatory?
The answer to the question whether or not eosinophils have pro or anti-inflammatory effects in airway inflammation (or indeed are simply bystanders) may have to await a more definitive inhibitor of eosinophil tissue accumulation than anti-IL-5, although the effects of this drug in HES do suggest that eosinophils can be disease causing in at least some circumstances (91,92). Eosinophils certainly have the capacity to contribute to inflammatory mechanisms. On one hand, they are involved in host defence and tissue destruction; on the other hand, they also correspond to remodelling and repair mechanisms. Any assessment of the role of eosinophils needs to take into account that they act primarily in the tissue. With their ability to interact with nerves, eosinophils highlight a new paradigm contributing to neuroimmune interactions in allergy. Thus, the eosinophil granulocyte is more than just a killer effector cell. Future investigations will hopefully highlight other exciting and new potentials of this interesting granulocyte.