The incidence of asthma within the borders of developed nations has increased dramatically since the 1950s. Rates of people experiencing strong allergic reactions, including allergic asthma has been on the rise, as there are fewer infectious diseases for the immune system to fight. Thus, we have the problem of autoimmune conditions that promote extreme reactivity against environmental triggers that are harmless to most individuals. The prevailing opinion in the medical community is that allergic asthma is simply a maladaptive immune response to otherwise harmless inhaled substances in individuals genetically predisposed to this condition. However, that stance is overly simplistic and further avenues need to be investigated.
In 1879, Paul Erlich first described eosinophils as effector cells in asthma pathogenesis—and this assumption had been heavily inspected and tested since it was first proposed, leading to various discoveries. These bilobed granulocytes possess secondary granules containing four extremely toxic primary cationic proteins including: major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO).
Eosinophilia is a prominent feature of the asthmatic lung. Asthma is a chroniccdisorder that predominantly affects lung function. During this disease a combination of inflammation and structural changes commonly termed airway remodelling, usually lead to theddevelopment of airwayhhyper- responsiveness (AHR) and obstruction, which is usually reversible. Whilst science is still unsure of the primary cause of asthma, a combination of genetic and environmental factors are believed to contribute to its pathology. There have been several genes identified and linked to increased susceptibility to asthma, which have been implicated in regulating both airway remodelling and inflammation. Moreover, the compound actions of various leukocytes and their mediators may be the reason for the variations seen in asthmatics lung pathology.
It is often common for the immune system to launch an attack against harmless particles, especially in the case of asthmatics. This is an IgEiimediated process leading to the activation of mastccells, basophils and macrophages, inducing the release of inflammatory agents such ashhistamine, eicosanoids and ROS (reactive oxygen species). Such agents induce micro-vascular leakage and oedema within the airway walls, leading to the constriction of the airway lumen. This may lead to plasma accumulation in the lumen of the airway, causing damage to the epithelium hence hindering mucus clearance. Moreover, the early phase pro-inflammatory mediators also induce airway smooth muscle cell (SMC)=contraction and mucus secretion. The various chemo-attractants produced lead to the proliferation of leukocytes, comprising of CD4 T-cells and eosinophils; the activation of these cells and their subsequent pro-inflammatory mediators, specifically Th-2 like cytokines, is held as an integral component of asthma pathology.
Given the extensive range of secretory products and receptors expressed by eosinophils, the central role of these granulocytes in the pathogenesis of asthma is of particular interest.
Eosinophils constitute about 1-3% of the total leukocyte population, and originate from iCD34++haematopoietic progenitor cells residing in the bone marrow. The differentiation of the eosinophillisiinducedbby the synchronized interplay and unique co-ordination of transcriptionNfactorsIGATA-1,IPU.1 and the enhancer-binding-protein-family (C/EBP) with specific cytokines leading to the electic development of eosinophils. Changing levels of PU.11expression, determines the differentiation of myelocytic or lymphocytic cells (DeKoter & Singh 2000; Du et al. 2002); meanwhile, C/EBPs have shown to contribute to eosinophil development through interactions with GATA-1. The interactions between EBP and transcription factor GATA-1 enhances the transactivation of theeeosinophil specific MBPPpromoter; however, the C/EBPε variant antagonistically affects GATA-1/PU.1 union and limits myeloid gene expression (Du et al. 2002). GATA-11is a selective promoter of eosinophil development; it acts via a high affinity “palindromic GATA site”, situated in the downstreamg GATA-1ppromoter as well as in eosinophil specific genes including3CCR3, MBP and IL-5Rα (Yu 2002).
Furthermore eosinophil development is also influenced by cytokines such as IL-3,IIIL-5 andGGM-CSF. These cytokines together with transcriptional regulators lead to theddevelopment of various cells with a myeloid lineage (Sanderson 1992). In addition to eosinophil development, the signals induced by these three cytokines affect eosinophil adhesion, activation and survival. Amongst these three cytokines,IIL-5 is the most selective for eosinophil differentiation, proliferation and maturation in the bone marrow.
In atopic asthmatic, the amounts of CD34+,cCD45+ and IL-5Rααeosinophil progenitors are elevated both in the peripheral blood and the bone marrow (Schragge et al. 1996), also a similar response is seen in mice models of airway inflammation (Johansson et al. 2004). This indicates that the allergen challenged bronchial mucosa must transmit signals to the bone marrow which leads to the increased numbers of eosinophils ready to function.
Interestingly, evidence suggests that bone marrow progenitor cells themselves can also migrate to the location of the inflamed tissue and undergo in-situ differentiation. This is based on observations that in bronchial mucosal of atopic asthmatics the levels of CD34+/ IL-5Rα messenger RNA cells are increased (Robinson et al. 1999). Similar results have been seen in lung tissue from mice, where hourssafter allergen challenge; prior to the increase in number ofeeosinophils in the bronchoalveolar lavage, the number of corresponding progenitor cells increase (Southam et al. 2005). This double trafficking of both mature eosinophils and progenitor cells is an indication of a mechanism for sustaining prolonged periods of inflammation in the tissues.
Eosinophil trafficking and recruitment in the lungs
The first eosinophil specific chemokine to be discovered was eotaxin. Eotaxin was previously identifieddas themmajor chemoattractants in the BALLfluid of guinea-pigs (Griffiths-Johnson et al. 1993). Further investigations led to the identification of two more functionally similar CC chemokines, which were subsequently named eotaxin 2 and 3 (Forssmann et al. 1997).
In asthmatics and allergen challenged mice, eotaxins are up regulated, and their production from sources such as mast cells, alveolar macrophages and most importantly epithelial cells is amplified (Gutierrez-Ramos et al. 1999). Based on human studies, eotaxin (eotaxin-1) is thought to be a key factor for the initial response to allergens, whereaseeotaxin-2 and perhaps eotaxin-3 may be necessary for the prolongation of eosinophiliaa(Ravensberg et al. 2005).
Eotaxins signalling occurs via theCCCR3 receptor, which is predominantly found on mast cells (Ochi 1999), basophils (Uguccioniiet al. 1997), and Th-2 cells (Sallusto et al. 1997). This receptor can also be found on mature eosinophils and CD34+ progenitor cell (Sehmi et al. 2003). CCR3 expression is up regulated in response to an inflammatory stimulus (Sehmi et al. 2003). The ligationnof eosinophil CCR3 by eotaxin, leads to the calciummmobilisation, actinnpolymerisation and structural changes as well as chemotaxis (Zimmermann & Rothenberg 2003). Due to the eosinophil-specific actions of the CCR3/eotaxin axis, this pathway is of high importance in the pathogenesis of asthma and as we will discuss later it could be a possible therapeutic target in asthma.
The cytokineIIL-5 is essential for eosinophil mobilisation from the bone marrow, representing the first stage of eosinophil trafficking. Even though eotaxin is primarily associated with eosinophil recruitmenttinto the lung, the synergy between IL-5 and eotaxin also enhances eosinophil mobilisation from thebbone marrow. Once the eosinophils depart from the bone marrow, they begin their migration to the lungs; this process is also regulated by the IL-5/eotaxin axis. Blood samples from asthmatic patients usually display elevated levels of IIL-5, IL-33 andGGM-CSF, which together with mediators including platelet-activatinggfactor (PAF) and complementtfactor 5a (C5a) are held essential in eosinophil priming (AJ Wardlaw et al. 1992).
Once eosinophils are partially primed, their departure from the vasculature into the tissue relies on the binding of various adhesion receptors, such as the eosinophil P-selectin glycoprotein ligand-1, α4β1 and αAβ2 to endothelial P-selectin VCAM-1 and ICAM-1, respectively (A J Wardlaw 1999).
The synergy between IL-5 and eotaxin within the lung is important for eosinophil-specific recruitment. In a study it was shown that in IL-5-KO mice, eotaxin alone cannot induce tissue eosinophilia, and IL-5 is required for this process to occur (Mould et al. 1997). In addition to IL-5’s direct connections with eotaxin, it has also been associated with Th-2 in further enhancing eosinophil recruitment to the lungs.
Th-2 cells are central to the eosinophilic inflammation seen in asthma. T-cell deficient mice are immune from lungeeosinophilia and AHRR(Gavett et al. 1994), whereas asthmatic patient have increased number of Th-2 cells in their BAL fluid (Bentley et al. 1992). Eotaxin is responsible for the early recruitment ofTTh-2 cells in allergen challenged murine modelssof airway inflammationn(Coyle & Gutierrez-Ramos 2000). Th-2 derived cytokinesIIL-4 andIIL-13 stimulate thepproduction of eotaxin which ultimately interacts with IL-5 in the selectivee recruitment of eosinophils (Gutierrez-Ramos et al. 1999). Figure 1 displays a summary of processes involved in eosinophil trafficking from the bone marrow to the lung.
Eotaxin knockout animals show marked reduction in eosinophilic inflammation (Rothenberg 1997), but not a complete inhibition of inflammation, suggesting that other factors may be present that can too influence this process. Studies have shown that apart from eotaxin other similar factors theCCCL1/CCR8 axis may also be important in selective recruitment of eosinophils to the lungs during allergen challenge (Lloyd 2003).
Furthermore, asthmatic patients exhibit elevated mRNA expression of MCP-1 (monocyte chemotactic protein-1), RANTES (regulated on activation, normal T cell expressed and secreted) and MIP-1α (macrophage inflammatory protein 1α), which all get bound by the chemokine receptor CCR1 that is expressed on the eosinophil (Alam et al. 1996); moreover, the blockade MIP-1α leads to a reduction in the eosinophil count of a subgroup of asthmatic patients (Sabroe et al. 1999), thus suggesting that CCR1 may be a good target for blocking eosinophils actions. Nevertheless, studies have shown that the variety of interactions within the chemokine system is crucial for eosinophil trafficking, however amongst these pathways, the actions of eotaxin via the chemokine receptor 3 (CCR3) has been marked as one of the most important in eosinophil trafficking, since CCR3-KO mice show a significant reduction in eosinophilia (Pope et al. 2005).
Eosinophils and airway inflammation
The established inflammatory networks within the asthmatic lung are largely influenced by eosinophils. During inflammation, primed eosinophils are further activated by cytokines and stimulatory factors including IL-3, GM-CSF, IL-5 and secretory IgA. Other factors such as β2-integrins are central to eosinophil activation and degranulation (Gleich 2000). Classical exocytosis, compound exocytosis and piece-meal degranulation are three highly regulated mechanisms through which eosinophils selectively release their stored mediators. Degranulation can also occur through cytolysis, during which the eosinophil ruptures and releases its entire contents (Logan et al. 2003).
Classical exocytosis, although not showed in airway tissue, involves the release of single secretory granules (Erjefält & Persson 2000); in contrast, compound exocytosis entails the combination of several intracellular granules followed by a directed secretion on the target cells at the site of adhesion (Hafez et al. 2003). On the other hand, piecemeal degranulation is an unconventional secretory pathway, characterised by partiall and selective release of granular contents. This selective release is achieved through the transfer of granules into smaller packets, which then travel to the cell surface and subsequently get released through exocytosis; a good example of this is the selective release of RANTESSfrom IFN-γ stimulated human eosinophils, which occurs independent of bothmMBP andeEPO (Lacy et al. 1999).
Figure 1 | A schematic summarising the development and trafficking of eosinophils. Initially hematopoieticcstem cells (HSC) and the common myeloiddprogenitor cells (CMP) differentiate into+CD34+/IL-5Rα+ (eosinophil progenitor cells); this process is governed by the transcription factors GATA, PU.1 and C/EBP in association with IL-3, IL-5 and GM-CSF. IL-5 and eotaxin (CCL11) produced due to the allergic inflammatory response send signals to the bone marrow, heightening the production and mobilisation of eosinophils. Eosinophils are primeddin the circulation and travel across the vasculature using their specific integrins. Afterwards eotaxin highly regulates their recruitment to the lung. The build up eosinophils in the lung is associated with both AHR and remodelling, whilst Th-2 accumulation leads to increased IL5 and eotaxin production, hence enhancing the inflammatory response (adapted from Trivedi & Lloyd 2007).
The discharge of mediators by exocytosis is controlled by the formation of a docking complex made up of SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which are situated on both the target cell and the vesicle itself (Hoffmann ettal. 2001; Lacy et al. 2001).
Based on data obtained from nasal tissues of allergen challenged mice, it was discovered that 67% of the eosinophils undergo piecemeal degranulation with the remainder undergoing cytolysis. Hence, in allergic inflammation, piecemeal degranulation appears to be the predominant mechanism through which granules are released (Erjefält et al. 1999). Furthermore, a similar tissue eosinophil count is observed in mucosal tissues obtained from patients suffering from allergic conditions such as asthma and rhinitis, however the levels of piecemeal degranulation within these tissues vary (Erjefält et al. 2001).
During exocytosis, the liable mechanisms try to limit tissue damage, however in case of eosinophils, due to the cytotoxic nature of their granules, there is a high probability of epithelial damage (Motojima et al. 1989). These granules are capable of increasing vascularppermeability in vivo, at concentrations seen in pathological conditions (Minnicozzi et al. 1994). Notably, eosinophil granules can activate mast cellsaand initiate the release of pro-inflammatory mediators such as eicosanoids, histamineeand cytokines (Piliponsky et al. 2002). Eosinophils also support the growth and survival of mast cells by producing nerve growth factors and stem cell factors (Piliponsky et al. 2002).
Furthermore, eosinophils are capable of producing pro inflammatory cytokines such as IL-4 and IL-13. These pro inflammatory factors lead to the stimulation and rapid release of eotaxins, shadowed by a marked increase in RANTES and MCP-1 production, which altogether enhance eosinophils recruitment.
Eotaxin is capable of inducing respiratory burst and actin polymerisation in eosinophils, thereby not only contributing to tissue damage but also continually recruiting eosinophils and T cells.
During an allergen challenge in the lungs, a protective inflammatory response is initiated, throughout which eosinophils are extensively recruited to the affected tissues, and once the inflammatory response ceases, cells areccleared from the lungs. The process of inflammation is ahhighly organised and dynamic event, during which different mechanisms exist to limiteeosinophilic inflammation; the action of the regulatory cytokineIIL-10 is a good example of such mechanisms. Failure in these regulatory pathways may lead to a persistent inflammation. For instance, the inability to down regulate IL-3 is believed to instigate atypical inflammation.
The activation and survival of the eosinophil is highly dependent on the cytokines IL-3,IIL-5 andGGM-CSF. Upon stimulation of the mature eosinophils, these cytokines, in contrast to their early effects within the bone marrow, result in the down regulation of IL-5 receptor (IL-5Ra) and up regulation of the IL-3 receptor (IL-3Ra) (Gregory et al. 2003).
In a study using murine models of acute airway hyper-responsiveness (AHR), a hallmark of asthma, it was seen that IL-3 levels in the lung are elevated before the resolution of inflammation; however, this increase was concomitant with dampened levels ofIIL-3 and increased leukocyte apoptosis in the peri bronchiolar regions. Additionally, the inhibition of IL-3 in murine models of sustained AHR, leads to a marked decrease in eosinophilia and airway hyper responsiveness, accompanied with increased cell apoptosis within the lung (Lloyd et al. 2001).
Hence, IL-3 may act to promote inflammation through inhibition of apoptosis, but on the other hand endogenous pro-resolving signals may act to down regulateIIL-33and increased cell clearance through apoptosis. In addition to apoptosis, a more effective way of eosinophil extraction is by promoting egress into the airway lumen. Steroid treatment of murine models of allergic airway inflammation, results in a transendothelial egression of eosinophils into the lumen, highlighting the possible implications that this mode of clearance may have for targeting eosinophil in asthma (Uller et al. 2006).
Eosinophils and airway hyper-responsiveness
Airway hyper-responsiveness (AHR) is a characteristic feature seen in many inflammatory lung disorders. AHR refers to the narrowing of the airways due to increased sensitivity towards non-specific stimuli. This condition is a classical hallmark of asthma, and its severity is correlational with he severity of disease (O’Byrne 2003).
A similar correlation exists between the number of activated eosinophils and the severity of asthma. During allergen challenge, eosinophil recruitment is enhanced in both human and animal models of the disease (O’Byrne 2003). It is proposed that activated eosinophils may directly or indirectly cause AHR, either through release of basic granules, leukotriene and mediators or by interacting with other cell types. Figure 2 summarises the mechanisms through which eosinophils may contribute to AHR.
Despite this alleged correlation, there is still no firm evidence that proves eosinophils and their contents directly cause AHR in clinical asthma. Evidence against this proposed correlation comes from studies of eosinophilic bronchitis (EB). In EB, the distribution of eosinophils in the bronchial mucosa is similar to that of found in asthma, however, the absence of AHR in these patients casts doubt over the significance of eosinophils in onset of this characteristic condition; it is useful to bear in mind that human subjects for AHR studies usually have longstanding chronic disease, which may or may not have secondary effects on AHR and its pathogenesis in asthma.
Amongst the range of mediators released by eosinophils, there exist basic granules specific to these cells. Majority of these granules are highly toxic and are present within the lungs of asthmatic patients at concentrations that can induce damage to the airway epithelium and increase smooth muscle cell reactivity (Frigas et al. 1981). Airway smooth muscle cell tone is controlled via the actions of M3 and M2 muscarinic receptors, with the majority of receptors being of the M2 subtype (Roffel et al. 1990). Stimulation of M3 receptors (coupled to Gq) by acetylcholine results in smooth muscle cell contraction, while activation of M2 receptors (coupled to Gi) leads to inhibition of beta-adrenergic mediated relaxation (Hirshman et al. 1999). There is mounting evidence that suggests eosinophils are liable for the loss of M2 receptor function. Biopsies taken from lungs of asthmatic patients show distinct areas where eosinophils are clustered around the vagal nerve ganglia (Costello & Schofield 1997). To clarify the effect of vagal stimulation on bronchoconstriction, it has been shown that MBP expression (an allosteric antagonist for the M2 receptor) initiates bronchoconstriction by hindering vagal muscarinic M2 and M3 receptor function (Jacoby et al. 1993). Tests carried out on OVA challenged guinea pig ileum, showed that eosinophil depletion prevent the loss of M2 receptor function and improves the accompanying bronchoconstriction (Elbon et al. 1995).
The secretion of cysteinyl leukotriene by the eosinophil is understood to directly contribute to broncho constriction; in addition, leukotriene lead to increased vascular permeability and enhanced inflammatory cell recruitment. The use of leukotriene receptor antagonists has shown to partially effective in alleviating symptoms of asthma, that is, to a certain degree (Tulic et al. 2003). As stated previously, eosinophils can indirectly contribute to the development of AHR. This route of activation involves the mast cells and basophil degranulation, this leads to the production of factors such as prostaglandins, histamine and leukotrienes, all of which are culprits in induction of AHR (Shi et al. 2000).
According to the majority of studies, the eosinophil is still viewed as a central effector in allergic airway disease, particularly asthma. In favour of this belief, experiments on mice have shown that depletion of lung eosinophils through inactivation of the chemokine receptor type 3 (CCR3), which is expressed at high levels in eosinophils and basophils, results in complete reversal of allergen induced AHR, while leaving other cells unaffected (Justice et al. 2003).
On the other hand, there are studies focusing on the interactions between eosinophils and T-cells. For instance, studies on OVA-sensitisedIIL-5 KO mice has shown that adoptive transfer of eosinophils to these mice leads to the development of eosinophilia, Th-2 cytokine production and the development of airway hyper-responsiveness on a similar scale to that of WTTmice; however, the suppression of Th-2 activity using anti-CD4 antibody, leads to a marked reduction in effects of adoptive eosinophil transfer in AHR (Shen et al. 2003).
IL-133is a cytokine secreted by many cells types, particularly Th-2 cells. This cytokine has been shown to single-handedly induce AHR independent of eosinophilia (Grünig et al. 1998). Loss of function experiment using IL-5 and eotaxin KO mice has shown that AHR is diminished in these mice, this is believed to occur due to reduced T-cell ability in production of IL-13 (Mattes et al. 2002). In addition, it has been shown that the transfer of IL-13 producing T cells to naïve eosinophil deficient mice, results in induction of AHR, this study highlights a more indirect role for eosinophils in induction of AHR (Mattes et al. 2002). Although the majority of studies have a common theme revolving around the idea of a central role for eosinophils in development of AHR, they also commonly suggest that other important factors may contribute to AHR in shape of mediators and other cell types.
Despite the fact that eosinophilia is a shared feature amongst many asthmatic patients, its precise role in the pathology of the disease remains to be elucidated. This is partly due to the overlapping actions of various cells that permeate the lung during an allergic response, making it difficult to separately evaluate the sole contribution that eosinophils may have.
Despite the large number of studies that have focused on the role of eosinophils in the induction of AHR, this topic is still surrounded with much controversy. Eosinophilia has been the indicator of choice for many in vivo studies analysing the pathology of AHR, however these studies have frequently shown that in humans the correlation between eosinophilia and the severity of asthma, as measured by AHR remains weak at best, or even absent in non-atopic forms of asthma (Scott & Andrew Wardlaw 2006). As mentioned previously, in eosinophilic bronchitis there is a similar distribution of tissue eosinophils to that seen in asthma, however AHR is absent in EB patients, advocating a more significant role for mast cells than eosinophils in development of AHR (Brightling et al. 2002).
Since eosinophil derived MBP is held to be a direct contributor to AHR, we would expect a MBP-1-KO mice to develop a certain degree of resistance to AHR, however these mice do not develop protection against AHR or airway inflammation (Denzler et al. 2000). Furthermore, amongst studies there are indications that suggest a less significant role for eosinophils in the induction of AHR and reject the idea of eosinophils as AHR inducers. For instance, mice with ovalbumin induced airway inflammation have shown that even in the absence of T cells or AHR, CCL1 administration leads to the recruitment of eosinophils, conversely its neutralisation blocks eosinophil recruitment, however there are no changes seen in AHR severity despite varying numbers of eosinophils (Pégorier et al. 2006; Lloyd 2003).
CCR3 and IL-5 have long been the targets of choice for blocking eosinophil action, allowing researchers to delineate the importance of eosinophils in induction of AHR. CCR3 is the main chemokine receptor implicated in eosinophil attraction to the siteoof inflammation (Pégorier et al. 2006; Ponath et al. 1996); however, a downside of CCR3 targeting is that this chemokine is ubiquitously expressed on many cell types, such as Th-2 cells (Sallusto et al. 1997) and mast cells (Ochi 1999).
Studies on CCR3-KO allergen-challenged mice have revealed that eosinophils within the blood vessels are unable to pass any further than the endothelium; indicating that eotaxin andCCCR3 are both essential for the complete migration of eosinophils into the inflamed tissue (Humbles et al. 2002). In addition CCR3 is held to be an important factor for mast cell homing, CCR3-KO mice have amplified numbers of tracheal mast cells, which may be a consequence of the increased AHR seen in allergic mice (Humbles et al. 2002). This, in addition to the fact that CCR3-KO mice give opposing effects with different methods of sensitising, suggests a complicated mechanism through which eotaxin/CCR3 axis works in airway inflammation seen in asthma. Hence it may be difficult to define the exact role of eosinophils in the induction of AHR by using CCR3 knockout mice, this is even further complicated since CCR3 expression is not exclusive to eosinophils.
However, a recent study has shown that treatment with CCR3 antagonist yields a 50-60% reduction in tissue eosinophilia and leads to a reducedAAHR while inhibiting airway remodelling (Sehmi et al. 2003; Wegmann et al. 2007). But, since in this experiment macrophages and lymphocytes retained their normal rate of infiltration and local production of IL-5 and IL-13 stayed unchanged, it is suggested that the observed reduction inAAHR could be due to the CCR3 antagonism on mast cells. In addition, the evident reduction in number of eosinophils observed after CCR3 (Sallusto et al. 1997; Wegmann et al. 2007) and IL-5 (Flood-Page et al. 2003) antagonism implies that interplay and perhaps compensation by other factors may be important.
Although the correlation betweenIIIL-5 blockade andAAHR remains controversial at best, but due to the critical role of IL-5 in eosinophil trafficking, it is undoubtedly a key-contributing factor in eosinophilia. In a study it was shown that IL-5 overexpression in transgenic mice leads to the development of AHR, conversely AHR seems to be reduced in IL-5-KO mice (Foster et al. 1996), however this change was dependent on the mice strain, such that IL-5-KO mice on a BALB/c background had a less significant reduction in AHR (Mould et al. 1997; Hogan et al. 1998). Furthermore, the blockade of IL-4 in vivo, in a bid to inhibit mast cell response, did not control AHR in BALB/c mice, suggesting that AHR in this strain of mice may be regulated through various T-cell mediated mechanisms. Similarly, throughout the time, anti-IL-5 therapies amongst asthmatic patients have produce rather conflicting results as discussed later.
The generation of eosinophil-deficient mice has been a significant step forward in overcoming the difficulties associated with the interpretation of data acquired fromCCCR3 and IL-5-KO animals. Eosinophil lacking mice (Δdbl-GATA) are developed via the deletion of a high affinityGGATA binding site in theGGATA-1 promoter (Ochi 1999; Yu 2002). Following airway inflammation in both WT and Δdbl-GATA mice, there stood no significant difference in AHR severity (Foster et al. 1996; Humbles 2004). However, remarkably, transgenic mice subjected to eosinophil depletion using diphtheria-toxin, developed protection against AHR (J. J. Lee et al. 2004). More interestingly, a recent study using Δdbl- to mimic the acute worsening of allergic asthma discovered that AHR development is not conditional on recruitment of eosinophils (Siegle et al. 2006).
The variations observed in these studies could be due to the fact that Δdbl-GATA mice had a BALB/c background; whereas the wild type mice were on a C57VL/6 background; hence genetic variations may play an important part in these results. Moreover, the deletion of maturing eosinophils using diphtheria toxin by apoptosis, resulted in a marked increase in total leukocyte present in the peripheral blood (J. J. Lee et al. 2004), which may have-affected-the immune response in these mice. In addition, the amplified levels of apoptosis in the wild type model may cause a widespread immunosuppressive effect, which may be involved in the suppression of AHR (McDonald et al. 1999).
Several independent studies have been unsuccessful in detecting any residual eosinophils in the Δdbl-GATA mice; it is proposed that these residues may contribute to the insignificant difference seen in AHR severity between Δdbl-GATA and wild type mice (Voehringer et al. 2006); notably, even the backcrossing of Δdbl-GATA mice to the IL-5 transgenic mice does not induce AHR (Yu 2002). Recently Fulkerson et al. (2006) reported a minor population of eosinophil-like cells in the BAL fluid of Δdbl-GATA mice following an allergen challenge in the lungs. However, lung tissues extractions from these mice had negligible amounts of eosinophils, and the BAL eosinophils numbers were condensed to levels similar to that of CCR3-KO mice (Fulkerson et al. 2006). Hence it is unlikely that such a population could be the cause of the AHR observed by Humble et al. (2004). The use of effective eosinophil depleting agents in humans may be a more assertive approach in identifying the potential roles of eosinophils in AHR. Despite the wide array of controversies and uncertainties surrounding the role of eosinophils in the induction ofAAHR, emerging evidence suggests a possible and more important role for eosinophils in airway remodelling, which has been less acknowledged.
Eosinophils and Airway Remodelling
The inflammatory response can be viewed as a constructive mechanism, which acts to repair tissue damage and initiate quick reformation of tissue structure following damage. This is necessary for the preservation of organ integrity and function, therefore it should be of no surprise that the deregulation of this process may cause tissue fibrosis and consequently contribute to the disease state. Airway remodelling refers to the structural changes that occur in the airways of asthmatic patients, and is thought to occur due to defective regenerative processes and excessive repair in the lungs.
A characteristic feature of airway remodelling is the increased deposition of extracellular matrix (ECM) proteins such as collagen-1 and Tenascin. This increase is particularly evident within the reticular basement membrane and bronchial mucosa. Furthermore, during remodelling there is a marked increase in airway SMC mass in addition to goblet cell hyperplasia (Zhu et al. 1999).
Airway remodelling is linked to AHR and fixed airway flow obstruction and is held responsible for the accelerated decline in lung function over time in asthmatic patients compared to non-asthmatics (Kariyawasam & Robinson 2005). This decline in lung function, particularly in patients with chronic asthma, is not controllable even with aggressive anti-inflammatory therapy (Holgate 2004).
In a recent study by Pohunek et al. (2005) it was discovered that in children, airway remodelling could occur prior to the clinical diagnosis of bronchial asthma, suggesting that airway remodelling could be an ideal target representing an important strategy in controlling asthma. Despite the variety of mediators released by eosinophils that could contribute to airway remodelling, it is only in the past few years that studies have emerged exclusively investigating the link between eosinophils and airway remodelling.
The elementary role of eosinophils in airway remodelling was discovered by studies carried out on Δdbl-GATA mice. When both WT and Δdbl-GATA mice are subjected to a period of sustained allergen challenge, the WT mice display extensive airway remodelling accompanied with increased collagen deposition and airway smooth muscle proliferation in addition to hyperplasia, whereas these characteristics are significantly less apparent in the eosinophil-depleted Δdbl-GATA mice (Humbles 2004). Such observations in the Δdbl-GATA mice support human studies where anti-IL-5 treatments guard against airway remodelling (Flood-Page et al. 2003). Despite the highly disputed role of eosinophils in the induction of AHR, this study highlights the importance of eosinophil targeting in asthma and the possible benefits that it may have in prevention of remodelling.
Most fibrotic diseases share a common paradigm of prolonged inflammatory response with monocyte-leukocyte communications generating fibrogenic cytokine that can initiate and prolong fibrotic processes. Eosinophils are also able to synthesise a variety of profibrotic mediators that can contribute to the induction of airway remodelling. TGF-β is one such profibrotic cytokine, which eosinophils are thought to be an importance source of (Minshall et al. 1997); however, this cytokine is also produced by numerous other cell types such as platelets, smooth muscle, fibroblasts and epithelial cells (Boxall et al. 2006). Nevertheless, a considerable number of studies believe that eosinophil-derived TGF-β is a key factor in promoting remodelling mechanisms during asthma.
Figure-2| Mechanisms by which eosinophils may contribute to AHR, airway-remodelling and modulation of the immune response (adapted from Trivedi & Lloyd 2007).
TGF-β induces the synthesis of many ECM proteins, and is able to stimulate fibroblast proliferation. This proliferation consequently contributes to the accumulation of fibroblasts beneath the reticular basement membrane (Richter et al. 2001). TGF-β is also able to promote differentiation of myofibroblasts from resident fibroblasts and circulating precursor cells known as fibrocytes (Mori et al. 2005). It is also believed that the proliferation and differentiation of myofibroblasts into smooth muscle cell may be under the control of TGF-β (McMillan et al. 2005).
In a study by Humbles et al. (2004) it was shown that the progression of airway remodelling in mice that undergo prolonged allergen challenge, can be prevented by administration of anti-TGF-ββantibody. In addition, these animal experiments are further supported by the work of Flood-page et al. (2003), whose team showed that anti-IL5 therapy in asthmatic patients can lead to reduced ECM protein deposition within the lungs, followed by reduced levels of BAL TGF-β.
The fact that TGF-β may also induce the expression of other fibrotic factors such as plasminogen activator inhibitor (Hara et al. 2001), further complicates the process of understanding the exact role of the eosinophil derived TGF-β in airway remodelling, moreover the synergistic and antagonistic actions of TGF-β with other factors such as epidermal growth factor also adds to the difficulties in discovering the exact role of this cytokine in airway remodelling (Hara et al. 2001).
Whilst eosinophils could be the primary source of TGF-β expressing its mRNA at the early stages of the disease, many other cell types such as myofibroblasts also produce TGF-β, and are thought to be of more importance at the later stages of the disease (Tanaka et al. 2004). Using a mouse model of chronic ovalbumin allergen-induced airway remodelling, it was discovered that monocytes and macrophages are the primary source of TGF-β in the lungs, and using the same model, Δdbl-GATA mice exhibited protection against airway remodelling, although via a TGF-β independent pathway (Humbles 2004).
In 2006 Pégorier et al. showed that when the typical human bronchial epithelial cell is incubated with non-harmful concentrations of eosinophil granule portions MBP or EPO, them RNA expression of endothelin-1 platelet derived growth factor (PDGF) increases. In addition matrix metalloproteases MMP1 and MMP9 are also observed to increase; all these factors are held responsible in airway remodelling (Pégorier et al. 2006). Metalloprotease MMP9 is also linked to eosinophil recruitment, thus contributing to both inflammation and the remodelling process in asthma (Pégorier et al. 2006).
On the other hand, leukotrienes are naturally produced eicosanoid lipid mediators that eosinophils happen to be an important source of, particularly cysteinyl leukotriene LTC4 (Bandeira-Melo & Weller 2003). Mice that are genetically modified to become incapable of producing cysteinyl leukotrienes have shown protection against alveolar septal thickening and collagen deposition (Beller et al. 2004). Although leukotrienes such as LTD4 have been observed to interact with growth factors in induction of airway smooth muscle proliferation (Sehmi et al. 2003), there has also been growing evidence supporting a role for CysLTs such as LTC4 in airway remodelling. In vitro studies have shown that LTC4 is able to trigger structural cell activation as well as increasing fibroblast and airway epithelium proliferation (Sallusto et al. 1997).
In a study comparing the lung tissue from allergen challenged WT, CCR3-KO and Δdbl-GATA mice using microarray analysis, it was found that in eosinophil-deficient and CCR3-KO mice the genes for the adenosine receptor (A3) and the coagulation related genes PAI-2, factor IV and factor X are significantly reduced in relation to the WT mice (Fulkerson et al. 2006). This group of genes are held responsible in regulation of mucus and fibrin deposition, and therefore can be important targets in airway remodelling in the context of eosinophil-dependent genes.
The interactions between eosinophils and other inflammatory cells and the ways that they may propagate airway remodelling is an area of interest that may shed light on mechanisms involved in airway remodelling. Amongst the variety of mediators and molecules released by eosinophils, mast cell stem cell factor (SCF) is a crucial element secreted by eosinophils essential for mast cell survival, activation, chemotaxis and function. This may be a good indication as to why tissue eosinophilia is usually accompanied by mast cell recruitment.
One of the consequences of mast cell activation is the secretion of TNF-α, a potent stimulus that triggers the release of eosinophil derived TGF-β1. Mast cells alone can significantly contribute to airway remodelling, since they too are an important source of fibrogenic factors. Tryptase is one such mast cell-produced fibrogenic factor, which acts through the G-protein coupled protease activated receptor type-2 (PAR-2) present on inflammatory cells in addition to the epithelium, fibroblasts and the airway smooth muscle cells (Palframan et al. 1998). PAR-22activation is linked to structural cell activation and proliferation, which leads to fibroblasts secretion of GM-CSF and SCF, thus leading to the preservation of eosinophil and mast cell derived inflammation and repair in the tissue (Mould et al. 1997).
Moreover, increased vascularity within the airway directly correlates with the severity of asthma whilst being inversely proportional to the degree of airway obstruction and AHR (Ochi 1999). Eosinophils infiltration is linked to increased vascularity. This condition is caused by the excessive production of variety of growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) as well as angiogenin. Using double immunohistochemical staining techniques, eosinophils were found to be a rich cellular source of VEGF and FGF in addition to angiogenin within the asthmatic airways (Foster et al. 1996). In addition to the potent effect of VEGF on inflammatory and remodelling processes, it has also been shown to enhance antigen presentation and Th-2 mediated inflammation in asthma (J. J. Lee et al. 2004).
Based on current observations, it is fairly clear that eosinophils are a classical feature of the inflammatory infiltrate and are also heavily implicated in airway remodelling. Since eosinophils are granulocytes, a large proportion of their actions depend on the release of granule proteins. However, the efficacy of this mode of immune defence is shadowed by the fact that despite the ability of the granule proteins to eliminate a finite number of pathogens they also may damage the host cell itself, hence rendering the benefit from pathogen elimination inadequate (J. Lee 2005).
It is believed that throughout evolution, eosinophils have undergone various structural and functional modifications enabling them to combat parasitic infection, bearing in mind that eosinophils existed prior to the emergence of parasitic helminths (J. Lee 2005). In addition eosinophils are believed to have existed before the development of the acquired immune system, including the Th-2 response, which is now regarded as a key mediator in clearance of parasites.
It is clear that eosinophils significantly contribute to immuno-modulation, this manifests from the fact that eosinophils are able to synthesise, store and secrete a range of cytokine all of which have shown to significantly contribute to the propagation of asthma. In addition to the direct contribution of Th-2, IL-4 (Nonaka et al. 1995) and IL-13 (Woerly et al. 2002) in asthma, other factors such as Th1, IL-12 (Grewe et al. 1998) and IFG-γ (Lamkhioued et al. 1996) as well as regulatory cytokines such asIIL-10 (Nakajima et al. 1996) are also implicated in the pathogenesis of this disease.
Furthermore, the expression of MHC class II and co-stimulatory molecules in eosinophils, implies that in humans, these cells are able to function as antigen presenting cells (Shi 2004). Moreover, in vivo studies have exhibited eosinophils ability to travel to T-celll areas of the para-tracheal lymph nodes and stimulate lymphocyte production (Shi et al. 2000; Shi 2004). They can also prime for Th-2-mediated allergic disease of the lung in naïve animals (MacKenzie et al. 2001). These in vivo observations suggest that eosinophils may influence T cell proliferation, differentiation and apoptosis, as well as polarisation by production of indolamine 2,3-dioxygenease (IDO), which leads to conversion of tryptophan to kynurenines, which induces the apoptosis of Th1 cell (Odemuyiwa et al. 2004).
Recent findings from CCR3-KO mice have shown that gene transcripts of the leukotrieneBB4 receptor, Erg-2 and IL-4 (which are implicated in T cell activation and regulation) are significantly reduced in these eosinophil-deficient mice (Fulkerson et al. 2006). The unifying consensus in recent literature is that the cells implicated in the pathology of asthma including eosinophils, need to be evaluated with a broader perspective, one that incorporates the whole biological system (a “system biology” approach) (Jacobsen et al. 2007). Table 1 summarises many of the potential cell-cell interactions that occur in the lungs, which not only induce pulmonary immune responses, but also affect airway structure and allergen-mediated pathophysiology (Jacobsen et al. 2007).
The eosinophil as a therapeutic target
Since eosinophils are implicated in nearly all key characteristic features of asthma, these cells have been targeted as part of a novel therapeutic strategy in treatment of asthma. Eosinophils have been specifically targeted via IL-5 using anti-IL-5 antibodies, however these experiments have produced rather conflicting data. Although the use of anti-IL-5 antibody in both BALB/c and C57BL/6 mice (Hamelmann 2001) leads to diminished eosinophilia and reduced AHR, but such dramatic changes are not evident when treating established airway disease (Mathur et al. 1999).
Following experiments on mice, two monoclonal antibodies against IL-5 (SCH55700 and Mebolizumab) were developed and used in clinical trials. Mebolizumab treatments emerged to be effective in preventing maturation of eosinophils in the bone marrow as well as reducing the number of eosinophils in the bronchial mucosa (Menzies-Gow et al. 2003).
Furthermore, Leckie et al. (2000) showed that when allergen-challenged atopic asthmatic are administered a single infusion of Mebolizumab, they showed a marked reduction in blood and sputum eosinophil numbers, lasting for up to 30 days after the treatment. However similar to animal studies, once the allergen is introduced, the anti-IL5 antibody fails to make any improvement in the late asthmatic reaction or AHR (Leckie et al. 2000). These observations abound with previous studies led to the belief that eosinophils may not be necessary or important in the induction of airway hyper-responsiveness. In support of this notion, another study showed that in patients with severe asthma the administration of SCH55700 leads to a prolonged reduction in blood eosinophils, but without any evidence of improved lung function (Kips 2003).
The data obtained from these trials should be treated very cautiously, since both anti IL-5 antibodies used in these clinical trials were either tested for a very short time (SCH55700) or were criticised due to their lack of statistical power (Mebolizumab) (O’Byrne et al. 2001), thus failing to provide any conclusive evidence. Moreover subsequent studies have shown that repeated administration of Mebolizumab (SB240563) only leads to a 55% reduction in eosinophil numbers in the bronchial mucosa (Flood-Page et al. 2003), although this reduction still caused a noteworthy decline in factors involve in airway remodelling, such as reduced deposition of Tenascin, pro-collagen III and a decreased percentage of eosinophils that express the mRNA for TGF-β (Flood-Page et al. 2003).
Given the inefficacy of anti-IL5 therapies, targeting the CCR-3/eotaxin axis may be a more effective strategy in controlling asthma; since the blockade of this axis prevents eotaxin mediated eosinophil recruitment as well as affecting a variety of other cell types, such as Th-2 cells and mast cells, both of which are implicated in asthma.
In a recent study on murine models of allergic airway inflammation, it was demonstrated that the administration of a low molecular weight CCR3 antagonist results in diminution of AHR and subsequently prevents airway remodelling (Wegmann et al. 2007). DPC168, having completed phase I clinical trials for asthma and allergic rhinitis, is currently the most effective CCR3 antagonist, offering the most reliable and advanced data (De Lucca et al. 2005).
This chemokine/receptor axis has also been targeted using neutralising monoclonal antibodies directed at CCR3, which lead to the blockage of chemotaxis and CCR3 induced calcium influx in human eosinophils (Heath et al. 1997). Moreover, in vivo studies have shown that the administration of anti-CCR3 antibody in mice leads to improved asthma pathology (Jacoby et al. 1993). Bertilimumab, a human IgG4 monoclonal antibody specific for eotaxin-1, is currently the most advance anti-chemokine antibody used for possible treatment of allergic disorders such as allergic rhinitis and is currently in preclinical investigation for asthma (Ding & Li 2004).
Ever since the failure of anti-IL5 therapies in improving lung function in asthmatic patients, the pharmaceutical industry has been reluctant to invest in development of eosinophil specific therapies targeting asthma; in addition, the possibility that eosinophils may not even be significant in induction of AHR adds to their hesitation (Yu 2002).
Future trials that incorporate a wider combination of therapies such as those designed to target both CCR3 and IL-5 should be designed and tested before ruling out any potential benefit of eosinophil targeting in asthmatic patients, particularly when considering the possible role of eosinophils in airway remodelling during asthma.
The growing literature abounds with emergent studies demonstrating a more extensive and complicated role for eosinophils in the development of allergen mediated pulmonary pathology, particularly asthma. Despite the large number of studies that either support or provide evidence against the role of eosinophils in the pathology of this disease, the studies outlined in this review attest to a changing paradigm where eosinophils are no longer regarded as mere correlative factors in allergic pulmonary disease, and are believed to encompass a greater variety of functions than that of a basic granulocyte.
The eosinophil remains an important therapeutic target in the management of asthma, since these cells are likely to be an integral component of the pathways involved in the allergic immune response, as well as pathophysiologic instabilities affecting lung function. They are also implicated in the histopathologic changes, (i.e. airway remodelling) which occur both in the acute and chronic phases of asthma. In addition, the important immunomodulatory functions exhibited by eosinophils, adds to the significance of these cells as therapeutic targets.
Future studies examining the mechanism by which eosinophils may induce airway remodelling and modulate specific immune functions will aid our understanding of the extensive role of eosinophils in the pathogenesis of asthma, thus allowing us to identify and devise the best methods for targeting eosinophils and the best strategies for tackling this fast growing disease. Nevertheless, emerging evidences show that this cell has still to be recognised with, and it is likely that eosinophils will have more unexpected implications in the pathogenesis of this fast growing disease.
- Alam, R. et al., 1996. Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage fluid of allergic asthmatic patients. American journal of respiratory and critical care medicine, 153(4 Pt 1), pp.1398–1404.
- Bandeira-Melo, C. & Weller, P.F., 2003. Eosinophils and cysteinyl leukotrienes. Prostaglandins, Leukotrienes and Essential Fatty Acids, 69(2-3), pp.135–143.
- Beller, T.C. et al., 2004. Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis. Proceedings of the National Academy of Sciences of the USA, 101(9), pp.3047–3052.
- Bentley, A., Corrigan, C. & Durham, S., 1992. Predominant TH-2-like bronchoalveolar T-lymphocyte population in atopic asthma. The New England Journal of Medicine. 326(5), pp.298-304
- Boxall, C., Holgate, S.T. & Davies, D.E., 2006. The contribution of transforming growth factor-beta and epidermal growth factor signalling to airway remodelling in chronic asthma. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology, 27(1), pp.208–229.
- Brightling, C.E. et al., 2002. Mast-cell infiltration of airway smooth muscle in asthma. The New England journal of medicine, 346(22), pp.1699–1705.
- Costello, R. & Schofield, B., 1997. Localization of eosinophils to airway nerves and effect on neuronal M2 muscarinic receptor function. American Journal of Physiology. 273(1), pp.93-103.
- Coyle, A. & Gutierrez-Ramos, J., 2000. Cc Chemokine Receptor (Ccr)3/Eotaxin Is Followed by Ccr4/Monocyte-Derived Chemokine in Mediating Pulmonary T Helper Lymphocyte Type 2 Recruitment after Serial Antigen Challenge in Vivo. The Journal of Experimental Medicine. 191(2), pp.265-274.
- De Lucca, G.V. et al., 2005. Discovery of CC chemokine receptor-3 (CCR3) antagonists with picomolar potency. Journal of medicinal chemistry, 48(6), pp.2194–2211.
- DeKoter, R.P. & Singh, H., 2000. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science, 288(5470), pp.1439–1441.
- Denzler, K., Farmer, S. & Crosby, J., 2000. Eosinophil Major Basic Protein-1 Does Not Contribute to Allergen-Induced Airway Pathologies in Mouse Models of Asthma. The Journal of Immunology, 165, pp.5509-5517
- Ding, C. & Li, J., 2004. Bertilimumab Cambridge Antibody Technology Group. Current Opinion in Investigational Drugs. 5(11), pp.1213-8
- Du, J. et al., 2002. Novel combinatorial interactions of GATA-1, PU.1, and C/EBPepsilon isoforms regulate transcription of the gene encoding eosinophil granule major basic protein. Journal of Biological Chemistry, 277(45), pp.43481–43494.
- Elbon, C., Jacoby, D. & Fryer, A., 1995. Pretreatment with an antibody to interleukin-5 prevents loss of pulmonary M2 muscarinic… – Abstract – UK PubMed Central. Americal journal of respiratory cell and Molecular Biology. 12(3), pp. 320-8
- Erjefält, J.S. & Persson, C.G., 2000. New aspects of degranulation and fates of airway mucosal eosinophils. American journal of respiratory and critical care medicine, 161(6), pp.2074–2085.
- Erjefält, J.S. et al., 1999. Allergen-induced eosinophil cytolysis is a primary mechanism for granule protein release in human upper airways. American journal of respiratory and critical care medicine, 160(1), pp.304–312.
- Erjefält, J.S. et al., 2001. Degranulation patterns of eosinophil granulocytes as determinants of eosinophil driven disease. Thorax, 56(5), pp.341–344.
- Flood-Page, P. et al., 2003. Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics. Journal of Clinical Investigation, 112(7), pp.1029–1036.
- Forssmann, U. et al., 1997. Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leukocytes. The Journal of experimental medicine, 185(12), pp.2171–2176.
- Foster, P.S. et al., 1996. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. The Journal of experimental medicine, 183(1), pp.195–201.
- Frigas, E. et al., 1981. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clinic proceedings. Mayo Clinic, 56(6), pp.345–353.
- Fulkerson, P.C. et al., 2006. A central regulatory role for eosinophils and the eotaxin/CCR3 axis in chronic experimental allergic airway inflammation. Proceedings of the National Academy of Sciences of the United States of America, 103(44), pp.16418–16423.
- Gavett, S.H. et al., 1994. Depletion of murine CD4+ T lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia. American journal of respiratory cell and molecular biology, 10(6), pp.587–593.
- Gleich, G.J., 2000. Mechanisms of eosinophil-associated inflammation. Journal of allergy and clinical immunology, 105(4), pp.651–663.
- Gregory, B. et al., 2003. Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor … but up-regulate IL-3 receptor alpha expression. Journal of Immunology, 170(11), pp.5359–5366.
- Grewe, M. et al., 1998. Human eosinophils produce biologically active IL-12: implications for control of T cell responses. Journal of Immunology, 161(1), pp.415–420.
- Griffiths-Johnson, D.A. et al., 1993. The chemokine, eotaxin, activates guinea-pig eosinophils in vitro and causes their accumulation into the lung in vivo. Biochemical and biophysical research communications, 197(3), pp.1167–1172.
- Grünig, G. et al., 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science, 282(5397), pp.2261–2263.
- Gutierrez-Ramos, J.C., Lloyd, C. & Gonzalo, J.A., 1999. Eotaxin: from an eosinophilic chemokine to a major regulator of allergic reactions. Immunology today, 20(11), pp.500–504.
- Hafez, I., Stolpe, A. & Lindau, M., 2003. Compound exocytosis and cumulative fusion in eosinophils. Journal of Biological Chemistry, 278(45), pp.44921–44928.
- Hamelmann, E., 2001. IL-5-induced airway eosinophilia, key to asthma? Immunological Reviews. 179(1), pp.182-191.
- Hara, K. et al., 2001. Inhibitory role of eosinophils on cell surface plasmin generation by bronchial epithelial cells: inhibitory effects of transforming growth factor beta. Lung, 179(1), pp.9–20.
- Heath, H. et al., 1997. Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. Journal of Clinical Investigation, 99(2), pp.178–184.
- Hirshman, C.A., Lande, B. & Croxton, T.L., 1999. Role of M2 muscarinic receptors in airway smooth muscle contraction. Life sciences, 64(6-7), pp.443–448.
- Hoffmann, H.J. et al., 2001. SNARE Proteins Are Critical for Regulated Exocytosis of ECP from Human Eosinophils. Biochemical and biophysical research com. 282(1), pp.194–199.
- Hogan, S.P. et al., 1998. A novel T cell-regulated mechanism modulating allergen-induced airways hyperreactivity in BALB/c mice independently of IL-4 and IL-5. Journal of Immunology, 161(3), pp.1501–1509.
- Holgate, S.T., 2004. Epithelial-Mesenchymal Communication in the Pathogenesis of Chronic Asthma. Proceedings of the American Thoracic Society, 1(2), pp.93–98.
- Humbles, A.A., 2004. A Critical Role for Eosinophils in Allergic Airways Remodeling. Science, 305(5691), pp.1776–1779.
- Humbles, A.A. et al., 2002. The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proceedings of the National Academy of Sciences of the United States of America, 99(3), pp.1479–1484.
- Jacobsen, E. A., Ochkur, S. I., Lee, N. A., & Lee, J. J. (2007). Eosinophils and asthma. Current Allergy and Asthma Reports, 7(1), pp. 18–26.
- Jacoby, D.B., Gleich, G.J. & Fryer, A.D., 1993. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor. Journal of Clinical Investigation, 91(4), pp.1314–1318.
- Johansson, A.-K. et al., 2004. Allergen stimulates bone marrow CD34(+) cells to release IL-5 in vitro; a mechanism involved in eosinophilic inflammation? Allergy. 59(10), pp.1080–1086.
- Justice, J.P. et al., 2003. Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology. American journal of physiology. Lung cellular and molecular physiology, 284(1), pp.L169–78.
- Kariyawasam, H.H. & Robinson, D.S., 2005. Airway remodelling in asthma: models and supermodels? Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 35(2), pp.117–121.
- Kips, J.C., 2003. Effect of SCH55700, a Humanized Anti-Human Interleukin-5 Antibody, in Severe Persistent Asthma: A Pilot Study. American journal of respiratory and critical care medicine, 167(12), pp.1655–1659.
- Lacy, P. et al., 2001. Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-induced piecemeal degranulation in human eosinophils from atopic individuals. The Journal of allergy and clinical immunology, 107(4), pp.671–678.
- Lacy, P. et al., 1999. Rapid mobilization of intracellularly stored RANTES in response to interferon-gamma in human eosinophils. Blood, 94(1), pp.23–32.
- Lamkhioued, B., Gounni, A. & Aldebert, D., 1996. Synthesis of Type 1 (IFNyγ) and Type 2 (IL-4, IL-5, and IL-10) Cytokines by Human Eosinophils. Annals of the New York Academy of Sciences. 796, pp.203-208
- Leckie, M.J. et al., 2000. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet, 356(9248), pp.2144–2148.
- Lee, J., 2005. Eosinophil degranulation: an evolutionary vestige or a universally destructive effector function? Clinical & Experimental Allergy. 35(8), pp.986-994.
- Lee, J.J. et al., 2004. Defining a link with asthma in mice congenitally deficient in eosinophils. Science, 305(5691), pp.1773–1776.
- Lloyd, C., 2003. CC chemokine ligand 1 promotes recruitment of eosinophils but not Th-2 cells during the development of allergic airways disease. The Journal of Immunology. 170(9), pp.4810-4817.
- Lloyd, C.M. et al., 2001. Resolution of bronchial hyperresponsiveness and pulmonary inflammation is associated with IL-3 and tissue leukocyte apoptosis. Journal of Immunology, 166(3), pp.2033–2040.
- Logan, M.R., Odemuyiwa, S.O. & Moqbel, R., 2003. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. The Journal of allergy and clinical immunology, 111(5), pp.923–32.
- MacKenzie, J.R. et al., 2001. Eosinophils promote allergic disease of the lung by regulating CD4(+) Th-2 lymphocyte function. Journal of Immunology, 167(6), pp.3146–3155.
- Mathur, M. et al., 1999. TRFK-5 Reverses Established Airway Eosinophilia But Not Established Hyperresponsiveness in a Murine Model of Chronic Asthma. American Journal of Respiratory and Critical Care Medicine. 159(2), pp.580-587.
- Mattes, J. et al., 2002. 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. The Journal of experimental medicine, 195(11), pp.1433–1444.
- McDonald, P.P. et al., 1999. Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells. Journal of Immunology, 163(11), pp.6164–6172.
- McMillan, S.J., Xanthou, G. & Lloyd, C.M., 2005. Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the Smad signaling pathway. Journal of Immunology, 174(9), pp.5774–5780.
- Menzies-Gow, A. et al., 2003. Anti–IL-5 (mepolizumab) therapy induces bone marrow eosinophil maturational arrest and decreases eosinophil progenitors in the bronchial mucosa of atopic asthmatics. Journal of allergy and clinical immunology, 111(4), pp.714–719.
- Minnicozzi, M., Duran, W. & Gleich, G., 1994. Eosinophil granule proteins increase microvascular macromolecular transport in the hamster cheek pouch. The Journal of Immunology. 153(6), pp.2664-2670
- Minshall, E., Leung, D. & Martin, R., 1997. Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma. – Abstract – UK PubMed Central. American Journal of Respiratory Cell and Molecular Biology, 17(3), pp.326-33
- Mori, L. et al., 2005. Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Experimental cell research, 304(1), pp.81–90.
- Morita, E., Schröder, J.M. & Christophers, E., 1989. Differential sensitivities of purified human eosinophils and neutrophils to defined chemotaxins. Scandinavian journal of immunology, 29(6), pp.709–716.
- Motojima, S. et al., 1989. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. The American review of respiratory disease, 139(3), pp.801–805.
- Mould, A.W. et al., 1997. Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice. Journal of Clinical Investigation, 99(5), pp.1064–1071.
- Nakajima, H., Gleich, G.J. & Kita, H., 1996. Constitutive production of IL-4 and IL-10 and stimulated production of IL-8 by normal peripheral blood eosinophils. Journal of Immunology, 156(12), pp.4859–4866.
- Nonaka, M. et al., 1995. Distinct immunohistochemical localization of IL-4 in human inflamed airway tissues. IL-4 is localized to eosinophils in vivo and is released by peripheral blood eosinophils. Journal of Immunology, 155(6), pp.3234–3244.
- O’Byrne, P.M., 2003. Airway Hyperresponsiveness. Chest, 123(90030), pp.411S–416.
- O’Byrne, P.M., Inman, M.D. & Parameswaran, K., 2001. The trials and tribulations of IL-5, eosinophils, and allergic asthma. The Journal of allergy and clinical immunology, 108(4), pp.503–508.
- Ochi, H., 1999. T Helper Cell Type 2 Cytokinemediated Comitogenic Responses and CCR3 Expression During Differentiation of Human Mast Cells In Vitro. Journal of Experimental Medicine, 190(2), pp.267–280.
- Odemuyiwa, S.O. et al., 2004. Cutting edge: human eosinophils regulate T cell subset selection through indoleamine 2,3-dioxygenase. Journal of Immunology, 173(10), pp.5909–5913.
- Palframan, R.T. et al., 1998. Mechanisms of acute eosinophil mobilization from the bone marrow stimulated by interleukin 5: the role of specific adhesion molecules and phosphatidylinositol 3-kinase. The Journal of experimental medicine, 188(9), pp.1621–1632.
- Pégorier, S. et al., 2006. Eosinophil-derived cationic proteins activate the synthesis of remodeling factors by airway epithelial cells. Journal of Immunology, 177(7), pp.4861–4869.
- Piliponsky, A.M. et al., 2002. Effects of eosinophils on mast cells: a new pathway for the perpetuation of allergic inflammation. Molecular immunology, 38(16-18), p.1369.
- Pohunek, P. et al., 2005. Markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 16(1), pp.43–51.
- Ponath, P.D. et al., 1996. Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. Journal of Clinical Investigation, 97(3), pp.604–612.
- Pope, S.M. et al., 2005. The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia. Journal of Immunology, 175(8), pp.5341–5350.
- Ravensberg, A.J. et al., 2005. Eotaxin-2 and eotaxin-3 expression is associated with persistent eosinophilic bronchial inflammation in patients with asthma after allergen challenge. Journal of allergy and clinical immunology, 115(4), pp.779–785.
- Richter, A. et al., 2001. The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic unit in asthma. American journal of respiratory cell and molecular biology, 25(3), pp.385–391.
- Robinson, D., Damia, R. & Zeibecoglou, K., 1999. CD34(+)/interleukin-5Ralpha messenger RNA+ cells in the bronchial mucosa in asthma: potential… – Abstract – UK PubMed Central. American Journal of Respiratory Cell and Molecular Biology, 20(1), pp.9-13
- Roffel, A.F., Elzinga, C.R. & Zaagsma, J., 1990. Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle. Pulmonary pharmacology, 3(1), pp.47–51.
- Rothenberg, M.E., 1997. Targeted Disruption of the Chemokine Eotaxin Partially Reduces Antigen-induced Tissue Eosinophilia. Journal of Experimental Medicine, 185(4), pp.785–790.
- Sabroe, I. et al., 1999. Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways. Journal of Immunology, 162(5), pp.2946–2955.
- Sallusto, F., Mackay, C.R. & Lanzavecchia, A., 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science, 277(5334), pp.2005–2007.
- Sanderson, C., 1992. Interleukin-5, eosinophils, and disease. Blood.
- Schragge, W., O’Byrne, P. & Denburg, J., 1996. Increased levels of CD34+ hemopoietic progenitor cells in atopic subjects. American Journal of Respiratory Cell and Molecular Biology, 15(5), pp.645-55.
- Scott, K. & Wardlaw, Andrew, 2006. Eosinophilic Airway Disorders. Seminars in Respiratory and Critical Care Medicine, 27(2), pp.128–133.
- Sehmi, R. et al., 2003. Allergen-induced fluctuation in CC chemokine receptor 3 expression on bone marrow CD34+ cells from asthmatic subjects: significance for mobilization of haemopoietic progenitor cells in allergic inflammation. Immunology. 109(4), pp.1365-2567
- Shen, H.H. et al., 2003. A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse. Journal of Immunology, 170(6), pp.3296–3305.
- Shi, H.Z., 2004. Eosinophils function as antigen-presenting cells. Journal of leukocyte biology, 76(3), pp.520–527.
- Shi, H.Z. et al., 2000. Lymph node trafficking and antigen presentation by endobronchial eosinophils. Journal of Clinical Investigation, 105(7), pp.945–953.
- Siegle, J.S. et al., 2006. Airway hyperreactivity in exacerbation of chronic asthma is independent of eosinophilic inflammation. American journal of respiratory cell and molecular biology, 35(5), pp.565–570.
- Southam, D.S. et al., 2005. Increased eosinophil-lineage committed progenitors in the lung of allergen-challenged mice. Journal of allergy and clinical immunology, 115(1), pp.95–102.
- Tanaka, H. et al., 2004. Role of interleukin-5 and eosinophils in allergen-induced airway remodeling in mice. American journal of respiratory cell and molecular biology, 31(1), pp.62–68.
- Trivedi, S.G. & Lloyd, C.M., 2007. Eosinophils in the pathogenesis of allergic airways disease. Cellular and Molecular Life Sciences, 64(10), pp.1269–1289.
- Tulic, M., Liu, M. & Moqbel, R., 2003. Inflammatory cells in asthma: mechanisms and implications for therapy. Journal of Allergy and clinical immunology. 111(1), pp.5-17
- Uguccioni, M. et al., 1997. High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines. Journal of Clinical Investigation, 100(5), pp.1137–1143.
- Uller, L. et al., 2006. Effects of steroid treatment on lung CC chemokines, apoptosis and transepithelial cell clearance during development and resolution of allergic airway inflammation. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 36(1), pp.111–121.
- Voehringer, D. et al., 2006. Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system. The Journal of experimental medicine, 203(6), pp.1435–1446.
- Wardlaw, AJ, Cromwell, O. & Kurihara, K., 1992. Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects. Blood. 79(11), pp.2952-2959
- Wardlaw, A J, 1999. Molecular basis for selective eosinophil trafficking in asthma: A multistep paradigm. The Journal of allergy and clinical immunology, 104(5), pp.917–926.
- Wegmann, M. et al., 2007. Effects of a low-molecular-weight CCR-3 antagonist on chronic experimental asthma. American journal of respiratory cell and molecular biology, 36(1), pp.61–67.
- Woerly, G. et al., 2002. Human eosinophils express and release IL-13 following CD28-dependent activation. Journal of leukocyte biology, 72(4), pp.769–779.
- Yu, C., 2002. Targeted Deletion of a High-Affinity GATA-binding Site in the GATA-1 Promoter Leads to Selective Loss of the Eosinophil Lineage In Vivo. Journal of Experimental Medicine, 195(11), pp.1387–1395.
- Zhu, Z., Chupp, G. & Homer, R., 1999. JCI – Airway remodeling in asthma. Journal of Clinical Investigation, 104(8), pp.1001-1006
- Zimmermann, N. & Rothenberg, M.E., 2003. Receptor internalization is required for eotaxin-induced responses in human eosinophils. The Journal of allergy and clinical immunology, 111(1), pp.97–105.