Coexpression of IL-5 and eotaxin-2 in mice creates an eosinophil-dependent model of respiratory inflammation with characteristics of severe asthma

Mouse models of allergen provocation and/or transgenic gene expression have provided significant insights regarding the cellular, molecular, and immune responses linked to the pathologies occurring as a result of allergic respiratory inflammation. Nonetheless, the inability to replicate the eosinophil activities occurring in patients with asthma has limited their usefulness to understand the larger role(s) of eosinophils in disease pathologies. These limitations have led us to develop an allergen-naive double transgenic mouse model that expresses IL-5 systemically from mature T cells and eotaxin-2 locally from lung epithelial cells. We show that these mice develop several pulmonary pathologies representative of severe asthma, including structural remodeling events such as epithelial desquamation and mucus hypersecretion leading to airway obstruction, subepithelial fibrosis, airway smooth muscle hyperplasia, and pathophysiological changes exemplified by exacerbated methacholine-induced airway hyperresponsiveness. More importantly, and similar to human patients, the pulmonary pathologies observed are accompanied by extensive eosinophil degranulation. Genetic ablation of all eosinophils from this double transgenic model abolished the induced pulmonary pathologies, demonstrating that these pathologies are a consequence of one or more eosinophil effector functions.     

Eosinophil Granule Proteins: Form and Function

Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.     

IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation

Background

Thymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.

Methods

Eosinophil mRNA expression of TSLPR and IL-7R?? was examined by real-time quantitative PCR of human eosinophils treated with TNF?? and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNF?? and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.

Results

Eosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNF?? and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNF?? and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.

Conclusions

This study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.     

Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow

Allergic inflammation involves the mobilization and trafficking of eosinophils to sites of inflammation. Galectin-3 (Gal-3) has been shown to play a critical role in eosinophil recruitment and airway allergic inflammation in vivo. The role played by Gal-3 in human eosinophil trafficking was investigated. Eosinophils from allergic donors expressed elevated levels of Gal-3 and demonstrated significantly increased rolling and firm adhesion on immobilized VCAM-1 and, more surprisingly, on Gal-3 under conditions of flow. Inhibition studies with specific mAbs as well as lactose demonstrated that: 1) eosinophil-expressed Gal-3 mediates rolling and adhesion on VCAM-1; 2) alpha(4) integrin mediates eosinophil rolling on immobilized Gal-3; and 3) eosinophil-expressed Gal-3 interacts with immobilized Gal-3 through the carbohydrate recognition domain of Gal-3 during eosinophil trafficking. These findings were further confirmed using inflamed endothelial cells. Interestingly, Gal-3 was found to bind to alpha(4) integrin by ELISA, and the two molecules exhibited colocalized expression on the cell surface of eosinophils from allergic donors. These findings suggest that Gal-3 functions as a cell surface adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.     

Chemokine receptor inhibitor,Antileukinate, suppressed ovalbumin-induced eosinophilic inflammation in the airway

Accumulating evidence suggests that eosinophils play an important role in the pathogenesis of allergic diseases. An eosinophil-active chemokine, eotaxin, and its receptor, C-C chemokine receptor 3, are particularly attractive as novel targets of immunological intervention for the disease. In this study, we examine the effects of a hexa-peptide (Ac-RRWWCR-NH(2)), Antileukinate, which we have previously defined as a potent inhibitor of CXC chemokine receptor 1 and 2, on eotaxin in vitro and in vivo. Antileukinate inhibited the binding of 125I-labeled human eotaxin to human eosinophils with an IC(50) of approximately 10 microM and eosinophil chemotaxis to human eotaxin was significantly inhibited by 10 microM of Antileukinate. We examined the effects of Antileukinate on eosinophil accumulation induced by intraperitoneal administration of murine eotaxin, and confirmed that Antileukinate is also active in the murine system. When Antileukinate was tested in ovalbumin-induced airway inflammation model in vivo, Antileukinate significantly inhibited eosinophil accumulation and allergen-induced increase in total protein in bronchoalveolar lavage fluids. Furthermore, Antileukinate suppressed fibrous thickening of submucosal tissue induced by chronic antigen challenge. These results suggest that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation, and that Antileukinate may be a promising tool to control allergic diseases.     

Biological activities of ecalectin: a novel eosinophil-activating factor

Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for beta-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the beta-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs

Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.     

Prostaglandin E2 inhibits eosinophil trafficking through E-prostanoid 2 receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E(2) exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE(2) potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD(2), and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE(2) on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE(2), inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE(2) on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.     

A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse

Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.     

Does IgE bind to and activate eosinophils from patients with allergy?

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.     

Stimulation of degranulation from human eosinophils by platelet-activating factor

Platelet-activating factor (PAF) is a highly active mediator which has been implicated in allergic inflammation and bronchial asthma, possibly by interacting with eosinophils. We have examined the effect of PAF on activation of purified human eosinophils as measured by degranulation (eosinophil peroxidase, eosinophil cationic protein, arylsulfatase B, beta-glucuronidase, and alkaline phosphatase) and oxidative metabolism (superoxide anion production). PAF induced enzyme release at concentrations ranging from 1 pM to 10 microM in a rapid (t1/2 5 to 8 min), Ca2+-dependent and noncytotoxic manner from both the specific and small granules, whereas its biologic precursor and metabolite, lyso-PAF, had no effect. For all enzymes, maximal enzyme release occurred at 100 nM PAF with a mean ED50 value of 1.47 +/- 0.4 nM. At this concentration the mean percentage of total enzyme release by PAF from specific granules was 20.3 +/- 1.6% (17.9% for eosinophil peroxidase, 20.6% for beta-glucuronidase, 22.4% for alkaline phosphatase) and 28.8 +/- 2.2% from small granules (arylsulfatase B). Calcium ionophore A23187, PMA, and opsonized zymosan also induced eosinophil degranulation but their peak effect after 10-min incubation with maximal release 14.7%, 12.9%, or 14.1%, respectively, was lower when compared with PAF. Incubation of eosinophils with the PAF-antagonist WEB 2086 led to a parallel shift of the dose-response curve to the right, indicating a competitive antagonism. PAF also caused generation of superoxide anions by human eosinophils but this occurred at higher concentrations of PAF (1 microM to 30 microM) with an ED50 of 8.4 +/- 0.9 microM. Again, this effect was competitively inhibited by WEB 2086. These studies demonstrate that PAF activates human eosinophils to release granule constituents and generate superoxide anions. Since both PAF and eosinophil products are associated with pathogenesis of bronchial asthma our findings may be of particular pathophysiologic relevance.     

IgA-induced eosinophil degranulation

Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.     

Requirement for CD28 in the effector phase of allergic airway inflammation

Central to the pathogenesis of allergic airway inflammation are the activation and differentiation of T lymphocytes. This process requires the participation of the CD28 costimulatory receptor. Blockade of CD28 has been demonstrated to prevent inflammation and airway hyperreactivity in a murine model of asthma. Whether this is due specifically to defects in initial T cell activation or whether effector responses are also impaired has not been determined. Using adoptive transfer studies of Ag-specific lymphocytes, we demonstrate that CD28 has a critical role in both the induction and effector phase of allergic airway inflammation. Transfer of in vitro activated and Th2-differentiated Ag-specific lymphocytes from wild-type hosts restored inflammation, but not tissue eosinophilia in CD28-deficient recipients. Furthermore, similarly activated and differentiated CD28-deficient lymphocytes were ineffective at mediating inflammation in wild-type recipients. Secondary cytokine and proliferative responses of activated Th2 cells were highly dependent on CD28 in vitro. Moreover, eosinophil recruitment to both the lung and peritoneum is impaired by the lack of CD28, suggesting a generalized defect in the ability of eosinophils to accumulate at sites of inflammation in vivo. These data identify a novel role for CD28 in the effector phase of allergic airway inflammation and suggest that inhibition of this pathway may be a useful therapeutic intervention in previously sensitized individuals.     

Regulation of Ig-induced eosinophil degranulation by adenosine 3',5'-cyclic monophosphate.

We have investigated the effects of cAMP on Ig-induced human eosinophil activation. Stimulation of human normodense eosinophils with IgG- or secretory IgA (sIgA)-coated Sepharose beads induced cellular degranulation, as measured by the release of the granule protein, eosinophil-derived neurotoxin (EDN). Pretreatment with cAMP analogs (N6,O2,-dibutyryl adenosine-3,':5' cyclic monophosphate; 8-bromoadenosine 3':5' cyclic monophosphate; or N6-benzoyladenosine 3':5' cyclic monophosphate) or cAMP phosphodiesterase-inhibitors (theophylline or isobutylmethyl xanthine (IBMX] strongly inhibited Ig-induced human eosinophil degranulation. The beta-adrenoceptor agonists, isoproterenol and salbutamol, induced relatively low level increases in intracellular cAMP, and weakly suppressed EDN release induced by IgG-coated beads. However, cellular pretreatment with IBMX synergistically enhanced the inhibitory effects of isoproterenol or salbutamol on both IgG and sIgA-induced eosinophil degranulation. Similarly, PGE2 treatment increased intracellular cAMP concentrations in eosinophils and correspondingly inhibited the Ig-dependent cellular degranulation response: co-incubation with IBMX further enhanced both effects of PGE2. Finally, cholera toxin, which irreversibly activates the stimulatory guanine nucleotide-binding protein linked to adenylyl cyclase, strongly inhibited the release of EDN from IgG- or sIgA-stimulated eosinophils. The time-dependent accumulation of cAMP in cholera toxin-treated cells closely paralleled the time courses of inhibition of IgG- and sIgA-induced EDN release after toxin exposure. These data indicate that the cAMP-dependent signal transduction mechanism in eosinophils exerts a negative modulatory effect on the cellular degranulation responses induced by sIgA or IgG. The inhibitory effects of cAMP on eosinophil activation may provide an important physiologic and a clinically relevant therapeutic mechanism for limiting the release of eosinophil-derived cytotoxic proteins during certain allergic or inflammatory responses in vivo.     

Allergen-specific CTL require perforin expression to suppress allergic airway inflammation

Allergen-specific CTL have a protective effect on allergic airway inflammation, a function thought to be mediated by cytokines, especially IFN-γ. However, the contribution of cytotoxic function to this protective effect has not been investigated. We examined the contribution of cytotoxic function to the therapeutic effect of allergen-specific CTL in allergic airway inflammation. We used a murine model of allergic airway inflammation in which mice were sensitized to OVA and then challenged with the same Ag via the intranasal route. CTL were elicited in these mice by immunization with dendritic cells (DC) or by adoptive transfer of in vitro-activated CD8(+) T cells. Hallmark features of allergic asthma, such as infiltration of eosinophils in the bronchoalveolar lavage fluid and mucus production, were assessed. Suppression of allergic airway inflammation by allergen-specific CTL was critically dependent on the expression of perforin, a key component of the cytotoxic machinery. Both perforin-sufficient and perforin-deficient allergen-specific CTL were recovered from the lungs of allergen-sensitized mice and upregulated CD69 expression and secreted the cytokines IFN-γ and TNF-α upon intranasal allergen challenge. However, only perforin-sufficient CTL inhibited eosinophil infiltration in the airway, mucus production, and cytokine accumulation in the bronchoalveolar lavage fluid. Treatment with allergen-specific CTL, but not their perforin-deficient counterparts, was also associated with a decrease in the number of DC in the mediastinal lymph node. Our data suggest that the cytotoxic function of allergen-specific CD8(+) T cells is critical to their ability to moderate allergic airway inflammation.     

Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation

Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.     

α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells

Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.     

Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)β(2) integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)β(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.     

Transduction of phosphatase and tensin homolog deleted on chromosome 10 into eosinophils attenuates survival, chemotaxis, and airway inflammation

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN antagonizes the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. In the present study, we used a TAT fusion protein transduction system to elucidate the role of PTEN in eosinophils and airway inflammation. A small region of the HIV TAT protein (YGRKKRRQRRR), a protein transduction domain known to enter mammalian cells efficiently, was fused to the N terminus of PTEN. Flow cytometric analysis of annexin V- and propidium iodide-stained cells was used to assess eosinophil survival. A chemotaxis assay was performed using a Boyden chamber. Cell analysis in bronchoalveolar lavage fluid and histological examinations were performed using OVA-challenged A/J mice. We found that TAT-PTEN was successfully internalized into eosinophils and functioned as a phosphatase in situ. TAT-PTEN, but not a TAT-GFP control protein, blocked the ability of IL-5 to prevent the apoptosis of eosinophils from allergic subjects. The eotaxin-induced eosinophil chemotaxis was inhibited by TAT-PTEN in a dose-dependent manner. Intranasal pretreatment with TAT-PTEN, but not TAT-GFP, significantly inhibited the OVA-induced eosinophil infiltration in bronchoalveolar lavage fluid. Histological examination of the lung, including H&E and Alcian blue/periodic acid-Schiff staining, revealed that TAT-PTEN, but not TAT-GFP, abrogated eosinophilic inflammation and mucus production. Our results suggest that PTEN negatively regulates eosinophil survival, chemotaxis, and allergic inflammation. The pharmacological targeting of PTEN may constitute a new strategy for the treatment of eosinophilic disorders.