Induction of eosinophil apoptosis by the cyclin-dependent kinase inhibitor AT7519 promotes the resolution of eosinophil-dominant allergic inflammation

Background

Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo.

Methodology/Principal Findings

Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk).

Conclusions

Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.     

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.     

The IL-3/IL-5/GM-CSF common receptor plays a pivotal role in the regulation of Th2 immunity and allergic airway inflammation

The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils are regulated by the CD4 Th2 cytokines IL-3, IL-5, and GM-CSF, which all signal through a common beta receptor subunit (betac). Recent therapeutic approaches targeting IL-5 alone have not ablated tissue accumulation of eosinophils and have had limited effects on disease progression, suggesting important roles for IL-3 and GM-CSF. By using a mouse model of allergic airways inflammation, we show that allergen-induced expansion and accumulation of eosinophils in the lung are abolished in betac-deficient (betac-/-) mice. Moreover, betac deficiency resulted in inhibition of hallmark features of asthma, including airways hypersensitivity, mucus hypersecretion, and production of Ag-specific IgE. Surprisingly, we also identified a critical role for this receptor in regulating type 2 immunity. Th2 cells in the lung of allergen-challenged betac-/- mice were limited in their ability to proliferate, produce cytokines, and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus, the betac plays a critical role in allergen-induced eosinophil expansion and infiltration and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy.     

Digestion of the fifth component of complement by eosinophil lysosomal enzymes. Production of eosinophil specific chemotactic activity

Recently our laboratory has shown that neutrophils contain enzymatic activity within their lysosomal granules which will generate chemotactic activity for neutrophils and tumor cells from the fifth component of complement (C5). We have now expanded this initial observation and have demonstrated that eosinophils can release enzymatic activity from their lysosomal granules upon stimulation with immune complexes or opsoninized zymosan, but not with C5a or synthetic chemotactic peptides. Furthermore, the enzymatic activity released from the eosinophil lysosomal granules can cleave C5 into eosinophil-specific chemotactic activity. The generation of the eosinophil chemotactic activities from C5 is blocked by prior treatment of the eosinophil preparations with a number of protease inhibitors. The eosinophil-derived C5 cleaving activity possesses a pH optimum of 7.2, thus suggesting the enzymatic activity is a neutral protease. The demonstration that enzyme activities derived from eosinophils have the ability to generate eosinophil chemotactic factor(s) from C5 may explain why eosinophils are the predominant inflammatory cell in both nasal polyps and in the nasopharynx and bronchi of patients with allergic conditions such as hay fever and asthma.     

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.     

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.     

Neuropeptides modulate human eosinophil chemotaxis.

To investigate the role of neuropeptides in allergic inflammation, we examined the effect of peptides on eosinophil chemotaxis. Eosinophils were purified from the blood of allergic and normal subjects using a discontinuous Percoll density gradients. Chemotaxis was induced by platelet-activating factor (PAF) and leukotriene B4, and was assayed by a modified Boyden's chamber technique. Four neuropeptides were examined in this study: substance P (SP), neurokinin A, calcitonin gene-related peptide (CGRP), and cholecystokinin octapeptide. Peptides alone (10 nM to 10 microM) were not chemotactic for eosinophils. However, when eosinophils were pre-treated with peptides (100 nM) at 37 degrees C for 30 min, chemotactic response to PAF (10 nM) was significantly enhanced (p < 0.01) in allergic subjects; % control by SP, neurokinin A, CGRP and cholecystokinin octapeptide was 269 +/- 42, 243 +/- 32, 227 +/- 21, and 251 +/- 42, respectively (n = 8). Similar results were obtained in leukotriene B4-induced eosinophil chemotaxis. In contrast, no enhancement was observed in normal subjects. Potentiating effect of SP and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by SP antagonist [D-Pro2,D-Trp7,9]-SP and human CGRP (8-37) receptor antagonist, respectively. Neutral endopeptidase inhibitors (phosphoramidon, leupeptin, and bestatin) failed to significantly augment the PAF-induced eosinophil chemotaxis when the cells were pretreated with various peptides and neutral endopeptidase inhibitors. The C-terminal fragment of SP (SP6-11) had an effect similar to that of the intact SP molecule, whereas no potentiating effect by the N-terminal of SP (SP1-9) was observed. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.     

Cutting edge: chemoattractant receptor-homologous molecule expressed on Th2 cells plays a restricting role on IL-5 production and eosinophil recruitment

PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.     

Novel Basophil- or Eosinophil-Depleted Mouse Models for Functional Analyses of Allergic Inflammation

Basophils and eosinophils play important roles in various host defense mechanisms but also act as harmful effectors in allergic disorders. We generated novel basophil- and eosinophil-depletion mouse models by introducing the human diphtheria toxin (DT) receptor gene under the control of the mouse CD203c and the eosinophil peroxidase promoter, respectively, to study the critical roles of these cells in the immunological response. These mice exhibited selective depletion of the target cells upon DT administration. In the basophil-depletion model, DT administration attenuated a drop in body temperature in IgG-mediated systemic anaphylaxis in a dose-dependent manner and almost completely abolished the development of ear swelling in IgE-mediated chronic allergic inflammation (IgE-CAI), a typical skin swelling reaction with massive eosinophil infiltration. In contrast, in the eosinophil-depletion model, DT administration ameliorated the ear swelling in IgE-CAI whether DT was administered before, simultaneously, or after, antigen challenge, with significantly lower numbers of eosinophils infiltrating into the swelling site. These results confirm that basophils and eosinophils act as the initiator and the effector, respectively, in IgE-CAI. In addition, antibody array analysis suggested that eotaxin-2 is a principal chemokine that attracts proinflammatory cells, leading to chronic allergic inflammation. Thus, the two mouse models established in this study are potentially useful and powerful tools for studying the in vivo roles of basophils and eosinophils. The combination of basophil- and eosinophil-depletion mouse models provides a new approach to understanding the complicated mechanism of allergic inflammation in conditions such as atopic dermatitis and asthma.     

IL-33 induces Th17-mediated airway inflammation via mast cells in ovalbumin-challenged mice

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.     

Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is alpha 4 beta 1 (very late antigen-4) dependent.

Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to IL-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and 20 h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation.     

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.     

Regulatory effects of eotaxin,eotaxin-2, and eotaxin-3 on eosinophil degranulation and superoxide anion generation

Eosinophilic leukocytes have been implicated as primary effector cells in inflammatory and allergic diseases. When activated by cytokines, human eosinophils secrete and produce a variety of proinflammatory or tissue damaging substances. Although well known for their chemoattractant effects, little is known about the precise contribution of the eosinophil-selective chemokines, eotaxin, eotaxin-2, and eotaxin-3 to the effector functions of eosinophils. This forms the central focus of these investigations for which clone 15-HL-60 human eosinophilic cells were used as the in vitro model. Investigation results suggest that all three subtypes of eotaxin directly stimulate eosinophil superoxide anion generation that is inhibited by neutralizing eotaxin antibody or pretreatment of cells with the receptor antibody anti-CCR3. Pretreatment or co-treatment with each of the eotaxins augmented phorbol myristate-induced superoxide generation. Concentration-dependent degranulation of eosinophil peroxidase was noted for all three chemokines, and potentiation of calcium ionophore-induced degranulation was observed with eotaxin pretreatments. Results of interleukin-5 pretreatment studies suggest that the eotaxin chemokines may act cooperatively to enhance effector functions of eosinophils. Collectively, the present studies have advanced knowledge of the eotaxin family of chemokines to include eosinophil priming and modulation of eosinophil activation and secretion effector functions.     

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.     

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.     

Mechanisms underlying eosinophil trafficking and their relevance in vivo

After their formation in the bone marrow, eosinophils circulate with a short half-life and are distributed throughout the body, especially in mucosal and sub-mucosal regions. Although a small amount of these cells are normally seen in healthy tissue, blood and tissue eosinophilia is a hallmark of helminthic and allergic diseases. The role of eosinophils in the normal physiology of mucosal tissues is not understood, but there is good evidence to demonstrate that these cells protect the host at least against some intestinal helminths, specially those with a lung cycle. In addition, there are now many data that support a role for eosinophils in the pathophysiology of allergic diseases, such as asthma. Because helminthic diseases have been largely controlled in developed countries, there has been much interest in the development of drugs which affect eosinophil migration and/or activation in the tissue and which may, thus, be useful in the treatment of allergic conditions. The understanding of the mechanisms controlling eosinophil trafficking and/or activation are essential in the development of anti-eosinophil-based therapeutic strategies. The present paper reviews aspects of eosinophil biology with emphasis on the role of eosinophils in parasitic infections and allergy, the basic mechanisms underlying the trafficking of eosinophils into tissue and how these can be modulated pharmacologically.     

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.     

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil.