Stem cell factor plays a major role in the recruitment of eosinophils in allergic pleurisy in mice via the production of leukotriene B4

The understanding of the mechanisms underlying eosinophil migration into tissue is an essential step in the development of novel therapies aimed at treating allergic diseases where eosinophil recruitment and activation are thought to play an essential role. In this study, we have examined the effects of the in vivo administration of stem cell factor (SCF) on eosinophil recruitment and tested whether endogenous SCF was involved in mediating eosinophil recruitment in response to Ag challenge in sensitized mice. The intrapleural injection of SCF induced a time- and concentration-dependent recruitment of eosinophils in mice. In allergic mice, SCF message was expressed early after Ag challenge and returned to baseline levels after 8 h. In agreement with the ability of SCF to induce eosinophil recruitment and its expression in the allergic reaction, an anti-SCF polyclonal Ab abrogated eosinophil recruitment when given before Ag challenge. SCF increased the levels of leukotriene B4 (LTB4) in the pleural cavity of mice and an LTB4 receptor antagonist, CP105,696, abrogated the effects of SCF on eosinophil recruitment. Similarly, recruitment of eosinophils in the allergic reaction was virtually abolished by CP105,696. Together, our data favor the hypothesis that the local release of SCF following Ag challenge may activate and/or prime mast cells for IgE-mediated release of inflammatory mediators, especially LTB4. The mediators released in turn drive the recruitment of eosinophils. Inhibition of the function of SCF in vivo may reduce the migration of eosinophils to sites of allergic inflammation and may, thus, be a relevant principle in the treatment of allergic diseases.     

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.     

Studies on the adhesive interaction between purified human eosinophils and cultured vascular endothelial cells

Many recent studies have established the eosinophil as a primary effector cell in the pathology of allergic diseases. However, relatively little is known about the mechanisms by which eosinophils accumulate and are activated at local sites of tissue inflammation in allergic or other eosinophil-dependent pathologic states. Because the adherence of leukocytes to vascular endothelial cells (VEC) is a critical initial event in eosinophil infiltration, we have studied the interaction of purified human eosinophils with cultured human umbilical vein endothelial cells. Treatment of VEC with stimuli known to activate endothelial cells, including purified human IL-1, rTNF-alpha, bacterial endotoxin LPS, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate resulted in time- and dose-dependent increases (from two- to fourfold) in adhesiveness for eosinophils. Adherence induced by optimal concentrations of IL-1 (2 U/ml), TNF (1 micrograms/ml), and LPS (1 microgram/ml) is dependent upon the CD18 leukocyte cell surface adherence glycoproteins, because a mAb (60.3) directed against the common beta-subunit of the complex inhibits adherence induced by these stimuli. Several agents directly activated eosinophils to display increased adhesiveness to both VEC and gelatinized plates. The bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine (10(-8) to 10(-6) M), TNF (1 to 1000 ng/ml), and 12-O-tetradecanoyl-phorbol-13-acetate (0.3 to 3 ng/ml) all increased eosinophil binding to VEC by two to fivefold. Platelet-activating factor (PAF; 10(-8) to 10(-6) M), but not lyso-PAF, caused approximately a twofold increase in eosinophil binding to both VEC and gelatinized tissue culture plates, suggesting that activation of eosinophils may be responsible for the known ability of PAF to induce eosinophilic responses. These results suggest that the initiation of an eosinophilic infiltrate in vivo can result from activation of endothelial cells, activation of eosinophils, or activation of both cell types.     

Characterisation of the biological activity of recombinant equine eotaxin in vitro

The chemokine eotaxin (CCL11) is a key player in the trafficking of eosinophils to normal tissues and in the tissue eosinophilia associated with human allergic disease. We have recently cloned equine eotaxin and here we report the production of rEq eotaxin, with and without a C-terminal fusion peptide, in a novel expression system utilising stably transfected insect cells. rEq eotaxin induced equine eosinophil migration and superoxide production in vitro. A shape change in human eosinophils that could be blocked by 7B11, a monoclonal antibody against human CCR3, was also observed. Biological activity was not dependent on an intact eotaxin C-terminus. These results suggest that equine eotaxin, like its human ortholog, may play a role in eosinophil accumulation and activation in the horse.     

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.     

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.     

Apoptosis in human eosinophils. Programmed cell death in the eosinophil leads to phagocytosis by macrophages and is modulated by IL-5.

Eosinophils are believed to injure tissues in a variety of allergic disease by virtue of their highly histotoxic contents and metabolites. They are readily observed in tissues during the allergic response yet the mechanisms governing the duration of tissue residence and route of removal remain obscure. We have previously reported in vitro and in vivo evidence that neutrophils undergo apoptosis (programmed cell death) and are recognized and ingested as intact cells by macrophages. We report that eosinophils, purified from the peripheral blood of asymptomatic healthy atopics, undergo apoptosis in vitro. After 72 to 96 h in culture, 57.0 +/- 6.2% (mean +/- SE) of the eosinophil population showed characteristic morphologic changes of apoptosis. Electrophoresis of the DNA from these cells demonstrated the typical "ladder" pattern of internucleosomal DNA cleavage, the hallmark of apoptosis-associated endonuclease activation. The rate of eosinophil apoptosis, slower than that reported for neutrophils, was delayed (by 80 +/- 6 h) in the presence of recombinant human IL-5, a cytokine previously reported to prolong eosinophil life in vitro but not known to modulate apoptosis. Aged, apoptotic eosinophils, but not fresh or aged preapoptotic eosinophils, were recognized and ingested as intact cells by macrophages. Apoptosis and ingestion by macrophages may represent a mechanism whereby the tissue longevity and removal of eosinophils is controlled.     

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.     

Human eosinophils exert TNF-α and granzyme A-mediated tumoricidal activity toward colon carcinoma cells

Peripheral blood and tissue eosinophilia is a prominent feature in allergic diseases and helminth infections. In cancer patients, tumor-associated tissue eosinophilia is frequently observed. Tumor-associated tissue eosinophilia can be associated with a favorable prognosis, notably in colorectal carcinoma. However, underlying mechanisms of eosinophil contribution to antitumor responses are poorly understood. We have in this study investigated the direct interactions of human eosinophils with Colo-205, a colorectal carcinoma cell line, and show that eosinophils induce apoptosis and directly kill tumor cells. Using blocking Abs, we found that CD11a/CD18 complex is involved in the tumoricidal activity. Coculture of eosinophils with Colo-205 led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin as well as TNF-α secretion. Moreover, eosinophils expressed granzyme A, which was released upon interaction with Colo-205, whereas cytotoxicity was partially inhibited by FUT-175, an inhibitor of trypsin-like enzymatic activity. Our data present the first demonstration, to our knowledge, that granzyme A is a cytotoxic mediator of the eosinophil protein arsenal, exerting eosinophil tumoricidal activity toward Colo-205, and provide mechanistic evidence for innate responses of eosinophil against tumor cells.     

An alternate STAT6-independent pathway promotes eosinophil influx into blood during allergic airway inflammation

Background

Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4⁺ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.

Methodology/Principal Findings

We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4⁺ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4⁺ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4⁺ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.

Conclusions

These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.     

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.     

Differential activation of CC chemokine receptors by AOP-RANTES

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.     

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.     

Human endothelial cells prolong eosinophil survival. Regulation by cytokines and glucocorticoids

Many recent studies have established the eosinophil as an active proinflammatory participant in a variety of disease states, most notably in allergic and helminthic disorders. In order to understand the effector role of eosinophils, factors which promote a selective eosinophilic infiltrate must be delineated. Eosinophil adherence to vascular endothelium is the first step in the formation of such an infiltrate. However, studies thus far have failed to identify factors which selectively activate the adherence of eosinophils. We have therefore speculated that the selective enrichment of eosinophils may result from nonselective recruitment of several leukocyte types combined with the production of local factors that promote the survival of eosinophils and not of other cells. We report that endothelial cell-conditioned medium selectively prolongs eosinophil survival up to 6 days in culture in a dose- and time-dependent manner. Stimulation of human vascular endothelial cells with IL-1 caused an increase in the generation of eosinophil survival-promoting activity, whereas stimulation with platelet-activating factor did not. Supernatants from human vascular endothelial cells cultured for 48 h in the presence of the glucocorticoid, dexamethasone, were less active in promoting eosinophil survival than control supernatants. These results suggest that factors produced locally in the vascular microenvironment may selectively promote eosinophil survival and may be under the regulation of cytokines and glucocorticoids.     

Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.     

Activation of mitogen-activated protein kinase regulates eotaxin-induced eosinophil migration.

Eotaxin is a potent eosinophil chemoattractant that plays an important role in regulating eosinophil tissue levels both in healthy individuals and in diseases associated with significant eosinophil infiltrates, such as the allergic inflammation observed in asthma. Here, we demonstrate that treatment of eosinophils with eotaxin induces the phosphorylation of the mitogen-activated protein kinases (MAPKs) p42 and p44, leading to kinase activation. Blockade of MAPK activation by the MAPK kinase inhibitor PD98059 leads to a dramatic decrease in eotaxin-induced eosinophil rolling in vivo and chemotaxis in vitro. This blockade in the leukocyte migration process is consistent with the observed inhibition of actin polymerization and rearrangement within the eosinophil following treatment with MAPK inhibitor. It is suggested, therefore, that the intrinsic mechanism of eotaxin-induced eosinophil rolling and migration involves activation of the p42/p44 MAPK, possibly through regulation of the cytoskeletal rearrangements necessary for chemotaxis.     

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.     

Selective suppression of leukocyte recruitment in allergic inflammation

Allergic diseases result in a considerable socioeconomic burden. The incidence of allergic diseases, notably allergic asthma, has risen to high levels for reasons that are not entirely understood. With an increasing knowledge of underlying mechanisms, there is now more potential to target the inflammatory process rather than the overt symptoms. This focuses attention on the role of leukocytes especially Th2 lymphocytes that regulate allergic inflammation and effector cells where eosinophils have received much attention. Eosinophils are thought to be important based on the high numbers that are recruited to sites of allergic inflammation and the potential of these cells to effect both tissue injury and remodelling. It is hoped that future therapy will be directed towards specific leukocyte types, without overtly compromising essential host defence responses. One obvious target is leukocyte recruitment. This necessitates a detailed understanding of underlying mechanisms, particularly those involving soluble chemoattractants signals and cell-cell adhesion molecules.     

Human peripheral blood eosinophils induce angiogenesis

Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.     

Regulation of eosinophil trafficking by SWAP-70 and its role in allergic airway inflammation

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70-deficient (Swap-70(-/-)) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70(-/-) eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70(-/-) eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70(-/-) eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70(-/-) mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.