IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.     

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.     

Stem cell factor-induced leukotriene B4 production cooperates with eotaxin to mediate the recruitment of eosinophils during allergic pleurisy in mice.

The understanding of the mechanisms underlying eosinophil recruitment in vivo may aid in the development of novel strategies for the treatment of allergic disorders. In this study, we investigated the role of chemokines in the cascade of events leading to eosinophil recruitment in a stem cell factor (SCF)- and leukotriene B(4) (LTB(4))-dependent allergic pleurisy model in mice. The intrapleural administration of the eosinophil-active chemokines eotaxin, RANTES, and macrophage-inflammatory protein 1alpha (MIP-1alpha) induced a time- and dose-dependent eosinophil recruitment. Pretreatment with anti-eotaxin, but not anti-RANTES or anti-MIP-1alpha, blocked the recruitment of eosinophils following Ag challenge of sensitized animals, and significant eotaxin immunoreactivity was detected in the pleural cavity of these animals. Similarly, only the anti-eotaxin inhibited the eosinophil recruitment induced by injection of SCF in naive animals. However, blockade of SCF did not inhibit the release of eotaxin after Ag challenge of sensitized mice. Akin to its effects on SCF and in the allergic reaction, eotaxin-induced eosinophil recruitment was blocked by the LTB(4) receptor antagonist CP105696. Nevertheless, SCF, but not eotaxin, appeared to regulate the endogenous release of LTB(4) after Ag challenge. Finally, we show that low doses of eotaxin synergized with LTB(4) to induce eosinophil recruitment in the pleural cavity. Overall, the present results show that eotaxin and SCF-induced LTB(4) cooperate to induce eosinophil recruitment into sites of allergic inflammation. Cooperation between inflammatory mediators must be an important phenomenon in vivo, explaining both the ability of lower concentrations of mediators to induce a full-blown functional response and the effectiveness of different strategies at inhibiting these responses.     

Eotaxin and the attraction of eosinophils to the asthmatic lung

Eosinophilic leukocytes accumulate in high numbers in the lungs of asthmatic patients, and are believed to be important in the pathogenisis of asthma. A potent eosinophil chemoattractant is produced in the asthmatic lung. This small protein, the chemokine eotaxin, is synthesized by a number of different cell types, and is stimulated by interleukin-4 and interleukin-13, which are produced by T-helper (Th)2 lymphocytes. Low molecular weight compounds have been developed that can block the eotaxin receptor C-C chemokine receptor (CCR)3, and prevent stimulation by eotaxin. This provides the potential for orally available drugs that can prevent eosinophil recruitment into the lung and the associated damage and dysfunction.     

Inhibitory effects of Schizandrae Fructus on eotaxin secretion in A549 human epithelial cells and eosinophil migration

Eosinophilia have been implicated in a broad range of diseases, most notably allergic conditions (e.g. asthma, rhinitis and atopic dermatitis) and inflammatory diseases. These diseases are characterized by an accumulation of eosinophils in the affected tissue. Defining the mechanisms that control the recruitment of eosinophil is fundamental to understanding how these diseases progress and identifying a novel target for drug therapy. Accordingly, this study was conducted to evaluate the regulatory effect of Schizandrae Fructus (SF) on the expression of eotaxin, an eosinophil-specific chemokine released in respiratory epithelium following allergic stimulation, as well as its effects on eosinophil migration.To accomplish this, human epithelial lung cells (A549 cell) were stimulated with a combination of TNF-α (100 ng/ml) and IL-4 (100 ng/ml) for 24 h. The cells were then restimulated with TNF-α (100 ng/ml) and IL-1β (10 ng/ml) to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis for another 24 h. Next, the samples were treated with various concentrations of Schizandrae Fructus (SF) (1, 10, 100, 1000 μg/ml) or one of the major constituents of SF, schizandrin (0.1, 1, 10, 100 μg/ml), after which following inhibition effect assay was performed triplicates in three independence.The levels of eotaxin in secreted proteins were suppressed significantly by SF (100 and 1000 μg/ml, p<0.01) and schizandrin (10 and 100 μg/ml, p<0.01). In addition, SF (1, 10, 100 and 1000 μg/ml) decreased mRNA expression levels in A549 cells significantly (p<0.01). Eosinophil recruitment to lung epithelial cells was also reduced by SF, which indicates that eotaxin plays a role in eosinophil recruitment. Furthermore, treatment with SF suppressed the expression of another chemokine, IL-8 (0.1 and 1 μg/ml SF, p<0.01), as well as intercellular adhesion molecule-1 (10 and 100 μg/ml SF, p<0.01) and vascular cell adhesion molecule-1 (0.1 and 1 μg/ml SF, p<0.05), which are all related to eosinophil migration. Taken together, these findings indicate that SF may be a desirable medicinal plant for the treatment of allergic diseases.     

Expression of the chemokine eotaxin and its receptor, CCR3, in human endometrium

Eosinophils are present in human endometrium only immediately before and during menstruation, suggesting a role in that process. The expression of the eosinophil chemoattractant, eotaxin, and its receptor, CCR3, within the human endometrium were investigated by immunohistochemical analysis of tissue sections spanning the entire menstrual cycle. Eotaxin was localized to perivascular cells in the late secretory phase, and it was also identified in eosinophils. However, the highest levels of this chemokine were present in both luminal and glandular epithelial cells during the proliferative and secretory phases of the cycle. Treatment of endometrial tissue with monensin, which blocks protein secretion, increased epithelial immunoreactive eotaxin, substantiating synthesis in these cells. Although the CCR3 receptor was expressed by eosinophils, it was also strongly expressed by endometrial epithelial cells. The CCR3 receptor on purified, cultured endometrial epithelial cells was functional, as assessed by a transient Ca(2+) flux in response to eotaxin. These analyses demonstrate that eotaxin is expressed by endometrial cells and may therefore be involved in the recruitment of eosinophils into this tissue premenstrually. However, the observation that this chemokine and the CCR3 molecule are strongly expressed by epithelial cells throughout the cycle suggests that these proteins may have additional important functions within the endometrium.     

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES).

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.     

Eotaxin selectively binds heparin. An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo

An important feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo. These interactions play an important role in modulating the action of chemokines by facilitating the formation of stable chemokine gradients within the vascular endothelium and directing leukocyte migration, by protecting chemokines from proteolysis, by inducing chemokine oligomerization, and by facilitating transcytosis. Despite the importance of eotaxin in eosinophil differentiation and recruitment being well established, little is known about the interaction between eotaxin and GAGs and the functional consequences of such an interaction. Here we report that eotaxin binds selectively to immobilized heparin with high affinity (K(d) = 1.23 x 10(-8) M), but not to heparan sulfate or a range of other GAGs. The interaction of eotaxin with heparin does not promote eotaxin oligomerization but protects eotaxin from proteolysis directly by plasmin and indirectly by cathepsin G and elastase. In vivo, co-administration of eotaxin and heparin is able to significantly enhance eotaxin-mediated eosinophil recruitment in a mouse air-pouch model. Furthermore, when heparin is co-administered with eotaxin at a concentration that does not normally result in eosinophil infiltration, eosinophil recruitment occurs. In contrast, heparin does not enhance eotaxin-mediated eosinophil chemotaxis in vitro, suggesting protease protection or haptotactic gradient formation as the mechanism by which heparin enhances eotaxin action in vivo. These results suggest a role for mast cell-derived heparin in the recruitment of eosinophils, reinforcing Th2 polarization of inflammatory responses.     

Cell-type-dependent induction of eotaxin and CCR3 by ionizing radiation

Eotaxin is an eosinophil-specific C-C chemokine that is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma and atopic dermatitis, by acting specifically on its receptor CCR3. Using RT-PCR analysis, we show that the expression of eotaxin is upregulated upon treatment with ionizing radiation (IR) in human dermal fibroblasts, but not in the bronchial epithelial cell line A549. In contrast, the gene encoding CCR3 is markedly induced in both cell types. None of the genes coding for other CCR3 ligands are significantly induced by IR in these cell types. cDNA array analysis of irradiated versus nonirradiated A549 cells and human dermal fibroblasts confirm and extend these results, and support the observation that regulation of eotaxin/CCR3-induction by IR occurs in a selective and cell-type-dependent manner. They further suggest that the induction of signaling via eotaxin and CCR3 may be an important step leading to eosinophilia in patients with radiation exposure.     

The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3

Th1 and Th2 lymphocytes express a different repertoire of chemokine receptors (CCRs). CXCR3, the receptor for I-TAC (interferon-inducible T cell alpha-chemoattractant), Mig (monokine induced by gamma-interferon), and IP10 (interferon-inducible protein 10), is expressed preferentially on Th1 cells, whereas CCR3, the receptor for eotaxin and several other CC chemokines, is characteristic of Th2 cells. While studying responses that are mediated by these two receptors, we found that the agonists for CXCR3 act as antagonists for CCR3. I-TAC, Mig, and IP10 compete for the binding of eotaxin to CCR3-bearing cells and inhibit migration and Ca(2+) changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-3, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A hybrid chemokine generated by substituting the first eight NH(2)-terminal residues of eotaxin with those of I-TAC bound CCR3 with higher affinity than eotaxin or I-TAC (3- and 10-fold, respectively). The hybrid was 5-fold more potent than I-TAC as an inhibitor of eotaxin activity and was effective at concentrations as low as 5 nm. None of the antagonists described induced the internalization of CCR3, indicating that they lack agonistic effects and thus qualify as pure antagonists. These results suggest that chemokines that attract Th1 cells via CXCR3 can concomitantly block the migration of Th2 cells in response to CCR3 ligands, thus enhancing the polarization of T cell recruitment.     

Eotaxin activates T cells to chemotaxis and adhesion only if induced to express CCR3 by IL-2 together with IL-4

The transmigration and adherence of T lymphocytes through microvascular endothelium are essential events for their recruitment into inflammatory sites. In the present study, we investigated the expression of CC chemokine receptor CCR3 on T lymphocytes and the capacities of the CC chemokine eotaxin to induce chemotaxis and adhesion in T lymphocytes. We have observed a novel phenomenon that IL-2 and IL-4 induce the expression of CCR3 on T lymphocytes. We also report that CC chemokine eotaxin is a potent chemoattractant for IL-2- and IL-4-stimulated T lymphocytes, but not for freshly isolated T lymphocytes. Eotaxin attracts T lymphocytes via CCR3, documented by the fact that anti-CCR3 mAb blocks eotaxin-mediated T lymphocyte chemotaxis. In combination with IL-2 and IL-4, eotaxin enhances the expression of adhesion molecules such as ICAM-1 and several integrins (CD29, CD49a, and CD49b) on T lymphocytes and thus promotes adhesion and aggregation of T lymphocytes. The eotaxin-induced T lymphocyte adhesion could be selectively blocked by a specific cAMP-dependent protein kinase inhibitor, H-89, indicating that eotaxin activates T lymphocytes via a special cAMP-signaling pathway. Our new findings all point toward the fact that eotaxin, in association with the Th1-derived cytokine IL-2 and the Th2-derived cytokine IL-4, is an important T lymphocyte activator, stimulating the directional migration, adhesion, accumulation, and recruitment of T lymphocytes, and paralleled the accumulation of eosinophils and basophils during the process of certain types of inflammation such as allergy.     

Novel co-operation between eotaxin and substance-P in inducing eosinophil-derived neurotoxin release

Eosinophils, chemokines, and neuropeptides are thought to play effector roles in the pathogenesis of allergic diseases such as rhinitis. Eotaxin is a novel C-C chemokine with a potent and relatively specific eosinophil chemoattractant activity that binds selectively to CCR3 receptor, however, its activity in inducing eosinophil granules proteins release is poorly characterized. This study was performed to determine whether eotaxin primes eosinophil exocytosis and whether this co-operates with the sensory neuroimmune-axis. In the present communication, we show that 10 ng/ml eotaxin primed normal human eosinophil for exaggerated eosonophil-derived neurotoxin (EDN) release stimulated by 10(-8) M Substance-P (SP). This novel priming was blocked by; 7B11 and Herbimycin A (HA), the CCR3 antagonist and the tyrosine kinase inhibitor, respectively. SDS-Page studies showed significant tyrosine phosphorylation of several protein residues induced by 10(-8) M SP only after priming with 10 ng/ml eotaxin. These results demonstrate a novel co-operation between eotaxin and SP in inducing eosinophil cytotoxicity, which at least in part involves tyrosine kinases pathway(s).     

Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of > or =30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-alpha. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 micro M), which inhibits ERK phosphorylation, abolished the synergism between TNF-alpha and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of > or =10 ng/ml caused an approximately 50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE(2) release and did not augment the COX-2 expression that was induced by IL-1beta. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.     

Postnatal mammary gland development requires macrophages and eosinophils

Interactions between mammary epithelial and mesenchymal cells including fibroblasts and adipocytes are crucial for the proper postnatal development of the mammary ductal tree. Often overlooked, however, are the migrant cells that enter tissues at different stages of development. In this paper we identify two such cell types, macrophages and eosinophils, that are recruited around the growing terminal end buds (TEBs) during postnatal development. An important role for leukocytes in mammary gland ductal outgrowth is first demonstrated by depleting mice of leukocytes using sub-lethal (gamma)-irradiation. This treatment results in a curtailment of mammary gland epithelial development that is completely rescued by bone-marrow transplantation, concurrent with a restoration of macrophage and eosinophil recruitment around the growing ducts. Using mice homozygous for a null mutation in the gene for CSF1 (Csfm(op)/Csfm(op)), the major growth factor for macrophages, we show that in the absence of CSF1, the population of macrophages in mammary glands is depleted. In this mutant, the formation of TEBs, their outgrowth into the fat pad and the branching of the resultant ducts are all impaired. Similarly, by using mice homozygous for a null mutation in the gene for eotaxin, a major chemokine for local recruitment of eosinophils in tissue, we identify eotaxin as the necessary and sufficient chemokine responsible for eosinophil recruitment around TEBs. In the absence of eosinophils, mammary gland branch formation and to a lesser extent TEB formation are reduced. Our data show that CSF1-regulated macrophages, in collaboration with eotaxin-regulated eosinophils, have essential and complementary functions in regulating the branching morphogenesis of the mammary gland.     

Eotaxin/CCL11 is involved in acute,but not chronic,allergic airway responses to Aspergillus fumigatus

Eotaxin/CCL11 is a major chemoattractant for eosinophils and Th2 cells. As such, it represents an attractive target in the treatment of allergic disease. The present study addresses the role of eotaxin/CCL11 during acute and chronic allergic airway responses to the fungus Aspergillus fumigatus. Mice lacking the eotaxin gene (Eo-/-) and wild-type mice (Eo+/+) were sensitized to A. fumigatus and received either an intratracheal challenge with soluble A. fumigatus antigens (acute model) or an intratracheal challenge with live A. fumigatus spores or conidia (chronic model). Airway hyperresponsiveness and eosinophil, but not T cell, recruitment were significantly decreased at 24 h after the soluble allergen in A. fumigatus-sensitized Eo-/- mice compared with similarly sensitized Eo+/+ mice. In contrast, the development of chronic allergic airway disease due to A. fumigatus conidia was not altered by the lack of eotaxin. Together, these data suggest that eotaxin initiates allergic airway disease due to A. fumigatus, but this chemokine did not appear to contribute to the maintenance of A. fumigatus-induced allergic airway disease.