Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.     

IL-16 activates plasminogen-plasmin system and promotes human eosinophil migration into extracellular matrix via CCR3-chemokine-mediated signaling and by modulating CD4 eosinophil expression

Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4(+) cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4(+) expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.     

The CCR3 receptor is involved in eosinophil differentiation and is up-regulated by Th2 cytokines in CD34+ progenitor cells

The involvement of chemokines in eosinophil recruitment during inflammation and allergic reactions is well established. However, a functional role for chemokines in eosinophil differentiation has not been investigated. Using in situ RT-PCR, immunostaining, and flow cytometric analysis, we report that human CD34+ cord blood progenitor cells contain CCR3 mRNA and protein. Activation of CD34+ progenitor cells under conditions that promote Th2 type differentiation up-regulated surface expression of the CCR3. In contrast, activation with IL-12 and IFN-gamma resulted in a significant decrease in the expression of CCR3. Eotaxin induced Ca2+ mobilization in CD34+ progenitor cells, which could explain the in vitro and in vivo chemotactic responsiveness to eotaxin. We also found that eotaxin induced the differentiation of eosinophils from cord blood CD34+ progenitor cells. The largest number of mature eosinophils was found in cultures containing eotaxin and IL-5. The addition of neutralizing anti-IL-3, anti-IL-5, and anti-GM-CSF Abs to culture medium demonstrated that the differentiation of eosinophils in the presence of eotaxin was IL-3-, IL-5-, and GM-CSF-independent. These results could explain how CD34+ progenitor cells accumulate and persist in the airways and peripheral blood of patients with asthma and highlight an alternative mechanism by which blood and tissue eosinophilia might occur in the absence of IL-5.     

Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils

Eosinophils and basophils, when activated, become major sources of cysteinyl leukotrienes, eicosanoid mediators pertinent to allergic inflammation. We show that the C-C chemokines, eotaxin and RANTES (regulated upon activation normal T cell expressed and secreted), activate eosinophils and basophils for enhanced leukotriene C(4) (LTC(4)) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles. Chemokine-induced lipid body formation and enhanced LTC(4) release were both mediated by CCR3 receptor G protein-linked downstream signaling involving activation of phosphoinositide 3-kinase, extracellular signal-regulated kinases 1 and 2, and p38 mitogen-activated protein kinases. Chemokine-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC(4) production; and as demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid, lipid bodies were the predominant sites of LTC(4) synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases both elicits the formation of lipid body domains and promotes LTC(4) formation at these specific extranuclear sites.     

Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics

Atopic (AA) and nonatopic (NAA) asthma are characterized by chronic inflammation and local tissue eosinophilia. Many C-C chemokines are potent eosinophil chemoattractants and act predominantly via the CCR3. We examined the expression of eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), MCP-4, and CCR3 in the bronchial mucosa from atopic (AA) and nonatopic (intrinsic; NAA) asthmatics and compared our findings with atopic (AC) and nonatopic nonasthmatic controls (NC). Cryostat sections were processed for immunohistochemistry (IHC), in situ hybridization (ISH), and double IHC/ISH. Compared with AC and NC, the numbers of EG2+ cells and the cells expressing mRNA for eotaxin, eotaxin-2, RANTES, MCP-3, MCP-4, and CCR3 were significantly increased in AA and NAA (p < 0.01). Nonsignificant differences in these variants were observed between AA and NAA and between AC and NC. Significant correlations between the cells expressing eotaxin or CCR3 and EG2+ eosinophils in the bronchial tissue were also observed for both AA (p < 0.01) and NAA (p = 0.01). Moreover, in the total asthmatic group (AA + NAA) there was a significant inverse correlation between the expression of eotaxin and that of the histamine PC20 (p < 0.05). Sequential IHC/ISH showed that cytokeratin+ epithelial cells, CD31+ endothelial cells, and CD68+ macrophages were the major sources of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4. There was no significantly different distribution of cells expressing mRNA for these chemokines between atopic and nonatopic asthma. These findings suggest that multiple C-C chemokines, acting at least in part via CCR3, contribute to bronchial eosinophilia in both atopic and nonatopic asthma.     

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.     

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.     

Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways

To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1alpha induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37 degrees C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1alpha in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1alpha signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

Cutting edge: prostaglandin D2 enhances leukotriene C4 synthesis by eosinophils during allergic inflammation: synergistic in vivo role of endogenous eotaxin

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies-potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.     

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.     

Mast cell beta-tryptase selectively cleaves eotaxin and RANTES and abrogates their eosinophil chemotactic activities

Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by beta-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of beta-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified beta-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human beta-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. beta-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with beta-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of beta-tryptase on RANTES. These findings suggest that mast cell beta-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.     

The prostaglandin E agonist,misoprostol, inhibits airway IL-5 production in atopic asthmatics

BACKGROUND: Prostaglandin E2 is a potent immunomodulator that inhibits the early and late bronchoconstriction to inhaled allergen, as well as inhibiting the acute allergen-induced release of mediators into the human airway. To determine if the stable prostaglandin E agonist misoprostol could alter the late allergic formation of mediators we measured the appearance of eosinophils and key cytokines in the bronchoalveolar lavage fluid 24 h after allergen instillation. METHODS: Six atopic asthmatics underwent bronchoscopy, alveolar lavage and antigen instillation followed 24 h later by bronchoalveolar lavage. Eosinophil counts were done, together with measurements of IL-4, IL-5, eotaxin, RANTES and cysteinyl leukotrienes by immunoassay. The study was done in randomized blinded fashion while the volunteers took placebo or 600 microg of misoprostol four times a day (QID). RESULTS: Misoprostol significantly decreased the appearance of IL-5 late after allergen challenge. Eotaxin levels were reduced, but not statistically significantly. Eosinophil number, RANTES, eosinophil cationic protein and cysteinyl leukotrienes were not altered by misoprostol. CONCLUSIONS: Misoprostol reduces the formation of IL-5 late after allergen challenge, perhaps by inhibiting eosinophil, mast cell, and/or T lymphocyte production of IL-5. Despite decreases in IL-5 and eotaxin, eosinophils were recruited and activated by allergen.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

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.     

The role of chemokines in cutaneous allergic inflammation.

Eosinophils are the major effector cells that kill helminthic parasites and are - for unknown reasons present in the dermal part of atopic skin. This review summarizes our knowledge on the chemotactic factors involved in eosinophil tissue recruitment, focusing on the role of eosinophil-chemotactic chemokines. It is the current view that the chemokines RANTES and eotaxin represent the most important eosinophil-attracting chemokines. The inducibility of eotaxin in dermal fibroblasts only upon stimulation with Th2-cytokines IL-4 and IL-13 may explain why eosinophils appear only in the dermis and why the presence of Th2-cytokines is always linked with tissue eosinophilia.     

Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells

Airway inflammation associated with asthma is characterized by massive infiltration of eosinophils, mediated in part by specific chemoattractant factors produced in the lung. Allergen-specific Th2 cells appear to play a central role in asthma; for example, adoptively transferred Th2 cells induced lung eosinophilia associated with induction of specific chemokines. Interestingly, Th2 supernatant alone administered intranasally to naive mice induced eotaxin, RANTES, monocyte-chemotactic protein-1, and KC expression along with lung eosinophilia. We tested the major cytokines individually and found that IL-4 and IL-5 induced higher levels of macrophage-inflammatory protein-1alpha and KC; IL-4 also increased the production of monocyte-chemotactic protein-1; IL-13 and IL-4 induced eotaxin. IL-13 was by far the most potent inducer of eotaxin; indeed, a neutralizing anti-IL-13 Ab removed most of the eotaxin-inducing activity from Th2 supernatants, although it did not entirely block the recruitment of eosinophils. While TNF-alpha did not stimulate eotaxin production by itself, it markedly augmented eotaxin induction by IL-13. IL-13 was able to induce eotaxin in the lung of JAK3-deficient mice, suggesting that JAK3 is not required for IL-13 signaling in airway epithelial cells; however, eosinophilia was not induced in this situation, suggesting that JAK3 transduces other IL-13-mediated mechanisms critical for eosinophil recruitment. Our study suggests that IL-13 is an important mediator in the pathogenesis of asthma and therefore a potential target for asthma therapy.     

A novel human CC chemokine, eotaxin-3, which is expressed in IL-4-stimulated vascular endothelial cells, exhibits potent activity toward eosinophils.

IL-4 has been shown to be involved in the accumulation of leukocytes, especially eosinophils, at sites of inflammation by acting on vascular endothelial cells. To identify novel molecules involved in the IL-4-dependent eosinophil extravasation, cDNA prepared from HUVEC stimulated with IL-4 was subjected to differential display analysis, which revealed a novel CC chemokine designated as eotaxin-3. The human eotaxin-3 gene has been localized to chromosome 7q11.2, unlike most other CC chemokine genes. The predicted mature protein of 71 aa showed 27-42% identity to other human CC chemokines. The recombinant protein induced a transient increase in the cytosolic Ca2+ concentration and in vitro chemotaxis on eosinophils. Furthermore, in cynomolgus monkeys, the accumulation of eosinophils was observed at the sites where the protein was injected. Eotaxin-3 inhibited the binding of 125I-eotaxin, but not 125I-macrophage inflammatory protein-1alpha, to eosinophils and acted on cell lines transfected with CCR-3, suggesting that eotaxin-3 recognized CCR-3. IL-13 as well as IL-4 up-regulated eotaxin-3 mRNA in HUVEC, whereas neither TNF-alpha, IL-1beta, IFN-gamma, nor TNF-alpha plus IFN-gamma did. The expression profile of eotaxin-3 is different from those of eotaxin, RANTES, and monocyte chemoattractant protein-4, which are potent eosinophil-selective chemoattractants and are induced by either TNF-alpha or TNF-alpha plus IFN-gamma. These results suggest that eotaxin-3 may contribute to the eosinophil accumulation in atopic diseases.