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Background: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells., Methods: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay., Results: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter., Conclusions: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Eosinophils are pleiotropic multi-functional leukocytes involved in initiation and propagation of diverse inflammatory responses. Recent studies examining eosinophil biology have focused on delineating the molecular basis of FIP1L1/PDGRFalpha-fusion gene induced HES, the molecular steps involved in eosinophil recruitment in tumor-associated eosinophilia and EGID, and the role of eosinophils in asthma. In this review, these studies are summarized, focusing on the implications of these findings in the understanding the role of eosinophils in diseases.

Background: There is increasing evidence that both neutrophilic and eosinophilic inflammation persist in the airways of patients with severe asthma. We have reported a positive relationship between the concentrations of eosinophils and neutrophils in sputum from severe asthmatics, suggesting a possible role of eosinophils in regulating neutrophilic inflammation. The aim of this study was to investigate whether activated eosinophils modify the trans-basement membrane migration (TBM) of neutrophils., Methods: Eosinophils and neutrophils were isolated from peripheral blood drawn from healthy donors. The TBM of neutrophils in response to a variety of chemoattractants was evaluated in the presence or absence of eosinophils by using the chambers with a Matrigel-coated Transwell insert., Results: As expected, eotaxin (10 nM) and RANTES (10 nM), but not IL-8 (10 nM), induced the TBM of eosinophils. On the contrary, only IL-8 induced the TBM of neutrophils. When eosinophils were coincubated with neutrophils and stimulated with IL-8, the TBM of eosinophils was significantly augmented. On the other hand, when neutrophils were coincubated with eosinophils and stimulated with eotaxin or RANTES, the TBM of neutrophils was not modified., Conclusions: Neutrophils migrated by IL-8 may lead eosinophils to accumulate in the airways of patients with severe asthma. On the other hand, it is unlikely that eosinophils migrated by chemoattractants such as CC chemokines regulate neutrophilic inflammation.

Background: Objective assessment of inflammatory reactions in the gastrointestinal tract could be useful in the diagnosis of food hypersensitivity. The aim of the present study was to investigate the involvement of eosinophils and mast cells in the inflammatory response of patients with food hypersensitivity before and after food challenges., Methods: Eleven patients (4 with IgE-mediated allergy and 7 without) with food hypersensitivity and positive double-blind, placebo-controlled food challenge were subjected to food challenge in a single-blinded fashion. Four subjects with no known food hypersensitivity were recruited as controls. Placebo was given after a 1-week washout period followed by an active dose. Stool, urinary and serum samples were collected and symptoms were recorded in a diary. Fecal samples were analyzed for eosinophil protein X (F-EPX) and tryptase; urinary samples for EPX (U-EPX) and leukotriene E4 (U-LTE4) and serum samples were analyzed for eotaxin and food-specific IgE antibodies., Results: Patients with IgE-mediated food allergy had increased levels of F-EPX compared to controls and tended to have lower serum levels of eotaxin compared to non-allergic patients and controls. U-LTE4 was significantly higher in allergic patients compared to non-allergic patients after challenge. Moreover, F-EPX correlated to U-LTE4 (p = 0.011). Reported symptoms, abdominal pain, distension, flatulence and nausea were similar in the allergic and non-allergic patients., Conclusion: The results strongly indicate that eosinophils are activated in the gastrointestinal tract of food-allergic patients but not in patients with non-allergic food hypersensitivity. Due to the inconsistent pattern of symptoms after placebo and active food challenge, it was not possible to relate the levels of inflammation markers to the recorded symptoms., (Copyright 2006 S. Karger AG, Basel.)

Background: Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) are capable of eliciting kinase activity after ligand binding. In several cells, RTKs are activated via the G-protein-coupled receptor independent of the ligand-RTK interaction. We have previously found that EGFR is transactivated via CC chemokine receptor 3 in bronchial epithelial cells and that this pathway is important for mitogen-activated protein (MAP) kinase activation and cytokine production. It has recently been suggested that hypereosinophilic syndrome results from the fusion tyrosine kinase FIP1L1-PDGFRA. Although it is possible that the PDGFR signal is involved in eosinophil function, the details are still unclear., Methods: Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Expression of PDGFR mRNA was examined by RT-PCR. After stimulating eosinophils with eotaxin, the phosphorylation of MAP kinases was examined by Western blotting with the antiphosphospecific MAP kinase antibody. The eotaxin-induced eosinophil chemotaxis was studied using Boyden chambers., Results: Eosinophils expressed PDGFRbeta mRNA in 4 out of 8 donors, while PDGFRalpha mRNA was expressed in only 1 donor. Protein expression of PDGFR was also detectable in eosinophils from some donors. AG1295, a specific inhibitor of PDGFR, showed dose-dependent inhibition of eotaxin-induced MAP kinase phosphorylation in the eosinophils expressing PDGFRbeta mRNA. The chemotaxis of these eosinophils was significantly inhibited by AG1295 (n = 3)., Conclusions: Our results suggest that PDGFR modifies the CCR3-MAP kinase signaling pathway and chemotactic response in some donors. The pharmacological targeting of PDGFR may be a new strategy to treat eosinophilic disorders., (Copyright 2006 S. Karger AG, Basel.)

Background: 2-Arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that human eosinophilic leukemia EoL-1 cells and human peripheral blood eosinophils express the CB2 receptor. We also found that 2-AG induces the migration of these cells in a CB2 receptor-dependent manner. In this study, we investigated whether the 2-AG-induced migration of human eosinophils is due to chemotaxis or chemokinesis. We also compared the ability of 2-AG to induce the migration of eosinophils with those of other eosinophil chemoattractants., Methods: Eosinophils were separated from the peripheral blood of healthy donors. The migration of eosinophils to various stimulants was examined using Transwell inserts. In view of the fact that 2-AG is rapidly metabolized by cells, we employed 2-AG ether, an ether-linked nonhydrolyzable analog of 2-AG, instead of 2-AG to determine whether the 2-AG-induced migration is due to chemotaxis or chemokinesis., Results: 2-AG ether induced the migration of human eosinophils, like 2-AG. The 2-AG ether-induced migration was reduced by the coincubation of eosinophils with 2-AG ether in the upper compartment of the Transwell inserts, indicating that the migration is attributable to chemotaxis. The concentration of 2-AG required to induce the eosinophil migration appears to be pathophysiologically relevant, although the order of the pharmacologically effective concentration of 2-AG was approximately ten times lower than those of platelet-activating factor, RANTES and eotaxin., Conclusion: These results strongly suggest that 2-AG is involved in the infiltration of eosinophils during allergic inflammation., (Copyright 2006 S. Karger AG, Basel.)

Background: Evidence shows that leukotriene receptor antagonist (LTRA) can cause a partial reduction of eosinophils in the asthmatic airway. Although cysteinyl leukotrienes (CysLTs) can regulate the development of eosinophilic inflammation, LTRA might modulate the eosinophilic response to other inflammatory molecules involved in allergic inflammation. Montelukast is an LTRA that inhibits eosinophil transendothelial migration (TEM) in response to platelet-activating factor (PAF). The present study evaluates whether pranlukast (an LTRA) modifies eosinophil TEM in response to chemoattractants including PAF and C-C chemokines., Methods: Eosinophils isolated from the blood of healthy individuals were incubated with or without pranlukast. We then evaluated eosinophil transmigration across human umbilical vein endothelial cells in response to LTD(4), eotaxin, RANTES and PAF., Results: Pranlukast did not modify the spontaneous transmigration of eosinophils (n = 5). As reported, eosinophil TEM was significantly augmented by 0.1 microM LTD(4) and this enhancement was blocked by 1 microM pranlukast (p < 0.001; n = 6). On the other hand, pranlukast did not modify eosinophil transmigration in response to eotaxin, RANTES, or PAF (p > 0.1; n = 5)., Conclusion: The inhibitory effect of pranlukast on eosinophil transmigration is highly specific for the CysLT1-dependent pathway., (Copyright 2005 S. Karger AG, Basel.)

Background: C-C chemokines and adhesion molecules expressed on eosinophils play an important role in the pathology of allergic inflammatory disease. C-C chemokines such as eotaxin or RANTES are involved in beta(2) integrin expression on purified eosinophils; so far we have no data on unpurified eosinophils in the peripheral blood. We measured beta(1) and beta(2) integrin activation after stimulation with eotaxin or RANTES in vitro using whole-blood flow-cytometric analysis., Methods: Heparinized whole blood obtained from allergic patients with eosinophilia or normal subjects was diluted with the same volume of RPMI 1640, and then cells were incubated in the presence or absence of PMA/ionomycin or chemokines for 45 min at 37 degrees C. After hemolyzation with lysing solution, expression of CD11b, CD11a, CD18 and CD49d on eosinophils was measured using flow cytometry., Results: The expression of CD11b, CD11a and CD18 in allergic patients was significantly higher than that in normal subjects. CD11b and CD18 expression showed a significant increase after stimulation with C-C chemokines, which was remarkable in allergic patients., Conclusion: Eosinophils in the blood of allergic patients exhibited a higher expression of beta(2) integrins and were more sensitive to RANTES and eotaxin than those of normal subjects., (Copyright 2005 S. Karger AG, Basel.)

Background: Glucocorticoids are known to repress the expression of CC chemokine eotaxin in airway epithelial cells. We focused our study on the molecular mechanisms of the glucocorticoid, fluticasone, in the inhibition of the expression of the eotaxin gene in the cells., Methods: The airway epithelial cell line, BEAS-2B, was stably transfected with signal transducers and activators of transcription 6 (STAT6)-expressing vector and used in the following experiments to clarify the function of STAT6. Levels of eotaxin mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by the electrophoretic mobility shift assay and dual luciferase assay using eotaxin promoter-luciferase reporter plasmids., Results: Fluticasone significantly inhibited the induction of eotaxin protein stimulated with TNF-alpha and IL-4 in the cells. Fluticasone also repressed the induction of eotaxin mRNA with these stimuli. It partially inhibited the activity of eotaxin promoter; however, it did not inhibit the nuclear translocation and binding of transcription factors, nuclear factor-kappa B (NF-kappaB) or STAT6, to the DNA derived from the proximal promoter region of the eotaxin gene. Moreover, the inhibitory effect was also conserved in the experiments using the reporter plasmid of which the putative glucocorticoid-responsive element was mutated., Conclusions: Fluticasone inhibits the expression of eotaxin gene in airway epithelial cells in part through repression of the transcription. However, the mechanisms depend neither on the inhibition of transcription factors' translocation into nuclei nor the function of the putative glucocorticoid-responsive element in the promoter, indicating that other mechanisms would be related to the transcriptional repression of the eotaxin gene in airway epithelial cells., (Copyright 2004 S. Karger AG, Basel)

Background: Atopic dermatitis (AD) is a chronic relapsing inflammation characterized by pruritic and eczematous skin lesions usually observed in patients with a familial history of atopic diseases, but its exact etiology is unclear. An animal model is indispensable for the analysis of the pathogenesis and the development of new drugs to treat this disease. Here, we compare changes in gene expression profiles in the AD-like skin lesions of NC/Nga or BALB/c mice stimulated intradermally by mite antigen under specific pathogen-free (SPF) conditions., Methods: Mite Extract-Dp was injected intradermally into the right and left pinnae and into the skin of the back of NC/Nga or BALB/c mice in 2 places once per 3 days, and the clinical symptoms and the ear thickness were measured. On day 14 or day 28 after starting mite extract injection, we collected plasma and pinnae from NC/Nga or BALB/c mice. The amount of total immunoglobulin E (IgE) in plasma was assayed. We analyzed mRNA transcripts in pinnae using real-time quantitative PCR for the murine counterparts of several known allergy-related genes. Moreover, genome-wide gene expression in pinnae from NC/Nga mice was analyzed using high-density oligonucleotide arrays (GeneChip, Affymetrix)., Results: From 2 weeks after stimulation, marked skin inflammation was induced in the pinnae of NC/Nga but not BALB/c mice. However, IgE levels in sera rose equally in both strains. Quantitative PCR analysis and comprehensive GeneChip analysis of the AD-like pinna skin lesions revealed that their development was accompanied by changes in expression of more than 1,000 genes. These included cytokines, cytokine receptors, proteases, and adhesion molecules. Furthermore, genes thus far not reported in association with AD were also affected., Conclusion: From these results, the NC/Nga mouse model using mite sensitization under SPF conditions could be useful for elucidating the mechanisms of AD pathogenesis and developing more effective therapy for AD., (Copyright 2003 S. Karger AG, Basel)

Background: Both CXC and CC chemokines play an important role in leukocyte recruitment. However, a systematic examination of their production by human airway epithelial cells (HAECs) has not been carried out. The objective of this study was to investigate whether Th1- and Th2-type cytokines regulate chemokine production in HAECs., Methods: HAECs were grown from both nasal and bronchial tissue and subsequently stimulated with either Th1- or Th2-type cytokines., Results: Constitutive mRNA expression for gro-alpha, IL-8 and RANTES was seen in both human nasal and human bronchial epithelial cells. IL-4 was the strongest stimulus for both gene expression and protein production of the chemokines RANTES, IL-8 and gro-alpha, while both IL-13 and IFN-gamma were weaker inducers of these chemokines, with the exception of gro-alpha (IL-13 was a strong stimulus for gro-alpha production). TNF-alpha synergized with IL-4, and to a lesser extent with IFN-gamma and IL-13, to release RANTES, IL-8 and gro-alpha. IL-4 and to a lesser extent IL-13 and IFN-gamma stimulated the production of MCP-3 and -4, eotaxin and eotaxin-2 immunoreactivities. However, no induction of the mRNAs encoding these chemokines was observed, suggesting that they may be released from a preformed pool within the HAECs., Conclusion: These findings suggest that when released into the airways, Th2- and to a lesser extent Th1-type cytokines may stimulate recruitment of eosinophils and neutrophils through the release of CC (RANTES, MCP-3 and -4, eotaxin and eotaxin-2) and CXC chemokines (gro-alpha and IL-8)., (Copyright 2003 S. Karger AG, Basel)

Background: Airway eosinophilia is frequently observed during acute exacerbation of asthma. Interleukin-5 (IL-5) and eotaxin are directly involved in the airway eosinophilia found in persistent asthma. Interrelation between these cytokines is expected to occur in acute exacerbation of asthma. Thus, we evaluated the relevance of interaction between eotaxin and IL-5 in the airway inflammation of acute exacerbation., Methods: We measured the number of inflammatory cells and the amount of eotaxin and IL-5 in sputum from 22 healthy subjects, 21 asthmatics with acute exacerbation and 16 patients with mild persistent asthma, and reassessed these values in 7 subjects with acute exacerbation after 7 days' treatment with systemic steroid (2 mg/kg/day). Sources of IL-5 and eotaxin were investigated by immunohistochemical staining of sputum cells of 4 cases from each group., Results: Both IL-5 and eotaxin levels were higher in patients with acute exacerbation of asthma than in patients with persistent asthma and normal subjects. IL-5 and eotaxin levels were significantly correlated with eosinophil percentages in mild persistent asthma. Eotaxin staining was found mainly on macrophages and occasionally on eosinophils. Steroid treatment markedly decreased eosinophil percentages and IL-5 levels within 7 days but did not alter eotaxin levels., Conclusions: Both IL-5 and eotaxin are associated with acute exacerbation of asthma. IL-5 rather than eotaxin is effectively decreased by the inhibitory effect of steroid in acute exacerbation., (Copyright 2003 S. Karger AG, Basel)

Background: Although eosinophils undergo apoptosis and are thus eliminated from sites of inflammation, they survive longer if survival factors, such as IL-3, IL-5 and GM-CSF, are present. However, it is often observed that some eosinophils survive even when incubated without any survival factors (spontaneous survival). The aim of the present study was to investigate what kind of factor(s) is associated with this spontaneous survival of eosinophils., Methods: Eosinophils were purified from bronchoalveolar lavage of antigen-exposed Balb/c mice and stained with propidium iodide to detect apoptotic ones., Results: We found that the spontaneous survival of eosinophils was reduced by treatment of the cells with anti-CC chemokine receptor-3 (CCR3) antibody (Ab). Moreover, eotaxin-1, eotaxin-2, and eotaxin-3 all prolonged the survival of eosinophils in a dose-dependent fashion. The survival-prolonging effect of eotaxin-1 was enhanced in the presence of eotaxin-3, indicating that these chemokines might work synergistically., Conclusion: We speculate that eosinophils survive longer under the influence of CCR3-reactive chemokines, which aid the infiltration of these cells into the tissue and that eosinophils may survive even longer if they encounter survival factors at local inflammatory sites., (Copyright 2003 S. Karger AG, Basel)

Background: The chemokine eotaxin is a potent and selective chemoattractant for eosinophils. The production of eotaxin by corneal fibroblasts likely contributes to eosinophil infiltration into the corneal stroma. The regulation of eotaxin synthesis in these cells was investigated by examining the effect of interferon-gamma (IFN-gamma), a T helper cell 1-derived cytokine, on eotaxin expression in cultured human corneal fibroblasts., Methods: The release of eotaxin from cultured corneal fibroblasts was measured by enzyme-linked immunosorbent assay, and the abundance of eotaxin mRNA in these cells was determined by reverse transcription combined with real-time polymerase chain reaction analysis., Results: IFN-gamma inhibited in a dose-dependent manner the release of eotaxin induced by each of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta in corneal fibroblasts. IFN-gamma also inhibited the increase in the abundance of eotaxin mRNA induced by each of these cytokines. The synergistic increases in eotaxin release and in eotaxin mRNA abundance induced by the combination of TNF-alpha and the T helper cell 2-derived cytokine IL-4 were also both markedly inhibited by the treatment of cells with IFN-gamma., Conclusions: IFN-gamma inhibited eotaxin expression at both the protein and mRNA levels in cultured human corneal fibroblasts. This effect of IFN-gamma may contribute to the inhibition of eosinophil infiltration into the cornea. Exogenous IFN-gamma thus represents a potential new therapeutic agent for the treatment of corneal disorders associated with inflammatory ocular diseases such as vernal keratoconjunctivitis., (Copyright 2002 S. Karger AG, Basel)

Background: Differences in allergic airway inflammation induced by ovalbumin (OVA) + diesel exhaust particles (DEP) in various murine strains have already been reported. However, there is no report that different murine strains respond differently towards house dust mites or DEP, which are known to aggravate allergic asthma., Methods: The Dermatophagoides farinae allergens Der f (1 microg) or Der f (1 microg) + DEP (50 microg) were administered intratracheally to two different mouse strains (CBA/JN and C57BL/6N). Histological changes in the lung tissues, asthma-relevant cytokines in the lungs, and allergen-specific immunoglobulins in plasma were investigated., Results: Der f treatment led to the proliferation of goblet cells, production of mucus plugs, and the recruitment of eosinophils and lymphocytes to the airways of the mice. The manifestation of the airway inflammation in the C57BL/6N mouse was much greater than in the CBA/JN mouse. The protein levels of interleukin (IL)-4 and IL-5, regulated on activation, normal T cell expressed, and presumably secreted (RANTES), and eotaxin in the lung tissue of C57BL/6N mice were higher than those in CBA/JN mice by a factor of 1.26 (IL-4), 5.26 (IL-5), 2.07 (RANTES) and 3.27 (eotaxin). DEP aggravated the manifestations of the eosinophilic inflammation in CBA/JN mice through goblet cell proliferation. However, the exact effect of DEP could not be evaluated in C57BL/6N because of its severe enhancement of the inflammation. DEP enhanced the local expression of IL-5, RANTES, and eotaxin in the CBA/JN mouse, and consequently triggered an increased IgG1 production in both strains. Allergen-specific IgE antibodies were lower than 1 titer in both mice., Conclusion: The murine strain differences in the pathogenesis of allergic airway disease caused by mite allergen might be related to the local expression of the cytokines we screened. The aggravating effect of DEP may be mediated by an increase in the local expression of IL-5, RANTES, eotaxin, and the production of an antigen specific to IgG1., (Copyright 2002 S. Karger AG, Basel)

Background: Besides being highly effective in the treatment of allergic and nonallergic rhinitis with eosinophilia, intranasal corticosteroids appear to be useful in reducing nasal polypoid lesions and the likelihood of polyp recurrence after surgery. We evaluated the ability of fluticasone propionate to downregulate fibroblast functions related to nasal inflammation and remodeling., Methods: Primary nasal polyp tissue-derived fibroblasts were stimulated with tumor necrosis factor (TNF)-alpha or interleukin (IL)-4 or basic fibroblast growth factor (bFGF) in the presence of fluticasone propionate (0.1-100 nM). Fibroblast proliferation, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression and eotaxin release were then evaluated., Results: As compared with unstimulated cultures, a significant increase in fibroblast proliferation was observed when the cells were stimulated with bFGF (p < 0.05), but not with TNF-alpha or IL-4 (p > 0.05). TNF-alpha induced an upregulation of ICAM-1 expression (p < 0.05), which was not seen in fibroblasts cultured in the presence of IL-4 or bFGF. No changes in VCAM-1 expression were induced by TNF-alpha, IL-4 or bFGF, whereas both TNF-alpha and IL-4 increased eotaxin release (p < 0.05). Both bFGF-induced fibroblast proliferation and TNF-alpha-induced ICAM-1 expression were significantly reduced by fluticasone, starting at the dose of 1 and 10 nM, respectively (p < 0.05). Fluticasone at concentrations of 1-100 nM effectively inhibited eotaxin release by TNF-alpha- or IL-4-stimulated fibroblasts (p < 0.05)., Conclusions: The pharmacologic activity of fluticasone in patients with chronic upper airway inflammatory disease may include inhibition of resident fibroblast functions involved in airway inflammation and remodeling., (Copyright 2002 S. Karger AG, Basel)

Background: Dermatitis herpetiformis (DH) is a subepidermal bullous disease characterized by a neutrophilic and eosinophilic infiltrate, together with activated lymphocytes that show preferential polarization towards the Th2-like phenotype. To date, the role of neither soluble mediators, such as cytokines, nor serum levels of myeloperoxidase (MPO), eosinophil cationic protein (ECP) and tryptase (TRY) have been studied in the peripheral blood of DH. In this study we investigated 40 DH subjects for possible alterations in MPO, ECP, TRY, eotaxin, interleukin (IL) 4 and IL-5 concentrations to evaluate the role of neutrophils, eosinophils and mast cells in the induction of skin lesions. Sera from 20 healthy subjects were tested as controls., Methods: Eotaxin, IL-4 and IL-5 serum contents were determined by a solid-phase enzyme-linked immunosorbent assay. A double antibody radioimmunoassay was used to measure serum levels of MPO, ECP and TRY., Results: Normal levels of eotaxin and IL-4 were found in all DH subjects; increased IL-5 levels were found only in 1 subject. The MPO levels were significantly higher in DH subjects than in healthy controls (p < 0.01). There was also a significant difference between the serum ECP levels of DH and healthy controls (p < 0.05). TRY was not revealed in the serum of DH subjects., Conclusions: The determination of Th2-like cytokines and TRY serum levels is not important in quantifying the involvement of activated lymphocytes and mast cells in DH; however, the finding of high MPO and ECP serum levels seems to confirm that these products are good markers of the presence of activated neutrophils and eosinophils., (Copyright 2002 S. Karger AG, Basel)

Histamine and serotonin are important inflammatory mediators in the pathophysiology of asthma, and asthmatic patients have higher plasma histamine and serotonin levels than nonasthmatic control subjects. Eotaxin, a potent eosinophil-specific chemotactic factor, is increased in the lower respiratory tract of allergic patients. Recently, lung fibroblasts have been reported to produce eotaxin and are suggested to be the major cellular source of eotaxin. We postulated that lung fibroblasts might release eotaxin in response to histamine or serotonin. To test this hypothesis, we evaluated the potential of histamine or serotonin to induce the release of eotaxin by the human fetal lung fibroblast cell line, HFL-1. HFL-1 released eotaxin in response to histamine and serotonin in a dose- and time-dependent manner (p < 0.05). Histamine or serotonin treatment of HFL-1 augmented the expression of eotaxin mRNA. Eosinophil chemotactic activity by HFL-1 supernatant fluids was inhibited by anti-human eotaxin-neutralizing antibody. These findings lead to the hypothesis that lung-fibroblast-derived eotaxin may in part be responsible for the eosinophil infiltration observed in allergic disease of the airways., (Copyright 2002 S. Karger AG, Basel)

In this study, we postulated that repeated cycles of IgE passive sensitisation and antigen challenge may play a role in up-regulating eosinophil response in allergic conditions. Antigen-mediated stimulation of the pleural cavity of rats passively sensitised with a single injection of IgE anti-DNP resulted in mast cell degranulation, increase in vascular permeability and mild neutrophilia, but no pleural eosinophilia. In contrast, a second cycle of sensitisation and challenge, performed within 7 days, showed a marked eosinophilia in parallel with a lower plasma leakage and comparable neutrophilia. The eosinophilic phenomenon was not reproduced when (1) IgE sensitisation or antigen challenge was omitted in the first cycle, or (2) the first cycle was replaced by either a histamine and 5-HT dual challenge or a PAF challenge. Furthermore, we found an increase in eotaxin levels in animals subjected to two rather than one cycle of sensitisation and challenge. Treatment with the PAF receptor antagonist BN 52021 or with the lipoxygenase inhibitor zileuton, but not mast cell granule depletion, prevented the allergen-evoked eosinophil accumulation in rechallenged animals. Our results indicate that repeated cycles of IgE-driven inflammation may lead to eosinophil accumulation in a mechanism dependent on eotaxin, PAF and leukotrienes., (Copyright 2002 S. Karger AG, Basel)

Background: The accumulation of eosinophils in the lung is a hallmark of asthma. In addition to cytokines such as IL-5 which are essential, chemokines have been implicated in the recruitment of eosinophils to the airway. In particular, eotaxin has been shown to be a selective and potent eosinophil chemoattractant, important in the pathogenesis of allergic disease. The goal of the present study was to define the role of eotaxin-1 in the development of allergen-induced eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to inhaled methacholine (MCh)., Methods: Eotaxin-1-deficient mice were sensitized and exposed to a single challenge with allergen. Airway function and airway and tissue as well as peripheral blood and bone marrow eosinophilia were examined 18 and 48 h after the last challenge., Results: Following allergen sensitization and challenge, eotaxin-1-deficient mice developed levels of AHR to inhaled MCh at 18 and 48 h comparable to controls. Further, levels of bronchoalveolar lavage (BAL) and tissue eosinophilia at the same time points were comparable in the two strains of mice. Tissue eosinophilia, assessed by quantitating major basic protein staining cells, preceded BAL eosinophilia in a similar manner. Bone marrow and peripheral blood eosinophilia were unimpaired in deficient mice., Conclusion: The results demonstrate that the major eotaxin, eotaxin-1 is not essential for the development of airway eosinophilia or AHR, implying that other chemokines, alone or in combination, can overcome this deficiency., (Copyright 2001 S. Karger AG, Basel)

Signals via chemokine receptors play an important role in the accumulation of eosinophils at allergic inflammatory sites. Eosinophils constitutively express CC chemokine receptor 3 (CCR3) and, to a lesser extent, CCR1. CCR3 is mainly responsible for migration of resting eosinophils, and its specific ligand, eotaxin, represents the most potent chemoattractant for eosinophils. Some reports also suggest the expression of CXC chemokine receptor 1 (CXCR1) and/or CXCR2 in eosinophils. In addition, we recently reported the functional expression of CXCR4. The ligand of CXCR4, stromal cell-derived factor-1 (SDF-1), was able to induce a strong migratory response comparable to that by eotaxin. In contrast to the CCR3/eotaxin system which is mainly regulated at the level of ligand production, the CXCR4/SDF-1 system is regulated at the level of receptor expression. CXCR4 expression was completely attenuated by IL-4 and IL-5 and upregulated by IFN-gamma and dexamethasone, while CCR3 expression was only marginally affected. The balance between the biological effects of these chemokine systems may affect the distribution and migration of eosinophils., (Copyright 2001 S. Karger AG, Basel)

Emedastine difumarate (emedastine) is an antiallergic drug found among the derivatives which has a series of benzimidazole frames. It has been reported that emedastine can significantly inhibit the migration of eosinophils elicited by classical chemoattractants, including LTB4 or PAF. However, the effect of emedastine on the selective migration of eosinophils that have been stimulated with CC chemokines has not been examined. Emedastine at concentrations of 10 nM or higher strongly inhibited the eosinophil migration elicited by CC chemokines, including eotaxin, RANTES and MCP-3. Preincubation of the eosinophils with emedastine did not alter the expression of the CCR3 receptor, although a decrease in the concentration of intracellular calcium ions was observed after stimulation with 100 ng/ml of eotaxin. Herbimycin A, genistein, staurosporin and emedastine were all able to inhibit the eotaxin-elicited migration. Tyrosine kinase activity in the cytosol supernatant of eosinophils obtained after stimulation with eotaxin significantly decreased when the eosinophils were preincubated with emedastine. In addition, protein kinase A and protein kinase C activities in eotaxin-stimulated EoL-1 cell supernatants decreased significantly with emedastine pretreatment. These findings suggest that emedastine inhibits CC chemokine-elicited eosinophil migration by decreasing the activities of tyrosine kinase or protein kinases but does not alter CCR3 expression., (Copyright 2001 S. Karger AG, Basel)

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca(2+)](i)) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37 degrees C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils., (Copyright 2001 S. Karger AG, Basel)

Background: The aim of this study was to investigate whether human mast cells express functional active CCR3 receptors, which are activated by CC chemokines. These ligands include the CCR3-selective chemokines eotaxin and eotaxin-2 and the more promiscuous CC chemokines, MCP-4, MCP-3, MCP-2 and RANTES., Methods: Immunohistochemical analysis was performed on skin, gut and lung specimens. Double immunostaining was performed with anti-CCR3 and antitryptase, and anti-CCR3 and antichymase antibody (Ab) by using the avidin-biotin-peroxidase system with two different substrates. Mast cells were isolated and purified from human lung parenchyma (HLMC) by countercurrent elutriation followed by discontinuous Percoll density gradient. Flow-cytometric analysis of HLMC surface CCR3 expression was performed with the monoclonal Ab anti-CCR3 (7B11). Functional activation of HLMC was verified by the ability of cells to release histamine and/or migrate in response to eotaxin., Results: High percentages (>70%) of tryptase-positive cells showing CCR3 expression were found in the skin and in the intestinal submucosa, whereas much lower percentages (< or = 20%) were found in the intestinal mucosa and in the lung interstitium. Eotaxin (1-100 nM) neither induced histamine release from HLMC nor enhanced anti-IgE-induced histamine release. In contrast, eotaxin (10-100 nM) and RANTES (10-100 nM) induced HLMC chemotaxis in vitro. Preincubation of HLMC with antibody anti-CCR3 (5 microg/ml) before loading into the chemotaxis chamber abrogated chemotaxis elicited by eotaxin. Double immunostaining with anti-CCR3 and anti-chymase antibody showed that the vast majority of CCR3-expressing mast cells in the various human tissues examined were tryptase-chymase double-positive., Conclusions: These results indicate that CCR3 is expressed on human mast cells and that these cells are attracted by CCR3-binding chemokines., (Copyright 2001 S. Karger AG, Basel)

Background: The CC chemokine eotaxin not only attracts eosinophils to inflamed sites but also promotes adhesion, degranulation and reactive oxygen species production of eosinophils. Reactive oxygen species released from eosinophils are believed to injure epithelial cells at inflamed sites, resulting in airway hyperresponsiveness. Roxithromycin has been reported to have antiasthmatic effects, although its mechanism of action is not thoroughly understood. Therefore, the effect of roxithromycin on eotaxin-primed reactive oxygen species production from eosinophils was studied., Methods: Reactive oxygen species production by eosinophils cultured with or without roxithromycin was evaluated using luminol-dependent chemiluminescence., Results: Roxithromycin inhibited the release of reactive oxygen species from eosinophils evoked with the calcium ionophore A23187, regardless of pretreatment with or without eotaxin., Conclusion: Roxithromycin may protect epithelial cells at inflamed sites, at least partly by inhibiting the release of reactive oxygen species from eosinophils., (Copyright 2001 S. Karger AG, Basel)

We recently located a rare cytokeratin-positive (CK+) type of microvascular endothelial cell (MVEC) in the corpus luteum and aorta. Bovine corpus luteum MVEC are known to be involved in the cyclic accumulation of eosinophils and macrophages. Since leukocyte migration is specifically mediated by adhesion molecules and the release of cytokines, we compared the expression of these factors in basal and TNF-alpha-stimulated CK+ MVEC and in common cytokeratin-negative (CK-) MVEC in order to obtain an initial insight into the functional capacities of CK+ MVEC. CK- MVEC revealed significantly higher basal RANTES mRNA expression than CK+ MVEC, and TNF- alpha up-regulated RANTES mRNA in both types of MVEC. Only resting and stimulated CK- MVEC expressed granulocyte-macrophage colony-stimulating factor mRNA. Both MVEC types expressed monocyte colony-stimulating factor mRNA, but remained negative for eotaxin and interleukin (IL)-5 mRNA even after stimulation. Resting CK+ MVEC were positive for CD29, CD31, CD49a and CD49e, but expressed most of these antigens at a significantly lower density than did CK- MVEC. In contrast to CK- MVEC, CK+ MVEC failed to express CD49b or MHC class II. The activation of CK+ MVEC with TNF-alpha induced the expression of CD62P, but not of CD49b or MHC class II. In summary, phenotypically variable MVEC derived from the microvascular bed of one organ differ in their TNF-alpha-regulated expression of cytokine mRNA and adhesion molecules. Morphological heterogeneity is related to a particular specialisation of functional MVEC., (Copyright 2000 S. Karger AG, Basel)

Background: Viral infection is known to cause lung inflammatory disease, including bronchial asthma. The mechanisms of inflammatory cell accumulation into the airways after viral infection are not well understood. Eotaxin is a CC chemokine which is a potent and specific agonist for CC chemokine receptor 3 (CCR3). CCR3 is expressed on eosinophils, basophils and T lymphocytes. These cells are known to be key cells in the pathogenesis of asthma. Although it has recently been demonstrated that airway epithelial cells express eotaxin in vivo and in vitro, there are few data about its epxression in viral infection. We hypothesized that eotaxin may play an important role in attracting inflammatory cells to the airways after viral infection, and analyzed whether viral infection attracts eotaxin in bronchial epithelial cells in vitro., Methods: Human airway epithelial cells obtained from bronchial tissue at lobectomy for lung cancer were infected with influenza virus A (subtype H3N2). The cells and cultured media were collected 8, 24, and 48 h after infection. Eotaxin mRNA was analyzed with reverse transcriptase-polymerase chain reaction. Eotaxin protein levels in the culture media were analyzed by enzyme-linked immunosorbent assay. We also studied a blocking assay to analyze the intervention of proinflammatory cytokines in its production induced by influenza virus., Results: Eotaxin mRNA appeared to be expressed constitutively in uninfected cells but was expressed more clearly in infected cells. Eotaxin protein release into culture media significantly increased after infection. Anti-TNF-alpha and anti-IL-1beta antibodies did not alter the eotaxin protein levels after viral infection., Conclusions: These results suggest that influenza virus A infection in airway epithelial cells activates the expression of eotaxin and that eotaxin may participate in the pathogenesis of airway inflammatory disease caused by viral infection, such as infectious type asthma., (Copyright 2000 S. Karger AG, Basel.)

Normal human bronchial epithelial cells (NHBECs) spontaneously synthesize and store chemokines including eotaxin, RANTES, or MCP-3. We also observed the constitutive expression of mRNA specific for CC chemokine receptor-3 (CCR3) in these cells. The stimulation of these cells by eotaxin (1-100 ng/ml) dose-dependently increased eotaxin production. In addition, RANTES or eotaxin-2 but not MCP-3 at the higher concentrations (100 ng/ml), also induced slight eotaxin production. Eotaxin elicited the expression of eotaxin mRNA at 6 h after stimulation. Antibody against eotaxin or CCR3, and actinomycin D inhibited these effects of eotaxin. These antibodies also significantly inhibited the ability of eotaxin to produce further eotaxin. These findings strongly suggest that eotaxin which originates from inflammatory cells or cells resident in the airway of bronchial asthmatics can induce further production of eotaxin and participate in expansion of allergic inflammation., (Copyright 2000 S. Karger AG, Basel.)

Background: Accumulating evidence indicates that eotaxin is the primary mediator of tissue eosinophilia. In the present study, we analyzed the mechanisms of eotaxin generation by Th1-/Th2-derived cytokines in vitro., Methods: Human dermal fibroblasts, human umbilical vein endothelial cells and A549 human bronchial epithelial cell line cells were stimulated with TNF-alpha, IL-4, IFN-gamma or TNF-alpha in combination with IL-4 or IFN-gamma and the amount of eotaxin production was analyzed., Results: Fibroblasts produced nanogram/milliliter quantities of eotaxin. Proinflammatory cytokine TNF-alpha and Th2-type cytokine IL-4 each induced eotaxin production, and combination of them caused a marked synergistic increase in that production. On the other hand, Th1-type cytokine IFN-gamma inhibited eotaxin generation at the protein/mRNA levels., Conclusion: The Th2-derived cytokine upregulated while the Th1-derived cytokine inhibited eotaxin production by fibroblasts. In view of the Th1/Th2 paradigm, these results indicate that (1) eotaxin regulates eosinophil accumulation in the Th2-dominant state such as allergic disease, and (2) direct suppression of eotaxin production by IFN-gamma is one of the major mechanisms by which IFN-gamma suppresses eosinophilic inflammation., (Copyright 2000 S. Karger AG, Basel.)

Background: In severe allergic eye diseases, the breakdown of epithelial barrier function can lead to severe corneal damage such as erosions or ulcers which often resist treatment. Although eosinophils are thought to play a crucial role in corneal tissue damage in severe ocular allergy, the mechanisms of eosinophil recruitment to the cornea has not been fully clarified. Eotaxin has been found in tears of severe allergic patients with corneal ulcer. In this study, we investigated whether the Th2 cytokine interleukin-4 (IL-4) induces eotaxin production in human corneal epithelial cells and keratocytes., Methods: Primary cultures of human corneal epithelial cells and keratocytes were incubated with IL-4 and/or TNF-alpha for 48 h. Released eotaxin was measured by ELISA, and the eotaxin proteins were visualized by immunocytochemistry. Eotaxin mRNA expression in cultured cells was analyzed by RT-PCR., Results: IL-4 induced eotaxin production in keratocytes in a dose- and time-dependent manner which was enhanced by TNF-alpha. There was no detectable eotaxin produced by corneal epithelial cells (<5 pg/ml). The cytoplasm of keratocytes incubated with IL-4 stained positively against anti-eotaxin antibodies, while eotaxin mRNA was detected in keratocytes incubated with IL-4., Conclusions: Human corneal keratocytes, but not epithelial cells, are capable of producing eotaxin by stimulation with IL-4. Our results suggest that eotaxin production in keratocytes induced by IL-4 may play an important role in eosinophil recruitment to corneal ulcers in allergic ocular disease. Eotaxin production by keratocytes may explain the severity of allergic disease involving the corneal stroma., (Copyright 2000 S. Karger AG, Basel)

Background: Cynomolgus monkeys have a natural hypersensitivity to Ascaris suum antigen. Inhalation of antigen produces immediate and delayed allergic reactions and an influx of inflammatory cells into the lungs. This study investigated the production of nitric oxide (NO) and the chemokine eotaxin during this allergic response. The effect of bronchoscopy alone on lung inflammatory cells was also investigated along with the time course of the eosinophil influx into the lung., Methods: Allergic cynomolgus monkeys were challenged with antigen. Bronchoalveolar lavage (BAL) was performed before and after challenge, and end-tidal NO was measured before and 24 h after challenge. Eotaxin was measured in the BAL fluid 6, 24 and 72 h after challenge. One group of animals was treated with dexamethasone before challenge to block the influx of cells into the lung., Results: BLA alone induced an influx of neutrophils, but not eosinophils, into the lung 24 h later. A single antigen challenge produced a marked increase in BAL eosinophils that was apparent at 6 h but increased at 72 h after challenge. The increase at 6 h was largely blocked by dexamethasone. Three antigen challenges produced elevated BAL eosinophil levels that persisted for at least 8 weeks. Eotaxin levels rose dramatically 6 h after challenge and remained the same after 24 h. By 72 h, the eotaxin levels had returned to baseline. The increase in eotaxin at 6 h was nonsignificantly reduced by dexamethasone. Exhaled NO levels doubled 24 h after challenge and were not affected by dexamethasone., Conclusions: Eotaxin and NO production were increased after airway challenge in allergic monkeys. The rise in NO was not blocked by dexamethasone. The effects of bronchoscopy on the BAL can be avoided by using alternate lungs on consecutive occasions. Eosinophils persist in the BAL for many weeks after antigen challenge.

Background: Eosinophils develop from bone marrow (BM) progenitors, and interleukin-5 (IL-5) and eotaxin may act in expansion and mobilisation of BM eosinophils in asthma., Methods: We have examined phenotypic changes as CD34+ cells develop to the eosinophil lineage in vitro, and have evaluated BM eosinophils from asthmatic and control subjects for expression of the eotaxin receptor, CCR3., Results: Acquisition of receptors for IL-5 and CCR3 was an early event in eosinophil development. There were increased CD34+ cells, and mature and immature CCR3+ eosinophils in BM from asthmatics., Conclusion: These data suggest that IL-5 may act early in eosinophil development, and that eotaxin has the capacity to mobilise a BM eosinophil pool in asthma.

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine-induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met-RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP-3 and eotaxin was investigated. Met-RANTES had no intrinsic activity on [Ca2+]i transients in eosinophils and was able to dose-dependently inhibit [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP-3 and eotaxin. The results of this study lead to the conclusion that Met-RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.

Background: Several studies have shown that cultured eosinophils can be generated from human umbilical cord blood mononuclear cells (UCMC) in the presence of interleukin (IL)-3 and IL-5 in vitro. Other reports have indicated that cellular adhesion to hyaluronic acid (HA) enhances the proliferation of cultured eosinophils derived from CD34+ cells purified from UCMC. The aim of this study was to obtain large numbers of mature eosinophils from UCMC using IL-3, IL-5 and HA, and to investigate their functions., Methods: We examined several combinations of IL-3 and IL-5 and their effect on eosinophil development from UCMC in HA-coated on non-coated flasks. We also examined whether cultured eosinophils degranulated eosinophil-derived neurotoxin (EDN) induced by secretory immunoglobulin A conjugated to sepharose beads (sIgA-beads) and responded to eotaxin., Results: Culture with HA-coated flasks for 35 days (in the presence of IL-3 and IL-5, with IL-3 omitted after day 14 of culture) caused a 11.2-fold augmentation in the proliferation of UCMC. On day 35 of the culture, 98% of cultured cells were eosinophils judging from May-Grünwald and Giemsa staining and transmission electron micrographs. The EDN content of the cultured eosinophils on day 35 was 156 ng/105 cells. Cultured eosinophils degranulated EDN induced by sIgA-beads and responded to eotaxin by chemotaxis and intracellular Ca2+ mobilization., Conclusion: We found a useful culture system to obtain large numbers of eosinophils derived from UCMC, which may facilitate the investigation of eosinophil function, since there was no significant difference in response to sIgA-beads and eotaxin between cultured and peripheral eosinophils.

Background: The CC chemokine eotaxin is a selective chemoattractant for eosinophils. Eosinophils have been considered to be the major effector cells in allergic inflammation, since not only eosinophil-specific granule proteins but also reactive oxygen species (ROS) from eosinophils may cause the damage to the cells or tissue of the mucosal epithelium. In this study, we examined the effect of eotaxin on ROS from an eosinophil cell line, YY-1., Methods: ROS in luminol-dependent reaction were examined. Calcium ionophore A23187 were added to the mixture of YY-1 cells with luminol, and then ROS were determined., Results: Eotaxin primed the production of ROS from YY-1 cells. ROS from untreated YY-1 cells evoked with calcium ionophore A23187 in luminol-dependent chemiluminescence gave a maximal value of 1,928 +/- 223 intensity counts (IC; mean +/- SE, n = 4) and an integral value of 17.04 +/- 1. 51 IC (x10(-4)), while eosinophils that were treated with eotaxin gave a maximal value of 2,264 +/- 86 IC (10 nM), 2,691 +/- 124 IC (100 nM) and an integral value of 21.22 +/- 0.67 IC (x10(-4); 10 nM), 26.20 +/- 1.41 IC (x10(-4); 100 nM)., Conclusion: Eotaxin might play important roles in the pathogenesis of allergic inflammation through eosinophil activation by priming of eosinophil oxidative metabolism as well as involvement in selective eosinophil chemotaxis.

A common feature of Th2-mediated skin diseases is selective eosinophil (Eo) infiltration in the affected dermis. However, the mechanisms of selective Eo recruitment are not yet fully understood. Our recent findings indicated that dermal fibroblasts actively participate in the accumulation of Eo in Th2-mediated skin inflammation. Following stimulation with IL-4, dermal fibroblasts produce eotaxin, which is the only biologically active Eo attractant detected in the supernatants. Natural eotaxin is comprised of a mixture of N-terminal-truncated and O-glycosylated variants. The expression of eotaxin mRNA is upregulated within 1 h after stimulation. TNF-alpha markedly enhances eotaxin mRNA expression and release of the protein product from IL-4-stimulated fibroblasts. As mast cells and Th2 cells produce both cytokines, it is probable that Eo recruitment into sites of allergen-induced, Th2-mediated skin reactions is, at least in part, due to IL-4-stimulated induction of eotaxin in dermal fibroblasts.

Background: Eosinophil granule proteins deposition at the site of allergic inflammation contributes to the late-phase reaction of hypersensitivity diseases. In the present communication, we describe the effect of human eotaxin on normal human eosinophil exocytosis measured as degranulation of eosinophil-derived neurotoxin (EDN)., Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Purified eosinophils were stimulated with various concentrations of eotaxin and the amount of EDN released was analysed by radioimmunoassay. Flow cytometry was used to examine the surface expression of adhesion molecules on eosinophils., Results: Eotaxin significantly induced EDN release in a dose-dependent manner. The potency of eotaxin in this effect was equal to that of RANTES, and comparable to that of platelet-activating factor. Eotaxin-induced EDN release was blocked by cytochalasin B in a dose-dependent manner. The surface expression of CD11a, CD11b, CD18 and VLA-4 adhesion molecules on normal human eosinophils were not modulated by eotaxin stimulation., Conclusions: These results indicate that eotaxin may play an important role not only as a selective chemotaxin for the cell type but also as a secretagogue. Furthermore, they demonstrate a degranulation mechanism(s) involving cytoskeletal changes which is probably independent of the quantitative expression of adhesion molecules.

Background: The mediators involved in eosinophil accumulation in diseases such as allergy continue to be an area of interest, even though little is known regarding the signaling involved in the human cell type recruitment. In the present study, we demonstrate a novel modulatory role of tyrosine kinase and tyrosine phosphatase activities on normal human eosinophil chemotaxis induced by different groups of chemoattractant., Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Chemotactic activities against platelet-activating factor (PAF), vasoactive intestinal peptide (VIP) and eotaxin were assessed using a 48-well microchemotaxis chamber assay. Purified eosinophils were pretreated with herbimycin A, erbastatin or pervanadate to examine the role of tyrosine kinase in chemoattractant signaling., Results: Pretreatment of eosinophils with the tyrosine kinase inhibitors herbimycin A and erbstatin significantly blocked chemotaxis induced by eotaxin whilst both inhibitors augmented chemotaxis induced by VIP; however, they had no effect on PAF-induced chemotaxis. On the other hand, pretreatment of eosinophils with the phosphotyrosine phosphatase inhibitor pervanadate resulted in augmentation of eotaxin-induced chemotaxis and inhibition of VIP-induced chemotaxis, but it had no effect on PAF-induced chemotaxis., Conclusions: These results suggest that protein kinase plays a modulatory role in eosinophil chemotaxis induced by various chemoattractants.