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Type 2-high asthma is a chronic inflammatory disease of the airways which is increasingly prevalent in countries where helminth parasite infections are rare, and characterized by T helper 2 (Th2)-dependent accumulation of eosinophils in the lungs. Regulatory cytokines such as TGF-β can restrain inflammatory reactions, dampen allergic Th2 responses, and control eosinophil activation. The murine helminth parasite Heligmosomoides polygyrus releases a TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β despite bearing no structural similarity to the mammalian protein. Here, we investigated if Hp-TGM could alleviate allergic airway inflammation in mice exposed to Alternaria alternata allergen, house dust mite (HDM) extract or alum-adjuvanted ovalbumin protein (OVA). Intranasal administration of Hp-TGM during Alternaria exposure sharply reduced airway and lung tissue eosinophilia along with bronchoalveolar lavage fluid IL-5 and lung IL-33 cytokine levels at 24 h. The protective effect of Hp-TGM on airway eosinophilia was also obtained in the longer T-cell mediated models of HDM or OVA sensitisation with significant inhibition of eotaxin-1, IL-4 and IL-13 responses depending on the model and time-point. Hp-TGM was also protective when administered parenterally either when given at the time of allergic sensitisation or during airway allergen challenge. This project has taken the first steps in identifying the role of Hp-TGM in allergic asthma and highlighted its ability to control lung inflammation and allergic pathology. Future research will investigate the mode of action of Hp-TGM against airway allergic eosinophilia, and further explore its potential to be developed as a biotherapeutic in allergic asthma., (© 2022 The Authors. Immunology published by John Wiley & Sons Ltd.)

Background and Aims: Recent studies highlight the role of chemokines for the attraction of inflammatory cells in liver injury and fibrogenesis. The CC chemokine ligand 11, eotaxin (CCL11), is up-regulated in senescent human hepatic stellate cells and crucial in animal models of T-cell mediated hepatitis. The aim of this study was to analyze the role of eotaxin in chronic liver disease., Methods: Plasma eotaxin levels of 111 patients with chronic liver disease were correlated with clinical presentation, laboratory parameters, liver histology and clinical course in a 6-year follow-up., Results: Eotaxin concentrations were significantly up-regulated in patients with liver cirrhosis and increased according to Child-Pugh and model of end-stage liver disease (MELD) score. Eotaxin correlated with the hepatic biosynthetic capacity and other inflammatory cytokines. High eotaxin was associated with hepatic necroinflammation and fibrosis in liver histology. In patients with typical clinical complications of cirrhosis, eotaxin was found to be increased. High eotaxin indicated an unfavorable prognosis in 6-year follow-up., Conclusions: High eotaxin expression may be involved in the pathogenesis of chronic liver diseases. Plasma eotaxin levels correlate with the degree of liver cirrhosis and could serve as an additional biomarker indicating histological hepatic necroinflammation and fibrosis as well as an adverse clinical course.

Background: Back-titration of inhaled corticosteroid (ICS) dose in well-controlled asthma patients is emphasized in clinical guidelines, but there are few published data on the airway cell and cytokine changes in relation to ICS reduction. In our study, 20 mild-to-moderate persistent (inspite of low-moderate dose ICS treatment) asthmatic subjects prospectively rendered largely asymptomatic by high-dose ICS were assessed again by clinical, physiological, and airway inflammatory indices after 4-8 weeks of reduced ICS treatment. We aimed at assessing the underlying pathological changes in relation to clinical deterioration., Methods: Patients recorded daily symptom scores and peak expiratory flows (PEF). Spirometry and airways hyperreactivity (AHR) were measured and bronchoscopy was performed with assessment of airway biopsies (mast cells, eosinophils, neutrophils, and T lymphocytes), bronchoalveolar lavage (BAL) IL-5 and eotaxin levels and cellular profiles at the end of high-dose ICS therapy and again after ICS dose reduction. Baseline data were compared with symptomatic steroid-free asthmatics (n=42) and non-asthmatic controls (n=28)., Results: After ICS reduction, subjects experienced a variable but overall significant increase in symptoms and reductions in PEF and forced expiratory volume in 1 s. There were no corresponding changes in AHR or airways eosinophilia. The most relevant pathogenic changes were increased CD4(+)/CD8(+) T cell ratio, and decreased sICAM-1 and CD18 macrophage staining (potentially indicating ligand binding). However, there was no relationship between the spectrum of clinical deterioration and the changes in cellular profiles or BAL cytokines., Conclusions: These data suggest that clinical markers remain the most sensitive measures of early deterioration in asthma during back-titration of ICS, occurring at a time when AHR and conventional indices of asthmatic airway inflammation appear unchanged. These findings have major relevance to management and to how back-titration of ICS therapy is monitored.

Background: The effects of protease-activated receptor-2 (PAR-2) stimulation on inflammation mechanisms of chronic rhinosinusitis (CRS) are still unknown., Methods: PAR-2 receptor expression was investigated by immunohistochemistry and Taqman mRNA analysis in the mucosa of different rhinosinusitis entities. In primary nasal epithelial cell cultures, the function of PAR-2 and its ability to produce CXC, CC chemokines, and IL-6 were measured by calcium mobilization and stimulation tests. Inhibition tests were performed using cortisone, serine protease inhibitors, cysteine protease inhibitors, Pertussis toxin (PTX) and nuclear transcription factor (NF-kappaB) inhibition (BAY 11-7085). Signal transduction pathways were analysed by electromobility shift assays (EMSA) and NF-kappaB binding studies., Results: The expression of PAR-2 was found to be increased in CRS specimens. The activation of PAR by trypsin or PAR-2-specific activating peptide (AP) caused an increase in cytosolic calcium, as well as the release of the CXC chemokines IL-8 and growth-related oncogene (GRO)-alpha, but not the release of CC chemokines or IL-6. AP-induced CXC chemokine was sensitive to PTX and activation of NF-kappaB was inhibited by BAY11-7085. Furthermore, a serine protease inhibitor significantly inhibited chemokine synthesis stimulated by trypsin and culture supernatants of staphylococci, whereas steroids and cysteine protease inhibitors had little effect., Conclusion: PAR-2 plays a role in serine protease-mediated regulation - staphylococcal and non-staphylococcal origin - of IL-8 and GRO-alpha in nasal epithelial cells, but not in the regulation of CC chemokines. PAR-2 may therefore be involved in the pathophysiology of CRS and NP at different sites of activation, namely (i) proteases, (ii) the PAR-2 receptor itself or (iii) the application of novel agents that block NF-kappaB/IkappaB-alpha signalling.

Background: Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation., Objective: The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils., Methods: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay)., Results: NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils., Conclusion: Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.

Background: A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. It is generally considered that eosinophilic inflammation and hyper-reactivity to methacholine in airways are fundamental to the pathophysiology of bronchial asthma., Objective: This study was designed to determine whether the Rho/Rho-kinase pathways are involved in the eosinophil recruitment and airway hyper-reactivity. We investigated inhibitory effects of fasudil, a specific inhibitor of Rho-kinase, on acute allergic inflammation in mice., Methods: BALB/c mice were sensitized and challenged with ovalbumin (OVA). OVA-challenged mice were treated orally with fasudil (3, 10, 30 mg/kg) or saline before each OVA challenge. Total cell counts, differential cell counts, cytokines, and chemokines levels were measured in bronchoalveolar lavage (BAL), and lungs were examined histologically. Moreover, respiratory resistance in response to methacholine was measured., Results: When fasudil was administrated to OVA-challenged mice, increased cell numbers of total cells and eosinophils were significantly attenuated in a dose-dependent manner. However, inflammatory cells other than eosinophils were not affected by fasudil. Fasudil caused a dose-dependent inhibition in increased levels of IL-5, IL-13, and eotaxin in BAL fluid by OVA challenges. Histological analysis of the airways revealed that both infiltration of inflammatory cells and goblet cell hyperplasia were significantly suppressed in fasudil treatment. Furthermore, fasudil significantly suppressed the augmented responsiveness to methacholine induced by OVA challenges., Conclusion: Oral administration of fasudil inhibits eosinophil recruitment, goblet cell hyperplasia and airway hyper-reactivity by allergen challenges. These effects of this agent may be mediated by suppressing a chemokine and cytokines related to the pathophysiology of bronchial asthma such as eotaxin, IL-5, and IL-13. Our findings provide evidence that inhibition of the Rho/Rho-kinase pathway may be beneficial for bronchial asthma.

The collectin surfactant protein D (SP-D) is an important component of the pulmonary innate host defence. Up to now, little is known about the regulation of eosinophil function by SP-D. Various murine models of pulmonary hypersensitivity suggest that SP-D may be a potent anti-allergic protein. We investigated the modulation of eosinophil chemotaxis and degranulation by human SP-D. SP-D markedly inhibited the chemotaxis of eosinophils triggered by eotaxin, a major tissue-derived CC-chemokine, as shown in a modified Boyden chamber assay. In addition, degranulation of ECP in response to Ca2+ ionophore, immobilized IgG and serum from allergic patients was inhibited by SP-D. In a fixed-cell enzyme linked immunosorbent assay and in flow cytometry, SP-D bound to eosinophils. This binding was saturable and was inhibited by the addition of maltose and ethylenediaminetetraacetic acid, suggesting the involvement of the carbohydrate recognition domain of SP-D. In addition, flow cytometry showed significant interaction of SP-D with CD32 (FcgammaII receptor) on eosinophils, which might explain the inhibitory effect of SP-D on the IgG and serum-triggered eosinophil cationic protein degranulation of eosinophils. Our data further support the concept of an anti-inflammatory function of SP-D in the lung of patients with allergic diseases.

Background: IL-25, a novel member of the IL-17 cytokine family, promotes CD4+ T-helper 2 lymphocyte-like (Th type-2) inflammatory responses in the lung. Although IL-25 up-regulates IL-13 in the lung, the contribution of this and other type 2 cytokine signalling pathways to the induction and persistence of airways hyper-reactivity (AHR) and allergic inflammation are unclear., Objective: To determine the downstream factors employed by IL-25 to induce Th type-2 pulmonary inflammation and AHR., Methods: IL-25 was delivered to the airways of BALB/c mice by intra-tracheal (i.t.) instillation and AHR and Th type-2 inflammatory responses were characterized in wild type (WT) and Th type-2-cytokine and -signalling pathway-deficient (-/-) mice., Results: IL-25 treatment resulted in AHR, eosinophilic inflammation, mucus hypersecretion and a progressive increase in the production of Th type-2 cytokines in the lungs. Levels of arginase-I (arg-I) and eotaxin were also elevated by IL-25 treatment. A significant reduction in AHR, and attenuation of mucus production was observed in IL-25-treated IL-13-/-, IL-4 receptor alpha (IL-4Ralpha-/-)- and signal-transducer-and-activator-of-transcription-factor-6 (STAT6-/-)-deficient mice. AHR was also inhibited in IL-4(-/-)- and IL-5/eotaxin(1)(-/-)- deficient mice treated with IL-25, however, mucus hypersecretion was not completely ablated. IL-25 promoted Th type-2 responses by directly acting on naïve T cells., Conclusion: IL-25 potently (single dose) induces sustained AHR and acute pulmonary inflammation with eosinophilia. IL-25-induced AHR is dependent on the production of Th type-2 cytokines, and removal of IL-13 and its signal transduction pathway prevents IL-25-induced airways inflammation and AHR. IL-25 potently induces inflammatory cascades that may exacerbate allergic airways inflammation by promoting Th type-2 cytokine responses in conjunction with the up-regulation of factors (eotaxin and arg-I) that can amplify inflammation associated with allergic disorders. Dysregulation in IL-25 production may predispose to features of allergic airways disease.

Background: The role of bacterial enterotoxins like Staphylococcus aureus enterotoxin B (SEB) in allergic asthma remains unknown. We used a mouse model of airway allergy to study the effects of nasal or bronchial contact with SEB on bronchial allergic inflammation., Methods: The features of allergic asthma were induced in ovalbumin (OVA)-sensitized mice (days 1-13) by repeated exposures to nebulized OVA (days 33-37). Nasal or bronchial application of SEB was performed on three occasions (days 33-35-37), and the effects on bronchial inflammation, IgE titres and expression levels of mRNA for T helper type 2 cytokines and other inflammatory mediators were evaluated., Results: Both nasal and bronchial SEB enhanced the allergen-induced bronchial inflammation, as reflected by more eosinophilic inflammation in the airway lumen and in bronchial tissue. Aggravation of experimental asthma correlated with higher expression of mRNA for IL-5, IL-4, IFN-gamma, IL-12 p40, eotaxin-1 and TGF-beta in bronchi. In addition, nasal SEB elevated concentrations of IL-4, IL-5 and IFN-gamma in serum and bronchial SEB increased titres of OVA-specific and total IgE in serum., Conclusion: Our data illustrate the potential of both nasal as well as bronchial SEB to aggravate several features of allergic asthma in a mouse model.

Background: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease., Objective: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model., Methods: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release., Results: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS., Conclusion: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

Background: Asthma is the most common medical condition during pregnancy. While increased production of T helper cytokines has been reported to occur in both asthma and pregnancy, the effect of T-helper type 2 (Th2) polarization on asthma symptoms during pregnancy has not been well-characterized., Objective: We hypothesized that systemic Th2 cytokine and chemokine polarization occurs among asthmatics to a greater extent during their pregnancy, and is associated with more severe asthma and increased Th2 polarization in the newborn., Methods: Fifty-six pregnant asthmatics were recruited from prenatal clinics affiliated with New York Presbyterian Hospital. Systemic production of interleukin-4, interferon-gamma, eotaxin and IP10 were measured by intracytoplasmic staining or ELISA at recruitment, peripartum and post-partum, and in the cord blood. The frequency of asthma symptoms was measured by questionnaires and compared with Th biomarkers., Results: The chemokine ratio (IP10/eotaxin) declined over the course of pregnancy (from 3.3 +/- 1.3 to 1.4 +/- 0.2, P = 0.016), but IP10 and eotaxin increased post-partum. The decrease in the chemokine ratio was associated with more frequent asthma symptoms. A non-significant trend towards decreased interferon-gamma and increased interleukin-4 production was detected. Cord blood eotaxin levels correlated with maternal levels (r = 0.35, P = 0.03). Other peripartum biomarkers were not associated with Th2 polarization nor with subsequent respiratory symptoms in the newborn., Conclusion: IP10/eotaxin declined over the course of pregnancy and was associated with worse asthma symptoms. Alterations of Th1/Th2 chemokine balance during pregnancy may identify women prone to more severe asthma during pregnancy.

Background: Several cytokines are involved in the recruitment and activation of inflammatory cells in ocular allergic diseases. The purpose of the study was to assay multiple cytokines and chemokines in tears, to compare subgroups of allergic conjunctivitis (AC) with controls, and in culture supernatants to determine whether conjunctival fibroblasts produce some of these cytokines., Methods: Fifty to one hundred microlitre tears were obtained from patients with active seasonal allergic conjunctivitis (SAC; n=12), vernal keratoconjunctivitis (VKC; n=18), atopic keratoconjunctivitis (AKC; n=6) and non-atopic controls (n=14). Primary conjunctival fibroblasts grown in vitro were stimulated with IL-4, IL-13 or TNF-alpha for 24 h. Cell-free tear and culture supernatants were assayed for IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IFN-gamma, TNF-alpha, eotaxin, MCP-1 and RANTES using multiplex bead analysis. Induction of chemokine gene expression was determined by PCR., Results: IL-1beta, IL-2, IL-5, IL-6, IL-12, IL-13, MCP-1 were increased in all tears groups compared with controls, with highly significant correlations between many of these molecules. In addition IL-4, IFN-gamma, and IL-10 were elevated in SAC and VKC, while eotaxin and TNF-alpha were only increased in VKC. IL-6, IL-8, MCP-1, RANTES and eotaxin were detected from fibroblasts cultures, and were all up-regulated by TNF-alpha. By PCR, fibroblasts expressed MCP-1 transcripts constitutively, whereas IP-10 and Mig were up-regulated by TNF-alpha., Conclusions: Differential cytokine levels support tears as a useful indicator of immune mechanisms occurring during AC. The striking similarities in chemokine profiles between tears and fibroblasts suggest these cells as likely sources of chemokines in tears.

Background: beta-Tryptase is a multifunctional mast cell serine protease released during mast cell degranulation and tryptase/trypsin inhibitors are a novel potential therapeutic approach for allergic inflammatory diseases., Objectives: This study was performed to assess the effects of RWJ-58643 on nasal symptoms, eosinophil influx, and cytokine and chemokine release following nasal allergen challenge (NAC)., Methods: Male patients with grass pollen allergic rhinitis (n=16) out of season received single doses of RWJ-58643 (100, 300, 600 microg) or matched placebo given 30 min before NAC in a double-blind, randomized crossover design. A single dose of 200 microg budesonide was studied in an open-label extension phase. NAC was performed with Timothy grass pollen (ALK) via a nasal device, and nasal lavage was performed at times 0 (pre-drug, pre-allergen), 0.5 (30 min post-drug, pre-NAC) 1.5, 2.5, 4.5, 6.5, 8.5, and 24 h after drug administration. Nasal lavage mediators were analysed using a sensitive multiplexed bead immunoassay system., Results: Low-dose RWJ-58643 (100 microg) and budesonide (200 microg) significantly reduced symptoms, eosinophils and levels of IL-5 following NAC. However, higher doses of RWJ-58643 (300 and 600 microg) caused a late eosinophilia and preceding increases in IL-5 compared with placebo., Conclusions: This study suggests that combined beta-tryptase and trypsin inhibition has therapeutic potential in allergic inflammation, however, this property is dose responsive and higher doses are ineffective and may cause eosinophilia.

Background: Asthma is associated with esoinophilic airway inflammation and overproduction of T-helper type 2 (Th2) lymphocyte-related cytokines., Objective: This study assessed the eosinophil chemoattractant eotaxin and Th2-specific macrophage-derived chemokine (MDC) in the adult asthmatic airway. Eotaxin and MDC levels were determined in exhaled breath condensate (EBC) obtained from adult patients with asthma., Methods: Fifty-four asthmatics (20 male, mean (SD) age 40 (12) years and percentage predicted forced expiratory volume in 1 s (FEV(1)) 81.7 (20.8)) and 20 age- and sex-matched controls were studied. EBC was collected using EcoScreen by 10 min of tidal breathing with a nose clip. Concentrations of eotaxin and MDC were measured by ELISA., Results: Asthma patients on inhaled corticosteroid (ICS) had a higher median interquartile range (IQR) level of eotaxin than the steroid-naïve asthmatics (18.5 (17.7-20.1) vs. 17.9 (17.0-18.6) pg/mL, P=0.02) and controls (18.5 (17.7-20.1) pg/mL vs 17.4 (16.3-18.0) pg/mL, P=0.001). Eotaxin level in EBC had a significant negative correlation with the FEV(1)/forced vital capacity ratio (r=-0.43, P=0.03) in steroid-naïve asthmatics. EBC MDC level was higher in subjects on ICS than the steroid naïve asthmatics (120 (118-125) vs. 117 (116-119) pg/mL, P=0.01) and the controls (120 (118-125) vs. 117 (116-120) pg/mL, P=0.02)., Conclusions: Eotaxin and MDC could be measured in EBC of adults with asthma. EBC eotaxin and MDC levels were higher in asthmatics on ICS than the steroid-naïve asthmatics or controls. Exhaled chemokines may be potential non-invasive markers for assessing airway inflammation in asthmatics.

Background: Steroid treatment of allergic eosinophilic airway diseases is considered to attenuate cell recruitment by inhibiting several chemokines and to cause eosinophil clearance through inducement of apoptosis of these cells. However, roles of these mechanisms in the actions of steroids in vivo have not been fully established. Also, as regards clearance of tissue eosinophils other mechanisms than apoptosis may operate in vivo., Objective: This study explores anti-inflammatory effects of steroids instituted during either development or resolution of airway allergic inflammation., Methods: Immunized mice were subjected to week-long daily allergen challenges (ovalbumin). Steroid treatment was instituted either amidst the challenges or exclusively post-allergen challenge. CC chemokines, goblet cell hyperplasia, occurrence of eosinophil apoptosis, and airway tissue as well as lumen eosinophilia were examined at different time-points., Results: Daily steroids instituted amid the allergen challenges non-selectively attenuated a range of chemokines, permitted egression of tissue eosinophils into airway lumen to increase, and reduced development of lung tissue eosinophilia. Steroid treatment instituted post-challenge selectively inhibited the CC-chemokine regulation upon activation, normal T cell expressed and secrted (RANTES), permitted continued egression of eosinophils into airway lumen, and resolved the tissue eosinophilia. Eosinophil apoptosis rarely occurred at development and resolution of the allergic eosinophilic inflammation whether the animals were steroid treated or not. However, anti-Fas monoclonal antibodies given to mice with established eosinophilia post-challenge produced apoptosis of the tissue eosinophils indicating that apoptotic eosinophils, if they occur, are well detectible in vivo., Conclusion: Airway tissue eosinophils are likely eliminated through egression into airway lumen with little involvement of apoptosis and phagocytosis. Our data further suggest that therapeutic steroids may resolve airway inflammation by permitting clearance of tissue eosinophils through egression and inhibiting RANTES-dependent cell recruitment to lung tissues.

Background: Second-generation antihistamines are H(1) receptor antagonists and may have additional anti-inflammatory effects., Objective: The aims of the study were to evaluate the effect of desloratadine (DL) on cytokine secretion by epithelial cells from both nasal mucosa (NM) and polyps (NP), and on eosinophil survival primed by epithelial cell secretions., Methods: Epithelial cells were cultured and stimulated with fetal bovine serum (FBS), IL-1beta or TNF-alpha with and without DL for 24 h. Culture supernatant cytokines concentration were measured by ELISA. Peripheral blood eosinophils were incubated with human epithelial cell conditioned media (HECM) and DL. Eosinophil survival was assessed by Trypan blue dye exclusion. Results are expressed as mean+/-SEM of cytokine concentration (pg/mL) or eosinophil survival index (%)., Results: FBS increased granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), IL-6, IL-8, and TGF-beta(1) secretion in epithelial cell cultures from both NM and NP. Only GM-CSF secretion was significantly (P<0.05) inhibited by a dose-response of DL compared with positive controls, in both NM (10(-5) m: 125+/-36 pg/mL, 10(-6) m: 95+/-22 pg/mL vs. control: 256+/-91 pg/mL, n=6) and NP (10(-5) m: 80+/-29 pg/mL, 10(-6) m: 109+/-45 pg/mL vs. control: 333+/-212 pg/mL, n=6). DL also showed an inhibitory effect on HECM-induced eosinophil survival from both NM and NP. At 72 h, DL significantly (P<0.01) inhibited eosinophil survival induced by HECM from NM (10(-5) m: 19.9+/-5.5%, n=9; 10(-6) m: 28.7+/-7.7%, n=9) and NP (10(-5) m: 6.2+/-2.8%, n=11) compared with HECM alone (NM: 42.1+/-7.3%; NP: 45.3+/-8.1%)., Conclusion: The inhibitory effects of DL on epithelial cell GM-CSF secretion and on eosinophil survival induced by epithelial cell secretions, suggest that this H(1) antagonist may regulate eosinophil inflammation in upper airways.

Background: Patients with intractable otitis media associated with bronchial asthma have an extensive accumulation of eosinophils in the effusion and mucosa of the middle ear; this condition is called eosinophilic otitis media (EOM). It remained to be determined how eosinophils accumulate in the middle ear., Objectives: To clarify the pathogenesis of middle ear diseases, we measured the concentration of eosinophil chemoattractants in middle ear effusion (MEE), and carried out immunohistochemical studies of middle ear mucosa specimens to demonstrate the expression of eosinophil chemoattractants., Methods: Middle ear effusion samples were obtained from 15 EOM patients with bronchial asthma and from six controls for the measurement of eosinophil cationic protein (ECP), IL-5, eotaxin and regulated on activation, normal T expressed and secreted concentrations. Middle ear mucosa samples were also taken from 14 EOM patients and 16 controls for immunohistochemical study. In 10 EOM patients, the numbers of immunoreactive cells as well as apoptotic cells were determined before and after the topical application of triamcinolone acetonide into the middle ear., Results: In EOM, significantly higher ECP and IL-5 concentrations were detected in MEE than in serum, and ECP, IL-5 and eotaxin concentrations in MEE were higher in the EOM patients than in the controls. ECP concentration positively correlated with that of IL-5. Immunohistochemically, the numbers of cells positive for EG2 and ecalectin were significantly higher in the EOM patients than in the controls. After the topical application of triamcinolone acetonide, the numbers of infiltrating cells and immunoreactive cells distinctly decreased, whereas the number of apoptotic cells significantly increased., Conclusion: In EOM, locally produced IL-5 may play a crucial role in the accumulation of eosinophils in the middle ear. Chemokines such as ecalectin and eotaxin are also produced in the middle ear, and help activate and enhance the survival of eosinophils to induce the intractable condition in the middle ear. The topical application of triamcinolone acetonide induces the apoptosis of not only eosinophils but also eosinophil chemoattractant-producing cells, thereby improving the middle ear condition.

Background: Monocytes/macrophages have a cysteinyl leukotriene 1 (CysLT1) receptor, but its function is poorly understood. Objective To elucidate the biological function of the CysLT1 receptor of human monocytes/macrophages., Methods: We examined the production of TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and eotaxin induced by CysLTs (leukotriene (LT)C4, -D4, and -E4) in THP-1 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages. Moreover, we examined the effect of CysLTs on the expression of beta-chemokine receptor 2B (CCR2B) as the receptor of MCP-1 by Western blot analysis., Results: ELISA revealed that CysLTs induced MCP-1 in THP-1 cells and peripheral blood CD14+ monocytes/macrophages, but not other cytokines. PCR demonstrated that CysLTs increased MCP-1 mRNA expression in THP-1 cells, and Western blotting showed that CysLTs increased the expression of CCR2B in THP-1 cells. Moreover, we demonstrated that pranlukast, a CysLT1 receptor antagonist, blocked MCP-1 production by CysLTs in THP-1 cells almost completely, and partially inhibited MCP-1 release by CysLTs in peripheral blood CD14+ monocytes/macrophages and CCR2B expression by CysLTs in THP-1 cells., Conclusion: CysLTs induce MCP-1 and increase CCR2B expression in human monocytes/macrophages.

Background: Eotaxin-1 (CCL11) is a CC chemokine whose nasal eosinophilic chemotactic activity in vivo and in vitro has been demonstrated primarily using nasal allergen challenge models. The extension of these challenge findings to the in vivo setting has been limited., Objective: To obtain nasal lavage fluid from volunteers with perennial and seasonal (in- and out-of-season) allergic rhinitis (AR) and non-atopic non-rhinitic controls for the measurement of eotaxin-1 concentrations and to relate these findings to the symptomatic disease severity, the percentage of lavage eosinophils, and to alpha(2)-macroglobulin (alpha(2)-MG) lavage concentrations, as a marker of vascular permeability and an index of airway inflammation., Methods: Thirty-seven volunteers with AR (16 seasonal and 21 perennial) and 20 non-atopic non-rhinitic volunteers were recruited and phenotyped. Nasal lavage fluid was obtained by standardized protocol. The nasal lavage fluid concentrations of eotaxin and alpha(2)-MG were measured by ELISA, and differential cell counts performed on cytospins., Results: Eotaxin-1 nasal lavage fluid concentrations were significantly higher in both the perennial and seasonal (in-season) AR groups compared with the controls, and significantly related to the severity of symptom expression and to the percentage of lavage eosinophils. The lavage eosinophil counts were significantly higher in both the symptomatic rhinitis groups compared with the control groups and correlated with the lavage concentrations of alpha(2)-MG. alpha(2)-MG levels were significantly increased in seasonal (in-season) rhinitics compared with both non-atopic controls and seasonal (out-of-season) rhinitics. A significant correlation was observed between the levels of alpha(2)-MG and levels of eotaxin in the symptomatic allergic rhinitic groups., Conclusions: This study clearly demonstrates the relevance of eotaxin-1 to the pathogenesis of naturally occurring AR.

Background: We have recently isolated two distinct components from Ascaris suum adult worms with different effects on the immune system: the allergenic protein of A. suum (APAS-3), which induces IgE antibody production, and suppressive protein of A. suum (PAS-1), which inhibits humoral and cellular immune responses induced by unrelated antigens. In this study, we investigated the immunomodulatory effect of PAS-1 on a murine model of asthma induced by APAS-3., Methods: BALB/c mice were immunized twice with APAS-3 or APAS-3 plus PAS-1 by the intraperitoneal and subcutaneous route (on days 0 and 7) and challenged twice with the same antigens intranasally (days 14 and 21). Two days after the last challenge, the allergic airway inflammation was evaluated by cellular migration, eosinophil peroxidase (EPO) activity, cytokine and chemokine production and pulmonary mechanical parameters., Results: The allergenic properties of APAS-3 were confirmed by the stimulation of anaphylactic IgE and IgG1 antibody production and eosinophilic airway inflammation and hyper-responsiveness. On the other hand, PAS-1-treated mice showed a marked suppression of cellular migration and EPO activity that correlated well with a significant reduction in the levels of IL-4, IL-5, eotaxin and RANTES in the bronchoalveolar lavage (BAL) fluid. In contrast, considerable amounts of IL-10 were observed in the BAL fluid of PAS-1-treated mice. Airway hyper-responsiveness was obtained in APAS-3-immunized mice, but the conductance of the respiratory system was restored to normal values in the presence of PAS-1., Conclusion: These results indicate that A. suum allergenic protein APAS-3 induces a T helper 2-type immune response and, consequently, eosinophilic airway inflammation and hyper-responsiveness. Moreover, the modulatory protein PAS-1 has a marked suppressive effect on this response, and the inhibition of cytokine (IL-4, IL-5) and chemokine (eotaxin and RANTES) release, probably because of the presence of IL-10, may contribute to this effect.

Background: Surfactant protein D (SP-D) is involved in the innate immunity within the lung and may have important roles in modulating the inflammatory process of asthma., Objective: To examine the potential immunomodulating role of SP-D on the allergic response in mice, and its interaction with the alveolar macrophages (AMs) during allergic inflammation., Methods: A recombinant 60 kDa fragment of human SP-D (rfh SP-D), Survanta, and budesonide were administrated, respectively, to Der p-sensitive BALB/c mice before or after allergen challenge (AC). Total and differential cell counts, levels of cytokines in bronchoalveolar lavage fluids(BALFs), and levels of Der p-specific IgE and IgG1 antibodies in sera, were assayed. The production of nitric oxide (NO), and inducible NO synthase (iNOS) expression, in AMs, were determined by ELISA and RT-PCR, respectively., Results: Instillation of rfh SP-D to sensitized mice 6 h after AC (therapeutic), but not 24 h before AC (preventive), markedly reduced infiltration of eosinophils, and also reduced levels of IL-4, IL-5, eotaxin, and TNF-alpha but elevated levels of IFN-gamma in the BALF. These effects were comparable with those obtained with budesonide treatment, whereas Survanta did not have a suppressive effect, either before or after AC. There was significant inhibition of NO production in the rfh SP-D pre-treated AMs of allergen-sensitized mice, but not in naive mice., Conclusions: These results indicate that rfh SP-D has a therapeutic effect on allergen-induced bronchial inflammation, and that this might be because of its inhibitory effect on NO and TNF-alpha production by AMs, and it thus prevents the development of T-helper type 2 cytokine response.

Background: Airway inflammation and remodelling are important pathophysiologic features of chronic asthma. Although current steroid use demonstrates anti-inflammatory activity, there are limited effects on the structural changes in the lung tissue., Objective: We have used a mouse model of prolonged allergen challenge that exhibits many of the salient features of airway remodelling in order to investigate the anti-remodelling effects of Budesonide., Methods: Treatment was administered therapeutically, with dosing starting after the onset of established eosinophilic airway inflammation and hyper-reactivity., Results: Budesonide administration reduced airway hyper-reactivity and leukocyte infiltration in association with a decrease in production of the Th2 mediators, IL-4, IL-13 and eotaxin-1. A reduction in peribronchiolar collagen deposition and mucus production was observed. Moreover, our data show for the first time that, Budesonide treatment regulated active transforming growth factor (TGF)-beta signalling with a reduction in the expression of pSmad 2 and the concomitant up-regulation of Smad 7 in lung tissue sections., Conclusions: Therefore, we have determined that administration of Budesonide modulates the progression of airway remodelling following prolonged allergen challenge via regulation of inflammation and active TGF-beta signalling.

Eosinophil recruitment to the airways, including involvement of haemopoietic eosinophil-basophil progenitors (Eo/B-CFU), is primarily regulated by interleukin-5 (IL-5) and eotaxin. In this study, we investigated the haemopoietic mechanisms in upper and lower airway eosinophilic inflammation. Ovalbumin (OVA) sensitized and challenged BALB/c mice were used to establish isolated upper (UAC), isolated lower (LAC), or combined upper and lower airway (ULAC) inflammation. Airway, blood and bone marrow responses were evaluated in each model. Numbers of airway eosinophils and CD4(+) cells were increased significantly in the nasal mucosa in UAC and ULAC mice, and in the lung tissue in LAC and ULAC groups. Levels of IL-5 and eotaxin were increased significantly in the nasal lavage fluid (NL) in UAC and ULAC mice, and in the bronchoalveolar lavage fluid (BAL) in LAC and ULAC groups. The proportion of IL-5-responsive bone marrow Eo/B-CFU was significantly higher than the control in all treatment groups, but peaked much earlier in the ULAC group. Kinetic studies revealed that IL-5 and eotaxin in NL, BAL and serum peaked between 2 and 12 hr after OVA challenge in ULAC mice, and at 24 hr in UAC mice, related to the timing of maximal progenitor responses. These data support the concept that the systemic mechanisms linking rhinitis to asthma depend on the location and extent of airway allergen exposure.

Background: Activation of signal transducer and activator of transcription (STAT)6 by IL-4 and IL-13 is essential in many key epithelial responses in the asthmatic airway including expression of numerous chemokines, goblet cell differentiation and mucus production and expression of other allergic inflammatory genes. While these responses are all inhibited by glucocorticoids (GC) administered systemically or by inhalation, the inhibitory mechanisms are unknown., Objective: To test the hypothesis that GC suppress allergic responses by blocking IL-4-induced STAT6 signalling in airway epithelial cells., Methods: Western blotting and reporter gene assays were used to determine whether GC could inhibit STAT6 production, phosphorylation or nuclear translocation, or whether GC could affect STAT6 transcriptional activity in the BEAS-2B airway epithelial cell line., Results: Our results showed that GC had no inhibitory effect on the total cellular or nuclear levels of STAT6 or phospho-STAT6. GC did not inhibit transcription from three different STAT6-driven reporter constructs, indicating that GC also did not inhibit STAT6 function., Conclusion: We conclude that airway epithelial STAT6 is not the central target of GC in allergic inflammation and that the inhibitory effect of GC on STAT6-mediated IL-4- and IL-13-induced responses is exerted by targeting pathways distinct from STAT6.

Background: Eosinophils are major effector cells in allergic diseases. After their recruitment to sites of inflammation, they contribute to the pathophysiology of the disease by releasing granule proteins and cytokines. Suplatast tosilate (IPD-1151T), a new anti-allergic agent, has shown beneficial effect in the treatment of asthma, associated with reduced bronchoalveolar lavage eosinophil infiltration and eosinophilic cationic protein (ECP) release in serum and sputum., Objective: We investigated whether suplatast tosilate could exert direct effects on human eosinophil activation., Methods: Eosinophils from hypereosinophilic patients or normal donors were purified by Percoll gradient and the magnetic cell separation system. Chemotaxis was studied using the Boyden chamber technique using three chemoattractants, formyl-methionine-leucine-phenylalanine (fMLP), IL-5 and eotaxin. Oxidative metabolism was determined by a luminol-dependent chemiluminescence assay after activation with eotaxin or secretory IgA (sIgA). The release of ECP and eosinophil derived neurotoxin (EDN) was measured by radioimmunoassay and cytokine production was determined by ELISA following activation with sIgA or anti-CD28., Results: The chemotactic response to fMLP, IL-5 and eotaxin was significantly inhibited by IPD-1151T. Suplatast tosilate was partially inhibiting the release of reactive oxygen species (ROS) induced by eotaxin and sIgA. Activation by sIgA and CD28 ligation resulted in the release of ECP and EDN, which was inhibited by IPD-1151T. Upon activation by anti-CD28, only IL-13 production was inhibited by IPD-1151T, whereas release of IL-2 and IFN-gamma was not affected. IL-10 release induced by sIgA was also inhibited by IPD-1151T. Additionally, the pro-inflammatory cytokine IL-6, which was secreted following anti-CD28 and sIgA stimulation, was strongly inhibited by IPD-1151T., Conclusion: Through inhibition of chemotaxis, IPD-1151T might limit the number of eosinophils at the inflammation site. Furthermore, it could reduce the pathological potential of eosinophils by inhibiting the release of ROS and cationic proteins, main inflammatory mediators produced by eosinophils. Moreover, the inhibition of immunoregulatory cytokines released by eosinophils could locally modify the immune response.

Background: The mechanism whereby allergen induces eotaxin expression at the site of allergic inflammation is incompletely understood. Structural cells, including endothelial cells, are a major source of eotaxin., Objective: We have investigated, in vivo and in vitro, the relationship between mast cell activation and the expression of eotaxin (eotaxin 1) by endothelial cells., Methods: The effects of intradermal allergen challenge and histamine injection on eotaxin mRNA and protein generation were studied in atopic subjects using immunofluorescence, immunohistochemistry and in situ hybridization. Histamine-induced expression of eotaxin mRNA and protein by endothelial cells was also measured, as was histamine-induced eosinophil adhesion to cultured endothelial cells., Results: A rapid increase in degranulating cutaneous mast cells, together with a concomitant increase in eosinophils, was observed 60 min after allergen challenge. This was accompanied by the appearance of immunoreactive eotaxin that peaked at 1 h around blood vessels and at 3 h within the tissue. Intradermal histamine injection produced an increase in the number of eotaxin+ cells in the tissues, which was maximal at the 3-h time-point. In vitro, endothelial cells produced eotaxin mRNA and protein product in a dose- and time-dependent fashion following incubation with histamine, an effect that was blocked by levocetirizine. Pre-incubation of endothelial cells with histamine also induced a significant increase in eosinophil adherence, an effect that was inhibited with an anti-eotaxin blocking monoclonal antibody., Conclusion: The antigen-induced expression of eotaxin by endothelial cells and the adherence and subsequent migration of eosinophils from the microvasculature to the tissues are rapid events partially under the control of histamine released from degranulating mast cells.

Background: Type 2 helper T lymphocyte-specific chemokines including macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and eotaxin are important mediators for allergic airway inflammation., Objective: We investigated whether these chemokines can be detected in exhaled breath condensate (EBC) and their relation to childhood asthma., Methods: Asthmatics recruited from paediatric clinics of a university teaching hospital were classified into intermittent asthma (IA) and persistent asthma (PA) according to Global Initiative for Asthma guidelines. EBC was collected by a disposable collection kit, whereas fractional exhaled nitric oxide (FENO) was measured by a chemiluminescence analyser. Concentrations of MDC, TARC and eotaxin in both EBC and plasma were measured using sandwich enzyme immunoassay. The intra-subject reproducibility of exhaled chemokine measurements was determined by co-efficients of variation (CV)., Results: Forty-eight patients with PA, 36 children with IA and 18 controls were recruited. MDC and eotaxin were present in EBC from nearly all subjects, whereas TARC could be measured in EBC from 33 (32%) subjects only. The median MDC concentration in EBC was higher in PA (117 pg/mL) as compared with IA (106 pg/mL) and controls (105 pg/mL; P=0.003 for both). The median plasma MDC concentration in PA (648 pg/mL) was also higher than that in IA (520 pg/mL; P=0.002) and controls (490 pg/mL; P=0.008). The median plasma TARC concentration was also increased in PA as compared with IA (72 pg/mL vs. 35 pg/mL; P=0.004). MDC concentrations in EBC were lower in patients with PA who received high-dose inhaled corticosteroid (P=0.005). FENO was significantly higher in asthmatics than controls (P<0.0001), but it was not associated with chemokines in EBC or plasma. The mean (range) CV for measuring MDC, TARC and eotaxin in EBC (n=6) were 5.5 (2.0-7.2%), 8.8 (3.6-14.4%) and 5.2 (2.8-7.9%), respectively., Conclusions: Our results suggest that MDC in EBC and MDC and TARC in plasma are increased in children with PA as compared with IA or control. MDC concentrations in EBC are suppressed in patients on high-dose inhaled corticosteroid treatment.

Background: There is growing evidence that eotaxin is a key mediator in the development of tissue eosinophilia. Fibroblasts are a major source of eotaxin. The severity of diseases with eosinophilic inflammation like nasal polyposis, atopic dermatitis and asthma, where Th2-type cytokines (IL-4 and IL-13) and TGF-beta are expressed locally, was shown to correlate with bacterial factors such as lipopolysaccharide (LPS) rather than allergen., Objective: We examined eotaxin production by nasal fibroblasts stimulated with IL-4 or IL-13 alone or in combination with LPS, and the effect of TGF-beta(1) on it. Moreover, we compared the magnitude of eotaxin produced by nasal fibroblasts with that produced by lung or skin fibroblasts., Methods: Fibroblast lines were established from human biopsy tissue. The expression of eotaxin mRNA was evaluated by RT-PCR. The amount of eotaxin in the supernatants was measured by ELISA., Results: IL-4, but not IL-13, synergized with LPS to produce eotaxin in a dose- and time-dependent manner. Sequential treatment of nasal fibroblasts with IL-4 and LPS did not have any effect. But when IL-4 and LPS were added together, synergy for eotaxin production was observed. Moreover, this synergy was observed in nasal and skin fibroblasts, but not in lung fibroblasts. The production of eotaxin by IL-4 and LPS was modulated by TGF-beta(1)., Conclusion: These results suggest that a co-stimulus like LPS is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammations such as nasal polyposis. Modulation by TGF-beta(1) may have important implications for the development of eosinophilic inflammation.

Background: H1-receptor antagonists are often effective in the treatment of allergic disorders such as atopic dermatitis. Cetirizine, a putative H1-receptor antagonist, has recently been shown to have anti-inflammatory properties through the inhibition of leucocyte recruitment and activation, and by the reduction of ICAM-1 expression on keratinocytes., Objective: To further elucidate the anti-inflammatory properties of cetirizine, we first examined its effects on antigen-induced eosinophilia and neutrophila in vivo. We then examined the anti-inflammatory effects of cetirizine on a human keratinocyte A431cell line., Methods: Mice were sensitized subcutaneously with ragweed pollen and were challenged intraperitoneally with the allergen. Cetirizine diluted in sterile water (0-20 mg/kg) or only sterile water was administered orally. Peritoneal cells were obtained at 8 and 24 h after challenge. The eosinophilia and neutrophilia induced by ragweed pollen extract were quantitated. Macrophage migration inhibitory factor (MIF), macrophage inflammatory protein 2 (MIP-2) and eotaxin contents of peritoneal fluid were also measured by mouse ELISA. The effects of cetirizine on MIF-induced IL-8 production in A431 cells were examined by ELISA. The effects of cetirizine on MIF expression and production in A431 cells were examined by human MIF ELISA and Northern blot analysis., Results: Eosinophilia and neutrophilia induced by ragweed pollen extract were found to be significantly reduced in cetirizine-treated mice (20 mg/kg). MIF, a pleuripotent cytokine, was significantly decreased at 8 and 24 h in the peritoneal fluid by cetirizine treatment. MIP-2 and eotaxin were also decreased 8 and 24 h, respectively, after challenge in the peritoneal fluid with cetirizine treatment. MIF stimulates IL-8 production in A431 cells. We found that MIF production in A431 cells was inhibited by 10 microm cetirizine. Consistent with this, cetirizine significantly inhibited MIF-induced IL-8 production., Conclusion: These results suggest that cetirizine exerts its anti-inflammatory effects by inhibiting MIF as well as IL-8 production, such as those involved in inflammatory allergic skin disease, suggesting a broad spectrum of action beyond its mere H1-receptor-antagonistic function.

There is increasing evidence that primitive progenitors migrate from the bone marrow (BM) via the peripheral circulation to tissue sites where they undergo in situ differentiation to provide a continued source of effector cells, such as eosinophils, during an allergic inflammatory response. To study mechanisms of progenitor cell mobilization in allergic reactions, we investigated fluctuations in the expression of the eotaxin receptor, CC chemokine receptor 3 (CCR3), on CD34+ cells from stable asthmatics following allergen (i.e. antigen) challenge. BM aspirates were taken from seven early responder (ER) and 10 dual responder (DR) asthmatics who, following antigen challenge developed only an early bronchoconstrictor response and an early and late- bronchoconstrictor response, respectively. Expression of CCR3 was detected on primitive (CD34+ cells) and eosinophil-lineage committed progenitors (CD34+ interleukin-5 receptor alpha-subunit+ cells) by flow cytometry and confirmed by co-localization of CCR3 messenger RNA to CD34 immunopositive cells using in situ hybridization. When preantigen levels were compared to 24-hr postantigen levels, significant increases in BM CD34+ CCR3+ cells were detected in DR, who also developed a significant sputum and blood eosinophilia and increased methacholine airway responsiveness. In contrast, a significant attenuation of BM CD34+ CCR3+ cells was observed in ER. In a dose-dependent manner eotaxin, but not interleukin (IL)-5, stimulated CD34+ progenitor cell migration in vitro. This migrational response to eotaxin was abrogated by anti-CCR3 monoclonal antibody and primed by preincubation with IL-5. We propose that fluctuations in CCR3 expression on human BM CD34+ cells may facilitate chemokine-mediated progenitor cell mobilization to the peripheral circulation and the resultant development of pulmonary eosinophilia, a cardinal feature of asthma.

Interleukin-5 (IL-5) and eotaxin are the most important cytokines/chemokines responsible for regulating eosinophil locomotion and are known to play a co-operative role in the selective recruitment of eosinophils to inflamed tissues. Following exposure to chemoattractants, eosinophils undergo a series of events, including reorganization of actin filaments and subsequent rapid shape changes, culminating in chemotaxis. In this study we examined the signalling pathways for eosinophil shape change regulated by eotaxin and IL-5, primarily using a gated autofluorescence/forward-scatter assay. Eotaxin and IL-5 were able to elicit shape change with peaks at 10 and 60 min, respectively, and IL-5 triggered the shape change more efficiently than eotaxin. The pharmacological inhibitors of mitogen-activated protein kinase (MAP kinase) and p38 blocked both eotaxin- and IL-5-induced eosinophil shape change in a dose-dependent manner. In addition, depletion of intracellular Ca2+ and inhibition of protein kinase A (PKA) strongly reduced eosinophil shape change. In contrast, even when used at high concentrations, protein tyrosine kinase (PTK) inhibitors caused only a slight reduction in the ability to change shape. However, treatment with protein kinase C (PKC) inhibitors, such as GF109203X and staurosporine, resulted in a striking inhibition of eosinophil shape change by IL-5, but not eotaxin. Data from the inhibition of activation and chemotaxis of the extracellular signal-regulated kinases (ERK1/2) by the PKC inhibitors were also consistent with findings from the experiments on shape change. Collectively, two eosinophil-selective cytokines/chemokines probably regulate eosinophil shape change via a largely overlapping signalling pathway, with involvement of PKC restricted to the IL-5 signal alone.

Background: Kimura's disease (KD) is a chronic inflammatory disorder characterized by tumours in the head and neck region, enlarged lymph nodes, increased eosinophil counts andhigh serum IgE. Mast cells are known to play a central role in IgE-mediated allergic diseases through the release of inflammatory mediators like IL-4, IL-5 and chemokines. We hypothesized that mast cells may play a role in the pathogenesis of KD by regulating eosinophilic infiltration and IgE synthesis., Objective: In order to investigate the role of mast cells in the pathogenesis of KD, we examined the expression of cytokines/chemokines in the lesions of KD., Methods: We examined the number of tryptase+ cells, EG2+ cells, CD3+ cells, IL-4+ cells, IL-5+ cells, eotaxin+ cells, RANTES+ cells and CCR3+ cells in five specimens of KD versus normal tissues by immunohistochemistry. The sources of IL-4, IL-5, eotaxin and RANTES and the expression of CCR3 were examined by immunostaining of serial sections with antibodies to IL-4, IL-5, eotaxin, RANTES and CCR3, and antibodies to tryptase, ECP (EG2) and CD3., Results: Mast cells, activated eosinophils, T cells, IL-4+ cells, IL-5+ cells, eotaxin+ cells, RANTES+ cells and CCR3+ cells were all increased in the lesions of KD as compared with those in normal tissue. Mast cells and T cells were the major source of IL-4, whereas mast cells, T cells and activated eosinophils were the main source of IL-5. Mast cells, T cells and activated eosinophils were the main source of eotaxin and RANTES., Conclusions: The number of IL-4, IL-5, eotaxin and RANTES-expressing mast cells and T cells were increased in the lesions of KD. As mast cells are lesional resident cells, these cells may play an important role in the pathogenesis of KD by regulating IgE synthesis and orchestrating eosinophilic infiltration.

Background: The increase of atopic disorders in developed countries has been associated with the decline of infectious diseases, including helminthic infections. We have already demonstrated that adult worm extracts from Ascaris suum (ASC) suppress the IgE antibody production against unrelated antigens., Objective: Here we investigated the influence of ASC on the development of pulmonary eosinophilic inflammation in a murine model of asthma., Methods: Heat-coagulated egg white alone (EWI) or mixed with ASC (EWI + ASC) was implanted subcutaneously in B10.A or C57BL/6 mice, and 14 days later they were challenged intratracheally with OVA or exposed to aerosolized OVA for 4 days., Results: The suppressive effect of ASC was demonstrated on the accumulation of cells into airways, with reduction of eosinophil numbers and of eosinophil peroxidase activity in EWI + ASC-immunized mice. This effect correlated with a marked reduction of IL-5 and IL-4 levels in the BAL from C57BL/6 and B10. A mice, respectively, and of eotaxin in BAL and lung tissue from both strains. OVA-specific IgG1 and IgE levels were also impaired in serum and BAL from these mice. Airway hyper-reactivity to methacholine was obtained in B10. A mice sensitized with EWI, but the respiratory mechanical parameters returned to normal levels in EWI + ASC-immunized mice., Conclusion: These results indicate that ASC has a profound inhibitory effect on lung inflammation and hyper-responsiveness and that suppression of IL-5 or IL-4 and of eotaxin contributes to this effect.

Background: Several second-generation antihistamines have documented anti-inflammatory effects which appear independent of H1-receptor blockade. We investigated the inhibitory effect of cetirizine and its active enantiomer levocetirizine on eosinophil transendothelial migration (TEM) through monolayers of normal human dermal microvascular endothelial cells (HMVEC-d) or human lung microvascular endothelial cells (HMVEC-l)., Methods: HMVEC-d or HMVEC-l were grown to confluence on micropore filters in transwells inserted into a 24-well tissue culture dish. Eosinophils were isolated by density gradient centrifugation and negative immunomagnetic selection. Untreated eosinophils or eosinophils pre-incubated (30 min at 37 degrees C) with a concentration range of cetirizine or levocetirizine (10-5 to 10-9 m) were added to the upper chamber of the transwell which was incubated for 60 min at 37 degrees C. Both spontaneous eosinophil TEM and TEM to 100 ng/mL of human eotaxin in the lower chamber were assessed., Results: Between 8 and 10% of the eosinophils added to the upper chamber underwent spontaneous TEM through HMVEC-d or HMVEC-l. The addition of eotaxin to the lower chamber enhanced eosinophil TEM through HMVEC-d or HMVEC-l monolayers to over 20%, i.e. an enhanced TEM of approximately 100% in each case. Pre-incubation of eosinophils with cetirizine or levocetirizine dose-dependently inhibited eosinophil TEM to eotaxin through both HMVEC-d or HMVEC-l with total inhibition of eotaxin-induced TEM observed at 10-8 m for HMVEC-d and 10-7 m for HMVEC-l. Both drugs gave a reduced but significant inhibition of eosinophil TEM at lower concentrations. No concentration of cetirizine or levocetirizine had any significant effect on expression of CD11b, CD18 or CD49d by either resting or eotaxin-stimulated eosinophils. Furthermore, no effect on spontaneous eosinophil TEM, or eosinophil viability was seen with any concentration of cetirizine or levocetirizine., Conclusion: Levocetirizine inhibits eotaxin-induced eosinophil TEM through both dermal and lung microvascular endothelial cells suggesting that, like cetirizine, levocetirizine has potential anti-inflammatory effects.

Background: Matrix metalloproteinases (MMPs) are likely to be relevant mediators of the extracellular matrix (ECM) degradation and airway remodelling., Objective: We have compared the levels of MMPs, eotaxin and soluble interleukin 2 receptor (IL-2R) in the plasma of healthy subjects, atopic patients and asthmatic patients., Methods: The asthmatic patients were separated into two groups, either well controlled on inhaled therapy or acute severe asthma. Patients with acute severe disease had all received systemic corticosteroids from 12 to 48 h before the blood was taken. Blood was recovered in EDTA tubes, incubated with either f MLP, PMA or vehicle for 10 min and centrifuged. MMP-9, TIMP-1, IL-2R and eotaxin levels were measured in the plasma by ELISA. Moreover, the activity of MMPs was also evaluated by zymography., Results: An increased basal level of MMP-9 and IL2-R was observed in acute severe asthma. Following stimulation with f MLP and PMA there was an enhanced production of MMP-9 in the plasma of all groups of patients. However, the MMP-9 level was significantly enhanced in acute severe asthma, compared with the others. No difference was found for the TIMP-1 level between the patients. The eotaxin level in plasma was found to be significantly lower in acute severe asthmatics compared with the others groups. Zymography technique showed a significant increased activity of MMP-9 (92 kDa) but not MMP-2 (66 kDa) in the plasma of patients with acute asthma., Conclusion: The increased in MMP-9 production and activity observed in the present study suggests a process of extracellular matrix degradation in acute severe asthmatic patients and proposes MMP-9 as a non-invasive systemic marker of inflammation and airway remodelling in asthma.

Background: Eotaxin is a chemokine that attracts and activates eosinophils. The present study examines the occurrence of eotaxin in nasal mucosal surface liquids in patients with seasonal allergic rhinitis without allergen exposure and during repeat allergen challenge with and without topical glucocorticosteroid treatment. The number of subepithelial eosinophils and mucosal outputs of bulk plasma (alpha2-macroglobulin) and eosinophil cationic protein (ECP) are also examined., Methods: Twelve patients underwent daily allergen challenges for 6 days. Separately, 14 patients, who were receiving budesonide and placebo in a parallel group design, also underwent allergen challenge for 6 days. Nasal biopsies were obtained before and 24 h after the allergen challenge series, and lavages were carried out before and 15 min after selected allergen challenges., Results: At baseline nasal lavage fluid levels of eotaxin correlated to levels of alpha2-macroglobulin and ECP. After the first allergen challenge there was a correlation between nasal lavage fluid levels of eotaxin and ECP. Repeat allergen exposure increased the mucosal output of eotaxin (P <0.05) and ECP (P <0.01) as well as eosinophil numbers (P <0.01), but no correlation was found between increased eosinophil numbers and eotaxin. Budesonide reduced eotaxin levels during repeat allergen challenge (P <0.05)., Conclusions: Repeat allergen exposure in allergic rhinitis is associated with increased mucosal output of eotaxin. Topical budesonide attenuates this effect, suggesting the possibility that inhibitory effects on mucosal eotaxin may contribute to anti-eosinophilic actions of topical glucocorticosteroids.

Background: To further elucidate mechanisms of human allergic rhinosinusitis, we studied the induction, distribution and modulation of allergen-induced upper airway inflammation in a BALB/c mouse model., Methods: Allergic inflammation induced with ovalbumin (OVA) by intraperitoneal (IP) injection in alum was compared to repeated intranasal instillation. The type and distribution of inflammatory cells was compared in the respiratory and olfactory epithelial compartments. Eosinophil distribution was assessed using Scarlet Red stain and a polyclonal antibody recognizing eosinophil major basic protein (MBP). The role of interleukin (IL)-5 in upper airway inflammation was tested by administration of polyclonal anti-IL-5 antibody during the sensitization protocol., Results: Unsensitized control mice receiving saline failed to develop upper airway eosinophil infiltration. IP OVA-sensitized mice developed marked upper airway mucosal eosinophil infiltration after aerosol OVA challenge, whereas repeated intranasal instillation of OVA produced qualitatively similar, but less intense eosinophil infiltration. Using either sensitization protocol, eosinophil infiltration was seen in areas of the lower portion of the nasal septum, the floor and the lower lateral walls of the mid-caudal region of the nasal cavity. Immunofluorescence staining for MBP confirmed this distribution of eosinophils but also demonstrated some eosinophils in the maxillary sinuses and in circumscribed regions of the ethmoturbinates. All areas of eosinophil infiltration were lined by respiratory epithelium. The selective infiltration of respiratory but not olfactory epithelium by eosinophils was unassociated with a measurable induction of epithelial ICAM-1 or eotaxin expression. OVA-induced upper airway eosinophil infiltration was found to be IL-5 dependent, since administration of a polyclonal anti-IL-5 antibody (TRFK-5) during OVA sensitization resulted in a marked modulation (80% decrease) in eosinophil infiltration in response to subsequent OVA challenge., Conclusion: The mouse upper airway, specifically in areas containing respiratory epithelium, is a target for OVA-induced allergic inflammation. This selective infiltration of respiratory, but not olfactory, epithelium is, in part, dependent upon IL-5. This model is useful for further dissection of the inflammatory response with genetic manipulations and targeted immunological approaches.

Background: Human rhinoviruses (RVs) are the most common precipitants of asthma exacerbations. RV infection of bronchial epithelium results in local airway inflammation inducing eosinophil recruitment and activation. Induction of eosinophil chemoattractants could represent a central mechanism, as well as a prime target for intervention., Objective: To assess the effect of RV infection on mRNA expression and production of eosinophil chemoattractants by bronchial epithelial cells in-vitro., Methods: BEAS-2B cells were infected with major and minor RVs and the mRNA expression of IL-8, RANTES, MIP-1alpha, eotaxin, eotaxin-2, MCP-2, MCP-3 and MCP-4 was assessed by reverse transcription PCR. In cases where mRNA induction was observed, a fluoroimmunoassay was used to confirm protein production. To assess the virus-specificity of the observed reactions, cells were also exposed to inactivated RVs., Results: RV infection was able to up-regulate mRNA expression of IL-8, RANTES, MIP-1alpha, eotaxin and eotaxin-2, did not affect MCP-4, while MCP-2 and MCP-3 were not expressed either at baseline or after virus infection. Protein production was confirmed for IL-8, RANTES and eotaxin, but not for MIP-1alpha. When RVs were inactivated cytokine up-regulation was almost completely lost., Conclusion: Infection of bronchial epithelial cells with RVs results in the production of a wide array of mediators that are able to chemoattract eosinophils. These include the eosinophil-specific molecules eotaxin and eotaxin-2, in addition to IL-8 and RANTES, which are the most abundant. Eosinophil recruitment after RV infection of bronchial epithelium could represent a central event in the pathogenesis of virus-induced asthma exacerbations.

Background: Respiratory virus is one of the most common causes of airway inflammation, but its pathogenic mechanisms are not well understood. Eotaxin is a potent eosinophil chemoattractant and is a selective agonist for C-C chemokine receptor 3 (CCR3). Although it has recently been demonstrated that epithelial cells express eotaxin, both in vivo and in vitro, there are few data concerning the expression in viral infection., Objects: We hypothesized that eotaxin may play an important role in attracting inflammatory cells into the airway after viral infection and analysed whether viral infection induces eotaxin in nasal epithelial cells in vitro., Methods: Nasal epithelial cells obtained from polypectomy for nasal polyp were infected with influenza virus A (subtype H3N2). The cells and supernatants were collected 8, 24 and 48 h after infection. Eotaxin mRNA was analysed by RT-PCR. Eotaxin concentration in the supernatants was analysed by enzyme-linked immunosorbent assay. We also examined a blocking assay to analyse the intervention of pro-inflammatory cytokines, TNF-alpha and IL-1beta in eotaxin production induced by influenza virus., Results: The results showed that eotaxin was expressed constitutively in uninfected cells, but was up-regulated for both mRNA and protein levels in infected cells. Blocking experiments using anti-TNF-alpha and anti-IL-1beta antibodies showed no effects of these agents on the level of eotaxin. In addition, UV-inactivated virus did not enhance the expression of eotaxin., Conclusions: These results suggest that influenza virus A infection in nasal epithelial cells stimulates the expression of eotaxin, and may play an important role in the pathogenesis of airway inflammation by inducing eotaxin.

Background: Allergic rhinitis is a common disease characterized by infiltration of eosinophils into the nasal mucosa during the periods of symptoms. Among chemokines, which attract cells to the site of inflammation, eotaxin is relatively specific for eosinophils., Objective: We examined the influence of grass pollen season on nasal eotaxin expression in patients with seasonal allergic rhinitis, as well as the effect of a nasal glucocorticoid on this eotaxin expression., Methods: Nineteen patients with allergic rhinitis received treatment with either nasal beclomethasone (400 microgram/day) or placebo over a grass pollen season. In these patients, nasal biopsies were taken prior to and during the peak of the pollen season and stained immunohistochemically for eotaxin and EG2 + eosinophils. Five healthy subjects served as controls and gave nasal biopsies once prior to the pollen season., Results: Prior to pollen season, there was no significant difference in nasal eotaxin expression between patients with allergic rhinitis and healthy subjects. Grass pollen season induced significant increase in eotaxin expression in placebo-treated (P = 0.04; n = 9) but not in beclomethasone-treated rhinitis patients (P = 0.8; n = 10). During peak grass pollen season, the eotaxin expression in placebo-treated patients was significantly higher compared with healthy subjects outside season (P = 0.03). There was no significant correlation between the expression of eotaxin and the number of EG2 + eosinophils in nasal mucosa. The serum levels of eotaxin in rhinitis patients remained stable over the pollen season., Conclusion: Expression of eotaxin in nasal mucosa of grass-pollen allergic rhinitis patients is upregulated during pollen season and treatment with a nasal glucocorticoid protects against this upregulation.

Background: There is increasing evidence that eotaxin is a key mediator in the development of tissue eosinophilia. However, the mechanism involved in the production of eotaxin has yet to be clarified. Most recently, it has been shown that interleukin (IL) -4 induces eotaxin in dermal fibroblasts. A novel cytokine termed IL-13, which binds to the alpha-chain of the IL-4 receptor, shares many biological activities with IL-4. It is known that fibroblasts express the IL-4 receptor and produce collagen type I upon stimulation with IL-4., Objective: We investigated whether IL-13, as well as IL-4, are able to induce eotaxin production in human nasal mucosal fibroblasts (HNMFs). Furthermore, we investigated the effect of costimulation of IL-13 and TNFalpha on eotaxin production., Methods: HNMFs, isolated from inferior nasal mucosa samples, were stimulated by various kind of cytokines for 1-36 h at 37 degrees C in 5% CO2. The change in the expression of eotaxin mRNA was then evaluated by reverse transcriptase-polymerase chain reaction and the Southern blot analysis. The amount of eotaxin in the culture media was measured by ELISA., Results: IL-13 as well as IL-4 dose-dependently induced eotaxin expression in HNMFs. Furthermore, IL-13 and TNFalpha synergistically induced eotaxin expression in HNMFs, while they hardly induced eotaxin expression in endothelial cells, epithelial cells or eosinophils. The synergy was observed when pre-incubation of HNMFs with IL-13 was followed by a stimulation with TNFalpha, or HNMFs were simultaneously stimulated with IL-13 and TNFalpha., Conclusion: These results strongly indicate that IL-13, as well as IL-4, may be important in eotaxin-mediated eosinophilic inflammation in nasal mucosa. In addition, in nasal mucosa, fibroblasts are the major cell source for eotaxin.

Background: A selective recruitment of eosinophils to sites of allergic inflammation is suggested to be controlled by regulation of cytokines, chemokines and adhesion molecules., Objective: The aim of this study was to examine whether allergen challenge in skin chambers, applied on patients with allergic rhinitis and mild asthma, results in a selective influx of activated eosinophils and detectable levels of cytokines/chemokines related to eosinophil recruitment, such as interleukin (IL)-5 and eotaxin., Methods: A skin blister was induced on the volar aspect of each forearm; one contained PBS-heparin buffer (control) and the other was challenged with relevant allergen. Peripheral blood was drawn before the allergen was applied to the skin chamber, and the expression of CD9, CD11b and EG2-epitope on intracellular eosinophil cationic protein (ECP) was analysed in eosinophils. Chamber fluid was collected 8 h after allergen application and analysed for differential cell counts, expression of eosinophil activity markers, the presence of ECP, eotaxin, and IL-5., Results: The number of recruited leucocytes was equal in the allergen-challenged chambers and in controls. However, the number of eosinophils was significantly increased in the allergen-challenged chambers, and elevated levels of released ECP were measured. Moreover, the eosinophils recruited were activated, as shown by increased expression of EG2 and CD11b, and decreased expression of CD9, in comparison with blood eosinophils. In the skin chamber fluids, higher levels of eotaxin were detected in the allergen-challenged chambers than in controls, but there were no detectable levels of IL-5., Conclusion: We have demonstrated a selective recruitment of eosinophils, and higher levels of released ECP and eotaxin, in skin chambers stimulated with allergen, as compared with control chambers. Allergen challenge in skin chambers is a useful tool for studies of eosinophil recruitment, their state of activation, and their involvement in the allergic inflammatory response.

In previous studies we found that guinea pigs demonstrate an increase in airway reactivity and eosinophil numbers 4 days after a respiratory infection with parainfluenza-3 (PI3) virus. Clinical data support the possible involvement of eosinophils in virus-induced airway hyperresponsiveness. Eotaxin, a newly discovered chemokine, could be involved in eosinophil migration to the airways. In this study, eosinophil numbers were counted in blood and bronchoalveolar lavage (BAL) fluid and related with eotaxin concentrations in BAL fluid 1, 2, 3, and 4 days after intratracheal PI3 virus administration. On day 1, blood eosinophils increased by more than 200% (P < 0.01). The number of eosinophils were only slightly enhanced from day 2 to day 4 (40%-70%). BAL fluid eosinophils were not increased on day 1 but were significantly elevated on day 2 (180%) and remained high on days 3-4 (>300%, P < 0. 05). This increase in lung eosinophils correlated well with eotaxin levels measured in BAL fluid. There was no significant increase in eotaxin on day 1 following PI3 infection; however, on days 2-4 eotaxin levels in BAL fluid were significantly elevated (four-sixfold increase) when compared with medium inoculated controls. Eotaxin appears to play an important role in eosinophil accumulation in guinea pig lung following PI3 infection.

Eotaxin is a potent eosinophil-specific CC-chemokine, which has been shown to play a role in the selective induction of eosinophil accumulation in a number of allergic models of inflammation. Many aspects of the mechanism by which eotaxin induces eosinophil accumulation in vivo remain unresolved. In the present study, we investigated the direct effect of synthetic human eotaxin on leucocyte/endothelial cell interactions within rat mesenteric venules, as quantified by intravital microscopy. Topical eotaxin (30 pmol) induced rapid firm adhesion and extravasation of leucocytes within the rat mesentery, the extravasated leucocytes all being eosinophils, as determined by histological analysis. Whilst eotaxin was unable to stimulate the interaction of rat eosinophils with vascular cell adhesion molecule-1 (VCAM-1) under static conditions in vitro, eotaxin-induced responses in vivo were significantly suppressed by anti-alpha4 integrin and anti-VCAM-1 monoclonal antibodies (mAbs). The anti-alpha4 integrin mAb, HP2/1 (3.5 mg/kg), inhibited the eotaxin-induced firm adhesion and extravasation, 60 min postapplication of the chemokine, by 89% and 84%, respectively. In the same set of experiments, the anti-VCAM-1 mAb, 5F10 (3.5 mg/kg), inhibited leucocyte adhesion and extravasation by 61% and 63%, respectively. These results demonstrate that eotaxin-induced migration of eosinophils through rat mesenteric venules in vivo is dependent on an alpha4 integrin/VCAM-1 adhesion pathway, the significance of which may only be evident under flow conditions and/or following the ligation of other adhesion molecules expressed on eosinophils.