Eosinophil Chemotactic Activity Research Articles

Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation.

Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F1t-PhytoPs (PPF1-I), 9-F1t-PhytoPs (PPF1-II), 16-E1t-PhytoPs (PPE1-I) and 9-D1t-PhytoPs (PPE1-II), and 16(RS)-9-epi-ST-Δ14-10-PhytoFs. Interestingly 16-E1t-PhytoP and 9-D1t-PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.

Galectin-9-like from Angiostrongylus cantonensis young adult worms modulates eosinophil chemotaxis invitro.

Eosinophils are recruited to the brain of mice after infection with Angiostrongylus canonensis. Several factors produced by infected mice are well known playing the role to chemoattract eosinophils from the blood into the brain. The purpose of this study is to investigate whether Angiostronylus cantonensis young-adult worms (AcYA) have components which have eosinophilic chemotactic activity. Eosinophil chemotactic activity of AcYA was tested by Boyden blind-well chamber technique. The components of AcYA were analysed by SDS-PAGE and Mass spectrometry. Furthermore, galectin-9 in the cerebrospinal fluid (CSF) of infected mice and galectin-9-like in AcYA were measured by ELISA technic and also were recognized by western blot analysis respectively. Excretory-secretory products of AcYA did not show eosinophil chemotactic activity. However, the extracts of AcYA showed protein concentration-dependent eosinophil chemotactic activity and reached the peak at the 24μg/ml. The eosinophil chemotactic activity was significantly reduced by lactose. The components of AcYA at molecular weights of approximatively 15kDa and 35kDa showed several galectins component in Mass spectrometric analysis. Furthermore, galectin-9-like in AcYA was recognized by ELISA and western blot analysis. In parallel with increase of galectin-9 in the CSF, eosinophils were also significantly increased in mouse after infected with A. cantonensis. Galectin-9-like in AcYA and galectin-9 in mouse CSF were confirmed demonstrating eosinophil chemotactic activity both invitro study and in the infection of mouse in this study.

Soluble antigen derived from IV larva of Angiostrongylus cantonensis promotes chitinase-like protein 3 (Chil3) expression induced by interleukin-13.

Angiostrongyliasis caused by Angiostrongylus cantonensis (A. cantonensis) is an emerging food-borne parasitic disease, which refers basically to eosinophilic meningitis. Chitinase-like protein 3 (Chil3), a member of chitinase-like protein family which has chemotactic activity for eosinophils, is reported to be highly upregulated in brain of mouse infected with A. cantonensis. The mechanisms of high expression of Chil3 and the association between A. cantonensis and Chil3 are rarely reported. In order to understand the mechanism of high expression of Chil3 in A. cantonensis-infected mouse, we measured the level of Chil3 in RAW 264.7 and BV2 cell lines stimulated with soluble antigen of A. cantonensis by qPCR and ELISA. To explore the role of Chil3 in inflammation caused by A. cantonensis, we extracted and cultured brain mononuclear cells (BMNCs) and detected the eosinophil chemotactic activity of Chil3 using transwell assay and flow cytometer. Furthermore, we treated the infected mice by injection with rmChil3 and then counted the number of larvae in brains of infected mice and treated mice to examine the association between the worm and Chil3. Our results showed the soluble antigen from A. cantonensis could promote the Chil3 expression in macrophage and microglial cell lines induced by interleukin-13. In conclusion, we supposed that high expression of Chil3 enhanced by soluble antigens from A. cantonensis might be the reason of serious eosinophil infiltration in mouse brain after A. cantonensis infection.

Allergen-induced resistin-like molecule-α promotes esophageal epithelial cell hyperplasia in eosinophilic esophagitis

Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-β, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4(+) and CD4(-) T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4(+) and CD4(-) T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE.

Response to Treatment in Individuals with Late‐Onset Asthma

Despite the fact that the onset of asthma can occur at any age, there have been few studies specifically assessing whether the therapeutic effects of asthma medications vary according to age of onset.1, 2 The present study was constructed to compare the responses to a combination of fluticasone (FP) and salmeterol (SAL) in terms of symptoms, airflow obstruction, and airway inflammation between individuals with asthma with early (<40) and late (≥65) onset. Various factors, including current medications, smoking history, asthma duration, comorbid conditions, adherence, and inhalation technique, may modify response to asthma treatment.1, 2 This prospective parallel-group study was designed to control for these modifiers. Never-smoking untreated individuals from both age groups whose asthma duration was <3 years were included. Individuals were excluded if they had other pulmonary or cardiac diseases or had been treated with any form of corticosteroid in the 8 weeks before the study. Hydrofluoroalkane (HFA)-FP/SAL (250/50 μg) was administered twice daily, and instructions were repeated monthly to all participants. Asthma control questionnaire (ACQ), spirometry, and fraction of exhaled nitric oxide (FENO) were assessed before and 3 months after HFA-FP/SAL therapy. FENO was measured using an online electrochemical nitric oxide analyzer (NIOX MINO; Aerocrine AB, Solna, Sweden) according to standard procedures.3, 4 Statistical analysis was performed using Mann–Whitney U-tests. The local ethics committee approved this trial, which was registered with the university hospital Medical Information Network (UMIN000006291). Informed written consent was obtained from each participant. Participants consisted of 16 younger individuals (seven male, nine female, 14 with atopy) with an average onset age of 27.4, and 16 elderly individuals (eight male, eight female, eight with atopy) with an average onset age of 72.3. At baseline, individuals with late onset had lower forced expiratory volume in 1-s percentage of predicted (%FEV1; 89.3 ± 7.3%) than those with early onset (80.9 ± 12.8%, P = .03), but no differences in ACQ score (1.3 ± 0.5 vs 1.4 ± 0.6, P = .7) of FENO (50 ± 18 vs 57 ± 38 ppb, P = .48) were seen between the two age groups. As shown in Figure 1, HFA-FP/SAL therapy significantly improved ACQ,%FEV1, and FENO equally in participants with early and late onset, and there was no significant difference in these outcomes between the groups 3 months after treatment (0.2 ± 0.3 vs 0.3 ± 0.3, P = .45; 94.7 ± 8.4% vs 90.1 ± 12.5%, P = .14; and 26 ± 11 vs. 24 ± 18 ppb, P = .99, respectively). Two points from the preliminary data should be emphasized. First, untreated individuals with late-onset asthma have the same degree of airway inflammation detected using FENO as those with early onset. Second, individuals with early- and late-onset asthma respond equally well to HFA-FP/SAL therapy in terms of asthma symptoms, airflow obstruction, and airway inflammation. Because inhaled corticosteroids are capable of reducing airway inflammation, thereby reducing symptoms and improving lung function, the present study indicates that airway inflammation is an important therapeutic target in persistent asthma regardless of the age of onset. It has been shown that peripheral blood eosinophil adhesion and chemotactic activities were comparable in individuals with early and late-onset asthma, and the percentages of sputum eosinophils were similar in the two age groups.5 There is also evidence that regular use of inhaled corticosteroids improves asthma-related outcomes in elderly adults.6-9 The findings of the current study suggest that regular antiinflammatory treatment and instructions are an important therapeutic strategy in asthma for all ages, and the implementation of this strategy can be expected to improve health outcomes in elderly adults. The authors thank Mr. Brent Bell for reading the manuscript. This study was supported by Grant 22591097 from the Japan Society for the Promotion of Science. No potential conflicts of interest exist with any companies whose products or services may be discussed in this article. Conflict of Interest: The editor in chief has reviewed the conflict of interest checklist provided by the authors and has determined that the authors have no financial or any other kind of personal conflicts with this paper. Author Contributions: Kazuto Matsunaga: study design and preparation of manuscript. Masakazu Ichinose: interpretation of data. Keiichiro Akamatsu and Tsunahiko Hirano: acquisition of subjects and data. Sponsor's Role: None.

Allergen Immunotherapy: A Centenary Celebration

In 1911 and long before the availability of antiallergic drugs, Leonard Noon demonstrated that prophylactic subcutaneous inoculation with a grass pollen extract was effective in suppressing immediate conjunctival sensitivity to grass pollen.1 Noon’s coworker, John Freeman, continued to practice immunotherapy and in 1930 published the first rush immunotherapy protocol.2 William Frankland, a colleague of Freeman, performed the first controlled clinical trial of grass pollen immunotherapy in 1954.3 He used a whole grass pollen extract that was compared with its partially purified proteins, the corresponding ultrafiltrate that contained no protein and a phenol-containing diluent. Both the whole extract and the purified grass pollen proteins were effective compared with the ultrafiltrate and the diluent control alone (Fig. 1). Frankland’s study established a firm scientific foundation for the practice of allergen immunotherapy. Noon, Freeman, and Frankland were all physicians at St. Mary’s Hospital, Paddington, now affiliated to Imperial College London, United Kingdom. Frankland in his 99th year continues to practice and play a major role in the allergy community; much admired and respected by his colleagues, he represents our centenary link with the origins of the practice of immunotherapy (Fig. 2). Major advances in allergen immunotherapy have resulted from parallel studies performed in the United States. Lowell and Franklin in 1964 were the first to clearly demonstrate that a single allergen (ragweed) in a multiallergen mixture was effective in reducing seasonal allergic symptoms.4 Philip Norman and Larry Lichtenstein in 1978 convincingly demonstrated the allergen-specificity of ragweed immunotherapy in patients with dual sensitivity to ragweed and grass pollen.5 Johnstone first highlighted the possibility that immunotherapy in children might confer protection against development of asthma,6 a concept supported by the more recent Preventive Allergy Treatment (PAT) study that identified a 2–3 fold risk reduction for developing asthma after 3 years treatment in children with seasonal pollinosis, protection that persisted for a further 7 years after discontinuation of immunotherapy.7 Hunt and colleagues8 demonstrated the efficacy of purified venom over whole insect body extract and placebo in patients with anaphylaxis to the stings of hymenoptera. Studies9 have confirmed the dose-dependency of allergen immunotherapy whereas the long-term benefits of allergen immunotherapy, with persistence of efficacy for several years after discontinuation10 have been illustrated for both venom11 and grass pollen immunotherapy, the latter both for subcutaneous and sublingual routes of therapy.12 Our increasing knowledge of the mechanisms of immunotherapy has informed both novel approaches and the development of putative biomarkers that might predict the clinical response to immunotherapy. Prausnitz and Kustner13 published in 1921 that a serum factor (‘reagin’) could transfer immediate allergen sensitivity as shown by skin testing was followed Robert Cooke’s observation in 1935 that serum obtained after pollen immunotherapy could confer ‘immunity and hypersensitivity.’14 These seminal observations long preceded the discovery of IgE antibody as reagin by the Ishizakas,15 Johansson and Bennich,16 and the concept of IgG ‘blocking antibodies.’17 The suppressive effect of ragweed immunotherapy on nasal eosinophils as a local marker of allergic inflammation was shown by Creticos in 1984,18 whereas Passalacqua and Canonica similarly demonstrated decreased local eosinophilia and adhesion molecule expression during mite sublingual immunotherapy.19 Warner in 197820 and Rak21 in 1991 observed decreases in allergeninduced late asthmatic responses and associated bronchial inflammation, respectively, in children and adults. A link between altered T-cell responses and immunotherapy was first shown by Rocklin22 whereas the critical role of regulatory T cells and IL-10 was highlighted by Akdis and colleagues.23 The concept of immune deviation of allergenspecific TH2 responses in favor of TH1 responses in both the periphery and in target organs has developed in parallel.24–26 From the Royal Brompton National Heart & Lung Institute, London; and National Jewish Health, Denver, CO. Correspondence to: Stephen R. Durham, MA, MD, FRCP, Royal Brompton Hospital and Imperial College, Dovehouse Street, London SW3 6LY United Kingdom. Telephone: 442073518024. Fax: 442073497780. E-mail: s.durham@ imperial.ac.uk. Copyright © 2011 by World Allergy Organization FIGURE 1. Table II shows the results of the first immunotherapy controlled trial, as published in: Frankland AW, Augustin R. Prophylaxis of summer hay-fever and asthma: a controlled trial comparing crude grass-pollen extracts with the isolated main protein component, The Lancet, May 24, 1954.

Involvement of Galectin-9 in Lung Eosinophilia in Patients with Eosinophilic Pneumonia

Background: Although we first found galectin-9 (Gal-9) as an eosinophil chemoattractant, its role in eosinophilic inflammation is still obscure. The purpose of the present study is to clarify the role of Gal-9 in human eosinophilic pulmonary inflammation in comparison with eotaxin (CCL11). Methods: We measured the levels of Gal-9 and eotaxin in the bronchoalveolar lavage fluid (BALF) of patients with acute and chronic eosinophilic pneumonia (AEP and CEP). Furthermore, the biological activities (chemotaxis and apoptosis) of Gal-9 were compared with those of eotaxin using interleukin-5-primed or -unprimed eosinophils. Results: The levels of Gal-9 and eotaxin in the BALF from patients with AEP and those with CEP were higher than those found in the controls. Although there was little difference in Gal-9 level between patients with AEP and patients with CEP, the eotaxin level was significantly lower in patients with CEP. In patients with AEP, the eosinophil number correlated well with both the Gal-9 and eotaxin levels. However, in patients with CEP, the eosinophil number only correlated well with the Gal-9 level. Moreover, the Gal-9 level correlated with the eotaxin level in patients with AEP, but there was no significant correlation between those levels in patients with CEP. Anti-Gal-9 antibody treatment strongly reduces eosinophil chemotactic activity in the BALF of patients with AEP and in that of patients with CEP, whereas the anti-CCR3 (receptor for eotaxin) antibody strongly reduces this activity in the BALF of patients with AEP but not in that of patients with CEP. Furthermore, Gal-9 exhibited both chemotactic and proapoptotic activities for activated eosinophils, though eotaxin only exhibited chemotactic activity. Conclusions: The present results provide two possibilities: that Gal-9 is involved in pulmonary eosinophilia in patients with AEP and CEP, and that Gal-9 exhibits regulatory functions for activated eosinophils at the site of inflammation.

Observation of eosinophil chemotactic activity in sera and bronchoalveolar lavage fluid in patients with eosinophilic pneumonia

Observation of eosinophil chemotactic activity in sera and bronchoalveolar lavage fluid in patients with eosinophilic pneumonia

Clinical Data and Inflammation Parameters in Patients with Cypress Allergy Treated with Sublingual Swallow Therapy and Subcutaneous Immunotherapy

The clinical efficacy of immunotherapy, either by high dose sublingual-swallow therapy (SLIT) or subcutaneous immunotherapy (SCIT), has been demonstrated in patients with pollinosis but few studies have been carried out analysing differences in these treatments in terms of an improvement of clinical and allergic phlogosis parameters. The aim of this double-blind placebo-controlled study is to investigate the efficacy of high dose SLIT and SCIT using a purified standardized Juniperus ashei extract in a population of allergic patients monosensitized to cypress. Forty patients with cypress-allergic rhino conjunctivitis were administered therapeutic or placebo SLIT or SCIT for 12 months. Laboratory parameters were studied, namely the eosinophil cationic protein (ECP) level in nasal lavage and in serum, as well as the number of eosinophils (EOS) in peripheral blood and in nasal lavage and the level of eosinophil chemotactic activity (ECA). These parameters were correlated with clinical symptoms, evaluated by means of the clinical symptoms score (CSS). After SCIT and SLIT the levels of ECP and ECA were reduced in nasal lavage. We also observed a significant reduction in the values of ECP in serum in the patients treated with SLIT. EOS were unchanged in peripheral blood, but significantly reduced in nasal lavage. These data were in accordance with the improvement of clinical symptoms, supported by the close correlation between CSS and laboratory parameters. Our data confirm a clinical improvement correlated with a decline in inflammation parameters after one year of immunotherapy, supporting the hypothesis that treatment with a major allergen of cypress is able to change the course of allergic rhinitis.

TP receptor-mediated release of eosinophil chemotactic activity from human bronchial smooth muscle cells

There are reports indicating that thromboxane A 2 receptors (TP receptors) may stimulate the eosinophil accumulation in the lower airways of asthmatics, however, the mechanisms behind such an effect remain unknown. We quantified the synthesis of eosinophil chemotactic activity and eosinophilic CC chemokines, including CCL5, CCL7, CCL8, CCL11, CCL13, CCL24, and CCL26 in primary cultures of human bronchial smooth muscle cells (BSMC) stimulated with a prostanoid TP receptor agonist, IBOP (10 − 9 –10 − 7 M). The activation of prostanoid TP receptors in BSMC induced the release of potent eosinophil chemoattractant(s) in the presence of interleukin (IL)-4. CCL11/eotaxin-1 was the only synthesis significantly increased by IBOP co-stimulated with IL-4, and pretreatment with an anti-CCL11 antibody abrogated the eosinophil chemotactic activity released from IBOP/IL-4-stimulated BSMC. The effect of IBOP was also completely blocked by pretreatment with a prostanoid TP receptor-specific antagonist, AA-2414. IBOP had no effect on the expression of IL-4 receptor-α, or on the IL-4-induced phosphorylation of STAT6 in BSMC. In conclusion, activation of prostanoid TP receptors in a Th2-dominant microenvironment might exacerbate the eosinophilic inflammation of the airways by synthesis and release of CCL11 from BSMC.

Granulocyte Chemotactic Activity in Exhaled Breath Condensate of Healthy Subjects and Patients With COPD

Several chemoattractants have been measured in exhaled breath condensate (EBC) from patients with COPD. The aim of this study was to compare the eosinophil and neutrophil chemotactic activity contained in EBC from healthy subjects and patients with COPD. EBC collected using a commercially available condenser (EcoScreen; Erich Jaeger Viasys; Hoechberg, Germany) was compared in 45 COPD patients and 65 healthy subjects. EBC chemotactic activity for eosinophils and neutrophils was assessed using microchambers (Boyden; Neuro Probe; Cabin John, MD). Chemotactic index (CI) was used to evaluate cell migration. EBC from patients with COPD (CI, 2.21 +/- 0.16 [mean +/- SEM]) and healthy subjects (CI, 1.67 +/- 0.11) displayed significant neutrophil chemotactic activity (p < 0.0001 for both), which was however higher in patients with COPD (p < 0.001). Healthy smokers had a significantly raised CI for neutrophils by comparison with healthy nonsmokers (p < 0.01) and ex-smokers (p < 0.05). Likewise, current COPD smokers tended to have greater neutrophil CI than COPD who stopped smoking (p = 0.08). COPD ex-smokers had raised chemotactic activity by comparison with healthy ex-smokers (p < 0.05). Anti-interleukin-8 (10(-6) g/mL) antibodies reduced neutrophil chemotactic activity by 35.2% (p < 0.05). EBC also contained significant eosinophil chemotactic activity in healthy subjects (CI, 1.68 +/- 0.09; p < 0.0001) and patients with COPD (CI, 1.23 +/- 0.07; p < 0.01), with a significantly lower CI in patients with COPD as compared to healthy subjects (p < 0.001). Smoking did not influence eosinophil chemotactic activity in healthy subjects or patients with COPD. Current smoking favors neutrophil chemotactic activity. As compared to healthy subjects, EBC from patients with COPD displays a skewed chemotactic activity toward neutrophils vs eosinophils.

Involvement of Galectin-9 in Guinea Pig Allergic Airway Inflammation

Background: There is little information about the involvement of galectin-9 (Gal-9) in allergic inflammation. Thus, we investigated the role of Gal-9 in asthma model guinea pigs. Methods: Airway resistance (R_aw) was measured using a double-flow plethysmograph system. Gal-9 expression in the lung was assessed by Western blot and immunohistochemistry. Eosinophil chemotactic activity was evaluated in a chamber containing a polyvinylpyrolidone-free membrane. Cell apoptosis was analyzed on a flowcytometry with propidium iodide. Results: In cloning guinea pig Gal-9 we identified three isoforms that differ only in the length of their linker peptides, just as with human Gal-9. Guinea pig Gal-9 was found to be a chemoattractant for eosinophils and to promote induction of apoptosis in sensitized but not non-sensitized T lymphocytes. In allergic airway hypersensitivity model, a low level of Gal-9 expression was observed in the nonsensitized/nonchallenged group, but upregulation was detected at 7 h after challenge and sustained up to 24 h. Such upregulation correlated with elevation of eosinophil peroxidase activity but not with increased R_aw. Conclusions: The present results provide evidence that Gal-9 is not involved in airway hypersensitivity, but is partly involved in prolonged eosinophil accumulation in the lung.

TLR3-Mediated Synthesis and Release of Eotaxin-1/CCL11 from Human Bronchial Smooth Muscle Cells Stimulated with Double-Stranded RNA

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

Isoproterenol suppresses cytokine-induced RANTES secretion in human lung epithelial cells through the inhibition of c-jun N-terminal kinase pathway

It has been reported that β 2-agonists may potentially exert some anti-inflammatory action in addition to bronchodilation that may contribute to their beneficial effects on asthma control. Bronchial epithelial cells are well known to respond to a range of stimuli by producing various biologically active mediators that can influence airway inflammation. RANTES (regulated on activation, normal T cells expressed and secreted) plays an important role in the pathophysiology of airway inflammation of asthmatics through its chemotactic activity for eosinophils. In this study, the authors investigated whether cytokine-induced RANTES release from BEAS-2B human bronchial epithelial cells could be modulated by β-agonist isoproterenol (ISO). The possible involvement of c-jun N-terminal kinase (JNK) pathway was also studied. Combination of tumor necrosis factor-α and interleukin-1β (cytokine mix) increased RANTES release from BEAS-2B cells and stimulated JNK activity. Similar to JNK inhibitor SP600125, ISO inhibited not only the production of RANTES but also the activation of JNK pathway in cytokine mix-stimulated BEAS-2B cells. The effect of ISO was mediated by the β 2-adrenoceptor, since it was blocked by ICI 118,551, a selective β 2-receptor antagonist, but not by atenolol, a selective β 1-receptor antagonist. Adenylyl cyclase activator forskolin reproduced the effects of ISO. Isoproterenol was found to inhibit the release of RANTES from the human bronchial epithelial cells, at least in part, through the inhibition of JNK signaling pathway.

Increased expression of Th2-associated chemokines in bullous pemphigoid disease. Role of eosinophils in the production and release of these chemokines

Bullous pemphigoid is an inflammatory disease of the skin associated with eosinophil infiltration and the presence of high levels of Th2 cytokines in the associated blister fluid. Little is known about the contribution of chemokines in this disease. We found that eotaxin and MCP-4 mRNA and immunoreactivity were expressed in all biopsies of BP patients and were mainly localized to the epidermis and eosinophils. The expression of eotaxin and MCP-4 was enhanced in eosinophils following IL-5 treatment. Subsequent stimulation of IL-5-primed eosinophils with Ig-immune complexes, results in increase secretion of eotaxin and MCP-4 in the supernatants. Using immunostaining, these two chemokines were localized to the granules of eosinophils. BF was found to contain chemotactic activity for eosinophils, neutrophils and T cells. The chemotactic effect of BF for eosinophils was more effective when eosinophils were stimulated with IL-5 or IL-4. We also found that the levels of Th2-associated chemokines (eotaxin and MCP-4) in BF were significantly higher than the Th1-associated chemokines (MIP-1β and IP-10). This was consistent with the increased chemotaxis of polarized Th2 cells toward BF, when compared to Th1-differentiated T cells. Our results support the involvement of Th2-associated chemokines in the pathogenesis of BP disease.

Mast Cell β-Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities

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

Cysteinyl‐leukotrienes contribute to sputum eosinophil chemotactic activity in asthmatics

Cysteinyl-leukotrienes are lipid derived mediators involved in asthma. They are able to stimulate eosinophil chemotaxis in vitro. Induced sputum from asthmatics has been shown to contain eosinophil chemotactic activity. The purpose of our study was to evaluate the contribution of cysteinyl-leukotrienes to sputum eosinophil chemotactic activity in asthmatics and to seek whether there might be differences between asthmatics free of inhaled corticosteroids vs those regularly receiving this treatment. Twenty-two patients (11 corticosteroid free, mean FEV1 99% predicted, 11 corticosteroid-treated, mean FEV1 77% predicted) recruited from our asthma clinic underwent a sputum induction. Sputum was processed according to standard procedure. Eosinophil chemotactic activity contained in the fluid phase was assessed using Boyden microchamber model and expressed as chemotaxis index (CI). Cysteinyl-leukotrienes were measured in sputum supernatant by ELISA and their role in sputum eosionophil chemotactic activity was evaluated by using montelukast, a selective antagonist of a cys-LT1 receptor. Cysteinyl-leukotrienes were well detectable in sputum supernatants from both steroid-naive (247 +/- 42 pg/ml) and steroid-treated (228 +/- 26 pg/ml) asthmatics. Sputum eosinophil chemotactic activity was indiscriminately present in both corticosteroid-naive (CI: 2.61 +/- 0.22) and corticosteroid-treated (2.98 +/- 0.35) asthmatics. Montelukast (100 microM) significantly inhibited the eosinophil chemotactic activity in both groups achieving a mean inhibition of 54.2 +/- 9.2% (P < 0.001) and 64.7 +/- 7.8% (P < 0.001) in steroid-naive and steroid-treated asthmatics respectively. Cysteinyl-leukotrienes actively participate in sputum eosinophil chemotactic activity found in asthmatics irrespective of whether they are or not under treatment with inhaled corticoids.

Prostaglandin D2 Induces Chemotaxis in Eosinophils Via Its Receptor CRTH2 and Eosinophils May Cause Severe Ocular Inflammation in Patients With Allergic Conjunctivitis

Eosinophils are known to have important roles in the pathogenesis of allergic conjunctivitis. Prostaglandin (PG) D2, which has been implicated as a factor in allergic diseases, is known to have chemotactic activity for eosinophils. Its receptor, chemoattractant receptor homologous molecule expressed on TH2 (CRTH2), serves as a receptor for PGD2 and has been reported to mediate PGD2-dependent migration of eosinophils. In the present study, both eosinophil toxic activity for corneal epithelial cells and chemotaxis induced by PGD2 in normal volunteers were investigated. Expression of CRTH2 in normal subjects was also measured. Primary cultured corneal epithelial cells and eosinophils in serum from normal volunteers were used and a human corneal epithelial cell line was established. Studies were performed with/without amniotic membrane. CRTH2 expression on eosinophils was assessed by flow cytometry. Chemotaxis experiments were performed using a modified Boyden chamber technique. Corneal epithelial cells cultured with eosinophils showed higher floating epithelial cells and epithelial defect than those cultured in the absence of eosinophils. Flow cytometry analysis revealed that eosinophils expressed CRTH2. PGD2 induced chemotaxis of eosinophils. Corneal epithelial damage might be caused by eosinophils, which are recruited by PGD2 secretion via CRTH2 expressed on eosinophils.

Reducing airways inflammation to prevent exacerbations in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is amajor cause of handicap, mortality and health careexpenses (1). Altered quality of life (QOL) is a hallmarkof the disease, and relates mainly to dyspnoea andexacerbations: both QOL at steady-state and its rate ofdecline over time correlate to the frequency of acuterespiratory events (2, 3). In parallel, hospitalizations forexacerbations in the most severe patients are importantdeterminants of health care costs (4). Thus, preventingexacerbations is a major goal of care for COPD patients,both from an individual and collective point of view.Inflammation and COPD exacerbationsIn most cases, COPD occurs in smokers and ex-smokers,in whom airflow limitation is the consequence of variousdegrees of emphysema, chronic bronchiolitis and mucusplugging (5). These components of the disease have beenlinked to inflammatory and remodelling processes in theairways and lung parenchyma, involved inflammatorycells being mainly neutrophils, B and T lymphocytes andmacrophages (5, 6). Airways inflammation is moreintense during exacerbations, as shown by increases inthe levels of (i) expression of neutrophil elastase, CXCL-5, CXCL-8 and CXCR1/2 in biopsies (7), (ii) exhaledLTB4 and 8-isoprostane in breath condensates (8), (iii)interleukin (IL)-8 and tumour necrosis factor (TNF)-a insputum (9).Infections are a leading cause of exacerbations andfurther amplify airway inflammation (10). However,about one half of exacerbations occur in the absence ofany detectable infective pathogen, which may not preventinflammation from increasing (11). Besides, higher levelsof inflammatory markers in the airways of patients atsteady-state are also associated with more frequentexacerbations (12). Studies of the East London COPDcohort even suggest the existence of a particular subgroupof COPD patients called frequent exacerbators, whoconsistently have more than 2–3 exacerbations per year(13). In these patients, QOL is more altered, forcedexpiratory volume at 1 s (FEV

Schistosoma mekongi: A prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.