Human airway submucosal glands augment eosinophil chemotaxis during rhinovirus infection

BACKGROUND: Asthma exacerbations are frequently associated with rhinovirus (RV) infections. However, the contribution of airway submucosal gland (SMG) to exacerbations of asthma in RV respiratory infection has not been studied. OBJECTIVE: This study was undertaken to examine whether RV-infected human respiratory SMG cells produce pro-inflammatory cytokines and chemokines for eosinophils, and augment eosinophil transmigration across human airway epithelium. METHODS: We infected cultured human tracheal SMG cells with RV14, collected culture media at 1, 3, and 5 days after infection, and measured the chemotactic activity for eosinophils in the culture supernatant using a 48-well microchemotaxis chamber and a (51)Cr-labelled eosinophil transmigration assay. RESULTS: Exposing a confluent human tracheal SMG cell monolayer to RV14 consistently led to infection. Human SMG cells with RV infection secreted soluble factors activating human eosinophil chemotaxis into the culture supernatant in a time-dependent manner, and the culture supernatant significantly augmented the transmigration of (51)Cr-labelled eosinophils through human airway epithelial cell layers from the basal to mucosal side. These effects were completely abolished by a mixture of a monoclonal antibody regulated on activation, normal T cells expressed and secreted (RANTES) and an antibody to granulocyte macrophage-colony stimulating factor (GM-CSF). CONCLUSION: These results suggest that human respiratory SMG cells may augment eosinophil transmigration across the airway epithelium through the secretion of RANTES and GM-CSF after RV infection, and may contribute to exacerbations of asthma.     

Induction, distribution and modulation of upper airway allergic inflammation in mice

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.     

CC chemokines and transmigration of eosinophils in the presence of vascular cell adhesion molecule 1.

BACKGROUND: Interaction between eosinophil alpha4 integrin and vascular cell adhesion molecule 1 (VCAM-1) expressed on activated endothelial cells may be a key step in the selective recruitment of eosinophils from the circulation to sites of inflammation. OBJECTIVE: To investigate the factor(s) that induces transmigration of eosinophils after firm adhesion via the alpha4 integrin/VCAM-1 pathway. METHODS: We examined the effects of a variety of inflammatory mediators on the migration of eosinophils across recombinant human (rh) intracellular adhesion molecule 1- or rhVCAM-1-coated Transwell filters or VCAM-1-expressing human pulmonary microvascular endothelial cells (HPMECs) that had been stimulated with interleukin 4 (IL-4) and tumor necrosis factor alpha. The number of eosinophils that had transmigrated was evaluated by measuring eosinophil peroxidase activity. RESULTS: The CC chemokines RANTES (regulated on activation, normal T-cell expressed, and secreted), eotaxin, eotaxin 2, monocyte chemotactic protein 3 (MCP-3), and MCP-4 each increased eosinophil transmigration across rhVCAM-1-coated filters compared with fetal calf serum-blocked or rh intracellular adhesion molecule 1-coated filters (P < .01). On the other hand, platelet-activating factor, C5a, formyl-methionyl-leucil-phenylalanine, granulocyte-macrophage colony-stimulating factor, IL-5, and IL-8 did not enhance migration across rhVCAM-1. The enhancement of migration by RANTES in the presence of rhVCAM-1 was blocked by an anti-alpha4 integrin monoclonal antibody. CC chemokines augmented eosinophil transmigration across VCAM-1-expressing HPMECs compared with resting HPMECs (P < .01). Conversely, the transmigration induced by platelet-activating factor, C5a, formyl-methionyl-leucil-phenylalanine, or IL-8 was not modified by the expression of VCAM-1 on HPMECs. CONCLUSIONS: CC chemokines induce transendothelial migration of eosinophils after interaction between eosinophil alpha4 integrin and endothelial VCAM-1.     

Eotaxin and RANTES enhance 5-oxo-6,8,11,14-eicosatetraenoic acid-induced eosinophil chemotaxis

BACKGROUND: The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of human eosinophils and, among lipid mediators, is the most active chemoattractant for these cells. Studies have demonstrated the importance of 5-lipoxygenase products in allergen-induced pulmonary eosinophilia. Because CC chemokines such as eotaxin and RANTES also play critical roles in this phenomenon, it would seem likely that members of both classes of mediators contribute to this response. OBJECTIVE: The study was designed to directly compare the effects of 5-oxo-ETE on eosinophils with those of eotaxin and RANTES and to determine whether these chemokines could enhance the chemotactic response to 5-oxo-ETE. METHODS: Eosinophil chemotaxis was measured with microchemotaxis chambers. CD11b, L-selectin, and actin polymerization were measured by flow cytometry. Calcium mobilization was measured by fluorescence. RESULTS: 5-Oxo-ETE stimulated eosinophil chemotaxis with a potency between those of eotaxin and RANTES and a maximal response about 50% higher than that of eotaxin. Threshold concentrations of eotaxin and RANTES increased the chemotactic potency of 5-oxo-ETE by more than 4-fold. 5-Oxo-ETE and eotaxin were approximately equipotent in mobilizing cytosolic calcium in eosinophils. Eotaxin was more potent in inducing CD11b expression and actin polymerization, but the maximal responses to 5-oxo-ETE were about 50% higher. 5-Oxo-ETE strongly induced L-selectin shedding, whereas eotaxin elicited only a weak and variable response. CONCLUSION: 5-Oxo-ETE is a strong activator of human eosinophils with a chemotactic potency comparable to those of eotaxin and RANTES, both of wwhich enhance 5-oxo-ETE-induced chemotaxis. 5-Oxo-ETE and CC chemokines may combine to induce pulmonary eosinophilia in asthma.     

Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells

Eotaxin is a newly discovered C-C chemokine which preferentially attracts and activates eosinophil leukocytes by acting specifically on its receptor CCR3. The airway inflammation characteristic of asthma is believed to be, at least in part, the result of eosinophil-dependent tissue injury. This study was designed to determine whether there is increased expression of eotaxin and CCR3 in the bronchial mucosa of asthmatics and whether this is associated with disease severity. The major sources of eotaxin and CCR3 mRNA were determined by co-localization experiments. Bronchial mucosal biopsy samples were obtained from atopic asthmatics and normal non-atopic controls. Eotaxin and CCR3 mRNA were identified in tissue sections by in situ hybridization (ISH) using radiolabeled riboprobes and their protein product visualized by immunohistochemistry (IHC). Co-localization experiments were performed by double ISH/IHC. Eotaxin and CCR3 (mRNA and protein) were significantly elevated in atopic asthmatics compared with normal controls. In the asthmatics there was a highly significant inverse correlation between eotaxin mRNA⁺ cells and the histamine provocative concentration causing a 20% fall in FEV₁ (PC₂₀). Cytokeratin-positive epithelial cells and CD31⁺ endothelial cells were the major source of eotaxin mRNA whereas CCR3 co-localized predominantly to eosinophils. These data are consistent with the hypothesis that damage to the bronchial mucosa in asthma involves secretion of eotaxin by epithelial and endothelial cells resulting in eosinophil infiltration mediated via CCR3. Since selective (eotaxin) and non-selective C-C chemokines such as RANTES, MCP-3 and MCP-4 all stimulate eosinophils via CCR3, this receptor is potentially a prime therapeutic target in the spectrum of diseases involving eosinophil-mediated tissue damage.     

Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 microM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 +/- 3.4%) than with eosinophils from normal subjects (38.7 +/- 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by < or = 10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by < or = 44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 +/- 2.4%; 10% FBS: 40.2 +/- 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 +/- 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system.     

Effect of a specific cysteinyl leukotriene-receptor 1-antagonist (montelukast) on the transmigration of eosinophils across human umbilical vein endothelial cells

BACKGROUND: Leukotrienes have been implicated in the selective infiltration of eosinophils into the bronchial mucosa in asthma. OBJECTIVE: We studied whether eosinophil transmigration through cultured human umbilical vein endothelial cells (HUVECs) can be blocked by a specific cysteinyl LT1-receptor-antagonist. METHODS: Unstimulated and stimulated eosinophils from patients with asthma and normal controls were subjected to confluent human umbilical vein endothelial cell (HUVEC) monolayers separating the upper and lower chamber of Transwell culture plates. Unstimulated eosinophils or cells pre-incubated in the presence of the eosinophil activating cytokines GM-CSF or IL-13 were placed in the upper chambers while PAF, a potent chemoattractant factor for eosinophils, was added to the lower chamber. Migration of eosinophils was quantified by a beta-glucuronidase assay. RESULTS: The assumption that eosinophils express CysLT1 (cysteinyl-leukotriene 1)-receptors was based on our demonstration of mRNA-expression for the CysLT-1-receptor by polymerase chain reaction on purified eosinophils. The chemotactic response to PAF was significantly reduced when eosinophils were pre-incubated with montelukast for 15 min. When eosinophils were pre-incubated with GM-CSF and/or IL-13, the migratory response to PAF was also significantly reduced by montelukast. CONCLUSION: From these data we conclude that the specific cysteinyl LT1-receptor antagonist montelukast can inhibit PAF-induced eosinophil transmigration through cultured HUVEC monolayers.     

Interleukin-5 enhances eosinophil adhesion to bronchial epithelial cells

BACKGROUND: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types. OBJECTIVES: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion. METHODS: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied. RESULTS: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins. CONCLUSIONS: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.     

Increased eosinophil transmigration after nasal allergen challenge in children with allergic asthma and rhinitis

BACKGROUND: Eotaxin and interleukin-5 together provide the signal essential for eosinophil transmigration to airway tissue in allergic reactions. However, it is not known whether peripheral blood eosinophils (PBE) possess an increased transmigration capacity in vitro after allergen challenge in vivo before they leave the circulation. We aimed to determine whether PBE in cat-sensitized children have increased spontaneous and/or eotaxin-induced transmigration capacity in vitro, and to what extent allergen challenge alters this feature. METHODS: Fourteen cat-allergic children and four healthy controls underwent nasal challenge with cat-allergen. Blood samples were drawn prechallenge and at 2 h and 24 h postchallenge. We analyzed the in vitro transmigration of PBE, with and without eotaxin as a chemoattractant. We used a transmigration assay with fibronectin-coated membranes. Eosinophil cationic protein (ECP) and PBE counts were run in parallel. RESULTS: The spontaneous transmigration capacity of eosinophils in vitro was significantly higher at 2 h after allergen challenge (P < 0.01 vs. prechallenge) and returned to prechallenge levels at 24 h postchallenge (P < 0.02 vs. 2 h postchallenge). Addition of eotaxin further augmented the increased transmigration. In concordance, no accompanying changes were measured in the levels of eosinophils in blood or ECP in serum. Furthermore no spontaneous or eotaxin-induced eosinophil transmigration was detected in healthy controls. CONCLUSION: PBE possess increased spontaneous (and eotaxin-induced) capacity to transmigrate as early as 2 h after allergen challenge in allergic children, without accompanying signs of eosinophil activation in terms of increased PBE count or ECP level. This is probably due to the increased stage of activation of the eosinophil, often referred to as "priming".     

Eotaxin expression by epithelial cells and plasma cells in chronic asthma

Chemoattractants such as eotaxin are believed to play an important role in the recruitment of eosinophils into the airways in asthma. We investigated expression of eotaxin in the airway wall in a model of chronic human asthma, in which systemically sensitized mice were exposed to low mass concentrations of aerosolized antigen for 6 weeks. In these animals, the number of intraepithelial eosinophils in the airways was significantly increased 3 hours after exposure and declined by 24 hours. In parallel, immunoreactivity for eotaxin was strikingly up-regulated in airway epithelial cells and in inflammatory cells in the lamina propria. The latter were identified as plasma cells by double immunofluorescent labeling. Increased expression of eotaxin by epithelial cells and plasma cells was also demonstrated in a case of fatal human asthma. In contrast, sensitized mice that received a single exposure to a high mass concentration of aerosolized antigen exhibited delayed eosinophil recruitment, which did not correlate with eotaxin expression. Furthermore, in sensitized chronically exposed interleukin-13-deficient mice there was virtually no recruitment of eosinophils into the airways, although eotaxin expression was greater than or equal to that in wild-type mice. These results indicate that there are striking differences between acute and chronic exposure models in the time course of eotaxin expression and eosinophil recruitment. Although high eotaxin levels alone are not sufficient to cause recruitment of eosinophils into the airways, recurrent exposure may generate or up-regulate additional signals required for eosinophil chemotaxis.     

Expression of eotaxin in induced sputum of atopic and nonatopic asthmatics

BACKGROUND: The chemokine eotaxin has been implicated in airway eosinophilia in atopic asthma. We have compared airway eosinophils and eotaxin expression in induced sputum from well-matched atopic and nonatopic asthmatics. METHODS: Eosinophil numbers, eosinophil cationic protein (ECP), and the expression of eotaxin were examined in induced sputum from atopic asthmatics (AA = 11), nonatopic asthmatics (NAA = 11), and atopic (AC = 12) and normal (NC = 10) controls. Slides were prepared for differential cell counts by Romanowsky stain, and ECP levels were measured by RIA. Eotaxin expression was detected by in situ hybridization, with 35S-labelled riboprobes and immunocytochemistry. RESULTS: The numbers of eosinophils and ECP concentration were increased in the sputum of AA and NAA compared with AC and NC (P < 0.05). The numbers of eotaxin mRNA+ and immunoreactive cells were increased in NAA, but not AA, when compared with controls (P < 0.05). Eotaxin immunoreactive cells in NAA were significantly higher than in AA (P < 0.05). Eotaxin was expressed predominantly by macrophages, eosinophils, and epithelial cells. In NAA, but not AA, the numbers of eotaxin mRNA+ cells were correlated with histamine PC20 (r = -0.81, P < 0.01) and eosinophil numbers in sputum (r = 0.7, P < 0.05). CONCLUSIONS: Eotaxin production by macrophages, eosinophils, and epithelial cells may play a more pronounced role in airway eosinophilia in nonatopic than in atopic asthma.     

Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2

BACKGROUND: Epithelium is considered an active participant in allergic inflammation. Proteinase-activated receptor (PAR) 2 is expressed in a variety of cell types, including epithelial cells, and has been implicated in inflammation. OBJECTIVE: PAR-2-mediated activation of airway epithelial cells induces the release of mediators that could promote eosinophil survival and mediate eosinophil recruitment. METHODS: PAR-2-activating peptides were used to activate the human airway epithelial cell line A549, as well as primary cultures of small airway epithelial cells (SAECs). Human peripheral blood eosinophils were cultured in the presence or absence of epithelial cell supernatants. Survival was assessed by using an Annexin V apoptosis detection kit. GM-CSF and eotaxin were measured by using ELISA. RESULTS: Eosinophils undergo apoptosis in the absence of growth factors. Supernatants from PAR-2-activated A549 epithelial cells increased eosinophil survival. Supernatants from resting SAECs also increased eosinophil survival, but supernatants from PAR-2-activated SAECs showed a greater effect. The effect of PAR-2-activated epithelial cell supernatants on eosinophil survival was completely inhibited by a neutralizing anti-GM-CSF antibody but not an anti-IL-5 antibody. Resting A549 cells did not release any detectable GM-CSF, whereas PAR-2-activated cells released 35 pg/10(6) cells. Resting SAECs released 754.3 pg/10(6) cells of GM-CSF, which was further increased to 1360.5 pg/10(6) cells after PAR-2-mediated activation. Budesonide inhibited this PAR-2 effect. PAR-2-activated epithelial cells also released eotaxin. CONCLUSION: PAR-2-mediated activation of airway epithelial cells induced release of GM-CSF, which promoted eosinophil survival and activation. It also induced release of eotaxin, which could mediate eosinophil recruitment to the airways.     

Mast cells and T cells in Kimura''s disease express increased levels of interleukin-4, interleukin-5, eotaxin and RANTES

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.     

Transepithelial migration of activated eosinophils induces a decrease of E-cadherin expression in cultured human nasal epithelial cells

BACKGROUND: The damage of respiratory epithelium in allergic diseases has a close correlation with the extent of eosinophil infiltration. It seems to be a good possibility that eosinophil infiltration could induce the changes in the expression of the epithelial cell adhesion molecules, which play a key role in the maintenance of structural and functional rigidity of epithelium. OBJECTIVE: We observed the expression of E-cadherin in cultured human nasal epithelial cells (HNECs) to study whether could it be affected by transepithelial migration of inflammatory cells, especially eosinophils. METHODS: In vitro study of the transmigration assay was designed using various types of inflammatory cells and HNEC monolayers. Various assays of each experimental group were done under the stimulation of interleukin-5 (IL-5) and/or platelet activating factor (PAF). Subsequently immunohistochemistry for E-cadherin was performed in the HNECs. The intensity of immunofluorescence of E-cadherin was quantified using confocal laser scanning microscopy (CLSM) system and compared before and after the transmigration. RESULTS: The mean intensity of immunofluorescence for E-cadherin decreased significantly after the transmigration of any types of inflammatory cells. Above all, the migration of eosinophils treated with IL-5 and PAF had an eminent effect on the decrease, whereas the degranulation extracts derived from eosinophils activated by IL-5 and secretory IgA (sIgA) did not affect the intensity. CONCLUSION: This work suggests that transepithelial migration of inflammatory cells can directly induce the decrease in epithelial E-cadherin expression. Furthermore, the most prominent change was induced by transmigration of activated eosinophils, which might be caused by some mechanisms independent of the eosinophil contents. The decrease in E-cadherin expression may trigger the damage of epithelial barrier, which contributes to the pathogenesis of allergic diseases.     

The CC chemokine receptor antagonist met-RANTES inhibits eosinophil effector functions

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

Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation

The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.     

alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice

We investigated the roles of eosinophil infiltration and activation induced by the eosinophil-selective chemokine eotaxin, and of the expression of eosinophil alpha4 and beta2 integrins in causing bronchial hyperresponsiveness (BHR) in interleukin (IL)-5 CBA/Ca transgenic mice. These mice did not show BHR, despite the presence of some eosinophils in the lungs. Intratracheal mouse recombinant eotaxin (3 micrograms) did not induce BHR in wild-type mice. In IL-5 transgenic mice, eotaxin (3 and 5 micrograms) increased responsiveness at 24 h and increased eosinophils in bronchoalveolar lavage (BAL) fluid by 9.4- and 14-fold by 24 h, respectively, together with augmentation of eosinophil peroxidase activity and eosinophil infiltration in the airway submucosa. Using flow cytometry, the expression of alpha4, CD11b, and CD18 was upregulated in BAL, but not in blood, eosinophils. A rat anti-alpha4 antibody inhibited eotaxin-induced BHR and eosinophil migration and activation, but an anti-CD11b antibody had no significant effects on BHR. A combination of both antibodies was more effective. IL-5 and eotaxin synergize in the induction of BHR and airway eosinophilia, effects that are dependent on the induction of eosinophil alpha4 integrin. Expression of BHR depends on the recruitment and activation of eosinophils.     

The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca²⁺]_i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 μg/ml and 1 μg/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 μg/ml and 3 μg/ml. Moreover, eotaxin-induced [Ca²⁺]_i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca²⁺]_i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 μg/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 μg/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.     

Priming of eosinophil migration across lung epithelial cell monolayers and upregulation of CD11b/CD18 are elicited by extracellular Ca2+

In patients with asthma, eosinophils are primed and massively infiltrate lung tissues and migrate across epithelia into airways. Using blocking monoclonal antibodies, we found that eosinophil transmigration across a lung epithelial cell monolayer depended on the functions of alphaMbeta2 integrin CD11b/CD18. To study the role of Ca2+ in eosinophil priming and transepithelial migration, we treated eosinophils with eotaxin or thapsigargin (TG), reagents that increase cytoplasmic free Ca2+ concentrations by receptor- or nonreceptor-mediated mechanisms, respectively. Pretreatment of eosinophils with TG enhanced CD11b/CD18-dependent transmigration across lung epithelium. Within minutes, TG time- and dose-dependently upregulated the expression of CD11b/CD18 but did not upregulate the expression of alphaL (CD11a) or beta1 (CD29) integrin. The upregulation of CD11b/CD18 expression by eotaxin or TG was prevented when Ca2+ entry was blocked. The priming of eosinophil transmigration by TG was also abrogated by the blockade of Ca2+ entry. Our results indicate that induction of Ca2+ entry by the depletion of Ca2+ from intracellular stores upregulates CD11b/CD18 expression on eosinophils and primes eosinophil transmigration across lung epithelium. Both responses are therefore elicited by extracellular Ca2+. We suggest that, as an important priming signal for human eosinophil functional responses, store-operated Ca2+ entry may be one of the underlying mechanisms of eosinophilic inflammation in asthma.