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Notes: Background We previously demonstrated that low concentrations of a new antihistamine levocetirizine inhibited eosinophil transmigration through human microvascular endothelial cells.Objective Here, the inhibitory effect of levocetirizine on eosinophil adhesion to recombinant human vascular cell adhesion molecule-1 (rhVCAM)-1 was examined under conditions of shear stress using an in vitro model of the post-capillary venules.Methods Eosinophils isolated from normal subjects were pre-incubated with a concentration range of levocetirizine (10−6–10−10 m) or negative dilution control. Resting or granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated cells were pumped through rhVCAM-1 (10 μg/mL) coated capillary tubes using a microfluidic syringe pump at a precise and constant flow rate (1 dyn/cm2). Images of rolling and firmly adherent eosinophils were captured using real-time video microscopy.Results Levocetirizine significantly inhibited resting eosinophil adhesion to rhVCAM-1 with maximal effect at 10−8 M with an EC50 of 10−9 m. Levocetirizine almost abolished resting eosinophil adhesion by the 15 min time-point. GM-CSF significantly enhanced eosinophil adhesion and their ability to flatten on rhVCAM-1. Both phenomena were inhibited by levocetirizine in a dose-dependent manner, at both 5 and 15 min (optimal concentration of 10−8 m with an EC50 of 10−9 m). Real-time imaging revealed that the effect of levocetirizine on post-adhesion behaviour (detachment, flatness) contributed to its inhibitory action on eosinophil adhesion to rhVCAM-1. In contrast, very late antigen (VLA)-4 mAb inhibited eosinophil adhesion to rhVCAM-1 from the earliest time-points.Conclusion Physiologically relevant concentrations of levocetirizine inhibit resting and GM-CSF-stimulated firm eosinophil adhesion to rhVCAM-1 under flow conditions.

Notes: Background We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils.Objectives To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes.Results Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (αvβ3, αvβ5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors.Conclusion These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.

Notes: Immunological release of histamine and lipid mediators is known to occur when basophils, contained in whole blood human leucocytes, are incubated with anti-IgE (reversed anaphylaxis). In the present study we show that IgE-dependent stimulation of basophils was associated with activation of bystander eosinophils and neutrophils, as assessed by enhanced complement (C3b) and IgG (Fc) rosettes, and increased cytotoxicity for complement-coated schistosomula of Schistosoma mansoni. These changes in eosinophil and neutrophil function were totally inhibited in a dose-dependent fashion by prior incubation with disodium cromoglycate (DSCG). In all in vitro systems examined, complete inhibition of enhancement was observed with concentrations as low as 10-7 moles/1. In contrast, DSCG had no effect on histamine release, or the percentage of rosettes or cytotoxicity prior to anti-IgE stimulation. These results suggest that DSCG inhibits activation of inflammatory cells consequent to an IgE-dependent stimulus.

Notes: 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 °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 °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.

Notes: Background Eosinophils and their secreted mediators are heavily implicated as effector cells in asthma and other allergic diseases. Comparisons were made between expression of CD45, CD45RA, CD45RB and CD45RO by eosinophils from asthmatic patients and non-asthmatic atopic and non-atopic, non-asthmatic control subjects.Methods Twenty-seven patients with asthma and 33 control subjects were recruited for the study. Eosinophil expression of CD45, CD45RA, CD45RB and CD45RO was established by immunostaining and flow cytometry was performed on whole leucocyte samples. Eosinophil apoptosis in response to CD45 and CD45 isoform monoclonal antibody (mAb)-dependent receptor ligation was assessed by binding of annexin V and flow cytometry.Results Eosinophils from patients with asthma expressed significantly (P〈0.05) higher levels of pan-CD45 and CD45RO compared with eosinophils from non-asthmatic, non-atopic subjects. No significant correlations were found between expression of either pan-CD45 or CD45RO and the degree of symptoms in the asthmatic patients as defined by lung function (FEV1 and FEF25–75) and methacholine PD20. Increased expression of pan-CD45 or CD45RO did not appear to be a consequence of the atopic phenotype. Higher expression of pan-CD45 or CD45RO by eosinophils from asthmatic patients was not associated with greater sensitivity to CD45 and CD45RO mAb receptor ligation-induced eosinophil apoptosis.Conclusion Higher expression of CD45 and CD45RO by eosinophils from asthmatic patients appeared to be a consequence of asthma rather than atopy and further supports a role for activated eosinophils in asthma.