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Background: Antenatal factors including maternal diet may predispose to airway disease, possibly by impacting on fetal airway development., Objective: This cohort study tested the hypothesis that maternal vitamin D and E status in early pregnancy is associated with airway epithelial cell (AEC) responses in new born infants and examined constitutive and TNFα/IL-1β, house dust mite (HDM) extract or lipopolysaccharide (LPS)-stimulated neonatal AEC responses in vitro., Methods: Maternal dietary vitamin D and E intakes (plasma 25[OH]D3 or α-tocopherol) were characterized at 10-12 weeks gestation. Neonatal nasal AECs were collected soon after birth and cultured to tertiary passage. Constitutive and stimulated - TNFα/IL-1β, HDM extract or LPS - secretory responses (VEGF, RANTES, MCP-1, IL-17A, IFN-γ, GM-CSF, eotaxin, MIP1-α, MIP1-β, ICAM, IL-6, IL-8, IL-10, TNF) in 139 AEC cultures were quantified., Results: AEC mediator release was greater following TNF-α/IL-1β, HDM or LPS stimulation compared to constitutive release. Increased maternal dietary vitamin D was associated with significant increases in IL-10 release by AEC after stimulation with TNF-α/IL-1β (P = 0.024) or HDM (P = 0.049). Maternal plasma α-tocopherol at 10-12 weeks gestation was positively associated with MIP1α (Spearman's rho 0.242, P = 0.009) and IL-3 (ρ 0.189, P = 0.043) responses after TNF-α/IL-1β stimulation and negatively associated with TNF (ρ -0.404, P = 0.011) and MIP1β (ρ -0.322, P = 0.046) responses after LPS stimulation., Discussion: Neonatal AECs respond to pro-inflammatory and allergenic stimuli in vitro demonstrating their potential to function as components of the innate immune response. Our findings suggest that associations exist between maternal micronutrient intake during early pregnancy and aspects of stimulated neonatal airway epithelial cell secretory function that may in turn impact on the development of asthma and/or allergic rhinitis in later life., (© 2015 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

Aims: This study tested inhibitory effects of in vitro Montelukast treatment on nasal airway epithelial cells (AEC) cultured from asthmatic patients treated with Montelukast with and without concomitant allergic rhinitis. We further examined the effect of Montelukast withdrawal in these patients on cytokine release from cultured nasal AEC., Methods: Nasal AEC were collected by brushings from subjects with a history of stable (no exacerbations or change in medication for ≥ 1 month) physician confirmed mild/moderate asthma whose asthma symptoms were documented to benefit from Montelukast treatment (NCT01230437). Release of the following mediators by nasal AEC were measured: IL-8, IL-6, IL-10, GM-CSF, RANTES, eotaxin and IFN-γ. Nasal AEC were cultured before and one week after withdrawal of their Montelukast treatment., Results: Forty two asthmatics were recruited. Nasal AEC were successfully cultured in 17 at the first assessment, 14 at the second assessment and in 10 individuals at both assessments. Nasal AEC release was no different between asthmatics with or without allergic rhinitis. Montelukast significantly suppressed the release of IL-8 (p = 0.016), IL-6 (p = 0.006), RANTES (p = 0.002) and IFN-γ (p = 0.046), in a dose dependent manner in unstimulated cultures but not in those stimulated with IL-1/TNF. Withdrawal of Montelukast treatment, was associated with increased IL-8 and RANTES secretion in unstimulated nasal AEC cultured from subjects with asthma and allergic rhinitis but not with asthma alone., Conclusions: Montelukast treatment for asthma symptoms reversibly suppresses nasal AEC release of pro-inflammatory mediators (i.e. IL-8 and RANTES) but only in those cells cultured from subjects with concomitant allergic rhinitis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Eosinophil accumulation in the lung is an important feature of airway inflammation in asthma. There is therefore much interest in developing novel therapies to prevent this process. Accumulating evidence suggests that statins have anti-inflammatory properties, including inhibition of leucocyte accumulation. We therefore assessed the ability of five statins to inhibit human eosinophil adhesion to recombinant human inter-cellular adhesion molecule (rhICAM)-1 under physiologically relevant flow conditions., Methods: Purified eosinophils were pre-treated with a panel of statins before elucidation of the adhesion profiles of resting and granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated cells to rhICAM-1-coated microchannels at a flow rate of 0.5 dynes/cm(2). Images were recorded in real-time at 1 min intervals and analysed using Ducocell software., Results: Fluvastatin and lovastatin (both 10 nm) significantly inhibited GM-CSF-stimulated eosinophil adhesion to rhICAM-1 after 2 min (34.4+/-3.0% inhibition and 37.8+/-12.6% inhibition, respectively, n=4, P<0.05) but had no significant inhibitory effect on unstimulated eosinophil adhesion. Mevastatin, simvastatin, and pravastatin (all 10 nm) had no significant effect on GM-CSF-stimulated eosinophil adhesion to rhICAM-1. A concentration range of fluvastatin and lovastatin inhibited GM-CSF stimulated eosinophil adhesion with significant (P<0.05) inhibition observed at low concentrations of 1 nm for both drugs. Mevalonate (100 nm) reversed fluvastatin-mediated but not lovastatin-mediated inhibition of eosinophil adhesion., Conclusions: Inhibition of eosinophil adhesion to ICAM-1 by fluvastatin and lovastatin under physiological shear stress represent novel actions by these drugs that may inform the development of anti-inflammatory therapy for allergic disease.

Eosinophils are major pro-inflammatory cells that make a major contribution to diseases that affect the upper and lower airways, skin and gastrointestinal tract. Interleukin (IL)-5 is central to their maturation and release from the bone marrow together with their subsequent accumulation and activation in the tissues. Mepolizumab is a humanized monoclonal antibody (mAb) with potent IL-5 neutralizing effects that represents a potential treatment for eosinophilic diseases. Several clinical trials with mepolizumab reported that treatment of patients with mild to severe asthma resulted in a substantial reduction in blood and sputum eosinophil numbers. However, clinical outcomes were disappointing as there were no significant effects on airway hyper-reactivity or the late asthmatic reaction to inhaled allergen challenge. More recently two studies, one in in patients with refractory eosinophilic asthma with a history of recurrent severe exacerbations and the other in patients with persistent sputum eosinophilia and symptoms despite systemic treatment with prednisone treatment, reported that monthly intravenous mepolizumab reduced sputum/blood eosinophilia, asthma exacerbations together with improvments in quality of life. Mepolizumab also appears to be an effective therapy for hypereosinophilic syndrome while other trials have shown efficacy of mepolizumab therapy in eosinophilic esophagitis. This review will consider the current status of the clinical development of mepolizumab for diseases with a significant eosinophilic component to their pathology.

The inappropriate accumulation of eosinophils and the subsequent release of their potent pro-inflammatory mediator arsenal are thought to be important contributors to the pathogenesis of asthma and other allergic diseases. It is also becoming apparent that eosinophils may play a role in the orchestration of immune responses in the asthmatic lung. There is therefore much interest in the development of strategies to limit or prevent eosinophil-induced toxicity. The mechanisms by which eosinophils accumulate in the peribronchial tissues of the lung are complex and include enhanced differentiation and release from the bone marrow, selective adhesion and transendothelial migration, directed movement in response to specific chemotactic mediators and finally prolonged survival as a consequence of delayed apoptosis. Thus it can be appreciated that there are many points at which the toxicity of eosinophils can be limited or even prevented. Important areas for potential advances in glucocorticoid (GC) development include efforts to dissociate their anti-inflammatory effects from unwanted side effects. Other areas include the development of humanized monoclonal antibodies against IL-4, IL-13 and IL-5 together with the inhibition of adhesion pathways and/or chemokines responsible for eosinophil accumulation in the asthmatic lung. Several avenues of research are currently underway in an attempt to define mechanisms by which pro-inflammatory cells such as eosinophils can be safely removed from the asthmatic lung through apoptosis induction and their subsequent ingestion by phagocytes. This review will discuss both the potential and shortcomings of these diverse approaches to limit eosinophil toxicity in the asthmatic lung.

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 microg/mL) coated capillary tubes using a microfluidic syringe pump at a precise and constant flow rate (1 dyn/cm(2)). 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 EC(50) 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 EC(50) 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.

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- (alphavbeta3, alphavbeta5, 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.

This study was carried out to investigate the relationship between induced sputum eosinophil apoptosis and clinical severity score, airway obstruction and symptom scores in patients with chronic stable asthma. Altogether, 41 chronic stable asthmatic subjects of varying severity defined by Aas score and 17 control subjects underwent spirometry, symptom questionnaire and successful sputum induction. Sputum was processed and cytospins prepared for light microscopy to determine normal and apoptotic eosinophils. Mild asthmatic subjects had a significantly lower percentage sputum eosinophils and a significantly higher eosinophil apoptotic ratio (AR) than moderate or chronic severe asthmatics. Severe asthmatic subjects had a significantly greater age, duration of asthma and sputum eosinophil count x mL(-1) than mild asthmatic subjects. Asthmatic subjects' symptom scores, severity scores and age inversely correlated with AR and the percentage of sputum eosinophils. Baseline forced expiratory volume in one second inversely correlated with percentage sputum eosinophils and positively correlated with AR. The study demonstrates a relationship between reduced sputum eosinophil apoptosis and increased clinical severity of chronic stable asthma, providing additional evidence that eosinophil apoptosis may be important in the resolution of eosinophilic airway inflammation in asthma.

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.

Anti-inflammatory therapy in asthma is reliant on corticosteroids, particularly in their inhaled form. However, steroids are rather non-specific in their actions and they also raise concerns regarding compliance and side-effect Issues. Furthermore, a small proportion of patients with asthma fail to respond to oral glucocorticoids even at high doses. This Article will review the role that steroids and membrane receptor ligation play in the induction of eosinophil apoptosis together with the mechanisms by which corticosteroids enhance the disposal of apoptotic eosinophils by both professional and non-professional phagocytes. Eosinophils are thought to be the major pro-inflammatory effector cell in asthma and their persistence in the airways is probably enhanced by the presence of several asthma-relevant cytokines that prolong eosinophil survival by inhibition of apoptosis (interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor, IL-9, IL-13, IL-15). In contrast, a number of signals have been described that accelerate apoptosis in human eosinophils including corticosteroids or ligation of membrane receptors (CD95, CD45, CD69). Thus, the load of lung eosinophils in asthmatic disease is likely to be related to a balance in the tIssue microenvironment between pro- and anti-apoptotic signals. Furthermore, removal of apoptotic eosinophils by phagocytosis by alveolar macrophages or bronchial epithelial cells in a specific receptor-mediated way is as important as the process of apoptosis induction. Corticosteroids enhance the recognition and engulfment of apoptotic eosinophils by macrophages or bronchial epithelial cells. Caspases are key intracellular molecules in the control of apoptosis and defects in caspase-induced apoptosis in eosinophils from steroid-resistant individuals may contribute to the molecular mechanisms underlying glucocorticoid insensitivity in these cells. These findings point the way to new and more targeted anti-inflammatory therapy for asthma and may provide important clues for the development of alternative therapies for glucocorticoid resistance.

Background: Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions., Objectives: The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro., Methods: Purified human peripheral blood CD34+ cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2TM light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation., Results: Positive immunostaining for MBP and ECP was detectable in a proportion (15-20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of approximately 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE., Conclusion: These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

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: We previously demonstrated receptor-mediated apoptotic-eosinophil engulfment by human small airway epithelial cells, which may represent a potentially important mechanism in the resolution of allergic and asthmatic inflammation., Objective: A549 cells were selected as being representative of alveolar epithelial cells, and their ability to ingest human apoptotic eosinophils was examined in terms of the effects of dexamethasone treatment and the receptor-mediated recognition mechanisms important in this process., Methods: A549 epithelial-cell expression of alpha(v)beta3, alpha(v)beta5, CD36, and the phosphatidylserine receptor was established by using immunostaining and flow cytometry, and inhibition assays were examined by using the role of these receptors in apoptotic-eosinophil recognition by resting and dexamethasone-treated A549 epithelial cells. Electron microscopy confirmed engulfment of apoptotic eosinophils, and receptor clustering around apoptotic eosinophils was examined by using immunofluorescent labeling., Results: A549 epithelial cells recognized and engulfed apoptotic eosinophils but not freshly isolated cells. Dexamethasone enhanced the number of A549 cells ingesting apoptotic eosinophils and dose dependently increased their capacity for ingestion. The tetrapeptide RGDS significantly inhibited apoptotic-eosinophil uptake by A549 cells, indicating a role for integrins in the recognition process. A549 cells expressed alpha(v)beta3, alpha(v)beta5, beta5, CD36, and the phosphatidylserine receptor, and expression of these receptors was not significantly increased after dexamethasone treatment. Engulfment of apoptotic eosinophils by A549 cells involved alpha(v)beta3-, CD36-, alpha(v)beta5-, and phosphatidylserine receptor-mediated recognition mechanisms and was associated with the formation of integrin focal adhesion complexes around apoptotic eosinophils., Conclusions: These data further suggest a nonpassive role for airway epithelium in the resolution of eosinophilic inflammation in asthma.

The eosinophil has a potent armory of proinflammatory mediators with considerable potential to initiate and sustain an inflammatory response. These include cytotoxic granule proteins, cytokines, chemokines, and lipid mediators. Eosinophils are considered important in the immune response to infection with helminthic parasitic worms. Incrementally increasing evidence supports a critical role for their proinflammatory activities in diverse human conditions, most notably in allergic diseases such as asthma. In these conditions severe tissue damage is a consequence of an inappropriate accumulation of eosinophils and the subsequent release of their highly toxic granule proteins. In addition, release of granule-associated products such as chemokines and cytokines at the sites of inflammation is likely to have significant paracrine and autocrine relevance. This review will update recent developments in understanding the role that eosinophil granule proteins play in human disease, particularly those of the respiratory tract.

Second generation antihistamines are recognised as being highly effective treatments for allergy-based disease and are among the most frequently prescribed and safest drugs in the world. However, consideration of the therapeutic index or the benefit/risk ratio of the H1 receptor antagonists is of paramount importance when prescribing this class of compounds as they are used to treat non-life threatening conditions. There are many second generation antihistamines available and at first examination these appear to be comparable in terms of safety and efficacy. However, the newer antihistamines in fact represent a heterogeneous group of compounds, having markedly differing chemical structures, adverse effects, half-life, tissue distribution and metabolism, spectrum of antihistaminic properties, and varying degrees of anti-inflammatory effects. With regard to the latter, there is growing awareness that some of these compounds might represent useful adjunct medications in asthma therapy. In terms of safety issues, the current second generation grouping includes compounds with proven cardiotoxic effects and others with the potential for adverse drug interactions. Moreover, some of the second generation H1 antagonists have given cause for concern regarding their potential to cause a degree of somnolence in some individuals. It can be argued, therefore, that the present second generation grouping is too large and indistinct since this was based primarily on the concept of separating the first generation sedating compounds from nonsedating H1 antagonists. Although it is too early to talk about a third generation grouping of antihistamines, future membership of such a classification could be based on a low volume of distribution coupled with a lack of sedating effects, drug interactions and cardiotoxicity.

Background: Eosinophils are important effector cells in asthma pathogenesis, and an understanding of the mechanisms involved in eosinophil apoptosis induction might thus be relevant to the resolution of asthmatic inflammation., Objective: Our aim was to determine the role of the common leukocyte antigen CD45 and the isoforms CD45RA, CD45RB, and CD45RO in human eosinophil apoptosis induction., Methods: Immmunostaining and flow cytometry were used to assess CD45 and CD45 isoform expression by eosinophils purified with use of density gradients and immunomagnetic negative selection. Apoptosis induction was measured by binding of fluorescein isothiocyanate-labeled annexin V to eosinophils cultured for 20 hours alone or with saturating quantities of mAb against CD45, CD45RA, CD45RB, CD45RO, CD9, CD11b, and isotype-matched controls in the presence or absence of GM-CSF., Results: Freshly isolated eosinophils had high expression of CD45 and CD45RO, modest expression of CD45RB, and low expression of CD45RA. Eosinophils cultured alone for 20 hours were found to be approximately 20% to 25% apoptotic. Incubation with mAb against CD45, CD45RA, and CD45RB resulted in significant (P <.005) enhancement (>100%) of their constitutive rate of apoptosis. Incubation with CD45RO, CD11b, CD9 mAb, or isotype controls had no significant effect on the rate of eosinophil constitutive apoptosis. The addition of GM-CSF inhibited eosinophil apoptosis but did not prevent CD45, CD45RA, or CD45RB mAb-dependent apoptosis induction., Conclusion: These data indicate that ligation of CD45, CD45RA, or CD45RB represents a novel pathway for the induction of apoptosis in human eosinophils.

Eosinophils, which are prominent cells in asthmatic inflammation, undergo apoptosis and are recognized and engulfed by phagocytic macrophages in vitro. We have examined the ability of human small airway epithelial cells (SAEC) to recognize and ingest apoptotic human eosinophils. Cultured SAEC ingested apoptotic eosinophils but not freshly isolated eosinophils or opsonized erythrocytes. The ability of SAEC to ingest apoptotic eosinophils was enhanced by interleukin-1alpha (IL-1alpha) or tumor necrosis factor alpha (TNFalpha) in a time- and concentration-dependent fashion. IL-1alpha was found to be more potent than TNFalpha and each was optimal at 10(-10) mol/L, with a significant (P <.05) effect observed at 1 hour postcytokine incubation that was maximal at 5 hours. IL-1alpha stimulation not only increased the number of SAEC engulfing apoptotic eosinophils, but also enhanced their capacity for ingestion. The amino sugars glucosamine, n-acetyl glucosamine, and galactosamine significantly inhibited uptake of apoptotic eosinophils by both resting and IL-1alpha-stimulated SAEC, in contrast to the parent sugars glucose, galactose, mannose, and fucose. Incubation of apoptotic eosinophils with the tetrapeptide RGDS, but not RGES, significantly inhibited their uptake by both resting and IL-1alpha-stimulated SAEC, as did monoclonal antibody against alphavbeta3 and CD36. Thus, SAEC recognize apoptotic eosinophils via lectin- and integrin-dependent mechanisms. These data demonstrate a novel function for human bronchial epithelial cells that might represent an important mechanism in the resolution of eosinophil-induced asthmatic inflammation.

Eosinophils play a protective role in host immunity to infections by parasitic worms and, detrimentally, are involved in the pathophysiology of asthma and other allergic diseases. Airway inflammation is central to the pathology of asthma and is characterized by infiltration of the bronchial mucosa by large numbers of proinflammatory cells, amongst which the eosinophil is prominent despite being a minority constituent of circulating leukocytes. Crucial steps in eosinophilic inflammation include augmented production of eosinophils in the bone marrow, their increased release into the circulation, and their selective accumulation in the conducting airways. The eosinophil has a potent armory of proinflammatory mediators, including cytotoxic granule proteins, cytokines and lipid mediators with considerable potential to initiate and sustain an inflammatory response. Thus there is much interest in the elucidation of the mechanisms responsible for eosinophil accumulation, persistence, activation and ultimate fate. This article reviews our current understanding of the role of the eosinophil in human disease and the immunobiology of this important proinflammatory cell.

The Bcl-2 family has been shown to be vital regulators of programmed cell death in numerous systems. To investigate the role of such proteins in the regulation of apoptosis of eosinophils, the expression of Bcl-2 and homologues Bcl-xL (death antagonists), Bax, and Bcl-xS (death agonists) were examined by immunoblot, flow cytometry, and reverse transcriptase-polymerase chain reaction analysis. Potential modulation of apoptosis-associated molecules during spontaneous apoptosis and in the presence of interleukin (IL)-5 was also investigated. Peripheral blood eosinophils were found to express constitutively Bax and Bcl-x, but Bcl-2 was absent. Analysis of mRNA revealed that the bcl-xL isoform predominated, although bcl-xS was also detectable. Spontaneous apoptosis due to culturing in the absence of cytokines for 24 h did not result in modulation of any of the Bcl-2 homologues examined. Culturing eosinophils in the presence of 100 pg/ml IL-5 for 24 h significantly reduced apoptosis (P < 0.01) to 10.7 +/- 2.6% compared with 46.8 +/- 7.4% in the absence of IL-5, and induced Bcl-2 mRNA and protein expression, with no detectable change in Bax, Bcl-x, or beta-actin as a control. This investigation indicates a specific profile of apoptotic molecules in eosinophils distinct from that of neutrophils, and indicates that survival-enhancing IL-5 modulates the expression of Bcl-2 in vitro.

Clearance of apoptotic neutrophils (polymorphonuclear leukocyte [PMN]) by macrophages is thought to play a crucial role in resolution of acute inflammation. There is increasing evidence that ingestion of apoptotic cells modulates macrophage behavior. We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself. Rat bone marrow-derived macrophages (BMDM) ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor (TNF) and attenuated by interferon (IFN)-gamma, interleukin (IL)-4, and IL-10. It was inhibitable by the tetrapeptide arg-gly-gln-ser (RGDS), therefore implicating the alphavbeta3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for 30 minutes, 48 hours before rechallenge reduced uptake of apoptotic PMN by 50% compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of preexposure. Comparable and sustained attenuation of uptake was obtained by ligating alphavbeta3 with the monoclonal antibody (MoAb), F11, after a delay of more than 90 minutes, whereas MoAbs to CD25 and CD45 had no effect. Ligation of alpha6beta1 and alpha1beta2, integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11. Therefore, apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.

The functional properties of infiltrating macrophages (Mphi) must be tightly regulated to facilitate appropriate responses to complex conditions in an inflammatory focus. This study was designed to ascertain whether uncommitted Mphi that have been exposed to combinations of cytokines with opposing functions develop properties dictated by one cytokine or by cytokine mixtures. Uncommitted rat bone marrow-derived Mphi (BMDMs) were incubated with IFN-gamma, TNF-alpha, TGF-beta, IL-4, IL-6, and IL-10 alone or sequentially in combinations. After 48 h, function was assessed by nitric oxide (NO) generation, uptake of apoptotic neutrophils, and beta-glucuronidase expression. IFN-gamma followed 4 h later by TNF-induced NO generation. The pretreatment of BMDMs before IFN-gamma priming with TNF, TGF-beta, and IL-4 suppressed NO generation by 87%, 92%, and 85%, respectively; IL-10 had no effect. The same cytokines administered at 4 h after IFN priming had no effect on NO generation. The uptake of apoptotic polymorphonuclear leukocytes was augmented by TNF (40% vs 29% controls; p < 0.05) and decreased by IFN-gamma, IL-10, and IL-4. The TNF response was unaffected by subsequent treatment with IFN-gamma, IL-4, or IL-10. Similarly, the decreased polymorphonuclear leukocyte uptake induced by IFN-gamma, IL-4, or IL-10 was unaffected by the subsequent addition of TNF. Beta-glucuronidase expression was increased by TGF-beta and decreased by IFN-gamma. These responses were not modified by cytokines with the opposing function. Thus, the functional response of BMDMs to complex mixtures of cytokines was determined by the first cytokine to which they were exposed. Once activated, BMDMs become unresponsive to alternative activating signals, a finding which has obvious implications for Mphi function in vivo.

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells. Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS.

The antiproliferative effect of BCX-34 was tested in normal human peripheral blood mononuclear cells (PBMCs) induced to proliferate with OKT3, tetanus toxoid, the mixed lymphocyte reaction, or IL-2. In the case of OKT3, tetanus toxoid, or the MLR the IC50s ranged between 0.7 and 4 microM. With IL-2, the IC50 was 14.6 microM. In T-cells purified by rosetting the IC50 with IL-2 was 0.62 microM. In CD4 or CD8 cells obtained by magnetic activated cell sorting the IC50s with IL-2 were 0.24 and 0.62 microM, respectively. BCX-34 inhibition of proliferation in human PBMCs may not depend entirely upon the accumulation of intracellular dGTP because tetanus toxoid-induced proliferation was inhibited in the absence of deoxyguanosine and was not reversed by deoxycytidine. BCX-34 did not inhibit IL-2 release from PBMCs and did not alter PBMC viability. The results of these studies show that BCX-34 is a potent inhibitor of normal human T-cell proliferation induced by antigenic or IL-2 stimulation. BCX-34 in normal human T-cells has a deoxyguanosine-independent mechanism to suppress in vitro proliferation. BCX-34 appears to have little effect on T-cell viability. The data suggest that BCX-34 may be useful in the treatment of T-cell proliferative disorders.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

A series of 94 benzoic acid derivatives was synthesized and tested for its ability to inhibit influenza neuraminidase. The enzyme-inhibitor complex structure was determined by X-ray crystallographic analysis for compounds which inhibited the enzyme. The most potent compound tested in vitro, 5 (4-acetylamino)-3-guanidinobenzoic acid), had an IC50 = 2.5 x 10(-6) M against N9 neuraminidase. Compound 5 was oriented in the active site of the neuraminidase in a manner that was not predicted from the reported active site binding of GANA (4) with neuraminidase. In a mouse model of influenza, 5 did not protect the mice from weight loss due to the influenza virus when dosed intranasally.

Background: Eosinophil autocrine generation of viability-promoting cytokines resulting from interaction with extracellular matrix (ECM) proteins may be important in promoting their persistence in allergic inflammation., Objective: We examined eosinophil interaction with fibronectin, laminin, collagen, fibrinogen, hyaluronate, or vitronectin in terms of adhesion and for their ability to promote prolonged eosinophil survival by autocrine generation of IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor (GM-CSF). The effect of dexamethasone was examined because it may inhibit integrin-dependent autocrine generation of viability-enhancing cytokines., Methods: Microtiter wells were coated with different ECM proteins, and assays were performed to determine their levels of eosinophil adhesion, and tests were performed to determine their prolonged survival in culture. The receptors involved in these processes, the cytokines generated, and the effect of a dose range of dexamethasone were all examined., Results: Eosinophils cultured for 3 days on laminin or tissue fibronectin exhibited dose-dependent prolonged survival, which was not seen with any other ECM protein. Unstimulated eosinophils adhered to wells coated with laminin or fibronectin but not to wells coated with any of the other ECM proteins that were tested. Laminin-dependent eosinophil adhesion and prolonged survival were inhibited by specific monoclonal antibodies to beta1 and alpha6beta1. Laminin-dependent survival was inhibited by a neutralizing antibody to GM-CSF, an anti-IL-5 antibody achieved a measurable but insignificant inhibition of survival, and an antibody to IL-3 had no effect. Increasing concentrations of dexamethasone (maximal at 10(-7) mol/L) inhibited both laminin- and fibronectin-dependent enhanced eosinophil viability at 3 days. Eosinophils cultured on either laminin or fibronectin for 24 or 72 hours generated picogram quantities of GM-CSF, which were inhibited by the addition of dexamethasone (10(-7) mol/L)., Conclusion: We have confirmed that eosinophils adhere to laminin in an alpha6beta1-dependent manner, and this interaction results in the autocrine generation of GM-CSF, which enhances eosinophil survival in culture. Both laminin and tissue fibronectin-dependent autocrine GM-CSF generation were inhibited by physiologically relevant concentrations of dexamethasone. This may represent an important mechanism by which glucocorticoids reduce the tissue eosinophilia in allergic disease.

Eosinophils, with their potentially harmful armoury of toxic products, have available a mechanism by which they can be cleared from the tissues while still intact in the absence of an undesirable pro-inflammatory response. However, our understanding of the induction and control of apoptosis in eosinophils is still incomplete and a great deal of work in this area remains to be done. In other cell types, important regulators of apoptosis have been well characterized. These include the proto-oncogene bcl-2 and related molecules, the family of proteases which share homology with IL-1 beta converting enzyme (ICE) and another protein family which interact with products of the proto-oncogene c-myc. These regulators act at various points on the apoptosis pathway and it appears their interactions are vital in determining whether a cell will survive or die. In particular, the ICE-like proteases have been implicated as a universal apoptosis effector which is associated with the downstream events of programmed cell death [62]. Whether these molecules are involved in the cell death pathway(s) of eosinophils remains to be determined. Much of eosinophilic inflammation might be a result of defects in pathways controlling eosinophil apoptosis and/or their clearance by phagocytes resulting in necrosis and the release of toxic products. A greater understanding of the processes involved in the induction and control of eosinophil apoptosis might provide novel approaches for therapeutic intervention.

9-(3-Pyridylmethyl)-9-deazaguanine (BCX-34), a new purine nucleoside phosphorylase inhibitor, has selective immunosuppressive activity with potential therapeutic value in T-cell-mediated disease. We now report a sensitive, specific and reproducible method for measurement of 9-(3-pyridylmethyl)-9-deazaguanine in biological fluids using high-performance liquid chromatography (HPLC). 9-(3-Pyridylmethyl)-9-deazaguanine was extracted from plasma using perchloric acid precipitation followed by passage through Sep-Pak C18 cartridges (average extraction efficiency, 64.6%). Standard curves were linear over the range of interest (28-1120 ng/ml in plasma and 200-4000 ng/ml in urine, r2 > 0.999). Within-day and between-day coefficients of variation were less than 8%. The limit of quantitation was 28 ng/ml in plasma and 200 ng/ml in urine. This HPLC method should be useful in future clinical studies with this drug.

Considerable progress has been made in our understanding of the molecular mechanisms involved in eosinophil migration into sites of allergic inflammation. A number of differences between eosinophils and neutrophils have been observed in their pattern of adhesion interactions. These include expression of alpha 4 beta 1, alpha 4 beta 7, and alpha 6 beta 1 by eosinophils but not neutrophils and structural differences between eosinophil and neutrophil PSGL-1 associated with increased eosinophil binding to P-selectin. On the basis of current evidence the various receptors and mediators involved are summarized in FIGURE 1. Once in the tissues eosinophils may persist for several days or weeks, surviving under the influence of locally generated cytokines and this persistence may also partly explain selective tissue accumulation of eosinophils. Understanding the molecular mechanisms involved in leucocyte migration offers the possibility of selective and effective antagonists to treat allergic disease by preventing cell migration. Results in a number of animal models already offer the hope that this approach may be successful. The development of drugs that can be tested in the clinic are awaited with considerable interest.

BCX-34 inhibits RBC PNP in vitro from humans, rats, and mice with IC50S ranging from 5 to 36 nM. BCX-34 also, in the presence but not in the absence of deoxyguanosine, inhibits human CCRF-CEM T-cell proliferation with an IC50 of 0.57 microM but not rat or mouse T-cell proliferation up to 30 microM. Inhibition of human T-cell proliferation is accompanied by an accumulation of intracellular dGTP with an associated reduction in GTP. These nucleotide changes do not occur in BC16A mouse T-cells and explain why proliferation is not inhibited by PNP inhibitors in this case. Reduction in intracellular GTP is not essential for the antiproliferative action of BCX-34. Oral bioavailability of BCX-34 in rats is 76%. BCX-34 is orally active in elevating plasma inosine in rats (2-fold at 30 mg/kg), in suppressing ex vivo RBC PNP activity in rats (98% at 3 h. 100 mg/kg), and in suppressing ex vivo skin PNP in mice (39% at 3 h, 100 mg/kg). The results demonstrate that BCX-34 inhibits human PNP and T-cell proliferation, is orally bioavailable in rodents, and pharmacologically active in vivo in rodents after oral dosing with no apparent side effects or toxicity. BCX-34 may, therefore, be useful in treating human T-cell proliferative inflammatory disorders.

We have investigated the contribution of integrin alpha 4 beta 7 to human peripheral blood eosinophil adhesive interactions. Immunofluorescence and flow cytometry demonstrated constitutive expression of alpha 4 beta 7 by eosinophils. Expression of alpha 4 beta 7 or alpha 4 beta 7 was not enhanced by eosinophil activation with platelet-activating factor (PAF). Expression of alpha 4 beta 7 was confirmed by immuno-precipitation of 125I-labeled lysates analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Approximately 20% of unstimulated eosinophils were adherent to L1-2 cells transfected with vascular cell adhesion molecule-1 (VCAM-1) cDNA, while very few resting eosinophils adhered to mouse mucosal adressin cell adhesion molecule-1 (MAdCAM-1) transfectants. Binding of unstimulated eosinophils to VCAM-1 transfectant was inhibited by HPI 2 (an antibody that blocks both alpha 4 beta 1 and alpha 4 beta 7 functions), but not Act-1, and alpha 4 beta 1 monoclonal antibody (mAb). PAF stimulation resulted in increased binding of eosinophils to MAdCAM-1 transfectants, which was inhibited by both HPI 2 and Act-1. In contrast, PAF did not enhance binding to VCAM 1 transfectants, although binding of PAE-stimulated eosinophils to VCAM-1 could be partially inhibited by Act-1. Stimulation of eosinophils with the beta 7-activating mAb TS2 16 resulted in enhanced binding of eosinophils to both VCAM-1 and MAdCAM-1 transfectants. The increased binding was largely alpha 4 beta 7-dependent. Unstimulated eosinophils bound to soluble recombinant human (rh) VCAM-1 and fibronectin (Fn), coated on 96-well plates in dose-dependent manner. Binding was inhibited by HPI-2 and 4b4, an anti-beta 1 mAb, but not by Act-1. TS2 16 treatment increased adherent cell numbers and this enhanced binding was inhibited by Act-1. We have therefore confirmed that alpha 4 beta 7 is functionally active on unstimulated eosinophils. In contrast, PAF-induced enhancement of eosinophils binding to VCAM-1 or MAdCAM-1 was alpha 4 beta 7-dependent. In addition treatment with TS2 16 resulted in a alpha 4 beta 7-dependent enhancement of eosinophil binding to VCAM-1, MAdCAM-1 and Fn. We therefore hypothesize that alpha 4 beta 7 may have an important role in eosinophil localization in diseases such as asthma and inflammatory bowel disease.

Our recent studies have indicated an important role for P-selectin in eosinophil adhesion. We have therefore compared eosinophil and neutrophil binding with nasal polyp endothelium as well as purified P-selectin. We have also compared the structure and expression of the eosinophil and neutrophil P-selectin ligands. Using the frozen section assay, eosinophils bound to 2-fold more blood vessels within the nasal polyp tissue than neutrophils. Up to 10-fold more eosinophils than neutrophils bound per unit length of endothelium. Neutrophil and eosinophil binding was inhibited by a mAb against P-selectin and a P-selectin chimera which binds to the P-selectin ligand. Eosinophils bound with approximately 2-fold greater avidity to purified P-selectin under flow conditions. Using SDS-PAGE we characterized the eosinophil P-selectin ligand as a sialylated, homodimeric glycoprotein consistent with the known structure of PSGL-1. However, expression of PSGL-1 by eosinophils was significantly greater than on neutrophils. The eosinophil ligand had a calculated molecular mass by SDS-PAGE of approximately 10 kDa greater than the neutrophil ligand, which was not due to differences in N-glycosylation. Eosinophils expressed the 15-decapeptide repeat form of PSGL-1 compared with neutrophils that have the 16-decapeptide repeat form. The increased binding of eosinophils, compared with neutrophils, to P-selectin in both an ex-vivo and in vitro assay suggests that P-selectin may have a role directing the specific migration of eosinophils in diseases such as asthma. The increased avidity may be due to increased expression of PSGL-1 by eosinophils, differences in the peptide backbone, or post-translational modifications.

Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti-CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF--cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo.

Background: Eosinophil-derived inflammatory mediators including cytokines are considered to be important in the pathogenesis of allergic inflammation. Fibronectin (Fn) has been shown to be a physiological trigger of autocrine cytokine production by human eosinophils. Fn is encoded by a single gene, but alternate splicing of the primary RNA transcript results in polypeptide diversity in a cell type-specific fashion. Thus, tissue Fn contains approximately 50% more of the CS-1 cell binding region recognized by the integrin alpha 4 beta 1 compared with plasma Fn., Objective: Since eosinophils are predominantly tissue-dwelling cells we compared the effect of tissue and plasma Fn on eosinophil survival in culture., Methods: The viability and cytokine generation of eosinophils (> 99.9% pure) cultured for up to 4 days in 96 well plates coated with tissue Fn, plasma Fn or BSA was compared., Results: There was a significant difference in the ability of tissue Fn to support eosinophil survival compared with plasma Fn (P < 0.01). Optimal survival with tissue Fn was seen at 25 micrograms/well (70% +/- 2.0% viability at 3 days vs 7% +/- 2.2% viability on BSA). Significant (P < 0.001) cell viability on tissue Fn was observed for up to 4 days in culture (54% +/- 6.0%) compared with BSA coated wells. Addition of autologous mononuclear cells (final concentration 0.5%, 1% or 2%) resulted in plasma Fn-dependent eosinophil survival comparable to that of 99.9% pure eosinophils adherent to tissue Fn. Tissue Fn-dependent survival was significantly inhibited by anti-interleukin-3, anti-granulocyte macrophage colony stimulating factor (GM-CSF) and anti-IL-5 monoclonal antibodies. Picogram quantities of these three cytokines were detected in supernatants from eosinophils cultured for 3 days on tissue Fn using specific enzyme-linked immunosorbent assays (ELISAs). Eosinophil survival on tissue Fn was significantly inhibited by anti-beta 1 and alpha 4 beta 1 monoclonal antibody (MoAb) and also by a MoAb specific for the CS-1 motif in the IIICS region of Fn., Conclusion: These observations show preferential survival of eosinophils cultured on tissue Fn as a result of alpha 4 beta 1-dependent interaction with the CS-1 region of tissue Fn triggering autocrine cytokine synthesis and release, thereby promoting their survival and persistence within the tissues.

Eosinophil adhesion has been studied in some detail in recent years, and a number of interesting observations have emerged. As with other aspects of eosinophil biology, there appears to be a greater similarity with basophils than with neutrophils in their pattern of adhesion interactions. A number of important differences with respect to neutrophils have emerged, which could be exploited for the treatment of eosinophil-mediated disease, including the observations that eosinophil adhesion can be modulated by selective cytokines such as IL-5 and that eosinophils, unlike neutrophils, express VLA-4 and alpha 4/beta 7. There is also tantalizing evidence emerging that eosinophils interact differentially with the selectins, with differing degrees of affinity of binding and possibly different counterreceptors. The extent to which these observations will be useful in treating allergic disease remains to be seen.

Considerable progress has been made in our understanding of the molecular mechanisms involved in eosinophil and basophil migration into sites of allergic inflammation. It is clearly a staged process, each stage offering a level of control over the cell specificity and degree of migration. On the basis of current evidence, the various receptors and mediators involved are summarized in Table 4. Once in the tissues, eosinophils may persist for several days or weeks, surviving under the influence of locally generated cytokines, and this persistence may also partly explain the selective tissue accumulation of eosinophils and basophils. Understanding the molecular mechanisms involved in leukocyte migration may lead to the discovery of selective and effective antagonists to treat allergic disease by preventing cell migration. Results in a number of animal models already suggest that this approach may be successful. The development of drugs that can be tested in the clinic is awaited with much interest.

Tissue eosinophilia is a characteristic feature of a number of inflammatory diseases including asthma and nasal polyposis. Eosinophil migration into tissues is controlled in part by interactions between eosinophil adhesion receptors and counter-structures on the vascular endothelium. To determine the receptors used by eosinophils to adhere to vascular endothelium in allergic inflammation we have adapted the Stamper-Woodruff frozen section assay (FSA) to study eosinophil adhesion to nasal polyp endothelium. Immunohistology indicated that intercellular adhesion molecule 1 (ICAM-1), E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas expression of vascular cell adhesion molecule 1 (VCAM-1) was weak or absent. Unstimulated human peripheral blood eosinophils adhered specifically to nasal polyp endothelium. Adherence was temperature and divalent cation-dependent and saturable at cell densities > 5 x 10(6) cells/ml. Eosinophil adhesion was almost completely inhibited by a monoclonal antibody (mAb) against P-selectin and by a chimeric molecule consisting of the Fc portion of human IgG and the lectin binding domain of P-selectin, which binds to the P-selectin ligand on leucocytes. Anti-Mac-1 mAb partially inhibited eosinophil adhesion whereas mAb against E-selectin, L-selectin, ICAM-1, VCAM-1, very late activation antigen 4, and lymphocyte function-associated antigen 1 had no effect. P-selectin is stored in intracellular granules within the endothelial cell and in vitro is only transiently expressed. To determine if P-selectin was expressed on the membrane of the nasal polyp endothelium we compared P-selectin expression in normal skin and nasal polyps after acetone fixation, which permeabilizes cells, and paraformaldehyde, which only allows staining of membrane expressed receptors. In the skin, good expression was seen with acetone fixation but no expression was seen after paraformaldehyde treatment, whereas in nasal polyps, similar expression was observed with both fixatives. In addition immunofluorescence with confocal microscopy demonstrated lumenal staining of nasal polyp endothelium indicating that P-selectin was located on the surface of endothelial cells while in skin only an intracellular granular distribution was apparent. Lastly, whereas eosinophils bound consistently to nasal polyp endothelium, no binding was observed to blood vessels in normal skin further supporting the idea that eosinophils were binding to membrane expressed and not intracellular P-selectin. The importance of P-selectin in eosinophil adhesion to nasal polyp endothelium suggests that P-selectin antagonists may be effective at inhibiting eosinophil accumulation at sites of allergic inflammation.

Human peripheral blood eosinophils adhered specifically to microtitre plates coated with plasma fibronectin (Fn) in a dose- and time-dependent fashion. Adhesion was optimal at 60 min at a concentration of 100 micrograms/ml. Adherence to Fn was up-regulated by platelet-activating factor (PAF; optimum concentration of 10(-6) M) and was significantly inhibited by a polyclonal anti-Fn antibody (P < 0.05). The following evidence suggested that eosinophil adhesion to Fn was mediated by alpha 4 beta 1: (1) eosinophil adherence to Fn was not inhibited by an Arg-Gly-Asp-Ser (RGDS) synthetic peptide; (2) there was a dose-dependent adherence of eosinophils to microtitre plates coated with the 40,000 MW proteolytic fragment of Fn that contains the CS-1 alpha 4 beta 1 binding region, whereas adherence to the 120,000 MW chymotryptic fragment of Fn, which contains the RGD-dependent binding site, was weak and only observed at high concentrations (> 250 micrograms/ml); (3) significant inhibition of eosinophil adherence to Fn was achieved by monoclonal antibodies (mAb) against the alpha chain of VLA-4 but not by a mAb against CD45 or a mouse myeloma antibody as negative controls. After adhesion to Fn, eosinophils were investigated for their capacity to release leukotriene C4 in response to stimulation with a suboptimal concentration of calcium ionophore (2 x 10(-6) M). Significant enhancement of release was detected with Fn-coated plates but not with the control bovine serum albumin (BSA) (P < 0.01). Furthermore, this enhancement was significantly inhibited by the alpha 4 beta 1 mAb HP2/1 (P < 0.05) but not by an anti-CD45 mAb. From these studies we conclude that (1) alpha 4 beta 1 (VLA-4) integrin is a major receptor for Fn on human eosinophils and (2) adhesion to Fn may prime eosinophils for mediator release during allergic inflammation.

We have investigated the effect of adhesion to fibronectin (Fn) on the survival of eosinophils in culture. Peripheral blood eosinophils from normal human donors were separated by immunomagnetic selection and cultured in RPMI on Fn- (100 micrograms/ml) coated microtiter plates for up to 96 h. Survival was measured by trypan blue exclusion. There was a significant enhancement of eosinophil survival with Fn as compared with both bovine serum albumin-coated and uncoated wells (p < 0.05-0.01). Fn-induced eosinophil survival was comparable to that obtained with exogenous interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and was inhibitable by antibodies against Fn, very late antigen 4 (VLA-4), IL-3, and GM-CSF. Supernatants from Fn-, but not BSA-coated wells contained picogram amounts of IL-3 and GM-CSF, and eosinophils cultured on Fn for 24 h expressed mRNA for GM-CSF as determined by in situ hybridization. Therefore, Fn prolongs eosinophil survival in culture by triggering autocrine generation of cytokines by eosinophils. Since neutrophils lack VLA-4, this could provide a partial explanation for the preferential accumulation of eosinophils at sites of allergic inflammation, as well as the predominant tissue localization of eosinophils in healthy individuals.