Airway inflammation resolution.
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Apoptosis↗
Biomedical subjects
G M Walsh
Publications and source records attributed to G M Walsh.
A new antihistamine levocetirizine inhibits eosinophil adhesion to vascular cell adhesion molecule-1 under flow conditions.
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.
Antibodies, Monoclonal↗
Control of eosinophil toxicity in the lung.
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.
Animals↗
Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells.
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.
Apoptosis↗
Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors.
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.
Adult↗
Eosinophils from patients with asthma express higher levels of the pan-leucocyte receptor CD45 and the isoform CD45RO.
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.
Adolescent↗
Reduced eosinophil apoptosis in induced sputum correlates with asthma severity.
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.
Adult↗
Corticosteroids, eosinophils and bronchial epithelial cells: new insights into the resolution of inflammation 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.
Apoptosis↗
Cetirizine and levocetirizine inhibit eotaxin-induced eosinophil transendothelial migration through human dermal or lung microvascular endothelial cells.
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.
Acetates↗
Eosinophil-epithelial cell interactions: a special relationship?
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Cell Communication↗
Human alveolar epithelial cells engulf apoptotic eosinophils by means of integrin- and phosphatidylserine receptor-dependent mechanisms: a process upregulated by dexamethasone.
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.
Apoptosis↗
Eosinophil granule proteins and their role in disease.
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.
Blood Proteins↗
New insights into the second generation antihistamines.
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.
Anti-Asthmatic Agents↗
Comparing the H1 profile of second-generation antihistamines.
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Cetirizine↗
The clinical relevance of the anti-inflammatory properties of antihistamines.
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Anti-Inflammatory Agents, Non-Steroidal↗
Eosinophil apoptosis: mechanisms and clinical relevance in asthmatic and allergic inflammation.
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Apoptosis↗
Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic 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.
Acetylglucosamine↗
Previous uptake of apoptotic neutrophils or ligation of integrin receptors downmodulates the ability of macrophages to ingest apoptotic neutrophils.
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.
Animals↗