Garry M. Walsh

Nasal Epithelial Cells as Surrogates for Bronchial Epithelial Cells in Airway Inflammation Studies

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.

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 ahererogeneous 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.

Effects of nedocromil sodium (Tilade®) on the activation of human eosinophils and neutrophils and the release of histamine from mast cells

The ability of nedocromil sodium, a new anti‐inflammatory agent for the treatment of asthma, to inhibit activation of human eosinophils and neutrophils in vitro, has been studied using an adherence reaction (the “rosette” technique) as well as a cytotoxicity assay. We have also investigated the capacity of nedocromil sodium to inhibit IgE‐ dependent histamine release from human lung mast cells. The drug was a potent inhibitor (IC50 approx 5 × 10‐9M) of fMLP‐induced enhancement of eosinophil and neutrophil complement (C3b) and IgG (Fc) rosettes. There was also a comparable inhibition of enhancement, by fMLP, of eosinophil and neutrophil cytotoxicity (for complement‐coated schistosomula of Schistosoma mansoni). Although nedocromil sodium also inhibited histamine release from human lung mast cells in a dose‐dependent fashion its activity was relatively weak (IC30 5 × 10‐6M) compared to its effect on granulocytes. These experiments support the view that the principal mode of action of nedocromil sodium is its capacity to inhibit the activation of inflammatory cells.

Advances in the Immunobiology of Eosinophils and Their Role in Disease

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.

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).

Eosinophil apoptosis: mechanisms and clinical relevance in asthmatic and allergic inflammation.

The eosinophil possesses a considerable array of histotoxic substances which contribute to the initiation and maintenance of the allergic inflammatory response; these include cytotoxic granule proteins, cytokines and lipid mediators, each of which plays a substantial though differing role. Although eosinophils are thought to be important in adaptive immunity to helminthic parasitic worms, there is now a wide consensus that eosinophil-derived products make a major contribution to allergic and asthmatic disease (Walsh, 1999). Infiltration of the bronchial mucosa by large numbers of proinflammatory cells is characteristic of the airway inflammation which is central to the pathogenesis of all forms of asthma. Despite being a minority constituent of circulating leucocytes, the eosinophil is a prominent feature of this infiltrate. Indeed, it has recently been suggested that there is as much as a 50to 100-fold increase in the accumulation in eosinophils over neutrophils in the airways of patients with asthma (Wardlaw, 1999). Importantly, there are many studies which have demonstrated a correlation between asthma severity and levels of eosinophils and their products in blood, induced sputum and in bronchial biopsy or lavage samples. Moreover, eosinophil granule-derived mediators are heavily implicated in bronchial epithelial cell damage; this leads to cilial dysfunction and cell loss, both of which are thought to be central to the development of bronchial hyper-responsiveness, one of the cardinal features of asthma (Bousquet et al, 1990). Recently, attention has focused on increasing our understanding of the factors involved in eosinophil clearance, not least because dissection of the factors which facilitate the apoptosis-induced clearance of eosinophils from the lung and their subsequent engulfment by phagocytes is a rational therapeutic aim for asthma. In the time since my previous reviews of the subject of eosinophil apoptosis (Walsh, 1997a, b), there has been a great increase in our knowledge, with the publication of some interesting and insightful studies. The current review is aimed at updating the field of eosinophil apoptosis and clearance ± including the relationship with prolonged eosinophil survival ± and will also discuss the relevance of these processes to allergic and asthmatic inflammation. EOSINOPHIL APOPTOSIS AND RECOGNITION BY PHAGOCYTES

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells

Background We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils.

Effect of disodium cromoglycate on activation of human eosinophils and neutrophils following reversed (anti‐IgE) anaphylaxis

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

Ligation of CD45 and the isoforms CD45RA and CD45RB accelerates the rate of constitutive apoptosis in human eosinophils

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.

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.