Morgan G. Blaylock

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.

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).

The effect of nitric oxide and peroxynitrite on apoptosis in human polymorphonuclear leukocytes

In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled nitric oxide is now a widely used therapy in patients with acute lung injury, and its effects on apoptosis may be important. We investigated the effect of nitric oxide and peroxynitrite on apoptosis in lipopolysaccharide stimulated polymorphonuclear leukocytes as a model of nitric oxide-treated lung injury. Cells were incubated for up to 16 h with and without 1.7 microg/ml lipopolysaccharide and the nitric oxide donor GEA-3162 or the peroxynitrite donor SIN-1. Apoptosis was assessed using flow cytometry following annexin-V staining, after 4, 6, 8, and 16 h. Data were assessed using Kruskal-Wallis analysis of variance or Mann-Whitney U-test as appropriate. Annexin-V staining increased spontaneously over 16 h in untreated cells (p = .0002) and incubation with either 1000 microM SIN-1 or 10 microM GEA-3162 increased annexin staining at early time points in nonactivated cells. Apoptosis was attenuated when cells were exposed to lipopolysaccharide and both nitric oxide and peroxynitrite dose dependently inhibited this suppression at all time points and was most apparent at 16 h (p = .004 and .001, respectively). Exposure of activated neutrophils to exogenous nitric oxide or peroxynitrite has marked influences on apoptosis. This work has implications for the modulation of neutrophil function within the lung in patients with lung injury who receive inhaled nitric oxide therapy.

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells

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

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.

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.

Sputum eosinophil apoptotic rate is positively correlated to exhaled nitric oxide in children.

RATIONALE FOR STUDY Exhaled nitric oxide (FE(NO)), a potential biomarker for asthma, is positively correlated with eosinophilic airway inflammation. Eosinophil apoptotic rate (AR) may be increased by NO but the relationship between eosinophil AR and NO has not been studied in vivo. This study tested the hypothesis that eosinophil AR will be positively related to FE(NO). METHODS Children with and without asthma were recruited and participated in an assessment that included FE(NO) measurement, skin prick reactivity, spirometry, and sputum induction. The absolute sputum eosinophil count and eosinophil AR were determined by morphology under light microscope after staining. RESULTS There were 31 children recruited, mean age 11 years, 21 were asthmatic and 19 were boys. The median FE(NO) (range) was 15.6 parts per billion (3.1-102.6), 17 were atopic and the mean (SD)% FEV(1) was 85 (10)%. Sputum eosinophil AR was determined in 19 children (16 asthmatics), mean (SD) value 0.49 (0.13). There were positive relationships between eosinophil AR and FE(NO) (Spearman rho = 0.46, P = 0.046), eosinophil AR and % eosinophil count (Spearman rho = 0.45, P = 0.050) and also FE(NO) and % eosinophil count (Spearman rho = 0.49, P = 0.024). CONCLUSION There is a positive relationship between FE(NO) and eosinophil AR. Nitric oxide may be involved in regulation of eosinophil AR in the airways.

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line

Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase‐3, ‐8, and ‐9 activities in receptor ligation‐dependent apoptosis induction in the differentiated human eosinophilic cell line EoL‐1. Differentiated EoL‐1 exhibited bi‐lobed nuclei, eosinophil‐associated membrane receptors, and basic granule proteins. Annexin‐V fluorescein isothiocyanate binding to EoL‐1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%±4.3), CD45RA (58%±2.3), CD45RB (68%±2.4), CD95 (47%±2.6), and CD69 (52%±2.1) compared with control (23%±1.6) or CD45RO mAb (27%±3.9). The pan‐caspase inhibitor Z‐Val‐Ala‐Asp‐fluoromethylketone (fmk) and inhibitors of caspase‐8 (Z‐Ile‐Glu‐Thr‐Asp‐fmk) and caspase‐9 (Z‐Leu‐Glu‐His‐Asp‐fmk) significantly inhibited mAb‐induced apoptosis of EoL‐1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag™ technique and flow cytometry. EoL‐1 treated with pan‐CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase‐3 and ‐9 activation at 12 h post‐treatment, which increased at 16 and 20 h. Activated caspase‐8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase‐3 activation, a modest but significant activation of caspase‐8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase‐9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation‐mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti‐inflammatory therapy for asthma.

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.

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.