Fiona A. Symon

Comparison of airway immunopathology of eosinophilic bronchitis and asthma

Background: Eosinophilic bronchitis is a condition characterised by a corticosteroid responsive cough, sputum eosinophilia, and normal tests of variable airflow obstruction and airway responsiveness. We performed a detailed comparative immunopathological study to test the hypothesis that the different airway function in patients with eosinophilic bronchitis and asthma reflects differences in the nature of the lower airway inflammatory response. Methods: Exhaled nitric oxide was measured and induced sputum, bronchoscopy, bronchial wash (BW), bronchoalveolar lavage (BAL), and bronchial biopsy were performed in 16 subjects with eosinophilic bronchitis, 15 with asthma, and 14 normal controls. Results: Both eosinophilic bronchitis and asthma were characterised by an induced sputum, BW and BAL eosinophilia, an increased number of epithelial and subepithelial eosinophils, and increased reticular basement membrane thickness. The median concentration of exhaled nitric oxide was higher in those with eosinophilic bronchitis (12 ppb) or asthma (8.5 ppb) than normal controls (2 ppb) (95% CI of the difference 5 to 16, p<0.0001 and 2 to 11.3, p=0.004, respectively). There were no group differences in epithelial integrity or the number of subepithelial T lymphocytes, mast cells or macrophages. Conclusion: With the exception of our previously reported association of smooth muscle mast cell infiltration with asthma, the immunopathology of eosinophilic bronchitis and asthma are similar which suggests that eosinophilic airway inflammation, increased exhaled nitric oxide, and increased basement membrane thickening are regulated independently of airway hyperresponsiveness.

Expression of Chemokine Receptors by Lung T Cells from Normal and Asthmatic Subjects

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and α4β7. No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.

Interleukin‐4 and ‐13 expression is co‐localized to mast cells within the airway smooth muscle in asthma

Background Airway smooth muscle infiltration by mast cells is a feature of asthma and not eosinophilic bronchitis. In asthma, Th2 cytokines have been implicated as playing a critical role in the development of airway inflammation and hyper‐responsiveness. Whether inflammatory cells within the airway smooth muscle release these cytokines is unknown.

Elevated Sputum Interleukin-5 and Submucosal Eosinophilia in Obese Individuals with Severe Asthma

RATIONALE The relationship between airway inflammation and obesity in severe asthma is poorly understood. OBJECTIVES We sought to determine the relationship between sputum mediator profiles and the distribution of eosinophilic inflammation and obesity in people with severe asthma. METHODS Clinical parameters and eight mediators in sputum were assessed in 131 subjects with severe asthma from a single center categorized into lean, overweight, and obese groups defined by their body mass index. In an independent group of people with severe asthma (n = 45) and healthy control subjects (n = 19) eosinophilic inflammation was enumerated in bronchial submucosa, blood, and sputum and related to their body mass index. MEASUREMENTS AND MAIN RESULTS Sputum IL-5 geometric mean (95% confidence interval) (pg/ml) was elevated in the obese (1.8 [1.2-2.6]) compared with overweight (1.1 [0.8-1.3]; P = 0.025) and lean (0.9 [0.6-1.2]; P = 0.018) subjects with asthma and was correlated with body mass index (r = 0.29; P < 0.001). There was no relationship among body mass index, the sputum cell count, or other sputum mediators. In the bronchoscopy group the submucosal eosinophil number in the subjects with asthma was correlated with body mass index (Spearman rank correlation, rs = 0.38; P = 0.013) and the median (interquartile range) number of submucosal eosinophils was increased in obese (19.4 [11.8-31.2]) (cells per square millimeter) versus lean subjects (8.2 [5.4-14.6]) (P = 0.006). There was no significant association between sputum or peripheral blood eosinophil counts and body mass index. CONCLUSIONS Sputum IL-5 and submucosal eosinophils, but not sputum eosinophils, are elevated in obese people with severe asthma. Whether specific antieosinophilic therapy is beneficial, or improved diet and lifestyle in obese asthma has antiinflammatory effects beyond weight reduction, requires further study.

Integrin alpha 4 beta 7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin.

We have investigated the contribution of integrin α4β7 to human peripheral blood eosinophil adhesive interactions. Immunofluorescence and flow cytometry demonstrated constitutive expression of α4β7 by eosinophils. Expression of α4β1 or α4β7 was not enhanced by eosinophil activation with platelet‐activating factor (PAF). Expression of α4β7 was confirmed by immunoprecipitation of 125I‐labelled 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 transfectants was inhibited by HP1/2 (an antibody that blocks both α4β1 and α4β7 functions), but not Act‐1, an α4β7 monoclonal antibody (mAb). PAF stimulation resulted in increased binding of eosinophils to MAdCAM‐1 transfectants, which was inhibited by both HP1/2 and Act‐1. In contrast, PAF did not enhance binding to VCAM‐1 transfectants, although binding of PAF‐stimulated eosinophils to VCAM‐1 could be partially inhibited by Act‐1. Stimulation of eosinophils with the β1‐activating mAb TS2/16 resulted in enhanced binding of eosinophils to both VCAM‐1 and MAdCAM‐1 transfectants. The increased binding was largely α4β7‐dependent. Unstimulated eosinophils bound to soluble recombinant human (rh)VCAM‐1 and fibronectin (Fn), coated on 96‐well plates in a dose‐dependent manner. Binding was inhibited by HP1/2 and 4b4, an anti‐β1 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 α4β1 is functionally active on unstimulated eosinophils. In contrast, PAF‐induced enhancement of eosinophils binding to VCAM‐1 or MAdCAM‐1 was α4β7‐dependent. In addition, treatment with TS2/16 resulted in a α4β7‐dependent enhancement of eosinophil binding to VCAM‐1, MAdCAM‐1 and Fn. We therefore hypothesize that α4β7 may have an important role in eosinophil localization in diseases such as asthma and inflammatory bowel disease.

Expression of CXCR6 and its ligand CXCL16 in the lung in health and disease

Background Chemokine receptors (CR) play an important role in T cell migration, but their contribution to lung trafficking is unclear.

P- and L-selectin mediate binding of T cells to chronically inflamed human airway endothelium.

The inflammatory process that underlies allergic diseases such as asthma is characterized by tissue infiltration of eosinophils and T cells. We have used the Stamper‐Woodruff frozen‐section assay to characterize the receptors involved in adhesion of human peripheral blood T cells to nasal polyp endothelium (NPE) as a model of T cell migration in allergic disease. T cells bound specifically to NPE in a temperature‐, cell concentration‐ and shear stress‐dependent fashion. Adhesion was inhibited by approximately 70 % by antibodies against P‐selectin and its counter‐receptor P‐selectin glycoprotein‐1 (PSGL‐1). In addition, a blocking monoclonal antibody (mAb) against L‐selectin caused significant although lesser inhibition. Cells adhering to NPE were primarily of the CD45RO+ memory subset. Although only a minority subset of peripheral blood T cells expressed functional PSGL‐1, as determined by binding of a P‐selectin Fc chimera, the majority of the P‐selectin chimera‐binding cells were found to be CD45RO+. This is consistent with the observation that memory T cells bind to NPE via P‐selectin. Using blocking mAb we also investigated which integrins and their counter‐structures were involved in T cell binding. A combination of anti‐β1 and β2 mAb was able to inhibit adhesion by almost 50 %. An antibody against intercellular adhesion molecule (ICAM)‐2 gave an inhibition similar to that by anti‐CD18 mAb, suggesting ICAM‐2 was the major counter‐receptor involved for the β2 integrin component. This study suggests that P‐selectin, and to a lesser extent L‐selectin, may be acting as specific homing receptors for the airway mucosa in the context of chronic allergic disease.

Human eosinophils preferentially survive on tissue fibronectin compared with plasma fibronectin

Background Eosinphil‐derived inflamatory mediators including cytolines are considered to be important in the pathogenesis of allergic inflammation. Fibronectin (Fn) has been shown to be a physiogical trigger of autocrine cytokihne 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 approxmately 50% more of the CS‐1 cell binding region recognized by the integrin α4 β1 compared with lasma Fn.

Characterisation of adhesion receptors mediating lymphocyte adhesion to bronchial endothelium provides evidence for a distinct lung homing pathway

Background: The lung is an important tertiary lymphoid organ and many lung diseases are associated with disordered lung immunity. Unlike the gut (α4β7 binding to MAdCAM-1) and skin (CLA+ve T cells binding to E-selectin) where the adhesion receptors involved in organ specific homing of T cells have been identified, the molecular pathways controlling lymphocyte migration to the lung are unclear. Using a modified version of the Stamper-Woodruff assay we have investigated the receptors mediating adhesion of peripheral blood lymphocytes to airway endothelium. Methods: Longitudinal frozen sections of bronchus (8 μm) obtained from lung resection specimens were incubated with T cell enriched peripheral blood mononuclear cells for 30 minutes under shaking conditions in the presence of a fluorescently labelled polyclonal anti-von Willebrand antibody to identify blood vessels. After fixation the percentage of blood vessels supporting adhesion was measured. Blocking monoclonal antibodies were used to determine the role of individual adhesion receptors in lymphocyte binding. Results: Specific cation dependent binding of lymphocytes to bronchial endothelium was observed which was significantly inhibited by antibodies against P-selectin, PSGL-1, L-selectin, LFA-1, ICAM-1 and ICAM-2 but not E-selectin, VLA-4, VCAM-1 or Mac-1. This was consistent with the pattern of endothelial expression of these receptors with strong expression of P-selectin and ICAM-1, but negligible expression of E-selectin on bronchial endothelium. Conclusion: This study suggests an important role for PSGL-1/P-selectin in T cell migration into the bronchi and provides further evidence for a pattern of recirculation for respiratory tract homing T cells distinct from the gut and skin.

Objective quantitative analysis of eosinophils and bronchial epithelial cells in induced sputum by laser scanning cytometry

BACKGROUND Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide. METHODS LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using α-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-α-human-MBP monoclonal antibody or mouse-α-human-cytokeratin antibody and goat-α-mouse Oregon Green conjugated second antibody. RESULTS Sputum induction provided a mean (SE) of 0.99 (0.2) × 106 cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation. CONCLUSION Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.