Characterisation of the range of neutrophil stimulating mediators in Cystic Fibrosis sputum
Abstract
Background: Most cystic fibrosis (CF) patients die of respiratory failure due to chronic infection and destructive neutrophilic inflammation.
Objective: To identify potential therapeutic targets by characterising the neutrophil stimulating mediators in the CF airway.
Methods: Spontaneously expectorated CF sputum was extracted in phosphate buffered saline for assays of neutrophil chemotaxis, intracellular calcium mobilisation and cell shape change. Mediators were purified by ion exchange, C18 reversed phase and size exclusion chromatography. Results: A pool of CF sputum contained considerable neutrophil stimulating activity but neutralisation of interleukin (IL)-8/CXCL8 had little inhibitory effect on neutrophil chemotactic (10149 ± 2023 migrating cells vs. 8661 ± 2597 at 62 mg sputum/ml, NS) or shape change (% FSC increase 46 ± 8 vs. 38 ± 5 at 19 mg sputum/ml, p<0.05) responses. Further, the CF sputum pool induced an elevation of intracellular calcium ions even after desensitisation of the neutrophils to IL-8. Chromatography identified contributions to the neutrophil shape change inducing activity from IL-8, other CXC chemokines, leukotriene (LT) B4 and two formyl peptides. There was also suggestive evidence for contributions from platelet activating factor (PAF) and C5a. Using non-chromatographed individual sputum samples, anti-IL-8 alone did have an inhibitory effect on neutrophil chemotaxis (median inhibition 41%, p = 0.0002). However, even in this experiment, there were clearly significantly important, non-IL-8 mediated effects of CF sputum on neutrophils and an inhibitor cocktail of anti-IL-8 plus CXCR2, LTB4, formyl peptide, PAF and C5a receptor antagonists inhibited chemotaxis by a median of 97% (p = 0.0002).
Conclusion: Many chemoattractants contribute to the neutrophil stimulating activity in CF sputum although the relative contribution of these mediators differs in different patients. The selective blockade of single mediators may not be sufficient to control neutrophil recruitment and activation in the CF airway.
