Lack of neutrophil degranulation in low-dose endotoxin inhalation based on a novel intracellular assay.

To study the nature of endotoxin or lipopolysaccharide (LPS) induced inflammation, we developed a method of quantifying intracellular human neutrophil elastase (HNE) in lysed sputum polymorphs as a means to study the degranulation status of LPS-recruited neutrophils. Induced sputum, blood and exhaled nitric oxide (NO) were collected from 10 healthy non-atopic human subjects after inhaling a single 15 microg dose of Escherichia coil LPS in an open study. At 6 hours, LPS inhalation caused significant increase of sputum and blood neutrophils but without parallel increase in myeloperoxidase, HNE or interleukin-8 (IL-8) in sputum sol and blood, or exhaled NO. Intracellular HNE in lysed sputum polymorphs or purified blood neutrophils did not show any significant changes between inhaled LPS and saline, nor was there any appreciable change in percentage HNE release induced by N-Formyl-Met-Leu-Phe (fMLP) in vitro. We concluded that in healthy humans, the transient neutrophilic inflammation induced by a single dose of inhaled 15 microg LPS is mainly characterized by cell recruitment, not enhanced secretion of granular mediators or increased exhaled NO based on our experimental conditions.

Sputum induction in corticosteroid-dependant asthmatics: risks and airway cellular profile.

Sputum induction with nebulized hypertonic saline is increasingly being used to evaluate airway inflammation. We investigated the procedure-associated risk in 16 asthmatics that were still symptomatic despite on high doses of regular corticosteroid (CS) therapy (7 on daily inhaled CS > or = 800 microg budesonide or equivalent; 9 on additional daily oral CS) and their sputum cellular profile. For comparison, 12 mild stable asthmatics and 10 normal healthy subjects were included. All subjects inhaled 3%, 4% and 5% hypertonic saline sequentially via ultrasonic nebulizer as a means to induce sputum. Maximal percentage fall of Forced Expiratory Volume on One Second (FEV1) during sputum induction was significantly greater in CS-dependent asthmatics (median % [IQR]: 16.0 [11.0-32.3]) than in mild asthmatics (5.3 [4.2-10.8], p = 0.002] and in normal subjects (4.6 [3.4-6.4]), p = 0.0001). The maximal percentage FEV1 fall was inversely correlated with baseline FEV1 (Rs= -0.69; p < 0.0001). Compared to mild asthmatics, induced sputum from CS-dependant asthmatics had proportionately fewer eosinophils (2.2 [0.8-7.0] versus 23.3% [10.7-46.3], p = 0.003) and greater neutrophils (64.2 [43.9-81.2] versus 28.7 [19.0-42.6], p = 0.009). Sputum neutrophils showed a significant inverse correlation to FEV1 (Rs = -0.51, p = 0.01). We concluded that sputum induction using nebulized hypertonic saline should be performed with caution in CS-dependant asthmatics. The airway cellular profile observed suggests that the immunopathology underlying CS-dependant asthmatics may be different or a consequence of CS therapy.