Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man

Abstract

Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 µg LPS to the airways and 5 µg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 µg LPS targeted to the alveoli caused significantly stronger effects compared to 5 µg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.

Figure 1. Study protocol. 24 h prior to LPS inhalation blood samples were taken and analyzed for inflammatory parameters (CRP, neutrophil count).

In addition body temperature (BT) and lung function (LF) was assessed. Exhaled breath condensate (EBC) was collected and analyzed for H₂O₂ concentration and acidity (pH). After targeting of 1, 5 or 20 µg LPS to the airways or 5 µg LPS to the alveoli, inflammatory parameters in blood and BT, LF and EBC were assessed according the time scale.

Figure 2. Profile of exhaled CO₂ of one subject and determination of the phase-1 dead space (DS_Ph1), the Fowler dead space (DS_F) and the Bohr dead space (DS_B).

DS_B was used as threshold volume for airway (AW) and alveolar (AL) condensate sampling separation. In addition the first 50 mL of the exhaled air were discarded. The grey areas show the size and the penetration of the shallow and the deep LPS aerosol bolus for targeting the airways or the alveolar space.

Figure 3

A) Systemic response parameter ‘body temperature’ during 72 h after targeting different doses of LPS either to the airways (closed symbols) or to the alveoli (open symbols). B) Increase of 12 h - body temperature with increasing LPS dose targeted to the airways in comparison to 5 µg LPS targeted to the alveoli (open symbol). Data represent mean +/− SD (n = 7; *: p<0.05, **: p<0.01 compared to baseline; ⁺⁺: p<0.01 for 5 µg alveolar compared to 5 µg airway LPS).

Figure 4. Systemic inflammatory response parameters after targeting LPS either to the airways (1, 5 and 20 µg, closed symbols) or to the alveoli (5 µg, open symbol).

A: absolute blood neutrophils (6 h after LPS challenge, baseline value = 3.4+/−1.3*10³/µL), B: CRP (24 h after LPS challenge, baseline value = 1.4+/−0.9 mg/L) and C: IL-6 (baseline value 2.0+/−0.4 pg/mL). Data represent mean +/− SD (n = 7; *: p<0.05, **: p<0.01 compared to baseline; ⁺⁺: p<0.01 for 5 µg alveolar compared to 5 µg airway LPS).

Figure 5. Local response parameters lung function (A, normalized FEV₁) and hydrogen peroxide in airway-EBC (B) after targeting different doses of LPS either to the airways or to the alveoli.

Data represent mean +/− SD (n = 7, *: p<0.05, **: p<0.01 compared to baseline).

Figure 6. Summary of peaking times of the different study parameters assessed after LPS challenge and associated dose – response relationships (∼D: parameter is dose dependent).