Variations in endotoxin-, peptidoglycan-, and organic dust-induced cytokine responses in a whole blood assay and sputum inflammatory markers in subjects with chronic obstructive lung disease
Harting, JR; Gleason, A; Romberger, DJ; Von Essen, SG; Qiu, F; Alexis, NE; Poole, JA
Introduction: Exposure to bioaerosols as found in organic dust from various settings, particularly agricultural environments, can lead to chronic respiratory diseases. Ex vivo whole blood assay (WBA) measuring induced mediator release to lipopolysaccharide (LPS) has been utilized to assess individual susceptibility to lung disease development. However, non-endotoxin components, such as peptidoglycan (PGN), are highly prevalent in large-animal farming operations and may influence disease development. In population studies exploring agricultural-related respiratory health effects, optimizing the WBA to include critical bioaerosol components is necessary.
Objective: The aim of this study was to modify the WBA to include settled swine confinement facility organic dust extract (ODE) and its comparative components, LPS and PGN, as well as collect lung function and sputum inflammatory markers in subjects with chronic obstructive pulmonary disease (COPD) and healthy controls for use in future population studies.
Methods: Twelve subjects with COPD as defined by the GOLD criteria and ten healthy control subjects between ages of 50 to 75 years were consented with IRB approval. All subjects participated in spirometry, an induced sputum protocol, and completed a questionnaire. Induced sputum samples were evaluated for cell counts/differentials and TNF-a, IL-6 and IL-8. Blood samples were obtained and ex vivo stimulated with ODE (0.1%, 1%), and comparable concentrations of LPS (10, 100 ng/ml) and PGN (1, 10 µg/ml) for 24 hours. Cell-free supernatants in sputum and blood were assayed by ELISA for TNF-α, IL-6 and IL-8.
Results: Sputum neutrophils (mean + SEM, [COPD] 39.7% + 8.4% vs. [control] 12.9% + 2.8%; p=0.01) and sputum IL-8 (729.4 pg/ml + 120.8 vs. 340.0 pg/ml + 58.9; p=0.01) were significantly elevated in COPD subjects. Collectively, ex vivo whole blood stimulation with ODE, LPS, and PGN resulted in detection of TNF-a, IL-6, and IL-8, but PGN and ODE resulted in significantly enhanced TNF-a, IL-6, and IL-8 release as compared to LPS stimulation. PGN- (1 mg/ml), LPS- (10 and 100 ng/ml) and ODE- (0.1%) induced IL-8 release was significantly increased in subjects with COPD as compared to healthy controls.
Conclusions: Subjects with COPD demonstrated increased IL-8 release following ex vivo whole blood stimulation with organic dust and microbial components. This suggests that circulating blood cells in subjects with COPD may be primed and might respond greater to microbial/inflammatory insult. In addition, PGN, but not LPS, closely resembled swine confinement facility organic dust-induced mediator release, suggesting that non-endotoxin components in the dust influence cellular responses.
