Free, soluble interleukin-17 protein during severe inflammation in human airways

Study design

The study protocol was approved by the Ethics Committee at Karolinska Institute. Fifteen clinically-healthy, nonsmoking volunteers of both sexes gave informed consent to participate in the study. All the subjects were nonatopic and nonasthmatic as determined by history and a questionnaire. These subjects were exposed to a swine dust confinement for 3 h while assisting a farmer weighing pigs in a swine confinement with ∼900 pigs, as described previously 12–14. This procedure is known to aerosolize organic dust; exposure was assessed as described previously 12–14. A normal lung function at baseline was ascertained by conducting spirometry (as described later) in all subjects prior to the first BAL and 2 weeks prior to exposure to a swine confinement. At this time point, it was also ascertained that no subject displayed airway hyperresponsiveness, by conducting methacholine provocation. BAL (see later) was performed 2 weeks prior to (baseline) and 24 h after the start of exposure to a swine confinement.

Lung function

Spirometry was performed in accordance with the criteria of the American Thoracic Society using a low-resistance rolling-seal spirometer (OHIO model 840; Airco, Madison, WI, USA) 15. The reference values by Hedenstrom and co-workers were used 16, 17.

Bronchial responsiveness

The methacholine provocation test was conducted in a standardized manner as described previously 18. Briefly, inhalation of the diluent (placebo) was followed by inhalation of doubling concentrations of methacholine starting at 0.5 mg·mL⁻¹. The challenge was stopped either at a forced expiratory volume in one second (FEV₁) decrease of 20%, compared to the value obtained after inhalation of diluent, or after inhalation of the highest methacholine concentration (64 mg·mL⁻¹). The cumulative dose causing a 20% decrease in FEV₁ (PD₂₀) was calculated.

Bronchoalveolar lavage

Bronchoscopy and BAL were performed according to standard procedures as described previously 18, 19. Briefly, after premedication with morphine-scopolamine, a flexible fibreoptic bronchoscope (Olympus Type 4B2; Olympus Optical Co. Ltd, Shinjukuku, Tokyo, Japan) was inserted through the mouth or the nose under local anaesthesia with lidocaine (Xylocain®, Astra, Södertälje, Sweden). The bronchoscope was wedged in a middle lobe subsegmental bronchus and 250 mL of sterile saline at 37°C was instilled in five aliquots of 50 mL. After each installation, the BAL suspension was gently aspirated and collected in a siliconized plastic bottle kept on ice.

After filtering through a single layer of gauze, the BAL suspension was centrifuged at 200×g for 10 min at 4°C. The cell-free supernatant was kept frozen at −70°C until analysis. The cell pellet was resuspended in Tris-Hanks balanced salt solution at pH 7.4 and the total cell number (i.e. concentration) was determined for each sample in a Bürkner chamber. Differential cell counts were performed by flow cytometry (Epics Profile II; Coulter Electronics Inc., Hialeah, FL, USA). The samples were prepared in the Coulter Q‐PREP®, and incubated for 10 min with CD14-CD45 monoclonal antibodies (Mo2-RD1/Kc56-Fitc; Cyto-Stat/Coulter Clone, Coulter Corp., Miami, FL, USA).

Analyses of bronchoalveolar lavage fluid

For IL-17 protein measurements, cell-free BAL fluid was concentrated 200 times using ultrafiltration (Centricon® Plus-20, cut-off 5000 nominal molecular weight limits; Millipore, Bedford, MA, USA) according to the manufacturer's instructions. The IL-17 concentration was determined using a commercial IL-17 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe, Abington, UK). Concentrations below the detection limit of the ELISA were assigned a value of 3.9 pg·mL⁻¹ (the mean of zero and the lowest IL-17 standard of the ELISA). The concentrations of IL‐6, IL‐8 and TNF‐α were determined in unconcentrated, cell-free BAL fluid using commercial ELISA for IL‐6, IL‐8 and TNF‐α, respectively (all from R&D Systems Europe).

Data analysis

All results are presented as median (range) values unless otherwise stated. Only nonparametric tests were utilized. The Wilcoxon signed rank test was used for comparisons between two dependent groups and the Spearman rank correlation for detecting a relationship between two variables. A p‐value <0.05 was considered as statistically significant.

Clinical baseline characteristics

Nine males and six females with a median (range) age of 26 (22–46) yrs were included in the study. These subjects displayed an FEV₁ of 100 (71–126)% predicted and a vital capacity of 95 (75–111)% pred. The included subjects displayed a median PD₂₀ value for methacholine of 2.35 (0.66–64.00).

Exposure to a swine confinement

For the exposures conducted in this study, the median (range) total dust and endotoxin content per m³ air was 24 (14–43) mg and 1.2 (0.3–1.5) µg, respectively.

Interleukin-17 in bronchoalveolar lavage fluid

Exposure to a swine dust confinement caused no pronounced change in BAL recovery volume; the recovery volume was 195 (145–220) mL prior to and 188 (162–203) mL after exposure. Prior to this exposure, 14 out of 15 subjects displayed nondetectable concentration (<7.8 pg·mL⁻¹) of IL-17 protein in BAL fluid. Exposure to a swine confinement significantly increased the IL-17 concentration in 13 out of 15 subjects (fig. 1⇓).

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Fig. 1.—

Effect of 3‐h exposure to a swine confinement on the concentration of free, soluble interleukin (IL)-17 protein in bronchoalveolar lavage fluid (cell-free, concentrated 200×) harvested 24 h after exposure from 15 healthy volunteers and measured using an enzyme-linked immunosorbent assay. Data are presented as individual IL-17 concentrations (log scale) prior to (baseline) and after exposure (post-exposure) to the swine confinement. After exposure, the median concentration of IL-17 was 26.9 (3.9–247.4) pg·mL⁻¹ and this increase was statistically significant (p<0.005).

Number of inflammatory cells in bronchoalveolar lavage fluid

Exposure to the swine confinement caused a 51-fold increase in BAL neutrophils and a 2–3-fold increase in BAL lymphocytes and macrophages (table 1⇓). There was no correlation between the level of IL-17 protein in BAL fluid and the concentration of BAL neutrophils after exposure to the swine confinement (data not shown).

Levels of interleukin‐6, ‐8 and tumour necrosis factor‐α in bronchoalveolar lavage fluid

Exposure to the swine confinement caused a statistically significant (Wilcoxon signed rank test: p<0.005, n=15) increase in IL‐6 (from 1.3 (0.2–2.5) to 32.4 (6.7–187.2)), IL‐8 (from <31.3 (<31.3–191.1) to 99.5 (42.3–431.1)) and TNF‐α (from 0.5 (<0.25–2.4) to 4.0 (0.5–16.6)) in cell-free BAL fluid. However, in spite of visual trends, there was no statistically significant correlation between the concentration of IL-17 and that of IL‐6, IL‐8 or TNF‐α (fig. 2⇓).

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Fig. 2.—

Scattergrams showing individual concentrations of interleukin (IL)-17 protein versus a) IL‐6, b) IL‐8 and c) tumour necrosis factor (TNF)‐α protein in cell-free bronchoalveolar lavage fluid harvested from previously-healthy human volunteers, 24 h after exposure to a swine confinement, measured using enzyme-linked immunosorbent assay. There was no statistically significant correlation between IL-17 and IL‐6 (rho=0.4, p=0.2, n=15); IL‐8 (rho=0.3, p=0.3, n=14); or TNF‐α (rho=0.1, p=0.6, n=15).