Interleukin-6 displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury

Abstract

Background: Interleukin 6 (IL-6) is a predictive factor of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, its acute pulmonary hemodynamic effects and role in lung injury have not been investigated in a clinically relevant murine model of ARDS.

Methods: We used adult C57Bl6 wild-type (WT) and IL-6 knock-out (IL-6KO) mice. The animals received intravenous recombinant human IL-6 (rHuIL-6) or vehicle followed by intratracheal lipopolysaccharide (LPS) or saline before undergoing low tidal volume mechanical ventilation (MV) for 5 h. Before sacrifice, right ventricular systolic pressure and cardiac output were measured and total pulmonary resistance was calculated. After sacrifice, lung inflammation, edema and injury were assessed with bronchoalveolar lavage (BAL) and histology. In other experiments, right ventricular systolic pressure was recorded during hypoxic challenges in uninjured WT mice pretreated with rHuIL-6 or rHuIL-6 + non-selective nitric oxide synthase inhibitor L-NAME or vehicle.

Results: IL-6KO_(LPS+MV) mice showed a faster deterioration of lung elastic properties and more severe bronchoalveolar cellular inflammation as compared to WT_(LPS+MV). Treatment with rHuIL-6 partially prevented this lung deterioration. Total pulmonary resistance was higher in IL-6KO_(LPS+MV) mice and this increase was abolished in rHuIL-6-treated IL-6KO mice. Finally, rHuIL-6 reduced hypoxic pulmonary vasoconstriction in uninjured WT mice, an effect that was abolished by co-treatment with L-NAME.

Conclusions: In a double-hit murine model of ARDS, IL-6 deficient mice experienced more severe bronchoalveolar cellular inflammation as compared to wild-type littermates. Furthermore, IL-6 deficiency caused marked acute pulmonary hypertension, which may be, at least partially, due to vasoactive mechanisms. A dysregulation of nitric oxide synthase may account for this observation, a hypothesis that will need to be investigated in future studies.

Keywords: Acute lung injury; Acute respiratory distress syndrome; Interleukin-6; Mechanical ventilation; Nitric oxide synthase; Pulmonary hypertension.

Fig. 1

Bronchoalveolar lavage data and lung histological analysis in wild-type adult mice subjected to lipopolysaccharide (LPS) aspiration, followed by 5-h low tidal volume mechanical ventilation (MV) and their control groups (four groups: control, MV, LPS and LPS + MV; five comparisons: control vs. MV, control vs. LPS, control vs. LPS + MV, MV vs. LPS + MV, LPS vs. LPS + MV). The lung injury score, total cell count, and total protein, IL-6, TNFα and MIP-2 concentrations in BAL fluid were assessed. The symbols §, ‡ and † denote Benjamini-Hochberg corrected p values <0.05 of Mann-Whitney pairwise comparisons (after Kruskal Wallis test) between the group so marked and the group that received MV alone, LPS alone and the combination LPS + MV, respectively. Data are presented as box and whiskers plots. N = 8–10 animals per group

Fig. 2

Respiratory mechanics during 5-h (H0 to H5) low tidal volume mechanical ventilation (MV) following either lipopolysaccharide (LPS) or vehicle (saline) aspiration in wild type (WT) and interleukin-6 knock-out (IL-6KO) mice receiving either recombinant human interleukin-6 (rHuIL-6) or vehicle (saline). The following respiratory system properties are shown A. Peak inspiratory pressures. B. Quasi-static compliance of the respiratory system calculated using a pressure-volume curve. C. Dynamic compliance of the respiratory system calculated using the single frequency forced oscillation technique. D. Dynamic elastance of the respiratory system calculated using the single frequency forced oscillation technique. Volume history standardization (VHS) consisted in two inflations to an airway pressure of 30 cmH2O. The letters a, b, c, d, e, f, g, h and i denote Benjamini-Hochberg corrected p values <0.05 of Mann-Whitney pairwise comparisons (following Kruskal Wallis test): WT_(MV) vs. WT_(LPS+MV), WT_(MV) vs. WT + rHuIL-6_(LPS+MV), WT_(LPS+MV) vs. WT + rHuIL-6_(LPS+MV), IL-6KO_(MV) vs. IL-6KO_(LPS+MV), IL-6KO_(MV) vs. IL-6KO + rHuIL-6_(LPS+MV), IL-6KO_(LPS+MV) vs. IL-6KO + rHuIL-6_(LPS+MV), WT_(MV) vs. IL-6KO_(MV), WT_(LPS+MV) vs. IL-6KO_(LPS+MV) and WT + rHuIL-6_(LPS+MV) vs. IL-6KOrHuIL-6_(LPS+MV) respectively. Data are presented as mean and standard error of the mean. N = 8 to 10 animals per group

Fig. 3

Bronchoalveolar lavage data at the end of a 5-h low tidal volume mechanical ventilation (MV) following either a lipopolysaccharide (LPS) or vehicle (saline) aspiration in wild type (WT) and interleukin-6 knock-out (IL-6KO) mice receiving either recombinant human interleukin-6 (rHuIL-6) or vehicle (saline). a Total cell count of BAL fluid (cell/μL). b TNFα concentration in the BAL fluid (pg/mL). c MIP-2 concentration in the BAL fluid (pg/mL). d IL-6 concentration in the BAL fluid (pg/mL). e Total protein concentration in the BAL fluid (μg/mL). The symbol * denotes Benjamini-Hochberg corrected p value < 0.05 of Mann–Whitney pairwise comparisons (following Kruskal Wallis test). Data are presented as box and whiskers plots. N = 6 to 8 animals per group

Fig. 4

Lung histological analysis at the end of a 5-h low tidal volume mechanical ventilation (MV) following either a lipopolysaccharide (LPS) or vehicle (saline) aspiration in wild type (WT) and interleukin-6 knock-out (IL-6KO) mice receiving either recombinant human interleukin-6 (rHuIL-6) or vehicle (saline). a Global lung injury score. b Lung edema score. c Alveolitis score. d Lung leukocyte infiltration score. e Lung congestion score. The symbol * denotes Benjamini-Hochberg corrected p value < 0.05 of Mann-Whitney pairwise comparisons (following Kruskal Wallis test). Data are presented as box and whiskers plots. N = 6 to 8 animals per group

Fig. 5

Lung histological sections after a 5-h low tidal volume mechanical ventilation (MV) following either a lipopolysaccharide (LPS) aspiration or vehicle (saline) in wild type (WT) and interleukin-6 knock-out (KO) mice receiving either recombinant human interleukin-6 (rHuIL-6) or vehicle. Lungs underwent fixation (paraformaldehyde 4%), paraffin embedding, staining with hematoxylin and eosin, and examination under a light microscope. Magnification x10 on the two uppers rows of images and X40 on the lower row of pictures. Scale bar: 50μm. N = 6 to 8 animals per group

Fig. 6

Hemodynamic data obtained at the end of a 5-h low tidal volume mechanical ventilation (MV) following either a lipopolysaccharide (LPS) or vehicle (saline) aspiration in wild type (WT) and interleukin-6 knock-out (IL-6KO) mice receiving either recombinant human interleukin-6 (rHuIL-6) or vehicle (saline). a Right ventricular systolic pressure (mmHg). b Total pulmonary resistances (mmHg.min/mL). c Heart rate (bpm). Right ventricular systolic pressure was measured during a short end-expiratory pause using an ultra-miniature catheter inserted into the right jugular vein and advanced into the right ventricle. Cardiac output was measured by the transpulmonary thermodilution technique. Total pulmonary resistances were calculated as the ratio of right ventricular systolic pressure to cardiac output (measured by the transpulmonary thermodilution technique). The symbol * denotes Benjamini-Hochberg corrected p value < 0.05 of Mann–Whitney pairwise comparisons (following Kruskal Wallis test). Data are presented as box and whiskers plots. N = 6 to 8 animals per group

Fig. 7

Hemodynamic data obtained during hypoxic challenges. WT mice received either recombinant human interleukin-6 (rHuIL-6) or rHuIL-6 in association with the non-specific inhibitor of nitric oxide synthase Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Control mice received vehicle (saline). Five hours later, mice were intubated for mechanical ventilation and an ultra-miniature catheter was immediately inserted into the right jugular vein and advanced into the right ventricle. a After 30 min of stabilization, the right ventricular systolic pressure was continuously recorded during four consecutive hypoxic challenges (FiO2 of 0.08 during 2 min, followed by 5 min of reoxygenation). Points represent the mean and errors bars represent the standard error of the mean. b The delta of right ventricular systolic pressure was calculated as the mean difference from peak to baseline right ventricular systolic pressure during the four consecutive hypoxic challenges. The symbol * denotes Benjamini-Hochberg corrected p value < 0.05 of the Mann–Whitney pairwise comparisons (following Kruskal Wallis test). Columns represent the mean and error bars represent the standard error of the mean. N = 4 animals per group