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. 2012;7(6):e38917.
doi: 10.1371/journal.pone.0038917. Epub 2012 Jun 11.

Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation

Jochen Grommes  1 , Santosh Vijayan, Maik Drechsler, Helene Hartwig, Matthias Mörgelin, Rolf Dembinski, Michael Jacobs, Thomas Andreas Koeppel, Marcel Binnebösel, Christian Weber, Oliver Soehnlein
Affiliations

Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation

Jochen Grommes et al. PLoS One. 2012.

Expression of concern in

Abstract

Introduction: Treatment of acute lung injury (ALI) remains an unsolved problem in intensive care medicine. As simvastatin exerts protective effects in inflammatory diseases we explored its effects on development of ALI and due to the importance of neutrophils in ALI also on neutrophil effector functions.

Methods: C57Bl/6 mice were exposed to aerosolized LPS (500 µg/ml) for 30 min. The count of alveolar, interstitial, and intravasal neutrophils were assessed 4 h later by flow cytometry. Lung permeability changes were assessed by FITC-dextran clearance and albumin content in the BAL fluid. In vitro, we analyzed the effect of simvastatin on neutrophil adhesion, degranulation, apoptosis, and formation of reactive oxygen species. To monitor effects of simvastatin on bacterial clearance we performed phagocytosis and bacterial killing studies in vitro as well as sepsis experiments in mice.

Results: Simvastatin treatment before and after onset of ALI reduces neutrophil influx into the lung as well as lung permeability indicating the protective role of simvastatin in ALI. Moreover, simvastatin reduces the formation of ROS species and adhesion of neutrophils without affecting apoptosis, bacterial phagocytosis and bacterial clearance.

Conclusion: Simvastatin reduces recruitment and activation of neutrophils hereby protecting from LPS-induced ALI. Our results imply a potential role for statins in the management of ALI.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Simvastatin reduces LPS-induced acute lung injury by interference with neutrophil recruitment.
Mice were challenged with LPS via inhalation and sacrificed 4 hours later. In addition, neutrophils were depleted by antibody injection or mice were treated with simvastatin (2 µg/g bodyweight) 12 hours and one hour before or one hour after LPS exposure as indicated. A: Quantification of alveolar (top), interstitial (middle), and intravascular neutrophils (bottom) in mice treated as indicated. B: FITC-dextran clearance (top), albumin concentration (middle), and elastase activity (bottom) in BAL fluids in mice treated as indicated (n = 8–10 for each bar). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference compared to LPS-treated animals.
Figure 2
Figure 2. Simvastatin prevents LPS-induced structural changes in the lung tissue.
Representative histological (left) and scanning electron microscopic (right) images of lungs from mice treated as indicated. Scale bars indicate 50 µm for scanning electron microscopy and 250 µm for histology. Quantification of histological lung sections (bottom) (n = 4 for each bar). Statistical significance was tested using ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference compared to LPS-treated animals.
Figure 3
Figure 3. Simvastatin impairs neutrophil adhesion.
Isolated human neutrophils were pre-treated with simvastatin (3 hours, 1 or 10 µM) and then activated with fMLP. Neutrophils were perfused over immobilized recombinant ICAM-1 or fibronectin at 1 dyne/cm2 and the number of adherent cells was enumerated. n = 8–10 for flow chamber experiments (repetition of the experiment = 4). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test.* indicates significant difference of fMLP treated samples compared to each other samples.
Figure 4
Figure 4. Simvastatin blocks ROS formation in neutrophils.
Isolated human neutrophils were pre-treated with Simvastatin (3 hours, 1 or 10 µM), stained with ROS-sensitive H2-DCFDA and then activated with fMLP. In addition, neutrophils were pre-treated with simvastatin as indicated and fluorescence was measured by flow cytometry FACS Canto (Becton Dickinson, San Jose, CA) every ten minutes following fMLP stimulation. n = 6 for each group; repetition = 4. Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference in comparison to the control group at each different time point.
Figure 5
Figure 5. Influence of simvastatin on neutrophil degranulation in vitro (A–C) and in vivo (D+E).
Isolated human neutrophils were pre-treated with Simvastatin (3 hours, 1 or 10 µM) and then activated with fMLP. MFI of surface expression of CD11b (A), CD29 (B) and FPRL1 (C) as measured by flow cytometry after staining with directly conjugated antibodies (n = 3–6; repetition = 4). D+E In vivo degranulation after LPS inhalation. Mice were challenged with LPS via inhalation and sacrificed 4 hours later. After isolation of neutrophils from the blood and the lung, MFI of surface expression of CD11b and CD29 as measured by flow cytometry after staining with directly conjugated antibodies (n = 4–6). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference in comparison to the fMLP or the LPS group.
Figure 6
Figure 6. Simvastatin does not affect neutrophil antimicrobial activity in vitro (A + B) and in vivo (C). A:
Bacterial uptake of fluorescent IgG- or complement-opsonized E. coli by activated or resting neutrophils as assessed by flow cytometry. Neutrophils were activated with fMLP and pretreated with simvastatin as indicated n = 4; repetition = 4. Statistical significance was tested using two way ANOVA with with Bonferroni posttest. * indicates significant difference to the control group without fMLP. B: Formation of colony forming units (CFU) after hypotonic lysis of neutrophils that had phagocytozed E. coli (BL21) for 20 min. (n = 4). C No negative effect on neutrophil bacterial clearance in acute lung injury. 24 h after cecal ligation and puncture (CLP), the supernatant of the harvested lungs was spread out on Luria Bertani agar over night and the colony forming units (CFU) were enumerated. The CFU did not increase after simvastatin treatment in comparison to the CLP group indicating no negative effect on the bacterial clearance (n = 5). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference to the control group without neutrophils.
Figure 7
Figure 7. Simvastatin does not affect neutrophil apoptosis.
Apoptosis of neutrophis in presence or absence of simvastatin was measured using Annexin V with flow cytometry after 3h and 24 hours respectively (n = 6; repetition = 3). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference to the control group without neutrophils.
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This study was supported by the Deutsche Forschungsgemeinschaft (SO876/3-1, FOR809 TP2, TP9), the German Heart Foundation, the Else-Kröner-Fresenius Foundation, and the B. Braun Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.