Lung endothelial ADAM17 regulates the acute inflammatory response to lipopolysaccharide

Abstract

Acute lung injury (ALI) is associated with increased vascular permeability, leukocyte recruitment, and pro-inflammatory mediator release. We investigated the role of the metalloproteinase ADAM17 in endotoxin-induced ALI with focus on endothelial ADAM17. In vitro, endotoxin-mediated induction of endothelial permeability and IL-8-induced transmigration of neutrophils through human microvascular endothelial cells required ADAM17 as shown by inhibition with GW280264X or shRNA-mediated knockdown. In vivo, ALI was induced by intranasal endotoxin-challenge combined with GW280264X treatment or endothelial adam17-knockout. Endotoxin-triggered upregulation of ADAM17 mRNA in the lung was abrogated in knockout mice and associated with reduced ectodomain shedding of the junctional adhesion molecule JAM-A and the transmembrane chemokine CX3CL1. Induced vascular permeability, oedema formation, release of TNF-α and IL-6 and pulmonary leukocyte recruitment were all markedly reduced by GW280264X or endothelial adam17-knockout. Intranasal application of TNF-α could not restore leukocyte recruitment and oedema formation in endothelial adam17-knockout animals. Thus, activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events most likely independently of endothelial TNF-α release leading to enhanced vascular permeability and leukocyte recruitment.

Figure 1. Influence of LPS challenge on ADAM10 and ADAM17 activity and mRNA expression in vitro

Data represent means ± SEM (n = 3 per group, C–E). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test (in D,E) or by two-way ANOVA followed by the Bonferroni post-test for double-treated cells (in C) and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate control. A-B. HMVEC-L were transduced with lentivirus encoding shRNA (LV-scramble, LV-antiA10 or LV-antiA17). Downregulation of ADAM10 (A) or ADAM17 (B) (black line) was analysed by surface staining with antibodies to ADAM10 or ADAM17 compared to isotype controls (light grey tinted) and surface stained scramble transduced cells (black tinted) followed by flow cytometry. Representative histograms of three independent experiments are shown. C. HMVEC-L were stimulated for 24 h with LPS (1 µg/ml) or vehicle control (PBS). Cell lysates were analysed for sheddase activity using a fluorogenic peptide cleavage assay. Results were expressed as percentage of sheddase activity in relation to the unstimulated control receiving the vehicle DMSO only (= 100%). D-E. HMVEC-L were stimulated for 0–4 h with LPS (1 µg/ml) or vehicle control (PBS) in the presence of 100 ng/ml LBP and in the absence of serum. ADAM17 (D) and ADAM10 (E) mRNA expression were examined by RT-qPCR analysis. Data are expressed as change of expression rate compared to control cells.

Figure 2. Role of ADAM10 and ADAM17 in lung microvascular endothelial cells in vitro

HMVEC-L were transduced with lentivirus encoding shRNA (LV-scramble, LV-antiA10 or LV-antiA17). Data represent means ± SEM (n = 3). Significance was calculated using one-way ANOVA followed by the Newman-Keuls post-test (in B and C) or by two-way ANOVA followed by the Bonferroni post-test for double-treated cells (in A) and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate control.

1. Transduced HMVEC-L were grown in transwell inserts and stimulated for 24 h with LPS (1 µg/ml) or were left unstimulated (PBS) in the presence or absence of GW280264X (10 µM). Permeability was measured by TRITC-dextran diffusion. Data are shown as percentage of TRITC-dextran permeability in relation to the unstimulated DMSO-treated control (= 100%).

2. HMVEC-L were stimulated for 4 or 24 h with LPS (1 µg/ml) or were left unstimulated (PBS) in the presence of 100 ng/ml LBP and in the absence of serum. Cells were treated either with GW280264X (10 µM) or with vehicle control (0.1% DMSO). Conditioned media were analysed by immunoblotting followed by densitometric quantification. A representative immunoblot of three independent experiments is shown below the graph.

3. Transduced HMVEC-L were pre-treated with or without GW280264X (10 µM) for 1.5 h and examined for IL-8-induced (10 ng/ml) transmigration of neutrophils. Experiments were performed with neutrophils from three different donors. Results were expressed as percentage of transmigration in relation to the LV-scramble-transduced control (C) receiving no IL-8.

Figure 3. Effect of ADAM10 and ADAM17 inhibition on permeability, oedema formation and cytokine secretion in LPS-induced lung inflammation

Mice were intranasally treated with LPS (400 µg/kg) or vehicle control (PBS) with or without the metalloproteinase inhibitor GW280264X (40 µg/kg). Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test and indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate control. A. Whole protein content of BAL fluid was determined 4 h after LPS application. B. Lung wet–dry-ratio was determined 24 h after intranasal LPS application. C-E. Release of CX3CL1 (C), TNF-α (D) and IL-6 (E) into BAL fluid was determined by ELISA 4 h after intranasal application.

Figure 4. Influence of ADAM10 and ADAM17 inhibition on leukocyte recruitment in LPS-induced lung inflammation

Mice were intranasally treated with LPS or vehicle control (PBS) with or without the metalloproteinase inhibitor GW280264X. After 24 h, lungs were lavaged and the lung tissue was enzymatically disintegrated. Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate PBS-treated controls. A-D. The total leukocyte cell number (A) as well as the number of macrophages (B) and neutrophils (C) in BAL fluid and the amount of neutrophils in lung tissue suspension (D) were determined by flow cytometry. Results were expressed as cell number per ml BAL fluid (A–C) or as percentage of the total number of lung tissue cells examined (D).

Figure 5. Role of endothelial ADAM17 on shedding processes in LPS-induced lung inflammation

Control and Tie2-adam17^−/− mice were intranasally treated with LPS or vehicle control (PBS). Data represent means ± SEM (n = 3 per group). Significance was calculated using Student's t-test (in A and B) or using one-way ANOVA followed by the Newman–Keuls post-test (in C–F) and is indicated by asterisks (*p < 0.05, **p < 0.01). Asterisks without line indicate significance to the appropriate PBS-treated controls. A-B. After challenge for 24 h, BAL and vascular perfusion, ADAM17 (A) and ADAM10 (B) mRNA expression in lung tissue were examined by RT-qPCR analysis. Data are expressed as percentage of expression in relation to PBS-treated controls. C-D. Release of soluble JAM-A into BAL fluid was measured by ELISA 4 and 24 h after intranasal application. E-F. Release of soluble CX3CL1 into BAL fluid was determined by ELISA 4 and 24 h after intranasal application.

Figure 6. Role of endothelial ADAM17 for permeability changes and tissue damage in LPS-induced lung inflammation

Control and Tie2-adam17^−/− mice were intranasally treated with LPS or vehicle control (PBS). Data represent means ± SEM (n = 3 per group for A and B and n = 10 per group for D). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test and is indicated by asterisks (**p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate PBS-treated controls. A. Whole protein content of BAL fluid was determined 4 h after application. B. Lung wet–dry-ratio was examined 24 h after application. C-D. After 24 h, 3 µm-sections of formalin-fixed and paraffin-embedded lung tissue were stained with hematoxylin–eosin. Representative images are shown in panel (C). Ten images per animal were analysed for the thickness of interalveolar septa using AixoVision software (D).

Figure 7. Role of endothelial ADAM17 for cytokine secretion in LPS-induced lung inflammation

Control and Tie2-adam17^−/− mice were intranasally treated with LPS or vehicle control (PBS). Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate PBS-treated controls. A-C. After 4 h of challenge, release of IL-6 (A), TNF-α (B) and KC (C) into BAL fluid was determined by ELISA measurement. D. Control and Tie2-adam17^−/− mice were compared for TNF-α-induced lung inflammation. The wet-dry-ratio of lung tissue was determined after 24 h of intranasal TNF-α-challenge.

Figure 8. Role of endothelial ADAM17 for leukocyte recruitment in LPS-induced lung inflammation

Control and Tie2-adam17^−/− mice were intranasally treated with LPS or vehicle control (PBS). Data represent means ± SEM (n = 3 per group for A–D and F and n = 10 per group for E). Significance was calculated using one-way ANOVA followed by the Newman–Keuls post-test and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks without line indicate significance to the appropriate PBS-treated controls. A-D. Twenty-four hours after application, the number of total leukocytes (A), neutrophils (B) and macrophages (C) in BAL fluid and the amount of neutrophils in lung tissue suspension (D) were determined by flow cytometry. Results were expressed as cell number per ml BAL fluid (A–C) or as percentage of the total number of lung tissue cells investigated (D). E. Ten images of hematoxylin–eosin stained 3 µm-sections per animal were analysed for invading neutrophils using the AixoVision software. F. Control and Tie2-adam17^−/− mice were compared for TNF-α-induced lung inflammation. The amount of neutrophils in BAL fluid was determined by flow cytometry after 24 h of intranasal TNF-α-challenge.