Lung endothelial ADAM17 regulates the acute inflammatory response to lipopolysaccharide

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.

CD16/32 Fc-Block was obtained from eBiosciences. Rabbit polyclonal antibody against βcatenin was from Santa Cruz Biotechnology, Inc (Delaware, USA). The mouse monoclonal antibody against human JAM-A was from BD Pharmingen. Lipopolysaccharide (LPS) from E. coli 0127:B8 was from Sigma-Aldrich (Munich, Germany), human and murine TNFα and IFNγ were from R&D Systems. Human LBP was from Peprotech (Rocky Hill, USA). The metalloproteinase inhibitor GW280264 was synthesized and assayed for inhibition of human and mouse ADAM17 and ADAM10 as described (2). The Complete Protease Inhibitor was from Roche (Munich, Germany).

Primary cells and cell culture
Lung human microvascular endothelial cells (HMVEC-L) (Lonza, Belgium) were cultured in EGM-2 MV medium (Lonza) and subcultured following the manufacturer's protocol. Cells were used in passage 4 for viral transduction and passage 5 to 6 for all assays. Recombinant lentiviruses were produced by transient transfection of 293T packaging cells according to standard protocols (3) as described earlier (4). The targeting sequences were: 5'-ACAGTGCAGTCCAAGTCAA-3'annealing from bp 1947 to bp 1965 of ADAM10 mRNA (LV-anti10) and 5'-AGGAAAGCCCTGTACAGTA-3'annealing from bp 2061 to bp 2079 of ADAM17 mRNA (LV-anti17). A sequence of 5'-CCGTCACATCAATTGCCGT-3' served as scramble control (LV-scramble). After 48 to 72 h, transduction was examined by fluorescence microscopy. Alveolar macrophages were isolated out of BAL fluid and seeded for 2 hours (5).
Before stimulation with 1 µg/ml LPS, non-adherent cells were removed, and after 4 hours the TNFα release to the cell culture supernatant was measured by ELISA.

Immunohistochemistry
HMVEC-L were grown on 48-well tissue culture plates to complete monolayers. Cells were washed with PBS and fixed for 15 min in 4% PFA, followed by 10 min incubation with 50 mM NH 4 Cl and permeabilization in 1% Triton X-100 in PBS for 4 min. After washing, cells were blocked in PBS/5% FBS for 30 min, followed by incubation with rat anti-m-βcatenin antibody for 30 min (6). Cells were washed, incubated with Alexa555-coupled anti-rat antibody for 30 min, DAPI-stained and at least covered with Immu-Mount (ThermoScientific, USA). Fluorescence microscopy was performed using the Leica DMI14000B (Leica Microsystems, Germany) with an image analysis package (DISKUS, Technisches Büro Carl H. Hilgers, Königswinter, Germany).

Peptide cleavage assay
Cell lysates were prepared in extraction buffer (0.15 M sodium citrate, 1% Tween 80, pH 8.0). After removal of cell debris by centrifugation at 16000 g and 4°C for 10 min, lysates were corrected for same protein amount (determined by BC assay kit), diluted 1:2 in reaction buffer (25 mM Tris-HCl, 0.01% Triton-X 100, pH 8.0) with or without GW280264 (10 µM) for monitoring ADAM-dependent activity. After incubation for 20 min, a fluorogenic peptidebased substrate mimicking the α-cleavage site of amyloid-precursor protein (APP, Ac-RE(EDANS-VHHQKLVF-K(DABCYL)-R-OH, 5 µM, Calbiochem) was added. Complete inhibition of general protease activity was obtained by using an inhibitor mix (Complete, Roche). The generation of the fluorescent cleavage product was monitored at 37°C over time in a spectral fluorimeter (Fluostar Optima, BMGLabtech, Offenburg, Germany) at 355 nm excitation and 510 nm emission wavelength. Linearity was determined by a serial dilution of a defined standard.

Transmigration assay
Neutrophils were isolated as described before (4). HMVEC-L were seeded on collagen-coated polycarbonate transwell filters with 5 µm-pores (Costar, Lowell, USA) and grown to confluence. 24-well culture plates were filled with 600 µl/well DMEM containing 0.2% BSA with or without 10 ng/ml IL-8 (R&D Systems). The transwells were transferred into the wells and filled with 100 µl of neutrophil suspension (2x10 5 neutrophils per well). After incubation for 30 min at 37°C and 5% CO 2 , transwells were carefully removed and transmigrated cells were quantified by measurement of endogenous glucuronidase activity as describe before (7).
For inhibition of ADAM10 and ADAM17, GW280264 (10 µM) was added to HMVEC-L layers 1.5 h prior to the assay.

Permeability assay
HMVEC-L were seeded on collagen-coated polycarbonate transwell filters with 5 µm-pores (Costar) and grown to confluence. For lentivirally-transduced HMVEC-L expressing green fluorescent protein (GFP), confluence was controlled by fluorescence microscopy. After 24 h stimulation with LPS (1 µg/ml) or/and GW280264 (10 µM), the cell culture medium in the upper chamber was replaced by 70 kDa TRITC-dextran suspension (Sigma-Aldrich, 1 mg/ml in PBS with 0.2% BSA,). After 90 min incubation at 37°C and 5% CO 2 , permeability was analyzed by measurement of the fluorescence intensity in the lower chamber at 535 nm excitation and 595 nM emission wavelength using XFluor4 Genios (Tecan, Grödig, Austria).

JAM-A cleavage assay
HMVEC-L were seeded on 6-wells and grown to confluence. JAM-A release to cell culture supernatant was performed as previously described (8). Values were standardized on equal volumes of BAL recovery.

Determination of protein content
The protein content of BAL fluid, tissue lysates and cell lysates (each in equal volumes of recovery) was determined using a commercial BC assay kit (Interchim) following the manufacturer's protocol. Linearity was determined using a bovine serum albumin (0 to 3000 ng/ml) standard in the aquivalent probe solution.

Flow cytometry
PBS supplemented with 1% FCS and 5 mM EDTA (sterile filtered) was used as assay buffer, and all steps of the staining process were performed at 4°C. L-HMVEC cells were detached from culture dishes using accutase (PAA, Cölbe, Germany) and analyzed for expression of ADAM10 and ADAM17 by incubation with mouse monoclonal antibodies against ADAM10 and ADAM17 (1 µg/ml and 5 µg/ml resp.) followed by incubation with a APC-conjugated anti-mouse antibody (1:100). Isotype controls for mouse IgG1 and mouse IgG2a, respectively,