ADAM15 deficiency attenuates pulmonary hyperpermeability and acute lung injury in lipopolysaccharide-treated mice

Abstract

ADAM15 is a disintegrin and metalloprotease recently implicated in cancer and chronic immune disorders. We have recently characterized ADAM15 as a mediator of endothelial barrier dysfunction. Whether this molecule contributes to acute inflammation has not been evaluated. The purpose of this study was to investigate the role of ADAM15 in mediating pulmonary microvascular leakage during acute inflammatory injury. Immunofluorescent staining and Western blotting revealed that the endothelium was the main source of ADAM15 in lung tissue. In a mouse model of acute lung injury induced by lipopolysaccharide (LPS), upregulation of ADAM15 was observed in association with pulmonary edema and neutrophil infiltration. The LPS-induced inflammatory injury, as demonstrated by bronchoalveolar lavage neutrophil count, lung wet-to-dry weight ratio, and myeloperoxidase activity, was significantly attenuated in Adam15(-/-) mice. Studies with primary cell culture demonstrated abundant ADAM15 expression in endothelial cells (ECs) of mouse lung but not in neutrophils. Deficiency of ADAM15 in ECs had no obvious effect on basal permeability but significantly attenuated hyperpermeability response to LPS as evidenced by albumin flux assay and measurements of transendothelial electrical resistance, respectively. ADAM15 deficiency also reduced neutrophil chemotactic transmigration across endothelial barriers in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). Rescue expression of ADAM15 in Adam15(-/-) ECs restored neutrophil transendothelial migration. These data indicate that ADAM15 upregulation contributes to inflammatory lung injury by promoting endothelial hyperpermeability and neutrophil transmigration.

Fig. 1.

The endothelium is a predominant source of ADAM15 in lung. A: ADAM15 colocalizes with CD31 in lung tissue. Immunofluorescence microscopy demonstrates colocalization of CD31 and ADAM15. Lung sections were labeled with CD31 and ADAM15 antibody, followed by rhodamine and FITC-conjugated second antibody, respectively. Nuclei are counterstained with Hoechst 33342 (blue). Images are captured with Zeiss Axio Observer 200M inverted microscope equipped with Zeiss AxioVision software (scale bar = 25 μm). B: FACS analysis: primary lung endothelial cells (EC) or non-EC (NEC) isolated from wild-type (WT) mice were labeled with an anti-CD31 antibody or IgG isotype control. C: FACS analysis of ADAM15 demonstrated coexpression with VE-cadherin positive endothelial cells vs. NEC. D: after CD31 selection, EC cells were cultured to confluence and assayed for VE-cadherin and ADAM15 expression as shown.

Fig. 2.

ADAM15 is upregulated in the lung tissue of mice receiving LPS. A: Western blot analysis of ADAM15 expression in mouse lung tissue. Bottom, representative blot from C57/BL6 WT mice 24 h after intraperitoneal injection of LPS (10 mg/kg body wt) or an equivalent volume of PBS. Top, quantification (n = 4; *P < 0.05 vs. PBS treated). B: immunofluorescence microscopy shows increased ADAM15 staining in LPS-treated compared with PBS-treated lungs. Lung sections were labeled with ADAM15 antibody, followed by phycoerythrin-conjugated second antibody. C: Western blotting and densitometry quantification (top, n = 3, *P < 0.01) of ADAM15 expressed by in vitro cultured endothelial cells with or without LPS stimulation (12 h.). D: immunocytochemistry of untreated (left) of LPS-treated cells (as in C). Green, ADAM15; red, VE-cadherin; blue, nuclei.

Fig. 3.

ADAM15 contributes to LPS-induced pulmonary edema and inflammatory injury in mice. Adam15^−/− mice and their Adam15^+/+ littermates were intraperitoneally injected with LPS (10 mg/kg body wt) or PBS vehicle. A: the ratio of lung tissue wet to dry weights (W/D ratio) is assessed as an indicator of edema (n = 12; *P < 0.05 vs. Adam15^+/+). B: neutrophil counts in bronchial alveolar lavage (n = 6; *P < 0.05 vs. Adam15^+/+). C: myeloperoxidase (MPO) activity in lung tissues (n = 6; *P < 0.05 vs. Adam15^+/+). D: comparison of the lung injury score (n = 6, *P < 0.05 vs. Adam15^+/+). E: compared with vehicle (PBS)-treated, LPS-treated lung shows thickened alveolus-capillary membrane and neutrophil infiltration as indicated with hematoxylin and eosin staining (arrowheads pointing to neutrophils in alveoli). The LPS-induced inflammatory injury is less pronounced in Adam15^−/− lungs. Scale bar = 50 μm.

Fig. 4.

ADAM15 expression is detectable in endothelial cells but not in neutrophils. A: flow cytometric evidence of cell identity and purity. Left, primary lung microvascular EC (MLEC) from Adam15^+/+ or Adam15^−/− or mice labeled with an anti-CD31 antibody or IgG isotype control. Right, neutrophils from mouse bone marrow (mPMN) are labeled with an anti-Ly-6G antibody, and granulocytes are gated. Isotype control is also shown. The data represent 3 independent experiments from different mice. B: ADAM15 expression in MLEC and mPMN. Shown are representative blots from 3 independent experiments demonstrating abundant ADAM15 in Adam15^+/+ EC but not in neutrophils.

Fig. 5.

ADAM15 deficiency attenuates LPS-induced endothelial barrier dysfunction. A: Transwell assays that measure the flux of FITC-albumin across endothelial monolayers show no significant difference in basal albumin permeability (P_a) between Adam15^−/− and Adam15^+/+ MLEC monolayers (P > 0.05). B and C: electrical cell impedance sensing (ECIS) studies that measure the dynamic transepithelial resistance changes indicate ADAM15 deficiency in MLEC attenuates LPS-induced endothelial hyperpermeability (n = 3, *P < 0.05 vs. Adam15^+/+ MLECs).

Fig. 6.

Endothelial ADAM15 mediates neutrophil transendothelial migration. A: mPMN transmigration across Adam15^−/− MLECs, especially in the presence of fMLP (10 nM), is significantly attenuated (n = 3; *P < 0.05 vs. Adam15^+/+ without fMLP; #P < 0.05 vs. Adam15^+/+ with fMLP). B: rescue expression of ADAM15 in Adam15^−/− MLECs is confirmed with Western blotting (left) and flow cytometry (right). C: rescue expression of ADAM15 in Adam15^−/− MLECs increased neutrophil transendothelial migration. Adam15^−/− MLECs were transfected with pcDNA/Adam15 or mock. At 72 h posttransmigration, fMLP-induced neutrophil transendothelial migration was assayed as above (n = 3, *P < 0.05 vs. mock transfected).