Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice

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Abstract

Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment.

Highlights

►Mer expression is restored by TAPI-0 treatment in bleomycin-stimulated lung. ►Mer signaling is enhanced by TAPI-0 treatment in bleomycin-stimulated lung. ►TAPI-0 enhances efferocytosis and promotes resolution of lung injury.

Introduction

Bleomycin is a chemotherapeutic drug used clinically for a variety of human malignancies. Administration of a high dose of bleomycin often leads to life-threatening pneumonitis that can progress to interstitial pulmonary fibrosis (Chandler, 1990). Severe acute lung injury occurs at 5–7 days post-bleomycin instillation in murine models (Goto et al., 2010, Koshika et al., 2005). Their pathology includes acute alveolitis and interstitial inflammation, characterized by recruitment of neutrophils and macrophages, with epithelial cell injury. Fibrotic responses subsequently occur, which are characterized by increased fibroblast proliferation and extracellular matrix synthesis. The early changes are very important because the development of fibrosis is directly influenced by the extent of initial injury (Shen et al., 1988). The histologic similarities have been demonstrated between early-stage bleomycin-induced lung injury in the rodent model and clinically acute lung injury in humans (Colombo et al., 2007).

The Mer receptor tyrosine kinase (Mer) belongs to the Tyro-3/Axl/Mer (TAM) receptor subfamily that share common ligands, including growth arrest-specific protein 6 (Gas6) and protein S. Ligand interaction with TAM receptors leads to receptor phosphorylation and activation of downstream signaling pathways that affect cell survival, proliferation, cytoskeletal reorganization, and cell migration. These receptors are crucial for many aspects of immune function by mediating engulfment of apoptotic cells (efferocytosis). Mer-deficient mice have multiple defects in monocyte function, which can lead to the development of autoimmune disorders (Lu and Lemke, 2001, Scott et al., 2001). A recent study reported that cytokine-dependent activation of TAM signaling stimulates an antiinflammatory pathway under the regulation of Toll-like receptors (Rothlin et al., 2007). Indeed, in response to lipopolysaccharide (LPS), Gas6-induced Mer activation was responsible for the reduction of inflammatory cytokine expression only in cell expressing Mer (Alciato et al., 2010). These results highlight the importance of Mer in immune system cells, particularly in macrophages. However, the contribution of Mer to the resolution of inflammation and the repair process of damaged tissues in vivo has not yet been characterized.

A recent report by Sather et al. (2007) indicates that the proteolytic cleavage of Mer producing soluble Mer (sMer) is significantly increased, resulting in a reduction of membrane-bound Mer protein, in response to stimuli such as LPS or phrobol 12-myristate 13-acetate (PMA). Thorp et al. (2011) conclusively showed that ADAM 17 is the key protease required for the sMer shedding. ADAM17 is a member of the ADAM (a disintegrin and metalloproteinase) family of proteases (Doedens and Black, 2000), which plays a central role ectodomain shedding (O'Bryan et al., 1995, Schlöndorff and Blobel, 1999). The soluble cleavage product may act as a decoy receptor and sequester ligand. This mechanism of Mer regulation inhibits receptor function in macrophage-mediated engulfment of apoptotic cells (Sather et al., 2007). However, the in vivo physiological relevance of Mer cleavage during diseases of inflammation and defective efferocytosis has not been determined.

In the present study, we investigated whether cleavage of Mer during bleomycin-induced acute lung injury is enhanced, and if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates early lung inflammation and apoptosis using the ADAM inhibitor TAPI-0. TAPI-0 is a peptide-based compound in which the hydroxamic group (a strong chelating moiety) interacts with the catalytic zinc of the ADAM family of proteases and consequently inhibits their activities (Antczak et al., 2008, Balakrishnan et al., 2006). To confirm the role of Mer, coadministration of specific Mer-neutralizing antibodies will be used in an attempt to reverse the effects of TAPI-0.

Section snippets

Animal protocols

Specific pathogen‐free male C57Bl/6 mice (Orient Bio, Sungnam, Republic of Korea) weighing 20–22 g were used in all experiments. The Animal Care Committee of the Ewha Medical Research Institute approved the experimental protocol.

Mouse pharyngeal aspiration was used for administration of bleomycin (5 U/kg, Sigma Chemical Company, St. Louis, MO) (Rao et al., 2003). 50 mg/kg TAPI-0 (N-{d,l-[2-(hydroxyaminocarbonyl)methyl]‐4-methyl-pentanoyl}L-3-(2-naphtyl)-alanyl-l-alanine, 2-aminoethyl amide; TNF-α

Membrane-bound Mer is reduced and sMer production is enhanced after bleomycin treatment

To determine the change of membrane-bound Mer levels after bleomycin treatment, Mer protein expression in lung tissue homogenates was analyzed by Western blot using anti-Mer antibody. Membrane-bound Mer was detected around at 180 kDa from the saline control lung tissue, consistent with previous reports (Linger et al., 2009). However, at days 1, 3, and 7 after bleomycin treatment, membrane-bound Mer expression in lung tissue was decreased for up to 7 days post-bleomycin treatment (Fig. 1A).

Discussion

Mer receptor tyrosine kinase plays a critical role in many aspects of the immune response by mediating clearance of apoptotic cells and intrinsic antiinflammatory pathways. Within cells of the lung, Mer expression has been detected in macrophages, and epithelial cells (Kazeros et al., 2008, Lee et al., 2012, Lu and Lemke, 2001). One mechanism of regulating the activation of tyrosine kinase receptors is through shedding of the membrane-bound cell surface protein. Data from our present study

Conflict of interest statement

The authors declare that there are no conflicts of interest.

The following is the supplementary data related to this article.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; 2010‐0029353).

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