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

Toxicology and Applied Pharmacology 263 (2012) 61–72

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Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice Ye-Ji Lee a, c, Seung-Hae Lee a, Young-So Youn a, c, Ji-Yeon Choi a, c, Keung-Sub Song a, Min-Sun Cho b, Jihee Lee Kang a, c,⁎ a b c

Department of Physiology, School of Medicine, Ewha Womans University, Seoul, Republic of Korea Department of Pathology, School of Medicine, Ewha Womans University, Seoul, Republic of Korea Tissue Injury Defense Research Center, School of Medicine, Ewha Womans University, Seoul, Republic of Korea

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Article history: Received 22 February 2012 Revised 1 May 2012 Accepted 31 May 2012 Available online 9 June 2012 Keywords: Mer receptor tyrosine kinase Bleomycin Apoptotic cell clearance Lung injury TAPI-0

a b s t r a c t 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 bleomycininduced 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. © 2012 Elsevier Inc. All rights reserved.

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

Abbreviation: Mer, Mer receptor tyrosine kinase; ADAM, a disintegrin and metalloproteinase; TAM, Tyro-3/Axl/Mer; Gas6, growth arrest-specific protein 6; LPS, lipopolysaccharide; sMer, soluble Mer; PMA, phrobol 12-myristate 13-acetate; BAL, bronchoalveolar lavage; PI, phagocytic index; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor-α; IL-1β, interleukin β; MIP-2, macrophage inflammatory protein-2; HGF, hepatocyte growth factor; TGF-β, transforming growth factor-β. ⁎ Corresponding author at: Department of Physiology, Tissue Injury Defense Research Center, School of Medicine, Ewha Womans University, 911‐1 Mok-6-dong, Yangcheonku, Seoul 158‐056, Republic of Korea. Fax: +82 2 2650 5850. E-mail address: [email protected] (J.L. Kang). 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2012.05.024

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-

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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. Materials and methods 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 protease inhibitor-0, Peptides International, Louisville, KY) was diluted in 25 μl dimethyl sulfoxide and further diluted with 275 μl of phosphate buffered saline. TAPI-0 or vehicle was given intraperitoneally 1 h before bleomycin or saline treatment, and then once a day thereafter (Terao et al., 2010). Mice were euthanized on day 1, 3, or 7 following bleomycin treatment. For anti-Mer antibody inhibition experiments, 2.0 mg/kg goat polyclonal anti-mouse Mer Ab (AF591, R&D Systems, Minneapolis, MN), or control goat IgG Ab (R&D Systems) in a volume of 200 μl phosphate buffered saline was coadministered intraperitoneally with TAPI-0 (50 mg/kg) 1 h before bleomycin treatment, and then once a day thereafter (Lee et al., 2012). The antibodies reach the lung through the blood stream (Huynh et al., 2002; Lee et al., in press; Shreffler, 2007). Mice were euthanized on day 3 following bleomycin treatment. Bronchoalveolar lavage (BAL) cells, lung tissue, and cell counts BAL was performed through a tracheal cannula using 0.7-ml aliquots of ice-cold Ca 2 +/Mg 2 +-free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM dextrose, pH 7.4) to a total of 3.5 ml for each mouse. Cell counts were determined using an electronic Coulter Counter fitted

with a cell sizing analyzer (Coulter Model ZBI with a channelizer 256; Coulter Electronics, Bedfordshire, UK) (Castranova et al., 1990; Moon et al., 2010). Neutrophils and alveolar macrophages were identified by their characteristic cell diameters. BAL cells were isolated and cytospins were made to assess phagocytic indices. Alveolar macrophage isolation and culture Alveolar macrophages were isolated by adhesion (60 min) and cultured in serum-free X-vivo medium (Biowhittaker, Walkersville, MA). Suspended alveolar macrophages from mice were over 95% viable as determined by trypan blue dye exclusion. Induction of apoptosis Human T lymphocyte Jurkat cells were obtained from the American Type Culture Collection (Rockville, MD). Jurkat T cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum supplemented with 100 μg/ml streptomycin, and 100 U/ml penicillin at 37 °C and 5% CO2. Jurkat T cells were exposed to ultraviolet irradiation at 254 nm for 10 min and incubated for 2.5 h before use. Cells were approximately 70% apoptotic by evaluation of nuclear morphology using light microscopy (Hoffman et al., 2001). Ex vivo phagocytosis assays Alveolar macrophages (105) from mice treated with saline+ vehicle, bleomycin + vehicle, or bleomycin + TAPI-0 were plated in each well of a 4-chamber slide glass plate at 37 °C and cultured overnight. Apoptotic Jurkat T cells were added to macrophage cultures in a ratio 10:1 in 1 ml media for 90 min. Samples were washed prior to Wright's Giemsa staining to remove uningested apoptotic cells. Phagocytosis was quantified using the phagocytic index [(number of apoptotic bodies)/ (200 total macrophages) × 100] as previously described (Hoffman et al., 2001; Moon et al., 2010). Each condition was tested in duplicate and the reader was blinded to the sample identification during analysis. ELISA BAL fluid samples were assayed in duplicate using TNF-α, IL-1β, MIP-2, TGF-β, and HGF ELISA kits (R&D Systems) according to the manufacturer's instructions. Concentrations of these cytokines were calculated as picograms per milliliter, based on the appropriate standard curve. ADAM17 activity Lung tissue lysates were prepared and assayed in duplicate for ADAM17 activity using a fluorogenic peptide-based assay kit from R&D Systems following the manufacturer's instructions. The enzymatic activity was expressed in relation to a recombinant ADAM17 standard that was serially diluted and run in parallel. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from lung tissue using TRIzol reagent according to the manufacturer's instructions. The concentrations and purities of the RNA samples were evaluated by spectrophotometry. Reverse transcription was conducted for 60 min at 42 °C with 1 μg of total RNA using Advantage RT-for-PCR kits (BD Biosciences, San Jose, CA). HGF-mRNA levels were determined using relative quantitative RT-PCR kits (Intron, Seoul, Republic of Korea). The primer sequences used were for mouse-specific TNF-α (sense 5′-CATGGATCTCAAAGACAACCAACTAGTG-3′ and anti-sense 5′-CCTTCTCCAGCTGGAAGACTCC-3′), mouse-specific IL-1β (sense 5′-GCTGAAAGCTCTCCACCTCAATGGA-3′ and anti-sense 5′-GCTCTGCTTGTGAGGTGCTGATGT-3′), mouse-specific

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MIP-2 (sense 5′-CAGTGAACTGCGCTGTCAATG-3′ and anti-sense 5′CTCTCAGACAGCGAGGCACATCAGGTA-3′), mouse-specific TGF-β (sense 5′-CTTCAGCTCCACAGAGAAGAACTGC-3′ and anti-sense 5′CACAA-TCATGTTGGACAACTGCTCC-3′), mouse-specific HGF (sense 5′-CCAAACTTCTGCCGGTCCTGTTGC-3′ and anti-sense 5′-AACGAAGGCCTTGCA-AGTGAACG-3′), and β-actin (sense 5′-CCTGACAGACTACCTCATGAAGATCCT-3′ and anti-sense 5′-CCACATCTGCTGGAAGGTGGAC-3′). cDNA was denatured for 5 min at 94 °C and amplification was achieved in a thermocycler (Perkin Elmer GeneAmp PCR System 2400, Foster City, CA). A total of 5 μl of each PCR sample was loaded on a 1.5% agarose gel stained with ethidium bromide. The relative fluorescence of each gene versus β-actin was analyzed by densitometry. Measurement of total protein Protein concentrations of the BAL samples were used as indicators of blood–pulmonary epithelial cell barrier integrity. Total protein content was measured in duplicate according to the method of Hartree (1972) using bovine serum albumin as a standard. Lactate dehydrogenase (LDH) assays LDH activity in the first aliquot of the acellular BAL fluid was measured in duplicate. LDH activity was determined by monitoring the LDH-catalyzed oxidation of pyruvate coupled with the reduction of nicotinamide adenine dinucleotide at 340 nm using a commercial kit (Roche Diagnostic Systems, Montclair, NJ). Western blot analysis Lung tissue homogenates and total cell lysates (50 μg protein/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. Separated proteins were electrophoretically transferred onto nitrocellulose paper and blocked for 1 h at room temperature with Tris-buffered saline containing 3% bovine serum albumin. Blocked membranes were incubated at room temperature for 1 h with an anti-phospho-Mer (Fab Gennix, Frisco, TX)/Mer (Santa Cruz, CA), ADAM17, or β-actin antibody and visualized by chemiluminescence. Lung histology Lung tissue was fixed with 10% buffered formalin with gentle perfusion through the trachea for 24 h and was then embedded in paraffin. Sections (3-μm thick) were stained with hematoxylin–eosin.

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excitation wavelength of 400 nm and an emission wavelength of 505 nm. Each condition was tested in duplicate. Statistical analysis Values are expressed as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) was applied for multiple comparisons and Tukey's post hoc test was applied where appropriate. Student's t test was used for comparisons of two sample means. A p value of b0.05 was considered statistically significant. Results 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). Interestingly, phosphorylation of Mer in lung tissue slightly increased on days 1 through 7 after bleomycin treatment in spite of decreased expression of this protein (Fig. 1A). Likely, the levels of membrane-bound Mer protein in alveolar macrophages from bleomycin-treated mice at the same time points were reduced (Fig. 1B), but phosphorylation was slightly enhanced (Fig. 1B). Moreover, phosphorylation of Akt, a downstream molecule of Mer signaling pathways, was also increased a little (Fig. 1C). In contrast to the changes of membrane-bound Mer levels, sMer production in BAL fluid after bleomycin treatment was increased for up to 7 days post-bleomycin treatment (Fig. 1D). In fact, among ADAM family proteases, ADAM17/TACE is only known to cleave Mer (Weinger et al., 2009). The cleavage of Mer by ADAM17 results in the release of sMer protein (Sather et al., 2007; Thorp et al., 2011). Thus, ADAM17 activity and protein expression in lung tissue after bleomycin treatment were assayed. In bleomycin-stimulated lungs, ADAM17 activities and expression substantially increased on days 1–7 correlated with increased levels of sMer (Figs. 1E and F). TAPI-0, an ADAM inhibitor, recovers membrane-bound Mer, and enhances Mer activation during bleomycin-induced acute lung injury

DNA damage and apoptosis in lung tissue DNA fragmentation in apoptotic cells was detected in lung tissues using TUNEL kits (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, a section of formalin-fixed, paraffin-embedded lung tissue from each mouse was examined by TUNEL. Samples were biotinylated at 37 °C for 1 h and endogenous peroxidases were blocked by 0.3% H2O2. Samples were then treated with streptavidin-HRP at room temperature for 30 min and with 3,3′-diaminobenzinine. Cells were counted using light microscopy. Positive staining was indicated by a black–brown color. The number of TUNEL-positive cells in a field from each section was counted using a microscope at1000 × magnification. Caspase-3 and caspase-9 activities The bioactivities of caspase-3 and caspase-9 were measured with a Fluorometric Assay Kit (ABcam, Cambridge, UK). Briefly, 25-μg lung homogenate samples were added to reaction buffer and then incubated with caspase-3 substrate DEVD-AFC or caspase-9 substrate LEHD-AFC. The fluorescence of the cleaved substrates was determined at an

In vitro experiments have previously demonstrated that increases in the production of sMer by macrophages in response to stimuli, such as LPS or PMA, are virtually eliminated in the presence of the ADAM inhibitor TAPI-0 (Sather et al., 2007). In the present study, TAPI-0 was thus used to prevent the enhanced cleavage of Mer after bleomycin treatment. Membrane-bound Mer expression in lung tissue was fully recovered on days 1–7 post-bleomycin treatment (Fig. 1A) and bleomycin-induced production of sMer was abrogated (Fig. 1D). Phosphorylation of Mer was further enhanced the day after bleomycin treatment (day 1) and the increased levels were maintained up to 7 days post-bleomycin treatment (Fig. 1A). Consistent with these results, TAPI-0 treatment resulted in the recovery of membrane-bound Mer expression in alveolar macrophages treated with bleomycin, and Mer phosphorylation was enhanced on days 1–7 post-bleomycin treatment by TAPI-0 treatment (Fig. 1B). Moreover, phosphorylation of Akt in lung tissue was also increased up to 7 days post-bleomycin treatment (Fig. 1C). As predicted, TAPI-0 suppressed ADAM17 activity and expression in bleomycin-stimulated lungs at these time points (Figs. 1E and F). In contrast, the levels of membranebound Mer expression and phosphorylation in lung tissue from mice

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Fig. 2. TAPI-0 enhances apoptotic cell clearance by alveolar macrophages. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, or 7 days post-BLM instillation. (A, B) BAL was performed, cytospins were stained, and alveolar macrophage ingestion of apoptotic cells was quantified by calculating the phagocytic index (PI). (C, D) Alveolar macrophages were cultured overnight and treated with ex vivo apoptotic Jurkat T cells for 90 min, and phagocytosis was quantified by calculating the PI. Representative photomicrographs show (B) cytospin-stained BAL cells and (D) alveolar macrophages cultured ex vivo with apoptotic cells at 3 days after bleomycin treatment. Arrowheads indicate alveolar macrophages with engulfed apoptotic cells or fragments. Scale bars: all 10 μm. Values represent means ± SEM of results from five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05.

treated with only TAPI-0 were not changed at each day compared with saline controls (Supplemental Fig. 1A). TAPI-0 treatment enhances apoptotic cell clearance in vivo and ex vivo Mer plays a critical role in the engulfment and efficient clearance of apoptotic cells. We thus examined whether recovered membranebound Mer and increased Mer activation after TAPI-0 treatment would result in enhancement of apoptotic cell clearance in vivo and ex vivo. The phagocytic index (PI) in BAL cytospin alveolar macrophages obtained from bleomycin-stimulated lungs was increased over the levels seen with saline controls at 1–7 days post-bleomycin treatment. Furthermore, TAPI-0 treatment significantly enhanced the PI in BAL cytospin alveolar macrophages at each day post-treatment compared with bleomycin + vehicle treatment (Fig. 2A). This data suggests that the phagocytic efficiency of alveolar macrophages was significantly increased at each day post-treatment due to enhanced Mer activation by increased membrane-bound Mer protein levels. Thus, phagocytic activities of alveolar macrophages obtained on days 1–7 post-bleomycin treatment were also assessed ex vivo using an approach that controls for the number and ratio of alveolar macrophages to apoptotic cells (Moon et al., 2010). Freshly obtained alveolar macrophages from saline or bleomycintreated lungs were cultured overnight prior to addition with apoptotic cells. The PI in alveolar macrophages taken from bleomycin-stimulated lungs was significantly enhanced when compared with saline controls

at each day post-bleomycin treatment. TAPI-0 treatment significantly enhanced the ability of alveolar macrophages to phagocytose apoptotic Jurkat cells ex vivo at each day post-treatment compared with the bleomycin + vehicle group (Fig. 2C). Two samples of enhanced apoptotic cell clearance in the bleomycin +TAPI-0 group at 3 days after bleomycin treatment in vivo and ex vivo are shown in Figs. 2B and C, respectively. TAPI-0 treatment reduces proinflammatory mediators but increases TGF-β1 and HGF Mer signaling down-regulates the proinflammatory pathway, which inhibits proinflammatory cytokine expression (Alciato et al., 2010; Rothlin et al., 2007). Thus, in the present study, we examined whether recovered expression and enhanced activation of Mer by TAPI-0 treatment down-regulates proinflammatory mediator expression at the protein and gene levels during bleomycin-induced acute lung injury. Protein levels of the proinflammatory cytokines, TNF-α and IL-1β, and a chemokine, MIP-2, were measured by ELISA. Cytokine levels in BAL fluid were significantly increased at days 1 and 3, and declined to basal levels at day 7 after bleomycin treatment. TAPI-0 significantly suppressed bleomycin-induction of TNF-α (approximately 78 and 59% inhibition at days 1 and 3 postbleomycin treatment, respectively; Fig. 3A), IL-1β (approximately 42 and 55% inhibition at days 1 and 3 post-bleomycin treatment, respectively; Fig. 3B), and MIP-2 (approximately 74 and 50% inhibition

Fig. 1. TAPI-0 recovers membrane-bound Mer expression and enhances Mer activation through reduction of ADAM 17 activity and sMer production. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, or 7 days post-BLM instillation. Western blots probed with anti-Mer antibody (Ab) or anti-phosphorylated Mer were employed to monitor the protein expression and phosphorylation of Mer in lung tissue (A) and in alveolar macrophages (B). The relative densitometric intensity was determined for each band and normalized to tubulin. (C, D, F) Western blots probed with antiphospho-Akt, anti-Akt, anti-Mer Ab, or anti-ADAM17 were employed to monitor the phosphorylation state of Akt, ADAM17 expression in lung tissue and sMer production in BAL fluid, respectively. Densitometric results are expressed as fold increase. (E) ADAM17 activity in lung tissue was analyzed on a given day, using a specific fluorogenic substrate as previously described and was expressed as relative fluorescence units (rfu). Values represent means ± SEM of results from five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05.

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Fig. 3. Changes in bleomycin-induced proinflammatory mediators, TGF-β1, and HGF after TAPI-0 treatment. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, and 7 days post-BLM instillation. (A) TNF-α, (B) IL-1β, (C) MIP-2, (D) TGF-β1, and (E) HGF protein levels in the BAL fluid were quantified by ELISA. The values represent the means ±SEM of results from at least five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05.

at days 1 and 3 post-bleomycin treatment, respectively; Fig. 3C) in BAL fluid. To evaluate the mRNA expression of TNF-α, IL-1β, and MIP-2, semiquantitative RT-PCR was performed using mRNA extracted from lung tissue. Similar to protein expression, TNF-α, IL-1β, and MIP-2 mRNA expression in lung tissue significantly increased at days 1 and 3 and declined at day 7 post-bleomycin treatment (Figs. 4A–C). However, TAPI-0 treatment significantly down-regulated the levels of mRNA of TNF-α and MIP-2 at days 1 and 3 post-bleomycin treatment (Figs. 4A and C). IL-1β mRNA expression was slightly decreased, but this change was not significant at these time points post-bleomycin treatment (Fig. 4B). The levels of TGF-β1 protein and mRNA in BAL fluid and lung tissue, respectively, peaked at day 1, and declined into basal levels by day 7 post-bleomycin treatment (Figs. 3D and 4D). TAPI-0 treatment significantly enhanced bleomycin-induced TGF-β1 protein only at day 1 postbleomycin treatment. However, the effects of TAPI-0 on the mRNA levels were not significant at days 1–7 post-bleomycin treatment. Interestingly, HGF protein and mRNA levels in BAL fluid and lung tissue gradually increased up to7 days after bleomycin (Figs. 3E and 4E). TAPI-0 treatment further enhanced the HGF protein and mRNA levels on days 3 and 7 after bleomycin treatment. In contrast, the levels of HGF in BAL fluid were not changed in mice treated with only TAPI-0 at each day compared with saline controls (Supplemental Fig. 1B). TAPI-0 treatment suppresses bleomycin-induced lung inflammatory responses and apoptosis TAPI-0 treatment significantly inhibited bleomycin-induced increases in intra-alveolar neutrophils at days 3 and 7 (Fig. 5A) and

macrophages at day 7 post-bleomycin treatment (Fig. 5B). TAPI-0 treatment also inhibited bleomycin-induced levels of BAL protein at days 3 and 7 and LDH activity at days 1–7 post-bleomycin treatment (Figs. 5C and D). Histological sections of lung tissue from these mice on day 7 after bleomycin treatment confirmed BAL findings. Hematoxylin–eosin staining of lung tissue fixed with formalin revealed significantly less parenchymal and intra-alveolar cells in the lungs after bleomycin + TAPI-0 treatment than from those treated with bleomycin only (Fig. 5E). Thus, these anti-inflammatory responses in keeping with down-regulation of proinflammatory mediator production following TAPI-0 treatment were associated with restored Mer expression and enhanced efferocytosis over the course of bleomycin-induced lung inflammation. Apoptosis of pulmonary epithelial cells reflects the degree of lung injury and plays a key role in the initiation of the lung fibrotic process (Selman et al., 2004). Thus, proapoptotic changes in murine lungs were assessed following TAPI-0 treatment by examining the activity of caspase-3 and caspase-9 in lung homogenates. Activities of caspase-3 and caspase-9 increased up to 7 days post-bleomycin treatment. However, TAPI-0 treatment inhibited these increases in caspase 3 and caspase-9 activities at each time point post-bleomycin treatment (Figs. 6A and B). DNA damage and apoptosis levels in lungs were assessed using TUNEL staining. The majority of TUNEL-positive cells at 3 days post-bleomycin treatment were alveolar epithelial cells (Fig. 6C). No TUNEL-positive cells were detected in saline control mice. The number of TUNELpositive signals was abrogated in lung tissues of TAPI-0-treated mice (Figs. 6C and D). These data suggest that enhanced Mer activation down-regulates apoptotic pathways and mediates protection from apoptotic cell death when lungs are exposed to bleomycin.

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Fig. 4. TAPI-0 treatment suppresses mRNA expression of proinflammatory mediators, TGF-β1, and HGF that are usually induced by bleomycin treatment. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, and 7 days postBLM instillation. (A–E) mRNA expression of TNF-α, IL-1β, MIP-2, TGF-β1, and HGF in lung homogenates was analyzed by semi-quantitative RT-PCR and normalized to β-actin mRNA levels. The values represent the means ± SEM of results from at least five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05.

Enhanced apoptotic cell clearance, antiinflammatory and antiapoptotic effects of TAPI-0 are mediated by Mer activation To determine if the increase in apoptotic cell clearance and the down-regulation of lung inflammation and apoptosis were mediated by increased activation of Mer, specific anti-Mer neutralizing antibodies or isotype antibodies were coadministered intraperitoneally with TAPI-0 on a daily basis. This antibody specifically blocks the Mer activation (no cross-reactivity for Axl and Tyro-3) through directing against the Mer extracellular domain both in in vitro and in vivo studies (Alciato et al., 2010; Eken et al., 2010; Gould et al., 2005; Sen et al., 2007; Wallet et al., 2008). Mice were assessed on day 3 post-bleomycin treatment as this time point represented the most significant increases in inflammatory responses, including inflammatory mediator production and caspase activities. The anti-

Mer antibody did not influence membrane-bound Mer expression, but significantly inhibited phosphorylation of Mer in lung tissue when compared with the BLM + TAPI-0 group or the BLM + TAPI-0+ isotype IgG group (Fig. 7A). The anti-Mer antibody completely abrogated the enhanced alveolar macrophage PI (Fig. 7B) and the reduction in TNF-α, IL-1β, and MIP-2 production (Figs. 7C–E) in the BLM + TAPI0 group. Similarly, reduction of mRNA expression of these inflammatory cytokines, such as TNF-α and MIP-2, was also abrogated after coadministration of the anti-Mer antibody (Figs. 7F and H). Additionally, reducing the bleomycin-induced inflammatory cell recruitment, the levels of protein, LDH activity in BAL fluid (Figs. 8A–C), caspase-3 activity, and caspase-9 activity (Figs. 8D and E) in the BLM+ TAPI-0 group were recovered similar to the levels seen in bleomycin-treated mice when anti-Mer antibody was coadministered with TAPI-0. In contrast, nonspecific IgG antibodies did not affect the effects of TAPI-0 (Figs. 7

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Fig. 5. TAPI-0 treatment reduces bleomycin-induced inflammation. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, and 7 days post-BLM instillation. (A) Neutrophil and (B) alveolar macrophage numbers in the BAL fluid. (C) Total protein levels in BAL fluid were analyzed by protein assay kit. (D) Lactate dehydrogenase (LDH) activity in BAL fluid was measured with a commercial assay kit and expressed as U/L using an LDH standard. The values represent the means ± SEM of results from at least five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05. (E) Hematoxylin–eosin stains of lung sections at 7 days after BLM (original magnification: × 400). Scale bars: all 10 μm. Representative results from five mice are shown for each group.

and 8). The isotype antibody had no effects. These findings suggest that TAPI-0 enhances apoptotic cell clearance by alveolar macrophages and antiinflammatory and antiapoptotic effects that are mediated by Mer activation. 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 suggest that increased ADAM17 activity after bleomycin treatment is responsible for cleavage of Mer, resulting in reduced membrane-bound Mer protein expression and enhanced sMer production. ADAM17 is an important regulator of ectodomain shedding and originally identified for its ability to shed the membrane-bound precursor form of TNF-α, releasing soluble TNF-α from cells (Black et al., 1997). To date, ADAM17 has also been shown to shed Axl and many other cell-binding proteins. However, in vitro, ADAM10 most efficiently cleaves Axl and therefore, is most likely responsible for Axl shedding in vivo (Budagian et al., 2005; Weinger et al., 2009). ADAM17 seems to prefer a site with a hydrophobic residue at the membrane-

proximal side (Wetzker and Böhmer, 2003). Increased ADAM17 activity and/or expression are associated with several disease states, including renal inflammation and fibrosis (Melenhorst et al., 2009), stroke (Katakowski et al., 2007) and multiple sclerosis (Comabella et al., 2006). Moreover, increased ADAM17 activity appears to participate in early inflammatory event following bleomycin instillation in the development of lung fibrosis (Pottier et al., 2007). Interestingly, data using ADAM17-null mice from Arndt et al. (2011) suggest that ADAM17 activity promotes inflammation in LPS-stimulated lungs, and thus this protease may be a potential target in the design of pharmacologic therapies for acute lung injury. Although no totally specific ADAM17 inhibitor is available yet, TAPI-0 has been demonstrated as a potential inhibitor of ADAM17 proteolysis in vivo and in vitro (Comabella et al., 2006; Kaup et al., 2002; Menschikowski et al., 2009). Moreover, this protease inhibitor specifically inhibited cleavage of Mer enhanced by in vitro treatment with LPS and PMA (Sather et al., 2007). In the present study, in vivo treatment with TAPI-0 was thus used to inhibit cleavage of membrane-bound Mer. As expected, TAPI-0 inhibited ADAM17 activity and expression during the early inflammatory response following bleomycin treatment. In concordance, production of sMer in BAL fluid was attenuated, and the expression of the membrane-bound Mer protein in alveolar macrophages and lung tissue treated with bleomycin was recovered. Although ADAM17 plays as a key role for the Mer cleavage, because of the not complete selectivity of TAPI-0, additional experiments in bleomycin-

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Fig. 6. TAPI-0 treatment reduces bleomycin-induced apoptosis. Mice were instilled with intratracheal bleomycin (BLM). Either TAPI-0 or vehicle was administered intraperitoneally 1 h before BLM and every 24 h thereafter. Mice were euthanized at 1, 3, and 7 days post-BLM instillation. (A, B) Caspase-3 and caspase-9 activities in lung tissues. (C) TUNEL stains of lung sections 3 days post-bleomycin treatment (original magnification: × 1000). Arrowheads indicate TUNEL-positive cells. Scale bars: all 5 μm. Representative results from five mice are shown for each group. (D) The number of TUNEL-positive cells per 200× field was averaged across 30 fields for each mice. The values represent the means ± SEM of results from at least five mice in each group. ⁎Significantly different from saline control, p b 0.05. +Significant differences between the BLM + TAPI-0 group and the BLM group at a given time, p b 0.05.

treated mice with a targeted deletion of ADAMs are required to understand the individual contribution of ADAM members to Mer cleavage. Relative to saline control, phosphorylation of Mer was slightly enhanced during the development of acute lung injury in spite of decreased levels of membrane-bound Mer protein. It is possible that overexpressed endogenous Mer ligands, such as Gas6 and protein S, ectosome released from neutrophils, and enhanced apoptotic neutrophils may activate Mer during bleomycin-induced lung injury. After TAPI-0 treatment, the total available number of Mer receptors for these endogenous stimulants could be increased. Indeed, further enhanced activation of Mer and the downstream molecule Akt in the presence of TAPI-0 was seen through 1–7 days after bleomycin treatment. Clearance of apoptotic cells is a critical regulator of lung homeostasis, which is defective in smoke-induced emphysema in mice (Iizuka et al., 2005), and patients with chronic obstructive pulmonary disease and cystic fibrosis (Hodge et al., 2003; Vandivier et al., 2006), suggesting a role in disease pathogenesis. Recently, we reported that there is a relative suppression of apoptotic cell clearance under oxidant stress in LPS-induced acute lung injury in mice (Moon et al., 2010). In the present study, clearance of apoptotic cells by alveolar macrophages from bleomycin-treated mice was enhanced on days 1–7 post-bleomycin treatment relative to the saline control group. This may be because apoptosis of recruited neutrophils and alveolar epithelial cells occurs rapidly and alveolar macrophages may be

globally activated in response to bleomycin at these time points (Moon et al., 2010; Vandivier et al., 2006). A critical role for Mer in the efficient clearance of apoptotic cells has been described in macrophages from Mer −/− mice that express a cytoplasmic truncation of Mer (Rothlin et al., 2007; Scott et al., 2001). Data from our study support this concept as in vivo and ex vivo clearance of apoptotic cells by alveolar macrophages with recovered Mer expression after TAPI-0 treatment was markedly enhanced at 1–7 days post-bleomycin treatment. It has been known that Mer activation mediates efferocytosis through the post-receptor signaling cascade involving Src-mediated tyrosine phosphorylation of FAK, the recruitment of FAK to the alpha beta5 integrin, and increased formation of the p130(CAS)/CrkII/Dock180 complex to activate Rac1 (Singh et al., 2007; Tibrewal et al., 2008). Concomitant with efferocytosis, apoptotic cells elicit potent antiinflammatory responses. In vitro and in vivo experiments on macrophages from LPS-stimulated lungs demonstrate that ingestion of apoptotic cells suppresses proinflammatory processes and up-regulates the production of antiinflammatory mediators TGF-β1 and prostaglandin E2 (Fadok et al., 1998; Huynh et al., 2002). Recent studies indicate that Gas6, the Mer ligand, modulates macrophage cytokine secretion, triggering the Mer-dependent antiinflammatory pathway, involving, PI3K/Akt/GSK3β and NF-κB (Alciato et al., 2010). In the present study, the inhibitory effects of Mer signaling on the production of proinflammatory mediators were confirmed since pretreatment with TAPI-0 inhibited bleomycin-induced production

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Fig. 7. Anti-Mer antibody abrogates the effects of TAPI-0 on Mer activation and apoptotic cell clearance and proinflammatory mediator expression. Mice were administered with vehicle 1 h before intratracheal instillation of bleomycin (Control). Goat neutralizing anti-Mer antibody (anti-Mer Ab) or normal goat IgG (Isotype Ab) was coadministered intraperitoneally with TAPI-0 1 h before bleomycin and every 24 h thereafter. Mice were euthanized 3 days after bleomycin instillation. (A) Western blots probed with anti-Mer or antiphosphorylated Mer were used to monitor the phosphorylation of Mer in lung tissue. The relative densitometric intensity was determined for each band and normalized to tubulin. (B) BAL was performed, cytospins were stained, and alveolar macrophage ingestions of apoptotic cells were quantified by calculating the phagocytic index (PI). (C–E) TNF-α, IL-1β, and MIP-2 in the BAL fluid were quantified by ELISA. (F–H) mRNA expressions of TNF-α, IL-1β, and MIP-2 in lung homogenates were analyzed by semi-quantitative RT-PCR and normalized to β-actin mRNA levels. Values represent means ± SEM of results from five mice in each group. +p b 0.05.

of TNF-α, IL-1β, and MIP-2 in BAL fluid. However, TGF-β1, an antiinflammatory and profibrotic cytokine, was enhanced at an early time point (day 1), but HGF, an epithelial growth and antifibrotic factor, was steadily enhanced up to 7 days post-bleomycin treatment. Similar to the regulation of the cytokine proteins, significant down-regulation of TNF-α and MIP-2 mRNA expression and the up-regulation of HGF mRNA in lung tissue from bleomycin + TAPI-0 mice were also demonstrated. However, greater expression of TGF-β1 mRNA in lung tissue from bleomycin + TAPI-0 mice was not shown, indicating no effect of TAPI-0 on regulation of TGF-β1 at the mRNA level. Consistent with these results, our in vitro study indicates that TGF-β1 production mediated by Mer activation in response to apoptotic cells is only regulated at the protein level (data not shown). These inhibitory effects of TAPI-0 on

inflammatory mediators suggest an in vivo role of Mer in mediating antiinflammatory responses. We previously found that the Mer signaling pathway up-regulates HGF mRNA and protein expression in macrophages after in vitro exposure to apoptotic cells or Gas6 (Park et al., 2011). These in vitro and in vivo data on HGF production suggest an additional role of Mer in epithelium repair during bleomycin-induced acute lung injury by mediating production of the growth factor through apoptotic cell recognition processes. Treatment with TAPI-0 resulted in a significant reduction of inflammatory cell recruitment and levels of protein at 3 and 7 days and LDH activity in BAL fluid at 1 to 7 days after bleomycin treatment. The enhanced resolution of inflammation was also confirmed by lung histology on day 7 after bleomycin treatment, which showed reduced

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Fig. 8. Anti-Mer antibody abrogates the antiinflammatory and antiapoptotic effects of TAPI-0. Mice were administered with vehicle 1 h before intratracheal instillation of bleomycin (Control). Goat neutralizing anti-Mer antibody (anti-Mer Ab) or normal goat IgG (Isotype Ab) was coadministered intraperitoneally with TAPI-0 into bleomycin-treated mice. Mice were euthanized 3 days after bleomycin instillation. (A) Neutrophil, (B) total protein levels, and (C) lactate dehydrogenase (LDH) activity in BAL fluid. (D, E) Caspase-3 and caspase9 activities in lung tissues. Values represent means ± SEM of results from five mice in each group. +p b 0.05.

infiltration of inflammatory cells in alveolus and lung interstitium. In regarding with the previous findings, these data suggest that Mer activation following TAPI-0 treatment contributes to anti-inflammatory responses directly through induction of anti-inflammatory pathway, leading to down-regulation of inflammatory mediator expression, and indirectly through macrophage-mediated efferocytosis. In the present study, increased activities of caspase-3 and caspase-9 after bleomycin treatment were significantly suppressed by TAPI-0, indicating down-regulation of apoptotic pathways. Furthermore, the number of TUNEL-positive cells, mainly alveolar epithelial cells, in lung sections 3 days after bleomycin treatment was reduced by TAPI-0 treatment. These observations suggest that TAPI-0 prevents apoptosis of alveolar epithelial cells through the effect of Mer on down-regulating TNF-α and IL-1β expression (Pantano et al., 2007; Zhang et al., 1997). Whether Mer expression enhances directly alveolar epithelial survival needs to be evaluated in future studies. Importantly, Terao et al. (2010) have reported that TAPI-1, another inhibitor of ADAMs, significantly suppressed bleomycin-induced skin fibrosis and the number of myofibroblasts. Whether the antiinflammatoy and antiapoptotic effects of TAPI-0 contribute to the prevention of the progressive fibrotic process after bleomycin treatment needs to be further studied. Previously our study demonstrated that i.v. injection of anti-Mer antibody reduced significantly LPS-induced phosphorylation of Mer in lung tissue and alveolar macrophages (given that full-length Mer protein expression in suppressed), and downstream molecules of Mer, such as Akt and FAK, in lung tissue (Lee et al., 2012). These data confirm the action of anti-Mer antibody on alveolar macrophages and lungs, which specifically blocks the Mer activation through directing against the Mer extracellular domain. Moreover, anti-Mer antibody augmented LPS-induced proinflammatory mediator production, inflammatory cell accumulation and destruction of blood–pulmonary epithelial cell barrier integrity (Lee et al., 2012). Thus, these data support the potential contribution of Mer in triggering antiinflammatory pathway in vivo in acute lung injury. In the present study, to confirm the role of Mer in TAPI-0induced increase in apoptotic cell clearance and the down-regulation of lung inflammation and apoptosis, this neutralizing antibody was coadministered with TAPI-0 before bleomycin treatment. Expectedly, coadministration with anti-Mer antibody inhibited Mer activation, which correlated with the abrogation of efferocytosis by alveolar

macrophages, and a reduction of proinflammatory cytokine expression. The inhibitory effects of TAPI-0 on inflammatory responses, as well as apoptotic activities, were also reversed by coadministration of the anti-Mer blocking antibody. Thus, evidence was provided that apoptotic cell clearance as well as the antiinflammatory and antiapoptotic effects of TAPI-0 were mediated by enhanced Mer activation. In summary, our present study demonstrates that recovered membrane-bound Mer protein expression in alveolar macrophages and lung tissue, in concordance with reduced ADAM17 activity and expression, by TAPI-0 promotes efferocytosis and down-regulates sustained lung inflammatory responses and epithelial cell apoptosis early in bleomycin-induced lung injury. Our findings suggest that the pharmacological control of Mer activity might be of therapeutic value for the resolution of lung injury after bleomycin treatment. Conflict of interest statement The authors declare that there are no conflicts of interest. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2012.05.024. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; 2010‐ 0029353). References Alciato, F., Sainaghi, P.P., Sola, D., Castello, L., Avanzi, G.C., 2010. TNF-alpha, IL-6, and IL-1 expression is inhibited by GAS6 in monocytes/macrophages. J. Leukoc. Biol. 87, 869–875. Antczak, C., Radu, C., Djaballah, H., 2008. A profiling platform for the identification of selective metalloprotease inhibitors. J. Biomol. Screen. 13, 285–294. Arndt, P.G., Strahan, B., Wang, Y., Long, C., Horiuchi, K., Walcheck, B., 2011. Leukocyte ADAM17 regulates acute pulmonary inflammation. PLoS One 6, e19938. Balakrishnan, A., Patel, B., Sieber, S.A., Chen, D., Pachikara, N., Zhong, G., Cravatt, B.F., Fan, H., 2006. Metalloprotease inhibitors GM6001 and TAPI-0 inhibit the obligate intracellular human pathogen Chlamydia trachomatis by targeting peptide deformylase of the bacterium. J. Biol. Chem. 281, 16691–16699. Black, R.A., Rauch, C.T., Kozlosky, C.J., Peschon, J.J., Slack, J.L., Wolfson, M.F., Castner, B.J., Stocking, K.L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K.A.,

72

Y.-J. Lee et al. / Toxicology and Applied Pharmacology 263 (2012) 61–72

Gerhart, M., Davis, R., Fitzner, J.N., Johnson, R.S., Paxton, R.J., March, C.J., Cerretti, D.P., 1997. A metalloproteinase disintegrin that releases tumour-necrosis factoralpha from cells. Nature 385, 729–733. Budagian, V., Bulanova, E., Orinska, Z., Duitman, E., Brandt, K., Ludwig, A., Hartmann, D., Lemke, G., Saftig, P., Bulfone-Paus, S., 2005. Soluble Axl is generated by ADAM10dependent cleavage and associates with Gas6 in mouse serum. Mol. Cell. Biol. 25, 9324–9329. Castranova, V., Jones, T., Barger, M.W., Afshari, A., Frazer, D.J., 1990. Pulmonary responses of guinea pigs to consecutive exposures to cotton dust. In: Jacobs, R.R., Wakelyn, P.J., Domelsmith, L.N. (Eds.), Proceedings of the 14th Cotton Dust Research Conference. National Cotton Council, Memphis, TN, pp. 131–135. Chandler, D.B., 1990. Possible mechanisms of bleomycin-induced fibrosis. Clin. Chest Med. 11, 21–30. Colombo, G., Gatti, S., Sordi, A., Turcatti, F., Carlin, A., Rossi, C., Lonati, C., Catania, A., 2007. Production and effects of alpha-melanocyte-stimulating hormone during acute lung injury. Shock 27, 326–333. Comabella, M., Romera, C., Camiña, M., Perkal, H., Moro, M.A., Leza, J.C., Lizasoain, I., Castillo, M., Montalban, X., 2006. TNF-alpha converting enzyme (TACE) protein expression in different clinical subtypes of multiple sclerosis. J. Neurol. 253, 701–706. Doedens, J.R., Black, R.A., 2000. Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme. J. Biol. Chem. 275, 14598–14607. Eken, C., Martin, P.J., Sadallah, S., Treves, S., Schaller, M., Schifferli, J.A., 2010. Ectosomes released by polymorphonuclear neutrophils induce a MerTK-dependent antiinflammatory pathway in macrophages. J. Biol. Chem. 285, 39914–39921. Fadok, V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott, J.Y., Henson, P.M., 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF. J. Clin. Invest. 101, 890–898. Goto, H., Ledford, J.G., Mukherjee, S., Noble, P.W., Williams, K.L., Wright, J.R., 2010. The role of surfactant protein A in bleomycin-induced acute lung injury. Am. J. Respir. Crit. Care Med. 181, 1336–1344. Gould, W.R., Baxi, S.M., Schroeder, R., Peng, Y.W., Leadley, R.J., Peterson, J.Y., Perrin, L.A., 2005. Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic response. J. Thromb. Haemost. 3, 733–741. Hartree, E.F., 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422–427. Hodge, S., Hodge, G., Scicchitano, R., Reynolds, P.N., Holmes, M., 2003. Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells. Immunol. Cell Biol. 81, 289–296. Hoffman, P.R., deCathelineau, A.M., Ogden, C.A., Leverrier, Y., Bratton, D.L., Daleke, D.L., Ridley, A.J., Fadok, V.A., Henson, P.M., 2001. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 155, 649–659. Huynh, M.L., Fadok, V.A., Henson, P.M., 2002. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation. J. Clin. Invest. 109, 41–50. Iizuka, T., Ishii, Y., Itoh, K., Kiwamoto, T., Kimura, T., Matsuno, Y., Morishima, Y., Hegab, A.E., Homma, S., Nomura, A., Sakamoto, T., Shimura, M., Yoshida, A., Yamamoto, M., Sekizawa, K., 2005. Nrf2-deficient mice are highly susceptible to cigarette smoke‐ induced emphysema. Genes Cells 10, 1113–1125. Katakowski, M., Chen, J., Zhang, Z.G., Santra, M., Wang, Y., Chopp, M., 2007. Strokeinduced subventricular zone proliferation is promoted by tumor necrosis factoralpha-converting enzyme protease activity. J. Cereb. Blood Flow Metab. 27, 669–678. Kaup, M., Dassler, K., Weise, C., Fuchs, H., 2002. Shedding of the transferrin receptor is mediated constitutively by an integral membrane metalloprotease sensitive to tumor necrosis factor alpha protease inhibitor-2. J. Biol. Chem. 277, 38494–38502. Kazeros, A., Harvey, B.G., Carolan, B.J., Vanni, H., Krause, A., Crystal, R.G., 2008. Overexpression of apoptotic cell removal receptor MERTK in alveolar macrophages of cigarette smokers. Am. J. Respir. Cell Mol. Biol. 39, 747–757. Koshika, T., Hirayama, Y., Ohkubo, Y., Mutoh, S., Ishizaka, A., 2005. Tacrolimus (FK506) has protective actions against murine bleomycin-induced acute lung injuries. Eur. J. Pharmacol. 515, 169–178. Lee, Y.J., Han, J.Y., Byun, J., Park, H.J., Park, E.M., Chong, Y.H., Cho, M.S., Kang, J.L., 2012. Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury. J. Leukoc. Biol. 91, 921–932. Lee, Y.J., Moon C., Lee, S.H., Park, H.J., Seoh, J.Y., Cho, M.S., Kang, J.L., in press. Apoptotic cell instillation after bleomycin attenuates lung injury through HGF induction. Eur. Respir. J. Linger, R.M., DeRyckere, D., Brandão, L., Sawczyn, K.K., Jacobsen, K.M., Liang, X., Keating, A.K., Graham, D.K., 2009. Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia. Blood 114, 2678–2687. Lu, Q., Lemke, G., 2001. Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science 293, 306–311. Melenhorst, W.B., Visser, L., Timmer, A., van den Heuvel, M.C., Stegeman, C.A., van Goor, H., 2009. ADAM17 upregulation in human renal disease: a role in modulating TGF-alpha availability? Am. J. Physiol. Renal Physiol. 297, F781–F790.

Menschikowski, M., Hagelgans, A., Eisenhofer, G., Siegert, G., 2009. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways. Exp. Cell Res. 315, 2673–2682. Moon, C., Lee, Y.J., Park, H.J., Chong, Y.H., Kang, J.L., 2010. N-acetylcysteine inhibits RhoA and promotes apoptotic cell clearance during intense lung inflammation. Am. J. Respir. Crit. Care Med. 181, 374–387. O'Bryan, J.P., Fridell, Y.W., Koski, R., Varnum, B., Liu, E.T., 1995. The transforming receptor tyrosine kinase, Axl, is post-translationally regulated by proteolytic cleavage. J. Biol. Chem. 270, 551–557. Pantano, C., Anathy, V., Ranjan, P., Heintz, N.H., Janssen-Heininger, Y.M., 2007. Nonphagocytic oxidase 1 causes death in lung epithelial cells via a TNF-RI–JNK signaling axis. Am. J. Respir. Cell Mol. Biol. 36, 473–479. Park, H.J., Choi, Y.H., Kang, J.L., 2011. Mer tyrosine kinase regulates transcriptional production of hepatocyte growth factor in response to apoptotic cells and Gas6 through the RhoA-dependent pathwayAvailable online at: http://www.atsjournals.org 2011. Pottier, N., Chupin, C., Defamie, V., Cardinaud, B., Sutherland, R., Rios, G., Gauthier, F., Wolters, P.J., Berthiaume, Y., Barbry, P., Mari, B., 2007. Relationships between early inflammatory response to bleomycin and sensitivity to lung fibrosis: a role for dipeptidyl-peptidase I and tissue inhibitor of metalloproteinase-3? Am. J. Respir. Crit. Care Med. 176, 1098–1107. Rao, G.V.S., Tinkle, S., Weissman, D.N., Antonini, J.M., Kashon, M.L., Salmen, R., Battelli, L.A., Willard, P.A., Hoover, M.D., Hubbs, A.F., 2003. Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx. J. Toxicol. Environ. Health A 66, 1441–1452. Rothlin, C.V., Ghosh, S., Zuniga, E.I., Oldstone, M.B., Lemke, G., 2007. TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 131, 1124–1136. Sather, S., Kenyon, K.D., Lefkowitz, J.B., Liang, X., Varnum, B.C., Henson, P.M., Graham, D.K., 2007. A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation. Blood 109, 1026–1033. Schlöndorff, J., Blobel, C.P., 1999. Metalloprotease-disintegrins: modular proteins capable of promoting cell–cell interactions and triggering signals by protein-ectodomain shedding. J. Cell Sci. 112, 3603–3617. Scott, R.S., McMahon, E.J., Pop, S.M., Reap, E.A., Caricchio, R., Cohen, P.L., Earp, H.S., Matsushima, G.K., 2001. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411, 207–211. Selman, M., Thannickal, V.J., Pardo, A., Zisman, D.A., Martinez, F.J., Lynch III, J.P., 2004. Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 64, 405–430. Sen, P., Wallet, M.A., Yi, Z., Huang, Y., Henderson, M., Mathews, C.E., Earp, H.S., Matsushima, G., Baldwin, A.S., Tisch, R.M., 2007. Apoptotic cells induce Mer tyrosine kinasedependent blockade of NF-κB activation in dendritic cells. Blood 109, 653–660. Shen, A.S., Haslett, C., Feldsien, D.C., Henson, P.M., Cherniack, R.M., 1988. The intensity of chronic lung inflammation and fibrosis after bleomycin is directly related to the severity of acute injury. Am. Rev. Respir. Dis. 137, 564–571. Shreffler, W.G., 2007. CD137-mediated immunotherapy for allergic asthma. Pediatrics 120, s153. Singh, S., D'mello, V., van Bergen en Henegouwen, P., Birge, R.B., 2007. A NPxYindependent beta5 integrin activation signal regulates phagocytosis of apoptotic cells. Biochem. Biophys. Res. Commun. 364, 540–548. Terao, M., Murota, H., Kitaba, S., Katayama, I., 2010. Tumor necrosis factor-alpha processing inhibitor-1 inhibits skin fibrosis in a bleomycin-induced murine model of scleroderma. Exp. Dermatol. 19, 38–43. Thorp, E., Vaisar, T., Subramanian, M., Mautner, L., Blobel, C., Tabas, I., 2011. Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cδ, and p38 mitogenactivated protein kinase (MAPK). J. Biol. Chem. 286, 33335–33344. Tibrewal, N., Wu, Y., D'mello, V., Akakura, R., George, T.C., Varnum, B., Birge, R.B., 2008. Autophosphorylation docking site Tyr-867 in Mer receptor tyrosine kinase allows for dissociation of multiple signaling pathways for phagocytosis of apoptotic cells and down-modulation of lipopolysaccharide-inducible NF-kappaB transcriptional activation. J. Biol. Chem. 283, 3618–3627. Vandivier, R.W., Henson, P.M., Douglas, I.S., 2006. Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest 129, 1673–1682. Wallet, M.A., Sen, P., Flores, R.R., Wang, Y., Yi, Z., Huang, Y., Mathews, C.E., Earp, H.S., Matsushima, G., Wang, B., Tisch, R., 2008. MerTK is required for apoptotic cellinduced T cell tolerance. J. Exp. Med. 205, 219–232. Weinger, J.G., Omari, K.M., Marsden, K., Raine, C.S., Shafit-Zagardo, B., 2009. Up-regulation of soluble Axl and Mer receptor tyrosine kinases negatively correlates with Gas6 in established multiple sclerosis lesions. Am. J. Pathol. 175, 283–293. Wetzker, R., Böhmer, F.D., 2003. Transactivation joins multiple tracks to the ERK/MAPK cascade. Nat. Rev. Mol. Cell Biol. 4, 651–657. Zhang, H.Y., Gharaee-Kermani, M., Phan, S.H., 1997. Regulation of lung fibroblast alpha-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1β. J. Immunol. 158, 1392–1399.