Abstract
Mer receptor tyrosine kinase (Mer) signaling plays a central role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Previously, we found that lung Mer protein expression decreased after lipopolysaccharide (LPS) treatment due to enhanced Mer cleavage. The purpose of the present study was to examine whether pharmacologically restored membrane-bound Mer expression upregulates the Mer signaling pathways and suppresses lung inflammatory responses. Pretreatment with the ADAM17 (a disintegrin and metalloproteinase-17) inhibitor TAPI-0 (tumor necrosis factor alpha protease inhibitor-0) reduced LPS-induced production of soluble Mer protein in bronchoalveolar lavage (BAL) fluid, restored membrane-bound Mer expression, and increased Mer activation in alveolar macrophages and lungs after LPS treatment. TAPI-0 also enhanced Mer downstream signaling, including phosphorylation of protein kinase b, focal adhesion kinase, and signal transducer and activator of transcription 1. As expected from enhanced Mer signaling, TAPI-0 also augmented suppressor of cytokine signaling-1 and -3 mRNA and protein levels and inhibited nuclear factor κB activation at 4 and 24 hours after LPS treatment. TAPI-0 suppressed LPS-induced inflammatory cell accumulation, total protein level elevation in BAL fluid, and production of inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, and macrophage inflammatory protein-2. Additionally, the effects of TAPI-0 on the activation of Mer signaling and the production of inflammatory responses could be reversed by cotreatment with specific Mer-neutralizing antibody. Restored Mer protein expression by treatment with TAPI-0 efficiently prevents the inflammatory cascade during acute lung injury.
Introduction
Mer is a cell membrane–bound receptor tyrosine kinase receptor that, together with Axl and Tyro3, constitutes the TAM receptor family (Lemke and Rothlin, 2008). The growth arrest–specific protein 6 (Gas6) and anticoagulant protein S are important biologic ligands for the TAM receptors (Lai et al., 1994; Godowski et al., 1995; Stitt et al., 1995). Ligand interaction with TAM receptors leads to receptor phosphorylation and activation of downstream signaling pathways that affect numerous functions, including cell survival, thrombosis, proliferation, cell migration, and phagocytosis of apoptotic cells. A recent study reported that cytokine-dependent activation of TAM signaling stimulates an anti-inflammatory pathway under the regulation of Toll-like receptors (TLRs) (Rothlin et al., 2007). Mer, in particular, is the primary phagocytic receptor for apoptotic cells in macrophages. Mer-deficient mice have multiple defects in monocyte function that can lead to the development of autoimmune disorders (Cohen et al., 2002; Behrens et al., 2003). Indeed, in response to lipopolysaccharide (LPS), Gas6-induced Mer activation was responsible for the reduction of inflammatory cytokine expression only in macrophages expressing Mer (Alciato et al., 2010). These results highlight the importance of Mer in immune cells, particularly macrophages. Our in vivo findings and others’ in vitro findings suggest that this immunomodulatory role of Mer signaling occurs through two modes. By acting on the type 1 interferon receptor (IFNAR)-signal transducer and activator of transcription 1 (STAT1) cassette upon TLR stimulation (Rothlin et al., 2007), Mer increases protein synthesis of suppressor of cytokine signaling (SOCS)-1 and -3. Also, Mer signaling has an immediate effect on the PI3K/protein kinase b (Akt) pathway, leading to inhibition of nuclear factor κB (NF-κB) translocation (Alciato et al., 2010; Eken et al., 2010).
Proteolytic cleavage of membrane-bound protein has emerged as an important posttranslational mechanism to regulate the functions of receptor tyrosine kinases. Recently, Sather et al. (2007) reported that the membrane-bound Mer protein is cleaved in the extracellular domain via ADAM17, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases. Moreover, cleavage of Mer was enhanced by treatment of macrophages with LPS and phorbol 12-myristate 13-acetate and was specifically inhibited by the ADAM17 inhibitor TAPI-0 (Sather et al., 2007). 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 (Balakrishnan et al., 2006; Antczak et al., 2008). Consistently, we found that in vivo exposure of lungs to LPS enhanced the production of soluble Mer protein (sMer) but decreased membrane-bound Mer expression in alveolar macrophages in spite of increased Mer mRNA expression (Lee et al., 2012a). ADAMs, including ADAM17, also known as tumor necrosis factor-α (TNF-α)-converting enzyme, and ADAM10, are involved in normal processes such as wound repair and tissue remodeling. They are associated with disease states, including renal inflammation and fibrosis (Melenhorst et al., 2009), stroke (Katakowski et al., 2007), and multiple sclerosis (Comabella et al., 2006). Although both of these proteases have been shown to cleave Axl and Mer (Weinger et al., 2009), ADAM17 is the key protease required for sMer shedding (Thorp et al., 2011). The soluble cleavage product may act as a decoy receptor to sequester ligand, which has been shown to inhibit receptor function in apoptotic cell engulfment during inflammation (Sather et al., 2007; Thorp et al., 2008).
The acute respiratory distress syndrome, a clinically important complication of severe acute lung injury (ALI) in humans, is a significant cause of morbidity and mortality in critically ill patients (Ware and Matthay 2000; Goss et al., 2003; Mendez and Hubmayr, 2005; Rubenfeld et al., 2005). Histologically, acute lung injury/acute respiratory distress syndrome in humans is characterized by a severe acute inflammatory response in the lungs and neutrophilic alveolites (Ware and Matthay 2000). Inflammatory stimuli from microbial pathogens such as LPS have been used to induce pulmonary inflammation in animal models of ALI (Kitamura et al., 2001; Matute-Bello et al., 2004; Gharib et al., 2006; Moon et al., 2010). Interestingly, alveolar neutrophil numbers and lung inflammation in ADAM17-null mice were reduced overall when compared with control mice, implicating this protease as a target in pharmacologic therapies for ALI (Arndt et al., 2011). In the present study, we investigated whether preventing the cleavage of Mer with TAPI-0 enhances the activation of Mer and downstream signaling transducers, including Akt, focal adhesion kinase (FAK), and STAT1, and consequently downregulates lung inflammatory responses in LPS-induced acute lung injury. To confirm that this effect is dependent on Mer signaling, coadministration of specific Mer-neutralizing antibodies was used in an attempt to reverse the effects of TAPI-0.
Materials and Methods
Reagents.
LPS (Escherichia coli lipopolysaccharide, 055:B5) was purchased from Sigma Aldrich (St. Louis, MO). The antibodies used in this study were Mer-blocking antibody, anti-IgG antibody (R&D Systems, Minneapolis, MN), anti-phospho-Mer, anti-Mer (Fab Gennix, Frisco, TX), anti-phospho-Akt, anti-Akt, anti-phospho-FAK (pY397), anti-FAK, anti-phospho-STAT1, anti-STAT1, anti-SOCS1, anti-SOCS3, and anti-phospho-IκB-α (pS32), anti-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA). The [α-32P]-dATP was obtained from Amersham (Waltham, MA), and the DNA polymerase Klenow fragment and dNTPs were obtained from Intron Biotechnology (Seoul, South Korea).
Animal Protocols.
Pathogen-free male BALB/C mice (Orient Bio, Sungnam, South Korea) weighing 19–21 g were used in all experiments. The Animal Care Committee of the Ewha Medical Research Institute approved the experimental protocol. Mice were cared for and handled in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mouse pharyngeal aspiration was performed as described by Rao et al. (2003). Animals were anesthetized with a mixture of ketamine and xylazine (45 mg/kg and 8 mg/kg i.p., respectively). The test solution (30 μl) containing LPS (1.5 mg/kg) was placed posterior in the throat and aspirated into the lungs. Control mice were administered sterile saline (0.9% NaCl). TAPI-0 (50 mg/kg; Peptides International, Louisville, KY) or vehicle was given intraperitoneally 1 hour before LPS treatment (Mohler et al., 1994; Wheeler et al., 2003; Terao et al., 2010). Mice were euthanized at 4 or 24 hours after LPS treatment. For the anti-Mer antibody inhibition experiments, 2.0 mg/kg goat anti-mouse Mer Ab (AF591; R&D Systems) (Sen et al., 2007; Wallet et al., 2008) or control goat IgG Ab (R&D Systems) was coadministered intraperitoneally with TAPI-0 (50 mg/kg) 1 hour before LPS treatment. Mice were euthanized 24 hours after LPS treatment.
Isolation of BAL cells, Lung Tissue, and Cell Counts.
BAL was performed through a tracheal cannula using 0.8-ml aliquots of ice-cold Ca2+/Mg2+-free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM dextrose, at pH 7.4) for a total of 2.4 ml per mouse. The number of neutrophils and alveolar macrophages were determined according to their unique cell diameter, using an electronic Coulter counter fitted with a cell-sizing analyzer (Coulter Model ZBI with a Channelizer 256; Coulter Electronics, Bedfordshire, UK).
Primary Cell Isolation and Culture.
The suspended alveolar macrophages were over 95% viable, as determined by Trypan blue dye (Sigma-Aldrich) exclusion. The alveolar macrophages were isolated by adhesion (60 minutes), and the resident peritoneal macrophages were isolated using 5 ml of ice-cold sterile Hanks’ balanced salt solution to lavage the peritoneum. Macrophages were plated at 5 × 105 cells per well and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/ml of streptomycin, and 100 U/ml of penicillin in a humidified 10% CO2 incubator at 37°C. Before stimulation, the medium was replaced with serum-free X-vivo 10 medium (Biowhittaker, Wakersville, MA). The suspended macrophages were over 95% viable, as determined by Trypan blue dye exclusion.
Measurement of Total Protein in BAL Samples.
BAL protein concentration was used as an indicator of blood-pulmonary epithelial cell barrier integrity. Total protein was measured using the method devised by Hartree (1972), using bovine serum albumin as a standard.
Enzyme-Linked Immunosorbent Assay (ELISA).
BAL fluid was analyzed using TNF-α, interleukin-1β (IL-1β), and macrophage inflammatory protein-2 (MIP-2) ELISA kits (R&D Systems) according to the manufacturer’s instructions. The concentrations of TNF-α, IL-1β, and MIP-2 were determined as picograms per milliliter based on the appropriate standard curve. Myeloperoxidase (MPO) is an enzyme that is found predominantly in the azurophilic granules of neutrophils. Tissue MPO activity statistically significantly correlates with the number of neutrophils determined histochemically in inflamed tissues (Bradley et al., 1982), so it is frequently used to estimate tissue neutrophil infiltration. The activity of MPO in lung homogenates was estimated by using a specific ELISA kit.
ADAM17 Activity.
Lung tissue lysates were prepared and analyzed for ADAM17 activity using a fluorogenic peptide-based assay kit from R&D Systems following the manufacturer's instructions.
Western Blot Analysis.
Lung tissue homogenates, total cell lysates (10–50 μg protein/lane), or BAL fluid (100 μg protein/lane) were separated on an 8–10% SDS-PAGE. Separated proteins were electrophoretically transferred onto nitrocellulose paper and were blocked for 1 hour at room temperature with Tris-buffered saline containing 3% bovine serum albumin. The membranes were then incubated with an anti-phospho-Mer/Mer, anti-rabbit phospho-Akt/Akt, anti-phospho-FAK/FAK, anti-phospho-STAT1/STAT1, anti-rabbit phospho-IκB-α (pS32)/IκB-α, or anti-ADAM17 at room temperature for 1 hour and were visualized using enhanced chemiluminescence.
RNA Isolation and Reverse-Transcriptase Polymerase Chain Reaction Analysis.
Total RNA was isolated from the lung homogenates by use of TRIzol (Invitrogen, Carlsbad, CA). The concentration and purity of RNA were evaluated by spectrometry at 260 and 280 nm. Reverse transcription (RT) was conducted for 60 minutes at 42°C with 1 μg of total RNA using the Power cDNA Synthesis kit (Intron Biotechnology, Seoul, South Korea). Mer, SOCS1, and SOCS3 mRNA levels were determined by use of a relative quantitative polymerase chain reaction (PCR) kit (Intron Biotechnology). The sequences of the primers used for the PCR were as follows: SOCS1 (sense 5′-TCGACTGCCTTTTCGAGCTG-3′ and anti-sense 5′-AGGCATCTCACCCTCCA CAA-3′), SOCS3 (sense 5′- CCCTCCAGCATCTTTGTCGG-3′ and anti-sense 5′-CAGGTC CTGCCAGCTCTACT-3′), and β-actin (sense 5′-GATGACGATATCGCTGCGCTG-3′ and anti-sense 5′-GTA CGACCAGAGGCATACAGG-3′). The cDNA was denatured for 5 minutes at 95°C, and the amplification was achieved in a temperature cycler (GeneAmp PCR system 2400; PerkinElmer, Waltham, MA) with 33 cycles of temperature (95°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds), followed by a 10-minute final extension at 72°C. A total of 10 µl of each PCR sample was loaded on a 1.5% agarose gel and stained with ethidium bromide. The relative densities of the cytokine versus β-actin were analyzed by densitometry.
Nuclear Extracts.
Nuclear extracts were prepared from lung tissue samples using a modification of a method described previously elsewhere (Kang et al., 2001). Aliquots of frozen tissue were placed in a Precellys tissue homogenizer (Bertin Technology, Saint-Quentin-en-Yvelines, France), and the cells were lysed with 0.5 ml of buffer A (100 mM HEPES, pH 7.9, 10 mM KCl, 0.1 M EDTA, 0.5 mM dithiothreitol, 0.6% Nonidet P-40, and 0.5 mM phenylmethylsulfonyl fluoride). Supernatants containing intact nuclei were incubated on ice for 5 minutes, then centrifuged for 10 minutes at 5000 rpm. Nuclear pellets were resuspended in buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.42 M NaCl, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride) for 30 minutes on ice. The supernatants containing nuclear proteins were collected by centrifuging at 10,000 rpm for 2 minutes and stored at –70°C.
Electrophoretic Mobility Shift Assay.
Binding-reaction mixtures (10 μl) containing 5 μg (4 μl) of nuclear extract protein, 2 μg poly(dI-dC) (Sigma-Aldrich), and 40,000 cpm 32P-labeled probe in binding buffer (4 mM HEPES, pH 7.9, 1 mM MgCl2, 0.5 mM dithiothreitol, 2% glycerol, and 20 mM NaCl) were incubated for 30 minutes at room temperature. Protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 1× Tris/borate/EDTA (TBE) buffer and were visualized by autoradiography. Autoradiograph signals of activated NF-κB were quantified by densitometry using an UltroScan XL laser densitometer (Model 2222-020; LKB Produkter AB, Bromma, Sweden) to determine the band intensities. The oligonucleotide (5′-CCTGTGCTCCGGGAATTTCCCTGGCC-3′) used as a probe for the electrophoretic mobility shift assay was a double-stranded DNA fragment containing the NF-κB consensus sequence (underlined) and labeled with [α-32P]-dATP (Amersham, Buckinghamshire, UK) using the DNA polymerase Klenow fragment (Life Technologies, Gaithersburg, MD). Cold competition and nonspecific binding were performed by adding 100 ng of unlabeled double-stranded probe to the reaction mixtures.
Lung Histology.
Lungs were fixed with 10% buffered formalin at room temperature for 48 hours, dehydrated, and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin. Blinded analysis of lungs was performed using a light microscope.
Statistical Analysis.
Values are expressed as mean ± S.E.M. Analysis of variance 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. P < 0.05 was considered statistically significant.
Results
TAPI-0 Treatment Reduces sMer Production and Restores Membrane-Bound Mer Expression in LPS-Stimulated Lungs.
Previously, we had demonstrated that sMer production was enhanced, but membrane-bound Mer protein was reduced in the alveolar macrophages and lung tissue from LPS-treated mice (Lee et al., 2012a). In our present study, we examined whether treatment with TAPI-0, an ADAM17 inhibitor, would prevent cleavage of Mer and restore levels of membrane-bound Mer protein in the LPS-stimulated lung.
TAPI-0 treatment reduced production of sMer in BAL fluid (Fig. 1A) and fully restored membrane-bound Mer expression in alveolar macrophages and lung tissue at 4 and 24 hours after LPS treatment (Fig. 1, D and E). In addition to increased full-length Mer protein, phosphorylation of Mer was also enhanced up to 24 hours after LPS treatment (Fig. 1, D and E). As expected, ADAM17 activity and expression were statistically significantly inhibited in the lung tissue of TAPI-0-treated mice at these time points (Fig. 1, B and C). In contrast, the levels of sMer in BAL fluid, ADAM17 activity, membrane-bound Mer expression, and phosphorylation in lung tissue from mice treated with only TAPI-0 were not changed at each time point compared with saline controls (Supplemental Fig. 1, A–C). Moreover, TAPI-0 treatment into naïve mice did not affect membrane-bound Mer expression and phosphorylation in alveolar macrophages (Supplemental Fig. 1D). Recovered membrane-bound Mer expression and enhanced phosphorylation of Mer by TAPI-0 were confirmed in vitro in alveolar macrophages and peritoneal macrophages that were exposed to LPS for 2 hours (Fig. 1F).
TAPI-0 Treatment Enhances Activation of the Downstream Molecules of Mer in LPS-Stimulated Lungs.
It is well established in vitro that TLR induction of the IFNAR-STAT1 cassette upregulates Mer expression and that subsequent Mer activation mediates the productin of the STAT1-dependent TLR suppressors, SOCS1 and SOCS3 (Rothlin et al., 2007). On the other hand, upon stimulation with Gas6, Mer triggers an anti-inflammatory pathway involving PI3K/Akt/GSK3β signaling and NF-κB inhibition in immune cells (Alciato et al., 2010; Eken et al., 2010). In addition, Mer mediates the induction of phosphorylation of cytoskeletal proteins p130cas and FAK, which have been previously implicated in apoptotic cell phagocytosis (Wu et al., 2005; Singh et al., 2007). Previously, our in vivo study found that blocking the interaction of Mer with its ligand using anti-Mer neutralizing antibody results in decreased LPS-induced Mer activation (given that full-length Mer protein expression is suppressed) as well as downstream signaling (Lee et al., 2012a). In the present study, changes in the activation of these downstream molecules in lung tissue after TAPI-0 treatment were examined. Phosphorylation of Akt and FAK was further enhanced by TAPI-0 treatment at 4 and 24 hours after LPS treatment compared with the group treated with LPS alone (Fig. 2, A and B). Furthermore, TAPI-0 treatment enhanced phosphorylation of STAT1 (Fig. 2C) as well as mRNA and protein expression of SOCS1 (Fig. 2, D and E) and SOCS3 (Fig. 2, F and G) at these time points after LPS treatment.
TAPI-0 Treatment Reduces Activation of NF-κB and Levels of Proinflammatory Mediators in LPS-Stimulated Lungs.
NF-κB activation in lungs peaked at 2 hours after LPS treatment and progressively decreased thereafter. However, its activity at 4 to 24 hours was statistically significantly greater than those of saline-treated mice (Kang et al., 2001). In the present study, to investigate the in vivo effect of TAPI-0 on the NF-κB pathway in the development of LPS-induced ALI, we examined phosphorylation and degradation of IκB-α and DNA activity of NF-κB binding in lung tissue. TAPI-0 treatment inhibited LPS-induced phosphorylation and degradation of IκB-α as well as NF-κB activation (Fig. 3, A–C).
The proinflammatory cytokines TNF-α and IL-1β, and chemokine MIP-2 are representative proinflammatory mediators that play major roles in neutrophil influx and lung damage. The regulation of the corresponding genes is known to be NF-κB-dependent. Protein levels of TNF-α, IL-1β, and MIP-2 in BAL fluid were measured by ELISA. TAPI-0 treatment significantly inhibited LPS-induced production of TNF-α (30 and 50% inhibition), IL-1β (58 and 60% inhibition), and MIP-2 (35 and 59% inhibition) at 4 and 24 hours after LPS treatment (Fig. 3, D–F). In contrast, the levels of TNF-α, IL-1β, and MIP-2 in BAL fluid from naive lungs were not significantly changed by TAPI-0 treatment at each time point (Supplemental Fig. 2, A–C).
TAPI-0 Treatment Reduces Inflammatory Cell Recruitment and Protein Levels in LPS-Stimulated Lungs.
LPS produces a well-characterized model of ALI associated with the activation of macrophages, neutrophil accumulation, increased permeability of the alveolar-capillary barrier, and parenchymal injury (Ware and Matthay, 2000). Indeed, neutrophil cell numbers in BAL fluid after intratracheal injection of LPS are remarkably increased. In contrast, changes of neutrophil cell numbers in blood are trivial compared with those in BAL fluid (Wiesel et al., 2000; Yamada et al., 2008). Moreover, monocyte cell numbers in blood are little changed (Hashimoto et al., 2004). In addition, the levels of TNF-α, IL-1β, and MIP-2 in serum after intratracheal administration of LPS were low and showed no statistically significant differences compared with saline-treated group (Hashimoto et al., 2004; Quinton et al., 2004). Thus, the number of neutrophils and levels of proinflammatory mediators in BAL fluid are usually explored to determine the effects on pathogenesis of ALI. Accordingly, in the present study, to determine the effects of TAPI-0 on lung inflammatory response, we evaluated these parameters in BAL fluid of mice challenged with intratracheal LPS.
TAPI-0 treatment reduced neutrophil cell numbers in BAL fluid by 45 and 55% at 4 and 24 hours after LPS treatment, respectively (Fig. 4A). Alveolar macrophage numbers were only significantly lower upon pretreatment with TAPI-0 by 45% at 24 hours after LPS treatment (Fig. 4B). TAPI-0 treatment significantly reduced MPO activities in lung tissue by 39% at 24 hours after LPS (Fig. 4C). TAPI-0 treatment also inhibited LPS-induced increases in levels of BAL protein levels by 79 and 69% at 4 and 24 hours after LPS, respectively (Fig. 4D). Hematoxylin and eosin staining of lung tissue fixed with formalin showed that levels of parenchymal and intra-alveolar cells were lower in the lung at 24 hours after LPS and TAPI-0 treatment than in lungs treated with LPS alone (Fig. 4E). In contrast, the number of neutrophils and alveolar macrophages in BAL fluid from naive lungs was not significantly changed by TAPI-0 treatment at each time point (Supplemental Fig. 3, A and B).
Anti-inflammatory Effects of TAPI-0 Are Mediated by Mer Activation.
To determine whether the upregulation of downstream signaling, induction of SOCS1/3 expression, and downregulation of lung inflammation were mediated by increased activation of Mer, specific anti-Mer neutralizing antibodies or isotype antibodies were coadministered intraperitoneally with TAPI-0. 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 (Sen et al., 2007; Wallet et al., 2008; Alciato et al., 2010; Eken et al., 2010). Mice were assessed at 24 hours after LPS treatment. The anti-Mer antibody did not influence membrane-bound Mer expression, but significantly inhibited phosphorylation of Mer in lung tissue when compared with the LPS group or the TAPI-0 treatment group with the isotype antibody control (Fig. 5A). The anti-Mer antibody completely abrogated the enhancement of downstream signaling, including phosphorylation of Akt and STAT1 as well as SOCS1/3 expression at the mRNA and protein levels in lung tissue compared with the LPS and TAPI-0 group or the LPS and TAPI-0 group with isotype antibody control (Fig. 5, B–G).
Reduction of NF-κB pathway activity, including phosphorylation and degradation of IκB-α and DNA binding activity of NF-κB, was also abrogated in lung tissue after coadministration of the anti-Mer antibody (Fig. 6, A–C). Similarly, the inhibition of TNF-α, IL-1β, and MIP-2 production in the LPS and TAPI-0 group was also abrogated after coadministration of the anti-Mer antibody (Fig. 6, D–F). Additionally, the reductions in LPS-induced inflammatory cell recruitment, MPO activity in lung tissue, and protein levels in BAL fluid in the LPS and TAPI-0 group were restored to the levels seen in LPS-treated mice when anti-Mer antibody was coadministered with TAPI-0 (Fig. 6, G–J). The isotype antibody had no effects. As described previously elsewhere (Lee et al., 2012b), anti-Mer antibody without TAPI-0 suppressed downstream signaling of Mer, and enhanced NF-κB signaling, the production of these proinflammatory mediators, accumulation of inflammatory cells, and the total protein levels in BAL fluid compared with control (the LPS group). However, the inhibitory effects of anti-Mer with TAPI-0 were greater than those of anti-Mer without TAPI-0. These findings suggest that the enhanced downstream signaling and anti-inflammatory effects generated by TAPI-0 treatment are mediated through the activation of Mer.
Discussion
Our previous in vivo data provided evidence that Mer expression in alveolar macrophages and lung tissue after intratracheal LPS treatment is reduced by enhanced Mer cleavage (Lee et al., 2012a). Moreover, our previous work had suggested that Mer acts as an intrinsic feedback inhibitor of TLR-driven immune responses during ALI. In addition, recent data from Thorp et al. (2011) demonstrated that LPS induces plasma sMer and that this is suppressed by ADAM17 deficiency in a mouse model of endotoxemia, indicating that ADAM17 is the key protease required for sMer shedding. Interestingly, following LPS inhalation, alveolar neutrophil levels, and lung inflammation in ADAM17-null mice were reduced overall, implicating ADAM17 as a target in the design of pharmacologic therapies for ALI. Based on these previous findings, we hypothesized that preventing cleavage of membrane-bound Mer by use of a pharmacologic ADAM17 inhibitor would restore Mer expression, leading to an enhancement of Mer signaling and a consequent suppression of lung inflammatory responses.
No inhibitor that is completely specific for ADAM17 is available yet. Instead, we used a general ADAM17 inhibitor, TAPI-0, in this study to inhibit the cleavage of membrane-bound Mer, because TAPI-0 is extensively used to block ADAM17 activity in vivo and in vitro (Katakowski et al., 2007; Sather et al., 2007). Moreover, its effect on the prevention of cleavage of Mer has been demonstrated in macrophages after in vitro exposure to LPS or phorbol 12-myristate 13-acetate (Sather et al., 2007).
As expected, the pretreatment with TAPI-0 inhibited ADAM17 activity 4 and 24 hours after LPS treatment, but not after saline treatment. In concordance with the decreased ADAM17 activity, 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 LPS was restored. Although ADAM17 plays a key role in Mer cleavage, additional experiments in LPS-treated mice with targeted ADAM deletions are required to understand the individual contribution of ADAM members to Mer cleavage, as TAPI-0 is not absolutely specific for ADAM17.
Because ADAM17 is considered the only Mer sheddase (Thorp et al., 2011), it is expected that ADAM17 activity correlates to soluble Mer production, as we indeed have shown in alveolar macrophages and lung tissue. TAM receptor signaling via the IFNAR-STAT1 cassette leads to increased expression of SOCS1 and SOCS3, which act to limit TLR signaling, resulting in resolution of lung injury (Rothlin et al., 2007). On the other hand, Mer activation is directly responsible for the reduction of cytokine (TNF-α, IL-6, and IL-1β) expression in macrophages stimulated with LPS (Alciato et al., 2010). Thus, in the present study, we examined whether activation of Mer and its downstream signaling molecules, including Akt, FAK, and STAT1, is enhanced by TAPI-0 treatment.
Our data demonstrated that treatment with TAPI-0 further enhanced phosphorylation of Mer as well as Akt and FAK at 4 and 24 hours after LPS treatment. LPS induction of STAT1 activation and consequent increases in SOCS1 and SOCS3 expression at the mRNA and protein levels in lung tissue at these time points were enhanced by TAPI-0 treatment. In contrast, TAPI-0 treatment downregulated LPS-induced phosphorylation and degradation of IκB-α and activation of NF-κB in lung tissue after LPS treatment.
These effects of TAPI-0 are exactly opposite to those of anti-Mer blocking antibody (Lee et al., 2012a). Treatment with this antibody in the LPS-stimulated lungs enhanced NF-κB activation and inhibited Akt and STAT1 activation and SOCS1/3 expression. Moreover, TAPI-0 treatment suppressed LPS induction of NF-κB-dependent secretion of proinflammatory cytokines, including TNF-α, IL-1β, and MIP-2.
Collectively, our findings have established that Mer activation triggers the STAT1 and Akt pathways. It is valuable to note that SOCS proteins are intracellular immune regulators that modulate TLR signaling cascades and negatively regulate inflammatory cytokine production, NF-κB-dependent transcription, and apoptosis (Jo et al., 2005; Yoshimura, 2009). In particular, SOCS3 has been linked to the resolution of TLR4-mediated ALI (Hilberath et al., 2011). Interestingly, these immunomodulatory effects of TAPI-0 in our experimental setting do not seem to be mediated by Gas6 induction because TAPI-0 did not change LPS-induced Gas6 expression in lung tissue (Supplemental Fig. 4).
In addition, increases in the recruitment of neutrophils into the lung and protein levels in BAL fluid at both 4 and 24 hours after LPS treatment were significantly attenuated by pretreatment with TAPI-0. Histologic findings support the improved alveolar and interstitial inflammatory changes after TAPI-0 treatment at 24 hours after LPS treatment. Interestingly, recent studies using ADAM17-null mice have suggested that MPO activity and alveolar macrophages were not different between the two groups of mice at 8 hours after LPS challenge (Arndt et al., 2011). However, in our study, alveolar macrophage numbers and MPO activity were significantly decreased by TAPI-0 treatment at the later time point (24 hours after LPS treatment). Thus, these different time points may explain the contrasting results obtained from the ADAM-null mice and TAPI-0 treated mice upon LPS challenge.
To date, ADAM17 has also been shown to shed Axl and many other cell membrane-bound proteins, including TNF-α, TNF receptors I and II (Bell et al., 2007), epidermal growth factor receptor (EGFR) ligand (Blobel, 2005), IL-6 receptor, IL-1 receptor, L-selectin (Peschon et al., 1998), and vascular cell adhesion molecule 1 (VCAM-1) (Garton et al., 2003). Wang et al. (2004) reported that inhibition of ADAM17 resulted in selective inhibition of soluble TNF-α release in brain after ischemic injury. It is likely that inhibition of ADAM17 prevents increased TNF-α serum levels in LPS-mediated shock (Horiuchi et al., 2007). In addition to a reduction in shedding TNF-α from its inactive cell-bound precursor, neutrophils and macrophages lacking functional ADAM17 produced fewer soluble TNF receptors I and II when stimulated with LPS (Bell et al., 2007). It is interesting that soluble TNF receptors are known to neutralize TNF-α and inhibit its effects (Terao et al., 2010). Activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events, most likely independent of endothelial TNF-α release, leading to enhanced vascular permeability and leukocyte recruitment (Dreymueller et al., 2012). Thus, a preference for specific cleavage substrates may be key to understanding distinct homeostatic and pathophysiologic contexts.
Previously, in the bleomycin-induced lung injury model, we also demonstrated that membrane-bound Mer protein expression in the lung was recovered in concordance with reduced ADAM17 activity by TAPI-0 treatment. Using specific anti-Mer neutralizing antibodies, our data previously had indicated that the downregulation effect of TAPI-0 on sustained lung inflammation after bleomycin treatment was mediated by increased activation of Mer (Lee et al., 2012b). An important distinction between the models of bleomycin and LPS-induced lung injury is the sequence and magnitude of the inflammatory events as well as the healing response. Within 24 hours of intratracheal bleomycin administration, there is an increase in BAL neutrophils, which then normalize toward day 11 (Matute-Bello et al., 2008). Proinflammatory cytokines peak between 24 and 72 hours (Serrano-Mollar et al., 2003; Lee et al., 2012b). The fibrotic response persists by days 14–28 (Shen et al., 1988).
In comparison, the intratracheal instillation of LPS is followed by an early phase (2–6 hours) characterized by profound increases in BAL fluid proinflammatory cytokines, neutrophils, and protein content and a later phase 24 to 48 hours after instillation characterized by normalization of the BAL fluid cytokine concentrations and large increases in the BAL neutrophils (O’Grady et al., 2001). Importantly, LPS is a potent activator of the innate immune responses via TLR4 pathways. Thus, the use of LPS provides information about the effects of host inflammatory responses, which occur in bacterial infections. In addition, acute respiratory LPS challenge models in animals have extensively been used for the testing of new anti-inflammatory drugs, although they do not reflect all features of humans, notably chronic, respiratory diseases (Seehase et al., 2012).
Considering these characteristics of different animal models of experimental lung injury, we examined here the effects of TAPI-0 on not only short-term inflammatory responses but also downstream signaling of Mer and TLR4 proximal signaling (NF-κB signaling) in the LPS-induced ALI model. In addition, novel findings were provided in the LPS model that coadministration of the anti-Mer antibody reversed the upregulation of Mer downstream signaling, STAT1 signaling, SOCS1/3 expression, and downregulation of NF-κB signaling as well as TLR4-driven inflammatory responses in the presence of TAPI-0. The inhibitory effects of anti-Mer with TAPI-0 were greater than those of anti-Mer without TAPI-0. These findings emphasize that the effects of TAPI-0 may be crucial in the resolution of TLR4-mediated acute lung injury due to preventing Mer cleavage and subsequent activation of downstream signaling transducers.
In conclusion, our data demonstrate that restored membrane-bound Mer expression by TAPI-0 treatment upregulates activation of Mer and the downstream signaling pathways, including the Akt and the STAT1 pathways, during LPS-induced ALI. In parallel, LPS-induced NF-κB signaling, proinflammatory mediator production, inflammatory cell accumulation, and destruction of blood-pulmonary epithelial cell barrier integrity were attenuated by TAPI-0 treatment. Thus, these data identify a strategy to restore Mer expression at the protein level through blocking the cleavage process, which may be a useful tool for the resolution of ALI.
Acknowledgments
The authors thank Dr. Min-Sun Cho for assistance with the histologic study.
Authorship Contributions
Participated in research design: Choi, Park, Lee, Kang.
Conducted experiments: Choi, Park, Lee, Byun, Youn.
Contributed new reagents or analytic tools: Choi, Woo.
Performed data analysis: Choi, Park, Lee, Kang.
Wrote or contributed to the writing of the manuscript: Kang.
Footnotes
This work was supported by a National Research Foundation (NRF) grant funded by the Korean government (MEST) (2012-0009844).
↵This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- ADAM
- a disintegrin and metalloproteinase
- Akt
- protein kinase b
- ALI
- acute lung injury
- BAL
- bronchoalveolar lavage
- ELISA
- enzyme-linked immunosorbent assay
- FAK
- focal adhesion kinase
- Gas6
- growth arrest–specific protein 6
- IFNAR
- type 1 interferon receptor
- IL-1β
- interleukin-1β
- LPS
- lipopolysaccharide
- Mer
- Mer receptor tyrosine kinase
- MIP-2
- macrophage inflammatory protein-2
- MPO
- myeloperoxidase
- NF-κB
- nuclear factor κB
- PCR
- polymerase chain reaction
- sMer
- soluble Mer
- SOCS
- suppressor of cytokine signaling protein
- STAT
- signal transducer and activator of transcription
- TAM
- Tyro-3/Axl/Mer
- TAPI-0
- tumor necrosis factor alpha protease inhibitor-0
- TBE
- Tris/borate/EDTA
- TLR
- Toll-like receptor
- TNF-α
- tumor necrosis factor-α
- Received August 31, 2012.
- Accepted November 28, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics