Smooth Muscle Cells Relay Acute Pulmonary Inflammation via Distinct ADAM17/ErbB Axes

Daniela Dreymueller, Christian Martin, Julian Schumacher, Esther Groth, Julia Katharina Boehm, Lucy Kathleen Reiss, Stefan Uhlig and Andreas Ludwig
J Immunol January 15, 2014, 192 (2) 722-731; DOI: https://doi.org/10.4049/jimmunol.1302496

Abstract

In acute pulmonary inflammation, danger is first recognized by epithelial cells lining the alveolar lumen and relayed to vascular responses, including leukocyte recruitment and increased endothelial permeability. We supposed that this inflammatory relay critically depends on the immunological function of lung interstitial cells such as smooth muscle cells (SMC). Mice with smooth muscle protein-22α promotor-driven deficiency of the disintegrin and metalloproteinase (ADAM) 17 (SM22-Adam17−/−) were investigated in models of acute pulmonary inflammation (LPS, cytokine, and acid instillation). Underlying signaling mechanisms were identified in cultured tracheal SMC and verified by in vivo reconstitution experiments. SM22-Adam17−/− mice showed considerably decreased cytokine production and vascular responses in LPS- or acid-induced pulmonary inflammation. In vitro, ADAM17 deficiency abrogated cytokine release of primary SMC stimulated with LPS or supernatant of acid-exposed epithelial cells. This was explained by a loss of ADAM17-mediated growth factor shedding. LPS responses required ErbB1/epidermal growth factor receptor transactivation by TGFα, whereas acid responses required ErbB4 transactivation by neuregulins. Finally, LPS-induced pulmonary inflammation in SM22-Adam17−/− mice was restored by exogenous TGFα application, confirming the involvement of transactivation pathways in vivo. This highlights a new decisive immunological role of lung interstitial cells such as SMC in promoting acute pulmonary inflammation by ADAM17-dependent transactivation.

Introduction

Acute respiratory distress syndrome (ARDS) develops as a result of acute pulmonary inflammation caused, for example, by bacteria or aspiration of acidic gastric juice (1). The inflammatory response, coordinated by cytokines, chemokines, and growth factors, leads to the recruitment and activation of inflammatory cells and diminished barrier function. Epithelial cells and alveolar macrophages are among the first cells to encounter inflammatory insults. To coordinate leukocyte recruitment and barrier permeability, signals from the activated airway epithelium must be relayed to the vascular endothelium through the interstitial cell layer, including vascular and airway smooth muscle cells (SMC). SMC are capable of releasing a large variety of proinflammatory factors in vitro and in vivo. This includes cytokines (TNF-α, IL-6), chemokines (CXCL8/IL-8/murine CXCL1), and growth factors (neuregulins [NRGs] and TGFα) (2). However, the significance of SMC and their mediators for acute pulmonary inflammation are essentially unknown. Several of these mediators are tightly regulated by the proteolytic cleavage of their transmembrane precursors into soluble forms by a disintegrin and metalloproteinase (ADAM) family member ADAM17 (3, 4), a process termed shedding. For example, shed TGFα or NRGs act both paracrine and autocrine by binding to epidermal growth factor receptor (EGFR)/ErbB1 or ErbB3 and ErbB4 receptors, respectively, leading to ErbB-mediated cell transactivation (5, 6). Whereas developmental and regenerative activities of ADAM17 have been linked to TGFα shedding using gene-targeted mice (7, 8), the modulation of inflammation by the protease has been attributed in part to shedding of TNF-α, TNFR, IL-6R, L-selectin, or junctional adhesion molecules by different cell types (9–11). Inhibition studies suggest a role of ADAMs in pulmonary inflammation (10, 12, 13), but to date a specific role in the lung has been demonstrated for endothelial cell–expressed ADAM17 only. In this study, we tested the hypothesis that SMC fulfill an important immunological function in acute pulmonary inflammation via their ADAM17 activity. Using mice with smooth muscle protein 22-α (SM22α) promotor-driven deficiency of ADAM17 in SMC (SM22-Adam17−/− mice), we demonstrate the importance of SMC-ADAM17 for edema formation and neutrophil recruitment in two murine models of ARDS caused by instilled endotoxin (LPS) or HCl into the upper respiratory tract. In vitro investigations analyzing the underlying signaling mechanism revealed the distinct importance of autocrine EGFR or ErbB4 transactivation upon LPS or acid exposure, respectively. In vivo, administration of soluble TGFα during endotoxin challenge reconstituted the inflammatory response in SM22-Adam17−/− mice. Together, our in vitro and in vivo data demonstrate that acute pulmonary inflammation critically depends on ADAM17-mediated growth factor shedding in SMC, resulting in the stimulus-specific transactivation of EGFR/ErbB1 or ErbB4. Therefore, selective and local targeting of ADAM17 might provide a novel intervention therapy for acute pulmonary inflammation.

Materials and Methods

Abs, ELISA, cytokines, and inhibitors

Abs are listed according to the supplier. R&D Systems (Wiesbaden, Germany): mouse mAb against human ADAM17 and mouse IgG1 isotype control. Jackson ImmunoResearch Laboratories: allophycocyanin-conjugated goat anti-mouse Ab; HRP-conjugated goat anti-mouse and goat anti-rabbit Ab. Miltenyi Biotec (Bergisch Gladbach, Germany): rat allophycocyanin-labeled mAb against murine Ly6G (allophycocyanin anti-mLy6G); rat mAb VioBlue-labeled anti-mCD45R. eBiosciences (NatuTec, Frankfurt, Germany): Armenian hamster mAb PeCy5 anti-mCD3e; mouse mAb allophycocyanin anti-human TLR4; CD16/32 Fc block. AbD Serotec (Duesseldorf, Germany): rat mAb FITC- or PE-labeled anti-mF4/80. Abcam: rabbit mAb anti-murine TGFα. BD Pharmingen (Heidelberg, Germany): rat monoclonals allophycocyanin-Cy7 anti-mCD4, allophycocyanin-Cy7 anti-mCD11b, PacificBlue anti-mCD8a, and anti-mCD31 (PE labeled and unlabeled); rat PE-Cy7 anti-mNK1.1; Cy5-conjugated PE (PE-Cy5) anti-mCD19. Cell Signaling (Hitchin, U.K.): polyclonal rabbit anti-human EGFR and anti-Tyr1068 phospho-human EGFR.

LPS from Escherichia coli 0127:B8 was from Sigma-Aldrich (Munich, Germany); human TNF-α and IFN-γ were from R&D Systems. Murine TNF-α, murine TGFα, murine CXCL1, human IL-1β, and human NRG1 were from PeproTech. The matrix metalloproteinase inhibitor GW280264 was synthesized and assayed for inhibition of human and mouse ADAM17 and ADAM10, as described (14). The Complete Protease Inhibitor was from Roche (Munich, Germany). The EGFR kinase inhibitor PD168393 (PD), the EGFR Ab cetuximab (Erbitux, cet), the p38 inhibitor SB203580 (S), and the ERK inhibitor U0126 (U) were obtained from Merck. The ErbB1/2/3 inhibitor AZD8931 (AZD) and the ErbB1/2/4 inhibitor PF299804 (PF) were obtained from Selleckchem (Houston, TX).

Murine TNF-α, murine IL-6, murine CXCL1 (KC), as well as human IL-6, human CXCL8, and human TGFα, were measured using R&D Systems DuoSet ELISA kits, according to the manufacturer’s protocols.

Animals

Animal experiments were approved by the local authorities (LANUV NRW, 87-51.04.2010.A026 and appendices). SM22-Adam17−/− mice [animals expressing Cre recombinase under control of the SM22α promotor crossed with floxAdam17 mice on C57BL/6 background (10)] were compared with Adam17+/+ mice not differing from Adam17flox/flox mice (10). Genotyping was performed by PCR for floxed Adam17 (primer sequences wild-type Adam17, 5′-TACTGGTGGGGAGGGGGAGAGATTACGAAGGC-3′, 5′-ATGTTCCCCCAGCTAGATTGTTTGCC-3′; primer sequences floxed Adam17, 5′-TACTGGTGGGGAGGGGGAGAGATTACGAAGGC-3′, 5′-TACTGCCGGGCCTCTTGCGGGG-3′) and SM22α-Cre (5′-GCGGTCTGGCAGTAAAAACTATC-3′, 5′-GTGAAACAGCATTGCTGTCACTT-3′). Animals were hosted in a pathogen-free environment and were transferred to individually ventilated cages conditions for experiments.

Models and analysis of ARDS

Animals were investigated in LPS-induced and acid-induced models of ARDS, respectively, as well as cytokine-induced pulmonary inflammation, as described before (10, 15). In brief, pulmonary inflammation was induced by intranasal (i.n.) application of 400 μg/kg LPS, 250 μg/kg TNF-α, 250 μg/kg CXCL1, or combined instillation of 400 μg/kg LPS and 250 μg/kg TGFα. Instillation of PBS or TGFα alone served as control.

In the acid-induced ARDS model, animals were anesthetized, and 50 μl HCl in 0.3% NaCl with pH 1.8 or vehicle (0.3% NaCl) was intratracheally (i.t.) instilled using a microsprayer system (Pencentury) and ventilated for 5.5 h (15). Thereafter, bronchoalveolar lavage fluid (BALF) was analyzed for cellular composition and the release of cytokines.

The leukocyte populations in BALF and lung tissue were analyzed by flow cytometry. The determination of BALF and tissue lysate protein content and of the release of cytokines into BALF was performed as described (10).

For histological examination, lungs were fixed by i.t. instillation of Roti-Fix (Roth, Germany), followed by bronchial ligation after 5 min. After 48 h of fixation, the tissue was dehydrated, embedded in paraffin, and cut in 3-μm slices. H&E staining was performed using standard protocols. Ten images per animal were taken with a Zeiss microscope (AxioLab.A1; Carl Zeiss MicroImaging) and analyzed for thickness of alveolar septa and influx of polymorphonuclear cells using the AxioVision software (Carl Zeiss MicroImaging).

Cell culture, lentiviral knockdown of ADAM17, and substrate–cleavage assay

Human tracheal SMC (htSMC; Promocell) were cultured in SMC basal medium 2 (Promocell) and subcultured following the manufacturer’s protocol. Cells were used in passage 3 for viral transduction and passage 4–6 for all assays.

For preparation of murine tracheal SMC (mtSMC), tracheae of Adam17+/+ and SM22-Adam17−/− mice were explanted and the surrounding tissue was removed. Cleaned tracheae were cut in pieces, covered with coverslips in SMC growth medium (Provitro), and selected for cell outgrowth after 9–14 d.

Lentiviral knockdown and sheddase activity analysis were specified earlier (10, 16).

Twenty-four hours before stimulation, cells were washed with PBS and starved in medium without supplements containing 0.5% FBS. Then cells were washed with PBS and treated with synthetic inhibitors or Abs 1 h before stimulation in starvation medium. Cells were washed again with PBS and stimulated with 0.1 μg/ml LPS or vehicle (PBS) in the absence or presence of inhibitors, as described in the figure legends.

Human bronchial epithelial cells (BEAS-2B; Lonza) were cultured in DMEM/F12-Ham’s (Sigma-Aldrich) supplemented with 20% FBS and 1% penicillin/streptomycin. BEAS-2B cells were treated with normal medium (pH 7.4) or medium acidified by HCl addition (pH 1.8) for 5 min, followed by 24-h cultivation in DMEM/F12-Ham’s supplemented with 0.5% FBS and 1% penicillin/streptomycin to obtain BEAS-2B–conditioned medium for htSMC stimulation. The supernatant of BEAS-2B cells did not contain detectable levels of CXCL8 or IL-6. As control for reaction to residual HCl in BEAS-2B–conditioned medium, htSMC were stimulated with pH 1.8 medium for 5 min, followed by 24-h cultivation with starvation medium.

Quantitative RT-PCR analysis

The mRNA levels for ADAM10, ADAM17, IL-6, and CXCL1 in murine lung tissue and isolated mtSMC, as well as human ADAM10, ADAM17, TGFα, NRG1-4, epiregulin (EREG), and heparin-binding epidermal growth factor–like growth factor (HB-EGF) in htSMC were quantified by quantitative RT-PCR analysis and normalized to the mRNA level of murine RPS29 or human GAPDH. RNA was extracted using RNeasy kit (Qiagen, Hilden, Germany) and quantified by spectrophotometry (NanoDrop, Peqlab, Germany). RNA (equal amounts within each data set) was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany), according to the manufacturer’s protocol. PCRs were performed using LightCycler480 SYBR Green I Master Mix (Roche), according to the manufacturer’s protocol. Following primers were used with the specific primer annealing time given in brackets: mAdam10 forward, 5′-AGCAACATCTGGGGACAAAC-3′, and mAdam10 reverse, 5′-TGGCCAGATTCAACAAAAC-3′ (57°C); mAdam17 forward, 5′-AAACCAGAACAGACCCAACG-3′, and mAdam17 reverse, 5′-GTACGTCGATGCAGAGCAAA-3′ (57°C); mCxcl1 forward, 5′-CAAACCGAAGTCATAGCCAC-3′, and mCxcl1 reverse, 5′-TGGGGACACCTTTTAGCATC-3′ (60°C); mIl-6 forward, 5′-CCAGAGATACAAAGAATGATGG-3′, and mIl-6 reverse, 5′-ACTCCAGAAGACCAGAGGAAAT-3′ (50°C); mRps29 forward, 5′-GAGCAGACGCGGCAA-3′, and mRps29 reverse, 5′-CCTTTCTCCTCGTTGGGC-3′ (61°C); hAdam17 forward, 5′-GAAGTGCCAGGAGGCGATTA-3′, and hAdam17 reverse, 5′-CGGGCACTCACTGCTATTACC-3′ (55°C); hAdam10 forward, 5′-GGATTGTGGCTCATTGGTGGGCA-3′, and hAdam10 reverse, 5′-ACTCTCTCGGGGCCGCTGAC-3′ (61°C); hGapdh forward, 5′-CGGGGCTCTCCAGAACATCATCC-3′, and hGapdh reverse, 5′-CCAGCCCCAGCGTCAAAGGTG-3′ (66°C); hTgfa forward, 5′-GAGAACAGCACGTCCCCG-3′, and human Tgfa reverse, 5′-CCAGAATGGCAGACACATGC-3′ (64°C); hNrg1 forward, 5′-TTTCCCAAACCCGATCCGAG-3′, and hNrg1 reverse, 5′-AGCCGATTCCTGGCTTTTCA-3′ (57°C); hNrg2 forward, 5′-GACGCTGGGGAGTATGTCTG-3′, and hNrg2 reverse, 5′-AGGACTTGGCTGTCTCGTTG-3′ (56°C); hNrg3 forward, 5′-TGTGGGACCAGCATATCAGC-3′, and hNrg3 reverse, 5′-ACCAGGTCCTTTTGCTCCAA-3′ (58°C); hNrg4 forward, 5′-GCTGTTGTCTGCGGTATTCA-3′, and hNrg4 reverse, 5′-TCTTGGTCAAGAGAGTAGGGTTG-3′ (64°C); hEreg forward, 5′-CTGCCTGGGTTTCCATCTTCT-3′, and hEreg reverse, 5′-gccacacgtggattgtcttc-3′ (64°C); hHB-EGF forward, 5′-GCTCTTTCTGGCTGCAGTTCT-3′, and hHB-EGF reverse, 5′-CAAGTCACGGACTTTCCGGT-3′ (60°C). All PCRs were run on a LightCycler 480 System (Roche) with the following protocols: 40 cycles of 10-s denaturation at 95°C, followed by 10-s annealing at the indicated temperature and 15-s amplification at 72°C. Standard curves were determined by a serial dilution of a defined cDNA standard within each data set. Data were obtained as cycle-crossing point values and calculated as Δ cycle-crossing point values using the LightCycler480 software and used for statistic analysis. Data are expressed as arbitrary units in relation to RPS29 reference gene for murine and GAPDH reference gene for human samples.

Western blot analysis

Cells were lysed in extraction buffer (20 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1-fold complete protease inhibitor, 1 mM benzamidine, 10 mM glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 30 mM NaF, 5 mM DTT, 10 mM p-NPP in H2O) for 20 min on ice and detached by scratching. The lysates were centrifuged for 10 min at 16,000 × g at 4°C, and the supernatant was used for Western blot analysis. A total of 50 μg complete protein per lane was separated by 10% SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes using the semidry-transfer system. Membranes were blocked in 5% BSA in TBST for 1 h at room temperature, followed by overnight incubation with primary Abs in 2% BSA in TBST at 4°C. At last, membranes were incubated with secondary Abs in TBST for 1 h at room temperature. Between incubation steps, membranes were washed three times in TBST. ECL Plus/Advanced Western Blotting Detection System (Amersham) was used for protein detection, according to the manufacturer’s protocol. Chemiluminescence was visualized using the LAS-3000 scanner (FUJIFILM Europe, Dusseldorf, Germany). For quantification of band intensity, AIDA Image Analyzer software (raytest) was used.

Flow cytometry

The flow cytometric analysis of lung tissue, BALF cells, and blood leukocytes (including detailed gating strategy) as well as the surface expression of human ADAM17 on htSMC was performed, as described (10). htSMC were analyzed for TLR4 expression by incubation with mouse mAb anti-TLR4 (1 μg/ml) or the allophycocyanin-labeled mouse IgG2a isotype control. The fluorescence signal was detected by flow cytometry (LRSII Fortessa; BD Biosciences) and analyzed with FlowJo 8.7.3 software (Tree Star).

Statistics

Quantitative data are shown as mean ± SEM calculated from at least three independent experiments/cell isolates/animals, if not indicated otherwise. Percentage data were arc sin transformed for statistical analysis. Data were statistically analyzed, as indicated in the figure legends, by either one-way ANOVA followed by Bonferroni correction, one-sample t test (hypothetical value 100%), or unpaired t test using GRAPH PAD PRISM 5.0 program (GraphPad Software, La Jolla, CA). Statistically significant differences between measured values are indicated by asterisks and lines. Asterisks without lines indicate significant differences to the appropriate vehicle control.

Results

Lung ADAM17 in SM22α-positive cells (SM22-ADAM17) attenuates tissue damage and leukocyte recruitment in response to LPS challenge

We investigated whether lung interstitial cells, especially SMC, contribute to acute pulmonary inflammation by ADAM17-mediated shedding using SM22-Adam17−/− mice. These mice lack an obvious phenotype in the absence of disease (17) and do not differ in lung architecture (Fig. 1A). We confirmed the ADAM17-KO in isolated mtSMC by quantitative RT-PCR (Supplemental Fig. 1A), which was not compensated by ADAM10 mRNA expression (Supplemental Fig. 1B). These isolated cells did not differ in morphology in comparison with mtSMC of Adam17+/+ mice (Supplemental Fig. 1C, 1D). Furthermore, Adam17+/+ and SM22-Adam17−/− mice did not differ in WBC composition (Supplemental Fig. 1E–H).

FIGURE 1.
FIGURE 1.

Role of SM22-ADAM17 for permeability, tissue damage, and leukocyte recruitment for LPS-induced pulmonary inflammation. Adam17+/+ and SM22-Adam17−/− mice were i.n. treated with 400 μg/kg LPS or vehicle (PBS). (A) Representative images of lung histology. H&E stain. Original magnification ×20. (B) Whole protein content of BALF determined after 4 h. (C) Lung wet/dry ratio determined after 24 h. (D) Thickness of intraalveolar septa was determined using AixoVision software. (E–H) Number of neutrophils/ml BALF (E, G) and percentage of neutrophils within lung tissue (F, H) were determined after 4 h (E, F) or 24 h (G, H) by flow cytometry. (I) Interstitial neutrophil infiltration was determined using AixoVision software. (A, B, D–I) Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA and the Bonferroni posttest. *p < 0.05, **p < 0.01, ***p < 0.001.

In Adam17+/+ mice, i.n. LPS instillation increased the protein content in the BALF (Fig. 1B) and the tissue wet/dry ratio (Fig. 1C) after 4 and 24 h of instillation, respectively. Both effects were completely suppressed in SM22-Adam17−/− mice to basal levels observed in PBS-challenged mice. This protection in edema formation was confirmed by histological examination of the lung tissue (Fig. 1A). LPS instillation increased the thickness of alveolar septa in Adam17+/+ mice (Fig. 1D), whereas SM22-Adam17−/− mice were protected. LPS instillation increased the neutrophil number in BALF and lung tissue moderately after 4 h (Fig. 1E, 1F) and prominently after 24 h (Fig. 1G, 1H). SM22-Adam17−/− mice showed reduced recruitment after 4 h, and almost no remaining recruitment after 24 h. Histological examination confirmed considerably less neutrophils in the absence of SM22-Adam17 (Fig. 1A, 1I). These data suggest that lung SM22-expressing cells contribute to LPS-induced pulmonary inflammation via ADAM17 activity.

Lung SM22-ADAM17 promotes proinflammatory cytokine secretion

Inflammation and neutrophil recruitment are mediated to a large extent by the proinflammatory cytokines TNF-α and IL-6 and the chemokine CXCL1. The release of TNF-α, IL-6, and CXCL1 was prominent in BALF 4 h after i.n. LPS instillation of Adam17+/+ mice but reduced (TNF-α, IL-6) or abrogated (CXCL1) in SM22-Adam17−/− mice (Fig. 2A–C). After 24 h, TNF-α and IL-6 levels were still elevated in LPS-treated Adam17+/+ mice, whereas SM22-Adam17−/− mice displayed only basal levels (Fig. 2D, 2E). At this time point, CXCL1 release was not detectable.

FIGURE 2.
FIGURE 2.

Role of SM22-ADAM17 for cytokine and chemokine secretion for LPS-induced pulmonary inflammation. Adam17+/+ and SM22-Adam17−/− mice were i.n. treated with 400 μg/kg LPS or vehicle (PBS). Release of soluble TNF-α (A, E), CXCL1 (B), and IL-6 (C, E) into BALF was measured after 4 h (A–C) or 24 h (D, E). (A–E) Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA and Bonferroni posttest. *p < 0.05, **p < 0.01, ***p < 0.001.

We investigated whether the reduced release of TNF-α and CXCL1 in SM22-Adam17−/− mice could be responsible for the observed protection. Instillation (i.n.) of exogenous TNF-α resulted in edema formation in both SM22-Adam17−/− and Adam17+/+ mice (Fig. 3A). In contrast, the TNF-α–induced increase in neutrophil recruitment into BALF (4-fold, Fig. 3B) was reduced in SM22-Adam17−/− mice (∼50%) compared with Adam17+/+ mice. In addition, the release of CXCL1 (Fig. 3C) and IL-6 (Fig. 3D) was significantly reduced. We then investigated whether the reduced release of CXCL1 caused by SM22-ADAM17 deficiency might be the reason for the reduced neutrophil recruitment in SM22-Adam17−/− mice. Instillation (i.n.) of exogenous CXCL1 resulted in edema formation in Adam17+/+ mice, which was attenuated in SM22-Adam17−/− mice (Fig. 3E), whereas the recruitment of neutrophils into the BALF was not affected by SM22-ADAM17 deficiency (Fig. 3F). Thus, SM22-ADAM17 promotes the release of TNF-α and CXCL1, which mediate edema formation and neutrophil recruitment, respectively.

FIGURE 3.
FIGURE 3.

SM22-ADAM17 regulates permeability and leukocyte recruitment by TNF-α and CXCL1 release. Adam17+/+ and SM22-Adam17−/− mice were i.n. treated with either 250 μg/kg TNF-α (A–D) or 250 μg/kg CXCL1 (E, F) and compared with vehicle (PBS). (A and E) Lung wet/dry ratio determined after 24 h. (B and F) Number of neutrophils/ml BALF determined by flow cytometry after 24 h. (C) Release of CXCL1 into BALF after 4 h. (D) Release of IL-6 into BALF after 4 h. (A–F) Data represent means ± SEM (n = 3 per group). Significance was calculated using one-way ANOVA and Bonferroni posttest. *p < 0.05, **p < 0.01, ***p < 0.001.

SM22-ADAM17 mediates proinflammatory cytokine expression/release in response to LPS

Focusing on SMC as potential relay between endothelial and epithelial cell layers in pulmonary inflammation, we compared isolated mtSMC from Adam17+/+ and SM22-Adam17−/− mice with respect to mRNA expression and inflammatory cytokine release in response to LPS exposure. ADAM17 activity was measured as cleavage of a fluorogenic peptide-based substrate mimicking the α-cleavage site of amyloid-precursor protein in the substrate–cleavage activity assay. Treatment of Adam17+/+ mtSMC with LPS increased the substrate cleavage by 34% (Fig. 4A), whereas this effect was abrogated in SM22-Adam17−/− mtSMC. LPS treatment induced mRNA expression of CXCL1 and IL-6 in Adam17+/+ mtSMC but not in SM22-Adam17−/− mtSMC (Fig. 4B, 4C). The reduction of mRNA expression in SM22-Adam17−/− mtSMC correlated with reduced release of CXCL1 (Fig. 4D) and abrogated IL-6 production in response to LPS exposure (Fig. 4E).

FIGURE 4.
FIGURE 4.

Role of ADAM17 for LPS-stimulated substrate–cleavage activity and inflammatory cytokine expression/secretion in mtSMC and htSMC. (A–E) Adam17+/+ or SM22-Adam17−/− mtSMC were stimulated with 0.1 μg/ml LPS or vehicle (PBS) for 4 h (B–E) or 24 h (A). Substrate–cleavage activity (A), mRNA expression of CXCL1 (B), mRNA expression of IL-6 (C), release of CXCL1 (D), and release of IL-6 (E). (F–H) LV-scramble and LV-A17 cells were stimulated with 0.1 μg/ml LPS or vehicle (PBS) for 24 h and investigated for substrate–cleavage activity (F), CXCL8 release (G), and IL-6 (H) release. (A–H) Data represent means ± SEM of three independent experiments [each three different animals in (A)–(E)]. Significance was calculated using one-way ANOVA and the Bonferroni posttest (B–E, G, H) or one sample t test [(A, F); control: PBS treatment)]. *p < 0.05, **p < 0.01, ***p < 0.001.

To investigate the underlying mechanism in more detail, we studied htSMC. ADAM17 expression was silenced in htSMC by transduction with lentivirus coding for ADAM17-specific short hairpin RNA (LV-A17) and controlled by FACS analysis in comparison with scramble-transduced cells (LV-scramble, Supplemental Fig. 2A). LPS did not enhance surface expression of ADAM17 (Supplemental Fig. 2B) or mRNA expression of ADAM17 (Supplemental Fig. 2C) or ADAM10 (Supplemental Fig. 2D), whereas LPS exposure of htSMC increased ADAM17 substrate cleavage, which was abrogated by ADAM17 deficiency (Fig. 4F). Furthermore, LPS induced release of CXCL8 (Fig. 4G) and IL-6 (Fig. 4H) in htSMC, which were both suppressed when ADAM17 expression was silenced. Thus, ADAM17 is essential for the LPS-induced expression and secretion of proinflammatory mediators in mtSMC and htSMC.

SMC-ADAM17 regulates the inflammatory response to LPS by EGFR/ErbB1 transactivation

Next, we focused on signaling pathways involved in the inflammatory action of SMC-ADAM17. ADAM17 could shed TLR4 and thereby affect binding and response to LPS (18), but ADAM17 downregulation did not affect TLR4 surface level (Supplemental Fig. 2E). Because ADAM17 cleaves many growth factors activating different members of the human EGFR family (ErbB, Her) (4), this autocrine and paracrine pathway could affect the responsiveness toward LPS. Treatment with the EGFR kinase inhibitor PD168393, the ErbB1/2/3 inhibitor AZD8931, the ErbB1/2/4 inhibitor PF299804, or cetuximab (block of EGFR ligand binding) reduced the LPS-induced release of CXCL8 (Fig. 5A, data not shown), suggesting a critical role of EGFR/ErbB1 in this response. Because EGFR signaling is mediated via the kinases ERK1/2 and could also involve p38 activation, we next tested the ERK inhibitor U0216 and the p38 inhibitor SB203580, indicating that only ERK1/2 is relevant for CXCL8 release (data not shown). We used the metalloproteinase inhibitor GW280264 to block the shedding of growth factors responsible for EGFR activation (receptor phosphorylation). EGFR activation increased over time of LPS exposure (Fig. 5B), and was significantly reduced by inhibitor treatment (Fig. 5C). Thus, the full proinflammatory action of SMC to LPS seems to require ADAM17-mediated EGFR signaling.

FIGURE 5.
FIGURE 5.

Role of ADAM17-mediated EGFR transactivation for the inflammatory response to LPS. (A) htSMC were pretreated with 50 μM PD168393 (PD), 50 μM PF299804 (PF), 50 μM AZD8931 (AZD), or vehicle (0.1% DMSO) 1 h prior to and during stimulation with 0.1 μg/ml LPS or vehicle (PBS) for 24 h, and investigated for release of CXCL8. (B) htSMC were stimulated with 0.1 μg/ml LPS for 2, 4, 8, and 24 h or vehicle (PBS/0 h) and analyzed for EGFR and EGFR phosphorylation (pEGFR). Below the graph, a representative blot of three independent experiments is shown. (C) htSMC were stimulated with 0.1 μg/ml LPS for 2, 4, and 8 h or vehicle (PBS/0 h) and analyzed for EGFR phosphorylation. Cells were treated with 10 μM metalloproteinase inhibitor GW280264 or vehicle (0.1% DMSO) 24 h prior and during stimulation. (A–C) Data represent means ± SEM of three independent experiments. Significance was calculated by one-way ANOVA and Bonferroni posttest (A) or one sample t test [(B, C); control: DMSO treatment)]. *p < 0.05, ***p < 0.001.

SM22-ADAM17–mediated TGFα release critically regulates LPS-induced pulmonary inflammation

TGFα is one major substrate of ADAM17 released during acute pulmonary inflammation in humans (19). A neutralizing Ab against TGFα abrogated LPS-induced CXCL8 release by htSMC (Fig. 6A). Untreated LV-scramble and LV-A17 cells showed the same level of basal TGFα mRNA expression, which was not altered upon LPS exposure (Supplemental Fig. 3A). Released TGFα was enhanced by LPS stimulation after 2 h (Supplemental Fig. 3B) and was detectable in the 24-h conditioned media of LPS-treated LV-scramble cells, whereas ADAM17 deficiency almost completely abrogated this TGFα release (Supplemental Fig. 3C). The release of TGFα was affected by the application of neither PD168393 nor cetuximab (Supplemental Fig. 3C). These data suggest an ADAM17-dependent release of TGFα by SMC, which could modulate cellular responsiveness toward LPS. Therefore, we aimed to reconstitute the inflammatory cytokine release in ADAM17-silenced LV-A17 cells by coadministration of exogenous TGFα during LPS exposure. LV-scramble cells did not respond to application of TGFα alone, and coapplication of TGFα and LPS led to the expected LPS-induced CXCL8 release without additional enhancement (Fig. 6B). In LV-A17 cells, LPS-induced release of CXCL8 was restored by coapplication of TGFα (Fig. 6B).

FIGURE 6.
FIGURE 6.

ADAM17-dependent transactivation during LPS stimulation in htSMC and in LPS-induced pulmonary inflammation. (A) LV-scramble cells were pretreated with Ab against TGFα or isotype control (both 2.5 μg/ml) for 1 h before 24-h stimulation with 0.1 μg/ml LPS or vehicle (PBS), and were investigated for CXCL8 release. (B) LV-scramble and LV-A17 cells were stimulated with 20 ng/ml TGFα, 0.1 μg/ml LPS, a combination of both stimuli or vehicle (PBS) for 24 h, and were investigated for CXCL8 release. (C–F) Adam17+/+ and SM22-Adam17−/− mice were i.n. treated with a combination of 400 μg/kg LPS and 250 μg/kg TGFα and analyzed after 24 h. The number of neutrophils/ml BALF (C) and the percentage of neutrophils within lung tissue (D) were determined by flow cytometry. Release of CXCL1 (E) and IL-6 (F) into BALF. The values of solely LPS-treated animals (compare Figs. 1, 2) are indicated by dashed lines for Adam17+/+ mice, and by dotted lines for SM22-Adam17−/− mice. (A–F) Data represent means ± SEM (three independent experiments, n = 3 per group). Significance was calculated using one-way ANOVA and Bonferroni posttest (A, B) or Student t test (C–F). **p < 0.01, ***p < 0.001.

We further investigated the influence of TGFα on LPS-induced pulmonary inflammation in vivo. The basal lung tissue level of TGFα was reduced in SM22-Adam17−/− mice compared with Adam17+/+ mice (Supplemental Fig. 3D, p = 0.0639). Instillation (i.n.) of TGFα alone, analyzed in Adam17+/+ mice, did not induce an inflammatory reaction (Supplemental Fig. 3E–G). When exogenous TGFα was coadministered (i.n.) with LPS, edema formation, neutrophil recruitment, and cytokine secretion in Adam17+/+ mice were similar to that seen in Adam17+/+ mice treated with LPS only (Fig. 6C–F). In SM22-Adam17−/− mice, edema formation (Supplemental Fig. 3H) and TNF-α release (Supplemental Fig. 3I) were still inhibited even upon coinstillation of LPS and TGFα, which was comparable to the effect seen in SM22-Adam17−/− mice treated with LPS only (compare Figs. 1, 2). By contrast, neutrophil recruitment into the alveolar space or lung tissue and release of CXCL1 and IL-6 upon coinstillation of LPS and TGFα were not significantly affected by SM22-ADAM17 deficiency (Fig. 6C–F), clearly different from the effect of SM22-ADAM17 deficiency observed in mice treated with LPS only (compare Figs. 1, 2). Thus, important components of the inflammatory response toward LPS in SM22-ADAM17–deficient mice can be restored by exogenous TGFα, suggesting that ADAM17-mediated TGFα release is a key factor in LPS-induced pulmonary inflammation.

SM22-ADAM17 deficiency attenuates acid-induced pulmonary inflammation

We questioned whether SM22-ADAM17 plays a comparable role in the model of acid aspiration-induced pulmonary inflammation. Mice were i.t. instilled with a nonlethal dose of hydrochloric acid (pH 1.8) or vehicle (NaCl, pH 7.2) and analyzed after 5.5 h. Alveolar protein influx (Fig. 7A, p = 0.0563) and TNF release (Fig. 7B, p = 0.0531) seemed to be reduced by SM22-ADAM17 deficiency. SM22-ADAM17 deficiency resulted in clearly reduced secretion of CXCL1 (Fig. 7C) into the BALF, accompanied by impaired neutrophil recruitment (Fig. 7D). We further analyzed the underlying mechanism in an in vitro model of acid-induced pulmonary inflammation. The supernatant of acid-exposed epithelial BEAS-2B cells induced considerable release of CXCL8 (Fig. 8A) and IL-6 (Supplemental Fig. 4A) in LV-scramble cells, which was profoundly reduced in LV-A17 cells. Treatment with supernatant from normal pH-treated BEAS-2B did not show any effect (Fig. 8A, Supplemental Fig. 4A). In contrast to LPS-induced cytokine release, acid-induced CXCL8 and IL-6 release from htSMC upon treatment with BEAS-2B supernatant was not affected by EGFR inhibition by PD168396 or cetuximab (Fig. 8B, Supplemental Fig. 4G, data not shown). Thus, acid-induced pulmonary inflammation involves signaling mechanisms distinct from LPS-induced pulmonary inflammation.

FIGURE 7.
FIGURE 7.

SM22-ADAM17 in acid-induced pulmonary inflammation. (A–D) Adam17+/+ and SM22-Adam17−/− mice were i.t. instilled with 50 μl 0.3% NaCl (pH 1.8) or vehicle (0.3% NaCl) and ventilated for 5.5 h. Whole protein content of BALF (A) and release of TNF-α (B) and CXCL1 (C) into BALF. The number of neutrophils/ml BALF (D) was determined by flow cytometry. (A–D) Data represent means ± SEM (Adam17+/+ mice: 8 acid, 4 vehicle; SM22-Adam17−/− mice: 5 acid, 3 vehicle). Significance was calculated using one-way ANOVA and Bonferroni posttest. *p < 0.05, ***p < 0.001.

FIGURE 8.
FIGURE 8.

ADAM17-dependent ErbB4 transactivation during acid-induced inflammation in htSMC. (A–D) LV-scramble/LV-A17 cells were stimulated with conditioned medium of nonstimulated BEAS-2B (normal medium, pH 7.4) or acid-exposed BEAS-2B (medium, pH 1.8) for 24 h and investigated for CXCL8 release. LV-scramble cells were treated with 50 μM PD, 50 μM PF299804 (PF), 50 μM AZD8931 (AZD), or vehicle (0.1% DMSO) 1 h prior to and during stimulation (B). LV-scramble and LV-A17 cells were stimulated with BEAS-2B supernatants in the absence and presence of 10 nM NRG1 (C), or cells were stimulated with 10 ng/ml IL-1β in the absence or presence of 10 nM NRG1 (D). Values below detection level were indicated as negative values. (A–D) Data represent means ± SEM of three independent experiments. Significance was calculated using one-way ANOVA and Bonferroni posttest. *p < 0.05, **p < 0.01, ***p < 0.001.

ADAM17-dependent ErbB4 transactivation regulates the acid-induced inflammatory response of htSMC

Besides cleaving TGFα, ADAM17 is involved in the cleavage of EREG and NRGs (6, 20). Our inhibition experiments indicated that the TGFα/EGFR axis was not relevant for acid-induced pulmonary inflammation. However, EREG and NRGs are capable of activating other ErbB receptors. The latter pathway is implicated in lung homeostasis (6) and may also contribute to the transactivation of SMC in acid-induced pulmonary inflammation. We found that NRG1, NRG2, TGFα, HB-EGF, and EREG were constitutively expressed at the mRNA level in cultured LV-scramble htSMC as well as in LV-A17 htSMC (Supplemental Fig. 4B–F) and were not changed in mRNA expression during the LPS- or acid-induced inflammatory response (data not shown). Expression of NRG3 and 4 could not be detected. As NRG1 and NRG2 are ligands for ErbB3 and ErbB4, we investigated the contribution of these receptors within the in vitro acid-exposure model. When LV-scramble cells were treated with BEAS-2B supernatant in the presence of the ErbB1/2/4 inhibitor PF299804, the induced CXCL8 and IL-6 release was inhibited or abrogated, respectively. However, the ErbB1/2/3 inhibitor AZD8931 showed no inhibition (Fig. 8B, Supplemental Fig. 4G), indicating an involvement of ErbB4. Therefore, we investigated whether application of exogenous NRG1 during treatment with BEAS-2B supernatant would reconstitute the inflammatory cytokine release in LV-A17 cells. Treatment with only NRG1 did not induce inflammatory cytokine secretion. Coapplication of NRG1 restored the induced CXCL8 release in LV-A17 cells, without further enhancement of the response in LV-scramble cells (Fig. 8C). Because IL-1β is released by acid-exposed epithelial cells (21), we questioned whether this cytokine could activate htSMC in an ADAM17-ErbB4–dependent manner. Treatment with IL-1β resulted in strong CXCL8 release by LV-scramble cells (Fig. 8D), which was profoundly reduced in LV-A17 cells. NRG1 coapplication with IL-1β restored the CXCL8 release in LV-A17 cells, without further enhancing the response in LV-scramble cells (Fig. 8D). Therefore, IL-1β also activates htSMC in an ErbB4-NRG–dependent manner (Fig. 9).

FIGURE 9.
FIGURE 9.

Model for ADAM17-dependent ErbB receptor transactivation in acute pulmonary inflammation. Acute pulmonary inflammation leads to activation of ADAM17, resulting in enhanced growth factor shedding. In LPS-induced pulmonary inflammation (dark grey pathway), release of TGFα results in the transactivation of EGFR/ErbB1, whereas in acid-induced pulmonary inflammation (light grey pathway) NRGs are released, resulting in the transactivation of ErB4. Both pathways lead to the release of inflammatory mediators, including IL-6, CXCL8, and TNF-α, and critically contribute to the development of acute pulmonary inflammation with edema formation, mediator secretion, leukocyte recruitment, and tissue damage. Options for pharmacological and genetic intervention are indicated.

Discussion

Combining in vivo and in vitro investigations, we show a decisive immunological role of lung interstitial cells, especially SMC, in acute pulmonary inflammation. These responses critically require ADAM17-mediated transactivation of ErbB receptors, which was demonstrated by pharmacological or transcriptional inhibition and application of exogenous TGFα or NRG1, compensating for the loss of ADAM17 in cultured SMC. Moreover, exogenous TGFα reconstituted the inflammatory response of SM22-Adam17−/− mice in LPS-induced pulmonary inflammation. Whereas lung LPS responses are mediated by ErbB1 transactivation via released TGFα, acid-induced inflammation is relayed by ErbB4 transactivation via NRGs such as NRG1 (Fig. 9).

ADAM17 was initially identified as TNF-α–converting enzyme, but during the last decades many more substrates have been identified. This includes the cleavage of proinflammatory mediators such as TNF-α itself and IL-6R, as well as the release of soluble TNFRs that act anti-inflammatory (7). Cleavage of other substrates such as CX3CL1 can be both pro- and anti-inflammatory (16, 22). Moreover, the anti- or proinflammatory balance of ADAM17-mediated cleavage can be cell, tissue, and injury specific (23, 24). For instance, on leukocytes the protease mediates shedding of L-selectins, resulting in reduced surface expression of the adhesion molecule, thus limiting leukocyte recruitment into the lung (25). On endothelial cells, however, ADAM17 is involved in the shedding of several adhesion molecules facilitating leukocyte recruitment and vascular permeability (10, 26). For chronic lung diseases, it has been postulated that interstitial cells, especially SMC, release a large variety of factors. These include many ADAM17 substrates, for example, TNF-α, TGFα, and epidermal growth factor, influencing the pathogenesis of chronic obstructive pulmonary disease and sarcoidosis (27). However, the role of ADAM17 on SMC in particular in acute inflammatory disorders had not yet been identified. We show that in LPS- and acid-induced acute pulmonary inflammation, SM22-ADAM17 contributes to edema formation, neutrophil recruitment, and proinflammatory mediator release. SM22α is predominantly expressed in lung SMC, but also in some pericytes within the wall of small distal pulmonary vessels (28). However, SMC may hold a more strategic position between the epithelial and endothelial cell layers than perivascular cells, highlighting their critical role in the inflammatory signal relay by SMC-ADAM17–dependent transactivation. The protection seen in the SM22-Adam17−/− mice in LPS-induced acute pulmonary inflammation can be explained to some extent by the reduced production of TNF-α (responsible for edema formation) and CXCL1 (responsible for leukocyte recruitment). Such a separation between both events has been observed before, without molecular explanation (29). The present findings may suggest the task of SMC to regulate pulmonary neutrophil sequestration and edema formation independently from each other.

ADAM17 is known as the main constitutive and PMA-induced sheddase of TGFα, amphiregulin, HB-EGF, EREG, and NRGs (5). A strong link between TGFα and ADAM17 activity is already indicated by the similar developmental phenotype of ADAM17- and TGFα-deficient mice (3). The signaling via TGFα release and EGFR activation is multifaceted. ADAM17-mediated TGFα shedding and EGFR transactivation are indispensible for the maintenance of the epithelium and its barrier function, as was also shown in TGFα transgenic mice (7, 8, 13. In contrast, TGFα- and ADAM17-dependent EGFR transactivation has been linked to the inflammatory reaction of cultured lung epithelial cells to particles or LPS (27). Furthermore, EGFR transactivation was reported to augment the development of fibrosis (13). Transactivation of ErbB receptors can occur in different lung diseases and can involve several growth factor ligands. Besides TGFα, NRGs are also shed in ARDS patients, which might also lead to transactivation of other ErbB receptors than EGFR/ErbB1 (20). Recent findings indicate that ADAM17 can shed different growth factors (e.g., TGFα and NRGs) via distinct substrate-selecting pathways induced depending on the type of cell stimulus (30). Our findings expand the role for growth factor shedding by SMC-ADAM17 in the lung. ADAM17-derived TGFα and autocrine EGFR activation constitute an important mechanism by which SMC may regulate acute pulmonary inflammation elicited by LPS. In acid-induced pulmonary inflammation, in contrast, NRGs—most likely NRG1 and NRG2—and autocrine activation of ErbB4 appear as a trigger of inflammatory signals from SMC. The use of different transactivating ligands and receptors by SMC could provide a means to adapt inflammatory responses to the type of injury. EGFR/ErbB1 sensing could be more important to the binding and clearance of bacteria (31), whereas the initiation of inflammation after acid exposure might require ErbB4 signaling, which is important for surfactant production and proliferation of lung epithelial cells (32). Moreover, we found that IL-1β induced CXCL8 release by htSMC in an ADAM17-NRG1–dependent manner. ADAM17 can be activated by IL-1β (33), and IL-1β–induced activation of airway epithelial cells involving ADAM17-mediated shedding of NRG1 has been proposed (6, 20, 21). Thus, in acid-induced acute pulmonary inflammation, epithelial cell–derived IL-1β may enhance ADAM17-dependent NRG release from lung cells, including SMC, leading to increased ErbB4 transactivation and initiation of the inflammatory response. As mentioned before, EGFR transactivation by ADAM17 signaling is also important for developmental and regenerative processes, including barrier function (7, 8). It was shown that ErbB receptor signaling influences alveolar epithelial injury and repair (6). Although ADAM17 in SMC clearly promotes the development of acute pulmonary inflammation, it remains to be clarified whether the protease may also hold protective functions at a later phase when inflammation either resolves or turns into a chronic disease.

The present study provides in vitro and in vivo evidence for a specific, immunological relay function of lung interstitial cells, especially SMC, in acute pulmonary inflammation. This pathway may be relevant for developing target- and time frame–specific treatments for ARDS by resolution of inflammation without inhibition of regenerative processes. Specific ADAM17 inhibitors, as well as disease-dependent targeting of ErbB signaling pathways, may be considered as systemic or inhalative treatments of acute pulmonary inflammation by interruption of the immunological relay function of interstitial cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Franz-Martin Hess for establishment of the lentiviral system. We thank Keisuke Horiouchi for generous support and constructive discussions. We thank Melanie Esser, Anke Kowallik, and Tanja Woopen for expert technical assistance.

Footnotes

  • This work was supported by the Interdisziplinaeres Zentrum fur Klinische Forschung Aachen of the Rheinisch-Westfaelische Technische Hochschule Aachen University and the German Research Foundation Lu869/5-1.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ADAM
    a disintegrin and metalloproteinase
    ARDS
    acute respiratory distress syndrome
    BALF
    bronchoalveolar lavage fluid
    EGFR
    epidermal growth factor receptor
    EREG
    epiregulin
    HB-EGF
    heparin-binding epidermal growth factor–like growth factor
    htSMC
    human tracheal smooth muscle cell
    i.n.
    intranasal
    i.t.
    intratracheal
    mtSMC
    murine tracheal smooth muscle cell
    NRG
    neuregulin
    SMC
    smooth muscle cell.

  • Received September 16, 2013.
  • Accepted November 10, 2013.

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