Smooth Muscle Cells Relay Acute Pulmonary
Inflammation via Distinct ADAM17/ErbB
Axes
This information is current as
of June 17, 2017.
Daniela Dreymueller, Christian Martin, Julian Schumacher,
Esther Groth, Julia Katharina Boehm, Lucy Kathleen Reiss,
Stefan Uhlig and Andreas Ludwig
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 192:722-731; Prepublished online 16
December 2013;
doi: 10.4049/jimmunol.1302496
http://www.jimmunol.org/content/192/2/722
The Journal of Immunology
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
A
cute 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-a, IL-6), chemokines (CXCL8/IL-8/murine CXCL1), and
growth factors (neuregulins [NRGs] and TGFa) (2). However, the
significance of SMC and their mediators for acute pulmonary in-
Institute of Pharmacology and Toxicology, Rheinisch-Westfaelische Technische
Hochschule Aachen University, 52074 Aachen, Germany
Received for publication September 16, 2013. Accepted for publication November
10, 2013.
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.
Address correspondence and reprint requests to Dr. Daniela Dreymueller, Institute of
Pharmacology and Toxicology, Rheinisch-Westfaelische Technische Hochschule
Aachen University, Wendlingweg 2, 52074 Aachen, Germany. E-mail address:
[email protected]
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, heparinbinding 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.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302496
flammation 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 TGFa or NRGs act both paracrine and
autocrine by binding to epidermal growth factor receptor (EGFR)/
ErbB1 or ErbB3 and ErbB4 receptors, respectively, leading to ErbBmediated cell transactivation (5, 6). Whereas developmental and
regenerative activities of ADAM17 have been linked to TGFa
shedding using gene-targeted mice (7, 8), the modulation of inflammation by the protease has been attributed in part to shedding of
TNF-a, 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-a (SM22a) promotor-driven deficiency of ADAM17 in SMC (SM22-Adam172/2 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 TGFa during endotoxin challenge reconstituted the inflammatory response in SM22-Adam172/2 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.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
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-22a promotor-driven deficiency of the disintegrin and metalloproteinase (ADAM) 17 (SM22-Adam172/2) 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-Adam172/2 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 TGFa, whereas acid responses required ErbB4 transactivation by neuregulins. Finally,
LPS-induced pulmonary inflammation in SM22-Adam172/2 mice was restored by exogenous TGFa 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. The Journal of Immunology, 2014,
192: 722–731.
The Journal of Immunology
Materials and Methods
Abs, ELISA, cytokines, and inhibitors
Animals
Animal experiments were approved by the local authorities (LANUV NRW,
87-51.04.2010.A026 and appendices). SM22-Adam172/2 mice [animals
expressing Cre recombinase under control of the SM22a 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, 59-TACTGGTGGGGAGGGGGAGAGATTACGAAGGC-39, 59ATGTTCCCCCAGCTAGATTGTTTGCC-39; primer sequences floxed
Adam17, 59-TACTGGTGGGGAGGGGGAGAGATTACGAAGGC-39, 59TACTGCCGGGCCTCTTGCGGGG-39) and SM22a-Cre (59-GCGGTCTGGCAGTAAAAACTATC-39, 59-GTGAAACAGCATTGCTGTCACTT-39).
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 mg/kg LPS, 250 mg/kg TNF-a, 250
mg/kg CXCL1, or combined instillation of 400 mg/kg LPS and 250 mg/kg
TGFa. Instillation of PBS or TGFa alone served as control.
In the acid-induced ARDS model, animals were anesthetized, and 50 ml
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), followedby bronchial ligation after 5 min. After 48 hoffixation,
the tissue was dehydrated, embedded in paraffin, and cut in 3-mm 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-Adam172/2 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 mg/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,
TGFa, 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, 59-AGCAACATCTGGGGACAAAC-39, and mAdam10
reverse, 59-TGGCCAGATTCAACAAAAC-39 (57˚C); mAdam17 forward,
59-AAACCAGAACAGACCCAACG-39, and mAdam17 reverse, 59-GTACGTCGATGCAGAGCAAA-39 (57˚C); mCxcl1 forward, 59-CAAACCGAAGTCATAGCCAC-39, and mCxcl1 reverse, 59-TGGGGACACCTTTTAGCATC-39 (60˚C); mIl-6 forward, 59-CCAGAGATACAAAGAATGATGG-39,
and mIl-6 reverse, 59-ACTCCAGAAGACCAGAGGAAAT-39 (50˚C);
mRps29 forward, 59-GAGCAGACGCGGCAA-39, and mRps29 reverse,
59-CCTTTCTCCTCGTTGGGC-39 (61˚C); hAdam17 forward, 59-GAAGTGCCAGGAGGCGATTA-39, and hAdam17 reverse, 59-CGGGCACTCACTGCTATTACC-39 (55˚C); hAdam10 forward, 59-GGATTGTGGCTCATTGGTGGGCA-39, and hAdam10 reverse, 59-ACTCTCTCGGGGCCGCTGAC-39 (61˚C); hGapdh forward, 59-CGGGGCTCTCCAGAACATCATCC-39, and hGapdh reverse, 59-CCAGCCCCAGCGTCAAAGGTG-39
(66˚C); hTgfa forward, 59-GAGAACAGCACGTCCCCG-39, and human
Tgfa reverse, 59-CCAGAATGGCAGACACATGC-39 (64˚C); hNrg1 forward, 59-TTTCCCAAACCCGATCCGAG-39, and hNrg1 reverse, 59-AGCCGATTCCTGGCTTTTCA-39 (57˚C); hNrg2 forward, 59-GACGCTGGGGAGTATGTCTG-39, and hNrg2 reverse, 59-AGGACTTGGCTGTCTCGTTG-39 (56˚C); hNrg3 forward, 59-TGTGGGACCAGCATATCAGC39, and hNrg3 reverse, 59-ACCAGGTCCTTTTGCTCCAA-39 (58˚C); hNrg4
forward, 59-GCTGTTGTCTGCGGTATTCA-39, and hNrg4 reverse, 59TCTTGGTCAAGAGAGTAGGGTTG-39 (64˚C); hEreg forward, 59-CTGCCTGGGTTTCCATCTTCT-39, and hEreg reverse, 59-gccacacgtggattgtcttc-39 (64˚C); hHB-EGF forward, 59-GCTCTTTCTGGCTGCAGTTCT-39,
and hHB-EGF reverse, 59-CAAGTCACGGACTTTCCGGT-39 (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
D 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 ben-
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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 antimouse 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 VioBluelabeled 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 antimurine TGFa. 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 antiTyr1068 phospho-human EGFR.
LPS from Escherichia coli 0127:B8 was from Sigma-Aldrich (Munich,
Germany); human TNF-a and IFN-g were from R&D Systems. Murine
TNF-a, murine TGFa, murine CXCL1, human IL-1b, 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-a, murine IL-6, murine CXCL1 (KC), as well as human
IL-6, human CXCL8, and human TGFa, were measured using R&D
Systems DuoSet ELISA kits, according to the manufacturer’s protocols.
723
724
SMC TRANSACTIVATION RELAYS ACUTE PULMONARY INFLAMMATION
zamidine, 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 3 g at 4˚C,
and the supernatant was used for Western blot analysis. A total of 50 mg
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
Statistics
Quantitative data are shown as mean 6 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
FIGURE 1. Role of SM22ADAM17 for permeability, tissue
damage, and leukocyte recruitment
for LPS-induced pulmonary inflammation. Adam17+/+ and SM22Adam172/2 mice were i.n. treated
with 400 mg/kg LPS or vehicle (PBS).
(A) Representative images of lung
histology. H&E stain. Original magnification 320. (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 6
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.
Results
Lung ADAM17 in SM22a-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-Adam172/2 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-Adam172/2 mice did not
differ in WBC composition (Supplemental Fig. 1E–H).
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-Adam172/2 mice to basal levels
observed in PBS-challenged mice. This protection in edema
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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 mg/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).
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.
The Journal of Immunology
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 SM22Adam172/2 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). SM22Adam172/2 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.
725
Lung SM22-ADAM17 promotes proinflammatory cytokine
secretion
Inflammation and neutrophil recruitment are mediated to a large
extent by the proinflammatory cytokines TNF-a and IL-6 and the
chemokine CXCL1. The release of TNF-a, IL-6, and CXCL1 was
prominent in BALF 4 h after i.n. LPS instillation of Adam17+/+
mice but reduced (TNF-a, IL-6) or abrogated (CXCL1) in SM22Adam172/2 mice (Fig. 2A–C). After 24 h, TNF-a and IL-6 levels
were still elevated in LPS-treated Adam17+/+ mice, whereas
SM22-Adam172/2 mice displayed only basal levels (Fig. 2D, 2E).
At this time point, CXCL1 release was not detectable.
We investigated whether the reduced release of TNF-a and
CXCL1 in SM22-Adam172/2 mice could be responsible for the
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FIGURE 2. Role of SM22-ADAM17 for cytokine and chemokine
secretion for LPS-induced pulmonary inflammation. Adam17+/+ and
SM22-Adam172/2 mice were i.n. treated with 400 mg/kg LPS or vehicle
(PBS). Release of soluble TNF-a (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 6 SEM (n = 3 per group). Significance was calculated
using one-way ANOVA and Bonferroni posttest. *p , 0.05, **p , 0.01,
***p , 0.001.
FIGURE 3. SM22-ADAM17 regulates permeability and leukocyte recruitment by TNF-a and CXCL1 release. Adam17+/+ and SM22-Adam172/2
mice were i.n. treated with either 250 mg/kg TNF-a (A–D) or 250 mg/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 6 SEM (n = 3 per group). Significance was calculated using one-way
ANOVA and Bonferroni posttest. *p , 0.05, **p , 0.01, ***p , 0.001.
726
SMC TRANSACTIVATION RELAYS ACUTE PULMONARY INFLAMMATION
observed protection. Instillation (i.n.) of exogenous TNF-a resulted in edema formation in both SM22-Adam172/2 and Adam17+/+
mice (Fig. 3A). In contrast, the TNF-a–induced increase in
neutrophil recruitment into BALF (4-fold, Fig. 3B) was reduced in
SM22-Adam172/2 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-Adam172/2 mice. Instillation (i.n.) of exogenous CXCL1
resulted in edema formation in Adam17+/+ mice, which was attenuated in SM22-Adam172/2 mice (Fig. 3E), whereas the recruitment of neutrophils into the BALF was not affected by SM22ADAM17 deficiency (Fig. 3F). Thus, SM22-ADAM17 promotes
the release of TNF-a and CXCL1, which mediate edema formation and neutrophil recruitment, respectively.
SM22-ADAM17 mediates proinflammatory cytokine
expression/release in response to LPS
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-Adam172/2 mtSMC were stimulated with 0.1 mg/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 mg/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 6 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.
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Focusing on SMC as potential relay between endothelial and
epithelial cell layers in pulmonary inflammation, we compared
isolated mtSMC from Adam17+/+ and SM22-Adam172/2 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 a-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-Adam172/2 mtSMC.
LPS treatment induced mRNA expression of CXCL1 and IL-6 in
Adam17+/+ mtSMC but not in SM22-Adam172/2 mtSMC (Fig. 4B,
4C). The reduction of mRNA expression in SM22-Adam172/2
mtSMC correlated with reduced release of CXCL1 (Fig. 4D) and
abrogated IL-6 production in response to LPS exposure (Fig. 4E).
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
The Journal of Immunology
727
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
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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.
SM22-ADAM17–mediated TGFa release critically regulates
LPS-induced pulmonary inflammation
TGFa is one major substrate of ADAM17 released during acute
pulmonary inflammation in humans (19). A neutralizing Ab
against TGFa abrogated LPS-induced CXCL8 release by htSMC
(Fig. 6A). Untreated LV-scramble and LV-A17 cells showed the
same level of basal TGFa mRNA expression, which was not altered upon LPS exposure (Supplemental Fig. 3A). Released TGFa
was enhanced by LPS stimulation after 2 h (Supplemental Fig.
3B) and was detectable in the 24-h conditioned media of LPStreated LV-scramble cells, whereas ADAM17 deficiency almost
completely abrogated this TGFa release (Supplemental Fig. 3C).
The release of TGFa was affected by the application of neither
PD168393 nor cetuximab (Supplemental Fig. 3C). These data
suggest an ADAM17-dependent release of TGFa 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 TGFa during LPS exposure. LV-scramble cells did not respond to application of TGFa alone, and coapplication of TGFa
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 TGFa (Fig. 6B).
We further investigated the influence of TGFa on LPS-induced
pulmonary inflammation in vivo. The basal lung tissue level of
TGFa was reduced in SM22-Adam172/2 mice compared with
Adam17+/+ mice (Supplemental Fig. 3D, p = 0.0639). Instillation
(i.n.) of TGFa alone, analyzed in Adam17+/+ mice, did not induce
an inflammatory reaction (Supplemental Fig. 3E–G). When exogenous TGFa 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-Adam172/2 mice, edema formation
(Supplemental Fig. 3H) and TNF-a release (Supplemental Fig. 3I)
FIGURE 5. Role of ADAM17-mediated EGFR transactivation for the
inflammatory response to LPS. (A) htSMC were pretreated with 50 mM
PD168393 (PD), 50 mM PF299804 (PF), 50 mM AZD8931 (AZD), or
vehicle (0.1% DMSO) 1 h prior to and during stimulation with 0.1 mg/ml
LPS or vehicle (PBS) for 24 h, and investigated for release of CXCL8. (B)
htSMC were stimulated with 0.1 mg/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 mg/ml LPS for
2, 4, and 8 h or vehicle (PBS/0 h) and analyzed for EGFR phosphorylation. Cells were treated with 10 mM metalloproteinase inhibitor
GW280264 or vehicle (0.1% DMSO) 24 h prior and during stimulation.
(A–C) Data represent means 6 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.
728
SMC TRANSACTIVATION RELAYS ACUTE PULMONARY INFLAMMATION
were still inhibited even upon coinstillation of LPS and TGFa,
which was comparable to the effect seen in SM22-Adam172/2
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
TGFa were not significantly affected by SM22-ADAM17 deficiency (Fig. 6C–F), clearly different from the effect of SM22ADAM17 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 TGFa, suggesting that ADAM17mediated TGFa 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
FIGURE 7. SM22-ADAM17 in acid-induced pulmonary inflammation.
(A–D) Adam17+/+ and SM22-Adam172/2 mice were i.t. instilled with 50
ml 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-a (B) and CXCL1
(C) into BALF. The number of neutrophils/ml BALF (D) was determined
by flow cytometry. (A–D) Data represent means 6 SEM (Adam17+/+ mice:
8 acid, 4 vehicle; SM22-Adam172/2 mice: 5 acid, 3 vehicle). Significance
was calculated using one-way ANOVA and Bonferroni posttest. *p , 0.05,
***p , 0.001.
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 LPSinduced 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.
ADAM17-dependent ErbB4 transactivation regulates the acidinduced inflammatory response of htSMC
Besides cleaving TGFa, ADAM17 is involved in the cleavage of
EREG and NRGs (6, 20). Our inhibition experiments indicated
that the TGFa/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, TGFa, 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
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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 TGFa or isotype control (both 2.5
mg/ml) for 1 h before 24-h stimulation with 0.1 mg/ml LPS or vehicle
(PBS), and were investigated for CXCL8 release. (B) LV-scramble and LVA17 cells were stimulated with 20 ng/ml TGFa, 0.1 mg/ml LPS, a combination of both stimuli or vehicle (PBS) for 24 h, and were investigated
for CXCL8 release. (C–F) Adam17+/+ and SM22-Adam172/2 mice were
i.n. treated with a combination of 400 mg/kg LPS and 250 mg/kg TGFa
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-Adam172/2 mice.
(A–F) Data represent means 6 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.
The Journal of Immunology
729
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 LVscramble 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-1b
is released by acid-exposed epithelial cells (21), we questioned
whether this cytokine could activate htSMC in an ADAM17ErbB4–dependent manner. Treatment with IL-1b resulted in
strong CXCL8 release by LV-scramble cells (Fig. 8D), which was
profoundly reduced in LV-A17 cells. NRG1 coapplication with
IL-1b restored the CXCL8 release in LV-A17 cells, without
further enhancing the response in LV-scramble cells (Fig. 8D).
Therefore, IL-1b also activates htSMC in an ErbB4-NRG–dependent
manner (Fig. 9).
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 TGFa or NRG1, compensating for the
loss of ADAM17 in cultured SMC. Moreover, exogenous TGFa
reconstituted the inflammatory response of SM22-Adam172/2
mice in LPS-induced pulmonary inflammation. Whereas lung LPS
responses are mediated by ErbB1 transactivation via released
TGFa, acid-induced inflammation is relayed by ErbB4 transactivation via NRGs such as NRG1 (Fig. 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 TGFa results in
the transactivation of EGFR/ErbB1, whereas in acidinduced 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-a,
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.
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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 mM
PD, 50 mM PF299804 (PF), 50 mM 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-1b in the absence or presence of
10 nM NRG1 (D). Values below detection level were
indicated as negative values. (A–D) Data represent
means 6 SEM of three independent experiments. Significance was calculated using one-way ANOVA and
Bonferroni posttest. *p , 0.05, **p , 0.01, ***p ,
0.001.
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SMC TRANSACTIVATION RELAYS ACUTE PULMONARY INFLAMMATION
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-1b induced CXCL8 release by
htSMC in an ADAM17-NRG1–dependent manner. ADAM17 can
be activated by IL-1b (33), and IL-1b–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-1b 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.
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.
Disclosures
The authors have no financial conflicts of interest.
References
1. Matthay, M. A., and R. L. Zemans. 2011. The acute respiratory distress syndrome: pathogenesis and treatment. Annu. Rev. Pathol. 6: 147–163.
2. Tliba, O., and R. A. Panettieri, Jr. 2009. Noncontractile functions of airway
smooth muscle cells in asthma. Annu. Rev. Physiol. 71: 509–535.
3. Le Gall, S. M., P. Bobé, K. Reiss, K. Horiuchi, X. D. Niu, D. Lundell,
D. R. Gibb, D. Conrad, P. Saftig, and C. P. Blobel. 2009. ADAMs 10 and 17
represent differentially regulated components of a general shedding machinery
for membrane proteins such as transforming growth factor alpha, L-selectin, and
tumor necrosis factor alpha. Mol. Biol. Cell 20: 1785–1794.
4. Blobel, C. P. 2005. ADAMs: key components in EGFR signalling and development. Nat. Rev. Mol. Cell Biol. 6: 32–43.
5. Sahin, U., G. Weskamp, K. Kelly, H. M. Zhou, S. Higashiyama, J. Peschon,
D. Hartmann, P. Saftig, and C. P. Blobel. 2004. Distinct roles for ADAM10 and
ADAM17 in ectodomain shedding of six EGFR ligands. J. Cell Biol. 164: 769–
779.
6. Finigan, J. H., G. P. Downey, and J. A. Kern. 2012. Human epidermal growth
factor receptor signaling in acute lung injury. Am. J. Respir. Cell Mol. Biol. 47:
395–404.
7. Chalaris, A., N. Adam, C. Sina, P. Rosenstiel, J. Lehmann-Koch, P. Schirmacher,
D. Hartmann, J. Cichy, O. Gavrilova, S. Schreiber, et al. 2010. Critical role of the
disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. J. Exp. Med. 207: 1617–1624.
8. Franzke, C. W., C. Cobzaru, A. Triantafyllopoulou, S. Löffek, K. Horiuchi,
D. W. Threadgill, T. Kurz, N. van Rooijen, L. Bruckner-Tuderman, and
C. P. Blobel. 2012. Epidermal ADAM17 maintains the skin barrier by regulating
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
ADAM17 was initially identified as TNF-a–converting enzyme,
but during the last decades many more substrates have been
identified. This includes the cleavage of proinflammatory mediators such as TNF-a 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 antiinflammatory (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-a,
TGFa, 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, SM22ADAM17 contributes to edema formation, neutrophil recruitment,
and proinflammatory mediator release. SM22a 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-Adam172/2 mice in
LPS-induced acute pulmonary inflammation can be explained
to some extent by the reduced production of TNF-a (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 TGFa, amphiregulin, HB-EGF, EREG, and NRGs (5).
A strong link between TGFa and ADAM17 activity is already
indicated by the similar developmental phenotype of ADAM17and TGFa-deficient mice (3). The signaling via TGFa release
and EGFR activation is multifaceted. ADAM17-mediated TGFa
shedding and EGFR transactivation are indispensible for the
maintenance of the epithelium and its barrier function, as was also
shown in TGFa transgenic mice (7, 8, 13. In contrast, TGFa- 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 TGFa, 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.,
TGFa 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 TGFa 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
The Journal of Immunology
9.
10.
11.
12.
13.
14.
15.
17.
18.
19.
20.
21. Ishizuka, S., M. Yamaya, T. Suzuki, K. Nakayama, M. Kamanaka, S. Ida,
K. Sekizawa, and H. Sasaki. 2001. Acid exposure stimulates the adherence of
Streptococcus pneumoniae to cultured human airway epithelial cells: effects on
platelet-activating factor receptor expression. Am. J. Respir. Cell Mol. Biol. 24:
459–468.
22. Mizuno, T., J. Kawanokuchi, K. Numata, and A. Suzumura. 2003. Production
and neuroprotective functions of fractalkine in the central nervous system. Brain
Res. 979: 65–70.
23. Pruessmeyer, J., and A. Ludwig. 2009. The good, the bad and the ugly substrates
for ADAM10 and ADAM17 in brain pathology, inflammation and cancer. Semin.
Cell Dev. Biol. 20: 164–174.
24. Dreymueller, D., J. Pruessmeyer, E. Groth, and A. Ludwig. 2012. The role of
ADAM-mediated shedding in vascular biology. Eur. J. Cell Biol. 91: 472–485.
25. Long, C., M. R. Hosseinkhani, Y. Wang, P. Sriramarao, and B. Walcheck. 2012.
ADAM17 activation in circulating neutrophils following bacterial challenge
impairs their recruitment. J. Leukoc. Biol. 92: 667–672.
26. Koenen, R. R., J. Pruessmeyer, O. Soehnlein, L. Fraemohs, A. Zernecke,
N. Schwarz, K. Reiss, A. Sarabi, L. Lindbom, T. M. Hackeng, et al. 2009.
Regulated release and functional modulation of junctional adhesion molecule A
by disintegrin metalloproteinases. Blood 113: 4799–4809.
27. Ovrevik, J., M. Refsnes, A. I. Totlandsdal, J. A. Holme, P. E. Schwarze, and
M. Låg. 2011. TACE/TGF-a/EGFR regulates CXCL8 in bronchial epithelial
cells exposed to particulate matter components. Eur. Respir. J. 38: 1189–1199.
28. El-Bizri, N., L. Wang, S. L. Merklinger, C. Guignabert, T. Desai, T. Urashima,
A. Y. Sheikh, R. H. Knutsen, R. P. Mecham, Y. Mishina, and M. Rabinovitch.
2008. Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a
impairs cardiac contractility but protects against pulmonary vascular remodeling.
Circ. Res. 102: 380–388.
29. Reiss, L. K., U. Uhlig, and S. Uhlig. 2012. Models and mechanisms of acute lung
injury caused by direct insults. Eur. J. Cell Biol. 91: 590–601.
30. Dang, M., N. Armbruster, M. A. Miller, E. Cermeno, M. Hartmann, G. W. Bell,
D. E. Root, D. A. Lauffenburger, H. F. Lodish, and A. Herrlich. 2013. Regulated
ADAM17-dependent EGF family ligand release by substrate-selecting signaling
pathways. Proc. Natl. Acad. Sci. USA 110: 9776–9781.
31. Shea, P. R., K. Virtaneva, J. J. Kupko, III, S. F. Porcella, W. T. Barry,
F. A. Wright, S. D. Kobayashi, A. Carmody, R. M. Ireland, D. E. Sturdevant,
et al. 2010. Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus. Proc. Natl. Acad. Sci. USA 107:
4693–4698.
32. Liu, W., M. A. Volpe, K. Zscheppang, H. C. Nielsen, and C. E. Dammann. 2009.
ErbB4 regulates surfactant synthesis and proliferation in adult rat pulmonary
epithelial cells. Exp. Lung Res. 35: 29–47.
33. Hall, K. C., and C. P. Blobel. 2012. Interleukin-1 stimulates ADAM17 through
a mechanism independent of its cytoplasmic domain or phosphorylation at
threonine 735. PLoS One 7: e31600.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
16.
EGFR ligand-dependent terminal keratinocyte differentiation. J. Exp. Med. 209:
1105–1119.
Horiuchi, K., T. Kimura, T. Miyamoto, H. Takaishi, Y. Okada, Y. Toyama, and
C. P. Blobel. 2007. Cutting edge: TNF-alpha-converting enzyme (TACE/
ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock. J. Immunol. 179: 2686–2689.
Dreymueller, D., C. Martin, T. Kogel, J. Pruessmeyer, F. M. Hess, K. Horiuchi,
S. Uhlig, and A. Ludwig. 2012. Lung endothelial ADAM17 regulates the acute
inflammatory response to lipopolysaccharide. EMBO Mol. Med. 4: 412–423.
Arndt, P. G., B. Strahan, Y. Wang, C. Long, K. Horiuchi, and B. Walcheck. 2011.
Leukocyte ADAM17 regulates acute pulmonary inflammation. PLoS One 6:
e19938.
Le Cras, T. D., T. H. Acciani, E. M. Mushaben, E. L. Kramer, P. A. Pastura,
W. D. Hardie, T. R. Korfhagen, U. Sivaprasad, M. Ericksen, A. M. Gibson, et al.
2011. Epithelial EGF receptor signaling mediates airway hyperreactivity and
remodeling in a mouse model of chronic asthma. Am. J. Physiol. Lung Cell. Mol.
Physiol. 300: L414–L421.
Ishii, Y., S. Fujimoto, and T. Fukuda. 2006. Gefitinib prevents bleomycininduced lung fibrosis in mice. Am. J. Respir. Crit. Care Med. 174: 550–556.
Hundhausen, C., D. Misztela, T. A. Berkhout, N. Broadway, P. Saftig, K. Reiss,
D. Hartmann, F. Fahrenholz, R. Postina, V. Matthews, et al. 2003. The
disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage
of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion.
Blood 102: 1186–1195.
Reiss, L. K., A. Kowallik, and S. Uhlig. 2011. Recurrent recruitment manoeuvres
improve lung mechanics and minimize lung injury during mechanical ventilation
of healthy mice. PLoS One 6: e24527.
Schwarz, N., J. Pruessmeyer, F. M. Hess, D. Dreymueller, E. Pantaler,
A. Koelsch, R. Windoffer, M. Voss, A. Sarabi, C. Weber, et al. 2010. Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1.
Cell. Mol. Life Sci. 67: 4233–4248.
Weskamp, G., K. Mendelson, S. Swendeman, S. Le Gall, Y. Ma, S. Lyman,
A. Hinoki, S. Eguchi, V. Guaiquil, K. Horiuchi, and C. P. Blobel. 2010. Pathological neovascularization is reduced by inactivation of ADAM17 in endothelial
cells but not in pericytes. Circ. Res. 106: 932–940.
Zager, R. A., A. C. Johnson, S. Lund, and J. Randolph-Habecker. 2007. Toll-like
receptor (TLR4) shedding and depletion: acute proximal tubular cell responses
to hypoxic and toxic injury. Am. J. Physiol. Renal Physiol. 292: F304–F312.
Chesnutt, A. N., F. Kheradmand, H. G. Folkesson, M. Alberts, and
M. A. Matthay. 1997. Soluble transforming growth factor-alpha is present in the
pulmonary edema fluid of patients with acute lung injury. Chest 111: 652–656.
Finigan, J. H., J. A. Faress, E. Wilkinson, R. S. Mishra, D. E. Nethery, D. Wyler,
M. Shatat, L. B. Ware, M. A. Matthay, R. Mason, et al. 2011. Neuregulin-1human epidermal receptor-2 signaling is a central regulator of pulmonary epithelial permeability and acute lung injury. J. Biol. Chem. 286: 10660–10670.
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