Leukocyte ADAM17 Regulates Acute Pulmonary
Patrick G. Arndt1, Brian Strahan1, Yue Wang2, Chunmei Long2, Keisuke Horiuchi3, Bruce Walcheck2*
1Division of Pulmonary, Allergy and Critical Care Medicine, Department of Internal Medicine, University of Minnesota, St. Paul, Minnesota, United States of America,
2Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, United States of America, 3Department of Orthopedic Surgery, School of Medicine,
Keio University, Shinjuku-ku, Tokyo, Japan
The transmembrane protease ADAM17 regulates the release and density of various leukocyte cell surface proteins that
modulate inflammation, including L-selectin, TNF-a, and IL-6R. At this time, its in vivo substrates and role in pulmonary
inflammation have not been directly examined. Using conditional ADAM17 knock-out mice, we investigated leukocyte
ADAM17 in acute lung inflammation. Alveolar TNF-a levels were significantly reduced (.95%) in ADAM17-null mice
following LPS administration, as was the shedding of L-selectin, a neutrophil-expressed adhesion molecule. Alveolar IL-6R
levels, however, were reduced by only <25% in ADAM17-null mice, indicating that ADAM17 is not its primary sheddase in
our model. Neutrophil infiltration into the alveolar compartment is a key event in the pathophysiology of acute airway
inflammation. Following LPS inhalation, alveolar neutrophil levels and lung inflammation in ADAM17-null mice were overall
reduced when compared to control mice. Interestingly, however, neutrophil recruitment to the alveolar compartment
occurred earlier in ADAM17-null mice after exposure to LPS. This decrease in alveolar neutrophil recruitment in ADAM17-
null mice was accompanied by significantly diminished alveolar levels of the neutrophil-tropic chemokines CXCL1 and
CXCL5. Altogether, our study suggests that leukocyte ADAM17 promotes inflammation in the lung, and thus this sheddase
may be a potential target in the design of pharmacologic therapies for acute lung injury.
Citation: Arndt PG, Strahan B, Wang Y, Long C, Horiuchi K, et al. (2011) Leukocyte ADAM17 Regulates Acute Pulmonary Inflammation. PLoS ONE 6(5): e19938.
Editor: Marco Idzko, University Hospital Freiburg, Germany
Received December 31, 2010; Accepted April 15, 2011; Published May 16, 2011
Copyright: ? 2011 Arndt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by research funding HL61613, AI083521 (BW) and HL84201 (PGA) from the National Institutes of Health. No additional
external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Acute lung injury (ALI), along with its more severe form acute
respiratory distress syndrome (ARDS), affects <200,000 persons
annually in the United States with mortality rates still unexpect-
edly high [1–3]. Various events can incite ARDS, and the release
of pleiotropic inflammatory mediators such as TNF-a plays a key
role in the lung inflammation that occurs [4,5]. TNF-a release
activates leukocytes, endothelial cells, and parenchymal cells in the
lung, and induces the production of various neutrophil chemoat-
tractants [6–8]. Neutrophil infiltration into the alveolar airspace is
a critical event in the pathophysiology of airway inflammation.
These cells release, in part, various proteases and reactive oxygen
species that facilitate progressive lung injury [9–15]. Accordingly,
the identification of mechanisms that regulate pulmonary
inflammation, and specifically the recruitment of neutrophils and
the release of TNF-a, is critical for determining therapeutic targets
to lessen lung injury.
During the inflammatory response, various cell surface proteins
undergo ectodomain shedding, typically at a juxta-membrane site
that leads to the release of a soluble extracellular domain fragment.
A number of leukocyte determinants that undergo this regulated
proteolytic process have an important role in modulating
inflammation. A disintegrin and metalloproteinase-17
(ADAM17), originally referred to as TNF-a converting enzyme
(TACE) [17,18], plays a broad role in mediating ectodomain
shedding . Hence, we hypothesized that ADAM17 may have
an important regulatory function in pulmonary inflammation.
However, examining the role of ADAM17 in vivo is challenging, as
homozygous deletion of the Adam17 gene results in perinatal
lethality [20,21]. To overcome this limitation, we have generated
conditional ADAM17-null mice with an ADAM17 deficiency in
all leukocytes . These animals are viable and we show here
that a deficiency of leukocyte-expressed ADAM17 markedly alters
neutrophil infiltration into the lung with an overall diminution in
their recruitment to the alveolar compartment during acute lung
inflammation. We also address the relevance of L-selectin, IL-6R,
and TNF-a as in vivo substrates of leukocyte ADAM17 in the lung.
ADAM17 regulates L-selectin, TNF-a and IL-6R levels in
the lung after LPS exposure
To examine if leukocyte ADAM17 can regulate pulmonary
inflammation, we generated ADAM17-null mice [Adam17flox/DZn/
Vav-Cre] with an ADAM17 deficiency in leukocytes only. Similar
to radiation chimeric mice reconstituted with ADAM17-deficient
leukocytes that we have previously described [23,24], our
ADAM17-null mice were viable . Cleavage of the well
described ADAM17 substrate L-selectin was greatly impaired in
PLoS ONE | www.plosone.org1May 2011 | Volume 6 | Issue 5 | e19938
neutrophils, monocytes, and lymphocytes from ADAM17-null
mice following their overt activation in vitro when compared with
the same leukocyte subsets from control mice (Fig. 1A, B). Despite
ADAM17-deficient leukocytes expressing higher surface levels of
L-selectin than control leukocytes, their levels of L-selectin mRNA
were equivalent (Fig. 1C), indicating differential L-selectin
shedding and not gene expression as a mechanism for their
increased cell surface L-selectin levels. Neutrophil migration from
the circulation into the underlying tissue at sites of inflammation
results in L-selectin shedding . We observed that alveolar
neutrophils from ADAM17-null mice after LPS inhalation
expressed significantly higher levels of surface L-selectin than
alveolar neutrophils from control mice (Fig. 1D). Surface L-
selectin levels were 5.462.5-fold higher on alveolar neutrophils
from ADAM17-null mice than from control mice after LPS
challenge (mean 6 SD, n=7 mice in each group). In contrast, the
non-cleavable, cell surface adhesion molecule Mac-1 (CD11b/
CD18) was expressed at equivalent levels by alveolar neutrophils
from ADAM17-null and control mice following LPS instillation
(Fig. 1D), demonstrating that ADAM17 deficiency did not cause a
global up-regulation in the expression of cell surface molecules.
Soluble L-selectin levels were also significantly reduced in the
bronchoalveolar lavage (BAL) fluid of ADAM17-null mice when
compared to control mice (Fig. 1E).
Neutrophils and macrophages are a primary source of soluble
TNF-a upon the induction of inflammation [26,27], and
ADAM17 is a well described sheddase of TNF-a by these cells
[17,20,21,24]. We determined the levels of TNF-a in the BAL
fluid of LPS exposed ADAM17-null and control mice, and found
it to be greatly decreased (.95%) in ADAM17-null mice when
compared to levels seen in control mice at time points that
encompass its peak production after LPS challenge (Fig. 2A).
Despite the near ablation of soluble TNF-a production in the
alveolar compartment, the levels of TNF-a in the lung interstitial
compartment were diminished by only <40% and 25% at 2 and
8 hours, respectively, following LPS inhalation in ADAM17-null
mice (Fig. 2A), which may be due in part to ADAM17 expression
by non-hematopoietic, lung parenchymal cells.
ADAM17 has been implicated as a key sheddase of the IL-6R as
well . We observed that soluble IL-6R levels were also
significantly decreased in the alveolar compartment of ADAM17-
null mice after LPS exposure (Fig. 2B), though this did not occur to
nearly the same extent as TNF-a. For instance, at 2 hours after
LPS exposure, soluble IL-6R levels in the airspace were <25%
lower in ADAM17-null mice than in control mice (Fig. 2B). In the
interstitial compartment, however, we did not observe a
statistically significant difference in IL-6R levels in ADAM17-null
mice after LPS exposure (Fig. 2B). It should be noted that our lung
homogenizing buffer contained a detergent, and thus a portion of
the TNF-a and IL-6R detected in the lung homogenates may
represent membrane forms of these molecules. Taken together, the
results above suggest that ADAM17 is the primary sheddase of L-
selectin and TNF-a by alveolar leukocytes, but not of the IL-6R
during acute pulmonary inflammation.
Targeting leukocyte ADAM17 alters the time course and
levels of pulmonary neutrophil recruitment
Neutrophil recruitment into the alveolar airspace is pivotal for
inducing lung damage associated with an increase in mortality
. We compared the levels of neutrophil recruitment in the
alveolar compartment of ADAM17-null and control mice. We
have shown that ADAM17-null mice have similar leukocyte subset
proportions in their peripheral blood and circulating neutrophil
counts as control mice [22–24]. We observed, however, that
2 hours after exposure to aerosolized LPS, alveolar neutrophil
counts were significantly higher in ADAM17-null mice (Fig. 3A).
These results reveal that the significantly decreased levels of
alveolar TNF-a and IL-6R observed in ADAM17-null mice at this
time point, as discussed above, were not due to an attenuated
influx of neutrophils into the alveolar compartment. Interestingly,
we found that alveolar neutrophil counts were considerably lower
in ADAM17-null mice at 8 hours after LPS inhalation (Fig. 3A),
which represents the peak in alveolar neutrophil recruitment in
our model [15,29]. Alveolar macrophages, however, were not
significantly different in ADAM17-null and control mice at either
time point following LPS inhalation (Fig. 3B). In contrast to the
alveolar air spaces, neutrophil levels in the interstitial compart-
ment, as assessed by myeloperoxidase (MPO), did not differ
between the two groups of mice at 2 or 8 hours after LPS
challenge (Fig. 3C). These data indicate that the greatest effect of
leukocyte ADAM17 was on regulating neutrophil movement to
the alveolar compartment, and overall there was a marked
decrease in acute lung inflammation, as revealed by histopathology
Alveolar levels of neutrophil chemoattractants are
decreased after exposure to LPS in ADAM17-null mice
TNF-a has been reported to induce the expression of
chemokines that promote pulmonary neutrophil recruitment
[7,8]. As levels of TNF-a were reduced in the alveolar and
interstitial compartments of the lung after LPS exposure in
ADAM17-null mice (Fig. 2A), this suggested that LPS-induced
chemokine levels may also be diminished, thereby providing a
mechanism for the decrease in alveolar neutrophil recruitment in
ADAM17-null mice. To determine whether the production of
chemokines important for neutrophil recruitment into the alveolar
airspace is altered in ADAM17-null mice upon LPS inhalation, we
examined the levels of CXCL1 (KC), CXCL2 (MIP-2), CXCL5
(LIX) in the BAL fluid. We observed that the alveolar
concentrations of CXCL5 were significantly lower in ADAM17-
null mice than control mice at 2 hours, but not 8 hours, after LPS
instillation (Fig. 4). CXCL1 alveolar levels were also significantly
lower in ADAM17-null mice, but only at 8 hours after LPS
instillation (Fig. 4). In contrast to CXCL1 and CXCL5, alveolar
levels of CXCL2 were not significantly different in the two groups
of mice at 2 and 8 hours after LPS instillation (Fig. 4). Lung levels
of CXCL5 were modestly lower at 2 hours, but not 8 hours, after
LPS instillation in ADAM17-null mice, whereas lung levels of
CXCL1 and CXCL2 were found not to differ between ADAM17-
null and control mice (Fig. 4). The latter findings correspond with
the similar level of neutrophils in the interstitial compartment after
LPS exposure in the two groups of mice (Figure 3B).
In this study we report for the first time that ectodomain
shedding by leukocyte ADAM17 participates in regulating
neutrophil migration into the inflamed lung. Pulmonary neutro-
phil recruitment is a critical mechanism of pulmonary inflamma-
tory disorders such as ARDS [9,10,12–15]. Excessive neutrophil
recruitment into the alveolar compartment in particular has been
associated with worsening lung damage and increased mortality
. We observed in ADAM17-null mice, whose leukocytes lack
functional ADAM17, that neutrophil accumulation in the alveolar
compartment, although occurring earlier, was overall significantly
decreased compared to control mice following their exposure to
LPS. This same pattern of rapid but attenuated neutrophil
accumulation in the lung air spaces also occurred upon
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org2 May 2011 | Volume 6 | Issue 5 | e19938
Figure 1. ADAM17 regulates alveolar neutrophil shedding of L-selectin. A: Bone marrow-derived leukocytes from ADAM17-null (KO) and
control mice (wild-type) were treated with PMA or without (resting) for 30 minutes, as indicated, and then double-stained for surface expression of L-
selectin and the neutrophil marker Ly-6G. B: Peripheral blood leukocytes from ADAM17-null (KO) and control mice (WT) were activated with PMA for
30 minutes and relative surface L-selectin expression levels were compared on neutrophils (Ly-6GhighCD11bhigh), monocytes (Ly-6G2CD11bhigh), B
cells (B220+), and T cells (CD3+), as indicated in the overlay plots. C: Detection of mouse L-selectin mRNA levels by semiquantitative RT-PCR was
performed as described in the Materials and Methods. RNA was isolated from bone marrow neutrophils harvested from ADAM17-null and control
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org3 May 2011 | Volume 6 | Issue 5 | e19938
intratracheal injection of ADAM17-null mice with the Gram-
positive bacterial constituent lipoteichoic acid (Arndt et al.
unpublished results), a functional equivalent to LPS that instead
induces neutrophil infiltration into the lung via TLR2. Neutrophil
migration into the lung interstitial compartment, however, was not
significantly decreased in ADAM17-null mice following LPS
challenge, indicating that the targeting of leukocyte ADAM17
does not impair neutrophil infiltration into the interstitium from
the pulmonary blood vessels, but does alter their advancement to
the alveolar compartment.
ADAM17 expressed by neutrophils is a primary sheddase of L-
selectin and TNF-a [23,24]; however, its relevancy in vivo by
neutrophils recruited to the inflamed lung has not previously been
examined. We show that the conversion of membrane L-selectin
and TNF-a to their soluble forms was greatly impaired in the
inflamed lungs of ADAM17-null mice. In contrast to these
substrates, the sheddase activity regulating the cleavage of IL-6R is
in the early stages of being understood. The cleaved form of the
IL-6R binds secreted IL-6 and enhances its pleiotropic activity
through the activation of cells that lack expression of IL-6R via
trans-signaling through the ubiquitously expressed glycoprotein
130 . In vitro studies have implicated ADAM17 in IL-6R
shedding , yet the biological relevance of ADAM17 in this
process has not been directly investigated in vivo. Our results reveal
that in the context of lung inflammation, ADAM17 participates in
the shedding of IL-6R, but in contrast to TNF-a and L-selectin,
ADAM17 is not the primary sheddase of leukocyte IL-6R.
ADAM10 has also been reported to cleave the IL-6R , and
thus it will be interesting to directly investigate its role in IL-6R
shedding in a setting of acute lung inflammation.
It is well recognized that TNF-a induces an extensive array of
downstream events that further promote inflammation, and thus
the greatly diminished production of soluble TNF-a by ADAM17-
deficient leukocytes likely contributed to the reduced levels of
alveolar neutrophils as lung inflammation progressed after LPS
exposure. For instance, TNF-a signaling has been directly shown
to induce the production of neutrophil-tropic chemokines in the
alveoli following LPS exposure [7,8]. As CXCL1, CXCL2, and
CXCL5 are major chemokines directing neutrophil recruitment
into the murine lung [32–35], we examined their levels in the
alveolar compartment of the lung in ADAM17-null and control
mice. CXCL2 levels were found to be similar in the two groups of
mice. However, CXCL5 and CXCL1 levels in ADAM17-null
mice were significantly decreased at 2 hours and 8 hours,
respectively, following LPS instillation. CXCL5 is primarily
expressed by activated alveolar type II cells , and attenuated
early production of soluble TNF-a by resident and recruited
leukocytes in ADAM17-null mice may have delayed the activation
of these cells and the initial production of CXCL5 in the airspace.
CXCL1 is secreted by a variety of cells including neutrophils
[34,37–40], and the time frame of its reduction in alveolar levels in
ADAM17-null mice corresponded with the marked reduction in
alveolar neutrophil numbers as inflammation progressed following
LPS exposure. In contrast to the alveolar spaces, only CXCL5 was
decreased in the interstitial compartment of the lung in ADAM17-
null mice. The greater reduction in neutrophil-tropic chemokines
Figure 2. Decreased alveolar levels of TNF-a and IL-6R levels in ADAM17-null mice. Protein concentrations for TNF-a (A) and IL-6R (B) were
measured by ELISA on BAL fluid (left panels) and lung homogenates (right panels) from ADAM17-null (KO) and control mice (WT) after LPS inhalation.
Levels of TNF-a and soluble IL-6R in both groups of mice prior to LPS exposure were below the detection sensitivity of the ELISA (data not shown).
* p,0.01 KO versus WT; # p,0.05 KO versus WT. Results are expressed as mean 6 SD of at least 5 mice in each group at each time point. For the
lung homogenates, data is expressed as pg/mg protein to normalize for the total amount of homogenate protein ‘‘loaded’’ in the ELISA well.
mice. PCR amplification was performed for L-selectin and hypoxanthine phosphoribosyltransferase (HPRT), and the PCR products generated were
280 bp and 320 bp, respectively. Data are representative of three experiments using bone marrow neutrophils from separate mice. D: BAL leukocytes
from ADAM17-null (KO) and control (WT) mice 2 hours after LPS inhalation were triple-stained for surface expression of Ly-6G, F4/80, and L-selectin or
Mac-1. Leukocytes with the phenotypic profile Ly-6GhighF4/802were considered to be neutrophils. For panels A, B, and D, relative staining levels of
10,000 labeled cells were determined by flow cytometry. For all histogram plots, isotype-matched negative control antibody staining is indicated
(Isotype). The y axis=cell number and the x axis=Log 10 fluorescence. Data are representative of at least 5 mice in each group. E: BAL fluid levels of
soluble L-selectin from ADAM17-null (KO) and control mice (WT) either exposed to LPS for 8 hours or not (control). Results are expressed as mean 6
SD of 4 mice in each group at each time point.
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org4May 2011 | Volume 6 | Issue 5 | e19938
Figure 3. ADAM17 regulates alveolar neutrophil recruitment during acute pulmonary inflammation. A: ADAM17-null (KO) and control
(WT) mice were exposed to aerosolized LPS. At the times indicated, mice were sacrificed and BAL was performed with total numbers of neutrophils
determined. Results are expressed as mean 6 SD of 5 mice in each group at each time point. * p,0.001 KO versus WT. B: Total numbers of BAL
macrophages were determined in ADAM17-null (KO) and control (WT) mice at the indicated times following their exposure to aerosolized LPS. Results
are expressed as mean 6 SD of at least 3 mice in each group at each time point. C: MPO assays were performed on lung homogenates obtained from
ADAM17-null and control mice exposed to LPS. MPO is expressed as activity per gram of lung tissue. D: Lungs from LPS exposed, ADAM17-null (left
panel) and control mice (right panel) were isolated 8 hours after LPS exposure as described in the Materials and Methods. Lung sections (5 mm) were
stained with hematoxylin and eosin. Note the increased number of cellular nuclei and thickened alveolar walls in the right panel. The stained tissue
sections are shown at 1006magnification. Images are representative of 4 mice per group.
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org5May 2011 | Volume 6 | Issue 5 | e19938
in the alveolar compartment of ADAM17-null mice implies a
specific molecular process accounting for the lack of neutrophil
transepithelial migration into the alveolar air spaces at the later
time point in our study. Of additional interest is that alveolar
neutrophil levels in ADAM17-null mice were initially enhanced
upon inducing lung inflammation when compared to control mice.
The reasons for this are not clear at this time, but may be the result
of still other neutrophil chemoattractants or an enhanced ability of
ADAM17-deficient neutrophils to respond to them.
In conclusion, our study demonstrates for the first time that
leukocyte ADAM17 regulates acute lung inflammation by
modulating intra-alveolar neutrophil levels and the shedding of
IL-6R, L-selectin, and TNF-a. It is known that preventing TNF-a
activity can increase host susceptibility to infection, and thus it will
be important to determine the role of leukocyte ADAM17 in
pulmonary defense against bacterial infection. Of interest is that
we have recently reported that ADAM17-null mice demonstrate
enhanced host resistance, including decreased hematogenous
Figure 4. Leukocyte ADAM17 deficiency alters expression of the neutrophil chemoattractants CXCL1 (KC) and CXCL5 (LIX), but not
CXCL2 (MIP-2), in the lung following LPS exposure. Protein concentrations for CXCL5, CXCL1, and CXCL2 were measured by ELISA on BAL fluid
(left column) and lung homogenates (right column) from ADAM17-null (KO) and control (WT) mice after exposure to LPS for the indicated times.
Levels of the chemokines in both groups of mice prior to LPS exposure were below the detection sensitivity of the ELISA (data not shown). * p,0.01
KO versus WT; # p,0.05 KO versus WT. Results are expressed as mean 6 SD of at least 5 mice in each group at each time point. For the lung
homogenates, data is expressed as pg/mg protein to normalize for the total amount of homogenate protein ‘‘loaded’’ in the ELISA well.
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org6 May 2011 | Volume 6 | Issue 5 | e19938
spread of bacterial to the lung, during E. coli-mediated abdominal
Materials and Methods
Generation of conditional ADAM17 knock-out mice
Experimental procedures involving animals were approved by
the Animal Care and Use Committee of the University of
Minnesota and performed in accordance with the United States
Public Health Service Policy on Humane Care and Use of
Laboratory Animals and the Animal Welfare Act. Taceflox/floxmice
, TaceDZn/+mice (DZn represents a targeted deletion of exon
11 that encodes the catalytic active site of the ADAM17
metalloprotease domain, resulting in a lack of enzymatic activity)
, and Vav1-cre transgenic mice [41,42] have all been previously
described. Adam17 instead of Tace is the current gene symbol and
will be used for our mouse colony nomenclature. Vav1-cre trans-
genic mice, which directs Cre-recombinase activity specifically in
all hematopoietic cells , were crossed with Adam17DZn/+mice
to produce Adam17DZn/+/Vav-Cre mice. These mice, which are of
mixed genetic background (129Sv, C57Bl/6), were then crossed
with Adam17flox/floxmice to generate Adam17flox/DZn/Vav-Cre and
Adam17flox/DZnmice. This breeding scheme was used to avoid Cre
activity in the germ line causing conversion of the floxed allele to a
null allele and non-tissue specific Adam17 gene deletion in the
offspring. Littermate Adam17flox/DZnmice were used as controls
since there is no described haploinsufficiency by Adam17 .
Herein, the Adam17flox/DZn/Vav-Cre and Adam17flox/DZnmice will
be referred to as ADAM17-null and control mice, respectively.
ADAM17-null and control mice were distinguished by PCR and
functional screening. For the latter, we assessed shedding of the
ADAM17 substrate L-selectin by peripheral blood neutrophils,
monocytes, and lymphocytes, as previously described [22,23].
ADAM17-null mice demonstrating essentially complete abroga-
tion of L-selectin shedding by their leukocytes were used in this
Aerosolized model of LPS-induced acute lung
Mice 8–12 weeks of age were used for our studies. LPS [E. coli
O111:B4; 300 mg/ml in 0.9% saline (Sigma, St. Louis, MO)] was
administered by aerosolization (20 minutes exposure period).
Based on previous experiments, 2 and 8 hour time points were
chosen as they represent the peaks in cytokine/chemokine levels
and neutrophil influx, respectively [15,29]. Animals were anes-
thetized with Avertin (240 mg/kg per mouse IP) 30 minutes prior
to intratracheal inoculation. Mice were sacrificed by an overdose
of sodium pentobarbital (100 mg/kg), blood was collected, BAL
was performed, bone marrow isolated, and lungs were collected,
snap frozen, and stored at 280uC until further analysis, as
previously described [15,43]. Total cell counts by hemocytometer
and cell differentials of Wright stained cytocentrifuged samples
were performed on BAL fluid. Endotoxin free glassware and
plasticware were used in all experiments. MPO levels, as a
quantification of the number of neutrophils recruited to the lung
after exposure to LPS, were performed as we have previously
described [15,43]. For lung histology, mice did not undergo BAL
and instead lungs were isolated, inflated, and fixed in 3.7%
formalin overnight at room temperature as previously described
ELISA for CXCL1, CXCL2, CXCL5, soluble IL-6R, L-
selectin, and TNF-a were performed on BAL fluid and lung
homogenate supernatants using commercially available kits (R&D
Systems, Minneapolis, MN) following the manufacturer’s proto-
cols. Lungs were homogenized in PBS containing leupeptin
(10 mg/ml), aprotinin (10 mg/ml), PMSF (300 mM), and 1%
Triton X-100, centrifuged 10 minutes at 2,040g with supernatants
collected and frozen at 280uC until assayed. For normalization of
results for lung total protein, protein estimation on lung
homogenates was performed using the Bradford assay kit following
the manufacturer’s instructions (Thermo Scientific, Rockford, IL).
Cell labeling and flow cytometry
Leukocytes mice were stained with various mAbs for flow
cytometry analysis, as we have previously described [23,24].
Detection of mouse L-selectin mRNA levels by semiquantitative
RT-PCR was performed as described with modifications .
Bone marrow neutrophils were harvested from ADAM17-null and
control mice and total cellular RNA was isolated from 56106cells
using a Qiagen RNeasy Mini Kit along with RNase-Free DNase to
remove residual amounts of DNA, which were performed
according to the manufacturer’s instructions (Qiagen, Valencia,
CA). Reverse transcription and PCR were performed sequentially
using a Qiagen OneStep RT-PCR Kit and mouse L-selectin gene-
specific primers, as per the manufacturer’s instructions. PCR
amplification was performed using the following primers (59 to 39):
L-selectin, CATTCCTGTAGCCGTCATGG and AGGAG-
GAGCTGTTGGTCATG; hypoxanthine phosphoribosyltransfer-
ase (internal control), GTTGGATACAGGCCAGACTTTGTTG
and GAAGGGTAGGCTGGCCTATAGGCT, which do not
amplify genomic DNA. The PCR conditions consisted of 95uC
for 15 min and 30 cycles of 94uC 30 sec; 57uC 30 sec; 72uC
40 sec, and a final 72uC for 10 min. Thirty cycles were
determined to be below the plateau phase of amplification for
all primers (data not shown), giving an accurate reflection of the
relative starting levels of mRNA. PCR products were detected by
1.5% agarose gel electrophoresis.
Statistical analysis was performed using Prism software (Prism 4;
GraphPad, San Diego, CA) using ANOVA and student’s t test
where appropriate. A p value of ,0.05 was considered significant.
We thank Dr. Carl Blobel (Hospital for Special Surgery, Weill Medical
College of Cornell University) for providing the Adam17flox/floxmice, Drs.
Roy Black and Jacques Peschon (Amgen, Inc.) for providing the
Adam17DZn/+mice, and Dr. Dimitris Kioussis (National Institute for
Medical Research, London) for providing the Vav1-Cre transgenic mice.
Conceived and designed the experiments: BW PGA. Performed the
experiments: BW PGA BS YW CL. Analyzed the data: BW PGA BS YW.
Contributed reagents/materials/analysis tools: KH CL. Wrote the paper:
1. Goss CH, Brower RG, Hudson LD, Rubenfeld GD (2003) Incidence of acute
lung injury in the United States. Crit Care Med 31: 1607–1611.
2. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, et al. (2005)
Incidence and outcomes of acute lung injury. N Engl J Med 353: 1685–1693.
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org7May 2011 | Volume 6 | Issue 5 | e19938
3. Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, et al. (2009) Has Download full-text
mortality from acute respiratory distress syndrome decreased over time? A
systematic review. Am J Respir Crit Care Med 179: 220–227.
4. Bhatia M, Moochhala S (2004) Role of inflammatory mediators in the
pathophysiology of acute respiratory distress syndrome. J Pathol 202: 145–156.
5. Mukhopadhyay S, Hoidal JR, Mukherjee TK (2006) Role of TNFalpha in
pulmonary pathophysiology. Respir Res 7: 125.
6. Neumann B, Machleidt T, Lifka A, Pfeffer K, Vestweber D, et al. (1996) Crucial
role of 55-kilodalton TNF receptor in TNF-induced adhesion molecule
expression and leukocyte organ infiltration. J Immunol 156: 1587–1593.
7. Calkins CM, Heimbach JK, Bensard DD, Song Y, Raeburn CD, et al. (2001)
TNF receptor I mediates chemokine production and neutrophil accumulation in
the lung following systemic lipopolysaccharide. J Surg Res 101: 232–237.
8. Elizur A, Adair-Kirk TL, Kelley DG, Griffin GL, Demello DE, et al. (2008)
Tumor necrosis factor-alpha from macrophages enhances LPS-induced clara
cell expression of keratinocyte-derived chemokine. Am J Respir Cell Mol Biol
9. Abraham E, Carmody A, Shenkar R, Arcaroli J (2000) Neutrophils as early
immunologic effectors in hemorrhage- or endotoxemia-induced acute lung
injury. Am J Physiol Lung Cell Mol Physiol 279: L1137–1145.
10. Savov JD, Gavett SH, Brass DM, Costa DL, Schwartz DA (2002) Neutrophils
play a critical role in development of LPS-induced airway disease. Am J Physiol
Lung Cell Mol Physiol 283: L952–962.
11. Li Q, Park PW, Wilson CL, Parks WC (2002) Matrilysin shedding of syndecan-1
regulates chemokine mobilization and transepithelial efflux of neutrophils in
acute lung injury. Cell 111: 635–646.
12. Abraham E (2003) Neutrophils and acute lung injury. Crit Care Med 31:
13. Yang KY, Arcaroli JJ, Abraham E (2003) Early alterations in neutrophil
activation are associated with outcome in acute lung injury. Am J Respir Crit
Care Med 167: 1567–1574.
14. Hollingsworth JW, Chen BJ, Brass DM, Berman K, Gunn MD, et al. (2005) The
critical role of hematopoietic cells in lipopolysaccharide-induced airway
inflammation. Am J Respir Crit Care Med 171: 806–813.
15. Arndt PG, Young SK, Lieber JG, Fessler MB, Nick JA, et al. (2005) Inhibition of
c-Jun N-terminal kinase limits lipopolysaccharide-induced pulmonary neutrophil
influx. Am J Respir Crit Care Med 171: 978–986.
16. Garton KJ, Gough PJ, Raines EW (2006) Emerging roles for ectodomain
shedding in the regulation of inflammatory responses. J Leukoc Biol 79:
17. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, et al. (1997) A
metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from
cells. Nature 385: 729–733.
18. Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, et al. (1997) Cloning of a
disintegrin metalloproteinase that processes precursor tumour-necrosis factor-
alpha. Nature 385: 733–736.
19. Reiss K, Saftig P (2009) The ‘‘a disintegrin and metalloprotease’’ (ADAM) family
of sheddases: physiological and cellular functions. Semin Cell Dev Biol 20:
20. Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, et al. (1998) An
essential role for ectodomain shedding in mammalian development. Science 282:
21. Horiuchi K, Kimura T, Miyamoto T, Takaishi H, Okada Y, et al. (2007)
Cutting Edge: TNF-a-Converting Enzyme (TACE/ADAM17) Inactivation in
Mouse Myeloid Cells Prevents Lethality from Endotoxin Shock. J Immunol 179:
22. Long C, Wang Y, Herrera AH, Horiuchi K, Walcheck B (2010) In vivo role of
leukocyte ADAM17 in the inflammatory and host responses during E. coli-
mediated peritonitis. J Leukoc Biol 87: 1097–1101.
23. Li Y, Brazzell J, Herrera A, Walcheck B (2006) ADAM17 deficiency by mature
neutrophils has differential effects on L-selectin shedding. Blood 108:
24. Bell JH, Herrera AH, Li Y, Walcheck B (2007) Role of ADAM17 in the
ectodomain shedding of TNF-alpha and its receptors by neutrophils and
macrophages. J Leukoc Biol 82: 173–176.
25. Jutila MA, Rott L, Berg EL, Butcher EC (1989) Function and regulation of the
neutrophil MEL-14 antigen in vivo: Comparison with LFA-1 and Mac-1.
J Immunol 143: 3318–3324.
26. Grivennikov SI, Tumanov AV, Liepinsh DJ, Kruglov AA, Marakusha BI, et al.
(2005) Distinct and nonredundant in vivo functions of TNF produced by T cells
and macrophages/neutrophils: protective and deleterious effects. Immunity 22:
27. Noulin N, Quesniaux VF, Schnyder-Candrian S, Schnyder B, Maillet I, et al.
(2005) Both hemopoietic and resident cells are required for MyD88-dependent
pulmonary inflammatory response to inhaled endotoxin. J Immunol 175:
28. Chalaris A, Rabe B, Paliga K, Lange H, Laskay T, et al. (2007) Apoptosis is a
natural stimulus of IL6R shedding and contributes to the proinflammatory trans-
signaling function of neutrophils. Blood 110: 1748–1755.
29. Nick JA, Young SK, Arndt PG, Lieber JG, Suratt BT, et al. (2002) Selective
suppression of neutrophil accumulation in ongoing pulmonary inflammation by
systemic inhibition of p38 mitogen-activated protein kinase. J Immunol 169:
30. Taga T, Kishimoto T (1995) Signaling mechanisms through cytokine receptors
that share signal transducing receptor components. Curr Opin Immunol 7:
31. Matthews V, Schuster B, Schutze S, Bussmeyer I, Ludwig A, et al. (2003)
Cellular cholesterol depletion triggers shedding of the human interleukin-6
receptor by ADAM10 and ADAM17 (TACE). J Biol Chem 278: 38829–38839.
32. McColl SR, Clark-Lewis I (1999) Inhibition of murine neutrophil recruitment in
vivo by CXC chemokine receptor antagonists. J Immunol 163: 2829–2835.
33. Zhang XW, Wang Y, Liu Q, Thorlacius H (2001) Redundant function of
macrophage inflammatory protein-2 and KC in tumor necrosis factor-alpha-
induced extravasation of neutrophils in vivo. Eur J Pharmacol 427: 277–283.
34. D’Ambrosio D, Panina-Bordignon P, Sinigaglia F (2003) Chemokine receptors
in inflammation: an overview. J Immunol Methods 273: 3–13.
35. Jeyaseelan S, Chu HW, Young SK, Worthen GS (2004) Transcriptional
profiling of lipopolysaccharide-induced acute lung injury. Infect Immun 72:
36. Jeyaseelan S, Manzer R, Young SK, Yamamoto M, Akira S, et al. (2005)
Induction of CXCL5 during inflammation in the rodent lung involves activation
of alveolar epithelium. Am J Respir Cell Mol Biol 32: 531–539.
37. Wolpe SD, Sherry B, Juers D, Davatelis G, Yurt RW, et al. (1989) Identification
and characterization of macrophage inflammatory protein 2. Proc Natl Acad
Sci U S A 86: 612–616.
38. Bozic CR, Kolakowski LF, Jr., Gerard NP, Garcia-Rodriguez C, von Uexkull-
Guldenband C, et al. (1995) Expression and biologic characterization of the
murine chemokine KC. J Immunol 154: 6048–6057.
39. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L (1995) Functional
characterization of the rat chemokine KC and its importance in neutrophil
recruitment in a rat model of pulmonary inflammation. J Immunol 154:
40. Driscoll KE, Hassenbein DG, Howard BW, Isfort RJ, Cody D, et al. (1995)
Cloning, expression, and functional characterization of rat MIP-2: a neutrophil
chemoattractant and epithelial cell mitogen. J Leukoc Biol 58: 359–364.
41. de Boer J, Williams A, Skavdis G, Harker N, Coles M, et al. (2003) Transgenic
mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol
42. Travis MA, Reizis B, Melton AC, Masteller E, Tang Q, et al. (2007) Loss of
integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice.
Nature 449: 361–365.
43. Arndt PG, Young SK, Worthen GS (2005) Regulation of lipopolysaccharide-
induced lung inflammation by plasminogen activator Inhibitor-1 through a
JNK-mediated pathway. J Immunol 175: 4049–4059.
44. Bai A, Hu H, Yeung M, Chen J (2007) Kruppel-like factor 2 controls T cell
trafficking by activating L-selectin (CD62L) and sphingosine-1-phosphate
receptor 1 transcription. J Immunol 178: 7632–7639.
Role of Leukocyte ADAM17 in Lung Inflammation
PLoS ONE | www.plosone.org8 May 2011 | Volume 6 | Issue 5 | e19938