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Milk Fat Globule-Epidermal Growth
Factor-Factor 8 Attenuates Neutrophil
Infiltration in Acute Lung Injury via
Modulation of CXCR2
Monowar Aziz, Akihisa Matsuda, Weng-Lang Yang, Asha
Jacob and Ping Wang
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 189:393-402; Prepublished online 25 May
2012;
doi: 10.4049/jimmunol.1200262
http://www.jimmunol.org/content/189/1/393
The Journal of Immunology
Milk Fat Globule-Epidermal Growth Factor-Factor 8
Attenuates Neutrophil Infiltration in Acute Lung Injury via
Modulation of CXCR2
Monowar Aziz,1 Akihisa Matsuda,1 Weng-Lang Yang, Asha Jacob, and Ping Wang
A
cute lung injury (ALI) and its more severe form, acute
respiratory distress syndrome (ARDS), which are characterized by an excessive inflammatory response, remain
as considerable clinical challenges to the intensive care medicine (1). Infectious etiologies, such as sepsis, pneumonia, and gut
ischemia/reperfusion (I/R), are among the leading causes of ALI/
ARDS (2). Despite extensive research in this field, only a few
Center for Immunology and Inflammation, Feinstein Institute for Medical Research,
Manhasset, NY 11030; and Department of Surgery, Hofstra North Shore–LIJ School
of Medicine, Manhasset, NY 11030
1
M.A. and A.M. contributed equally to this work.
Received for publication January 20, 2012. Accepted for publication April 26, 2012.
This work was supported in part by National Institutes of Health Grants R01 GM
057468 and R33 AI 080536 (to P.W.).
M.A., A.M., W.-L.Y., and P.W. conceived and designed the experiments; M.A., A.M.,
and W.-L.Y. performed the experiments; M.A., A.M., W.-L.Y., and A.J. analyzed the
data; M.A., A.M., W.-L.Y., and A.J. wrote the paper; and P.W. supervised the project.
All authors read and approved the final manuscript.
Portions of this work were presented in an abstract at the 41st Critical Care Congress
of the Society of Critical Care Medicine, February 4–8, 2012, Houston, TX.
Address correspondence and reprint requests to Dr. Ping Wang, Department of Surgery, Hofstra North Shore–LIJ School of Medicine, Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mail address:
pwang@nshs.edu
Abbreviations used in this article: Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amido4-methylcoumarin; ALI, acute lung injury; AMC, 7-amino-4-methylcoumarin; ARDS,
acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; BMDN,
bone marrow-derived neutrophil; EGF, epidermal growth factor; GRK2, G proteincoupled receptor kinase 2; I/R, ischemia/reperfusion; i.t., intratracheal(ly); MFG-E8,
milk fat globule-epidermal growth factor-factor 8; PI, propidium iodide; RGD,
arginine-glycine-aspartate; rm, recombinant murine; WT, wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200262
therapeutic strategies for ALI/ARDS have emerged, and specific
options for treatment remain limited.
Neutrophil recruitment is critical to the pulmonary inflammatory
responses associated with ALI (3, 4). Activated neutrophils release proteolytic enzymes, such as elastase and myeloperoxidase
(MPO), and reactive oxygen species, including hydrogen peroxide
and superoxide. Excessive production of these agents not only
kills invaded pathogens, but it also engages in disruption of endothelial barrier functions and promotes extravascular host tissue
damage during uncontrolled inflammation (4, 5). Neutrophil infiltration into the lungs is mediated by a local production of chemokines released by macrophages as well as other cell types
in response to inflammation (6, 7). CXC chemokines, such as
IL-8, are elevated significantly in the bronchoalveolar lavage fluid
(BALF) of patients with ARDS, and increased IL-8 levels are
associated with increased neutrophil infiltration (8, 9). In rodents,
the IL-8 homolog, CINC-1/2, and MIP-2 regulate neutrophil
recruitment into the lungs of experimental ALI via chemokine
receptor CXCR2 (10–12). CXCR2 is a seven transmembrane type
G protein-coupled receptor whose expression, localization, and
function in polymorphonuclear leukocytes are tightly regulated
by the intracellular G protein-coupled receptor kinase 2 (GRK2).
Upon activation, GRK2 phosphorylates CXCR2 and causes receptor desensitization and internalization, leading to downregulation of neutrophil chemotaxis (13–15).
Milk fat globule-epidermal growth factor-factor 8 (MFG-E8),
a secretary glycoprotein, is composed of an N-terminal cleavable
signal peptide, followed by two epidermal growth factor (EGF)-like
domains, a proline-threonine–rich motif and two C-terminal discoidin domains resembling the sequences of blood coagulation
factors V and VIII (16). The second EGF domain contains an
arginine-glycine-aspartate (RGD) motif that enables it to bind avb3
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Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factorfactor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular
functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed
to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and
Mfge82/2 mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the
neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and
myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in
neutrophils were measured by flow cytometry. Following LPS challenge, Mfge82/2 mice exhibited extensive lung damage due to
exaggerated infiltration of neutrophils and production of TNF-a, MIP-2, and myeloperoxidase. An increased number of apoptotic
cells was trapped into the lungs of Mfge82/2 mice compared with WT mice, which may be due to insufficient phagocytosis of
apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2–mediated
chemotaxis revealed higher migration of neutrophils of Mfge82/2 mice than those of WT mice via increased surface exposures to
CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and
downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-av integrin Abs. These studies
clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic
potential for ALI. The Journal of Immunology, 2012, 189: 393–402.
394
Materials and Methods
i.t. LPS instillation. The protocol was approved by the Institutional
Animal Care and Use Committee of the Feinstein Institute for Medical
Research.
Lung tissue histology
Lung tissues were fixed in 10% formalin and embedded in paraffin. Tissue
blocks were sectioned at a thickness of 5 mm and stained with H&E.
Morphological changes were scored by an independent pathologist as absent (0), mild (+1), moderate (+2), or severe injury (+3) based on the
presence of exudates, hyperemia/congestion, neutrophilic infiltrates, intraalveolar hemorrhage/debris, and cellular hyperplasia (20). The sum of
scores of different animals was averaged.
In situ TUNEL assay. DNA breaks occur late in the apoptotic pathway and
can be determined by performing the TUNEL assay. The presence of
apoptotic cells in lung tissues was determined using a TUNEL staining kit
(Roche Diagnostics, Indianapolis, IN). Briefly, lung tissues were fixed in
10% phosphate-buffered formalin and were then embedded into paraffin and
sectioned at 5 mm following standard histology procedures. Lung sections
were dewaxed, rehydrated, and equilibrated in TBS. The sections were
then digested with 20 mg/ml proteinase K for 20 min at room temperature.
Following this, the sections were washed and incubated with a mixture
containing TdT and fluorescence-labeled nucleotides and examined under
a fluorescence microscope (Nikon Eclipse Ti-S, Melville, NY).
Caspase-3 enzyme activity assay. The caspase-3 activity in lung tissues was
assessed using a fluorimetric assay kit (Sigma-Aldrich), which is based on the
principles of hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3, resulting in the
release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety. In
brief, lung tissues were homogenized in liquid nitrogen, and equal weights of
powdered tissues (∼50 mg) were dissolved in 500 ml lysis buffer (10 mM
HEPES [pH 7.4], 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 2 mM
each EDTA and EGTA) and subjected to sonication on ice. Protein concentration was determined by a protein assay reagent (Bio-Rad, Hercules,
CA). Equal amounts of proteins in a 5 ml vol were added to the 100 ml assay
buffer (20 mM HEPES [pH 7.4], 5 mM DTT, 2 mM EDTA, and 0.1%
CHAPS) containing 10 mM Ac-DEVD-AMC substrate and the changes of
fluorescence intensity with time at 37˚C were measured at excitation (370
nm) and emission (450 nm) in a fluorometer (Synergy H1; BioTek, Winooski,
VT). A standard curve was generated using various concentrations of AMC as
standard. The results are expressed as mM AMC/min/g protein.
Isolation of BALF and cell counts
Mice were euthanized and the trachea was cannulated using a PE50 catheter.
PBS (1 ml) was infused into the lungs to collect the lavage fluid five times.
The number of total cells in BALF was counted with a hemacytometer. To
identify cell population, BALF aliquots were subjected to cytospin and
stained with a Differential Quik Stain Kit (Polysciences, Warrington, PA).
Alternatively, the percentage of neutrophils in BALF was determined by
gating with cell size and positive staining of Abs allophycocyanin-Ly6G
and FITC-CD11b (BD Biosciences, San Jose, CA) in flow cytometric
analysis.
Isolation of bone marrow-derived neutrophils
Bone marrow-derived neutrophils (BMDNs) were isolated as described by
Boxio et al. (28). Mice were euthanized and femurs from both hind legs
were removed. The distal tip of each edge was cut off and bone marrow
cells were isolated by flushing the femur with HBSS. Cell suspensions
were filtered through a nylon membrane and centrifuged at 1000 rpm for
10 min. Cell pellets were resuspended in HBSS and laid on a three-layer
Percoll gradient of 78, 69, and 52% (Amersham Pharmacia, Uppsala, Sweden),
followed by centrifugation at 3000 rpm for 30 min without braking. Cells
at the 69/78% interface were carefully removed and washed with cold
PBS. The identification and purity of isolated BMDNs, which were stained
with Abs allophycocyanin-Ly6G and FITC-CD11b, were examined by
flow cytometric analysis.
Experimental model
Male (25–30 g) age-matched wild-type C57BL/6J (Taconic, Albany, NY)
and Mfge82/2 mice (a gift of Dr. Shigekazu Nagata, Kyoto University,
Kyoto, Japan) were anesthetized with isoflurane and then instilled with
40 ml sterile saline (PBS) without or with 5 mg/kg body weight LPS
(Escherichia coli serotype O55:B5; Sigma-Aldrich, St. Louis, MO) via
intratracheal (i.t.) injection using a 28-gauge U-100 insulin syringe. Although the present model of LPS-induced ALI is nonlethal, it can significantly induce lung injury. The rmMFG-E8 (R&D Systems, Minneapolis,
MN) was administered i.p. at 20 mg/kg body weight dose 2 h before
MPO staining and activity assay
Paraffin-embedded lung tissue sections were incubated with rabbit anti-MPO
Abs (Abcam, Cambridge, MA), followed by incubation with biotinylated
anti-rabbit IgG. Staining was developed by Vectastain ABC reagent and a
diaminobenzidine kit (Vector Laboratories, Burlingame, CA). For the negative control, the primary Ab was substituted with normal rabbit IgG. To
determine MPO activity, tissues were homogenized in KPO4 buffer containing 0.5% hexa-decyl-trimethyl-ammonium bromide and incubated at
60˚C for 2 h, followed by centrifugation. The supernatant was diluted in a
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integrin of macrophages, whereas the discoidin domains facilitate
opsonization of the apoptotic cells via recognizing phosphatidylserine, thus promoting their engulfment by macrophages (16).
Expression of MFG-E8 is ubiquitously found in spleen, lungs, liver,
kidneys, intestine, and mammary glands (17), whereas a marked
decrease in its content is noted in various inflammatory diseases,
causing exaggerated inflammation and abnormal tissue homeostasis (17, 18). The cellular expression pattern of MFG-E8 reveals
its localization into the macrophages and dendritic, epithelial, and
fibroblast cells from various organs (17). In normal lungs, MFG-E8
is present in the alveolar interstitium and pulmonary vasculature,
which are largely comprised of epithelial and endothelial cells
and a few resident macrophages (19). In our previous studies, we
noticed significant decrease of MFG-E8 expression in the lungs
after gut I/R injury (20). This decrease might be due to the exaggerated activation of immune-reactive cells since MFG-E8 expression is negatively regulated by TLR-mediated pathways (21).
Among several features, cellular apoptosis is markedly noticed in
ALI (7), thus predicting a scavenging role of MFG-E8 to get rid of
the deleterious effects of apoptotic cells before undergoing secondary necrosis (18). Phagocytosis of apoptotic cells can indirectly
regulate the proinflammatory milieu by modulating the activation
of the potent transcription factor NF-kB (22). However, in immunereactive cells we recently demonstrated a direct anti-inflammatory
role of MFG-E8 by inhibiting NF-kB–mediated proinflammatory
cytokine production, regardless of its effect in phagocytosis (23–
25). Although the concepts of clearance of apoptotic cells and the
downregulation of NF-kB may greatly improve our understanding
of the protective roles of MFG-E8 against ALI, the underlying
mechanisms of resolving excessive neutrophil infiltration remain
unexplored. Recently, the synthetic peptide RGD has been shown to
attenuate lung neutrophil chemotaxis in ALI by recognizing avb3
integrin and modulating downstream signaling events (26). Because MFG-E8 has a binding affinity for avb3 integrin through its
RGD motif (16), we therefore consider an additional mechanism by
which MFG-E8 may attenuate neutrophil migration during ALI.
Although the immune homeostatic functions of MFG-E8 have
been demonstrated in macrophages, dendritic cells, and epithelial
cells (22–25, 27), its effect in polymorphonuclear leukocytes is
completely unknown. Considering our initial findings of exaggerated accumulations of neutrophils in lungs of Mfge82/2 mice, we
hypothesize that MFG-E8 is a crucial factor for controlling neutrophil migration in LPS-induced ALI. Pertaining to our hypothesis, we report that Mfge82/2 mice exhibit detrimental impact in
experimental ALI due to excessive neutrophil infiltration, proinflammatory cytokine production, and extensive tissue damage and
apoptosis, which can be resolved by treatment with recombinant
murine (rm)MFG-E8. We further clarify the pivotal roles of MFGE8 as avb3 integrin-mediated regulation of neutrophil migration by
modulating the surface expression of CXCR2 via GRK2-dependent
pathways. Importantly, the present research identifies an outstanding additional role by which MFG-E8 decreases neutrophil
infiltration into the lungs and ameliorates LPS-induced ALI.
MFG-E8 ATTENUATES NEUTROPHIL INFILTRATION
The Journal of Immunology
reaction solution, and DOD was measured at 460 nm to calculate MPO
activity.
Quantitative real-time PCR analysis
Total RNA was extracted from lung tissues by TRIzol reagent (Invitrogen,
Carlsbad, CA) and reverse-transcribed into cDNA using murine leukemia
virus reverse transcriptase (Applied Biosystems, Foster City, CA). A PCR
reaction was carried out in a 25 ml final volume containing 0.08 mmol each
forward and reverse primer, cDNA, and 12.5 ml SYBR Green PCR Master
mix (Applied Biosystems). Amplification was conducted in an Applied
Biosystems 7300 real-time PCR machine under the thermal profile of 50˚C
for 2 min and 95˚C for 10 min, followed by 45 cycles of 95˚C for 15 s and 60˚
C for 1 min. The level of mouse b-actin mRNA was used for normalization.
Relative expression of mRNA was calculated by the 22DDCt method, and
results were expressed as fold change in comparison with control group. The
sequence of primers used for this study are: TNF-a (NM_013693), forward,
59-AGACCCTCACACTCAGATCATCTTC-39, reverse, 59-TTGCTACGACGTGGGCTACA-39; MIP-2 (NM_009140), forward, 59-CATCCAGAGCTTGATGGTGA-39, reverse, 59-CTTTGGTTCTTCCGTTGAGG-39; MFGE8 (NM_008594), forward, 59- CGGGCCAAGACAATGACATC-39, reverse,
59-TCTCTCAGTCTCATTGCACACAAG-39; and b-actin (NM_007393), forward, 59-CGTGAAAAG ATG ACCCAGATCA-39, reverse, 59-TGGTACGACCAGAGGCATACAG-39.
Levels of TNF-a and MIP-2 in the lung tissues, BALF, and cell culture
supernatants were measured using an ELISA kit specific for mouse TNF-a
(BD Biosciences) and MIP-2 (R&D Systems). The assay was carried out
according to the instructions provided by the manufacturers.
Western blot analysis
Lung tissues were homogenized and lysed in RIPA buffer (10 mM Tris-HCl
[pH 7.5], 120 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and
0.1% SDS) containing a protease inhibitor mixture (Roche Diagonstics,
Indianapolis, IN). Protein concentration was determined by a Bio-Rad
protein assay reagent. Lysates were electrophoresed on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes
were blocked with 5% nonfat dry milk in TBST buffer (0.1% Tween 20, 20
mM Tris-HCl [pH 7.5], and 140 mM NaCl) and incubated with primary Ab
against MFG-E8 (MBL International, Nagoya, Japan), followed by secondary Ab-HRP conjugate and detected using chemiluminescence (GE
Healthcare, Buckinghamshire, U.K.) and autoradiography. The immunoblot
was reprobed with anti–b-actin Abs as loading control.
Results
MFG-E8 deficiency augments pulmonary inflammation and
injury induced by LPS
To identify whether MFG-E8 played a role in ALI, we compared
the inflammatory parameters between WT and Mfge82/2 mice
subjected to i.t. injection of LPS. Within 4 h after LPS instillation,
robust induction of TNF-a was noted in the lungs of WT and
Mfge82/2 mice (Fig. 1A, 1B). However, the TNF-a mRNA and
protein levels in Mfge82/2 mice after 4 h LPS treatment were
found to be 1.50- and 1.45-fold higher, respectively, than those in
WT mice. Similar trends were also noted at 24 h after LPS exposure, where the Mfge82/2 mice showed 1.62- and 1.86-fold
higher amounts of TNF-a mRNA and proteins, respectively, than
did WT mice (p , 0.05). Moreover, the TNF-a levels in the
BALF collected from Mfge82/2 mice were significantly higher
than those from WT mice at 4 and 24 h (Fig. 1C). The histological
images of the lung tissues at 24 h after LPS instillation represented
increased alveolar congestion, exudates, interstitial and alveolar
neutrophilic infiltrates, intra-alveolar capillary hemorrhages, and
extensive damage of epithelial architecture in Mfge82/2 mice as
compared with the WT counterparts (Fig. 2A). These changes
were reflected in a higher lung tissue injury score in Mfge82/2
mice than in WT mice (10.9 versus 7.6; p , 0.05; Fig. 2B).
MFG-E8 deficiency leads to increased neutrophil infiltration to
the lungs
To further validate the neutrophil infiltration as observed in the
histological analysis (Fig. 2), we first carried out qualitative assessment of MPO, a marker of infiltrating granulocytes in lung
Flow cytometric analysis
To examine surface CXCR2 expression, BMDNs with various treatments
were stained with propidium iodide (PI) and Abs FITC-CD11b, allophycocyanin-Ly6G, and PerCP/Cy5.5-CXCR2 (BioLegend, San Diego,
CA) and subjected to FACSCalibur (BD Biosciences). CXCR2 levels were
analyzed in the cell population with PI2 CD11b+Ly6G+. To examine
intracellular GRK2 expression, cells were first stained with appropriate
fluorescence Abs to detect cell surface markers and then fixed and permeabilized with IntraPrep (Beckman Coulter, Fullerton, CA), followed by
staining with PE-GRK2 Abs (Abcam). After washing, the stained cells were
subjected to FACSCalibur. Data were analyzed by FlowJo software (Tree
Star, Ashland, OR) with 15,000 events per sample. Isotype controls were
used for all the samples.
In vitro neutrophil migration assay
The migration assays were conducted in a modified 24-well (3.0 mm)
Boyden chamber (BD Biosciences). Cells (3 3 105) were plated in the
upper well, and medium with 1 ng/ml rmMIP-2 (R&D Systems) was
placed in the lower well as a chemotactic stimulus. After 2 h incubation,
the upper surface of the filter was swabbed with cotton to remove nonmigratory cells. Migrated cells were fixed with 10% formalin and stained
with PI. Five random microscopic fields per well were counted. For
blocking experiments, cells were incubated with rmMFG-E8, anti–MFGE8 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-av neutralizing
Abs (EMD Biosciences, La Jolla, CA) for 2 h prior to plating.
Statistical analysis
All data are expressed as means 6 SE and compared by one-way ANOVA
and a Student–Newman–Keuls test. A Student t test was used when only
two groups were compared. Differences in values were considered significant with p , 0.05.
FIGURE 1. Assessment of TNF-a in lungs and BALF after ALI. WT and
Mfge82/2 mice were subjected to i.t. injection of LPS (5 mg/kg). At 4 and 24 h
after LPS instillation, lung tissues and BALF were collected. (A) The levels of
TNF-a mRNA in lungs were determined by real-time PCR. Results are normalized by b-actin as an internal control and are expressed as fold induction in
comparison with sham WT mice. The protein levels of TNF-a in (B) lungs and
(C) BALF were determined by ELISA. Data are expressed as means 6 SE (n =
5 mice/group). *p , 0.05 versus WT sham, #p , 0.05 versus WT LPS.
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Measurements of TNF-a and MIP-2 proteins
395
396
MFG-E8 ATTENUATES NEUTROPHIL INFILTRATION
tissues, by immunostaining, which revealed a stronger intensity of
MPO staining in Mfge82/2 mice than that in WT mice (Fig. 3A).
After quantitation by MPO activity assay, we noticed that its activity in Mfge82/2 mice was 1.85-fold higher than that in WT
mice after 24 h LPS instillation (Fig. 3B). We next analyzed the
cells isolated from BALF, which revealed no significant increase
in their numbers in either WT or Mfge82/2 mice at 4 h after LPS
instillation (Fig. 3C). However, at 24 h, the numbers of total cell
counts from WT mice reached 3.1 6 0.27 3 106 cells, whereas it
was 5.8 6 0.45 3 106 cells in Mfge82/2 mice (p , 0.05; Fig. 3C).
The major cell type in BALF of sham mice was alveolar macrophages, whereas neutrophils were predominantly found in both
WT and Mfge82/2 mice after ALI (Fig. 3D). Consistent with the
cytospin results, BALF isolated from WT and Mfge82/2 mice
after 24 h LPS instillation contained ∼80% neutrophils as determined by flow cytometry (data not shown). The number of neutrophil counts in Mfge82/2 mice was 2.0-fold higher than that in
WT mice at 24 h after LPS instillation (Fig. 3E). Furthermore, we
detected a significant increase in total protein levels in BALF,
which emerged to be comparatively higher in Mfge82/2 mice than
in WT mice with 24 h ALI (16.4 6 0.86 versus 11.6 6 0.85 mg/
ml; p , 0.05; Fig. 3F).
Accumulation of apoptotic cells in lungs after ALI
MFG-E8 was initially identified as a factor for engulfment of apoptotic cells by professional phagocytes, and its deficiency led to
the development of autoimmune disease (16, 27). In this study, we
carried out TUNEL assay in lung tissues, which revealed a significant increase in the number of apoptotic cells in Mfge82/2
mice as compared with WT mice after ALI (Fig. 4A, 4B), reflecting inadequate clearance of apoptotic cells by the phagocytes.
Additionally, following ALI, we noticed increased activation of
caspase-3 in the lungs of Mfge82/2 mice, which led to a greater
increase in cellular apoptosis in Mfge82/2 mice than in WT
counterparts (Fig. 4C). Collectively, these findings demonstrated
that the higher amounts of apoptotic cells in the lungs of Mfge82/
2
mice were due to reduced phagocytosis and/or an increased rate
of apoptosis mediated by the activation of caspase-3.
Supplement of rmMFG-E8 attenuates neutrophil infiltration to
the lungs during ALI
At 4 h after LPS instillation, MFG-E8 mRNA and protein levels in
the lungs decreased by 42 and 57%, respectively, compared with
FIGURE 3. Neutrophil infiltration to the lungs after ALI. WT and
Mfge82/2 mice were injected i.t. with LPS (5 mg/kg). After LPS instillation, lung tissues and BALF were collected. (A) Representative images of
lung tissues (24 h after LPS instillation) immuostained with MPO at
original magnification of 3100 and 3400 (inset). Scale bars, 100 mm. (B)
MPO enzymatic activity in lungs was determined at 24 h after LPS instillation. (C) Total cell numbers in BALF isolated at 4 and 24 h after LPS
instillation were counted with a hemacytometer. (D) Representative cytospin images of BALF stained with a Differential Quik Stain Kit. Yellow
arrows point to alveolar macrophages; white arrows point to neutrophils.
Original magnification 3100. (E) The number of neutrophils in BALF
isolated at 24 h after LPS instillation was determined by the ratio of cell
population in cytospin. N.D., not detectable. (F) The protein content in
BALF isolated at 24 h after LPS instillation was measured by a Bio-Rad
DC protein assay reagent. Data are expressed as means 6 SE (n = 5 mice/
group). *p , 0.05 versus WT sham, #p , 0.05 versus WT LPS.
the WT sham controls (Fig. 5). Although at 24 h MFG-E8 expression was rebounded, it was still significantly lower than the
WT sham controls (Fig. 5). Considering this fact, we sought to
determine the effect of rmMFG-E8 pretreatment to WT mice prior
to induction of ALI as a therapeutic regimen to salvage the deficits
of endogenous MFG-E8 that occur during ALI. Interestingly, the
numbers of total cells and neutrophil counts in BALF of rmMFGE8–pretreated WT mice were significantly lower than those in WT
mice without rmMFG-E8 treatment after LPS instillation (Fig. 6).
Based on this finding, we proposed that the excess in neutrophil
infiltration into the lungs was due to decreased production of
endogenous MFG-E8 during ALI, which could be ameliorated by
exogenous treatment of rmMFG-E8.
Exaggerated production of MIP-2 and TNF-a in lungs, BALF,
and alveolar macrophages of Mfge82/2 mice during
inflammation
After observing the excessive neutrophil infiltration in Mfge82/2
mice, we then examined the levels of MIP-2, a critical chemokine
responsible for neutrophil chemotaxis. MIP-2 mRNA and protein
levels in Mfge82/2 mice were 2.2- and 2.1-fold higher, respec-
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FIGURE 2. Histological assessment of lung tissue damage after ALI.
WT and Mfge82/2 mice were subjected to i.t. injection of LPS (5 mg/kg). At
24 h after LPS instillation, lung tissues were fixed and stained with H&E.
(A) Representative histological images at original magnification of 3100
and 3400 (inset). Scale bars, 100 mm. (B) Tissue injury was scored based on
the presence of exudates, hyperemia/congestion, neutrophilic infiltrates,
intra-alveolar hemorrhage/debris, and cellular hyperplasia. Data are
expressed as means 6 SE (n = 5 mice/group). *p , 0.05 versus WT LPS.
The Journal of Immunology
397
tively, than those in WT mice at 4 h, and 2.4- and 2.3-fold higher
at 24 h (p , 0.05; Fig. 7A, 7B). Similarly, the MIP-2 levels in
BALF from Mfge82/2 mice were significantly higher than those
from WT mice at 4 and 24 h (Fig. 7C). Because macrophages are
one of the major cell types for producing chemokines and cytokines, we therefore harvested alveolar macrophages from BALF of
sham mice and assessed MIP-2 and TNF-a levels in LPS-treated
conditions. After 4 h exposure to LPS, the MIP-2 mRNA and
protein levels were increased significantly in alveolar macrophages
from Mfge82/2 mice, being 1.8- and 1.9-fold higher than those
from WT mice (p , 0.05; Fig. 7D, 7E). Similarly, TNF-a mRNA
and protein levels in alveolar macrophages from Mfge82/2 mice
were also significantly higher than those from WT mice after LPS
stimulation (Fig. 7F, 7G).
MFG-E8 inhibits neutrophil migration through regulating
CXCR2 expression
We further examined whether MFG-E8 had a direct effect on the
neutrophil migration by isolating neutrophils from bone marrow of
WT and Mfge82/2 mice. The number of migrated neutrophils of
Mfge82/2 mice was 1.7-fold higher than that of WT mice (Fig.
8A). CXCR2 is the putative receptor expressed on neutrophils for
MIP-2–dependent chemotaxis (10). By using flow cytometric
analysis, we observed that CXCR2 surface levels of MFG-E8–
deficient neutrophils (CD11b+Ly6G+) were 30% higher than those
of neutrophils from WT mice (Fig. 8B). To validate the role of
MFG-E8 in regulating neutrophil migration, BMDNs were treated
with rmMFG-E8 before applying the migration assay. As shown in
Fig. 8C, the number of migrated cells in rmMFG-E8–treated
BMDN was reduced by 40% compared with vehicle-treated BMDNs.
Furthermore, cotreatment of anti–MFG-E8 neutralizing Abs with
rmMFG-E8 abrogated the functions of rmMFG-E8 for reducing
the migration of BMDNs, hence becoming comparable to the
vehicle control (Fig. 8C). Correspondingly, the rmMFG-E8–treated
BMDNs had a lower CXCR2 expression compared with the
vehicle control, and the reduction of CXCR2 expression was
rescued by cotreatment of anti–MFG-E8 Abs (Fig. 8D). Intracellular GRK2 is a major determinant for surface CXCR2 flip-flop in
neutrophils, whose activation leads to desensitization of CXCR2,
resulting in its intracellular translocation, and negative regulation
of the neutrophil migration (29–31). GRK2 expression in BMDNs
was increased by 44% after rmMFG-E8 treatment, compared with
the vehicle control, whereas cotreatment of anti–MFG-E8 Abs diminished such induction of GRK2 expression (Fig. 8E).
MFG-E8 interacts with avb3 integrin for inhibition of
neutrophil migration
It has been well characterized that MFG-E8 can bind avb3 integrin
in immune cells through its N-terminal domain (16). To examine
the utilization of avb3 integrin in MFG-E8–mediated suppression
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FIGURE 4. Infiltration of apoptotic cells
into the lungs after ALI. LPS (5 mg/kg)-induced experimental ALI was generated in
WT and Mfge82/2 mice, and at 24 h after
LPS exposure lung tissues were harvested.
(A) Formalin-fixed and paraffin-embedded
lung tissue sections were stained with FITCTUNEL (green) and counterstained with
DAPI (blue). Representative images at 3100
original magnifications are shown. Scale bars,
100 mm. (B) Green fluorescent TUNELpositive apoptotic cells were counted at five
random fields in a blinded fashion, and the
average numbers of cells per field are
shown. (C) Lung tissue homogenates were
used for caspase-3 fluorometric assay using
Ac-DEVD-AMC as substrate. The results
were generated by plotting the sample values to a multipoint standard curve. Data are
expressed as means 6 SE (n = 3). *p , 0.05
versus WT sham, #p , 0.05 versus WT LPS.
398
of neutrophil migration, anti-av integrin neutralizing Abs were
applied (32). As shown in Fig. 9A, BMDNs pretreated with antiav integrin Abs abrogated the functions of MFG-E8–mediated
inhibition of their migration. We further demonstrated that the
pretreatment of BMDNs with anti-av integrin Abs blocked the
FIGURE 7. Effects of LPS on MIP-2 and TNF-a expression in lungs,
BALF, and alveolar macrophages. (A–C) WT and Mfge82/2 mice were
subjected to i.t. injection of LPS (5 mg/kg). At 4 and 24 h after LPS instillation, lung tissues and BALF were collected. (A) The levels of MIP-2
mRNA in lungs were determined by real-time PCR. Results are normalized
by b-actin as an internal control and are expressed as fold induction in
comparison with sham WT mice. The protein levels of MIP-2 in (B) lungs
and (C) BALF were determined by ELISA. Data are expressed as means 6
SE (n = 5 mice/group). *p , 0.05 versus WT sham; #p , 0.05 versus WT
LPS. (D–G) Alveolar macrophages isolated from normal WT and Mfge82/2
mice were cultured and stimulated with LPS (10 ng/ml). After 4 h, total
RNA isolated from cells and culture supernatants was used to determine (D,
E) MIP-2 and (F, G) TNF-a mRNA and extracellular protein levels by realtime PCR and ELISA, respectively. Data are expressed as means 6 SE (n =
5 independent experiments). *p , 0.05 versus WT control, #p , 0.05 versus
WT plus LPS.
effects of rmMFG-E8–mediated downregulation of CXCR2 and
upregulation of GRK2 (Fig. 9B, 9C). Collectively, these features
clearly demonstrated the critical roles of MFG-E8 for GRK2dependent downregulation of surface CXCR2 expression in neutrophils through avb3 integrin-mediated pathway.
Discussion
FIGURE 6. Effects of rmMFG-E8 administration on neutrophil infiltration in lungs after ALI. WT mice were i.p. injected with rmMFG-E8 (20
mg/kg) 2 h prior to LPS (5 mg/kg) instillation. At 24 h after LPS instillation, BALF was collected. (A) Total cell numbers in the BALF were
counted with a hemacytometer. (B) The number of neutrophils in the
BALF was determined by the ratio of cell population in the cytospin. Data
are expressed as means 6 SE (n = 5 mice/group). *p , 0.05 versus sham,
#
p , 0.05 versus LPS. N.D., Not detectable.
In this study, we demonstrated the novel mechanism of MFG-E8 for
regulating chemokine-mediated neutrophil migration in an LPSinduced murine model of ALI, which revealed more severe lung
injury in MFG-E8–deficient mice than in WT counterparts. This
observation is consistent with our previous study showing the
beneficial effects of MFG-E8 for attenuating lung injury induced
by intestinal I/R (20). In this study, we noticed exaggerated infiltration of neutrophils into the lungs of Mfge82/2 mice, as
confirmed by increased levels of MPO in interstitial tissues, as
well as neutrophil numbers and protein contents in BALF in
comparison with WT mice. We further identified the potential
roles of MFG-E8 in inhibiting migration of neutrophils by com-
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FIGURE 5. Endogenous expression of MFG-E8 in lungs following ALI.
WT mice were injected with LPS (5 mg/kg) i.t., and after 4 and 24 h lung
tissues were collected. (A) Total RNA isolated from lung tissues was
subjected to real-time PCR to determine MFG-E8 mRNA levels. (B) Total
protein extracts isolated from lung tissues were subjected to Western
blotting to determine MFG-E8 protein levels. Results are normalized with
b-actin as an internal control and are expressed as fold induction in
comparison with sham. Data are expressed as means 6 SE (n = 3 mice/
group). *p , 0.05 versus sham.
MFG-E8 ATTENUATES NEUTROPHIL INFILTRATION
The Journal of Immunology
399
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FIGURE 8. Inhibition of BMDN migration by MFG-E8 is mediated through regulation of CXCR2 and GRK2. (A and B) BMDNs were isolated from WT
and Mfge82/2 mice. (A) BMDNs were placed in the upper well of a modified Boyden chamber for migration assays. The media containing rmMIP-2 (1 ng/
ml) was placed in the lower wells as chemotactic stimulus. After 2 h, migrated cells were fixed and stained with PI. Representative images of migrated
BMDNs are shown. Five random microscopic fields per well were counted. Original magnification 3100. (B) CXCR2 expression in BMDNs was analyzed
by flow cytometry. Granulocytes were gated according to forward and side scatter dot plots, followed by identification of neutrophils with staining of FITCCD11b and allophycocyanin-Ly6G Abs. Representative histograms of expression intensity of CXCR2 in CD11b+Ly6G+ cells stained with PerCP/Cy5.5CXCR2 Abs are shown. The average of mean fluorescence intensities (MFI) for CXCR2 in BMDNs is plotted. Data are expressed as means 6 SE (n = 5
mice/group). *p , 0.05 versus WT. (C–E) BMDNs isolated from WT mice were treated with isotype Abs (1 mg/ml) plus rmMFG-E8 (500 ng/ml) or anti–
MFG-E8 Abs (1 mg/ml) plus rmMFG-E8 (500 ng/ml) for 2 h before analysis. (C) BMDNs with various treatments were subjected to a migration assay.
Representative images of migrated BMDN with PI staining are shown. Five random microscopic fields per well were counted. Original magnification
3100. (D) CXCR2 expression in BMDNs with various treatments was analyzed by flow cytometry. (E) Intracellular GRK2 expression in BMDNs with
various treatments was analyzed by flow cytometry. Representative histograms of expression intensity of GRK2 in CD11b+Ly6G+ cells stained with PEGRK2 Ab are shown. The average of MFI for GRK2 in BMDNs is plotted. Data are expressed as means 6 SE (n = 5 independent experiments). *p , 0.05
versus PBS control, #p , 0.05 versus isotype Ab plus rmMFG-E8.
paring the BMDNs isolated from WT and Mfge82/2 mice and the
effect of rmMFG-E8 administration by modulating the surface
exposures of CXCR2 via GRK2-dependent pathways. Because
avb3 integrin is a putative receptor of MFG-E8 (16), we finally
revealed the novel mechanism of MFG-E8 for regulating neutrophil migration through recognizing avb3 integrin.
400
MFG-E8 ATTENUATES NEUTROPHIL INFILTRATION
FIGURE 9. MFG-E8–mediated inhibition of BMDN migration is through
avb3 integrin. BMDNs isolated from WT mice were treated with isotype
Abs (1 mg/ml) plus rmMFG-E8 (500 ng/ml) or anti-av integrin Abs (1 mg/ml)
plus rmMFG-E8 (500 ng/ml) for 2 h before analysis. (A) BMDNs with various treatments were subjected to a migration assay. Representative images
of migrated BMDNs with PI staining are shown. Five random microscopic
fields per well were counted. Original magnification 3100. (B) CXCR2 and
(C) GRK2 expression in BMDNs with various treatments was analyzed by
flow cytometry. Representative histograms of expression intensity of CXCR2
and GRK2 in CD11b+Ly6G+ cells are shown. The averages of mean fluorescence intensities (MFI) for CXCR2 and GRK2 in BMDN are plotted. Data
are expressed as means 6 SE (n = 5 independent experiments). *p , 0.05
versus PBS control, #p , 0.05 versus isotype Abs plus rmMFG-E8.
We previously demonstrated the roles of MFG-E8 in promoting
phagocytosis of apoptotic cells as one of the mechanisms of
ameliorating inflammation in animal models of sepsis, renal, and
FIGURE 10. Hypothesis scheme. Intratracheal LPS exposure triggers
MIP-2 expression in lungs, which serves as a potent chemoattractant for
neutrophil migration into the lungs by recognizing CXCR2, causing
widespread inflammation and tissue damage. In neutrophils, rmMFG-E8
recognizes avb3 integrin and then generates downstream signaling for
intracellular GRK2 activation, which ultimately downregulates the surface
CXCR2 expression, hence attenuating the MIP-2–dependent neutrophil
migration during LPS-induced ALI.
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gut I/R (18, 20, 24). We therefore monitored the well-characterized function of MFG-E8 for engulfment of apoptotic cells to
maintain the homeostatic balance and observed increased numbers
of apoptotic cells in lung tissues of Mfge82/2 mice after ALI (Fig.
4). We further noticed significant induction of caspase-3 activity in
Mfge82/2 mice as compared with the WT animals (Fig. 4), suggesting the increased occurrence of apoptosis in lung tissues following ALI. However, exaggerated infiltration of neutrophils into
the lungs is a hallmark of ALI. The initial cascade of the development of ALI is the promotion of neutrophils into the lung tissues,
which release inflammatory mediators, proteases, and reactive oxygen species to cause inflammation and epithelial cell apoptosismediated lung damage (33). Moreover, substantial research demonstrated that increased rates of epithelial cell death and decreased
rates of activated neutrophil apoptosis in lungs are the two potentially important pathological mechanisms of ALI (33). Uncontrolled migration of neutrophils due to ALI deteriorates the disease
status, whereas reducing their contents improves lung integrity.
Therefore, blocking the neutrophil migration to the lungs by MFGE8 to attenuate ALI precedes phagocytosis of apoptotic cells.
Correspondingly, we recently demonstrated reduced contents of
MPO in the lung tissues of rmMFG-E8–treated animals with gut I/R
injuries (20). Hence, delineating the direct effects of MFG-E8 on
regulation of neutrophil migration could be the dominant mechanism for attenuating ALI.
The therapeutic strategy for ALI is attained by attenuating
neutrophil chemotaxis toward the inflammatory sites. CXCR2 expressed on the surface of neutrophils functions as a sensor to lead
neutrophils at the inflamed sites (7, 10). The significant role of
CXCR2 in contributing to neutrophil infiltration to the inflamed
lungs has been well demonstrated by using CXCR2 knockout
mice (11). Inhibition or knockout of this chemokine receptor diminished neutrophil influx into the lungs and improved mortality
associated with ALI (11, 34, 35). Considering the putative roles of
The Journal of Immunology
Acknowledgments
We thank Mian Zhou, Weifeng Dong, and Cletus Cheyuo for technical help
and valuable discussion.
Disclosures
P.W. is an inventor of the pending Patent Cooperation Treaty application.
The other authors have no financial conflicts of interest.
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