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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Recombinant human activated protein C inhibits integrin-mediated neutrophil
migration
*Gwendolyn F. Elphick,1 *Pranita P. Sarangi,2 Young-Min Hyun,2 Joseph A. Hollenbaugh,2 Alfred Ayala,1 Walter L. Biffl,3
Hung-Li Chung,4 Alireza R. Rezaie,5 James L. McGrath,4 David J. Topham,2 Jonathan S. Reichner,1 and Minsoo Kim2
1Department of Surgery, Rhode Island Hospital and Brown Medical School, Providence; 2Department of Microbiology and Immunology, David H. Smith Center
for Vaccine Biology and Immunology, University of Rochester, NY; 3Department of Surgery, Denver Health Medical Center, CO; 4Department of Biomedical
Engineering, University of Rochester, NY; and 5Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, MO
Integrin-mediated cell migration is central to many biologic and pathologic processes. During inflammation, tissue injury results from excessive infiltration
and sequestration of activated leukocytes. Recombinant human activated protein C (rhAPC) has been shown to protect
patients with severe sepsis, although the
mechanism underlying this protective ef-
fect remains unclear. Here, we show that
rhAPC directly binds to ␤1 and ␤3 integrins and inhibits neutrophil migration,
both in vitro and in vivo. We found that
human APC possesses an Arg-Gly-Asp
(RGD) sequence, which is critical for the
inhibition. Mutation of this sequence abolished both integrin binding and inhibition
of neutrophil migration. In addition, treat-
ment of septic mice with a RGD peptide
recapitulated the beneficial effects of
rhAPC on survival. Thus, we conclude
that leukocyte integrins are novel cellular
receptors for rhAPC and the interaction
decreases neutrophil recruitment into tissues, providing a potential mechanism by
which rhAPC may protect against sepsis.
(Blood. 2009;113:4078-4085)
Introduction
Migration of leukocytes to infection sites is vital for pathogen
clearance and, thus, host survival.1 Interaction of cell surface
integrins with their counterpart ligands, which are expressed on the
endothelial surface, results in the localization and adherence of
circulating neutrophils to endothelial cells. This is followed by
neutrophil activation and directed migration to sites of infection
through the extracellular matrix. An important function of integrins
is to concentrate neutrophils at the infection site, ensuring that their
immune products and activities remain at this site, while minimizing unnecessary injury to uninfected tissues. Sustained or dysregulated integrin activation, resulting in abnormal neutrophil trafficking, as well as direct damage to the vasculature and the underlying
tissue, is known to contribute to sepsis.2-4
Recombinant human activated protein C (rhAPC), the only
FDA-approved drug for treating severe sepsis, is a vitamin
K–dependent serine protease that is derived from protein C (PC).
Activated protein C (APC) is most well known for its anticoagulant
functions. Although initial hypotheses to explain its efficacy in
preventing severe sepsis centered on the antithrombotic and
profibrinolytic functions of rhAPC,5-8 other agents including antithrombin III and tissue-factor pathway inhibitor, known to have
potent effects on such pathways, did not demonstrate the same
clinical efficacy in the treatment of severe sepsis as rhAPC,9,10
suggesting the ability of APC to improve several immunerelated functions independent of its anticoagulant functions.
Although regulation of leukocyte migration has been proposed
to underlie the protective effects of APC against sepsis,11-16 the
molecular mechanisms of the inhibitory effects of APC have not
been demonstrated.
Methods
Reagents
Recombinant human APC (rhAPC) was obtained from Eli Lilly (Indianapolis, IN). The protein C mutant containing Glu substitution in place of
Asp-222 (D222E) was constructed by overlap extension polymerase chain
reaction (PCR). The inner complementary primers were 5⬘-GACCAGGCGGGGAGAGAGCCCCTGGCAGGTG-3⬘ and 5⬘-CACCTGCCAGGGGCTCTCTCCCCGCCTGGTC-3⬘. The outer primers corresponded to vector plasmid cDNA (pRC/CMV) bases 837 to 856 and 1038 to
1065. The second round PCR product was digested with HindIII and XbaI,
and inserted into pcDNA 3.1(⫹)/Hygro vector (Invitrogen). The mutant
zymogen was expressed in HEK293 cells, purified and converted to
activated protein C by thrombin, and separated from thrombin by an FPLC
Mono Q column as described.17 The RGD peptide (Ac-c[(Pen)-Tyr(Me)-AlaArg-Gly-Asp-Asn-Tic-Cys]NH2)18 was synthesized in Peptides International (Louisville, KY). Human tissue cDNA was purchased from Clontech
(Mountain View, CA).
DNA plasmids and constructs
For human endothelial protein C receptor–monomeric cyan fluorescence
protein (hEPCR-mCFP), PCR extensions were performed on wild-type
hEPCR cDNA in pSVzeo as a template. The upstream primer 5⬘ATATAAAGCTTGCCACCATGTTGACAACATTGCTGCC-3⬘ with a HindIII site and the downstream primer 5⬘-TATATACCGGTCCACATCGCCGTCCACCTGTGC-3⬘ with a AgeI site were used with hEPCR-pSVzeo.
After digestion with HindIII and AgeI, the PCR fragment was inserted into
HindIII and AgeI-digested pECFP.
For ␤1–monomeric yellow fluorescence protein (mYFP) with 5-aminoacid residue linker, PCR extension was performed using wild-type ␤1
subunit cDNA (GenBank BC020057) as a template with the upstream
Submitted September 24, 2008; accepted February 13, 2009. Prepublished
online as Blood First Edition paper, February 24, 2009; DOI 10.1182/blood2008-09-180968.
The online version of this article contains a data supplement.
*G.F.E. and P.P.S. contributed equally to this work.
© 2009 by The American Society of Hematology
4078
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
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BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
primer 5⬘-ATATACTCGAGGCCACCATGAATTTACAACCAATTTT-3⬘
containing XhoI site and the downstream primer 5⬘-TATATACCGGTCCTTTTCCCTCATACTTCGGAT-3⬘ containing AgeI site. After digestion with
XhoI and AgeI, PCR product and mYFP then ligated to generate ␤1
(5)-mYFP containing 5-amino-acid–residue linker of GPVAT. Monomeric
mCFP and mYFP mutants were generated by replacing Leu-221 at the
crystallographic dimer interface with a Lys.
Human neutrophil preparation
Blood was collected from healthy volunteers via antecubital vein puncture
in heparin or EDTA-containing vacutainers. Granulocytes and erythrocytes
were separated from whole blood by centrifugation through a Histopaque
1077 (Sigma-Aldrich, St Louis, MO) density gradient. Remaining erythrocytes were removed by hypotonic lysis, yielding a neutrophil purity of
greater than 98%. The Human Research Studies Review Board of the
University of Rochester approved this study, and informed consent was
obtained in accordance with the Declaration of Helsinki.
Underagarose migration assay
Delta T dishes (Bioptechs, Butler, PA) were coated with either with
fibronectin (FN). Plates were then rinsed with the appropriate media and
allowed to air-dry. Molten agarose (Seakem GTG; FMC Bioproducts,
Rockland, ME) was prepared as described.19 A subset of cells was treated
with 10 ␮g/mL rhAPC at 37°C for 20 minutes before being loaded into the
wells. The dishes were incubated for 60 minutes at 37°C and then fixed with
10% buffered formalin. Counts were then made of the number of cells that
had migrated 1 mm toward the right well (toward fMLP, directional
migration), 0.5 mm toward the left and right wells (“straddle”), and 1 mm
toward the left well (random migration). The experiments were repeated
3 times per condition.
Measurement of random migration and micropipette assay
Delta T dishes were coated with human FN (50 ␮g/mL; BD Biosciences,
San Jose, CA) for overnight at 4°C and then for 4 hours at room
temperature. Plates were then rinsed with L-15 media. Human neutrophils
(⬃500 000) were incubated, in the presence or absence of 10 ␮g/mL
wild-type rhAPC, rhPC, or mutant APC in 500 ␮L L-15 media containing
2 mg/mL glucose for 20 minutes at 37°C. The cells were then added to
FN-coated dishes in 1.5 mL L-15 with 2 mg/mL glucose. For neutrophil live
cell staining, 2 ␮M Green Cell Tracker CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes, Carlsbad, CA) was used. Temperature
was maintained at 37°C throughout the experiment using a FCS2 live-cell
imaging chamber (Bioptechs). Images were acquired for 30 to 60 minutes
under a 10⫻ objective lens using a Nikon TE-2000U inverted microscope.
Phase-contrast images were acquired every 5 seconds and fluorescence
images every 30 seconds. Cell paths were traced using MetaVue imaging
software (Molecular Devices, Sunnyvale, CA).
Random migration was investigated by adding fMLP or IL-8 to media at
a final concentration of 10⫺8 M and analyzed by manually tracing the
outline of each cell in selected frames (ie, at 50-second intervals). The x and
y coordinates of each cell were measured using ImageJ software and were
corrected so that the starting position was x ⫽ 0 and y ⫽ 0.
Directional migration was investigated using a micropipette assay to
analyze the dynamics of neutrophil migration on different integrin ligands.
Sterile Femtotips II micropipette tips (Eppendorf, Westbury, NY) were
loaded with 5 ␮L of 1 ␮M fMLP and then placed on the bottom of
FN-coated delta T dishes that had been prewarmed to 37°C. Image
acquisition began within 2 minutes of tip placement.
The time-lapsed images of migrating neutrophils were taken under
phase contrast microscopy. Each image was thresholded and binarized
based on brightness to define the centroids of the neutrophils. The closest
centroids between consecutive images were linked as a trajectory under the
assumption that these centroids were made by the same neutrophil. The
maximum distance between the closest centroids was also defined to
preclude the artifacts due to cells moving into and out of the field of view.
To demonstrate the randomness of chemokinesis and the distances that each
ACTIVATED PROTEIN C AND NEUTROPHIL MIGRATION
4079
cell travel, the starting points of the trajectories of the neutrophils were
translated to the origin.
Flow cytometric analysis
Neutrophils were pretreated with 10 ␮g/mL rhAPC in HBS (20 mM Hepes,
pH 7.5, 140 mM NaCl) ⫹ 1 mM Ca2⫹ and 1 mM Mg2⫹, or ⫹3 mM Ca2⫹
and 0.6 mM Mg2⫹ for 30 minutes at 37°C. LIBS mAbs (B44 and D3) were
added 5 minutes before fixation with 3.7% formaldehyde for 10 minutes at
room temperature. Cells were then washed and incubated with PE-labeled
goat anti–mouse IgG for 30 minutes at 4°C in the dark and then washed and
resuspended in phosphate-buffered saline (PBS) for flow cytometric
analysis. For EPCR, neutrophils were incubated with/without 1 ␮M fMLP
for 30 minutes at 37°C. Cells were then washed and labeled with EPCR
mAb (RCR-252) or control IgG (rat-IgG1).
Neutrophil adhesion assays
The adhesion assay was carried out essentially as described.20 Cover slips
were coated with FN (50 ␮g/mL). Residual binding sites were blocked by
incubation of the wells with 0.05% (wt/vol) polyvinylpyrrolidone in PBS
for 30 minutes at room temperature; 2.5 ⫻ 105/250 ␮L of neutrophils were
suspended in L15 medium plus 2 mg/mL glucose and pretreated for
15 minutes at 37°C with 10 ␮g/mL rhAPC, 50 ␮g/mL human EPCR
blocking mAb (RCR-252), or 10 ␮g/mL Gla-less rhAPC (Enzyme Research Laboratories, South Bend, IN). The cover slips were aspirated and
washed with L15 medium; 250 ␮L of L15/2 mg/mL glucose medium
containing 10 ␮g/mL rhAPC, 50 ␮g/mL human EPCR blocking mAb, or
10 ␮g/mL Gla-less rhAPC, with/without 20 nM fMLP was placed on each
cover slip and prewarmed for 15 minutes at 37°C. Cells (250 ␮L) were then
immediately added, and further incubated at 37°C for 15 minutes. Unbound
cells were then washed with warm L15 medium. The bound cells were then
fixed with formaldehyde. For each experimental condition from 3 independent donors, 5 random phase-contrast images were obtained, and the
number of cells in each well was scored from printed micrographs.
Integrin ligand binding assay
The solid phase binding assay was performed using purified soluble human
␣3␤1, ␣5␤1, and ␣V␤3 (United States Biological, Swampscott, MA);
2 ␮g/mL soluble integrins were added to microtiter wells for capture with
immobilized monoclonal antibody against ␤1 or ␤3 subunits (mAb TS2/6
for ␤1 and mAb BB10 [Millipore Bioscience Research Reagents, Temecula,
CA] for ␤3 integrins); 10 ␮g/mL rhAPC, 10 ␮g/mL RGE-APC, or 5 ␮g/mL
of human FN was incubated in the presence of 1 mM Ca2⫹ and Mg2⫹ plus
1 mM Mn2⫹ for 1 hour at room temperature. After washing, bound APC
was chromogenically detected by peroxidase–streptavidin conjugate anti-PC
(DiaPharma, West Chester, OH) or chromogenic substrate S-2366 (DiaPharma). To minimize dissociation, all wash buffers contained 1 mM Mn2⫹,
and less than 15 minutes elapsed between the end of binding and beginning
of color development. Functional blocking Abs for integrins (mAb P4C10
for ␤1, mAb B3A for ␤3 integrins) were from Millipore.
Intracellular Ca2ⴙ measurement
Human neutrophils (107 cells/mL) were labeled with 5 ␮M Fluo-4 AM
(Molecular Probes) at 37°C for 30 minutes, then at room temperature for an
additional 5 to 10 minutes. After wash twice, cells were resuspended to
4 ⫻ 106 cells/mL (6 mL) in F15 and incubated at RT for de-esterification.
For measurements, cells (4 ⫻ 106) were spun down in a microtube (1 mL),
resuspended in 1 mL F15 at 37°C, transferred to a quartz cuvette containing
a fluorometric stir bar and 1 mL of the same buffer at 37°C, and
fluorescence signals were measured in the fluorometer.
Reverse transcription PCR
Total RNA was prepared from human neutrophils using RNeasy Mini Kit
(QIAGEN, Valencia, CA). Isolated mRNA was reverse-transcribed and
amplified by PCR in one step using a QIAGEN One-Step reverse
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4080
ELPHICK et al
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
transcription (RT)–PCR kit with the following sense and antisense primers:
sense, 5⬘-ggcagtttcatcattgctgg-3⬘; antisense, 5⬘-ttgaacgcctcaggtgattc-3⬘.
Immunoblotting
Cells were lysed in cold buffer containing Hanks balanced salt solution, 2%
Triton X-100, 120 mM N-octyl ␤-D-glucopyranoside, and EDTA-free
mini-protease inhibitor cocktail (Roche, Indianapolis, IN). Insoluble cell
debris was removed by centrifugation. Laemmli buffer was added to the
lysates and boiled for 5 minutes; 20 ␮L of each sample was separated on a
15% gel (Cambrex, East Rutherford, NJ), and proteins were transferred to
PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were
probed with primary antibodies (anti-EPCR [R&D Systems, Minneapolis,
MN] and anti-GFP [Molecular Probes]) and appropriate peroxidaseconjugated secondary antibody. Immunoreactive bands were visualized by
chemiluminescence.
Fluorescence resonance energy transfer
Dynamic fluorescence resonance energy transfer (FRET) imaging was
carried out using a Nikon Eclipse TE2000-U microscope (Nikon, Melville,
NY) equipped with a Dual-View (MAG Biosystem, Tucson, AZ) and
CFP/YFP dual-band filter set (Chroma, Brattleboro, VT). Total internal
reflection fluorescence (TIRF) imaging was performed with a white light
TIRF aperture diaphragm and a 100⫻ TIRF 1.49 NA oil-immersion
objective coupled to a QuantEM EMCCD (Roper Scientific, Trenton, NJ).
The microscope was controlled by NIS element software (Nikon), and data
analysis was performed with AutoDeblur (Autoquant Imaging, Troy, NY)
by the sensitized emission method.21 Forty-eight hours after cotransfection
with hEPCR-mCFP and ␤1-mYFP DNA construct in a Delta T dish (Fisher
Scientific, Waltham, MA), HEK293 cells were washed with PBS and then
1 mL L15 medium supplemented with 2 mg/mL D-glucose. Cells were
equilibrated for approximately 10 minutes at 37°C in a FCS2 live cell
imaging chamber (Bioptechs). Each image of CFP, YFP, and bright field of
cells was then taken for 0.1 seconds for hEPCR-mCFP and 1 second for
␤1-mYFP with 2 ⫻ 2 binning through a 100⫻ oil-immersion objective lens
every 10 seconds for 15 minutes.
Neutrophil recruitment into airspace
Mice (C57BL/6, 6 to 8 weeks) were anesthetized with avertin and then
intranasally administered 30 ␮g lipopolysaccharide (LPS; Sigma-Aldrich);
2 hours later the mice were tail vein injected with rhAPC or RGE-APC
(10 ␮g/mouse) or PBS. Mice were killed 4 hours later, and bronchoalveolar
lavage was harvested. Cells were fixed with 4% formaldehyde for
20 minutes. Cells were counted using microbeads (Polysciences, Warrington, PA) by flow cytometry. Cells were stained with Gr-1 APC antibody
(eBioscience, San Diego, CA) and collected by flow cytometry. Data were
analyzed using FlowJo software (TreeStar, Ashland, OR). Experiments
were done in accordance with National Institutes of Health (NIH, Bethesda,
MD) guidelines and were approved by the University of Rochester
University Committee on Animal Resources.
Figure 1. rhAPC inhibits neutrophil adhesion and migration. (A) Binding of 10 nM
fMLP-treated neutrophils to immobilized FN in the presence of various concentrations of rhAPC. For each condition, binding was measured in triplicate and stated as
mean (⫾ SEM). *P ⬍ .05 versus fMLP-treated cells in the absence of rhAPC.
(B) Migration of human neutrophils on FN-coated cover glasses in the presence of
fMLP ⫾ rhAPC. Cells were tracked over a 30-minute period, and each line represents
one cell. Experiments were repeated on neutrophil preparations from 3 independent
donors. (C) Directional migration of neutrophils, as measured by the underagarose
migration assay. The number of control or rhAPC-treated neutrophils migrating to
fMLP (1 and 2) or to PBS (3) was counted. Results are expressed as mean ⫾ SEM of
3 experiments from 2 independent donors. (D) Center-zeroed tracks of control or
rhAPC-treated neutrophils migrating toward microtips containing fMLP. The scale of
each graph is in microns. The speed (S, ␮m s⫺1), migratory index (MI), and XD (mean
direction in which the population is moving, in degrees) are shown (mean ⫾ SEM).
(E) Migration of human neutrophils on FN-coated cover glasses in the presence of
IL-8 ⫾ rhAPC. (F) Cytosolic Ca2⫹ levels in stirred Fluo-4–labeled neutrophils were
continuously measured in a fluorometer. Control (PBS) or rhAPC-treated neutrophils
in Ca2⫹-containing buffer were sequentially stimulated with 10 nM fMLP and 40 ␮M
digitonin. The data are representative of at least 3 independent experiments.
Data analysis
All values are expressed as the mean plus or minus SEM. The differences
between all groups were analyzed by the Student t test. Survival curves
were analyzed by the Kaplan-Meier log-rank test. All statistics were
performed using the Prism program for Macintosh, version 4.0 (GraphPad
Software, La Jolla, CA).
Results
After transendothelial migration, neutrophils cross the basal lamina
and migrate through the extracellular matrix and into tissue or sites
of inflammation. To investigate the effects of rhAPC on neutrophil
adhesion to the matrix proteins, we performed a cell adhesion assay
on fibronectin (FN)–coated cover glass. Human neutrophils were
allowed to adhere to immobilized FN in the presence of N-formylMet-Leu-Phe (fMLP). The addition of rhAPC significantly reduced
fMLP-induced adhesion (Figure 1A). To investigate the effects of
rhAPC on the migration of neutrophils on matrix proteins, we
performed live-cell imaging of neutrophils migrating on FN-coated
cover glass in the presence of the chemoattractant fMLP. Cell
tracking analysis revealed that fMLP significantly increased the
random migration of neutrophils on FN, and the presence of rhAPC
dramatically reduced this effect (Figure 1B).
Directional migration of neutrophils toward the chemokine
gradients is critical to reach the site of infection. Therefore, we
tested the effect of rhAPC on neutrophil directional migration using
the under-agarose assay. Neutrophils were seeded in a well flanked
by 2 wells containing either fMLP or PBS (Figure 1C top panel).
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BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
The majority of neutrophils migrated toward the fMLP-containing
well, suggesting that the chemoattractant diffused toward the
middle well. As with random migration, rhAPC decreased directional migration of neutrophils on FN toward fMLP (Figure 1C).
The effect of rhAPC on the dynamics of directional neutrophil
migration was further investigated using the micropipette assay. In
this assay, a micropipette tip containing fMLP was placed in a field
of cells to establish a chemokine gradient. Consistent with results
obtained using the under-agarose assay, neutrophil migration on
FN toward the tip was dramatically decreased by rhAPC pretreatment (Figure 1D and Videos S1,S2, available on the Blood website;
see the Supplemental Materials link at the top of the online article).
Quantitative analysis of more than 30 neutrophils in each group
revealed that rhAPC decreased the average lateral migration speed
(S) more than 2.5-fold, without significantly altering the migratory
index ([MI], distance from origin/total distance traveled) or the
direction of movement (KD; Figure 1D). These results indicate that,
although neutrophils can sense chemoattractants and become
polarized toward chemoattractant gradients in the presence of
rhAPC, they cannot move toward the gradient. This is further
supported by measurement of intracellular calcium mobilization by
fMLP in the presence/absence of rhAPC. Dynamic measurement of
intracellular Ca2⫹ shows that rhAPC has no effect on the increase in
Ca2⫹ concentration by fMLP treatment (Figure 1F), suggesting that
rhAPC does not functionally alter the signaling pathway associated
with chemotaxis receptors on neutrophil.
Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium
to arrive at a site of infection. To investigate whether rhAPC
inhibits neutrophil migration induced by host-derived chemoattractants, we used IL-8 in the migration assay. Consistent with results
obtained using fMLP, neutrophil migration on FN by interleukin-8
(IL-8) was dramatically decreased by rhAPC pretreatment
(Figure 1E).
During leukocyte migration, integrins act as the “feet” by
supporting adhesion to the extracellular matrix or other cells. The
best characterized integrin-binding motif is the RGD sequence,
which is present in FN, fibrinogen, von Willebrand factor, vitronectin, and a variety of other adhesion proteins. Many of integrins that
bind to extracellular protein ligands, including ␣3␤1, ␣5␤1, ␣IIb␤3,
and ␣V␤3, specifically interact with the RGD sequence of their
target proteins. Amino acid sequence analysis revealed that the
catalytic domain of human protein C/APC also contains the RGD
sequence. Therefore, we hypothesized that rhAPC directly interacts
with neutrophil integrins to regulate cell migration. To test this
hypothesis, we performed rhAPC-binding assays with neutrophils
in suspension. The assay buffer containing 1 mM Ca2⫹ and 1 mM
Mg2⫹ was supplemented with 1mM Mn2⫹ to activate cell surface
integrins. This significantly increased rhAPC binding to the
neutrophil surface (Figure 2A). The addition of blocking mAbs
against ␤1 or ␤3 integrins partially displaced rhAPC from the cell
surface (Figure 2A). The presence of both ␤1 and ␤3 integrin
blocking mAbs further reduced rhAPC binding on neutrophil
surface (Figure 2A). Cyclic RGD peptide also significantly inhibited rhAPC binding, whereas blocking anti-␤2 integrin (non-RGD
binding integrin) mAb had no effect (Figure 2A). Collectively,
these results suggest that the major mechanism that controls rhAPC
binding to neutrophil surface is through direct interaction with the
integrins.
Upon ligand binding, integrins undergo pronounced conformational changes that result in the appearance of ligand-induced
binding sites (LIBSs), which can be detected by specific monoclo-
ACTIVATED PROTEIN C AND NEUTROPHIL MIGRATION
4081
Figure 2. Direct binding of rhAPC to neutrophil integrins. (A) Binding of rhAPC to
human neutrophils was assayed using chromogenic substrate S-2366 in the
presence of 1 mM MnCl2 ⫾ cyclic RGD peptide (20 ␮g/mL), ␤1 blocking Ab, ␤3
blocking Ab, or ␤2 blocking Ab (10 ␮g/mL each). *P ⬍ .05 versus MnCl2-treated cells.
(B) Binding of control or 10 nM fMLP-treated neutrophils to immobilized FN in the
presence of rhAPC (10 ␮g/mL) or Gla-less APC (10 ␮g/mL), or an equivalent amount
of PBS. (C) Binding of control or 10 nM fMLP-treated neutrophils to immobilized FN in
the presence of rhPC (10 ␮g/mL) or an equivalent amount of PBS. (D) Induction of
LIBS epitopes by rhAPC. Control (PBS) or rhAPC-treated neutrophils were incubated
with the indicated concentrations of MgCl2 and CaCl2. The LIBS of ␤1 and ␤3 integrins
were detected by B44 and D3 mAb, respectively. *P ⬍ .05 versus PBS-treated cells.
(E) Solid-phase binding of immobilized ␣3␤1, ␣5␤1, and ␣V␤3 to FN or rhAPC.
(F) Solid-phase binding of immobilized ␣3␤1, ␣5␤1, and ␣V␤3 to wild-type or mutant
rhAPC (RGE-APC). (G) Migration of human neutrophils on FN-coated cover glass in
the presence of fMLP ⫾ RGE-APC, rhPC, Gla-less APC, or S360A-APC. Experiments were repeated on neutrophil preparations from 3 independent donors. Results
in panels A and C through F are expressed as mean (⫾ SEM) of 3 independent
experiments.
nal antibodies. The direct binding of rhAPC to ␤1 and ␤3 integrins
on intact neutrophils was tested by measuring the appearance of
LIBS using B44 mAb (␤1 integrin LIBS Ab) and D3 mAb (␤3
integrin LIBS Ab). In the presence of 1 mM Ca2⫹ and Mg2⫹,
rhAPC strongly induced B44 and D3 binding (Figure 2D). The
binding of APC to EPCR is Ca2⫹-dependent, with ion concentrations of 3 mM CaCl2 and 0.6 mM MgCl2 being optimal for
binding.22 Ca2⫹, however, has been shown to exert a negative
regulatory effect on integrin-ligand binding.23 Consistent with this
finding, APC did not induce the LIBS Abs binding in the presence
of 3 mM CaCl2 and 0.6 mM MgCl2 (Figure 2D). These data
suggest that APC induces conformational changes in neutrophil
integrins through direct interaction with the integrins and not
through signals resulting from its interaction with EPCR on the
neutrophil surface.
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ELPHICK et al
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
Figure 3. Simultaneous binding of rhAPC to neutrophil integrins and EPCR. (A) EPCR RT-PCR on human neutrophils. Reverse-transcribed cDNAs from human heart and
lung served as positive controls (top). FACS analysis of cell-surface EPCR using mAb RCR252 (bottom). IgG control and fMLP-treated cells were exposed to fMLP for
30 minutes before staining with Rat IgG1 isotype and RCR252, respectively. As a negative control (Untreated), cells were incubated without fMLP and labeled with RCR252.
(B) Binding of 10 nM fMLP-treated neutrophils to immobilized FN in the presence of rhAPC (10 ␮g/mL) ⫾ EPCR mAb (50 or 100 ␮g/mL). (C) A hypothetical model for rhAPC
binding. Cells expressing hEPCR-mCFP and ␤1-mYFP will exhibit FRET only when these 2 molecules are brought into close proximity (100 Å) after rhAPC binding.
(D) Whole-cell lysates of HEK293 cells transiently transfected with hEPCR-mCFP and ␤1-mYFP K562 were subjected to SDS-PAGE and Western blotting with the indicated
antibodies. (E) Fluorescence images of transiently transfected HEK293 cells with hEPCR-mCFP and ␤1-mYFP demonstrate membrane localization of CFP and YFP signals.
(F) HEK293 cells were transfected with hEPCR-mCFP and ␤1-mYFP in a delta T dish. FRET images under TIRF microscopy were extracted from videos (time above images,
Videos S3-S5). FRET was measured by sensitized emission method and analyzed by AutoQuant software. FRET signals are shown as rainbow colors (red indicates highest
and blue, lowest).
Among the neutrophil integrins that recognize the RGD motif,
␣3␤1, ␣5␤1, and ␣V␤3 are key in regulating neutrophil chemotaxis.24-26 To test whether APC can bind directly to these integrins,
we performed an ELISA-like solid phase receptor-binding assay
(Figure 2E). Soluble ␣3␤1, ␣5␤1, and ␣V␤3 were captured onto
microtiter wells using mAbs specific for ␤1 or ␤3. Binding to FN or
rhAPC was then measured in buffer containing 1 mM Mn2⫹, 1 mM
Ca2⫹, and 1 mM Mg2⫹. As shown in Figure 2E, FN and rhAPC
bound to all 3 integrins, and this binding was specific, as shown by
its reversibility in the presence of integrin-blocking mAbs.
To determine whether rhAPC binds to ␤1 and ␤3 integrins
through its RGD sequence, the RGD sequence was mutated to RGE
(RGE-APC). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of RGE-PC zymogen before and
after activation by thrombin, enzyme assays using the chromogenic
substrate, S-2366, and permeability assay using EA.hy926 cell
monolayers indicated that the molecular weight and glycosylation,
enzymatic activity, and EPCR-dependent PAR1-mediated signaling of RGE-APC were similar to those of wild-type APC (data not
shown). Despite these similarities, the direct binding of RGE-APC
to soluble ␣3␤1, ␣5␤1, and ␣V␤3 integrins was significantly less than
that of wild-type APC, as measured by the solid-phase receptorbinding assays (Figure 2F). In random migration assay, RGE-APC
had no effect on the migration of neutrophils on FN, whereas the
presence of wild-type rhAPC dramatically reduced the migration
(Figure 1). To investigate whether activation of PC is required for
the inhibition of neutrophil migration, we tested the zymogen PC
(rhPC) in our adhesion and migration assays. Unlike rhAPC, rhPC
failed to block neutrophil adhesion and migration on FN (Figure
2C,G). The recombinant APC variant S360A-APC, however,
which lacks proteolytic activity,27 successfully inhibited neutrophil
migration (Figure 2G). Thus, these data suggest that the RGD
sequence in rhAPC is an essential feature of its direct interaction
with the neutrophil integrins and for inhibition of neutrophil
migration on matrix proteins but that APC’s enzymatic proteolytic
activity is not necessary for the inhibition.
EPCR was the first identified cellular APC receptor and is expressed
in healthy human neutrophils and monocytes.14 Because activated
neutrophils contribute to organ system dysfunction and mortality in
sepsis, we first compared EPCR expression between unstimulated and
fMLP-stimulated neutrophils. Reverse-transcription PCR revealed that
healthy neutrophils expressed EPCR mRNA (Figure 3A). Changes in
the transcript abundance measured by real-time PCR in fMLPstimulated human neutrophils were indiscernible (ratio to GAPDH,
0.54 ⫾ 0.79). Cell-surface expression of EPCR could also be detected in
these cells, although the overall levels of the receptor were very low
(Figure 3A). Stimulation with fMLP enhanced cell-surface expression
of EPCR (Figure 3A).
Previously, it was shown that neutrophil migration through
nitrocellulose micropore filter (without any protein coatings) was
inhibited by rhAPC and the inhibition was reversed by an EPCR
blocking Ab.14 In our neutrophil adhesion and migration assays on
immobilized FN, however, Gla-less APC, a mutant form of APC
that lacks the EPCR binding motif,28 inhibited adhesion and
migration to a slightly less but similar degree as the wild-type
protein (Figure 2B,G). To further investigate roles of EPCR in
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
neutrophil migration, we tested whether the anti-EPCR Ab could
relieve the rhAPC-induced inhibition of neutrophil adhesion on
FN. Blocking of neutrophil EPCR with high concentration of Ab
only partially but significantly reversed the inhibitory effect of
rhAPC on neutrophil binding to FN (Figure 3B). In endothelial
cells, EPCR-bound APC can mediate signals through PAR-129 and
S1P1 receptors.30,31 Agonists of PAR-1 (SFLLRNPNDKYEPF)
and S1P1 (SEW2871), however, did not inhibit neutrophil migration (data not shown). Thus, these results have led us to hypothesize
that rhAPC can bind to EPCR and ␤1/␤3 integrins simultaneously
on the neutrophil surface (Figure 3C), where EPCR provides a
supportive role for the integrin binding. To investigate the double
occupation of rhAPC on living cell surface, fluorescence resonance
energy transfer (FRET) analysis was used to measure the energy
transfer between monomeric yellow fluorescent protein (mYFP)
and monomeric cyan fluorescent protein (mCFP) as a function of
distance (Figure 3C). We constructed C-terminal CFP-tagged
EPCR (EPCR-mCFP) and YFP-tagged ␤1 integrin (␤1-mYFP;
Figure 3C,D). EPCR-mCFP and ␤1-mYFP were transiently cotransfected into the EPCR deficient HEK293, where they localized
predominantly to the plasma membrane (Figure 3E). To test
whether there is a decrease in the distance between EPCR and ␤1
integrins by rhAPC ligation, dynamic FRET was used to measure
changes in the relative proximity of these cell-surface proteins in
the presence of rhAPC, GLA-less APC, or RGE-APC. Because the
ligand will only bind to cell surface receptors, we confined our
optical measurements to the plasma membrane using through the
objective total internal reflection fluorescence (TIRF). Dynamic
FRET measurements on TIRF microscopy showed significant
increase in FRET efficiency after rhAPC treatment (Figure 3F and
Video S3). No obvious FRET change was observed with Gla-less
APC and RGE-APC (Figure 3F and Videos S4, S5). Therefore,
these data support the hypothesis that rhAPC recruits EPCR and ␤1
integrins in close proximity, within 100 Å, on cell membrane
through simultaneous binding to EPCR and the integrins.
Intravenous administration of rhAPC is known to reduce lipopolysaccharide (LPS)–induced pulmonary inflammation by attenuating neutrophil chemotaxis toward the alveolar compartment. To show that the
protective effect of rhAPC in vivo is associated with its interaction with
neutrophil integrins and its suppression of neutrophil migration, LPS
instillation induced neutrophil recruitment into bronchoalveolar lavage
fluid (BALF) was determined by flow cytometry. Neutrophil recruitment in the BALF was significantly reduced by rhAPC but not by
RGE-APC injection (Figure 4A). Administration of rhAPC significantly
reduced mortality in a subset of patients with severe sepsis,32 and
currently it is indicated for use in patients with sepsis involving acute
organ dysfunction and a high risk of death. To further prove that the
RGD sequence in rhAPC is important for its beneficial effects in sepsis
and to examine whether the RGD peptide alters mortality in “highgrade” sepsis, 90% mortality was induced by endotoxemia. When LPS
was given at LD90, a single dose of the RGD peptide significantly
reduced mortality to 50% (Figure 4B). No significant reductions in the
mortality were observed with control peptide (RGD 3 RAD; Figure
4B). These findings demonstrate that the blocking of integrins by rhAPC
is critical for the inhibition of neutrophil recruitment and for the
protection in sepsis.
Discussion
Given the complexity of the systemic inflammatory response to
infection, it is not surprising that many targeted therapies in sepsis
ACTIVATED PROTEIN C AND NEUTROPHIL MIGRATION
4083
Figure 4. The RGD sequence of rhAPC is critical for inhibition of neutrophil
migration in vivo. (A) Neutrophil counts in BALF from LPS-treated mice. Mice were
given rhAPC or RGE-APC (10 ␮g/mouse) or PBS 2 hours after LPS treatment.
Results are expressed as mean (⫾ SEM); n ⫽ 5 per group. (B) Survival curves for
mice (C57BL/6, male, 6-8 weeks) challenged with an LD90 of LPS; 200 ␮g of the RGD
peptide or the control (RAD) peptide was given via intravenous injection before
receiving 37 to 40 mg/kg LPS intraperitoneally (n ⫽ 15-25 per group). The statistical
significance of mortality was determined by the Kaplan-Meier log-rank test.
have not been able to improve survival. rhAPC was the first drug
approved by the FDA for this indication, but its broad application
has been questioned. Nonetheless, improvements in current APC
therapy and development of better targeted, more efficient antisepsis therapies have been hampered by a lack of understanding of the
exact mechanisms underlying the beneficial effects of rhAPC on
organ function and survival rate in sepsis. The results presented
here demonstrate that rhAPC inhibits neutrophil adhesion and
migration on extracellular matrix proteins by directly binding to
integrins (␤1 and ␤3 integrins) at the neutrophil surface. Therefore,
we conclude that leukocyte integrins are novel cellular receptors
for rhAPC and that specific APC-integrin interactions inhibit
neutrophil migration.
Consistent with our findings, recent in vitro and in vivo studies
suggest that the marginal reduction in mortality and organ dysfunction/failure of septic patients by rhAPC may not result solely from
its anticoagulation functions but also from its ability to reduce
accumulation of activated neutrophils in severely infected organs.12
Integrin-mediated leukocyte migration is critical for the proper
positioning of immunocompetent cells in the body. Chemokine/
cytokine-directed cell adhesion and migration allow circulating
neutrophils to access sites of inflammation and to phagocytose
invading foreign pathogens, as well as necrotic and apoptotic cells.
In response to a variety of mechanical, thermal, and chemical
stimuli at the inflammation site, neutrophils release proteolytic
enzymes such as elastase and myeloperoxidase, and reactive
oxygen species including hydrogen peroxide and superoxide.
Though all potent killers of foreign pathogens, these agents can
also participate in disruption of endothelial barrier functions and
promote extravascular host tissue damage during uncontrolled
inflammation such as sepsis. Based on this possibility, therapies
designed to inhibit neutrophil integrins have been proposed for use
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
4084
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
ELPHICK et al
in sepsis and septic shock.33-35 Identification of the RGD (Arg-GlyAsp) sequence as a key motif on APC for the interaction with
neutrophil integrins and for the inhibition of neutrophil migration
suggests that neutrophil ␤1 and ␤3 integrins and RGD sequence
contained in extracellular matrix components such as fibronectin,
laminin, and the collagens could be novel therapeutic targets for the
treatment of severe sepsis.
The RGD sequence occurs only in human PC. In contrast to
human PC, the mouse and rat homologues contain a QGD instead
of an RGD sequence. This change in the arginine position of the
RGD integrin-binding domain, however, is considered to be a
conservative substitution that maintains integrin binding.36,37 Recently, crystal structures of the complex of RGD peptide and
integrin ␣V␤3 have been solved.38 The peptide binds with its Arg
contacting the ␣ subunit/␤ propeller domain and its Asp ligated
to a Mn2⫹ held in the metal ion-dependent adhesion site of the
␤ subunit I–like domain. The reported buried area of the RGD
motif in the ␣V␤3 crystal is 355Å.38 We calculated the solvent
accessible surface area of rhAPC RGD motif, which is comparable
to the buried area of the complex. The solvent accessible surface
area of rhAPC RGD motif is 140Å.39 The small buried area in APC
suggests that the RGD sequence in wild-type APC is not fully
exposed for optimal integrin binding. The novel finding presented
here is that rhAPC binds to EPCR and ␤1/␤3 integrins simultaneously on the neutrophil surface (Figure 3). In addition, the
median steady-state level of APC in the plasma from the PROWESS
trial was approximately 50 ng/mL, whereas the normal concentration of endogenous APC is around 1 ng/mL.40 Thus, high plasma
concentration during therapeutic infusions and simultaneous binding to cell surface EPCR would facilitate and strengthen the
interaction of rhAPC with neutrophil integrins. Our data also
suggest that subtle conformational changes around the RGD area
can dramatically enhance the affinity of APC to the integrins.
Although Zymogen rhPC failed to block neutrophil adhesion and
migration on FN, proteolytic activity of APC is not required for the
inhibition. Therefore, activation of PC may result in a conformational change that exposes the RGD sequence for more favorable
integrin binding.
The involvement of ␤2 (CD18) integrins, such as LFA-1
(␣L␤2; CD11a/CD18) and Mac-1 (␣M␤2; CD11b/CD18) and
ICAM-1, their endothelial membrane counterpart ligand, in
systemic and local inflammation has been well documented both
in vitro and in vivo.41-44 Other studies indicate, however, that
neutrophil adhesion to pulmonary microvascular endothelial
cells and migration into inflamed lung tissue can also occur by
␤2 integrin–independent adhesion pathways.25,42,45-49 In animal
models, anti-␤2 integrin Abs blocked sepsis-induced neutrophil
emigration by only approximately 60%,42,50,51 suggesting an
important component of neutrophil migration involves ␤2
integrin-independent pathways. Indeed, ␤1 integrins on neutrophils are significantly up-regulated at the later stage of sepsis in
an animal model, whereas ␤2 integrin was elevated as early as
3 hours.52 Rapid elevation of ␤2 integrin expression may be
important for host defense and tissue repair at the early phase of
sepsis by recruiting circulating neutrophils into inflamed organs.
Enhanced expression and activation of ␤1 integrin at the later
stage may, however, alter the balance of leukocyte trafficking
and induce accumulation of activated neutrophils in tissue
during severe sepsis, which may lead to organ failures. Therefore, selective blocking of ␤1 integrins may inhibit only aberrant
neutrophil infiltration while preserving other immune responses
and minimizing potential immune suppression, which could be
induced by other, nonselective antiadhesion therapies.
Our data demonstrate that EPCR is not a major mediator of the
inhibitory effect of rhAPC on neutrophil adhesion and migration on
FN. These results, however, do not preclude the possibility that the
antiapoptotic and vascular permeability effects of APC mediated
through engagement of EPCR provide an important contribution to
its beneficial effects in sepsis.30 In agreement with this concept,
Kerschen et al53 showed that the beneficial effect of APC on the
septic survival was significantly decreased in mice genetically
altered to express very low amounts of EPCR (⬍ 10% of normal).
The authors proposed that the cell signaling activities of APC,
mediated by the receptors EPCR and PAR1, are important for
APC’s ability to reduce sepsis mortality. It is important to note that,
in the aforementioned study, in vivo survival assays were performed only with recombinant mouse APC, which lacks the RGD
sequence required for integrin binding. Given our observation that
recombinant human APC directly blocks neutrophil integrins and a
point mutation [RGD3RGE] abolished the inhibition, it is tempting to speculate that the primary effects of rhAPC on sepsis are
attributable both to protection of endothelium and to changes in
integrin-mediated neutrophil migration. If the beneficial effects of
rhAPC on sepsis are mediated, in part, by diminishing integrinmediated neutrophil infiltration into the inflamed tissue, then
anti-integrin reagents may be used as potential therapies for severe
sepsis or as a combination therapy with rhAPC to improve the
clinical outcomes of rhAPC treatment. Modification of rhAPC
may also enable the development of safer and better-targeted
anti-inflammation therapies, but such advances will depend upon
a better understanding of underlying molecular mechanisms
of its action.
Acknowledgments
We thank Rachel Spoonhower, Foon-Yee Law, Nicole Morin, Lii
Feng Chen, Brian LeBlanc, Liz Lavigne, and Sanguk Kim for their
technical assistance and Andrea Sant for comments on the manuscript. We thank Lisa Jennings for antibody (D3), Björn Dahlbäck
for human protein C cDNA, and Charles Esmon for human EPCR
cDNA.
This project was supported by National Institutes of Health
grants HL087088 (M.K.), HL18208 (M.K.), HL68571 (A.R.R.),
GM065085 (G.F.E.), and GM066194 (J.S.R.). W.L.B. was supported by Eli Lilly (Indianapolis, IN).
Authorship
Contribution: G.F.E, P.P.S., Y.-M.H., J.A.H., H.-L.C., and A.R.R.
designed and performed research; A.A., W.L.B., J.L.M., D.J.T., and
J.S.R. designed research and analyzed data; and M.K. conceived,
designed, and performed research, analyzed data, and wrote the
paper.
Conflict-of-interest disclosure: W.L.B. received research funding from Eli Lilly. The remaining authors declare no competing
financial interests.
Correspondence: Minsoo Kim, Department of Microbiology
and Immunology, David H. Smith Center for Vaccine Biology and
Immunology, University of Rochester, Rochester, NY 14642;
e-mail: [email protected].
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 23 APRIL 2009 䡠 VOLUME 113, NUMBER 17
ACTIVATED PROTEIN C AND NEUTROPHIL MIGRATION
4085
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2009 113: 4078-4085
doi:10.1182/blood-2008-09-180968 originally published
online February 24, 2009
Recombinant human activated protein C inhibits integrin-mediated
neutrophil migration
Gwendolyn F. Elphick, Pranita P. Sarangi, Young-Min Hyun, Joseph A. Hollenbaugh, Alfred Ayala,
Walter L. Biffl, Hung-Li Chung, Alireza R. Rezaie, James L. McGrath, David J. Topham, Jonathan S.
Reichner and Minsoo Kim
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