PHAGOCYTES, GRANULOCYTES,AND MYELOPOIESIS
PaulA. O’Connell,1Alexi P. Surette,1Robert S. Liwski,2Per Svenningsson,3,4and David M. Waisman1
Departments of1Biochemistry and Molecular Biology and2Pathology, Dalhousie University, Halifax, NS;3Center for Molecular Medicine, Karolinska Institutet,
Stockholm, Sweden; and4Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY
The plasminogen activation system plays
an integral role in the migration of macro-
phages in response to an inflammatory
stimulus, and the binding of plasminogen
to its cell-surface receptor initiates this
process. Although previous studies from
our laboratory have shown the impor-
S100A10 in cancer cell plasmin produc-
tion, the potential role of this protein in
macrophage migration has not been in-
vestigated. Using thioglycollate to induce
a peritoneal inflammatory response, we
demonstrate, for the first time, that com-
pared with wild-type (WT) mice, macro-
phage migration across the peritoneal
membrane into the peritoneal cavity in
decreased by up to 53% at 24, 48, and
72 hours. Furthermore, the number of
trated Matrigel plugs was reduced by
8-fold compared with their WT counter-
part in vivo. Compared with WT macro-
phages, macrophages from S100A10?/?
mice demonstrated a 50% reduction in
plasmin-dependent invasion across a
Matrigel barrier and a 45% reduction in
plasmin generation in vitro. This loss in
plasmin-dependent invasion was in part
the result of a decreased generation of
plasmin and a decreased activation of
pro-MMP-9 by S100A10-deficient macro-
phages. This study establishes a direct
involvement of S100A10 in macrophage
recruitment in response to inflamma-
Monocytes/macrophages play a central role in pathogenic inflam-
matory responses associated with atherosclerosis, restenosis, tumor
surveillance, and arthritis.1-3In response to changes in the cellular
environment, monocytes and monocytoid cells undergo extensive
phenotypic alterations, including marked changes in their fibrino-
lytic properties. Synthesis and activation of matrix-degrading
proteinases by monocytes and macrophages play an essential role
in their migration through tissue.Akey proteinase that participates
in pericellular proteolysis is the serine proteinase plasmin. Plasmin
is a broad substrate proteinase that is formed from the inactive
zymogen plasminogen (Plg) by the Plg activators, tissue Plg
activator (tPA) and urokinase-type Plg activator (uPA).4,5The
participation of plasmin in cell invasion and migration is dependent
on the ability of plasmin not only to degrade extracellular matrix
(ECM) proteins but to also activate other proteinases that have
matrix-degrading activity. Plasmin can degrade a variety of matrix
proteins, such as laminin and fibronectin, and appears to activate
matrix metalloproteinase-1 (MMP-1), MMP-3, and MMP-13 di-
rectly, and to activate MMP-2 and MMP-9 indirectly, thereby
facilitating cell migration through ECMs.6
The assembly of Plg and its activators on the cell surface is
facilitated by the protein S100A10 (also referred to as p11).
S100A10 is a member of the S100 family of calcium-binding
(p36) ligand as the heterotetrameric (S100A10)2-(annexin A2)2
complex, annexin A2 heterotetramer (AIIt).7,8The S100A10 sub-
unit of AIIt possesses a carboxy-terminal lysine residue that binds
tPA and Plg, resulting in the conversion of Plg to plasmin.9The
binding of plasmin to AIIt protects plasmin from inactivation by
?2-antiplasmin.10Removal of the carboxyl-terminal lysines from
S100A10 by carboxypeptidase B (CpB) results in the loss of Plg
binding and plasmin generation.11Loss of S100A10 from the
extracellular surface of cancer cells significantly reduces plasmin
generation, thus dramatically impacting on the cells capacity to
degrade ECM and infiltrate into surrounding tissue.12In contrast,
increased extracellular levels of S100A10 result in increased Plg
binding and increased production of plasmin.12
provides a mechanism to localize the proteolytic activity of plasmin to
the cell surface. Previous studies have established the presence of
S100A10 and its binding partner, annexin A2 (p36), on the surface of
murine macrophages.13,14These studies showed that knockdown of
annexin A2 resulted in decreased plasmin generation, matrix remodel-
ing, and a dramatic loss in directed migration.13,15However, because
been difficult to attribute these effects to annexinA2 or S100A10. Our
laboratory has previously shown that loss of S100A10 from the
significantly reduces plasmin generation and also results in a loss in the
ability of these cells to degrade ECM and also to infiltrate into
In the current report, we investigated the potential role of
S100A10 in the regulation of peritonitis-dependent macrophage
migration using the recently developed S100A10-deficient
(S100A10?/?) mouse.This study marks the first characterization of
macrophage function in the S100A10?/?mouse and establishes
that S100A10 plays a significant role in peritonitis-directed macro-
phage migration in vivo.
Submitted January 21, 2010; accepted April 18, 2010. Prepublished online as
Blood First Edition paper,April 27, 2010; DOI 10.1182/blood-2010-01-264754.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
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.
© 2010 by TheAmerican Society of Hematology
1136BLOOD, 19AUGUST 2010?VOLUME 116, NUMBER 7
For personal use only.on November 5, 2015. by guest
Adetailed description of the routine methods is presented in the supplemen-
tal data (available on the Blood Web site; see the Supplemental Materials
link at the top of the online article). Only nonroutine procedures and
specialized materials are described here.
A detailed description of the mice used in this study can be found in the
Supplemental data. All mouse experiments were performed in accordance
with protocols approved by the University Committee on Laboratory
Animals at Dalhousie University.
Mouse leukocyte collection
Wild-type (WT) and S100A10?/?mice were injected intraperitoneally with
2.5 mL of a 4% Brewers thioglycollate (TG) solution (Sigma-Aldrich).
Peritoneal leukocytes were collected each day for 6 days after injection of
TG (n ? 3-9) and cell viability determined by Trypan blue exclusion.
Resident leukocytes were collected from uninjected mice (n ? 6). Perito-
neal cells were collected by lavage with 5 mL complete RPMI media. The
lavage fluid was centrifuged, the supernatant aspirated, and the cell pellet
was resuspended in complete RPMI. Total numbers of leukocytes were
determined by cell counting with a hemocytometer. Cytospin slides were
also prepared at each time point using a Shandon Cytospin 2 (Thermo
Shandon). Slides were air dried, stained with May-Gru ¨nwald-Giemsa
(Sigma-Aldrich), and cellular morphology examined under the light
microscope. Cell differentials were obtained from morphologic analysis
(n ? 5), and values were expressed as mean plus or minus SD.
Mouse peritoneal macrophages
WT and S100A10?/?mice were injected intraperitoneally with 2.5 mLof a
4% Brewers TG solution (Sigma-Aldrich). After 4 days, when most
recruited cells are macrophages,17mice were killed and peritoneal cells
were collected by lavage with 5 mL complete RPMI media. The lavage
fluid was centrifuged, the supernatant aspirated, and the cell pellet
resuspended in complete medium. Macrophages were further purified
by adherence for 3 to 4 hours at 37°C, at which point all nonadher-
ent (nonmacrophage) cells were eliminated. Cells were maintained in
Analysis of protein expression
Proteins were analyzed by Western immunoblot, flow cytometry, and
cell-surface biotinylation as described in detail in the supplemental data.
Proteolyzed annexinA2 was prepared according to Kwon et al.18
Matrigel invasion and cell migration
Thioglycollate-elicited macrophages were loaded (1 ? 105to 3 ? 105
cells/well, where indicated) into the upper portion of Transwell chambers,
coated with Matrigel (invasion assays) or uncoated (migration assays; BD
Biosciences). Plg (0.5?M,American Diagnostica) was added in the absence
or presence of ?-amino caproic acid (?-ACA, 100mM; Sigma-Aldrich),
CpB (5 U/mL; Sigma-Aldrich), aprotinin (2.2?M; Sigma-Aldrich), MMP
inhibitor GM6001 (25?M; Millipore), the MMP-9 neutralizing antibody
(20 ?g/mL; Calbiochem), proMMP9 (2 ?g/mL; R&D Systems), plasmin
(0.3?M; Enzyme Research Laboratories), S100A10, annexinA2, or bovine
serum albumin (2 ?g/mL).As a chemoattractant, MCP-1 (10 ng/mL; R&D
systems), C5a (10 ng/mL; R&D Systems), plasmin (0.43 catalytically
active [CTA] U/mL), or thioglycollate (4% weight/volume; Sigma-Aldrich)
was added to the lower chamber for both the invasion and migration
assays.After 48 hours, cells on the underside of the membrane were stained
with hematoxylin and eosin (Sigma-Aldrich) and counted. Recombinant
S100A10 and annexin A2 were purified from bovine lung as described by
Khanna et al.19
WT and S100A10?/?thioglycollate-elicited macrophages (2.5 ? 105cells)
were plated in 96-well plates. Cells were incubated with or without uPA
(50nM) for 10 minutes at room temperature. Cells were washed 3 times
with incubation buffer (Hanks balanced salt solution [HBSS] containing
3mM CaCl2and 1mM MgCl2) and incubated with 0.5?M glu-Plg with or
without CpB (5 U/mL) for 10 minutes before the addition of 500?M
plasmin substrate S2251 (Chromogenix, Diapharma Group). The rate of
plasmin generation was measured using a spectrophotometer (405nM)
taking readings every minute for 2 hours.
Glu-Plg (2-5 mg/mL) was dialyzed against 0.1M carbonate buffer (pH 9),
and a 50M excess of fluorescein isothiocyanate (FITC) was added after
being dissolved in dimethyl sulfoxide. Plg and FITC were mixed for
16 hours in the dark and treated with 0.01% hydroxylamine to remove all
labile FITC-Plg bonds. Unincorporated FITC was removed by gel filtration
through an NAP-10 column using HBSS (20mM N-2-hydroxyethylpipera-
zine-N?-2-ethanesulfonic acid, 1mM CaCl2, and 1mM MgCl2;pH 7.4).
Typically, 2 FITC molecules were bound to each Plg molecule.
Plg binding assays
WTand S100A10?/?thioglycollate-elicited macrophages were washed and
cultured in the absence of serum for 2 hours before assay. Cells were
incubated with 200nM FITC Glu-Plg, either with or without ?-ACA
(100mM), for 1 hour at 4°C in HBSS (1mM MgCl2and 3mM CaCl2). Plg
binding was measured by fluorescence-activated cell sorter (FACS),
excluding cells that were positive for propidium iodide (Sigma-Aldrich).
Percentage binding refers to mean fluorescence intensity.
Detailed zymographic techniques are described in the supplemental data.
A total of 750 ?L growth factor–reduced Matrigel with 200 ng/mL basic
fibroblast growth factor (Invitrogen) and 60 U/mL heparin (Calbiochem)
added was injected subcutaneously into WT and S100A10?/?C57BL/6
mice. After 7 days, the Matrigel plug was removed and processed for
Histochemistry and immunohistochemistry
Matrigel plugs were fixed in 10% formalin and embedded in paraffin.
Paraffin sections were deparaffinized, blocked with horse serum (1:20;
Invitrogen) and incubated with an antibody against Mac-3 (55092; BD
Biosciences), F4/80 (MCA497; Abd Serotec), or normal mouse IgG1 (as
control; BD Biosciences) at room temperature overnight. Subsequently, a
peroxidase diaminobenzidine detection system (Dako North America) was
stained with hematoxylin. Sections were mounted using Cytoseal 60
mounting media (Richard-Allen Scientific) and viewed using either a
40?/0.75 NA or 10?/0.3 NA objective lens. Images were captured by the
Nikon Eclipse E600 microscope using a Nikon DXM1200F camera. Digital
acquisition of the images was performed using ACT-1 v2.7 software
(Nikon). Figures were generated using Adobe Photoshop CS3 v10 (Adobe
Systems Incorporated). The data were quantified using Image J v1.42q
software (National Institutes of Health).
Statistical significance was determined by Student t test or one-way analysis of
variance with Tukey multiple comparisons. Results were regarded as significant
S100A10AND MACROPHAGE INVASION1137BLOOD, 19AUGUST 2010?VOLUME 116, NUMBER 7
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1146 O’CONNELLet alBLOOD, 19AUGUST 2010?VOLUME 116, NUMBER 7
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online April 27, 2010
2010 116: 1136-1146
Paul A. O'Connell, Alexi P. Surette, Robert S. Liwski, Per Svenningsson and David M. Waisman
S100A10 regulates plasminogen-dependent macrophage invasion
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