Chemerin Peptides Promote

Phagocytosis in a ChemR23- and

Syk-Dependent Manner
This information is current as
of August 16, 2010 Jenna L. Cash, Annabel R. Christian and David R.
Greaves

J. Immunol. 2010;184;5315-5324; originally published

online Apr 2, 2010;
doi:10.4049/jimmunol.0903378
http://www.jimmunol.org/cgi/content/full/184/9/5315

Downloaded from www.jimmunol.org on August 16, 2010

Supplementary http://www.jimmunol.org/cgi/content/full/jimmunol.0903378/D
Data C1

References This article cites 50 articles, 24 of which can be accessed free

at: http://www.jimmunol.org/cgi/content/full/184/9/5315#BIBL

Subscriptions Information about subscribing to The Journal of Immunology is

online at http://www.jimmunol.org/subscriptions/

Permissions Submit copyright permission requests at

http://www.aai.org/ji/copyright.html

Email Alerts Receive free email alerts when new articles cite this article. Sign
up at http://www.jimmunol.org/subscriptions/etoc.shtml

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc., 9650
Rockville Pike, Bethesda, MD 20814-3994.
Copyright ©2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
 The Journal of Immunology

Chemerin Peptides Promote Phagocytosis in a ChemR23- and

Syk-Dependent Manner

Jenna L. Cash, Annabel R. Christian, and David R. Greaves

Chemerin peptides represent a recently identified component of the endogenous anti-inflammatory network that act via the
G protein-coupled receptor ChemR23. The role of the chemerin peptide/ChemR23 pathway in phagocytosis, the clearance of
apoptotic cells (efferocytosis), and the resolution of inflammation is unknown. In this article, we report that low picomolar
concentrations of the chemerin peptide chemerin15 (C15) enhance macrophage (MF) phagocytosis of microbial particles and
apoptotic cells by up to 360% in vitro. These prophagocytic effects of C15 are significantly impaired in ChemR232/2 MFs and are
associated with increased actin polymerization and localization of F-actin to the phagocytic cup. Importantly, pharmacological
inhibition of Syk activity completely abrogates the prophagocytic activities of C15 and associated changes in actin polymerization
and phagocytic cup formation, suggesting that C15 promotes phagocytosis by facilitating phagocytic cup development in a Syk-
dependent manner. During peritoneal inflammation, C15 administration (8 pg/mouse) enhances microbial particle clearance and
apoptotic neutrophil ingestion by MFs in wild-type but not ChemR232/2 mice, such that levels of apoptotic and necrotic cells at
the inflammatory site are profoundly reduced. In contrast, neutralization of endogenous chemerin species during peritoneal

Downloaded from www.jimmunol.org on August 16, 2010

inflammation significantly impairs MF ingestion of apoptotic neutrophils and zymosan. Our data identify a key role of the
chemerin peptide/ChemR23 axis in the efficient clearance of foreign material, efferocytosis, and, hence, the resolution of in-
flammation. Manipulation of the chemerin peptide/ChemR23 axis may represent a novel therapeutic approach for the treatment
of inflammatory pathologies, especially if failure to efficiently clear phagocytic targets has been implicated in their pathogenesis.
The Journal of Immunology, 2010, 184: 5315–5324.

M
acrophages (MFs) are innate immune cells that can because necrotic cell ingestion elicits MF activation, and necrotic
recognize, phagocytose, and kill microbial pathogens; cell lysis releases cytotoxic, proinflammatory, and immunogenic
as such, they represent an important component of the material (11–17). Thus, failure to efficiently clear apoptotic cells
body’s defense against infection (1–3). Efficient clearance of favors inflammatory and autoimmune reactions rather than in-
pathogenic material by MFs is important in limiting the magni- flammatory resolution, and it promotes a persistent state of in-
tude and duration of the ensuing inflammatory response, although flammation seen in systemic lupus erythematosus (SLE), as well as
recognition and engulfment of microbial particles by MFs typi- in atherosclerosis and diabetes mellitus (18–22). Studies in exper-
cally results in their activation and the secretion of inflammatory imental animal models combined with clinical evidence from
cytokines (4, 5). In contrast, MF ingestion of apoptotic cells is human inflammatory diseases, including SLE, highlight the im-
nonphlogistic (noninflammatory) because it does not provoke in- portance of efficient phagocytosis of apoptotic material during in-
flammatory mediator expression and is associated with active flammation and also suggest its potential as a therapeutic target for
suppression of proinflammatory mediator release and upregulation the treatment of certain inflammatory diseases (12, 19, 20, 22–24).
of anti-inflammatory mediator expression, including TGF-b (6–9). We previously reported that picogram quantities of C-terminal
Thus, apoptotic cell phagocytosis (efferocytosis) plays an impor- peptides derived from the chemoattractant chemerin, in particular
tant role in the resolution of inflammation and the maintenance of chemerin15 (C15; AGEDPHGYFLPGQFA), inhibit MF activation
peripheral immune tolerance (1, 2, 7, 9, 10). Inefficient clearance and suppress peritonitis induced by the yeast cell wall component
of apoptotic cells, resulting in the accumulation of secondary zymosan (25). We hypothesized that chemerin peptides may
necrotic cells, can result in the exacerbation of inflammation, achieve this effect, in part, by enhancing MF phagocytosis of the
inciting stimulus, zymosan, and/or modulating the nonphlogistic
Sir William Dunn School of Pathology, University of Oxford, Oxford, United King- ingestion of apoptotic cells at the site of inflammation.
dom In this study, we show for the first time that chemerin peptides
Received for publication October 14, 2009. Accepted for publication March 3, 2010. potently and profoundly enhance MF clearance of microbial
This work was supported by the British Heart Foundation Grants RG/05/011 (to particles and apoptotic cells in a nonphlogistic and ChemR23-
D.R.G.) and FS/05/121 (to J.L.C.). dependent manner, a process that requires Syk-dependent changes
Address correspondence and reprint requests to Dr. David R. Greaves, Sir William in F-actin polymerization and phagosome formation.
Dunn School of Pathology, Oxford University, South Parks Road, Oxford OX1 3RE,
United Kingdom. E-mail address: [email protected]
The online version of this paper contains supplemental material. Materials and Methods
Abbreviations used in this paper: ChAb, neutralizing anti-chemerin Ab; C15, chem- Animals
erin15; C15-S, scrambled C-15 peptide; GeoMFI, geometric mean fluorescence in-
tensity; MF, macrophage; OpZ, serum-opsonized zymosan; PEC, peritoneal exudate All animal studies were conducted with ethical approval from the Dunn
cell; PI, propidium iodide; PIC, piceatannol; RRI, relative recognition index; SLE, School of Pathology Local Ethical Review Committee and in accordance
systemic lupus erythematosus. with the U.K. Home Office regulations (Guidance on the Operation of
Animals, Scientific Procedures Act, 1986). ChemR232/2 mice on an
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Sv129Ev background were a kind gift of Takeda (Cambridge, U.K.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903378
5316 CHEMERIN PEPTIDES ARE POTENT PROPHAGOCYTIC MEDIATORS

MF culture and zymosan phagocytosis assays Calculation of the RRI

MFs were obtained and cultured, as previously described (25). Briefly, RRI was calculated using the equation RRI = % 3 (unknown geometric
Bio-Gel P100 polyacrylamide beads (1 ml 2% w/v in PBS; Bio-Rad, mean fluorescence intensity [GeoMFI])/(vehicle GeoMFI), where % is the
Hemel Hempstead, U.K.) were injected into the peritoneal cavities of 8– percentage of zymosan-FITC+ MFs, OpZ-FITC+ MFs, or CFSE-labeled
12-wk-old C57BL/6 mice. Mice were killed 4 d later, and peritoneal ex- apoptotic cell+ MFs, and “unknown” GeoMFI is the GeoMFI of C15-,
udate cells (PECs) were collected in PBS 2 mM EDTA (Lonza, Slough, C15-S–, or chemerin-treated samples.
U.K.). Harvested cells were centrifuged and resuspended in OptiMEM
supplemented with 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml Detection of secreted proteins by ELISA and Luminex
streptomycin (all from Invitrogen, Paisley, U.K.). Cells were plated in 24- TNF-a, IL-6 and -12 p40, and JE (MCP-1) concentrations in cell super-
well suspension plates (0.4 3 106/well; Greiner Bio-One, Stonehouse, natants were assessed by Luminex multiplex bead assay (Bio-Rad), ac-
U.K.) and allowed to adhere for 1 h at 37˚C in a humidified atmosphere cording to the manufacturer’s instructions. Detection limits were 5–20 pg/
containing 5% CO2 to purify MF populations by adherence. Nonadherent ml, depending on the cytokine in question. TGF-b concentrations were
cells and Bio-Gel beads were removed by washing with PBS after 1 h. determined by ELISA (R&D Systems; detection limit was 5 pg/ml).
MFs were preincubated with 10213–1029 M C15 (Biosynthesis, Lewis-
ville, TX), C15-S (scrambled C15 peptide; GLFHDQAGPPAGYEF; Bio- F-actin staining and phagocytic cup formation
synthesis), or chemerin (R&D Systems, Minneapolis, MN) for 45 min at MFs (1 3 106/well) were plated in six-well tissue culture plates (Costar)
37˚C and then transferred to ice for 10 min prior to the addition of zy- containing a 22-mm thickness 1 coverslip (VWR, Leighton, U.K.). Following
mosan-FITC (Invitrogen) or serum-opsonized zymosan (OpZ)-FITC in MF adherence and removal of nonadherent cells, MFs were treated with C15
a 10:1 ratio (zymosan: MF). Cells were challenged with zymosan/OpZ- for 45 min at 37˚C; challenged with zymosan-FITC, as described above, for
FITC for 5, 15, 30, 45, or 60 min at 37˚C, and the assay was terminated by 5 min; and then transferred to ice where media were removed; cells were
transferring cells to ice and vigorously washing off noningested material
washed; and actin staining solution (4% v/v formalin in PBS, 5 U/ml Alexa
with ice-cold PBS four times. Cells were lifted from tissue culture plastic
Fluor 546-phalloidin [Invitrogen] and 5 mg/ml lysopalmitoylphosphati-
using lidocaine-EDTA (Sigma-Aldrich, St. Louis, MO) and scraping and dylcholine [Sigma-Aldrich]) was added for 20 min. After 20 min on ice, the
were fixed using 4% v/v formalin. In control experiments, fluorescence staining solution was removed, and cells were washed three times with cold
from extracellularly bound FITC particles was neutralized by quenching PBS. Coverslips were mounted, and polymerized actin, phagocytic cups, and
with trypan blue. The addition of trypan blue and the reconstruction of phagosomes were visualized by confocal microscopy. Early and late phagocytic

Downloaded from www.jimmunol.org on August 16, 2010

individual Z-series confocal images confirmed that extracellularly bound
cups and early phagosomes were judged from phalloidin-Alexa Fluor 546–
particles were not present following stringent washing. Opsonized FITC-
stained images. Early phagocytic cups were defined as the presence of an actin
zymosan was generated by incubating zymosan-FITC (20 mg/ml) and cup-like structure around ∼10–25% of the circumference of the zymosan par-
100% mouse serum in a 1:1 v/v ratio for 1 h at 37˚C with gentle shaking. ticle. Late phagocytic cups were defined as the presence of an actin cup around
The OpZ was then washed three times in PBS to remove excess serum. ∼50% of the zymosan particle. Phagosomes were defined by 100% enclosure of
Where appropriate, the Syk inhibitor, piceatannol (PIC; 10 mM; Tocris the zymosan particle by F-actin where early phagosomes were located at the
Cookson, Bristol, U.K.), was administered 20 min before C15 pre- cells periphery, and late phagosomes were found near the center of the cell.
treatment. Results obtained with PIC were confirmed using a second Syk
inhibitor, BAY 61-3606 (1 mM; Sigma-Aldrich; data not shown). Lack of In vivo phagocytosis
cytotoxicity of 10 mM PIC and 1 mM BAY 61-3606 was determined using
CellTiter-Glo assay (Promega, Madison, WI). In separate experiments, the C57BL/6J mice were administered 500 ml C15 (0.32 ng/kg), C15-S (0.32 ng/
effect of C15 treatment on MF binding of zymosan was determined by kg), neutralizing anti-murine Chemerin Ab (ChAb, R&D Systems; 100 ng/
pretreating with C15 at 37˚C and then performing the above phagocytosis mouse), control IgG (R&D Systems; 100 ng/mouse), or vehicle (PBS; Lonza)
assay at 4˚C. All in vitro experiments were performed in OptiMEM sup- i.p. 1 h before injection of 500 ml 10 mg unlabeled zymosan (for Ly6G+ cell
plemented as described above, and all peptides were reconstituted in filter- phagocytosis; Sigma Aldrich) or zymosan-FITC (for zymosan phagocytosis;
sterilized PBS 0.1% BSA. Data from all in vitro phagocytosis assays are Invitrogen). For Ly6G+ cell phagocytosis, PECs were collected by lavage with
expressed as a relative recognition index (RRI), as described below. 5 ml PBS, 2 mM EDTA after 2, 4, 8, 16, or 24 h. For zymosan phagocytosis,
For collection of MF supernatants, the above zymosan-phagocytosis PECs were collected after 0.5, 1, 2, or 4 h. PECs (1 3 106) were fixed (4% v/v
assays were carried out in six-well plates (1 3 106 cells/well; Costar, formalin), permeabilized, and blocked (3% BSA [Sigma-Aldrich], 0.1%
Loughborough, U.K.) with C15 pretreatment (45 m), followed by stimu- Triton X-100 [Sigma-Aldrich], 2.5 mg/ml 2.4G2 FcgII/III [AbD Serotec,
lation with unlabeled zymosan (Sigma-Aldrich) in a 10:1 ratio for 15 min. Kidlington, U.K.]) prior to staining with F4/80-Alexa Fluor 647 (4 mg/ml; BD
Noningested zymosan was removed by washing on ice with cold PBS, Biosciences, Oxford, U.K.) and Ly6G-PE (2 mg/ml, only for Ly6G+ cell
after which 2 ml supplemented OptiMEM was added to each well, and phagocytosis; BD Biosciences). Zymosan+, F4/80+, and Ly6G+F4/80+ cells,
cells were incubated for 15 h at 37˚C. MF supernatants were then col- as a percentage of the total PECs and total F4/80+ cells, were obtained using
lected and stored at 280˚C until use in Luminex assays (Bio-Rad). FlowJo (Tree Star, Ashland, OR).
ChAb effect on leukocyte recruitment
Measurement of apoptosis
C57BL/6J mice were administered 500 ml ChAb (100 ng/mouse) or isotype
Jurkat cells were cultured in RPMI 1640 (PAA Laboratories, Yeovil, U.K.) control IgG (100 ng/mouse; both from R&D Systems) i.p. 1 h before i.p.
with 10% FCS (PAA Laboratories), 1 mM sodium pyruvate (Invitrogen), injection of 500 ml 10 mg zymosan (Sigma-Aldrich). After 2, 4, 8, 16, 24, or
10 mM HEPES (Invitrogen), 0.05 mM b-mercaptoethanol (Sigma-Al- 48 h and humane sacrifice, PECs were collected by peritoneal lavage with 5
drich), 50 U/ml penicillin, and 50 mg/ml streptomycin. Apoptotic Jurkat ml sterile PBS-2 mM EDTA. Total cell counts were determined, and the
cells were generated by UV irradiation (20–30 mJ/cm2), followed by a 4-h percentage of neutrophils and monocytes in the peritoneal lavage fluid were
incubation at 37˚C in serum-free media. The percentage of apoptosis was obtained by FACS analysis, as previously described (25). Briefly, cells (1 3
assessed by staining 5 3 105 cells for 15 min with 0.5 mg propidium iodide 105) were blocked with anti-mouse 2.4G2 FcgII/III (2.5 mg/ml) for 10 min
(PI; Sigma-Aldrich) and 5 ml Annexin V-FITC (BD Biosciences) and and stained for 15 min with FITC-conjugated anti-mouse 7/4 (10 mg/ml;
performing FACS analysis in accordance with published protocols. Typi- AbD Serotec) and PE-conjugated anti-mouse Ly6G (4 mg; BD Bio-
cally, 70% of Jurkat cells had undergone apoptosis using this protocol; sciences). Gates were constructed around two populations: the neutrophils
therefore, they were Annexin-V+PI2. (7/4high, Ly6Ghigh) and inflammatory monocytes (7/4high, Ly6Glow).
Apoptotic cell phagocytosis assays Statistics
Following the induction of apoptosis, Jurkat cells were stained with CFSE (1 Values for all measurements are expressed as mean 6 SEM. The Student t test,
mM; Sigma-Aldrich) for 5 min and delivered to the MFs in a 1:1 ratio after one-way ANOVAwith the Dunnett post hoc test, and two-way ANOVAwith the
45 min of pretreatment with 10214–10211 M C15, C15-S, or chemerin. Bonferroni post hoc test were performed to determine the levels of significance
MFs were exposed to apoptotic Jurkat cells for 1 h, and the assay was between groups using GraphPad Prism 5.0 software (GraphPad, San Diego, CA).
terminated as described above. In some cases, the serine and cysteine
protease inhibitor leupeptin (15 mg/ml; Sigma-Aldrich) was added 20 min Results
before chemerin pretreatment. For collection of MF supernatants, apo- C15 promotes phagocytosis of zymosan and OpZ in vitro
ptotic cell-phagocytosis assays were carried out, as described above but in
six-well plates (1 3 106 cells/well; Costar). MF supernatants were col- To determine the effect of C15 and control agents on MF
lected 15 or 24 h (TGF-b only) after removal of the phagocytic targets. phagocytosis of zymosan, MFs were pretreated with 0.1 pM–1
The Journal of Immunology 5317

nM C15 (AGEDPHGYFLPGQFA), C15-S (GLFHDQAGPPA- MF activation and the secretion of proinflammatory cytokines
GYEF; scrambled C15 peptide), or chemerin for 45 min and (5, 26, 27). Given the prophagocytic effect of C15 on zymosan
challenged with zymosan-FITC over a 5–60-min time course. C15 clearance, we determined whether C15 induces corresponding
elicited a dose-dependent increase in zymosan phagocytosis that increases in inflammatory cytokine release in association with its
was optimal at a dose of 10 pM (360% increase) at the 15-min effects on zymosan phagocytosis. In contrast, we found that
time point (Fig. 1A, 1C, Supplemental Fig. 1A). Zymosan particles C15-treated MFs, which have phagocytosed up to 360% more
were readily phagocytosed by MFs without opsonization; how- zymosan, released reduced levels of proinflammatory cytokines
ever, particles are unlikely to exist for extended periods in vivo and chemokines, including TNFa (53% decrease), IL-6 (48%
without becoming opsonized. Therefore, we tested the effect of decrease), IL-12 p40 (47% decrease), and JE (64% decrease), 15 h
C15 on phagocytosis of OpZ. C15 induced a dose- and time- postphagocytosis (Fig. 1E). These data demonstrate that low pi-
dependent increase in OpZ phagocytosis that was optimal with comolar doses of C15 peptide enhance zymosan phagocytosis in
10 pM C15 at the 15-min time point (230% increase; Fig. 1B, 1D). a nonphlogistic manner. To our knowledge, C15 peptide is the first
Maximal phagocytosis occurred in vehicle-treated MFs at mediator shown to induce phagocytosis of an inflammatory
45-min postzymosan or OpZ challenge, whereas C15 (10 pM)- stimulus in a nonphlogistic manner. This may halt inflammation
pretreated MFs reached the saturation point at 15 min (Fig. 1C, by removing the inciting stimulus and preventing the release of
1D). At the 45- and 60-min time points, when vehicle- and C15- further proinflammatory mediators by MFs.
treated MF phagocytosis had reached a plateau, a 45% and 34%
C15 promotes nonphlogistic MF ingestion of apoptotic cells
increase in zymosan and OpZ phagocytosis with C15 was noted
in vitro
(Fig. 1C, 1D). Thus, picomolar concentrations of C15 peptide
enhanced the rate of MF zymosan uptake and increased overall MF phagocytosis of apoptotic cells is the classic example of
MF phagocytic capacity for zymosan. The parent molecule, phagocytic clearance without the provocation of an inflammatory
chemerin, and the control peptide, C15-S, had no effect on zy- response and is an important determinant of the resolution of in-

Downloaded from www.jimmunol.org on August 16, 2010

mosan or OpZ phagocytosis by MFs (Fig. 1A, 1B). flammation (7, 9, 28). To probe the effect of chemerin-derived
peptides on apoptotic cell phagocytosis in vitro, MFs were pre-
C15 promotes zymosan phagocytosis in a nonphlogistic manner treated with C15 (0.01–10 pM) for 45 min and then exposed to
Phagocytosis of zymosan is a widely used model of microbial CFSE-labeled apoptotic Jurkat cells for 1 h. C15 enhanced MF
recognition by the innate immune system that typically results in phagocytosis of apoptotic cells with an optimal dose of 1 pM;

FIGURE 1. C15 promotes nonphlogistic phagocytosis of zymosan and OpZ in vitro. MFs were pretreated with C15, C15-S, chemerin (0.1 pM–1 nM), or
vehicle for 45 min, followed by a 15-min challenge with zymosan-FITC (A) or OpZ-FITC (B). MFs were pretreated with C15 (10 pM) or vehicle for 45 min,
followed by challenge with zymosan-FITC (C) or OpZ-FITC (D) for 5, 15, 30, 45, or 60 min. Noningested phagocytic targets were removed by thorough
washing, and quantification of phagocytosed zymosan was carried out by FACS analysis. RRI = percentage of zymosan-FITC+ MFs 3 GeoMFI normalized to
vehicle-treated samples; see Materials and Methods for a detailed description of the RRI. E, Effect of C15 on inflammatory cytokine production. MF su-
pernatants were collected 15 h after ingestion of zymosan-FITC. TNF-a, JE, and IL-6 and -12 p40 levels were determined in cell supernatants using multiplex
bead assays. Graphs show mean values 6 SEM from four to eight independent experiments. pp , 0.05; ppp , 0.01; pppp , 0.001, relative to vehicle-treated
samples. A and B, One-way ANOVA with the Dunnett post hoc test. C and D, Two-way ANOVA with Bonferroni post hoc test. E, Student t test.
5318 CHEMERIN PEPTIDES ARE POTENT PROPHAGOCYTIC MEDIATORS

a 247% increase in phagocytosis was observed (Fig. 2A, Supple- were completely abolished in ChemR232/2 MFs, indicating that
mental Fig. 1B). However, similar prophagocytic effects were still C15-enhanced clearance of zymosan requires the involvement of
seen with 0.1 pM C15: doses that are 103 and 1003 lower than ChemR23 (Fig. 3A). In contrast, ChemR232/2 MFs displayed
that required for optimal enhancement of zymosan and OpZ a 71% reduction in C15 enhancement of OpZ phagocytosis at the
phagocytosis (Fig. 1A, 1B). We postulate that reduced C15 con- optimal 10-pM dose (Fig. 3B). These data suggest the existence of
centrations are required for optimal enhancement of apoptotic cell an additional C15R that may be required for optimal enhancement
phagocytosis because C15 may act in concert with prophagocytic of OpZ clearance in vitro (Fig. 3B). Furthermore, the level of zy-
mediators that are known to be released by apoptotic cells, in- mosan (+/+, 22 RRI; 2/2, 20 RRI; Fig. 3A) and OpZ (+/+, 49 RRI; 2/2,
cluding nucleotides and Annexin A1 (29–31). Intriguingly, the 51 RRI; Fig. 3B) phagocytosis in untreated wild-type and
parent protein chemerin, which failed to enhance zymosan in- ChemR232/2 MFs was of equivalent magnitude.
gestion (Fig. 1A), promoted phagocytosis of apoptotic cells with an We next assessed the involvement of ChemR23 in mediating C15
optimal dose of 0.1 pM (Fig. 2B). These prophagocytic effects were enhancement of apoptotic cell clearance, finding that the pro-
abolished when chemerin was administered in the presence of the phagocytic effects of C15 on apoptotic cell phagocytosis were
serine and cysteine protease inhibitor (leupeptin), demonstrating completely abolished in ChemR232/2 MFs, indicating that C15-
that chemerin promotes phagocytosis in a proteolysis-dependent enhanced clearance of this phagocytic target also requires the
manner (Fig. 2B). These data mirror our previous observations that involvement of ChemR23 (Fig. 3C). In addition, the basal level of
chemerin suppresses MF activation in a proteolysis-dependent apoptotic cell ingestion in ChemR232/2 MFs (14.5 RRI) was
manner (25), suggesting that classically activated MFs and apo- significantly lower than that of wild-type MFs (22.7 RRI), pro-
ptotic cells are capable of releasing proteases that cleave chemerin viding the first indication of a phenotype for ChemR232/2 MFs
to generate anti-inflammatory and prophagocytic peptides. We in the absence of an inflammatory stimulus (25).
postulate that chemerin is cleaved during this assay by apoptotic
C15 enhances MF zymosan phagocytosis during peritonitis
cell-derived proteases, because chemerin did not exert any pro-

Downloaded from www.jimmunol.org on August 16, 2010

phagocytic effects on zymosan and OpZ phagocytosis (Fig. 1A, We previously showed that C15 ameliorates zymosan-induced
1B). In addition, C15-treated MFs, which had ingested up to 2.5- peritonitis, reducing leukocyte recruitment by up to 65%, with
fold more apoptotic cells, released reduced levels of proin- a concomitant suppression of inflammatory mediator expression
flammatory cytokines TNF-a (63%; Fig. 2C) and JE (54%; Fig. (25). Having demonstrated that C15 is capable of enhancing zy-
2D) and exhibited increased TGF-b expression (108%; Fig. 2E) mosan phagocytosis in vitro, we next assessed the effect of C15
with an optimal dose of 1 pM C15. (8 pg/mouse; 0.32 ng/kg) on MF zymosan phagocytosis in vivo in
the zymosan peritonitis model. PECs were analyzed for uptake of
ChemR232/2 MFs exhibit impaired phagocytosis in response zymosan-FITC and expression of the MF marker F4/80 (32). The
to C15 maximum level of MFs engaged in the clearance of zymosan
To assess the involvement of the G-protein coupled receptor occurred in control animals 1 h postzymosan challenge, where
ChemR23 in mediating C15 enhancement of zymosan and OpZ 23% of total cells were zymosan+F4/80+ (Fig. 4A, Supplemental
phagocytosis, we compared the effect of C15 on phagocytosis of Fig. 1C). A single 8-pg dose of C15 enhanced MF zymosan
these targets by wild-type and ChemR232/2 MFs at the 15-min time clearance by 38%, increasing the percentage of zymosan+F4/80+
point. The prophagocytic effects of C15 on zymosan phagocytosis PECs to 34% (Fig. 4A). C15 also enhanced MF zymosan

FIGURE 2. C15 and chemerin promote nonphlogistic phagocytosis of apoptotic cells in vitro. MFs were pretreated with vehicle, C15, or C15-S (0.01–10
pM) (A) or chemerin (0.01–10 pM) 6 leupeptin (10 mM) (B) for 45 min and then exposed to CFSE-labeled apoptotic Jurkat cells for 1 h. C–E, Effect of C15
on MF cytokine production following ingestion of apoptotic cells. TNF-a (C), JE (D), TGF-b (E), IL-6, and IL-12 p40 levels were determined in cell
supernatants 15 or 24 h (TGF-b) following phagocytosis of apoptotic cells using multiplex bead assays and ELISA. IL-6 and -12 p40 levels were below the
limit of detection for this assay (5 pg/ml). Noningested CFSE-labeled apoptotic cells and cell debris were removed by thorough washing, and quantification of
ingested CFSE-labeled apoptotic cells was carried out by FACS analysis. Bar graphs show mean values 6 SEM from four independent experiments. pp ,
0.05; ppp , 0.01; pppp , 0.001; relative to vehicle-treated (A, C–E) or leupeptin-treated (B) samples. A, One-way ANOVA with the Dunnett post hoc test. B,
Two-way ANOVA with Bonferroni post hoc test. C–E, Student t test.
The Journal of Immunology 5319

point by 88% (Fig. 4C, Supplemental Fig. 1D). C15 also increased
apoptotic neutrophil clearance at 2, 4, and 16 h (Fig. 4C, 4D),
whereas the control peptide C15-S was unable to promote Ly6G+
cell phagocytosis (Supplemental Fig. 2B).
C15 enhances MF phagocytosis during peritonitis through
ChemR23
We used ChemR232/2 mice to determine the ChemR23 de-
pendency of C15’s in vivo prophagocytic effects. C15 was unable
to enhance MF phagocytosis of apoptotic neutrophils or zymosan
in the absence of ChemR23, indicating that C15-enhanced clear-
ance of apoptotic neutrophils and zymosan requires the in-
volvement of ChemR23 (Fig. 4E, 4F). The basal level of MF
zymosan engulfment in ChemR232/2 mice (4.8% cells zymosan+
F4/80+) was indistinguishable from that of wild-type animals
(4.9% zymosan+F4/80+; Fig. 4E). In contrast, ChemR232/2 mice
exhibited a 27% deficit in apoptotic cell clearance compared with
their wild-type counterparts when measured as the percentage of
total cells. However, this effect is not seen when data are repre-
sented as the percentage of F4/80+ cells, indicating that the ob-
served effect could be an artifact caused by differences in the
numbers of F4/80+ cells in the peritoneal cavities of wild-type and

Downloaded from www.jimmunol.org on August 16, 2010

ChemR232/2 cells, rather than a direct effect of ChemR23 abla-
tion on phagocytosis (Fig. 4F). Taken together, our data support
the conclusion that the chemerin peptide C15 is a potent stimu-
lator of MF-mediated zymosan and apoptotic neutrophil phago-
cytosis in vivo through ChemR23.
C15 reduces levels of apoptotic and necrotic cells at the
inflammatory site
We have clearly demonstrated the ability of C15 to potently en-
hance the clearance of apoptotic neutrophils in vivo; therefore, we
next analyzed the effect of C15 on the level of apoptotic and
FIGURE 3. ChemR232/2 MFs exhibit impaired phagocytosis in re- necrotic cells at the inflammatory site during peritonitis. We used
sponse to C15. Wild-type and ChemR232/2 MFs on an Sv129 genetic the standard approach of PI and Annexin V staining to quantitate
background were pretreated with C15 (0.01 pM–1 nM) or vehicle for
apoptotic and necrotic cells in the peritoneal cavity. In control
45 min, followed by a 15-min challenge with zymosan-FITC (A) or OpZ-
FITC (B) or a 1-h exposure to CFSE-labeled apoptotic cells (C). Non-
animals 4 h postzymosan challenge, apoptotic cells represented
ingested phagocytic targets were removed by thorough washing, and 18% of total PECs, whereas necrotic cells accounted for 4%. When
quantification of ingested zymosan, OpZ, and apoptotic cells was carried out mice were pretreated with a single 8-pg dose of C15, the percentage
by FACS analysis. Graphs show mean values 6 SEM from four or five in- of apoptotic cells decreased by 31%, to 12% of total PECs (Fig. 5A,
dependent experiments. pp , 0.05; ppp , 0.01; pppp , 0.001, comparing Supplemental Fig. 1), whereas the necrotic cell population was
wild-type MF C15 responses with ChemR232/2 MF C15 responses; two- reduced by 53% to only 1.9% of total PECs (Fig. 5B, Supple-
way ANOVA with the Bonferroni post hoc test. +p , 0.05, comparing mental Fig. 1E). These data indicate that the prophagocytic effects
ChemR232/2 MFs treated with 10211 M C15 with vehicle-treated of C15 on apoptotic cell clearance result in a quantifiable re-
ChemR232/2 MFs; one-way ANOVA with the Dunnett post hoc test. duction in the level of apoptotic cells present at the inflammatory
site. This may then lead to the observed reduction in necrotic cells
ingestion at 2 and 4 h postzymosan administration (Fig. 4A, 4B); because fewer apoptotic cells escape phagocytic clearance and
this effect was not seen with the control peptide C15-S (Supple- undergo secondary necrosis.
mental Fig. 2A). Our data show for the first time that the chemerin peptide C15
can promote inflammatory resolution through its ability to potently
C15 enhances phagocytosis of apoptotic neutrophils during enhance apoptotic neutrophil phagocytosis during peritoneal in-
inflammation flammation in a ChemR23-dependent manner.
Constitutive apoptosis of neutrophils at inflammatory sites and
their subsequent clearance by MFs is a well-documented phe- Blockade of endogenous chemerin species results in impaired
nomenon and a critical determinant of inflammatory resolution. phagocytosis in vivo
Failure to efficiently clear apoptotic cells can exacerbate in- We assessed the role of endogenous chemerin peptides in
flammation because these cells will ultimately undergo secondary phagocytosis and leukocyte recruitment during peritoneal in-
necrosis and lyse to release their histotoxic, proinflammatory, and flammation using ChAb. We completed a zymosan-induced peri-
immunogenic material (12, 23, 33, 34). Therefore, we assessed the tonitis time-course in which animals received pretreatment with
effect of C15 on the clearance of apoptotic neutrophils in vivo control IgG or ChAb (100 ng/mouse) prior to a 0–48-h zymosan
during zymosan-induced peritoneal inflammation by analyzing challenge. Neutralization of endogenous chemerin species re-
PECs for the expression of the neutrophil marker Ly6G (35) and sulted in elevated neutrophil and monocyte recruitment at all time
the MF marker F4/80 (32). We found that a single 8 pg/mouse points, except for the earliest (2 h), with a maximum increase of
dose of C15 enhanced Ly6G+ cell phagocytosis at the 8-h time 170% (Fig. 6A, 6B). These data strongly support the existence of
5320 CHEMERIN PEPTIDES ARE POTENT PROPHAGOCYTIC MEDIATORS

Downloaded from www.jimmunol.org on August 16, 2010

FIGURE 4. C15 enhances MF phagocytosis of zymosan and apoptotic neutrophils during peritonitis through ChemR23. A and B, C57BL/6 mice were
dosed i.p with vehicle or C15 (0.32 ng/kg), followed by injection with zymosan-FITC 1 h later. PECs were harvested by peritoneal lavage at multiple time
points postzymosan-FITC injection, permeabilized, and stained with anti-F4/80-Alexa Fluor 647. Zymosan+F4/80+ cells are expressed as the percentage of
total cells (A) and as percentage of MFs (F4/80+ cells) (B), as determined by FACS analysis. C and D, C57BL/6 mice were dosed i.p with vehicle or C15
(0.32 ng/kg), followed by injection with zymosan 1 h later. PECs were harvested by peritoneal lavage at multiple time points postzymosan injection,
permeabilized, and stained with anti–Ly6G-PE and anti–F4/80-Alexa Fluor 647. Ly6G+F4/80+ cells are expressed as the percentage of total cells (C) and as
the percentage of MFs (F4/80+ cells) (D), as determined by FACS analysis. Graphs in A–D show mean values 6 SEM, with 5–10 mice per treatment group
(vehicle or C15) used at each time point. pp , 0.05; ppp , 0.01; pppp , 0.001, relative to vehicle-treated mice; two-way ANOVA with Bonferroni post
hoc test. Sv129 and ChemR232/2 (Sv129) mice were dosed i.p with vehicle or C15 (0.32 ng/kg), followed by injection with zymosan-FITC (E) or zymosan
(F) 1 h later. PECs were harvested by peritoneal lavage 4 h postzymosan-FITC administration to quantify zymosan+F4/80+ populations and 8 h post-
zymosan administration to evaluate Ly6G+F4/80+ cell populations. PECs were permeabilized, stained, and analyzed as described in A–D. Graphs show
mean values 6 SEM for 6–12 mice per group. ppp , 0.01; pppp , 0.001; ++p , 0.01, relative to vehicle-treated wild-type mice.

anti-inflammatory chemerin-derived peptides in vivo that are in- engulfment (37), it is likely that C15 enhances a common pathway
volved in modulating leukocyte recruitment during peritoneal of phagocytosis rather than having an effect on specific phagocytic
inflammation. In light of the potent prophagocytic effects of the receptor expression. This is particularly likely because FACS
chemerin peptide C15 on zymosan and apoptotic cell phagocy- analysis showed no significant C15-elicited changes in dectin-1
tosis, we postulated that endogenous chemerin peptides may expression during the course of the zymosan phagocytosis assay
modulate the inflammatory response by promoting clearance of (data not shown), and C15 exerted no effect on MF binding of
these phagocytic targets, in addition to suppressing leukocyte re- zymosan, as gauged by phagocytosis assays performed at 4˚C
cruitment. Neutralization of endogenous chemerin species re- (Supplemental Fig. 3).
sulted in a reduction of up to 46% in zymosan phagocytosis Remodeling of the actin cytoskeleton is a prerequisite for
(Fig. 6C, 6D) and Ly6G+ cell (neutrophil) phagocytosis (Fig. 6E, phagocytosis, enabling the formation of phagocytic cups and,
6F). These data provide evidence that endogenous chemerin subsequently, the phagosome to internalize the phagocytic target
species are an important component of the endogenous resolution (38–41). Therefore, we evaluated the effect of C15 on phagocytic
system, involved in mediating the clearance of microbial particles cup formation and F-actin polymerization and localization. In
and apoptotic cells in vivo. addition, we further probed the mechanism by which C15 pro-
motes phagocytosis using the Syk inhibitor PIC. Syk is a tyrosine
C15 enhances phagocytosis and phagocytic cup formation kinase required for lysosome–phagosome fusion in unopsonized
through Syk-dependent changes in F-actin polymerization yeast phagocytosis (42), but it is not required for yeast or apo-
Because C15 enhances the phagocytosis of multiple phagocytic ptotic cell internalization (43–45). In agreement with these ob-
targets, which are cargo for a variety of MF receptors, including servations, Syk inhibition had no effect on basal MF zymosan or
dectin-1 for zymosan ingestion (36) and CD36 for apoptotic cell apoptotic cell phagocytosis (Fig. 7A, 7B). However, pretreatment
The Journal of Immunology 5321

MFs predominantly possessed early phagocytic cups, whereas

higher levels of early phagosomes and late phagocytic cups were
observed following C15 treatment (Supplemental Fig. 4). These
data suggest that C15 may increase the kinetics of phagosome
formation in a Syk-dependent manner. C15 also enhanced F-actin
polymerization at the early phagocytic cup (Fig. 7D), the late
phagocytic cup (Fig. 7E), and the early phagosome (Fig. 7F). C15-
mediated alterations in local F-actin polymerization were in-
hibited by PIC, indicating an essential role for Syk in this process
FIGURE 5. C15 reduces levels of apoptotic and necrotic cells at the in- (Fig. 7D–F). In light of these observations, we assessed the effect
flammatory site. A and B, C57BL/6 mice were dosed i.p with vehicle or C15 of C15 on Syk activation, finding that C15 (10 pM) triggered
(0.32 ng/kg), followed by injection with 10 mg zymosan 1 h later. PECs were phosphorylation of Syk (Tyr352) in a time-dependent manner
harvested by peritoneal lavage 4 h postzymosan challenge, and the per- (Supplemental Fig. 5). Since phagocytosis, phagocytic cup for-
centages of live, apoptotic (A) and necrotic (B) cells were determined by mation, and F-actin polymerization at the phagocytic cups and
staining with annexin V-FITC and propidium iodide. Live cells were An- early phagosomes were enhanced by C15, and C15-mediated
nexin V2PI2, apoptotic cells were Annexin+PI2, and necrotic cells were effects were abrogated by the Syk inhibitor PIC, we suggest that
Annexin V+PI+. Graphs show mean values 6 SEM for five to seven mice per C15 enhances phagocytosis through Syk-mediated alterations in
treatment group. ppp , 0.01, relative to vehicle-treated mice; Student t test.
F-actin polymerization, which likely aid phagocytic cup formation
and, thus, enhances MF phagocytosis.

of MFs with PIC prior to C15 treatment resulted in complete

abrogation of C15-induced enhancement of zymosan (Fig. 7A) Discussion

Downloaded from www.jimmunol.org on August 16, 2010

and apoptotic cell phagocytosis (Fig. 7B). Therefore, these data In this study, we identify the chemerin peptide C15 as a new
demonstrate that C15 enhances zymosan and apoptotic cell mediator capable of promoting inflammatory resolution following
phagocytosis in a Syk-dependent manner. microbial challenge by stimulating MF clearance of the inciting
Subsequently, we probed the effect of C15 on phagocytic cup stimulus and apoptotic neutrophils in a nonphlogistic manner.
formation, finding that C15 treatment elicited a significant increase Efficient clearance of invading microorganisms at sites of in-
in the percentage of cells with phagocytic cups, from 25% of cells flammation is an indispensible role of MFs. The ability of C15 to
with vehicle treatment up to 40% of cells following treatment with potently enhance this process, in vitro and in vivo, further high-
10 pM C15. This increase in phagocytic cup formation was lights the dynamic nature of the MF and its capacity for phago-
completely abrogated by treatment with the Syk inhibitor PIC cytosis while providing novel insights into the regulation of innate
(Fig. 7C). Importantly, vehicle-treated and C15 + PIC-treated immune responses by endogenous anti-inflammatory pathways.

FIGURE 6. Neutralization of endoge-

nous chemerin species results in elevated
leukocyte recruitment and impaired phago-
cytosis in vivo. C57BL/6J mice were dosed
i.p with ChAb (100 ng/mouse) or control
IgG (100 ng/mouse), followed by injection
with zymosan (10 mg/cavity) 1 h later.
PECs were harvested at multiple time
points postzymosan injection (A, B, E, F) or
postzymosan-FITC injection (C, D). A and
B, Total PECs were quantified, and cellular
composition (neutrophils versus mono-
cytes) was determined by FACS analysis.
Cells were stained with Ly6G-PE and 7/4-
FITC, and gates were constructed around
two populations: neutrophils (A; 7/4high
Ly6Ghigh) and monocytes (B; 7/4highLy6-
Glow). C and D, PECs were permeabilized
and stained with anti–F4/80-Alexa Fluor
647, and the percentage of zymosan+F4/80+
cells was determined by FACS analysis. E
and F, PECs were permeabilized and
stained with anti–Ly6G-PE and anti–F4/80-
Alexa Fluor 647, and the percentage of
Ly6G+, F4/80+ cells was determined
by FACS analysis. Graphs show mean val-
ues 6 SEM for 5–10 mice per treatment
group (IgG control or ChAb) per time point.
pp , 0.05; ppp , 0.01; pppp , 0.001,
relative to control IgG-treated mice. Two-
way ANOVA with Bonferroni post hoc test.
5322 CHEMERIN PEPTIDES ARE POTENT PROPHAGOCYTIC MEDIATORS

Downloaded from www.jimmunol.org on August 16, 2010

FIGURE 7. C15 enhances phagocytosis and phagocytic cup formation through Syk-dependent changes in F-actin polymerization. A and B, MFs were
pretreated with vehicle or PIC (10 mM) for 30 min followed by treatment with C15 (10214–1029 M) or vehicle for 45 min. MFs were subsequently
challenged with zymosan-FITC for 15 min (A) or CFSE-labeled apoptotic Jurkat cells for 1 h (B). Samples were processed as in Fig. 1. C–F, MFs were
pretreated with vehicle or PIC for 30 min, followed by treatment with C15 (10 pM) or vehicle for 45 min and subsequent challenge with zymosan-FITC for
15 min (C) or 5 min (D–F). Cells were fixed and stained with Alexa Fluor 546-phalloidin to visualize polymerized actin, phagocytic cups, and phagosomes
and viewed using a 360 magnification. C, Early and late phagocytic cups were quantified and expressed as the percentage of total cells. Representative
images of early phagocytic cups (D), late phagocytic cups (E), and early phagosomes (F) are shown. Figures show mean values 6 SEM from three in-
dependent experiments. A and B, ppp , 0.01; pppp , 0.001, relative to PIC-treated samples; two-way ANOVA with the Bonferroni post hoc test. C, ppp ,
0.01, relative to vehicle-treated samples; #p , 0.05, relative to C15-treated samples. One-way ANOVA with the Bonferroni posttest. Results were confirmed
with a second Syk inhibitor BAY 61-3606 (data not shown).

Furthermore, this study indicates that manipulation of the chemerin Interestingly, the dose required for optimal enhancement of
peptide/ChemR23 axis may be an attractive avenue for therapeutic apoptotic cell phagocytosis in vitro by C15 was 10-fold lower than
intervention in inflammation. that required for other phagocytic targets. This observation may
Perhaps the most striking characteristic of C15 is the dose at reflect differences in the nature of these assays; phagocytosis of
which it exerts its effects. C15 suppresses leukocyte recruitment zymosan can be viewed as a proinflammatory event because MF
by up to 65% (25) and enhances phagocytosis in vivo by up to activation and the release of proinflammatory mediators ensues,
100% when administered in low picogram quantities. The lipid- whereas apoptotic cell engulfment is a nonphlogistic process.
resolution mediators resolvin E1, protectin D1, and lipoxin A4 Alternatively, apoptotic cells were shown to release prophagocytic
exert less dramatic effects at much higher doses (27). Although signals, including Annexin A1 and Annexin-derived peptides
these mediators enhance zymosan phagocytosis (27), it is un- (30), which could act in concert with the C15 peptide administered
known whether this occurs in a nonphlogistic manner, whereas the in this assay to promote MF clearance of apoptotic cells.
prototypical anti-inflammatory drug dexamethasone inhibits MF We have described for the first time a mechanism through which
zymosan phagocytosis (46). To our knowledge, C15 peptide is the C15 enhances MF phagocytosis, which involves Syk-dependent
first mediator shown to induce MF phagocytosis of an in- changes in phagocytic cup formation and F-actin localization.
flammatory stimulus in a nonphlogistic manner. This may halt Syk2/2 MFs, along with DAP122/2 MFs, have been shown to
inflammation by removing the inciting stimulus and preventing display a phenotype characterized by elevated proinflammatory
the release of further proinflammatory mediators by MFs. cytokine production in comparison with their wild-type counterparts
The Journal of Immunology 5323

(47). It is plausible that C15 and endogenous chemerin species may 9. Huynh, M. L., V. A. Fadok, and P. M. Henson. 2002. Phosphatidylserine-
dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the
harness this endogenous DAP12/Syk signaling pathway to elicit resolution of inflammation. J. Clin. Invest. 109: 41–50.
their anti-inflammatory and prophagocytic effects. If this is the 10. Neumann, J., S. Sauerzweig, R. Rönicke, F. Gunzer, K. Dinkel, O. Ullrich,
case, 0Syk-deficient mice could be rendered unresponsive to C15 M. Gunzer, and K. G. Reymann. 2008. Microglia cells protect neurons by direct
engulfment of invading neutrophil granulocytes: a new mechanism of CNS
treatment. immune privilege. J. Neurosci. 28: 5965–5975.
We have demonstrated that the anti-inflammatory and propha- 11. Silva, M. T., A. do Vale, and N. M. dos Santos. 2008. Secondary necrosis in
gocytic effects of C15 are absent in ChemR232/2 MFs and multicellular animals: an outcome of apoptosis with pathogenic implications.
Apoptosis 13: 463–482.
ChemR232/2 mice, but we were unable to detect a significant 12. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past:
difference between zymosan-elicited neutrophil and monocyte re- clearance of apoptotic cells regulates immune responses. Natl. Rev. Immunol. 2:
cruitment in wild-type and ChemR232/2 at the 4-h time point (25). 965–975.
13. Vandivier, R. W., P. M. Henson, and I. S. Douglas. 2006. Burying the dead: the
Therefore, it is intriguing that ChemR23 ablation also has no dis- impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory
cernable effect on phagocytosis in vitro or in vivo, when we have lung disease. Chest 129: 1673–1682.
14. Serhan, C. N., and J. Savill. 2005. Resolution of inflammation: the beginning
clearly demonstrated that neutralization of endogenous chemerin programs the end. Nat. Immunol. 6: 1191–1197.
species results in deficits in zymosan and apoptotic neutrophil 15. Haslett, C. 1992. Resolution of acute inflammation and the role of apoptosis in
phagocytosis. Our previous studies demonstrated that the full- the tissue fate of granulocytes. Clin. Sci. (Lond.) 83: 639–648.
16. Zhang, Z., G. Cherryholmes, and J. E. Shively. 2008. Neutrophil secondary necrosis
length protein chemerin is cleaved into anti-inflammatory peptides is induced by LL-37 derived from cathelicidin. J. Leukoc. Biol. 84: 780–788.
that act on ChemR23, in addition to other unidentified receptor(s) 17. Rock, K. L., and H. Kono. 2008. The inflammatory response to cell death. Annu.
(25). Thus, the absence of impaired phagocytosis in ChemR232/2 Rev. Pathol. 3: 99–126.
18. O’Brien, B. A., W. E. Fieldus, C. J. Field, and D. T. Finegood. 2002. Clearance
mice may be due to redundancy in the receptors for chemerin of apoptotic beta-cells is reduced in neonatal autoimmune diabetes-prone rats.
peptides in the anti-inflammatory/proresolution system. Additional Cell Death Differ. 9: 457–464.
19. Cohen, P. L., R. Caricchio, V. Abraham, T. D. Camenisch, J. C. Jennette,
receptors for chemerin, and potentially the chemerin peptides, are
R. A. Roubey, H. S. Earp, G. Matsushima, and E. A. Reap. 2002. Delayed ap-
GPR1 and CCRL2 (48, 49). Therefore, ablation of the chemerin optotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer

Downloaded from www.jimmunol.org on August 16, 2010

gene (RARRES-2) may result in mice with a more overt phenotype membrane tyrosine kinase. J. Exp. Med. 196: 135–140.
than the ChemR232/2 mice through the removal of chemerin-de- 20. Munoz, L. E., U. S. Gaipl, S. Franz, A. Sheriff, R. E. Voll, J. R. Kalden, and
M. Herrmann. 2005. SLE—a disease of clearance deficiency? Rheumatology
rived ligands for ChemR23 and other potential receptors. (Oxford) 44: 1101–1107.
Taken together, our novel data suggest that C15’s potent anti- 21. Gaipl, U. S., R. E. Voll, A. Sheriff, S. Franz, J. R. Kalden, and M. Herrmann.
2005. Impaired clearance of dying cells in systemic lupus erythematosus. Au-
inflammatory effects in vivo are a result of a combination of direct toimmun. Rev. 4: 189–194.
suppression of MF activation (25) and enhancement of MF 22. Gaipl, U. S., L. E. Munoz, G. Grossmayer, K. Lauber, S. Franz, K. Sarter,
phagocytosis of the inciting stimulus and apoptotic neutrophils. R. E. Voll, T. Winkler, A. Kuhn, J. Kalden, et al. 2007. Clearance deficiency and
systemic lupus erythematosus (SLE). J. Autoimmun. 28: 114–121.
We provide compelling evidence for the role of chemerin peptides 23. Schrijvers, D. M., G. R. De Meyer, M. M. Kockx, A. G. Herman, and
and ChemR23 in MF phagocytosis and the resolution of in- W. Martinet. 2005. Phagocytosis of apoptotic cells by macrophages is impaired
flammation. Therefore, manipulation of the chemerin peptide/ in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 25: 1256–1261.
24. Ait-Oufella, H., V. Pouresmail, T. Simon, O. Blanc-Brude, K. Kinugawa,
ChemR23 axis may represent a novel therapeutic approach for the R. Merval, G. Offenstadt, G. Lesèche, P. L. Cohen, A. Tedgui, and Z. Mallat.
treatment of inflammatory pathologies, such as atherosclerosis and 2008. Defective mer receptor tyrosine kinase signaling in bone marrow cells
promotes apoptotic cell accumulation and accelerates atherosclerosis. Arte-
SLE, in which failure to efficiently clear apoptotic cell debris has rioscler. Thromb. Vasc. Biol. 28: 1429–1431.
been implicated in their pathogenesis (50). 25. Cash, J. L., R. Hart, A. Russ, J. P. Dixon, W. H. Colledge, J. Doran,
A. G. Hendrick, M. B. Carlton, and D. R. Greaves. 2008. Synthetic chemerin-
derived peptides suppress inflammation through ChemR23. J. Exp. Med. 205:
Acknowledgments 767–775.
We thank E. McNeil, P. Taylor, and G. White for advice. 26. Damazo, A. S., S. Yona, R. J. Flower, M. Perretti, and S. M. Oliani. 2006. Spatial
and temporal profiles for anti-inflammatory gene expression in leukocytes during
a resolving model of peritonitis. J. Immunol. 176: 4410–4418.
Disclosures 27. Schwab, J. M., N. Chiang, M. Arita, and C. N. Serhan. 2007. Resolvin E1
and protectin D1 activate inflammation-resolution programmes. Nature 447:
The authors have no financial conflicts of interest. 869–874.
28. Ren, Y., and J. Savill. 1998. Apoptosis: the importance of being eaten. Cell
Death Differ. 5: 563–568.
References 29. Maderna, P., S. Yona, M. Perretti, and C. Godson. 2005. Modulation of
1. Metchnikoff, E. 1887. Ueber den Kampf der Zellen gegen Erysipelkokken, ein phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-
Beitrag zur Phagocytenlehre. Arch. Pathol. Anat. [Virchow’s Archiv.] 107: 209– treated macrophages and annexin-derived peptide Ac(2-26). J. Immunol. 174:
249. 3727–3733.
2. Gordon, S. 2007. The macrophage: past, present and future. Eur. J. Immunol. 37 30. Scannell, M., M. B. Flanagan, A. deStefani, K. J. Wynne, G. Cagney, C. Godson,
and P. Maderna. 2007. Annexin-1 and peptide derivatives are released by apo-
(Suppl. 1): S9–S17.
3. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- ptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages.
J. Immunol. 178: 4595–4605.
rophage activation. Natl. Rev. Immunol. 8: 958–969.
31. Elliott, M. R., F. B. Chekeni, P. C. Trampont, E. R. Lazarowski, A. Kadl,
4. Rossi, A. G., D. A. Sawatzky, A. Walker, C. Ward, T. A. Sheldrake, N. A. Riley,
S. F. Walk, D. Park, R. I. Woodson, M. Ostankovich, P. Sharma, et al. 2009.
A. Caldicott, M. Martinez-Losa, T. R. Walker, R. Duffin, et al. 2006. Cyclin-
Nucleotides released by apoptotic cells act as a find-me signal to promote
dependent kinase inhibitors enhance the resolution of inflammation by pro-
phagocytic clearance. Nature 461: 282–286.
moting inflammatory cell apoptosis. Nat. Med. 12: 1056–1064. 32. McKnight, A. J., A. J. Macfarlane, P. Dri, L. Turley, A. C. Willis, and S. Gordon.
5. Taylor, P. R., S. V. Tsoni, J. A. Willment, K. M. Dennehy, M. Rosas, H. Findon, 1996. Molecular cloning of F4/80, a murine macrophage-restricted cell surface
K. Haynes, C. Steele, M. Botto, S. Gordon, and G. D. Brown. 2007. Dectin-1 is glycoprotein with homology to the G-protein-linked transmembrane 7 hormone
required for beta-glucan recognition and control of fungal infection. Nat. Im- receptor family. J. Biol. Chem. 271: 486–489.
munol. 8: 31–38. 33. Baumann, I., W. Kolowos, R. E. Voll, B. Manger, U. Gaipl, W. L. Neuhuber,
6. Fadok, V. A., P. P. McDonald, D. L. Bratton, and P. M. Henson. 1998. Regulation T. Kirchner, J. R. Kalden, and M. Herrmann. 2002. Impaired uptake of apoptotic
of macrophage cytokine production by phagocytosis of apoptotic and post- cells into tingible body macrophages in germinal centers of patients with sys-
apoptotic cells. Biochem. Soc. Trans. 26: 653–656. temic lupus erythematosus. Arthritis Rheum. 46: 191–201.
7. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and 34. Ren, Y., J. Tang, M. Y. Mok, A. W. Chan, A. Wu, and C. S. Lau. 2003. Increased
P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro apoptotic neutrophils and macrophages and impaired macrophage phagocytic
inhibit proinflammatory cytokine production through autocrine/paracrine clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis
mechanisms involving TGF-beta, PGE2, and PAF. J. Clin. Invest. 101: 890–898. Rheum. 48: 2888–2897.
8. Fadok, V. A., D. L. Bratton, and P. M. Henson. 2001. Phagocyte receptors 35. Daley, J. M., A. A. Thomay, M. D. Connolly, J. S. Reichner, and J. E. Albina.
for apoptotic cells: recognition, uptake, and consequences. J. Clin. Invest. 108: 2008. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.
957–962. J. Leukoc. Biol. 83: 64–70.
5324 CHEMERIN PEPTIDES ARE POTENT PROPHAGOCYTIC MEDIATORS

36. Brown, G. D., and S. Gordon. 2001. Immune recognition. A new receptor for 44. Herre, J., A. S. Marshall, E. Caron, A. D. Edwards, D. L. Williams,
beta-glucans. Nature 413: 36–37. E. Schweighoffer, V. Tybulewicz, C. Reis e Sousa, S. Gordon, and G. D. Brown.
37. Fadok, V. A., M. L. Warner, D. L. Bratton, and P. M. Henson. 1998. CD36 is 2004. Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages.
required for phagocytosis of apoptotic cells by human macrophages that use Blood 104: 4038–4045.
either a phosphatidylserine receptor or the vitronectin receptor (alpha v beta 3). 45. Underhill, D. M., E. Rossnagle, C. A. Lowell, and R. M. Simmons. 2005. Dectin-
J. Immunol. 161: 6250–6257. 1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive
38. Sheterline, P., J. E. Rickard, and R. C. Richards. 1984. Fc receptor-directed oxygen production. Blood 106: 2543–2550.
phagocytic stimuli induce transient actin assembly at an early stage of phago- 46. Mlambo, G., and L. B. Sigola. 2003. Rifampicin and dexamethasone have
cytosis in neutrophil leukocytes. Eur. J. Cell Biol. 34: 80–87. similar effects on macrophage phagocytosis of zymosan, but differ in their ef-
39. Greenberg, S., J. el Khoury, F. di Virgilio, E. M. Kaplan, and S. C. Silverstein. fects on nitrite and TNF-alpha production. Int. Immunopharmacol. 3: 513–522.
47. Hamerman, J. A., N. K. Tchao, C. A. Lowell, and L. L. Lanier. 2005. Enhanced
1991. Ca(2+)-independent F-actin assembly and disassembly during Fc receptor-
toll-like receptor responses in the absence of signaling adaptor DAP12. Nat.
mediated phagocytosis in mouse macrophages. J. Cell Biol. 113: 757–767. Immunol. 6: 579–586.
40. Greenberg, S. 1999. Modular components of phagocytosis. J. Leukoc. Biol. 66: 48. Zabel, B. A., S. Nakae, L. Zúñiga, J.-Y. Kim, T. Ohyama, C. Alt, J. Pan, H. Suto,
712–717. D. Soler, S. J. Allen, et al. 2008. Mast cell-expressed orphan receptor CCRL2
41. May, R. C., and L. M. Machesky. 2001. Phagocytosis and the actin cytoskeleton. binds chemerin and is required for optimal induction of IgE-mediated passive
J. Cell Sci. 114: 1061–1077. cutaneous anaphylaxis. J. Exp. Med. 205: 2207–2220.
42. Majeed, M., E. Caveggion, C. A. Lowell, and G. Berton. 2001. Role of Src 49. Barnea, G., W. Strapps, G. Herrada, Y. Berman, J. Ong, B. Kloss, R. Axel, and
kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome- K. J. Lee. 2008. The genetic design of signaling cascades to record receptor
lysosome fusion. J. Leukoc. Biol. 70: 801–811. activation. Proc. Natl. Acad. Sci. USA 105: 64–69.
43. Canetti, C., B. Hu, J. L. Curtis, and M. Peters-Golden. 2003. Syk activation is 50. Aprahamian, T., I. Rifkin, R. Bonegio, B. Hugel, J. M. Freyssinet, K. Sato,
a leukotriene B4-regulated event involved in macrophage phagocytosis of IgG- J. J. Castellot, Jr., and K. Walsh. 2004. Impaired clearance of apoptotic cells promotes
coated targets but not apoptotic cells. Blood 102: 1877–1883. synergy between atherogenesis and autoimmune disease. J. Exp. Med. 199: 1121–1131.

Downloaded from www.jimmunol.org on August 16, 2010