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Induction of CCR2-dependent macrophage accumulation by oxidized phospholipids in the air-pouch model of inflammation.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 5, May 2009, pp 1362–1371
DOI 10.1002/art.24448
© 2009, American College of Rheumatology
Induction of CCR2-Dependent Macrophage
Accumulation by Oxidized Phospholipids in the
Air-Pouch Model of Inflammation
Alexandra Kadl, Elena Galkina, and Norbert Leitinger
and vascular cell adhesion molecule 1. OxPAPC increased the expression of the CCR2 ligands monocyte
chemotactic protein 1 (MCP-1), MCP-3, and MCP-5, as
well as RANTES and growth-related oncogene ␣
(GRO␣), while it down-regulated the expression of
CCR2 on macrophages. Moreover, oxidized
phospholipid–induced macrophage accumulation was
abrogated in CCR2ⴚ/ⴚ mice.
Conclusion. These data demonstrate that oxidized phospholipids trigger a type of inflammatory
response that leads to selective macrophage accumulation in vivo, a process relevant for the pathogenesis of
chronic inflammatory rheumatic diseases.
Objective. Macrophages are key players in the
pathogenesis of rheumatoid synovitis as well as in
atherosclerosis. To determine whether atherogenic oxidized phospholipids potentially contribute to synovial
inflammation and subsequent monocyte/macrophage
recruitment, we examined the effects of oxidized 1palmitoyl-2-arachidonoyl-sn-3-glycero-phosphorylcholine
(OxPAPC) on chemokine expression and leukocyte recruitment in a facsimile synovium in vivo using the
murine air-pouch model.
Methods. Air pouches were raised by 2 injections
of sterile air, and inflammation was induced by injecting either lipopolysaccharide (LPS) or OxPAPC into
the pouch lumen. Inflammation was assessed by analysis of inflammatory gene expression using reverse
transcription–polymerase chain reaction or immunohistochemical analysis, and leukocytes were quantified
in the lavage fluid and in the pouch wall after staining
with Giemsa or after enzymatic digestion followed by
fluorescence-activated cell sorter analysis.
Results. Application of OxPAPC resulted in selective recruitment of monocyte/macrophages into the airpouch wall, but not in the lumen. In contrast, LPS
induced both monocyte and neutrophil accumulation in
the pouch lumen as well as in the wall. LPS, but not
OxPAPC, induced the expression of adhesion molecules
E-selectin, P-selectin, intercellular adhesion molecule 1,
Patients with rheumatic diseases are at increased
risk of cardiovascular complications (1). In fact, cardiovascular disease accounts for nearly 40% of deaths in
rheumatoid arthritis (RA) patients (2); however, the
common pathogenic mechanisms remain unclear. We
hypothesized that “atherogenic” lipid oxidation products, which have been shown to cause vascular complications, also contribute to synovial inflammation.
The chronic inflammatory response in the RA
synovium shares many similarities with the inflamed
vascular wall during atherogenesis (3). Accumulation of
macrophages in the vascular wall is a hallmark of the
development of atherosclerotic lesions (4). In arthritic
joints, monocyte/macrophages accumulate in the synovial tissue (5), and neutrophils migrate into the synovial
cavity. In RA joints and in atherosclerotic lesions,
accumulating macrophages take up oxidized lipoproteins and develop into foam cells (6). Genetic deletions
of monocyte chemotactic protein 1 (MCP-1) or its
receptor CCR2 have been shown to affect macrophage
accumulation and joint destruction in experimental arthritis (7) and to decrease monocyte accumulation and
lesion formation in mice susceptible to atherosclerosis
(8,9). The factors that trigger monocyte recruitment and,
Dr. Kadl’s work was supported by a Postdoctoral Fellowship
from the Max Kade Foundation. Dr. Galkina’s work was supported by
a Scientist Development grant from the American Heart Association.
Dr. Leitinger’s work was supported by NIH grant R01-HL-084422-01.
Alexandra Kadl, MD, Elena Galkina, PhD, Norbert Leitinger, PhD: Robert M. Berne Cardiovascular Research Center,
University of Virginia, Charlottesville.
Address correspondence and reprint requests to Norbert
Leitinger, PhD, Robert M. Berne Cardiovascular Research Center,
University of Virginia, 409 Lane Road, Charlottesville, VA 22908.
E-mail: nl2q@virginia.edu.
Submitted for publication April 29, 2008; accepted in revised
form January 19, 2009.
1362
OXIDIZED PHOSPHOLIPIDS AND MACROPHAGE ACCUMULATION
thus, propagate chronic inflammatory processes are still
poorly understood.
Due to persistent increased production of freeradical species, lipid oxidation products accumulate at
sites of inflammation and exert a variety of biologic
activities. Increased concentrations of lipid oxidation
products have been found in synovial fluid (10) and
synovial membranes (6) from RA patients as well as in
atherosclerotic lesions (11). Biologically active oxidized
phospholipids that are present in oxidized lipoproteins
(12) and in the membranes of apoptotic cells (13) induce
inflammatory gene expression (14) that may contribute
to monocyte/macrophage recruitment and, thus, to the
progression of chronic inflammation.
Considerable advances have been made in dissecting the molecular structures of oxidized phospholipids, allowing for the experimental use of structurally
defined compounds rather than complex lipoproteins. In
vitro oxidation of the common phospholipid 1-palmitoyl2-arachidonoyl-sn-3-glycero-phosphorylcholine (PAPC)
yields a series of structurally identified oxidation products (OxPAPC) that have been shown to accumulate in
atherosclerotic lesions (11). The atherogenic potential of
OxPAPC has been demonstrated in cell culture studies,
as demonstrated by enhanced monocyte, but not neutrophil, binding to OxPAPC-stimulated endothelial cells, as
well as induction of MCP-1 and interleukin-8 (IL-8)
(15). Moreover, OxPAPC has been shown to induce
inflammatory gene expression in vivo when applied
intravenously or periadventitially in mice (14).
Whether oxidized phospholipids can induce
and/or propagate chronic inflammation in vivo remains
elusive due to the lack of data demonstrating oxidized
phospholipid–induced leukocyte accumulation in adequate animal models. In the present study, we used the
murine air-pouch model (16) to study the ability of
oxidized phospholipids to induce leukocyte recruitment
in vivo. Since the CCR2 chemokine receptor regulates
monocyte recruitment and has been shown to be involved in macrophage-dependent inflammatory responses in various chronic inflammatory diseases (7,9,
17–19), we also examined the involvement of CCR2 in
oxidized phospholipid–induced monocyte recruitment.
MATERIALS AND METHODS
Analysis of oxidized phospholipids. PAPC was purchased from Avanti Polar Lipids (Alabaster, AL) and oxidized
by exposure of dry lipid to air for 72 hours. The extent of
oxidation was monitored by positive ion–electrospray mass
spectrometry as described previously (11). Analysis of OxPAPC was performed by mass spectrometry using a Finnigan
1363
LCQ Classic mass spectrometer (Thermo Electron Corporation, San Jose, CA) connected to an HP HPLC 1100 series
system (Hewlett-Packard, McMinnville, OR). Phospholipids
were introduced to the ion source of the mass spectrometer by
flow injection, using a solvent consisting of acetonitrile/water/
formic acid (50:50:0.1 volume/volume/volume). Lipids were
stored at –70°C in chloroform and were used within 1 month
after testing. OxPAPC preparations were tested for endotoxin by the Limulus amebocyte assay (BioWhittaker, Walkersville, MD).
Preparation of the air-pouch model. The air-pouch
model was originally developed as a facsimile synovium for the
study of inflammatory processes that occur in RA. The model
allows the differential quantification of leukocyte species that
accumulate in the air-pouch wall (tissue) as well as those that
transmigrate into the air-pouch cavity (lavage), and it allows
the characterization of the chemokines and adhesion molecules responsible for diapedesis induced by a variety of inflammatory stimuli. Another advantage of this model over systemic
application is that it allows for local application of oxidized
phospholipids, thus avoiding rapid uptake by the liver and
degradation by serum enzymes.
Female CCR2⫺/⫺ mice (B6.129S4-Ccr2tm1Ifc/J) as well
as background control C57BL/6 mice (8–12 weeks old) were
obtained from The Jackson Laboratory (Bar Harbor, ME). All
animal experiments were approved by the Animal Care and
Use Committee of the University of Virginia. Air pouches
were raised by injection of 5 ml of sterile air into the skin on
the dorsum of each mouse on day 1 and were maintained by
reinjecting 3 ml of sterile air on day 4. Before the injection of
air, mice were briefly anesthetized with isoflurane. To avoid
pain after the creation of the pouch, animals received an
intraperitoneal injection of buprenorphine (2.0 mg/kg). On day
7, inflammation was induced by injecting into the air pouch
either 50 ␮g of LPS or 250 ␮g of OxPAPC, each of which was
dissolved in 1 ml of sterile 0.9% saline solution. Control
animals were injected with 1 ml of 0.9% saline solution. To
avoid stretching of the lining tissue, 1 ml of air was withdrawn
from the pouch prior to the injection of the test agent. Animals
were killed by CO2 asphyxiation and perfused with phosphate
buffered saline (PBS) containing 2% heparin via the right
ventricle. By extensive perfusion and microdissection, we ensured that the tissues we collected were not contaminated by
blood and nonadherent leukocytes.
Assessment of inflammation and inflammatory gene
expression. Inflammation was assessed by the following 4
methods: analyzing cells in the lavage fluid; counting the
leukocytes in the pouch wall after en face sections were prepared and stained with Giemsa; counting the leukocytes in the
membrane after enzymatic digestion followed by fluorescenceactivated cell sorting (FACS); and analyzing inflammatory
gene expression using reverse transcription–polymerase chain
reaction (RT-PCR) and immunohistochemistry. For the lavage, a shielded intravenous catheter (Becton Dickinson,
Mountain View, CA) was inserted into the air-pouch lumen,
which was then washed 3 times with 1 ml of 0.9% saline
solution while the catheter remained in place. The total fluid
was collected and used for cell counts.
To identify differences between LPS- and oxidized
phospholipid–induced inflammatory gene expression, the lining tissue from the air pouch was isolated, and initial screen-
1364
ings for gene expression were performed using a Mouse
Chemokines and Receptors Array (SuperArray Biosciences,
Frederick, MD), which allows screening for 67 genes that
encode chemokines and chemokine receptors (data not
shown). For time course experiments, OxPAPC or LPS was
injected into the air pouches, and animals were killed 1, 3, 6,
12, and 24 hours later. Messenger RNA (mRNA) was isolated
from the pouch tissues and analyzed by real-time PCR.
Tissue preparation and FACS analysis. For flow analysis, air-pouch tissue was enzymatically digested for 1 hour at
37°C with 125 units/ml of type XI collagenase, 60 units/ml of
type I-s hyaluronidase, 60 units/ml of DNase I, and 450
units/ml of type I collagenase (all from Sigma, St. Louis, MO)
in PBS containing 20 mM HEPES and then mashed through a
70-␮m strainer. This procedure yielded ⬃107 cells. For determination of the different leukocyte subtypes, the cell suspension was processed for FACS analysis using a FACSCalibur
(BD Immunocytometry Systems, San Jose, CA) or CyanADP
(Dako, Fort Collins, CO) system. Data were analyzed using
FlowJo software (Tree Star, Ashland, OR). Antibodies against
CD45, CD11b, Gr-1, CD3, and CD19 were obtained from
PharMingen (San Diego, CA), anti-CD68 from Serotec (Raleigh, NC), and anti-F4/80 from Caltag (South San Francisco,
CA). Cells expressing high levels of Gr-1 and CD11b and
being negative for F4/80 or I-Ab were characterized as neutrophils (20).
Monocyte/macrophages were defined as cells positive
for CD11b and expressing intermediate or low levels of Gr-1
(21,22). Cells that were negative for Gr-1 and CD11b were
defined as lymphocytes and were further characterized as
T cells (CD3⫹) or B cells (CD19⫹). We used spleen tissue to
confirm that the antigens were not degraded by the enzymes
used for tissue preparation (data not shown). Briefly, spleen
was cut into pieces and incubated separately either in enzyme
cocktail or in PBS. After 1 hour, cell suspensions from splenic
tissue were made and stained with antibodies. The expression
of antigens from the enzyme-treated cell suspensions was
compared with the antigen expression from untreated samples
(23).
RNA isolation and quantitative RT-PCR. Freshly harvested tissue was immediately immersed in ice-cold RNAlater
(Ambion, Austin, TX) and stored at ⫺70°C until the time of
analysis. For each determination, 100 ng of total RNA isolated
with TRIzol reagent (Invitrogen, Carlsbad, CA) was reverse
transcribed to complementary DNA (cDNA) using a GeneAmp RNA PCR core kit (Applied Biosystems, Foster City,
CA). Messenger RNA sequences of the investigated genes
were obtained from GenBank. PCR primers were designed
using Primer3 software from the Whitehead Institute for
Biomedical Research (Cambridge, MA). Amplified cDNA
regions were chosen to span 1 or more large introns in the
genomic sequence to avoid coamplification of genomic DNA.
Melting point analysis, agarose gel electrophoresis, and DNA
sequencing of the PCR products confirmed primer specificity.
(A list of the primer sequences used can be obtained from the
authors upon request.) Quantitative real-time PCR was performed using SYBR Green. Porphobilinogen deaminase or
␤2-microglobulin was used as an endogenous control. PCR
efficiency was determined for each primer pair from dilution
series of a typical sample of cDNA. Relative quantification of
gene expression was performed as described previously (24).
KADL ET AL
Immunohistochemistry. Immunostaining was performed on 5-␮m transverse cryosections. Sections were incubated with antibodies against F4/80 (eBioscience, San Diego,
CA), myeloperoxidase, or heme oxygenase 1 (HO-1; StressGen, San Diego, CA). Antigens were visualized with Alexa
488–conjugated secondary antibodies (Invitrogen). Sections
were counterstained with 4⬘,6-diamidino-2-phenylindole and
analyzed by epifluorescence microscopy (Olympus, Lake Success, NY).
Bone marrow–derived macrophages. Femurs and tibias from female C57BL/6 mice were harvested and flushed with
ice-cold PBS containing 20 units/ml of heparin. Bone marrow
cells were collected and incubated at room temperature with
sterile 0.843% ammonium chloride solution for 10 minutes to
lyse erythrocytes. A single-cell suspension of bone marrow
cells was obtained by straining the suspension through a 70-␮m
filter. Cells were seeded into 24-well culture dishes at a density
of 5 ⫻ 105/ml and maintained for 7 days in RPMI 1640 medium
(Sigma, St. Louis, MO) supplemented with 1% antibiotics,
10% fetal bovine serum (Gibco-BRL, Grand Island, NY), and
10% L929 cell supernatant as the source of macrophage
colony-stimulating factor (M-CSF). Nonadherent cells were
removed, and adherent cells were maintained for 12 hours in
medium without M-CSF. The cells were then stimulated with
OxPAPC (50 ␮g/ml) for 18 hours.
RESULTS
OxPAPC-induced selective monocyte recruitment into the air pouch. Previous studies have characterized the air-pouch model as a reliable experimental
approach to the study of inflammatory mechanisms that
occur in the synovium (25–28) as well as the accumulation of blood cells in general (16,29). Here, we used a
murine air-pouch model to investigate the effects of
OxPAPC on leukocyte accumulation in vivo.
Injection of 50 ␮g of LPS or 250 ␮g of OxPAPC
into the air-pouch cavity (5-ml volume) resulted in
accumulation of inflammatory cells in the surrounding
pouch lining tissue, or pouch wall. Transverse sections of
the pouch wall revealed that leukocytes also adhered to
the pouch wall on the luminal side (Figure 1A, arrows).
Treatment with LPS, but not OxPAPC, also caused
substantial edema (Figure 1A).
In order to assess cell counts microscopically, we
produced en face preparations of the air-pouch wall
after carefully dissecting the lining tissue from the
overlying skin. Staining with Giemsa facilitated counting
and enabled us to distinguish mononuclear (including
monocytes and T cells) from polymorphonuclear (PMN)
cells. We found that LPS induced the accumulation of
PMNs and mononuclear cells, whereas OxPAPC predominantly induced mononuclear cell accumulation
(⬎95%) (Figure 1B). Injection of nonoxidized PAPC
did not lead to recruitment of inflammatory cells (data
OXIDIZED PHOSPHOLIPIDS AND MACROPHAGE ACCUMULATION
Figure 1. Induction of mononuclear cell accumulation in air-pouch
tissue by injection of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphorylcholine (OxPAPC) into the air-pouch lumen. Air
pouches were raised in the dorsal skin of mice and then injected with
1 ml of 0.9% saline (control) or with 1 ml of 0.9% saline containing
either 250 ␮g of OxPAPC or 50 ␮g of lipopolysaccharide (LPS). After
24 hours, animals were euthanized, and the air-pouch tissue was
analyzed. A, Representative cross-sections of the air-pouch wall show
leukocytes adhering to the luminal side of the pouch membrane
(arrows in insets) after injection of OxPAPC or LPS (original magnification ⫻ 4; ⫻ 20 in insets). B, Adherent cells were counted in en face
preparations of air-pouch tissues from the 3 groups of mice. Cells were
differentiated by morphologic characteristics. Shown are the numbers
of total cells, monocytes (mono), and polymorphonuclear neutrophils
(PMNs). C, Lavage was performed on air pouches in the 3 groups of
mice, and the numbers of total cells, monocytes, and PMNs accumulating in the lumen were counted in the lavage fluid. D and E, Time
course analyses of adherent cells in air pouches injected with OxPAPC
(D) or LPS (E) were performed. Shown are the numbers of total cells,
monocytes, and PMNs. Values in B–E are the mean and SEM of 4
mice per group. ⴱ ⫽ P ⬍ 0.01 versus control in B; P ⬍ 0.05 versus
control in C, by analysis of variance. HPF ⫽ high-power fields. Color
figure can be viewed in the online issue, which is available at
http://www.arthritisrheum.org.
not shown). Analysis of air-pouch lavage fluid after 24
hours showed that LPS treatment also resulted in in-
1365
creased numbers of leukocytes in the pouch lumen,
whereas OxPAPC treatment did not result in accumulation of leukocytes in the lumen (Figure 1C).
Moreover, we found that the kinetics of accumulation of leukocytes in the air-pouch wall in response to
OxPAPC treatment differed from that induced by LPS.
Time course experiments showed that monocyte recruitment induced by OxPAPC reached a maximum at 24
hours after application (Figure 1D), whereas LPSinduced accumulation of leukocytes was more rapid and
peaked earlier (Figure 1E). Inflammation induced by
both agonists resolved at 48 hours.
Next, we wanted to further characterize the leukocyte subsets in the OxPAPC-treated pouch wall. Immunostaining for inflammatory cells in transverse sections of the air-pouch wall revealed that OxPAPC
caused the accumulation of F4/80-positive cells (Figure
2A). Myeloperoxidase-positive cells were present in
LPS-treated air-pouch walls (Figure 2B), some of which
were in the process of migrating into the pouch lumen
(Figure 2C, arrows).
We enzymatically digested the air-pouch wall to
obtain single-cell suspensions (23) and then further
characterized the accumulated leukocytes by FACS analysis. Prior to FACS, cells were stained for CD45,
CD11b, Gr-1, CD19, and CD3 to differentiate leukocyte
subtypes (Figure 2D). Treatment with either OxPAPC
or LPS significantly increased the accumulation of CD45⫹
cells in the air-pouch wall (Figure 2E). In the CD45⫹
population, OxPAPC specifically induced the accumulation of CD11b⫹/Gr-1low cells, indicating monocyte/
macrophages, whereas LPS induced the accumulation of
CD11b⫹/Gr-1low cells as well as CD11b⫹/Gr-1high cells,
indicating monocyte/macrophages and PMNs, respectively. No significant differences in the numbers of
CD19⫹ (B cells) or CD3⫹ (T cells) cells were seen,
indicating that the mononuclear cells that were counted
above (Figure 1) predominantly consisted of monocytes.
Inflammatory genes differentially regulated by
LPS and oxidized phospholipids. To study possible
differences in inflammatory gene expression that could
account for the differences in leukocyte subset recruitment induced by OxPAPC versus LPS, we examined the
expression of endothelial adhesion molecules, which are
known to control extravasation of inflammatory cells.
For time course experiments, OxPAPC or LPS was
injected into the air pouches, and animals were killed 1,
3, 6, 12, or 24 hours later. Studies using RNA isolated
from the air-pouch wall demonstrated that expression of
vascular cell adhesion molecule 1 (VCAM-1),
E-selectin, intercellular adhesion molecule 1 (ICAM-1),
1366
KADL ET AL
Figure 2. Selective induction of the recruitment of CD11b⫹/Gr-1low cells by injection of oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphorylcholine (OxPAPC). Leukocytes accumulating in the air-pouch wall were analyzed in tissues collected 24 hours
after injection with OxPAPC, lipopolysaccharide (LPS), or saline alone (control). A–C, Cross-sections of the air-pouch wall were
stained for antibodies against F4/80 (A) or myeloperoxidase (B and C). Some myeloperoxidase-positive cells in the LPS-treated
air-pouch wall are in the process of migrating into the pouch lumen (arrows). D, Tissue was weighed and enzymatically digested to
obtain a single-cell suspension, which was stained with leukocyte markers and subjected to flow cytometry. The CD45⫹ cells were then
analyzed for CD11b and Gr-1 expression. Polymorphonuclear neutrophils (PMNs) were defined as Gr-1high/CD11b⫹ cells and
monocyte/macrophages (MN/M⌽s) as Gr-1low/CD11b⫹. The remaining CD11b–/Gr-1– cells were defined as lymphocytes, which
stained positive for CD3 (T cells) or CD19 (B cells). SSC ⫽ side scatter; FSC ⫽ forward scatter. E, Absolute numbers of leukocytes,
monocyte/macrophages, PMNs, B cells, and T cells were obtained as the products of flow cytometry percentages and total cell counts
in samples from the 3 experimental groups. Values are the mean and SD of 4 mice per group. ⴱ ⫽ P ⬍ 0.01 by analysis of variance.
OxPL ⫽ oxidized phospholipids (i.e., OxPAPC).
and P-selectin were rapidly and potently induced by
LPS. In contrast, OxPAPC treatment did not result in
up-regulation of these genes (Figures 3A–D). These
findings are consistent with our previously published
results obtained in vitro (30).
Our previous study also demonstrated that HO-1
is differentially regulated by LPS and OxPAPC (31). In
the present study, we demonstrated that in contrast to
LPS, OxPAPC induced the expression of HO-1 protein
and mRNA in the air-pouch wall (Figures 3E and F).
Time course experiments showed that OxPAPC-induced
HO-1 mRNA expression peaked at 6 hours after stimulation (Figure 3F).
Different kinetics and potency of OxPAPCinduced versus LPS-induced chemokine expression in
air-pouch tissue. Treatment with OxPAPC induced the
expression of the CCR2 ligands MCP-1/JE, MCP-3, and
MCP-5 in the air-pouch wall. Interestingly, the expression of MCP-3 and MCP-5 induced by OxPAPC was
delayed and sustained as compared with LPS treatment,
OXIDIZED PHOSPHOLIPIDS AND MACROPHAGE ACCUMULATION
1367
LPS-induced expression. In all cases, LPS induced the
expression of these genes to a much greater extent than
did OxPAPC (Figure 4).
Mediation of oxidized phospholipid–induced
monocyte recruitment by CCR2. MCPs 1, 3, and 5 are all
ligands for CCR2. To investigate the role of CCR2 in
OxPAPC-induced monocyte recruitment, we treated
wild-type or CCR2⫺/⫺ mice with OxPAPC and examined monocyte accumulation in the air-pouch tissue by
quantitative FACS analysis. It was previously shown that
CCR2⫺/⫺ mice have lower numbers of circulating monocytes as a result of diminished egress from the bone
marrow (18). Accordingly, we found that basal levels of
resident macrophages were lower in CCR2⫺/⫺ mice than
in wild-type mice (2 ⫻ 105 versus 8 ⫻ 105) (Figure 5A).
Total leukocyte accumulation, as characterized by the
presence of CD45⫹ cells, was increased by 50% after
OxPAPC treatment of wild-type mice. In contrast, in
CCR2⫺/⫺ mice, the OxPAPC-induced increase in accumulation of CD45⫹ cells was abrogated (Figure 5A). In
addition, accumulation of F4/80⫹/CD68⫹ cells was sig-
Figure 3. Induction of heme oxygenase 1 (HO-1) expression, but no
increase in the expression of endothelial adhesion molecules, following
injection of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphorylcholine (OxPAPC). A–D, In contrast to lipopolysaccharide
(LPS; 50 ␮g), OxPAPC (250 ␮g) failed to induce the expression of
vascular cell adhesion molecule 1 (VCAM-1) (A), E-selectin (B),
intercellular adhesion molecule 1 (ICAM-1) (C), or P-selectin (D) in
air-pouch tissue analyzed at the indicated time points after injection, as
determined by reverse transcription–polymerase chain reaction analyses. E, Cross-sections of the air-pouch wall 24 hours after saline
(control) or OxPAPC injection show HO-1 protein in the OxPAPCinjected tissue (original magnification ⫻ 40). F, Time course analysis
of the expression of mRNA for HO-1. Values in A–D and F are the
mean and SD of 4 mice per group. Color figure can be viewed in the
online issue, which is available at http://www.arthritisrheum.org.
with MCP-5 showing a biphasic induction (Figure 4).
Similarly, OxPAPC induced the expression of
interferon-␥–inducible 10-kd protein (CXCL10), RANTES (CCL5), and BRAK (CXCL14). Expression of
these 3 chemokines was significantly delayed and sustained as compared with LPS-induced expression (Figure 4). The expression kinetics of OxPAPC-induced
MCP-1/JE, growth-related oncogene ␣ (CXCL1),
MIP-1␣ (CCL3), and MIP-1␤ (CCL4) overlapped with
Figure 4. Induction of chemokine expression following injection of
oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine
(OxPAPC). Animals were injected with either 1 ml of 0.9% saline
(control) or 1 ml of 0.9% saline containing 250 ␮g of OxPAPC or 50
␮g of lipopolysaccharide (LPS) into the air pouch and were euthanized
at the indicated time points. RNA was isolated from air-pouch tissue,
and the expression of monocyte chemotactic protein 1 (MCP-1)/JE,
MCP-3, MCP-5, interferon-␥–inducible 10-kd protein (IP-10),
RANTES, BRAK, growth-related oncogene ␣ (GRO␣), macrophage
inflammatory protein 1␤ (MIP-1␤), and MIP-1␣ was analyzed by
reverse transcription–polymerase chain reaction. Values are the mean
and SD fold increase over controls (n ⫽ 4 mice per group). Color
figure can be viewed in the online issue, which is available at
http://www.arthritisrheum.org.
1368
KADL ET AL
a significant decrease in CCR2 protein surface expression (Figure 5D).
DISCUSSION
Figure 5. Requirement of CCR2 for oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC)–induced
monocyte recruitment. A, F4/80-positive and CD68-positive cells in the
air-pouch wall from wild-type (W/T) mice (top) and from CCR2deficient (CCR2⫺/⫺) mice (bottom) injected with saline (control) or
OxPAPC were analyzed by flow cytometry. B and C, Expression of
CCR2 and CCR5 in the air-pouch tissue after treatment with OxPAPC
was analyzed by reverse transcription–polymerase chain reaction (B),
and a time course analysis of CCR2 expression (C) was performed. D,
Bone marrow–derived macrophages were stimulated with OxPAPC in
vitro, and CCR2 expression was analyzed by flow cytometry. Values in
A–C are the mean and SD of 4 mice per group. ⴱ ⫽ P ⬍ 0.05 versus
control in A and versus 0 hours in B and C, by analysis of variance.
nificantly increased by OxPAPC treatment in wild-type
mice, but not in CCR2⫺/⫺ mice (Figure 5A).
Moreover, we found that during the course of
OxPAPC-induced inflammation, the expression of
CCR2 in the pouch tissue was significantly decreased,
whereas CCR5 was not appreciably changed (Figure
5B). CCR2 mRNA reached a low at 12 hours and
returned to basal levels after 24 hours (Figure 5C). To
investigate the possibility that the down-regulation of
CCR2 expression was due to a direct effect of oxidized
phospholipids on macrophages, we isolated and cultured
murine bone marrow–derived macrophages. Treatment
of these macrophages with OxPAPC in vitro resulted in
The factors that trigger monocyte recruitment
and, thus, propagate chronic inflammatory processes in
RA are still poorly understood. A large body of data
obtained in vitro indicates that oxidized phospholipids
can be regarded as triggers of the inflammatory response
in the setting of chronic inflammation (15). Increased
concentrations of lipid peroxidation products and antibodies against oxidized lipoproteins have been found in
the synovial fluid of RA patients (32,33); however,
whether oxidized phospholipids contribute to synovial
inflammation and subsequent leukocyte recruitment has
not previously been demonstrated. This study of the
air-pouch model is the first to show that oxidized
phospholipids can be regarded as propagators of macrophage accumulation in a facsimile synovial tissue.
Chemokines serve a vital role in supporting the
inflammatory response in chronically inflamed tissues,
including rheumatoid synovium and atherosclerotic vessels (34,35). Monocytes that express CCR2 respond to
MCP-1 (CCL2), as well as MCP-3 (CCL7) and MCP-5
(CCL12), which are produced by a variety of cells during
inflammation (36). Consequently, CCR2⫺/⫺ mice have
profound defects in monocyte recruitment (18,37). In
our study, oxidized phospholipids were shown to induce
the expression of MCP-1/JE (CCL2) in various cell types
in vitro. Our data demonstrate that OxPAPC induced
the expression of a set of chemokines in the air-pouch
wall, including the CCR2 ligands CCL2 (MCP-1/JE),
CCL7 (MCP-3), and CCL12 (MCP-5). Although from
our results, we cannot deduce which cell type in the
air-pouch lining tissue contributed to the expression of
these chemokines, we hypothesized that their production would lead to CCR2-dependent monocyte recruitment. Indeed, we found that monocyte/macrophage
recruitment was abrogated in CCR2-deficient mice.
These results demonstrate an important role of CCR2 in
oxidized phospholipid–induced inflammation, consistent
with a role of CCR2 ligands in monocyte recruitment in
RA (7,17).
Of the 2 distinct subpopulations of circulating
monocytes in mice, Gr-1–/CCR2–/CX3CR1high monocytes can be found in normal tissues, while Gr-1⫹/
CCR2⫹/CX3CR1low cells represent “inflammatory”
monocytes that accumulate in inflamed tissues (38).
Interestingly, Gr-1high monocytes were not found in the
pouch wall after treatment with OxPAPC. It is possible
OXIDIZED PHOSPHOLIPIDS AND MACROPHAGE ACCUMULATION
that either OxPAPC does not induce the accumulation
of these cells or that Gr-1high monocytes quickly convert
to Gr-1low cells due to the oxidized phospholipid–rich
microenvironment in the pouch wall. Future experiments will address the question of whether oxidized
phospholipids induce a preferential recruitment of
Ly6Chigh or Ly6Cintermediate/low monocytes.
A major difference between oxidized phospholipid–
induced and LPS-induced inflammation was that inflammatory cells were absent in the air-pouch lumen in
animals treated with OxPAPC. In fact, macrophages
accumulated in the air-pouch wall but did not migrate
into the pouch lumen. This prolonged residence of
inflammatory macrophages in the tissue has important
implications for the progression of chronic inflammatory
tissue damage. The reasons for decreased emigration of
macrophages from inflamed tissue have not yet been
fully elucidated, but down-regulation of the expression
of chemokine receptors such as CCR2 by microenvironmental factors may cause prolonged arrest of macrophages (39). In this context, we showed that in macrophages, the expression of CCR2, but not CCR5,
was down-regulated by the direct action of oxidized
phospholipids. We conclude that down-regulation of
CCR2 expression on macrophages by oxidized phospholipids is at least partly responsible for the prolonged
arrest of inflammatory macrophages in damaged tissue,
which would contribute to the propagation of chronic
inflammation.
We and other investigators have previously
shown that OxPAPC selectively induces monocyte–
endothelial cell interactions in vitro (12,30,40–44). This
is in sharp contrast to LPS-induced endothelial cell
activation, since LPS caused monocyte adhesion as well
as neutrophil adhesion (45,46). There are striking differences in endothelial cell activation between oxidized
phospholipids and other proinflammatory mediators,
such as IL-1, tumor necrosis factor, or LPS. The latter
activate the classic NF-␬B pathway that leads to the
expression of the endothelial adhesion molecules
E-selectin, VCAM-1, or ICAM-1, resulting in adhesion
of monocytes as well as neutrophils. In contrast, oxidized
phospholipids stimulate endothelial cells to specifically
bind monocytes, but not neutrophils, a hallmark of
chronic inflammation (12,43). Recent studies indicate
that activation of MAP kinases, rather than the NF-␬B
pathway, by oxidized phospholipids mediates the expression of inflammatory genes that leads to specific monocyte adhesion (30). Furthermore, we have also shown
that OxPAPC potently inhibits LPS-induced neutrophil
accumulation (46), pointing to a potential mechanism by
1369
which oxidized phospholipids may determine monocyte
specificity. Indeed, when LPS and OxPAPC were coinjected into the air pouch, leukocyte accumulation in the
lumen was abrogated, whereas there was still significant
macrophage accumulation in the pouch wall (data not
shown).
In the present study, we confirmed major differences between oxidized phospholipid–induced and LPSinduced inflammation in vivo, including differences in
adhesion molecule and HO-1 expression. The relative
expression of these inflammatory genes determines the
type of inflammatory cell that infiltrates inflamed tissues. Our results showed that monocyte recruitment can
occur in the absence of increased expression of endothelial adhesion molecules. This indicates that the constitutive expression of certain adhesion molecules (e.g.,
ICAM-1) and increased expression of chemokines is
sufficient to promote monocyte emigration. Neutrophils,
in contrast, were shown to require increased expression
of endothelial adhesion molecules. In this context, it has
been shown that recruitment of inflammatory monocytes
does not require the prior influx of neutrophils (20).
Our results indicate that monocyte selectivity
induced by oxidized phospholipids cannot solely be
explained by the chemokine expression pattern, since we
found that chemokines with known neutrophil-activating
capacity were also induced by oxidized phospholipids.
Comparing the effects of LPS and OxPAPC, selectivity
for monocyte recruitment induced by OxPAPC could
have been brought about by the differential expression
of inflammatory genes, by differences in potency, and/or
by differences in the kinetics of inflammatory gene
expression during the course of the inflammatory response. While both OxPAPC and LPS induced the
expression of an overlapping set of chemokines in the
air-pouch tissue, we found major differences in the
extent and time course of expression. Whether these
differences account for selectivity in leukocyte recruitment remains to be determined.
The enzymatic activity of HO-1 has been shown
to limit overshooting inflammation and to contribute to
the resolution of acute inflammatory reactions (46–48).
It is tempting to speculate that HO-1 contributes to the
monocyte selectivity in oxidized phospholipid–induced
inflammation by actively inhibiting neutrophil accumulation. Moreover, it has been reported that resistance of
macrophages to apoptosis is essential for the development of RA (5). Our finding that oxidized phospholipids
up-regulate the antiapoptotic enzyme HO-1 supports
the concept of prolonged survival of macrophages that
accumulate in inflamed synovium.
1370
KADL ET AL
Our findings have important implications for
various chronic inflammatory diseases. Atherosclerotic
lesions as well as inflamed synovial tissue reflect chronic
inflammatory states, which are characterized by oxidative tissue damage and specific infiltration of monocytic
cells. Synovial inflammation is accompanied by angiogenesis, and macrophages and their products seem to
play an important role in this process as well (49). We
have recently shown that oxidized phospholipids may
increase the propensity of atherosclerotic lesions to
rupture by inducing angiogenesis (50). Whether oxidized
phospholipids can induce angiogenesis in inflamed synovium remains to be demonstrated.
The microenvironmental factors that induce the
inflammatory reactions that cause specific monocyte/
macrophage accumulation in chronically inflamed tissues have not been described. Based on our data, we
propose a model in which the formation of a defined
subset of oxidized phospholipids in tissues leads to a
persistent inflammatory response. Regulation of the
expression of chemokines and their receptors, in particular of the CCL2/CCR2 axis, mediates the selective
monocyte/macrophage accumulation and, thus, the
propagation of chronic inflammation. To fully understand the mechanisms that are involved in oxidized
phospholipid–induced chronic inflammatory processes,
structure–function relationships need to be further investigated. Identification of structures as well as receptors that recognize oxidized phospholipids will provide a
foundation for the development of novel therapeutic
strategies for the treatment of chronic inflammatory
disorders, including RA.
AUTHOR CONTRIBUTIONS
Dr. Leitinger had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Kadl, Galkina, Leitinger.
Acquisition of data. Kadl, Galkina, Leitinger.
Analysis and interpretation of data. Kadl, Galkina, Leitinger.
Manuscript preparation. Kadl, Leitinger.
Statistical analysis. Kadl, Leitinger.
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