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*
Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy;
Department of Obstetrics and Gynecology, S. Gerardo Hospital, Monza, Italy;
Department of Biotechnology, University of Brescia, Brescia, Italy;
Receptor Pharmacology, Leukosite, Inc., Cambridge, MA; and
¶
Biological Therapy Laboratory, Imperial Cancer Research Fund, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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restored the
CCR2 mRNA level in monocytes cultured in the presence of
ascitic fluid. The finding of defective CCR2 expression
in TAM, largely dependent on local TNF production, is consistent with
previous in vitro data on down-regulation of chemokine receptors
by proinflammatory molecules. Receptor inhibition may serve as a
mechanism to arrest and retain recruited macrophages and to prevent
chemokine scavenging by mononuclear phagocytes at sites of inflammation
and tumor growth. In the presence of advanced tumors or chronic
inflammation, systemic down-regulation of receptor expression by
proinflammatory molecules leaking in the systemic circulation may
account for defective chemotaxis and a defective capacity to mount
inflammatory responses associated with advanced
neoplasia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages represent a major component of the lymphoreticular infiltrate of tumors, and the percentage of TAM for each tumor is usually maintained as a relatively stable "individual" property during tumor growth and upon transplantation in syngeneic hosts (1, 5). The search for tumor-derived chemotactic factors, which may account for recruitment of mononuclear phagocytes in neoplastic tissues, lead to the identification of the monocyte chemotactic protein-1 (MCP-1, CCL2) as well as of other chemokines (1, 2, 6, 7, 8, 9, 10, 11, 12).
MCP-1 is a member of a superfamily of cytokines called chemokines. The
hallmark of this family is a conserved cysteine residue motif
(13, 14, 15). According to the relative position of the first
two cysteines, it is possible to distinguish two main families: the CXC
(or ) chemokines, which are active on neutrophils, T and B
lymphocytes (13, 14, 15), and the CC (or 
) chemokines that
exert their action on multiple leukocyte populations, including
monocytes, basophils, eosinophils, T lymphocytes, NK, and dendritic
cells (13, 14, 15, 16, 17, 18). A third type of protein (the C or 
chemokines) was described, which is active on T lymphocytes and NK
cells. This protein is characterized by the absence of the first
and third cysteines, but shows overall sequence identity with CC
chemokines (19, 20). More recently, fractalkine, CX3C (or
motif), was described as chemotactic for monocytes, T cells, and NK
cells (21, 22).
Chemokines, as well as classical chemotactic agonists, such as formylated peptides (of which fMLP is the prototype) and C5a, bind to and activate a family of rhodopsin-like, GTP-binding protein-coupled seven-transmembrane domain receptors (23, 24, 25). Nine receptors for CC chemokines, now named CCR1 through 9, have been identified and cloned (13, 14, 23, 24, 25, 26).
MCP-1 interacts with CCR2, of which two isoforms have been cloned and termed A and B (26). In monocytes and NK cells, CCR2 is expressed predominantly as B isoform, with vanishingly low levels of A transcripts (27). In addition to MCP-1, CCR2 recognizes MCP-2 and MCP-3 (28, 29, 30). Several lines of evidence, including studies in gene-targeted mice, indicate that MCP-1 and CCR2 are important for monocyte recruitment at the site of inflammation (31, 32). There is also evidence that in a variety of murine and human tumors, including ovarian cancer, MCP-1 is a major determinant of the degree of macrophage infiltration in neoplastic tissue (1, 2, 33, 34, 35, 36, 37, 38, 39, 40).
Recent results indicate that proinflammatory and anti-inflammatory
signals regulate chemokine receptor expression in human mononuclear
phagocytes (41, 42, 43, 44). In particular, certain primary
proinflammatory signals (e.g., LPS) rapidly inhibit chemokine receptor
expression (41). It was speculated that inhibition of
chemokine receptor expression may serve as a stop signal to arrest and
retain mononuclear phagocytes at sites of infection or inflammation
(41). As discussed elsewhere (2), tumors have
served as a paradigm of the in vivo function of chemokines in monocyte
recruitment with minimal activation. It was therefore important to
investigate chemokine receptor expression in TAM, since regulation of
receptor expression has emerged as a crucial set point for the action
of these molecules. In this study, we report that macrophages isolated
from the ascitic fluid or solid tumor from ovarian cancer patients
exhibit a drastic and selective defect of expression of the MCP-1
receptor (CCR2), which correlates with the lack of chemotaxis in
response to MCP-1. In addition, we identified TNF- as a protumor
factor which may contribute to the negative regulation of
CCR2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocytes were separated from peripheral blood of human healthy donors by Percoll gradient centrifugation (45). Monocytes (>98% pure as assessed by morphology) were resuspended at 107/ml in RPMI 1640 supplemented with 10% of FBS, 2 mM glutamine, and antibiotics. All reagents contained <0.125 endotoxin units/ml of endotoxin as checked by Limulus amebocyte lysate assay (Microbiological Associates, Walkersville, MD). Monocyte-derived macrophages (M-DM) were derived from freshly isolated monocytes (3–5 x 106cells/ml) after incubation for 5 days in RPMI 1640 medium supplemented with 10% of FBS, with 2 mM glutamine, antibiotics, and 40% autologous serum on hydrophobic plates (Petriperm Hydrophobic; Heraeus Instruments GmbH, Germany) as described previously (46). PBMC of healthy donors were obtained from buffy coats, whereas PBMC from patients with ovarian carcinoma were obtained from heparinized venous blood. Blood was diluted 1/5 with saline, and 40 ml was then placed on 10 ml of Ficoll (Seromed, Berlin, Germany) in 50-ml conical tubes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) for centrifugation at 400 x g for 20 min at room temperature. PBMC were collected at the interface, washed with saline, and suspended in complete medium at 2–5 x 106 cells/ml in 50-ml conical tubes.
TAM
Blood samples (5) and ascitic fluids (10) were collected from untreated patients with histologically confirmed epithelial ovarian carcinoma admitted to the Department of Obstetrics and Gynecology, San Gerardo Hospital (Monza, Italy). All patients had cancer classified as stage II, III, or IV. Ascitic fluid was collected and centrifuged. Cell pellets were resuspended in RPMI 1640 medium without serum and layered on top of a Ficoll-Hypaque cushion to prepare mononuclear cells. Purification of peritoneal macrophages was further conducted by two subsequent adherence steps for 45 min, each in RPMI 1640 medium without serum. After adherence procedures, cells were repeatedly washed with saline to remove all nonadherent cells. The adherent cells were cultured with complete medium over night at 37°C to rest and then stimulated as indicated above. To purify TAM by flow cytometry and sorting, ascitic fluids (four patients) were incubated with anti-CD68 mAb. A total of 5 x 107 cells was washed in saline with 1% human serum and then incubated in 0.5 ml of anti-CD68 diluted 1/5 for 30 min at 4°C. The cells were then washed three times in saline/1% human serum. The second incubation was conducted at 4°C for 30 min with FITC-conjugated goat F(ab')2 anti-mouse Ig. The sorting process was performed with a FACStarPlus apparatus (Becton Dickinson, Mountain View, CA). Purification of TAM from solid tumor was performed as described previously (34).
FACS analysis
Cell staining was performed using human mAb anti-CCR5 (clone
2D7; PharMingen, San Diego, CA) and its irrelevant control mouse,
IgG2a, 
(UPC10-Sigma) followed by FITC-conjugated affinity-purified,
isotype-specific goat anti-mouse Ab (Southern Biotechnology
Associates, Birmingham, AL). For phenotype analysis, indirect
immunofluorescence was performed with the human anti-CCR2 mAb Ab
(clone LS132.1D9) and PE-labeled goat anti-mouse Ig (Jackson
ImmunoResearch, West Grove, PA) using a FACStar (Becton Dickinson).
Cytokines and Abs
Human recombinant IL-1 was obtained through the courtesy of
Dr. J. E. Sims (Immunex, Seattle, WA) and was used at 20 ng/ml;
TNF- (BASF/Knoll, Ludwighafen, Germany) was used at 500 U/ml. Human
recombinant MCP-1, MCP-2, and RANTES (regulated on activation normal T
cell expressed and secreted) were from PeproTech (Rocky Hill, NJ).
Human recombinant MCP-3 and macrophage inflammatory protein-1
(MIP-1 ) were a kind gift from Dr. A. Minty (Sanofi, Labège,
France) and Dr. Czaplewski, respectively. The mAb against TNF-
(B154.2) was a kind gift from Dr. G. Trinchieri (Wistar Institute of
Anatomy and Biology, Philadelphia, PA). The mAb against IFN- (IFGCP)
was purchased from American Type Culture Collection (Manassas, VA).
IL-1ra mutant DoB 0039 was kindly donated by Dr. Diana Boraschi
(Dompe’, L’Aquila, Italy).
Migration assay
Cell migration was evaluated using a chemotaxis microchamber technique (47) as described previously (48). Twenty-seven microliters of chemoattractant solution or control medium (RPMI 1640 with 1% FCS) was added to the lower wells of a chemotaxis chamber (Neuroprobe, Pleasanton, CA). A polycarbonate filter (5-µm pore size; Neuroprobe) was layered onto the wells and covered with a silicon gasket and the top plate. A total of 50 µl of cell suspension (1.5 x 106/ml monocytes in PBMC) was seeded in the upper chamber. The chamber was incubated at 37°C in air with 5% CO2 for 90 min. At the end of the incubation, filters were removed, stained with Diff-Quik (Baxter S.P.A., Rome, Italy), and five high-power oil-immersion fields were counted.
Northern blot analysis
Cells were cultured in medium alone or supplemented with the
indicated agents, and total RNA was purified as described
(49). Ten micrograms of total RNA from each sample was
electrophoresed under denaturing conditions, blotted onto Nytran
membranes (Schleicher & Schuell, Keene, NH), and cross-linked by UV
irradiation. Membranes were prehybridized at 42°C in Hybrisol (Oncor,
Gaithersburg, MD) and hybridized overnight with 1 x
106cpm/ml of 32P-labeled
probe. Membranes were then washed three times at room temperature for
10 min in 0.2x SSC [1x SSC: 0.15 M NaCl, and 0.015 M sodium citrate
(pH 7.0)], 0.1% SDS, and twice at 60°C for 20 min in 0.2x SSC and
0.1% SDS before being autoradiographed using Kodak XAR-5 films
(Eastman, Rochester, NY) and intensifier screens at -80°C. cDNAs
were labeled by random priming using a commercial kit (Boehringer
Mannheim, Indianapolis, IN) and
[-32P]deoxycytidine 5'-triphosphate (3000
Ci/mmol; Amersham, Arlington Heights, IL). CCR2B cDNA was obtained by
PCR amplification of the reported sequence (27, 50). CCR1
and CCR5 cDNAs were obtained as described previously (51).
Densitometric analysis was performed with a scanning densitometer GS300
(Hoefer Scientific Instruments, San Francisco, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We investigated expression of chemokine R mRNA in TAM by Northern
blot analysis. Fig. 1
A shows
one representative experiment, and Fig. 1B summarizes a
densitometric analysis of the results obtained in the examined
populations. TAM from ascites (10 samples) or solid tumor (four
samples) showed little or no detectable CCR2 mRNA. This
defect was selective because CCR1 was normally expressed
and, as shown by densitometric analysis (Fig. 1B), only in a
few samples was the CCR5 mRNA level slightly decreased.
Because the defective expression of CCR2 in TAM may have been the
result of the separation procedure, resulting for instance in exposure
of mononuclear phagocytes to cellular debris, PCR analysis of the
CCR2 mRNA level was performed on 25 ovarian cancer
biopsies, including 19 serous adenocarcinomas, 2 clear cell carcinomas,
1 mucinous adenocarcinoma, 1 anaplastic carcinoma, 1 signet ring
carcinoma, and 1 endometrioid carcinoma. All of the examined specimens
were negative for CCR2A and CCR2B mRNAs
expression, whereas 80% of the biopsies were positive for
MCP-1 (data not shown). In addition, the THP-1 and MonoMac6
cell lines, which were included as part of the screen, displayed
detectable CCR2 mRNA. In two patients, mRNA data were confirmed by FACS
analysis (Fig. 1C). Although monocytes displayed high
positivity for CCR2, TAM showed almost undetectable levels of this
receptor. In contrast, CCR5 surface expression in TAM was only modestly
decreased.
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A,
circulating monocytes from five ovarian cancer patients (two, stage II;
two, stage IV; and one, stage III) consistently showed defective
CCR2 mRNA expression, but the defect was not as drastic
as in TAM. TAM do not migrate in response to MCP-1
Having established that TAM and peripheral mononuclear cells from
patients with ovarian carcinoma display absent or decreased expression
of the CCR2, we wanted to investigate whether this defect correlated
with impaired cell migration. MCP-1 (CCL2) is a high-affinity ligand
for the CCR2 receptor (26), thus migration of both TAM and
PBMC was assayed in chemotaxis microchamber in response to MCP-1. As
shown in Fig. 2A, MCP-1 did
not enhance the migration of TAM, whereas a classical chemotactic
stimulus such as C5a increased it. Despite the presence of CCR5 mRNA
expression, no significant increase over the spontaneous migration of
TAM was obtained in response to MIP-1, suggesting that other
determinants, such as receptor desensitization and/or internalization,
may control CCR5 activity in TAM (41, 42). As expected,
fresh monocytes displayed high responsiveness to the chemoattractants
used in the assay. As shown in Fig. 2B , in response to
MCP-1, migration of PBMC from tumor patients was defective (60% of
inhibition), but not as profound as that of TAM. These cells also
displayed reduced migration also toward MIP-1 (40% of inhibition).
In contrast, their migration in response to C5a was less affected (21%
of inhibition).
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of tumor ascites on the CCR2 mRNA expression by
monocytes
Based on these observations, it was important to understand
whether factors present in ascites from the ovarian cancer could
suppress CCR2 expression. Tumor ascites were reported to contain a
number of cytokines (1, 37, 52, 53). Some of these, such
as IL-1, TNF-, and IFN-, are potent inhibitors of CCR2
mRNA expression (41, 42). We observed that IL-6 and MCP-1
do not affect CCR2 mRNA expression in monocytes and M-DM
(data not shown). In Fig. 3A,
we investigated whether ascites from the ovarian carcinoma may cause
inhibition of the CCR2 mRNA expression. Freshly isolated
monocytes (Fig. 3A, lanes 1 and 2) and
M-DM (Fig. 3A, lanes 3 and 4) from healthy donors
were incubated for 4 h in the presence or absence of 20% of
ascitic fluid. Ascitic fluid elicited a strong inhibitory effect on the
expression of CCR2 mRNA. This inhibition was specific as no
alterations were observed for the levels of expression of the
CCR5 transcript. Based on this observation, the
identification of putative factors responsible for the suppression of
CCR2 mRNA expression was conducted by using blocking Abs
directed against proinflammatory cytokines. Anti-IFN-,
anti-TNF- Abs, and the IL-1ra were preincubated for 2 h at
room temperature with ascitic fluid in an attempt to block the
inhibitory activity for CCR2. As shown in Fig. 3B, in the presence of ascitic fluid, anti-TNF-
(lane 5) was able to efficiently preserve the levels
of expression of CCR2 mRNA, whereas the anti-IFN-
Ab (lane 3) and IL-1ra (lane 4) did
not. Coincubation with anti-IFN- and anti-TNF- Abs did
not synergize (Fig. 3B, lane 6). These results
strongly indicate TNF- present in the ascites of ovarian carcinoma
as a tumor-derived factor responsible for the inhibition of
CCR2 in TAM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The selective loss of CCR2 expression in TAM resembles
the specific and selective inhibition operated by LPS and certain
proinflammatory cytokines (e.g., IL-1, TNF-, and IFN-) on the
CCR2 expression in monocytes (41, 42). We
also reported that the anti-inflammatory cytokine IL-10 induces
CCR2, CCR5, and CCR1
expression (43), suggesting that proinflammatory and
anti-inflammatory signals have opposite and divergent actions on CC
chemokine receptor expression in monocytes. In this regard, ovarian
cancer is one epithelial malignancy in which there is evidence of a
complex network between the tumor microenvironment and the immune
system (37, 54), and there is strong evidence that TNF-
is a cytokine which strongly influences the biology of this tumor
(54, 55). Despite the expression of chemokine receptors,
lack of migration in response to a specific ligand has been reported in
cells activated with proinflammatory signals (41, 42, 43), a
condition likely shared by TAM. This may potentially be the result of a
number of mechanisms such as homologous or heterologous receptor
desensitization, internalization, and/or inhibition of receptor
signaling. In addition, the observed difference in the number of
migrated TAM vs migrated monocytes in response to C5a may be at least
partially due to cell differentiation.
Previous reports have indicated that ovarian cancer cells significantly
express TNF- (55) and we pointed to this cytokine as a
possible tumor-derived inhibitor of the CCR2 expression.
The validity of this hypothesis is substantiated by the observation
that inhibition of CCR2 mRNA expression by tumor ascites
is efficiently prevented by an anti-TNF- Ab. Despite this
observation, the levels of TNF- that we detected in tumor ascites
were quite low (data not shown) and may not fully explain the drastic
down-regulation of CCR2 mRNA levels, but rather suggest
possible synergism with other proinflammatory signals. Indeed, since
ovarian cancer cells are a rich source of cytokines (37),
it is likely that other proinflammatory cytokines within the tumor
microenvironment may strengthen the inhibitory action of TNF-. The
observed decrease of CCR2 mRNA levels in PBMC from
cancer patients may also imply the possibility that factors released
from growing tumors may, beyond a certain size, leak into the systemic
circulation to control excessive recruitment and reverse
transmigration. Thus, the drastic down-regulation of
CCR2 in comparison to the others receptors studied here
may assume particular relevance in view of evidence suggesting MCP-1 as
a main determinant of macrophage infiltration in tumors (1, 2) and normal tissues (31, 32).
We previously proposed (41) that reciprocal influences
exerted by proinflammatory molecules on chemokine agonist production
and receptor expression may represent a crucial set point in the
regulation of the chemokine system. Our data provide the first evidence
that this emerging paradigm has in vivo relevance in the
pathophysiology of tumors and point to TNF- as a tumor-derived
cytokine controlling the chemokine system of macrophages associated
with tumors.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Antonio Sica, Istituto di Ricerche Farmacologiche "Mario Negri," via Eritrea 62, 20157 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: TAM, tumor-associated macrophages; MCP-1, monocyte chemotactic protein-1; MIP-1, macrophage inflammatory protein-1; M-DM, monocyte-derived macrophage.
Received for publication June 15, 1999. Accepted for publication November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on melanocyte transformation. Oncogene 6:1115.[Medline]
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