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*
Istituto Ricerche Farmacologiche Mario Negri, Milan, Italy;
Hôpital Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U283, Paris, France;
Dipartimento Medicina Sperimentale e Patologia, Università La Sapienza, Rome, Italy;
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Sanofi Recherches, Labège, France; and
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Dipartimento Biotecnologie, Univ. Brescia, Brescia, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not against IL-4, inhibited rejection of
MCP-3-producing cells. An anti-polymorphonuclear mAb
caused only a retardation of MCP-3-elicited tumor rejection. Thus,
MCP-3 gene transfer elicits tumor rejection by activating
type I T cell-dependent immunity. It is tempting to speculate that
altered trafficking of APCs, which express receptors and respond to
MCP-3, together with recruitment of activated T cells, underlies
activation of specific immunity by MCP-3-transfected cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are a superfamily of cytokines that attract and activate leukocytes (4). Chemokines, and monocyte chemotactic protein-1 (MCP-1)3 in particular, are produced by a variety of tumor types in vitro and in vivo. There is evidence that MCP-1 plays an important role in the regulation of tumor-associated macrophages (TAM) in mice as well as in certain human tumors, including ovarian carcinoma (5), Kaposi’s sarcoma (6), cervical carcinoma (7), and melanoma (8).
Chemokine genes have been transduced in a variety of experimental tumors. In certain cellular contexts, transfer of genes encoding antiangiogenic C-X-C chemokines (9) has blocked tumorigenicity (10). C-C chemokine gene transfer has yielded variable results (11, 12, 13, 14, 15, 16). For instance, MCP-1 gene transfer has resulted in TAM recruitment and, concomitantly, no effect (11), destruction (12), growth retardation (13, 17), or even augmentation of metastasis (17).
Certain chemokines have the capacity to attract dendritic cells (DC) (18, 19, 20, 21, 22) and are likely to play an as yet undefined role in the complex trafficking of these cells (20). DC play a central role in activation of specific immunity including specific antitumor responses (23, 24, 25).
MCP-3 is a C-C chemokine identified in osteosarcoma supernatant and as an inducible gene in mononuclear cells (26, 27). It is structurally related to the prototypic C-C chemokine MCP-1, but with a distinct receptor usage and spectrum of action. MCP-3 binds to CCR1, CCR2, and CCR3 receptors (28, 29, 30, 31, 32). Accordingly, the spectrum of action of MCP-3 overlaps with, but is distinct from, that of MCP-1, which binds CCR2. MCP-3 activates mononuclear phagocytes, T cells, NK cells, and basophils, as MCP-1 does (33, 34, 35, 36). MCP-3 is also active on eosinophils and DC, which are not affected by MCP-1 (18, 36). MCP-3 elicits Ca2+ fluxes and chemotaxis in human neutrophils (31), although others have failed to observe polymorphonuclear neutrophil (PMN) chemotaxis (Ref. 26 and our unpublished data).
The broad spectrum of action of MCP-3 prompted us to investigate its potential to activate antitumor resistance in a gene transfer approach. MCP-3 gene transfer elicited tumor rejection by activating type I T cell-dependent responses. MCP-3 gene transfer and tumor rejection were associated with leukocyte recruitment (macrophages, neutrophils, and T cells) and changes in DC trafficking.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P815 mastocytoma cells were grown in RPMI 1640, 10% FCS and
antibiotics. Cells were transfected with the human MCP-3
gene (27) (P815/MCP3) or were mock transfected (P815). To produce
supernatants for the evaluation of MCP-3 production, 3 x
106 P815 and P815/MCP3 cells were cultured in 1 ml of
medium without serum overnight. P815/MCP3 cells produced 4.6 ± 0.9
(SD) ng/ml of MCP-3, measured with a commercial ELISA kit for human
MCP-3 (Biosource Bouty, Sesto S. Giovanni, Italy). Mock- and
MCP-3-transfected cells showed identical in vitro growth
rate, cell density, and marker expression (CD45). MCP-3
transfection did not cause the expression of other CC or CXC chemokines
(MCP-1/JE, KC, and Mip-1
) as assessed by Northern blot analysis
(data not shown).
DBA/2NCrlBR and Crl:nu/nu (CD-1) BR male mice were obtained from Charles River, Calco, Italy, and were used at 8 to 10 wk of age. Procedures involving animals and their care were conducted in conformity with institutional guidelines in compliance with national and international law and policies (EEC (European Economic Community) Council Directive 86/609, OJL 358,1 December 12, 1987; National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985).
Antibodies
Rat mAbs against CD8 (53-6-72), CD4 (GK1.5), and IL-4 (11B11)
were hybridomas from American Type Culture Collection (Manassas, VA);
anti-PMN (RB6-8C5) and anti-IFN- (AN18) hybridomas were kind
gifts from Dr. R. Coffman (DNAX Research Institute, Palo Alto, CA) and
Dr. S. Landolfo (University of Turin, Turin, Italy), respectively.
Anti-CD3 and Mac-3 were from PharMingen, San Diego, CA). Rat mAbs
DEC205 (anti-DC) and 2A1 (anti-DC/B cells) were kindly donated
by Dr. R. M. Steinman (Rockefeller University, New York,
NY).
Rat Ig concentration in ascites fluids was measured by a radial
immunodiffusion kit (The Binding Site, Birmingham, U.K.). In in vivo
experiments, mice were treated with 0.3 mg/mouse of mAbs on days 0
(administered i.v.) and 1 and 2 (i.p.), then with 0.2 mg/mouse i.p. two
(anti-CD4, -CD8, -PMN) or three times (anti-IL-4, -IFN-) a
week for 2 more weeks. Animals of the control group were injected with
saline. Mice were checked three times a week for tumor appearance;
tumor diameters were measured with a caliper on days reported and
survival was checked every day.
Immunohistochemistry
At least two mice from each group were analyzed at days 7 (nude) and 12 (DBA/2) after injection. The tumors were removed and embedded in OCT compound (Miles Laboratory, Elkhart, IN), snap frozen in liquid nitrogen, and stored at -80°C until used for immunohistochemistry. Five-micrometer cryostat sections were fixed in acetone and immunostained with rat anti-mouse mAbs against CD4, CD8, Mac-3, CD3, PMN, and DC. Endogenous peroxidase was inhibited by a 10-min incubation with 1% H2O2 in PBS at room temperature. Sections were preincubated with rabbit or hamster serum, and then sequentially with the optimal dilution of the primary Abs, biotinylated rabbit or hamster anti-rat or anti-hamster IgG and streptavidin-peroxidase (PharMingen). Each incubation lasted 30 min and was followed by a 10-min wash in PBS. Enzyme reaction was developed with 0.03% H2O2 and 0.06% 3.3'-diaminobenzidine (BDH Chemicals, Poole, U.K.) for 2 to 5 min, then washed in tap water and the sections counterstained with hematoxylin. The number of positive cells was evaluated by light microscopy at 400x enlargement in five fields on an mm2 grid and given as cells/mm2 (mean ± SD of one representative tumor).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P815 and P815/MCP3 cells were injected s.c. into DBA/2 mice. After
injection of 105 tumor cells of both lines, 100% of
the animals had palpable tumors by day 11 (Fig. 1
A). Tumor was rejected
by 1 of 6 of the P815- and 6 of 7 of the P815/MCP3-injected mice;
median survival time of the P815-injected mice was 30 days (range,
26–57 days). Mice injected with 5 x 105 P815/MCP3
tumor cells developed progressively growing tumors. Tumor volumes of
the P815/MCP3 (5 x 105 cells)-injected mice were
significantly smaller than those of the P815-injected animals, as shown
in Figure 1B. Thus, MCP-3 transduction substantially
decreased P815 tumorigenicity. To evaluate the mechanisms at the basis
of the resistance induced by MCP-3 transfection, P815 and
P815/MCP3 cells were transplanted in nude mice. No differences were
observed in tumor appearance (Fig. 1C) and tumor
growth (Fig. 1D) in the two groups, indicating that
tumor resistance is sustained by specific immune mechanisms.
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MCP-3 gene transfer profoundly altered leukocyte
recruitment in tumors grown in immunocompetent and nude mice. Twelve
days after tumor injection, which is the time when 100% of
immunocompetent mice are positive and tumors have reached the minimum
size to allow a histologic analysis, P815 tumors contained high
levels of TAM (Mac 3+), and these were augmented
in MCP-3-transfected lesions (Fig. 2). MCP-3-transfected tumors,
unlike the mock-transfected cells, also contained high numbers of T
cells (CD4+ and CD8+) and PMN (Fig. 2). An
increase in the number of eosinophils was also observed (Fig. 2).
DEC205+ cells were extremely rare in P815 lesions, and
there was little influence of MCP-3 gene transfer in the
tumor mass itself. However, in peritumoral tissues, DEC205+
cells were considerably increased in perivascular areas (Fig. 3). Similar results were obtained when
the mAb 2A1 staining intracytoplasmic granules was used to
identify DC.
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Having observed that MCP-3 gene transfer elicited T
cell-dependent antitumor resistance, we wanted to establish the
underlying critical cell populations and soluble mediators. As shown in
Figure 4A, anti-CD4 and
anti-CD8 mAb completely abolished the MCP-3-elicited rejection of
P815. The RB6-8C5 anti-PMN Ab, effective in other tumor systems
(37), delayed (about 30 days) but did not abrogate tumor rejection. In
an effort to establish whether the MCP-3-elicited specific immunity
depended primarily on a polarized type I or type II response,
anti-IFN- and anti-IL-4 mAbs were used. As shown in Figure 4B, anti-IFN- significantly
(p < 0.05) interfered with the capacity of
immunocompetent mice to reject P815-transfected cells, whereas
anti-IL-4 had no significant effect.
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), anti-IL-4-treated mice
were more able to resist the tumor (7 of 7) than controls injected with
MCP-3-transfected cells alone (5 of 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After early studies with MCP-1 (13, 14), gene transfer approaches have been widely used to investigate the role of chemokines in tumor immunobiology (10, 11, 15, 16). Chemokines such as IP-10 exert antitumor activity primarily by affecting angiogenesis (9). That an antiangiogenic action does not play a primary or major role in MCP-3-elicited tumor regression can be concluded from different evidences. MCP-3 does not affect the in vitro proliferation of endothelial cells from various sources (38). In our model, the tumor grew in immunocompetent mice to a size of 0.5 to 0.8 cm3 before being rejected, and MCP-3-transfected cells grew normally in nude mice. Finally, examination of the tumor vasculature using anti-CD31 (39) failed to reveal early alterations in MCP-3-transfected tumors.
MCP-3 is structurally related to MCP-1 and shares with it the CCR2 receptor (28, 40, 41). Accordingly, both MCP-1 and MCP-3 are active on monocytes, T cells, NK cells, and basophils (34, 35, 36). In addition, MCP-3, by interacting with CCR1 and CCR3 as well as with other as yet unidentified receptors, is active on other cell types including DC (18), eosinophils (42), and possibly neutrophils (see below). MCP-1 gene transfer has caused tumor regression (12), growth retardation (13), and even tumor promotion (17). In preliminary experiments, transfer of the mouse MCP-3 gene into the B78 line of B16 melanoma elicited antitumor resistance under conditions in which MCP-1 had modest activity (13). Under conditions in which MCP-1-transfected cells were destroyed in vivo, this effect was observed in nude mice and depended only on activation of innate resistance (12). The present results, as well as the distinct spectrum of action, suggest that interaction with DC and possibly PMN may play a role in the different activity of MCP-1 vs MCP-3 in gene transfer studies.
In general, members of the C-C and C chemokine families do not attract
PMN, and there is no evidence that they may do so in vivo. The in vitro
chemotactic activity of MCP-3 for PMN has been the object of
conflicting reports (26, 31). The results presented here show that PMN
accumulate in MCP-3-transfected tumors in immunocompetent as
well as in nude mice (not shown). Since the tumor grew normally in nude
mice, PMN recruitment is not secondary to tumor damage or host immune
responses. However, it has been recently observed that IFN-
up-regulates in vitro CCR1 and CCR3 expression in human neutrophils,
and IFN- exposure induces their migration in response to some CC
chemokine, including MCP3 (S. Sozzani et al., unpublished
observations). It is possible that in our model IFN-, locally
produced by T and/or NK cells, regulates neutrophil recruitment at the
tumor site by a similar mechanism.
MCP-3 has a wide spectrum of action that includes NK cells (35). CCR2 is expressed at low levels in resting NK cells and is augmented by IL-2 (43). A similar up-regulation is observed in T cells after activation (44). NK cells are present at low frequency in tumors (45). MCP-3 gene transfer did not elicit appreciable NK cell infiltration. Several explanations may account for this apparent discrepancy between in vitro and in vivo data. An early transient influx of NK cells may have escaped detection, or recruitment may be followed by rapid cell death in the tumor microenvironment. Alternatively, the low chemotactic activity of MCP-3 and other C-C chemokines for resting circulating NK cells may be insufficient to elicit extravasation.
PMN are frequently present at sites of cytokine-elicited tumor rejection (37). Direct evidence for an important role of recruited PMN in causing tumor rejection has been obtained after IL-4 and granulocyte-CSF gene transfer (3, 46). In the present study, the RB6-8C5 anti-PMN mAb (which effectively blocks rejection in the above systems (37)) caused only a retardation of tumor rejection after MCP-3 gene transfer, but no increase in tumor takes. Therefore, PMN recruited as a consequence of MCP-3 gene transfer exert some antitumor activity but do not play a pivotal role in rejection.
Rejection of MCP-3-transfected P815 cells depended on T
cells and was abrogated by anti-CD4 and anti-CD8 mAb. PA1 has
been identified on P815 cells as a tumor rejection Ag recognized by
cytotoxic CD8 T cells (47), and CD8 T cells likely are involved in the
rejection observed here. Previous studies on tumor resistance following
chemokine gene transfer did not analyze the role of polarized Th1 vs
Th2 responses, or they excluded a role for T cells (12, 13).
Anti-IFN- mAb, but not anti-IL-4 mAb, inhibited rejection of
MCP-3-transfected P815 cells. Interestingly, mice that had
rejected MCP-3-transfected cells and that had been treated
with anti-IL-4 exhibited increased resistance against subsequent
challenge with high numbers of P815 cells. Therefore, rejection of
MCP-3-transfected P815 cells is mediated by a type I
response, and orientation of immunity in this direction by
anti-IL-4 potentiates antitumor resistance.
The results presented here show that MCP-3 gene transfer
elicits tumor rejection by activating type I T cell responses.
MCP-3-elicited rejection is associated with alterations in leukocyte
accumulation within and around the tumor mass, including primary
recruitment of neutrophils and perivascular accumulation of cells with
a DC phenotype. In vitro-cultured human DC have been reported to
express a selected pattern of chemokine receptors (CCR1, CCR2, CCR5,
CXCR1, CXCR2, CXCR4). Active chemokines (MCP-1, MCP-3, RANTES,
MIP-1) did not appear to affect DC functions such as endocytosis and
Ag presentation (20). Also, MCP-3 does not directly induce IL-12
production (our unpublished results). In our preliminary Northern blot
experiments, bone marrow-derived mouse DC were found to express CCR1,
CCR2, CCR3 (our unpublished results). All of these receptors bind
MCP-3. On the basis of the in vivo observations in the P815/MCP3 model,
we speculate that altered trafficking of APCs plays an important role
in the activation of type I antitumor T cell responses by
MCP-3-transfected neoplastic cells. It will be of interest
to investigate the antitumor potential in this setting of the novel
macrophage-derived chemokine, which is more active on DC than on
mononuclear phagocytes (19).
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Alberto Mantovani, Dept. of Immunology and Cell Biology, Istituto Ricerche Farmacologiche Mario Negri, via Eritrea, 62-20157 Milano, Italy. E-mail address:
3 Abbreviations used in this paper: MCP-3, monocyte chemotactic protein-3; TAM, tumor-associated macrophages; PMN, polymorphonuclear neutrophil; DC, dendritic cells.
Received for publication September 22, 1997. Accepted for publication February 26, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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