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Immunology |
Third Department of Internal Medicine, University of Tokushima School of Medicine, Tokushima 770-8503 [H. N., H. Y., Y. N., S. Y., S. S.]; Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934 [N. M.]; and Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033 [K. M.], Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection of cytokine genes into tumor cells has been investigated widely for analysis of cytokine-mediated biological effects on tumor growth and is very important in successful cancer immunotherapy to attract effector cells such as monocyte-macrophages and lymphocytes into the tumor growth site (8 , 9) . Therefore, we examined the effect of MCP-1 gene transfection on tumor progression. Our recent reports (10 , 11) demonstrated that MCP-1 gene transfection into a human small cell lung cancer cell line (H69/VP) did not affect tumorigenicity or growth rates in the s.c. space of nude mice or SCID mice, although it caused greater macrophage infiltration into the tumors. Moreover, to evaluate the monocyte-mediated effect of MCP-1 more selectively, we used NK cell-depleted SCID mice pretreated with anti-IL-2 receptor ß chain Ab (TM-ß1) and showed that MCP-1 gene transfection into H69/VP cells again had no effect on metastasis formation in NK cell-depleted SCID mice (11) . However, MCP-1 gene transfection into H69/VP cells caused a remarkable enhancement of Ab-dependent cellular cytotoxicity reaction by augmentation of macrophage infiltration into metastatic lesions and hence augmented the therapeutic efficacy of anti-P-glycoprotein Ab on metastasis by P-glycoprotein-expressing H69/VP cells in multiple organs such as the liver, kidneys, and systemic lymph nodes (11) .
On the other hand, the NK cell-mediated effect of MCP-1 on metastasis has not been examined thus far. We developed a model for lung metastasis and PEs of human lung adenocarcinoma cells in NK cell-intact SCID mice (12) . To directly analyze the contribution of NK cells, we examined the metastatic potential of MCP-1 gene-transfected lung adenocarcinoma cells in NK cell-intact and NK cell-depleted SCID mice in this study.
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MATERIALS AND METHODS |
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Cell Cultures.
Human lung adenocarcinoma PC-14 cells were kindly supplied by Dr. N.
Saijo (National Cancer Institute, Tokyo, Japan), and K562 cells were
obtained from the American Type Culture Collection (Rockville, MD).
These cell cultures were maintained in RPMI 1640 supplemented with 10%
heat-inactivated FBS (CRPMI 1640) and gentamicin at 37°C in a
humidified atmosphere of 5% CO2 in air. YAC-1
cells were generously given by W. Chambers (University of Pittsburgh,
Pittsburgh, PA) and were maintained in CRPMI 1640 and 2 mM
glutamine, 100 µg/ml streptomycin, 100 IU/ml penicillin, and
5 x 10-5 M
2-mercaptoethanol (Life Technologies) at 37°C in a humidified
atmosphere of 5% CO2 in air.
Gene Transfection and Cloning of Transfected Cell Lines.
PC-14 cells were transfected with BCMGSNeoMCAF (13)
containing a 400-bp PstI fragment of human MCP-1 cDNA using
Lipofectin Reagents (Life Technologies), as described in detail
elsewhere (14)
. After 18 h, the medium was changed to
fresh CRPMI 1640 medium containing 800 µg/ml of G418 in 24-well
plates. Cell subclones resistant to G418 were assayed for the
expression of MCP-1 mRNA by Northern blotting. For the establishment of
cell lines that stably expressed MCP-1, the subclones positive for
MCP-1 mRNA were cloned by limiting dilution. Stable clones transfected
with BCMGSNeo were established by a similar method.
Preparation of Total Cellular RNA and Northern Blot Analysis.
Tumor cells were lysed in ISOGEN (Nippon Gene, Tokyo, Japan), a
mixture of guanidinium isothiocyanate and phenol. Total cellular RNA
was then extracted with chloroform and precipitated with isopropanol,
as described elsewhere (11)
. The denatured RNA (5 µg)
was electrophoresed in a 1% agarose gel containing 2.2 M
formaldehyde, transferred to a Hybond-N+ nylon
membrane, and hybridized with 32P-labeled probes.
The filters were washed twice with 0.1 x saline-sodium
phosphate-EDTA and 0.1% SDS for 1 h at 60°C. The membranes were
autoradiographed using a Fujix bioimaging analyzer BAS 1500 (Fuji
Photofilm Co., Tokyo, Japan). A cDNA probe of human MCP-1
was obtained by digesting BCMGSNeoMCAF plasmid vector with
XhoI and NotI. cDNA fragments of human
MCP-1 and ß-actin (Wako, Osaka, Japan) were labeled using
a random primer labeling kit (Takara Shuzo, Kyoto, Japan) with
[
-32P]dCTP (Amersham Pharmacia Biotech; 110
TBq/mmol).
Enzyme Immunoassay of Human MCP-1.
Tumor cells were plated at 5 x 105 cells/1 ml/well in 24-well plates. After
24 h, the supernatants were harvested and used for measuring MCP-1
protein. An ELISA of human MCP-1 was performed essentially as described
previously (15)
. Briefly, microtiter plates were coated
overnight with antihuman MCP-1 monoclonal Ab (ME61, 1 µg/ml) in 100
µl/well of 0.05 M carbonate buffer (pH 9.6) at 4°C. The
plates were then washed three times with PBS containing 0.05% Tween 20
(buffer A), blocked with a solution of 1% BSA in buffer A (buffer B)
at 37°C for 1 h, and washed again with buffer A. The standards
and samples diluted in buffer B were then incubated in the wells
overnight at 4°C. The wells were then washed three times and
incubated with 100 µl of rabbit antihuman MCP-1 Ab at 37°C for
2 h. They were then washed at least 10 times and incubated with
100 µl of alkaline phosphatase-conjugated antirabbit IgG (Tago code
6500; diluted 1:10,000 with buffer B) at 37°C for 2 h. Finally,
100 µl of enzyme substrate solution [1 mg/ml
p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, MO)
in 1 M diethanolamine (pH 9.8) supplemented with
0.5 mM MgCl2] were added
to each well, and the plates were incubated at room temperature for 30
min. The enzyme reaction was stopped by adding 100 µl 1
M NaOH, and A405
was determined using an ELISA plate reader (Titertek Multiscan; Flow,
Meckenheim, Germany). All samples were assayed in triplicate at least.
Analysis by Flow Cytometry.
Expression of surface antigens was measured by flow microfluorometry as
described in detail previously (11)
. Briefly, tumor cells
(1 x 106) were washed once with
PBS supplemented with 2% FBS and 0.05% sodium azide (2% FBS-PBS).
The washed cells were incubated for 30 min at 4°C in 2% FBS-PBS with
anti-CD44 (Cosmo Bio Co., Tokyo, Japan), anti-MHC class I (Immunotech,
Marseilles, France), anti-ICAM-1 Abs (Becton Dickinson, San Jose, CA),
or normal mouse serum as a negative control. They were then washed
twice with 2% FBS-PBS and treated with FITC-conjugated goat antimouse
IgG (H+L; Immunotech) for 30 min at 4°C. The cells were washed again,
and their fluorescence intensity was measured with a FACScan (Becton
Dickinson, Mountain View, CA).
In Vitro Proliferation Assays.
Tumor cells (1 x 105) were plated
into each well of six-well plates. After 12, 24, 48, 72, and 96 h
incubation at 37°C, the cells were trypsinized, and the number of
viable cells was counted using trypan blue stain (Life
Technologies). Triplicate wells were counted per time point.
Cell proliferation was estimated as the doubling time.
Animals.
Male SCID mice, 6–8 weeks of age, were obtained from Charles River
(Yokohama, Japan) and maintained under specific pathogen-free
conditions. Experiments were performed according to the guidelines of
our university (University of Tokushima).
Tumorigenicity and Experimental Metastasis.
For the production of tumors, parental PC-14 and PC-14 clones
transfected with the MCP-1 gene or Neo gene
(5 x 106 cells/200 µl
PBS/mouse) were injected s.c. into the flanks of SCID mice. The
formation and size of the tumors were monitored every 3–4 days until
day 49. Tumor volume (V) was calculated by the formula
V = 1/2ab2
,
in which a is the longest diameter and b is the
shortest diameter of the tumor.
For experimental metastases of human lung cancer cells, viable tumor cells suspended in 300 µl of PBS were injected into the lateral tail vein of SCID mice (12 , 16) . In some experiments, SCID mice received injections i.p. with TM-ß1 Ab (1 mg/mouse) 2 days before tumor inoculation to deplete NK cells (16) . After the indicated periods, the mice were sacrificed, and the numbers of metastatic lymph nodes were counted. Nodules in the liver, kidneys, and lungs were counted with the aid of a dissecting microscope.
Isolation of Human CD56+ NK Cells.
Leukocyte concentrates from healthy donors were separated into
peripheral blood mononuclear cells by density gradient centrifugation
in lymphocyte separation medium (10)
. Subsequently,
peripheral blood mononuclear cells were separated into lymphocytes and
monocytes by counterflow centrifugal elutriation in a Beckman JE-5.0
rotor (Beckman Instruments, Inc., Fullerton, CA) as reported previously
(10)
. The lymphocyte-rich fraction was collected at flow
rates of 12–16 ml/min at 2000 rpm. The purity of the lymphocyte
fraction, determined by morphological examination and nonspecific
esterase staining, was >99%. In these lymphocytes, highly purified
CD56+ cells were obtained by MACS (Miltenyi
Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s
instructions as described previously (17)
. Briefly, the
purified lymphocytes were incubated with MACS CD56 MicroBeads on ice
for 15 min. After washing, these cells were applied for the positive
selection column MiniMACS
MS+/RS+. The column was
washed five times, and CD56+ cells were flushed
out. The purity of CD56+ NK cells was >94%, as
determined by flow cytometry after staining with PE-conjugated
antihuman CD56+ Ab (Immunotech).
The Cytotoxicity Assay.
As effector cells, the spleen cells of SCID mice were also prepared as
described elsewhere (18)
. The purified human
CD56+ NK cells or murine spleen cells were
cultured in medium with various concentrations of rMCP-1, IL-2 (500
units/ml), or culture supernatants of tumor of PC-14neo13 or PC-14MCP2
cells. The cytotoxicity was determined using a standard
51Cr release assay as described previously
(10)
. The K562, YAC-1, or PC-14
cells were used as targets to evaluate NK activity. In brief, target
cells were labeled with 100 µCi of 51Cr for
1 h. After washing twice, these effector and target cells were
plated at an appropriate E:T ratio in 96-well round-bottomed plates.
The supernatant (100 µl) was collected after a 4-h (human
CD56+ NK cells) or an 8-h (murine spleen cells)
incubation, and the radioactivity was counted using a gamma counter.
Determinations were carried out in triplicate. The percentage of
specific cytolysis was calculated from the release of
51Cr from test samples and control samples, as
follows:
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in which E is the release in the test sample (cpm in the supernatant from target cells incubated with test effector cells), S is the spontaneous release (cpm in the supernatant from target cells incubated in medium alone), and M is the maximum release (cpm released from target cells lysed with 1 N HCl).
Distribution and Fate of [125I]IdUrd-labeled Tumor
Cells in Vivo.
To investigate the distribution and fate of mock-transfected cells or
MCP-1 gene-transfected cells, we compared the organ
distribution of these cells after tumor injection with a method
reported earlier (19)
. In brief, cells were labeled with
[125I]-IdUrd for 24 h. After washing
twice, [125I]IdUrd-labeled cells (1 x 106 cells/300 µl of PBS/mouse) were
injected into the lateral tail vein of SCID mice. After the indicated
periods, the mice were sacrificed, and the radioactivity of each organ
was counted using a gamma counter.
Statistical Analysis.
The significance of differences of in vitro data were
analyzed by Student’s t test (two-tailed), and the
significance of differences of in vivo data were analyzed by
Mann-Whitney U test or Wilcoxon test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Table 1
). We found no production of MCP-1 at
mRNA or protein level in parental PC-14 cells. After transfection with
BCMGSNeoMCAF or BCMGSNeo, we selected three clones that produce MCP-1
(PC-14MCP2, PC-14MCP10, and PC-14MCP12) and two neo control clones
(PC-14neo7 and PC-14neo13) for the following assays (Fig. 1)
. These
transfectants (PC-14MCP2, PC-14NCP10, and PC-14MCP12) secreted MCP-1
stably for at least 15 months in vitro. Transfection of the
MCP-1 gene into PC-14 cells did not cause any change in the
expression of surface antigens (MHC class I, CD44, and ICAM-1) or
in vitro proliferation (Table 1)
. Moreover, we examined
production of other cytokines, such as IL-1, IL-1ß, IL-6, IL-8,
granulocyte-CSF, granulocyte/macrophage-CSF, macrophage-CSF,
tumor necrosis factor-, IFN-
, or VEGF. PC-14 cells or
transfectants produced IL-6, IL-8, M-CSF, and VEGF, and there was no
difference in production of these cytokines (data not shown).
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. There was no significant difference
statistically in the in vivo growth rate between parental
tumors and transfectants (Fig. 2)
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. In contrast, MCP-1 gene
transfectants formed fewer lung metastases, and the incidence in the
formation of PE was remarkably less.
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, which
suggests that NK cells play a critical role in metastatic formation by
MCP-1 gene transfectants.
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).
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. In addition,
pretreatment of NK cells with the supernatant of PC-14neo13 cells also
stimulated NK activity. Because PC-14 cells produced various cytokines,
such as IL-6, IL-8, macrophage-CSF, and VEGF (data not shown), and IL-6
has been reported to stimulate NK activity (21)
, IL-6
(and/or other NK cell-activating molecules) secreted in the
supernatants might stimulate NK activity. Under these experimental
conditions, culture supernatants of PC-14MCP2 cells further augmented
NK activity compared with those of PC-14neo13 cells. Furthermore, human
rMCP-1 or culture supernatants of PC-14MCP2 cells also enhanced the
killer activity of murine spleen cells against YAC-1 or PC-14 cells
(Fig. 5)
. The cytotoxicity of murine
spleen cells cultured with recombinant human IL-2 against YAC-1 or
PC-14 cells was 73.2% or 26.4%, respectively.
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. However, at
24 h after tumor cell injection, six times more viable neo control
cells were detected in the lungs of NK cell-depleted SCID mice compared
with NK cell-intact SCID mice, which suggests that NK cells play an
important role in tumor cell eradication at this phase. In NK
cell-depleted SCID mice, there was no difference between neo control
cells and MCP-1 gene transfectants on viability in the lungs
up to 72 h after tumor cell injection. In contrast, a marked
difference in cell viability in the lung was observed at 24 and 72 h after tumor cell injection in NK cell-intact SCID mice. The viability
of MCP-1 gene transfectants in the lung was much lower than
that of neo control cells in NK cell-intact SCID mice.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because lymphocytes (T and B cells) are lacking in SCID mice, NK cells or monocyte-macrophages can be considered as mechanisms of the antimetastatic effect observed in SCID mice. NK cells, especially, have been reported to play an important role in metastasis formation (22) . We demonstrated previously that NK cell depletion promoted metastasis formation by human lung cancer cell lines (H69/VP small cell carcinoma and RERF-LC-AI squamous cell carcinoma) into multiple organs, and we extended this phenomenon using PC-14 adenocarcinoma cells in this study. To deplete NK cells, we pretreated SCID mice with an i.p. injection of antimouse IL-2 receptor ß chain Ab TM-ß1. This treatment successfully and selectively depletes NK cells in SCID mice at least for 6 weeks, irrespective of tumor cell injection (16 , 23) .
Although MCP-1 was discovered originally as a chemotactic and activating factor for monocytes, the chemotactic activity of MCP-1 for T lymphocytes was documented later. In addition, recent reports (4, 5, 6, 7) have shown that CC chemokines, including MCP-1, potentiate the function of NK cells. Loetscher et al. (6) found that CC chemokines induced chemotaxis, Ca2+ changes, and the release of granule enzymes from both cloned and blood NK cells. Consistent with these reports, we show in this study that MCP-1 enhanced the killer activity of human CD56+ NK cells or spleen cells of SCID mice against K562, YAC-1, or PC-14 cells in vitro. Moreover, MCP-1 gene transfection into PC-14 cells resulted in inhibition of lung metastasis only in NK cell-intact SICD mice but not in NK cell-depleted SCID mice. MCP-1 was reported to augment not only killer activity of NK cells but also migration of NK cells (4, 5, 6, 7) . These findings strongly suggest that MCP-1 secreted by transfectants augments the migration and killer activity of NK cells, hence inhibiting the lung metastasis of PC-14 cells.
The process of metastasis consists of several sequentially linked steps (24) . Although tumor cells bypassed the initial steps of metastasis (separation from the primary tumor, invasion, and release into blood vessels) in our experimental metastasis model, all subsequent steps (arrest at a distant site, extravasation, and proliferation as a secondary colony with neovascularization) must have occurred. To clarify further the mechanism of inhibition of lung metastasis of MCP-1 gene transfectants in NK cell-intact SCID mice, we determined at which steps this inhibition occurred. Our results show that MCP-1 gene transfectants and neo control cells were equally arrested at the capillary of the lung at 4 h after tumor cell injection and that a marked difference in cell viability between the transfectants and neo control was observed at 24–72 h after injection. Within 6 h after injection, extravasation can occur from the capillary plexus (25) , although it was not determined when PC-14 cells extravasated in this model. Therefore, it is possible that NK cells activated with MCP-1 secreted by transfectants eradicated these tumor cells just before or after extravasation from the lung capillary (at least after the arrest at the lung capillary).
An interesting point of this study is that MCP-1 gene transfection did not result in the inhibition of tumorigenicity or growth when inoculated into the s.c. space even in NK cell-intact SCID mice. The same phenomenon has been well documented by Hirose et al. (26) and ourselves (10 , 11) . Hirose et al. (26) demonstrated that MCP-1 gene transfection into Chinese hamster ovary cells had no effect on tumorigenicity in nude mice. We reported also that there was no effect of MCP-1 gene transfection on the tumorigenicity of human small cell lung cancer (H69/VP) cells in nude and SCID mice. As we proposed previously (20) , antitumor effects by cytokine gene modification can be regulated differentially by various organ microenvironments (including the lung and skin). In fact, NK cells are found predominantly in the blood and spleen (27) . Therefore, the microenvironments of the lungs and skin might differ especially in the accumulation or activation of NK cells induced by MCP-1.
In summary, MCP-1 gene transfection into human lung adenocarcinoma cells resulted in an inhibition of metastatic formation presumably via the activation of NK cells. Locally secreted MCP-1 can augment Ab-dependent cellular cytotoxicity reaction and the therapeutic efficacy of Ab through the accumulation of monocyte-macrophages into the tumors (11) . Therefore, an approach to facilitate the recruitment and activation of immuno-effector cells such as NK cells and monocyte-macrophages may be crucial for the development of a novel immunotherapy for lung cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Grant-in-Aid for Cancer Research
from the Ministry of Education, Science, Sports and Culture of Japan
and a Grant from the Ministry of Health and Welfare of Japan. 
2 To whom requests for reprints should be
addressed, at Third Department of Internal Medicine, University of
Tokushima School of Medicine, Kuramoto-cho 3, Tokushima 770-8503,
Japan. Phone: 81-88-633-7127; Fax: 81-88-633-2134; E-mail: ssone{at}clin.med.tokushima-u.ac.jp
3 The abbreviations used are: MCP, monocyte
chemoattractant protein; rMCP-1, recombinant MCP-1; NK, natural killer;
IL, interleukin; SCID, severe combined immunodeficient; PE, pleural
effusion; Ab, antibody; FBS, fetal bovine serum; ICAM-1, intercellular
adhesion molecule-1; Neo, neomycin resistant; MCAF, monocyte
chemotactic and activating factor; MACS, magnetic cell sorting; CSF,
colony-stimulating factor; VEGF, vascular endothelial growth factor;
IdUrd, iododeoxyuridine.
Received 4/12/00. Accepted 10/ 6/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol., 155: 3877-3888, 1995.[Abstract]
/ß secretion. J. Immunol., 150: 4844-4855, 1993.[Abstract]
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