Enhancement of tumoricidal activity of alveolar macrophages
via CD40-CD40 ligand interaction
Kazuyoshi
Imaizumi1,
Tsutomu
Kawabe1,
Satoshi
Ichiyama2,
Hitoshi
Kikutani3,
Hideo
Yagita4,
Kaoru
Shimokata5, and
Yoshinori
Hasegawa1
1 The First Department of
Internal Medicine and 5 Department
of Clinical Preventive Medicine, Nagoya University School of Medicine,
Nagoya 466-8550; 2 Department of
Clinical Laboratory Medicine, Kyoto University Hospital, Kyoto
606-8507; 3 Department of
Molecular Immunology, Research Institute for Microbial Diseases, Osaka
University, Osaka 565-0871; and
4 Department of Immunology,
Juntendo University School of Medicine, Tokyo 113-8421, Japan
 |
ABSTRACT |
CD40-CD40 ligand (CD40L) interaction was
originally defined as important molecules for the development of
humoral immunity. Thereafter, some investigations have focused on its
essential roles for the induction of cell-mediated immunity in host
defenses. Here we investigated the antitumor activity of murine
alveolar macrophages through CD40-CD40L interaction. The
CD40L gene was transfected into murine lung cancer cells (3LLSA), and CD40L-expressing clones (3LLSA-CD40L) were established. Stimulation of CD40 molecules on
the surface of alveolar macrophages with 3LLSA-CD40L cells induced the
production of nitric oxide, tumor necrosis factor-
, and
interleukin-12 and the tumoricidal activity of alveolar macrophages in
the presence of interferon-
, which increased the surface expression of CD40 molecules on alveolar macrophages. These findings were not
observed when alveolar macrophages were obtained from CD40-deficient mice. On the other hand, interleukin-6 production by alveolar macrophages did not depend on CD40-CD40L interaction. We also established a murine melanoma cell line expressing CD40L (B16 4A5-CD40L) that could induce tumoricidal activity of alveolar macrophages. Furthermore, when spleen cells were cocultivated with
3LLSA-CD40L cells, specific cytotoxic T lymphocytes for wild-type 3LLSA
cells could be induced. These results suggest that
CD40L gene transfer into tumor cells
may induce antitumor immunity in a tumor-bearing host and may offer a
new strategy for cancer gene therapy.
nitric oxide; cytokine production; cytotoxic T lymphocyte; lung
cancer; CD40-deficient mice
 |
INTRODUCTION |
CD40 IS A MEMBER of the tumor necrosis factor
(TNF)-receptor family of cell surface protein expressed on B cells,
dendritic cells, human thymic epithelial cells, human endothelial
cells, and several carcinoma cell lines (5, 19). CD40 binds a CD40 ligand (CD40L; CD154), which is an ~35-kDa glycoprotein, a member of
the TNF superfamily, and expressed on activated T cells, basophils, and
mast cells (5). CD40-CD40L interaction is important for cross talking
between T cells and B cells that is essential for B-cell immunoglobulin
class switching (5), avoidance of B-cell apoptosis (40), and formation
of germinal centers in secondary lymphoid organs (22). A recent study
(29) demonstrated the important role of this ligand-receptor pair in
cell-mediated immunity in addition to its function in the regulation of
humoral immunity. It has been reported (2, 29, 34, 39) that monocytes
and macrophages were activated by stimulation through CD40-CD40L
interaction and developed tumoricidal activity. CD40 also regulates the
activity of antigen-presenting cells (APCs) (29). Recently, several
reports demonstrated that CD40-CD40L interaction plays a critical role in the induction of antitumor immunity (26), especially the essential
role in the induction of cytotoxic T cells (CTLs) (6, 25, 30, 32).
Taking these findings into consideration, we hypothesized that
stimulation of macrophages and APCs through CD40 with CD40L-expressing
tumor cells could enhance the cytotoxic effect of macrophages and the
antitumor immunity of T cells. In the present study, we investigated
the antitumor immunity against lung cancer cells because lung cancer
cells generally express low antigenicity, and it seems to be difficult
to induce the lung cancer-specific cellular immunity, in contrast to
melanoma cells. We generated CD40L-expressing lung carcinoma cells by
transfection of CD40L cDNA, and we attempted to verify whether these
cells could induce alveolar macrophage activation and enhance its
tumoricidal effect against wild-type lung carcinoma cells.
 |
MATERIALS AND METHODS |
Mice. C57BL/6 mice
(H-2b) were supplied by the
Institute for Laboratory Animal Research (Nagoya University School of
Medicine, Nagoya, Japan). CD40-deficient mice used in this study were
generated by a gene-targeting technique as previously reported (22). A CD40(+/
) mouse was produced by backcrossing the originally
described CD40(
/
) mouse to a C57BL/6 mouse. The
heterozygous littermates were intercrossed to generate CD40(+/+),
CD40(+/
), and CD40(
/
) mice. These mice were
genotyped by a PCR of genomic DNA obtained from a tail biopsy with
primers to identify the rearranged CD40 locus as described previously
(22).
Cells. A murine lung carcinoma cell
line (3LLSA; Lewis lung cancer cells), which was originally established
from the lung of a C57BL/6 mouse bearing a tumor, was obtained from the
Japanese Cancer Research Resources Bank (Tokyo, Japan). A
murine melanoma cell line, B16 4A5, which also originated from a
C57BL/6 mouse (31, 35), was obtained from Riken Cell Bank (Tsukuba,
Japan). 3LLSA cells were maintained in RPMI 1640 medium supplemented
with 1% L-glutamine, 1%
penicillin-streptomycin, and 10% fetal calf serum (RPMI-10% FCS). B16
4A5 cells were maintained in Dulbecco's modified Eagle's medium
(DMEM; GIBCO BRL, Grand Island, NY) containing 10% FCS (DMEM-10%
FCS). Alveolar macrophages were collected from C57BL/6 or
CD40-deficient mice by bronchoalveolar lavage. A 26-gauge polystyrene
needle was inserted into the mouse trachea, and 1 ml of
phosphate-buffered saline (PBS) was injected into the lung. After the
inflated lung was gently massaged, the injected fluid was sucked up by
the syringe. These procedures were repeated three times in each mouse.
The collected fluid was centrifuged, and the cell pellet was suspended
in PBS. After being washed, the cells were cultured in RPMI-10% FCS.
After 90 min of incubation at 37°C, nonadherent cells were removed
by gentle washing, and adherent cells were used as an alveolar
macrophage population.
Reagents. Recombinant murine
interferon (IFN)-
was purchased from GIBCO BRL (Gaithersburg, MD).
Hamster anti-mouse CD40 monoclonal antibody (MAb) IgM (HM40-3) and
hamster anti-mouse CD40L MAb IgG (HM40L-1) were established as
previously described (16, 18). Fluorescein isothiocyanate
(FITC)-conjugated goat anti-hamster IgG
F(ab')2 fragment was
purchased from Caltag Laboratories (San Francisco, CA). FITC-conjugated
hamster anti-mouse CD40 antibody (HM40-3) and rat anti-mouse TNF-
antibody (MP6-XT3) were purchased from PharMingen (San Diego, CA).
NG-monomethyl-L-arginine
(L-NMMA) was obtained from
Calbiochem (La Jolla, CA).
Transfection. Murine CD40L cDNA
encoding the entire coding region was prepared by RT-PCR according to
the published sequence (4). CD40L cDNA was cloned into the pEF-BOS
mammalian expression vector (27), which contains the promoter lesion of
the human elongation factor-1
gene (pBOS-CD40L). pMC1neo Poly A,
which contains the
neor gene from
Tn5, the herpes simplex thymidine kinase promoter, and the enhancer
sequence from the polyoma virus Py F411, was purchased from Stratagene
(La Jolla, CA).
The expression vectors pBOS-CD40L and pMC1neo Poly A were cotransfected
into 3LLSA and B16 4A5 cells. Transfections were performed on 100-mm
plates with 10 µg plasmid DNA/plate with the lipofection method with
Lipofectace reagent (GIBCO BRL, Gaithersburg, MD) according to the
manufacturer's instructions. After 12 h of exposure, the cells were
washed three times with medium and cultured in 10 ml of complete
medium. Forty hours after the medium exchange, cells were selected in
medium containing 800 µg/ml of G418 (GIBCO BRL, Grand Island, NY).
After 2 wk of selection, G418-resistant clones were randomly selected
from the surviving colonies and used in the following experiments.
Mock-transfected clones (3LLSA-MOCK and B16 4A5-MOCK), which were
established by cotransfecting the pEF-BOS plasmid without a cDNA insert
and pMC1neo Poly A, were used in the experiments as a control.
Flow cytometry. After the cells were
incubated in PBS with 0.5 mM EDTA for 3 min at 37°C, they were
detached by vigorous pipetting. The cells were then harvested into
complete medium containing 10% FCS, centrifuged at 1,500 rpm for 3 min
at 4°C, and resuspended (5 × 105 cells/100 µl) in PBS. To
examine the expression of CD40L, the cells were incubated with hamster
anti-mouse CD40L IgG (HM40L-1; 25 µg/ml) or an isotype-matched
control IgG (25 µg/ml) for 30 min at 4°C. The cells were then
washed and stained with an FITC-conjugated goat anti-hamster IgG
F(ab')2 fragment (2.5 µg/ml) for 30 min at 4°C. For analyzing the CD40 expression of
alveolar macrophages, the cells were cultured with and without IFN-
(100 U/ml) for 24 h, then incubated with 2% rabbit serum and 2% goat
serum in PBS for 30 min at 4°C to block the nonspecific antibody
binding for Fc receptors. After being washed with PBS supplemented with 1% FCS and 0.1% NaN3, the cells
were incubated with FITC-conjugated hamster anti-mouse CD40 IgM (2.5 µg/ml) for 30 min at 4°C. Fluorocytometric analysis was done with
a Coulter Epics XL equipped with an argon-ion laser (Coulter
Electronics, Miami, FL).
RNA isolation and RT-PCR. Total RNA
was isolated from the cells with the Isogen RNA extraction kit (Nippon
Gene, Toyama, Japan) according to the manufacturer's instructions. For
PCR analysis of RNA, cDNA was prepared by RT from 1 µg of
each RNA sample in a 20-µl reaction volume containing 0.5 µg of
oligo(dT)12-18 primer, 10 mM
dithiothreitol, 0.5 mM deoxynucleotide triphosphates, 20 mM
Tris · HCl, 50 mM KCl, 2.5 mM
MgCl2, and 200 U/µl of reverse transcriptase (Superscript II, GIBCO BRL, Gaithersburg, MD). Then the
reaction mixture was incubated at 42°C for 50 min and heated at
70°C for 15 min to stop the RT. Amplification of cDNA was carried out with the PCR method with the sense primer 5'-AAG CGA AGC CAA CAG TAA TG-3' and the antisense primer 5'-GAC AAA CAC AGA
AGC ACC AG-3'. These primers result in a 337-bp cDNA encoding
murine CD40L. As an internal control, amplification of an 887-bp cDNA encoding murine
-actin was used (the sense primer was 5'-GCA AGA GAG GTA TCC TGA CCC TGA AG-3', and the antisense primer was 5'-CAT CTG CTG GAA GGT GGA CAG TGA GG-3'). The PCR
amplifications were performed in a 20-µl reaction volume containing 1 µg of each cDNA preparation, 0.5 µM each primer, 0.2 mM
deoxynucleotide triphosphates, 20 mM Tris · HCl, 50 mM KCl, 20 µM EDTA, 200 µM dithiothreitol, 0.01% Tween 20, 0.01%
Nonidet P-40, 1% glycerol solution (final concentration), and 1 U of
Taq polymerase (Takara Shuzo, Tokyo, Japan). PCR was performed in a thermal cycler (Astec, Fukuoka, Japan)
by 30 cycles of denaturation at 94°C for 1 min, annealing at
64°C for 1 min, and extension at 72°C for 2 min. PCR products were analyzed by electrophoresis on 3% agarose gels and visualized by
ethidium bromide staining.
Macrophage-mediated tumor cell lysis
assay. Alveolar macrophage-mediated tumor cell lysis
was assessed by measuring cytotoxicity against
51Cr-labeled 3LLSA cells as target
cells (15, 33). 3LLSA or B16 4A5 cells
(106 cells) were labeled with 100 µCi of
Na251CrO4
in 0.5 ml of RPMI-10% FCS for 1 h at 37°C. Then the cells were
washed three times with medium and finally resuspended in medium
containing 10% FCS. Alveolar macrophages as effector cells obtained
from C57BL/6 or CD40-deficient mice were preincubated in round-bottom
96-well microculture plates (1 × 105 cells/well) with and without
murine IFN-
(100 U/ml) for 24 h at 37°C and then stimulated via
CD40 for 12 h at 37°C. As for the stimulation via CD40, alveolar
macrophages were cultured with hamster anti-mouse CD40 IgM (10 µg/ml)
or 40 Gy-irradiated murine CD40L cDNA-transduced cells (3LLSA-CD40L and
B16 4A5-CD40L cells, respectively; 1 × 104 cells/well). 40 Gy-irradiated
3LLSA-MOCK or B16 4A5-MOCK cells (1 × 104 cells/well) were used as a
control. To inhibit the function of cell surface CD40L, hamster
anti-mouse CD40L IgG (20 µg/ml) was added to the culture medium.
Radiolabeled target cells (1 × 104 cells/well) were placed onto
the round-bottom 96-well microculture plates in which the alveolar
macrophages were cultured. The radioactivity released during a 36-h
incubation was determined with a gamma counter with 100 µl of culture
supernatant from each well. Experimental release was determined from
the amount of 51Cr released by the
target cells when they were incubated with the alveolar macrophages.
Spontaneous 51Cr release was
determined from cultures containing target cells alone. Total release
was determined from a 1% Nonidet P-40 lysate of the target cells. The
percentage of specific 51Cr
release was calculated as [(experimental release
spontaneous release)/(total release
spontaneous
release)] × 100. All tests were performed in triplicate,
and mean values were calculated.
Measurement of nitrite and cytokines.
Alveolar macrophages obtained from C57BL/6 or CD40-deficient mice were
incubated in round-bottom 96-well microculture plates (1 × 105 cells/well) with and without
murine IFN-
(100 U/ml) in combination with the stimulation of CD40
as mentioned in Macrophage-mediated tumor cell lysis
assay. After 48 h of incubation at
37°C, the supernatants were removed and applied for measurement of
nitrite, TNF-
, interleukin (IL)-6, and IL-12 (p70). The nitrite
concentration was measured with a calorimetric assay based on the
Griess reaction previously described (21). Briefly, 100 µl of
supernatant were incubated for 10 min at room temperature with an equal
volume of Griess reagent containing 0.5% sulfanilamide and 0.05%
N-(1-naphthyl)ethyleneamine dihydrochloride in 2.5% phosphoric acid. The optical density at 570 nm
was measured with a microtiter plate reader. The
NO
2 contents were quantified by
comparison with a standard curve generated with
NaNO2 in the range of 0-100
µM. Murine TNF-
, IL-6, and IL-12 (p70) concentrations were
determined with an enzyme-linked immunosorbent assay (ELISA) kit with
mouse TNF-
ELISA (Endogen, Woburn, MA), mouse IL-6 immunoassay (R&D
Systems, Minneapolis, MN), and mouse IL-12 (p70) ELISA kits (Genzyme,
Cambridge, MA). All kits were used according to the manufacturer's
instructions. The lower limits of detection were 10 pg/ml for TNF-
,
3.1 pg/ml for IL-6, and 5 pg/ml for IL-12.
CTL assays. Spleens were removed from
8- to 10-wk-old C57BL/6 mice. After a single-cell suspension was
prepared, 2 × 106
spleen cells were cocultivated with 40 Gy-irradiated 3LLSA-CD40L, 3LLSA-MOCK, or wild-type 3LLSA cells (2 × 105 cells) in 2 ml of RPMI-10%
FCS containing 1% nonessential amino acids and 1% sodium pyruvate in
a six-well tissue culture plate. Five days later, nonadherent cells
were harvested, counted, and used as effector cells. Wild-type 3LLSA or
B16 4A5 cells (5 × 105
cells) were labeled with 100 µCi of
Na251CrO4
in 0.5 ml of conditioned medium containing 10% FCS for 1 h at 37°C
and used as target cells. Then, labeled target cells (5 × 103) were cocultivated for 12 h
with effector cells at different effector-to-target cell ratios in
wells of round-bottom 96-well microculture plates. The radioactivity
released during incubation was determined with a gamma counter with 100 µl of culture supernatant from each well. The percentage of specific
51Cr release was calculated as
described in Macrophage-mediated tumor cell lysis
assay.
 |
RESULTS |
Expression of CD40L in cDNA-transfected murine lung
carcinoma cells. Expression of CD40L mRNA was studied
by RT-PCR. Total cellular RNA was extracted from the CD40L
cDNA-transfected 3LLSA clone (3LLSA-CD40L) and the
vector-alone-transfected clone (3LLSA-MOCK). Expression of CD40L mRNA
was detected only in 3LLSA-CD40L but not in 3LLSA-MOCK cells (Fig.
1A).
Expression of the cell surface protein of CD40L was analyzed by flow
cytometry with an anti-CD40L MAb. As shown in Fig.
1B, a high expression of CD40L was
obtained in CD40L gene-transfected
clones but not in the vector-alone-transfected clone. Thus we used
these clones in the following experiments.

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Fig. 1.
Expression of CD40 ligand (CD40L) in CD40L cDNA-transfected murine lung
carcinoma cells. A: expression of
CD40L mRNA was detected by RT-PCR (see MATERIALS AND
METHODS). PCR products were analyzed by
electrophoresis on 3% agarose gels and visualized by ethidium bromide
staining. Lane 1, detection of CD40L
cDNA from pBOS-CD40L plasmid; lane 2,
detection of CD40L mRNA from CD40L-transfected 3LLSA (3LLSA-CD40L)
cells; lane 3, detection of CD40L mRNA
from mock-transfected 3LLSA (3LLSA-MOCK) cells; lane
4, detection of -actin mRNA from 3LLSA-CD40L cells;
lane 5, detection of -actin mRNA
from 3LLSA-MOCK cells. *, Molecular markers.
B: expression of CD40L on cell surface
was detected by flow cytometry. Cells (5 × 105 cells/100 µl) were incubated
with hamster anti-mouse CD40L IgG and FITC-conjugated goat anti-hamster
IgG F(ab')2 fragment. Thin
black line, 3LLSA-MOCK cells; thick black line, 3LLSA-CD40L cells; gray
line, 3LLSA-MOCK cells stained with isotype-matched control IgG and
FITC-conjugated goat anti-hamster IgG
F(ab')2 fragment.
|
|
CD40 expression on alveolar
macrophages. To investigate the activation of alveolar
macrophages through CD40, we first studied CD40 expression on the cell
surface of murine alveolar macrophages collected by bronchoalveolar
lavage. The macrophages were analyzed by flow cytometry with an
FITC-labeled anti-CD40 MAb. Freshly isolated macrophages cultured in
medium alone for 24 h showed no significant expression of CD40 on the
cell surface (Fig. 2). However, macrophages
cultured in medium containing recombinant murine IFN-
(100 U/ml) induced a significantly higher expression of cell surface
CD40 (Fig. 2). These findings suggest that IFN-
will enhance the
CD40-CD40L-mediated activation of alveolar macrophages.

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Fig. 2.
Interferon (IFN)- induces CD40 expression on cell surface of
alveolar macrophages. Alveolar macrophages were cultured without (thin
black line) and with (thick black line) IFN- (100 U/ml) for 24 h,
and surface expression of CD40 was analyzed by flow cytometry. Cells (5 × 105 cells/100 µl) were
stained with FITC-labeled hamster anti-mouse CD40 IgM. Gray line,
nonstimulated control alveolar macrophages without staining.
|
|
Effect of CD40 stimulation on alveolar macrophage
tumoricidal activity. When murine alveolar macrophages
were incubated with the anti-CD40 IgM antibody or 3LLSA-CD40L cells
alone, we could not observe any tumoricidal activity of alveolar
macrophages (data not shown). Then we incubated the alveolar
macrophages with IFN-
(100 U/ml) in addition to the stimulation via
CD40. IFN-
activated macrophage tumoricidal activity by itself, and
stimulation of anti-CD40 IgM antibody (data not shown) or 3LLSA-CD40L
cells with IFN-
even further enhanced its cytotoxic activity against
wild-type 3LLSA cells (Fig.
3A). The
enhancement by 3LLSA-CD40L cells was blocked by the anti-CD40L blocking
antibody (Fig. 3A). In addition, macrophages obtained from CD40-deficient mice showed no enhancement of
tumoricidal activity against wild-type 3LLSA cells when they were
incubated with anti-CD40 IgM antibody (data not shown) or 3LLSA-CD40L
cells combined with IFN-
(Fig.
3B).

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Fig. 3.
Stimulation of CD40 molecules enhances tumoricidal activity of alveolar
macrophages. Alveolar macrophages from C57BL/6
(A and
C) and CD40-deficient mice
(B) were cultured in presence and
absence of murine IFN- (100 U/ml) for 24 h. CD40 molecules on
alveolar macrophages were stimulated for 12 h at 37°C with
irradiated 3LLSA-CD40L (A and
B) or B16 4A5-CD40L
(C) cells or irradiated 3LLSA-MOCK
(A and
B) or B16 4A5-MOCK
(C) cells.
51Cr-labeled 3LLSA cells
(A and
B) or B16 4A5 cells
(C) were cultured with stimulated
alveolar macrophages for an additional 36 h. CD40LIgG, anti-mouse
CD40L IgG. Amount of 51Cr released
in supernatants was measured. Data are means ± SE from 3 independent experiments. Spontaneous counts released in absence of
macrophages were always <30% of maximal counts released by 1%
Nonidet P-40 lysate of target cells.
|
|
We also investigated whether the enhancement of tumoricidal activity
via CD40-CD40L interaction would be demonstrated in another tissue
origin of the tumor cell line. We used the B16 4A5 murine melanoma cell
line originated from C57BL/6 mice and established the
CD40L gene-transfected clone
designated B16 4A5-CD40L, as was the clone for 3LLSA-CD40L. Although
parental B16 4A5 cells showed no expression of CD40L on their cell
surface, B16 4A5-CD40L cells expressed both CD40L mRNA and the cell
surface protein (data not shown). As shown in Fig.
3C, B16 4A5-CD40L could enhance
alveolar macrophage tumoricidal activity against the parental wild-type melanoma cells. These results demonstrated that CD40 stimulation activates the alveolar macrophage, resulting in the enhancement of
tumoricidal activity.
CD40 stimulation enhances nitric oxide and TNF-
production by alveolar macrophages. It is of interest
to know how alveolar macrophages exhibit the tumoricidal effect after
CD40 stimulation. We investigated the production of nitric oxide (NO)
and cytokines by alveolar macrophages under the stimulation of CD40. As
shown in Fig. 4, we detected significant
increases in NO and TNF-
production when alveolar macrophages were
stimulated with 3LLSA-CD40L cells in combination with IFN-
. In
contrast, these enhancements were not observed when alveolar
macrophages from CD40-deficient mice were used (data not shown). The
increase in NO and TNF-
production by stimulation with IFN-
and
3LLSA-CD40L cells was blocked by the anti-CD40L blocking antibody down
to the same level as the production by macrophages stimulated with
IFN-
and 3LLSA-MOCK cells. The production of NO and TNF-
by
alveolar macrophages stimulated with IFN-
and 3LLSA-CD40L cells was
much higher than that in macrophages stimulated with IFN-
and
anti-CD40 IgM antibody (data not shown). In addition, 3LLSA-MOCK cells
moderately enhanced the production of NO and TNF-
by alveolar
macrophages stimulated with IFN-
(Fig. 4). These findings suggest
that 3LLSA cells express some molecules other than CD40L and activate
alveolar macrophages to produce NO and TNF-
.

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Fig. 4.
Enhancement of nitric oxide (NO; nitrite; solid bars) and tumor
necrosis factor (TNF)- (open bars) production by alveolar
macrophages through CD40 stimulation. Alveolar macrophages from C57BL/6
mice were stimulated with irradiated 3LLSA-CD40L or irradiated
3LLSA-MOCK cells in presence and absence of IFN- (100 U/ml). After a
48-h culture, supernatants were removed, and concentrations of nitrite
and TNF- were measured. Data are means ± SE from 3 independent
experiments.
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|
Because the tumoricidal activity and production of NO and TNF-
seem
to be mutually related (Figs. 3 and 4), we speculate that NO and
TNF-
play important roles in the generation of macrophage tumoricidal activities through CD40-CD40L interaction. To confirm these
findings, we used an anti-TNF
antibody and
L-NMMA for inhibiting macrophage-mediated tumoricidal activity. As shown in Fig.
5, the anti-TNF-
antibody and
L-NMMA inhibited the
macrophage-mediated tumoricidal activity induced by CD40L. However, the
combination of the two agents could not block the tumoricidal activity
completely. These findings showed that both NO and TNF-
were major
factors contributing to macrophage tumoricidal activity. In
addition, we speculated that other molecules, including cell surface
proteins or factors except NO and TNF-
, may also contribute to the
development of the macrophage-mediated tumoricidal activity induced by
tumor cells.

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Fig. 5.
NO and TNF- play important roles in generation of macrophage
tumoricidal activity. Alveolar macrophages from C57BL/6 mice were
cultured in presence of murine IFN- (100 U/ml) for 24 h and then
cultured with irradiated 3LLSA-CD40L cells with and without
anti-TNF- antibody (200 ng/ml) and/or 0.5 mM
NG-monomethyl-L-arginine
(L-NMMA).
51Cr-labeled 3LLSA cells were
cultured with stimulated alveolar macrophages for an additional 36 h.
Amount of 51Cr released in
supernatants was measured. Data are means ± SE from 3 independent
experiments. Spontaneous counts released in absence of macrophages were
always <30% of maximal counts released by 1% Nonidet P-40 lysate of
target cells.
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Production of IL-6 by alveolar macrophages does not
depend on CD40-CD40L interaction. We also investigated
IL-6 production by alveolar macrophages. As shown in Fig.
6, anti-CD40 IgM with IFN-
could induce
IL-6 production by alveolar macrophages in both wild-type and
CD40-deficient mice (Fig. 6). Similarly, IL-6 was significantly
produced from alveolar macrophages cultured with 3LLSA-CD40L or
3LLSA-MOCK cells with and without IFN-
, and the anti-CD40L blocking
antibody could not inhibit IL-6 production (Fig.
6A). These findings were also
confirmed by the results from alveolar macrophages obtained from
CD40-deficient mice (Fig. 6B) in
which IL-6 production was stimulated by 3LLSA cells with and without
CD40L molecules on their cell surfaces. These results indicated that
IL-6 production by alveolar macrophages was mainly stimulated through
pathways other than CD40-CD40L interaction.

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Fig. 6.
Interleukin (IL)-6 production by alveolar macrophages. Alveolar
macrophages from C57BL/6 (A) or
CD40-deficient (B) mice were
cultured in presence and absence of IFN- (100 U/ml). CD40 molecules
on alveolar macrophages were stimulated with irradiated 3LLSA-CD40L or
irradiated 3LLSA-MOCK cells. After 48 h of culture, supernatants were
removed, and IL-6 concentration was measured. Data are means ± SE
from 3 independent experiments.
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CD40 stimulation enhances production of
IL-12. We investigated the effect of CD40-CD40L
interaction on IL-12 production by alveolar macrophages stimulated with
IFN-
(Fig. 7). 3LLSA-CD40L cells with
IFN-
but not 3LLSA-MOCK cells enhanced the production of IL-12.
Furthermore, the anti-CD40L antibody blocked the enhancement of IL-12
production by alveolar macrophages stimulated with 3LLSA-CD40L cells.
These findings were confirmed by the results with alveolar macrophages
obtained from CD40-deficient mice. Although IFN-
alone moderately
stimulated IL-12 production by alveolar macrophages from CD40-deficient
mice, neither anti-CD40 IgM nor 3LLSA-CD40L cells could enhance IL-12
production by macrophages stimulated with IFN-
(data not shown).

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Fig. 7.
Enhancement of IL-12 production by alveolar macrophages through CD40
stimulation. Alveolar macrophages from C57BL/6 mice were stimulated
with irradiated 3LLSA-CD40L or irradiated 3LLSA-MOCK cells in presence
and absence of IFN- (100 U/ml). After 48 h of culture, supernatants
were removed, and IL-12 concentration was measured. Data are means ± SE from 3 independent experiments.
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Induction of CTL response against wild-type lung
cancer cell in vitro. Because the
CD40L gene-transfected lung cancer
cells activate macrophages and produce cytotoxic factors or cytokines, we speculate that the activated macrophages or APCs may effectively present the tumor antigen to CTLs and induce tumor cell-specific killer
T lymphocytes. Figure 8 shows the CTL assay
with mouse spleen cells stimulated by 3LLSA-CD40L, 3LLSA-MOCK, and
wild-type 3LLSA cells. CTL activity against wild-type 3LLSA target
cells was induced when 3LLSA-CD40L cells were used as the stimulation. CTLs induced by 3LLSA-CD40L cell stimulation did not kill the B16 4A5
target cells (data not shown). These findings suggested that the
stimulation of macrophages through CD40 with CD40L-expressing tumor
cells could induce tumor-specific CTL activity.

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Fig. 8.
Induction of cytotoxic T-lymphocyte activity by CD40L-expressing lung
cancer cells in vitro. Spleen cells obtained from C57BL/6 mice were
stimulated with irradiated wild-type 3LLSA (wt), 3LLSA-CD40L (40L), or
3LLSA-MOCK (MOCK) cells. Five days later, nonadherent cells were
cocultivated with 51Cr-labeled wt
cells for 12 h at different effector-to-target cell (E:T) ratios.
Radioactivity released during incubation was determined as described in
MATERIALS AND METHODS. Data are means ± SE from 3 independent experiments.
|
|
 |
DISCUSSION |
We demonstrated in this study that stimulation of CD40 molecules on
alveolar macrophages enhanced the production of NO, TNF-
, and IL-12
as well as the tumoricidal activity under the stimulation of IFN-
.
Enhancement of the tumoricidal activity and NO, TNF-
, and IL-12
production were observed when macrophages were stimulated with
CD40L-transfected tumor (3LLSA-CD40L) cells but not with mock-transfected (3LLSA-MOCK) or wild-type tumor (3LLSA) cells. The
observed findings were blocked by an anti-CD40L blocking antibody. Furthermore, we could not observe these findings when we used alveolar
macrophages obtained from CD40-deficient mice.
Several reports have shown that cross-linking of CD40 molecules by
anti-CD40 antibodies or recombinant soluble CD40L activates monocytes
(2), splenic macrophages (39), and dendritic cells (8) to produce
various proinflammatory mediators and cytokines. These findings suggest
important roles for the upregulation of microbicidal activity (7, 17,
36), antigen presentation, and costimulation (29). Thus CD40-CD40L
interaction appears to induce multifunctional activating signals in
macrophages and monocytes in addition to the regulation of humoral
immunity (37). In fact, several reports (14, 20, 26, 28) suggested that CD40-CD40L interaction would be required for the induction of antitumor
immunity. However, there have been few reports demonstrating that
alveolar macrophages activated by CD40-CD40L signaling showed tumoricidal activity against syngeneic lung cancer cells. Furthermore, we demonstrated that CD40L
gene-transfected lung cancer cells could induce tumor-specific CTL
activity against the parental lung cancer cells, which generally
express low antigenicity. Thus we focused on alveolar macrophages that
play a central role for the host defense immunity in the alveolar space
and the lung and demonstrated that the expression of CD40L on lung
cancer cells enhanced the antitumor immunity of the tumor-bearing host.
Activation of alveolar macrophages through CD40 stimulation required
costimulation of IFN-
for the production of NO and TNF-
and the
generation of tumoricidal activity. We observed that surface expression
of CD40 on alveolar macrophages was detectable only after the
stimulation of IFN-
, suggesting that IFN-
increases the number of
CD40 molecules on the cell surface of alveolar macrophages and enhances
the signaling through CD40-CD40L interaction.
Previous reports showed that CD40 cross-linking induced the production
of NO (39), TNF-
, IL-1 (42), IL-6 (2), and IL-12 (3, 23, 34) by
macrophages or monocytes. We showed in this report that CD40
stimulation enhanced the production of NO, TNF-
, and IL-12 but not
of IL-6 by alveolar macrophages under the stimulation of IFN-
. CD40L
or lymphocyte function-associated antigen-1 on the cell surface of
activated T cells enhanced the production of NO by splenic macrophages
(39). In fact, 3LLSA-MOCK cells enhanced NO and TNF-
production by
itself by alveolar macrophages under the stimulation of IFN-
.
Furthermore, stimulation of CD40 on dendritic cells increased the
expression of adhesion molecules on their cell surfaces (8). Therefore,
we speculated that CD40 cross-linking could activate alveolar
macrophages not only by direct CD40 signaling but also by upregulating
other cell surface molecules. Because tumoricidal activity was
inhibited by the addition of a specific inhibitor of NO synthesis or an
anti-TNF-
antibody, we concluded that NO and TNF-
play an
important role in the generation of macrophage tumoricidal activities
through CD40-CD40L interaction.
We could not observe the enhancement of IL-6 production by alveolar
macrophages with CD40 stimulation, whereas Alderson et al. (2) reported
that human monocytes stimulated by CD40L with granulocyte-macrophage
colony-stimulating factor (GM-CSF), IFN-
, or IL-3 produced IL-6.
Although anti-CD40 IgM slightly enhanced its production, IL-6 produced
by alveolar macrophages is mainly induced by the tumor cell itself and
is not specific for CD40-CD40L interaction in our experimental
conditions. 3LLSA cells produced GM-CSF (12), and GM-CSF induced IL-6
production (38). These results suggest that IL-6 production by alveolar
macrophages is enhanced by soluble factors or surface molecules of the
tumor cells rather than by CD40-CD40L interaction.
IL-12 plays a critical role in the development of Th1 cells, which are
effective inducers of cellular immunity. IL-12, which is mainly
produced by monocytes and macrophages, induces the production of
IFN-
by natural killer (NK) or T cells, and enhances the lytic activity of CTLs, NK cells, and lymphokine-activated killer cells. Furthermore, dendritic cells also produce a high amount of IL-12 by
CD40 cross-linking (9, 24). These findings suggest that the CD40-CD40L
interaction may augment the tumoricidal activity of not only
macrophages and monocytes but also of CTLs, NK cells, and
lymphokine-activated killer cells. In addition, the CD40-CD40L interaction induces APC activation, including IL-12 production and B7
surface expression (29). These findings also support the fact that the
transduction of the CD40L gene into
tumor cells may have the advantage for inducing antitumor immunity.
Recent advances in immunology may lead to several strategies for cancer
treatment by enhancing antitumor immunity (11), including cytokine
gene transfer such as IL-2 (41), IFN-
(43), and GM-CSF (1, 13), into
tumor cells and gene transfer of costimulatory molecules such as B7,
which is a counterreceptor for T-cell surface molecules of CD28 and
CTLA-4 (10). Taken together, our study may offer the possibility of
inducing antitumor immunity by CD40L gene transfer combined with
IFN-
gene transfer into lung cancer
cells as one of cancer gene therapy strategies. We are now
investigating the induction of antitumor cellular immunity by the
CD40L and
IFN-
two-gene transduction into
lung cancer cells.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Hasegawa,
First Dept. of Internal Medicine, Nagoya Univ. School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (E-mail:
yhasega{at}tsuru.med.nagoya-u.ac.jp).
Received 23 April 1998; accepted in final form 8 March 1999.
 |
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