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Receptor I- and Fc
Receptor I-Mediated Tumor Cytotoxicity by Monocyte-Derived Macrophages
*
Medarex, Inc., Annandale, NJ 08801; and
Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) and the IgA FcR (Fc
RI) on monocyte-derived macrophages
(MDM) cultured in the presence of IFN-, M-CSF, or GM-CSF. Bispecific
Abs were used to target a Her2/neu breast carcinoma cell
line, SKBR-3, to FcRI or FcRI on MDM. Although FcRI and
FcRI share a common signaling pathway contingent on association with
the -chain (FcR subunit), a marked difference in their efficiency
in mediating tumoricidal functions was seen in response to specific
cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient
phagocytosis of SKBR-3 cells by FcRI and FcRI; however,
IFN--treated MDM phagocytosed tumor cells only with the
FcRI-directed bispecific Abs. Similarly, IFN--cultured MDM lysed
tumor cells more efficiently via FcRI then by FcRI as measured in
Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated
MDM mediated more efficient lysis of tumor cells via FcRI than
FcRI, while M-CSF-cultured MDM were relatively less efficient in
mediating Ab-dependent cellular cytotoxicity through either receptor.
With the exception of IFN--mediated enhancement of FcRI
expression and FcRI -chain complexes, the regulation of FcRI-
or FcRI-mediated activity occurred without significant change in
either receptor expression or total complexes with subunit. These
data suggest that the efficiency of Ab-mediated tumor therapy, which
depends on FcR effector cell functions, may be modified by the use of
specific cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI), and the high and low affinity Fc receptors
specific for IgG (FcRI, FcRIIA, and FcRIIIA). These FcR
mediate effector functions that are well documented, including
Ab-dependent cellular cytotoxicity
(ADCC)2 and
phagocytosis (3, 4, 5, 6, 7).
In contrast, polymorphonuclear cells (PMN), an other myeloid effector
cell with phagocytic and cytolytic capacity, constitutively express
FcRI, but not FcRI. However, FcRI expression can be induced in
vitro with IFN- treatment or in vivo with either G-CSF or IFN-
(8, 9, 10). The relative importance of these two FcR types in
promoting cytotoxic effector functions of various myeloid populations
(monocytes, macrophages, and PMN) has not been clearly delineated.
FcRI (CD64) binds monomeric human IgG1 and IgG3 with high affinity
(3, 4), and FcRIIIA (CD16) has intermediate affinity
for monomeric IgG. FcRIIA (CD32) efficiently binds to IgG immune
complexes and IgG-opsonized particles, but not to monomeric IgG
(3). On macrophages, a single class of IgA Fc receptor,
FcRI (CD89), has been characterized and binds both Ag-complexed and
monomeric IgA1 and IgA2 (5, 11). This suggests that in
vivo FcRI may be saturated with monomeric IgA in the same manner as
FcRI and FcRIIIA are significantly occupied with IgG.
mAb have been developed that bind to FcRI (mAb 32.2 and 22) and
FcRI (mAb A77) at sites distinct from their ligand binding domains
(11, 12, 13). ADCC mediated by bispecific Abs (BsAb) prepared
using these anti-FcR Abs is not blocked by human IgG or IgA
(6, 7, 14, 15, 16). In addition to triggering effector
functions under physiologically relevant conditions, BsAb made from
these mAbs bind exclusively to the targeted FcR and provide a suitable
method to study the capacity of individual FcR in a variety of
functional assays. Both FcRI and FcRI have been shown to be
functionally associated with the subunit, which mediates signaling
events following receptor clustering (17, 18). Although
these receptors share a common signaling component (the subunit),
their expression on the surface of effector cells is differentially
regulated. FcRI expression on monocytes can be enhanced by TNF-
(19, 20), IL-1ß, GM-CSF, and bacterial LPS
(20), whereas TGF-ß1 has been shown to decrease FcRI
expression (21). Monocyte FcRI expression is
up-regulated with IFN- and IL-10, and can be down-regulated with
IL-4 (8, 9, 22, 23).
Previous studies have demonstrated that treatment of monocyte-derived
macrophages (MDM) with M-CSF or IFN- differentially regulates
FcR-mediated phagocytosis and lysis of tumor cells (22, 24, 25). These data showed that that M-CSF-cultured MDM were
proficient in mediating phagocytosis of tumor cells via FcRII and
FcRIII, whereas cells incubated with IFN- were ineffective at
mediating phagocytosis of tumor cells via these two low affinity IgG
receptors. The reverse has been demonstrated with regard to ADCC. MDM
propagated in IFN- appear to be more efficient at mediating
FcRII- and FcRIII-dependent tumor cell lysis than untreated or
M-CSF-cultured MDM (22, 24). However, the FcRI-mediated
effector functions of MDM (ADCC and phagocytosis) can be enhanced with
IFN- (15, 26).
The cytotoxic capacity of the macrophage FcRI has not been fully
explored. Recently, we and Valerius et al. reported that FcRI on
monocytes, PMN, and macrophages is a potent trigger molecule for ADCC
and phagocytosis of tumor cells (6, 7). Because of the
role of IgA and FcRI in mucosal immunity, the potential for this
receptor to mediate anti-tumor activity through systemic treatment
has been largely overlooked. In this study we report for the first time
the ability of MDM to mediate ADCC via the FcR. In addition, we have
investigated the impact of several immune-modulating cytokines on the
ability of MDM to kill tumor cells by phagocytosis or ADCC through
FcRI and FcRI.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FcRI-specific mAb A77, anti-HER2/neu mAb
520C9, anti-FcRII (IV.3), anti-CD33 (251), anti-CD14
(AML-2-23), and anti-FcRI (H22, 32.2) mAbs were purified from
culture supernatants by protein A chromatography. The
HER2/neu-specific mAb 251.3 was provided by Dr. Paul Guyre,
Dartmouth Medical School (Hanover, NH). The SKBR-3 cell line that
overexpresses HER2/neu protooncogene was obtained from
American Type Culture Collection (Manassas, VA) and cultured in RPMI
supplemented with 10% FBS.
Bispecific Abs
The preparation of the BsAb A77 x 520C9 and H22 x 520C9 has previously been described in detail (7, 15). All the BsAb preparations were tested for endotoxin contamination by the chromogenic Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and were found to be free of endotoxin contamination (assay sensitivity limit, 0.1 EU/ml).
Phagocytosis assay
BsAb-mediated phagocytosis of SKBR-3 cells by (MDM) was examined
by a modification of the method described by Munn and Cheung
(27). Briefly, monocytes, purified from normal adult
source Leukopacs (Advanced Biotechnologies, Columbia, MD), were
differentiated in 24-well plates in macrophage serum-free medium (Life
Technologies, Grand Island, NY) supplemented with 10% FBS and either
M-CSF (2 ng/ml; R&D Systems, Minneapolis, MN), GM-CSF (10 ng/ml; R&D
Systems), or IFN- (1000 U/ml; Genzyme, Cambridge, MA) for 5–10
days. The cultures were given fresh medium containing the appropriate
cytokines every 3–4 days. The cytokine-containing medium was removed
before the phagocytosis assay. SKBR-3 cells were labeled with the
lipophilic red fluorescent dye, PKH 26 (Sigma, St. Louis, MO). The
labeled SKBR-3 cells were added to the wells containing MDM in the
absence or the presence of BsAb and incubated at 37°C overnight
(unless otherwise stated). MDM and nonphagocytized SKBR-3 cells were
recovered with trypsin (0.025% for 30–45 min at 37°C), transferred
to FACS tubes, and stained with a cocktail of FITC-labeled
anti-CD14 mAb (AML-2-23; 10 µg/ml) and anti-CD33-FITC (10
µg/ml) for 1 h on ice. Cells were washed and analyzed by
two-color fluorescence using the FACScan. The percent phagocytosis was
calculated as the number of dual-positive target cells (ingested by
MDM) divided by the total number of target cells. Assays were performed
in duplicate.
Confocal imaging
After fixation and flow cytometric analysis of the phagocytosis
samples, dual-positive cells were sorted with a
FACStarPlus flow cytometer (Becton Dickinson,
Mountain View, CA) and examined with a Bio-Rad MRC1024 laser scanning
confocal microscope (Hercules, CA). Cells were scanned for fluorescence
using the 488-nm line from a 15-mW KR/AR laser and two photodetectors
(522/32 nm dichroic for FITC fluorescence and 585 nm longpass for
PKH-26 fluorescence). A x63 Plan-Apo 1.4 NA objective (Carl Zeiss,
Thornwood, NY) was used in conjunction with an iris setting of 2.5,
which allowed for detection of optical sections of the fluorescence
image that were 
1.5 µm thick. A minimum of 100 cells/sample were
examined for quantitative evaluation of phagocytosis.
Tumor cell survival assay
To determine the tumor cell survival following phagocytosis, samples were obtained from the trypsin-harvested cells and plated in 96-well tissue culture plates. Live tumor cells were allowed to adhere overnight in RPMI containing 10% FBS. The medium was removed from each well, and the cells were fixed with 0.25% glutaraldehyde. Plates were blocked with 5% BSA solution, then reacted with mAb 251.03, which binds HER2/neu at a different site than 520C9 mAb. The 251.03 mAb was detected with goat anti-murine IgG Fc-specific alkaline phosphatase probe (The Jackson Laboratory, Bar Harbor, ME). The plates were developed using p-nitrophenyl phosphate and read at a wavelength of 405–650 nm. The relative number of SKBR-3 cells was calculated from the formula: % cell survival = [(sample OD - MDM only OD)/SKBR-3 only OD - MDM only OD)] x 100%. Samples were analyzed in duplicate.
Ab-dependent cellular cytotoxicity
SKBR-3 cells were cultured as described above and used as targets for lysis by MDM. Targets were labeled with 100 µCi of 51Cr for 1–2 h before combining with effector cells and BsAb in a U-bottom microtiter plate. After incubation for 16–18 h at 37°C supernatants were collected and analyzed for radioactivity. Cytotoxicity was calculated by the formula: % lysis = (experimental cpm - target leak cpm)/(detergent lysis cpm - target leak cpm) x 100%. Specific lysis = % lysis with BsAb - % lysis without BsAb. Assays were performed in triplicate.
FcR expression
Monocytes were differentiated in 24-well or
175-cm2 plates in the presence or the absence of
IFN-, M-CSF, or GM-CSF as described for the phagocytosis assays. The
cells were harvested with trypsin and incubated at 4°C for 60 min
with 10 µg/ml of IV.3, 32.2, A77, or 251 mAbs to stain for CD32,
CD64, CD89, and CD33, respectively. After incubation with anti-murine
IgG-FITC probe, cells were washed and fixed in 1% paraformaldehyde,
and their fluorescence was analyzed by FACScan.
Subunit association with FcRI and FcRI
Monocytes from three donors were prepared and differentiated
into macrophages exactly as described for the phagocytosis experiments,
except they were cultured in 175-cm2 tissue
culture flasks. Cells were harvested by scraping using cold 0.02% EDTA
solution, then were washed with cold PBS. A portion of these cells was
analyzed by flow cytometry. Macrophages were adjusted to
107/ml of lysis buffer (1% digitonin, 20 nM
triethanolamine, 150 nM NaCl, 0.12% Triton X-100, 2 mM PMSF, and 0.5
trypsin-inhibitory unit/ml aprotinin) and incubated for 1 h on
ice. The lysates were centrifuged at 15,000 x g for 30
min at 4°C, and the supernatants were adjusted to 1.0 mg/ml protein,
then frozen at -80°C. The lysates were added directly to microtiter
wells coated with A77 F(ab')2, M22
F(ab')2, or 520C9 F(ab')2
and incubated overnight at 4°C. After washing, the wells were
incubated for 2 h at ambient temperature with a 1/2,000 dilution
of subunit-specific rabbit serum (donated by Dr. Don Durden,
Indiana University School of Medicine, Indianapolis, IN). The assay was
developed with an alkaline phosphatase-conjugated goat anti-rabbit
IgG probe with p-nitrophenyl phosphate and read at a
wavelength of 405–650 nm.
Statistics
Statistical analysis was performed using Student’s t test and calculated by SIGMAPLOT software (Jandel, San Francisco, CA). p < 0.05 was considered significant. All experiments were performed a minimum of three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI and
FcRI. This two-color method employs a red lypophilic dye to stain
SKBR-3 carcinoma cells, and FITC-conjugated mAbs to label MDM.
Phagocytosis can be evaluated by quantitative analysis of the number of
single-colored cells (red or green) and the number of dual-colored
cells, which represent tumor cells engulfed by MDM. Initial experiments
using confocal microscopy were performed to confirm that dual-positive
cells represented phagocytosis of target cells and not merely binding
of target with effector cells. The BsAb A77 x 520C9 and H22
x 520C9, which specifically targeted the
HER2/neu-expressing SKBR-3 cells to FcRI and FcRI,
respectively, were used to mediate phagocytosis. These BsAb contained
only the Fab' of the individual mAbs; therefore, binding to FcR was
restricted to the specificity of the Ab.
As shown in Table I
, 20–25% of the
dual-positive events were E:T cell conjugates at 30 min. However, by
300 min this value was 3% or less, and >90% of dual-positive
macrophages contained partial or whole tumor cells. The images in Fig. 1 show conjugates from the 30 min and
phagocytosis at the 300 min points.
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RI-mediated phagocytosis (H22 x 520C9) under all
conditions (Fig. 2). M-CSF-treated MDM
were consistently the best effector cells for phagocytosis via FcRI
(maximum specific phagocytosis with H22 x 520C9: M-CSF, 36.8%;
GM-CSF, 31.4%; IFN-, 23.3%; without cytokine, 24.0%). The level
of phagocytosis via the FcRI (A77 x 520C9) was similar to that
observed with FcRI, except with IFN--treated MDM (maximum
specific phagocytosis with A77 x 520C9: M-CSF, 38.7%; GM-CSF,
26.6%; IFN-, 9.8%; without cytokine, 21.4%). Using the same
IFN--treated MDM, FcRI-mediated phagocytosis was significantly
less than FcRI-dependent phagocytosis (p <
0.05 at 0.1 and 1.0 µg/ml BsAb). Similarly, inhibition of
phagocytosis by IFN- treatment of MDM has been reported previously
for FcRII and FcRIII (24, 25).
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RI- and FcRI-mediated
phagocytosis using IFN--treated MDM, we studied tumor cell survival
following phagocytosis. Samples of cells from the phagocytosis assay
were taken (just before fixation), washed, and allowed to adhere
overnight to microtiter plates in growth medium. The ability of the
tumor cells to adhere was the primary requirement for viability, and
relative cell survival was assessed by a HER2/neu-specific
ELISA. The SKBR-3 cells cultured without MDM served as the 100%
survival control, and MDM cultured without SKBR-3 cells was used to
determine 0% survival. Fig. 3 shows that
the percent reduction in SKBR-3 cell survival was similar for FcRI-
and FcRI-targeted BsAb when MDM were cultured with M-CSF. Both BsAb
reduced cell survival 50–60% from control values. In contrast,
with IFN--treated MDM, the FcRI-targeted BsAb, and not the
FcRI-targeted BsAb, resulted in a significant loss of tumor cells
(50–60% reduction). These results make clear the divergence in
cytotoxic function of FcRI and FcRI under specific
conditions.
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). The FcRI-dependent lysis of SKBR-3
cells was most efficient using MDM cultured with GM-CSF (maximum
specific lysis: A77 x 520C9, 36.2%; H22 x 520C9, 22.0%).
Whereas, FcRI-mediated ADCC was greater with IFN--treated MDM
(maximum specific lysis: H22 x 520C9, 34.3%; A77 x 520C9,
14.3%). These differences in FcRI- and FcRI-mediated
phagocytosis were significant at 0.1 µg/ml BsAb
(p < 0.05). Essentially no ADCC was observed
with MDM not supplemented with cytokines, and MDM cultured in medium
containing M-CSF generated relatively low SKBR-3 lysis (maximum
specific lysis: A77 x 520C9, 20.0%; H22 x 520C9,
18.0%).
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significantly up-regulated
FcRI on myeloid cells (8, 9, 10), while FcRI levels
were relatively unaffected by IFN- (20). In contrast,
GM-CSF has been shown to increase FcRI on monocytes
(20). Fig. 5 illustrates the
relative expression of FcR compared with that by MDM cultured without
cytokines (averaged from six independent donors). The only
statistically significant change was the increase in FcRI expression
with IFN--treated MDM (4.8-fold increase over control,
p < 0.05). Treatment of MDM with various cytokines did
not result in significant differences in FcRI expression. No effect
was demonstrated on FcRII or CD33 expression, a nontriggering
macrophage surface receptor used as a control.
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subunit and
FcRI and FcRI. To study the level of association between the
subunit and FcRI and FcRI, lysates were prepared from
cytokine-differentiated MDM and were analyzed by ELISA. These assays
were performed by capturing FcR -chain complexes with
F(ab')2 of FcR-specific mAbs or an isotype
control, followed by detection of subunit with -specific rabbit
serum (Fig. 6). As expected, the results
from three independent MDM preparations showed a significant
enhancement of total FcRI -chain complexes in IFN--treated
MDM. Interestingly, GM-CSF-cultured MDM also had elevated FcRI
-chain complexes compared with MDM cultured without exogenous
cytokine or with M-CSF (p < 0.05). No
significant differences were observed for FcRI -chain complexes,
although MDM cultured in GM-CSF and IFN- had somewhat higher values
than control MDM or MCSF-treated MDM. There was no reactivity when
lysates were added to plates coated with irrelevant
F(ab')2 molecules (maximum
OD405 was <0.06 in all cases). These data imply
that most of the differential activities of cytokine cultured MDM are
not manifested by changes in total FcR -subunit complex formation.
However, the effect of IFN- on enhanced FcRI expression and
FcRI subunit complexes may play a significant role in the
FcRI-mediated tumoricidal activity of IFN--treated MDM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI and FcRI on macrophages
derived from monocytes in the presence of M-CSF, IFN-, or GM-CSF.
Although the cytotoxic effector functions of FcRI have been well
studied, relatively little is known regarding the ability of FcRI to
mediate tumor cell killing. To study the activities of individual FcRs,
we used chemically linked Fab' x Fab' BsAb, which excluded the
possibility of interactions with other FcR through Ab Fc domains.
Specifically, we used BsAb A77 x 520C9 and H22 x 520C9,
which targeted the HER2/neu-expressing SKBR-3 tumor cells to
FcRI and FcRI, respectively. These BsAb have been previously
described (7, 15), and H22 x 520C9 is currently
being investigated in clinical trials for treatment of
HER2/neu-expressing malignancies (31, 32). We
found significant differences in tumor cell cytotoxicity that were
dependent on which FcR was targeted as well as the presence of specific
cytokines during the differentiation of monocytes into MDM.
The most striking finding in this study was the lack of significant
ADCC or phagocytosis of SKBR-3 cells by the BsAb targeted to the
FcRI when MDM cultured in the presence of IFN- were used as
effector cells (maximum FcRI activity, 10% phagocytosis and 14%
ADCC). In contrast, the IFN--treated MDM mediated both phagocytosis
and ADCC through FcRI (maximum FcRI activity, 23% phagocytosis
and 34% ADCC). Decreased phagocytic function with IFN--treated MDM
have previously been reported with anti-tumor Abs that mediate
their activity via FcRII and FcRIII (22, 24, 25).
Interestingly, our results and those reported by Ely et al., which
demonstrate enhanced phagocytosis with IFN--treated MDM
(26), suggest that the FcRI is unique from other FcRs.
This observation is intriguing, because both FcRI and FcRI appear
to require association with the FcR subunit to mediate intracellular
signaling (17, 18).
The difference between FcRI and FcRI activity using
IFN--derived MDM was most pronounced when we examined tumor cell
survival following coculture of MDM with tumor cells. Addition of the
FcRI-targeted BsAb to these cultures resulted in 50–60% reduction
in SKBR-3 cells, whereas no significant loss of tumor cells was noted
(<10%) with the FcRI BsAb. These results correlated well with the
ability of FcRI on IFN--treated MDM to mediate both phagocytosis
and ADCC and the lack of such activity for FcRI.
We speculated that the variation in FcRI- and FcRI-mediated
activity of cytokine-cultured MDM may have been the result of changes
in their surface expression or the ability of -chain to functionally
associate with the FcR. Recent studies by Launay et al.
(33), which demonstrated the presence of FcRI with and
without -chain on the surface of monocytes and neutrophils,
implicated that receptor activity may be regulated by the magnitude of
-chain association. However, when we examined the effects of
cytokines on MDM FcR surface expression or total levels of FcR
subunit complexes, we did not find a complete correlation with these
parameters and the observed effects of cytokines on tumoricidal
function via FcRI and FcRI. Consistent with previous reports,
IFN- treatment of MDM resulted in increased levels of surface
FcRI and FcRI subunit complexes (8, 9, 10). These
effects probably contributed to FcRI-mediated phagocytosis and ADCC
with IFN--treated MDM. However, FcRI-mediated phagocytosis was
greater when MDM were generated in the presence of M-CSF and GM-CSF
despite lower FcRI expression and lower total FcRI subunit
complexes. The fact that FcRI-mediated phagocytosis with GM-CSF- or
M-CSF-cultured MDM was greater than phagocytosis with IFN--treated
MDM, supports the previous finding that IFN- treatment reduces the
overall phagocytic capacity of macrophages (22, 24, 25).
In contrast to FcRI, no significant change in the total level of
surface FcRI or FcRI subunit complexes were noted among MDM
cultured under the influence of different cytokines. Therefore, these
parameters were unable to explain the enhanced FcRI-mediated ADCC
with GM-CSF-treated MDM or the low tumoricidal activity of FcRI
using IFN--treated MDM. These data suggest that other molecules and
interactions probably contribute significantly to the differential
effect of cytokines on FcRI- and FcRI-mediated tumoricidal
activity of MDM.
Although IFN- treatment has been shown to promote ADCC activity, we
found that this did not apply for FcRI on MDM. We observed <15%
FcRI-dependent ADCC under these conditions. This result was
surprising considering our recent findings that demonstrated FcRI to
be a potent mediator of ADCC when fresh (untreated) monocytes or PMN
were used as effector cells (7). On the other hand, MDM
generated in the presence of GM-CSF were efficient at FcRI-mediated
ADCC. In fact, MDM cultured with GM-CSF mediated greater ADCC activity
via FcRI than via FcRI (p < 0.05),
although there was no significant up-regulation of FcRI on MDM
cultured with GM-CSF. Therefore, the positive or negative effects on
FcRI activity induced by cytokine treatment of MDM appear to work
downstream from receptor binding. These results further imply that
FcRI and FcRI (and probably other FcR) interact with different
molecules (in addition to the subunit), which uniquely regulate
their function.
FcRI and FcRI both mediated efficient phagocytosis of SKBR-3
cells when MDM were cultured with MCSF or GM-CSF. Ab-dependent
phagocytosis was also demonstrated when MDM were cultured without
exogenous cytokines. On the other hand, neither BsAb mediated efficient
lysis of the tumor cells with untreated MDM or M-CSF-cultured MDM. The
fact that MDM cultured without cytokines were unable to mediate
significant ADCC is intriguing, because monocytes (before
differentiation into MDM) have been shown to mediate efficient ADCC via
both FcRI and FcRI (7, 15). Previous studies have
demonstrated that monocytes rapidly lose ADCC function in culture
(30, 34). Therefore, the lack of MDM ADCC activity without
addition of cytokines does not imply that macrophages in vivo require
cytokines for this function. The data we have presented demonstrate
that MDM cultured under the influence of different cytokines result in
effector cells with varying capacity to mediate cytotoxic function via
FcRI and FcRI. These results suggest that Ab-dependent cytotoxic
activity of human macrophages is dependent on the FcR that can be
engaged by the anti-tumor Ab and the physical state of the
macrophages. Further, the data imply that the use of specific cytokines
to regulate macrophage function may enhance or reduce specific
cytotoxic mechanisms in a clinical setting.
This study has focused on macrophages; however, in the use of cytokines
in combination with Ab treatment for tumor therapy, other effector cell
populations that may be recruited for ADCC and phagocytosis need to be
considered. For example, the effects of G-CSF and IFN- (in vitro and
in vivo) on up-regulation of FcRI levels and cytotoxic activity on
granulocytes are well documented (8, 9, 10). On the other
hand, granulocytes constitutively express FcRI, which mediates
potent ADCC without addition of exogenous cytokines (6, 7). Further, because PMN constitute an abundant effector cell
population, they may play an important role in FcR-mediated
immunotherapy. Having a collective understanding of the important
effector cell populations and their different cytotoxic mechanisms will
allow for greater exploitation of immunotherapy. Further studies that
help optimize the effector functions of FcR in the course of
immunotherapy will probably lead to more successful protocols for
treatment of human cancers.
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Acknowledgments |
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subunit-specific
rabbit serum. |
Footnotes |
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2 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; MDM, monocyte-derived macrophages; BsAb, bispecific Ab; PMN, polymorphonuclear leukocytes.
Received for publication May 19, 1999. Accepted for publication March 16, 2000.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI (CD89) as a novel trigger molecule for bispecific antibody therapy. Blood 90:4485.RI, CD89) and tumor antigens efficiently promote cell-mediated cytotoxicity of tumor targets in whole blood. J. Immunol. 160:167.
receptor detected by receptor-specific monoclonal antibodies. J. Immunol. 148:1764.[Abstract]
-Ig receptor type I (FcRI) monoclonal antibody. J. Immunol. 155:4996.[Abstract]
R BsAb and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER2/neu overexpressing breast cancer. Cancer Res. 57:696. receptor type I-expressing effector cells. Cancer Res. 57:4008.RI bispecific antibodies. J. Immunol. 159:5629.[Abstract]
chain: molecular basis for CD89/FcR chain association. J. Biol. Chem. 270:29781. on the expression of FcIgG and IgA receptors, and other markers by cultured human blood monocytes and U937 cells. Scand. J. Immunol. 39:151.[Medline]
(rIFN-) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity. J. Immunol. 148:169.[Abstract]
R surface expression and cytotoxic activity. J. Immunol. 149:4048.[Abstract]
-interferon. Cancer Res. 53:2603.-Interferon inhibits Fc receptor II-mediated phagocytosis of tumor cells by human macrophages. Cancer Res. 54:2456.-primed macrophage-mediated phagocytosis of malignant non-Hodgkin’s lymphoma. Blood 87:3813. association and protects against degradation of bound ligand. J. Biol. Chem. 274:7216.This article has been cited by other articles:
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  S. N. Karagiannis, M. G. Bracher, J. Hunt, N. McCloskey, R. L. Beavil, A. J. Beavil, D. J. Fear, R. G. Thompson, N. East, F. Burke, et al. IgE-Antibody-Dependent Immunotherapy of Solid Tumors: Cytotoxic and Phagocytic Mechanisms of Eradication of Ovarian Cancer Cells J. Immunol., September 1, 2007; 179(5): 2832 - 2843. [Abstract] [Full Text] [PDF]
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 |
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 D. N. Forthal, G. Landucci, K. S. Cole, M. Marthas, J. C. Becerra, and K. Van Rompay Rhesus macaque polyclonal and monoclonal antibodies inhibit simian immunodeficiency virus in the presence of human or autologous rhesus effector cells. J. Virol., September 1, 2006; 80(18): 9217 - 9225. [Abstract] [Full Text] [PDF]
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 I. O. Gordon and R. S. Freedman Defective Antitumor Function of Monocyte-Derived Macrophages from Epithelial Ovarian Cancer Patients Clin. Cancer Res., March 1, 2006; 12(5): 1515 - 1524. [Abstract] [Full Text] [PDF]
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M. A. Otten, E. Rudolph, M. Dechant, C. W. Tuk, R. M. Reijmers, R. H. J. Beelen, J. G. J. van de Winkel, and M. van Egmond Immature Neutrophils Mediate Tumor Cell Killing via IgA but Not IgG Fc Receptors J. Immunol., May 1, 2005; 174(9): 5472 - 5480. [Abstract] [Full Text] [PDF]
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 R. Reljic, C. Crawford, S. Challacombe, and J. Ivanyi Mouse IgA inhibits cell growth by stimulating tumor necrosis factor-{alpha} production and apoptosis of macrophage cell lines Int. Immunol., April 1, 2004; 16(4): 607 - 614. [Abstract] [Full Text] [PDF]
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 A. P. Kumar, F. J. Piedrafita, and W. F. Reynolds Peroxisome Proliferator-activated Receptor {gamma} Ligands Regulate Myeloperoxidase Expression in Macrophages by an Estrogen-dependent Mechanism Involving the -463GA Promoter Polymorphism J. Biol. Chem., February 27, 2004; 279(9): 8300 - 8315. [Abstract] [Full Text] [PDF]
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C. A. Guyre, T. Keler, S. L. Swink, L. A. Vitale, R. F. Graziano, and M. W. Fanger Receptor Modulation by Fc{gamma}RI-Specific Fusion Proteins Is Dependent on Receptor Number and Modified by IgG J. Immunol., December 1, 2001; 167(11): 6303 - 6311. [Abstract] [Full Text] [PDF]
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I. Komuro, N. Keicho, A. Iwamoto, and K. S. Akagawa Human Alveolar Macrophages and Granulocyte-macrophage Colony-stimulating Factor-induced Monocyte-derived Macrophages Are Resistant to H2O2 via Their High Basal and Inducible Levels of Catalase Activity J. Biol. Chem., June 22, 2001; 276(26): 24360 - 24364. [Abstract] [Full Text] [PDF]
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) or H22 x 520C9
(
) was performed as described in Materials and
Methods. Before fixing cells for analysis by flow cytometry,
samples (50 µl) were removed from each tube and transferred to a
96-well microtiter plate in standard growth medium. After overnight
incubation at 37°C in 5% CO2, the adherent cells were
fixed to the plate, and the relative number of SKBR-3 cells was
determined by a HER2/neu-specific ELISA. One hundred
percent survival was defined as the OD405 values from wells
with SKBR-3 cells alone, 0% survival was defined as the
OD405 values from wells with MDM alone. The percentage of
BsAb-dependent reduction in tumor cell survival was calculated as: (%
SKBR-3 cell survival with BsAb)/(% SKBR-3 cell survival without
BsAb) x 100%. The data show the mean and SD of duplicates from a
representative experiment.
