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PHAGOCYTES
From the Immunotherapy Laboratory and Medarex Europe,
University Medical Center Utrecht, Utrecht, The Netherlands; Department
of Pathology, University Hospital, Nijmegen, The Netherlands; and
Department of Pathology, Harvard Medical School, Boston, MA.
Receptors for human immunoglobulin (Ig)G and IgA initiate potent
cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has
previously been implicated in receptor cooperation with Fc receptors
(FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was
characterized by studying normal human PMNs, Mac-1-deficient mouse
PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently
phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in
Mac-1 Antibodies have been studied extensively for their
use in immunotherapy of cancer.1-4 It is evident that
receptors for the Fc portion of immunoglobulins (FcRs) on myeloid cells
are critical in triggering antitumor cytotoxicity in
vivo.5,6 Antibody-dependent cellular
cytotoxicity (ADCC), considered crucial for antibody-mediated tumor
cell degradation, can be mediated by polymorphonuclear leukocytes (PMNs), monocytes/macrophages, eosinophils, and natural killer (NK)
cells.7,8 These effector cells use different cytotoxic mechanisms, depending on their activation state and the nature of the
target.7,9-12 PMNs, representing the most populous type of
white blood cell, exhibit fast recruitment activity in vivo. Potent and
very rapid (within 30 minutes) PMN cytotoxicity toward various tumor
targets has been documented.10,13-15 Two classes of
immunoglobulin (Ig)G receptors (Fc Mac-1 (CR3, CD11b/CD18) is a member of the Mac-1 was found to trigger Ab-dependent phagocytosis by Fc Antibodies
Isolation of human PMNs
Mouse PMNs CD11b knockout mice (Mac-1 /) were generated by
homologous recombination in C57bl/6 × 129SV
background.40 To study Mac-1 involvement in human
Fc RI-mediated functions, mice were crossed back with human FcRI
(CD89) transgenic (Tg) FVB/N mice,37 yielding 4 different
genotypes: nontransgenic (Ntg) Mac-1+/, Ntg
Mac-1/, Tg Mac-1+/, and Tg
Mac-1/. Mac-1 deficiency has no influence on PMN FcR
expression.37 To increase blood PMN counts, mice were
injected subcutaneously with 15 µg polyethylene glycol granulocyte
colony-stimulating growth factor (kindly provided by Dr J. Andresen,
Amgen, Thousand Oaks, CA), and blood was collected 3 days later.
Erythrocytes were removed by hypotonic lysis, followed by washing the
remaining leukocytes with RPMI 1640 medium (Gibco BRL, Grand Island,
NY) with 10% fetal calf serum (FCS). Cell viability was always more than 95%. Flow cytometry analysis on a FACScan (Becton Dickinson, San
Jose, CA) revealed leukocytes to consist of 55% to 60% PMNs, 35% to
40% lymphocytes, approximately 3% monocytes, and approximately 1% eosinophils.
C albicans phagocytosis and kill C albicans (ATCC 448585) phagocytosis was analyzed by flow cytometry and light microscopy, and fungicidal activity was assayed by radiometric killing assays, as previously described.41 Percentages of specific FcR-mediated phagocytosis and fungal death were calculated by subtracting control values (no antibody present).ADCC assays The capacity of PMNs to lyse tumor cells was evaluated in 51Chromium release assays.13 Briefly, 51Cr-labeled SK-BR-3 (human breast carcinoma) cells (ATCC, HTB-30) were plated in round-bottom 96-well plates (5 × 103 cells/well) in RPMI 1640 medium (with 10% FCS). Isolated human or mouse PMNs were added in the absence or presence of 0.5 µg/mL BsAb [A77 × 520C9] or 2 µg/mL mAb 520C9, giving different effector (E)/target (T) ratios, and incubated for 4 hours at 37°C after which 51Cr-release was measured in supernatants. In blocking experiments, human PMNs were incubated with anti-Mac-1 mAb 44a (10 µg/mL), mAb M1/70 (10 µg/mL), or 0.1M N-acetyl-D-glucosamine (NADG; Sigma) during ADCC. All experiments were performed in the absence of serum complement to exclude complement-dependent cytotoxicity.Cellular adhesion and spreading Mouse PMNs were incubated with SK-BR-3 cells (ratio 50:1) in RPMI 1640 medium (with 10% FCS) with or without BsAb [A77 × 520C9] (0.5 µg/mL) on glass coverslips (Marienfeld, Lauda-Königshofen, Germany) at 37°C for 30, 60, or 120 minutes. Cells were fixed, stained (Diff-Quick staining, Dade Behring, Düdingen, Germany), and analyzed randomly by light microscopy to determine attachment indices (AIs; number of attached PMNs per 100 tumor cells).To study PMN spreading, SK-BR-3 cells were cultured on glass coverslips overnight at 37°C and incubated with mouse PMNs (with or without BsAb) for 30 minutes at 37°C. After carefully washing away unbound cells, cells were fixed in 3.8% paraformaldehyde, and actin was stained with phalloidin-fluorescein isothiocyanate (FITC; 1:200; Sigma) for 15 minutes at 20°C. In an additional set of experiments, glass slides were coated with 100 µg/mL human serum IgA (ICN, Aurora, OH) or IgG (CLB, Amsterdam, The Netherlands) for 3 hours at 37°C and blocked with 0.5% (wt/vol) bovine serum albumin (BSA). Isolated PMNs were plated on the coated slides for 30 minutes at 37°C, fixed, and stained with phalloidin-FITC. Samples were mounted, and cell spreading was analyzed by confocal laser scanning microscopy using a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The Netherlands). Cell morphology was imaged just above (0.2 µm) the coated surfaces in x/y and x/z directions, sectioning both PMN and underlying surfaces. Neutrophil degranulation Human PMNs were incubated with SK-BR-3 cells (with or without 0.5 µg/mL BsAb [A77 × 520C9]) in Hepes+ buffer (20 mM Hepes pH 7.4 with 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM NaH2PO4, 1 mM CaCl2, 5 mM glucose, and 0.5% [wt/vol] BSA) for 30 minutes at 37°C. Cytochalasin B (10 µg/mL) (Sigma) was added for 10 minutes at 4°C to detach PMNs from tumor cells. In addition, PMNs were incubated in IgA- and IgG-coated 96-well plates (see above) in Hepes+ buffer for 30 minutes at 37°C. As positive controls, PMNs were stimulated with 100 ng/mL phorbol myristate acetate (PMA) (Sigma) or with 106 M FMLP (Sigma) and cytochalasin B for
10 minutes at 37°C. In Mac-1-blocking experiments, mAb 44a (10 µg/mL) was added during PMN stimulation. In all cases, supernatants
were collected for  -glucuronidase and lactoferrin analysis. The
-glucuronidase activity was measured by incubating supernatants (in
triplicate) with 1.1 mM phenolphthalein--glucosiduronic acid (Sigma)
in 0.1 M acetate buffer (pH 4.5) overnight at 37° C. Reactions were
stopped by 0.1 M glycine (pH 10.2) and measured by spectrophotometry at 560 nm. Lactoferrin release was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) by using rabbit antilactoferrin IgG (1:2500; Sigma) to coat 96-maxisorp plates (Nunc, Roskilde, Denmark) and alkaline phosphatase-conjugated rabbit antilactoferrin IgG (1:5000; Sigma) to detect bound lactoferrin. Total lactoferrin and
-glucuronidase contents of PMNs were determined by lysing cells with
0.2% Triton X-100.
PMN fractionation assays PMN fractionation assays42 were adjusted for mouse cells. Twenty-four-well plates were coated (see above) with IgA or BSA. Isolated PMNs (Ntg Mac-1+/, Tg Mac-1+/, and
Tg Mac-1/) were stimulated with PMA (100 ng/mL), or
incubated on BSA-coated plates, or IgA-coated plates
(5 × 106 PMN/well) for 30 minutes at 37°C. Cells were
detached with PBS containing 100 mM EDTA at 4°C, washed with PBS, and
resuspended in 1 mL ice-cold oxidase buffer (75 mM NaCl, 10 mM Hepes,
170 mM sucrose, 1 mM MgCl2, 0.5 mM EGTA, 10 µM ATP and 2 mM azide, pH 7.0). The cells were then homogenized in a "cell
cracker" (EMBL, Heidelberg, Germany). After centrifugation (10 minutes at 800g), 800 µL postnuclear supernatants were
layered on discontinuous sucrose gradients in SW60 tubes (Beckman, Palo
Alto, CA) containing layers of 10% sucrose, 35% sucrose, 50%
sucrose, and 60% sucrose each of 800 µL in oxidase buffer. After
ultracentrifugation (45 minutes at 100 000g), 600 µL
supernatant (cytosol fraction), 600 µL of the 10%/35% interphase
(plasma membrane), 600 µL of the 35%/50% interphase (specific
granules), and 600 µL of the 50%/60% interphase (azurophilic
granules) were harvested. Protein contents of fractions were determined
with Bradford protein assays (Biorad, München, Germany). For
immunodetection, fractions (2 µg of protein) were separated on 10%
reducing SDS-polyacrylamide gel electrophoresis. P22-phox
was detected by Western blotting, using rabbit polyclonal Ab (a kind
gift from Dr M. C. Dinauer, Indiana University, Indianapolis, IN43), followed by horseradish peroxidase (HRP)-conjugated
swine antirabbit IgG (Dako, Glostrup, Denmark).
Respiratory burst measurements The (iso)luminol-enhanced chemiluminescence method was used for analysis of real time respiratory burst activity. Luminol (membrane permeable) was used for detection of total (intracellular and extracellular) oxygen radical production, isoluminol (membrane impermeable) served as the substrate for oxygen radical detection outside membrane-enclosed compartments.44 Mouse PMNs (5 × 105) were gently centrifuged (40g for 3 minutes at 4°C) on SK-BR-3 cells (1 × 104) opsonized with or without BsAb [A77 × 520C9] in RPMI 1640 medium (with 10% FCS) and were placed in a 953 LB Biolumat (Berthold, Wildbad, Germany). Luminol (150 µM) or isoluminol (56 µM) (Sigma) was injected in all tubes, and light emission was recorded continuously for 30 minutes at 37°C. Respiratory burst measurements using isoluminol were performed in the presence of HRP (4 U; Sigma) to overcome the limited availability of amplifying peroxidase outside membrane-enclosed compartments.44 As positive controls PMNs, were stimulated with PMA, as negative controls, PMNs were incubated with HRP and (iso)luminol only.Analysis of immunologic synapse formation Freshly grown SK-BR-3 cells (approximately 2 × 106) were biotinylated with 2 mg/mL sulfo-NHS-biotin (Sigma) in PBS for 10 minutes at 4°C. Tumor cells were subsequently incubated in PBS with 50 mM NH4Cl at 4°C to quench reactions. After washing in PBS, cell viability was checked by trypan blue exclusion, and cells were adhered to glass coverslips for 1 hour at 37°C. Subsequently, mouse PMNs were incubated on tumor cells (with or without 0.5 µg/mL BsAb [A77 × 520C9]) for 30 minutes at 37°C. After washing to remove unbound PMNs, cells were fixed in methanol. Samples were washed, incubated with streptavidin-FITC (1:100; Dako) for 10 minutes at 4°C for plasma membrane staining, and incubated with propidium iodide (1:1000; Sigma) for 1 minute at 4°C for nuclei staining. Slides were mounted, and tumor cell membrane FITC staining between PMNs and tumor cells was analyzed by confocal laser scanning microscopy (see above). Sections of approximately 0.75 µm were acquired, recording 8 images from top to bottom of cells. For quantification, at least 50 PMNs that adhered to tumor targets were analyzed randomly per sample for the presence or absence of membrane FITC staining at tumor cell-PMN interaction sites.Electron microscopy For transmission electron microscopy, SK-BR-3 cells were cultured to confluence in RPMI 1640 medium (10% FCS) on 6.5-mm 0.4-µm pore-size transwell filters (Costar, Cambridge, MA). Mouse PMNs (Tg Mac-1+/, Tg Mac-1/, and Ntg
Mac-1+/) were incubated on the tumor cells with and
without 0.5 µg/mL BsAb [A77 × 520C9] for 40 minutes at 37°C.
After the filters were rinsed with RPMI 1640 medium, cells were fixed
with 2.5% glutaraldehyde in cacodylate (Sigma) buffer (pH 7.2)
overnight. Subsequently, filters were washed in cacodylate buffer,
postfixed in cacodylate-buffered 1% OsO4 (Johnson Matthey
Chem, Roystone, United Kingdom) for 30 minutes, and embedded in Epon
812 (Merck, Darmstadt, Germany). Ultrathin sections were cut on an
ultratome (LKB Instruments, Bromma, Sweden) and contrasted with 3%
aqueous uranyl acetate for 45 minutes and lead citrate for 2 minutes at
20°C. The sections were examined in a Jeol 1200EX electron microscope
(Jeol, Tokyo, Japan).
Statistical analysis Unpaired 2-tailed Student t tests were used to determine differences. Significance was accepted at the P < .05 level.
To assess the role of Mac-1 in human FcR-mediated functions, we
analyzed PMNs from Mac-1-deficient mice crossed with human FcR
transgenic mice. This enabled us to selectively study Mac-1, in the
presence of other Mac-1-deficient PMNs potently mediate phagocytosis and killing via FcRs To examine the role of Mac-1 in FcRI-mediated uptake of
microorganisms, phagocytosis of BsAb [A77 × Can]-opsonized
C albicans by PMNs (Tg Mac-1+/ and Tg
Mac-1/) was monitored at different times. Light
microscopic analyses revealed both Mac-1+/ and
Mac-1/ PMNs to be efficient in uptake of C
albicans (Figure 1A).
FcRI-mediated phagocytosis, assessed by FACS analysis, was
quantified on subtraction of uptake in the absence of BsAb. No
differences were observed in FcRI-mediated phagocytosis of C
albicans between Mac-1+/ and Mac-1/
PMNs (Figure 1B). To assess whether Mac-1 was important in PMN microbicidal activity following FcRI-mediated uptake, C
albicans killed by Mac-1+/ and
Mac-1/ PMNs was analyzed after 2 hours (Figure 1C).
Both Mac-1+/ and Mac-1/ PMNs
killed C albicans with equal efficacy (28% ± 7% versus
25% ± 7%, n = 4). Similar results were obtained with
IgG-coated C albicans. Ntg Mac-1+/ PMNs
mediated neither phagocytosis nor killing of BsAb-coated C
albicans (data not shown). These results indicate that Mac-1 is
not required for FcR-mediated PMN phagocytosis and intracellular killing of C albicans.
Mac-1 is crucial for PMN ADCC To study Mac-1 involvement in PMN FcR-mediated extracellular cytotoxicity, lysis of human breast carcinoma cells (SK-BR-3 cells) by both mouse and human PMNs was examined. Mac-1-expressing Tg mouse PMNs efficiently killed tumor targets in the presence of FcRI-directed
BsAb [A77 × 520C9] at different E/T ratios (Figure 2). However, ADCC capacity of
Mac-1-deficient Tg mouse PMNs was absent. Similarly, control ADCC
experiments performed with mouse IgG1 (mAb 520C9) demonstrated tumor
cytotoxicity of Mac-1+/ PMNs but not of
Mac-1/ PMNs (data not shown). Ntg
(Mac-1+/) mediated cytotoxicity toward tumor cells in the
presence of 520C9 but not in the presence of FcRI-directed BsAb (not
shown). These results show that dependence of ADCC on Mac-1 is not
restricted to cytotoxicity mediated via FcRI. ADCC mediated via
mouse Fc R or human FcRI (tested in FcRI-Tg
mice15) was similarly dependent on Mac-1. Furthermore, the
Mac-1 requirement for ADCC was observed for different tumor cell lines
(not shown). To further assess the requirement for Mac-1, experiments
with human PMNs in the presence of anti-Mac-1 antibodies were
performed. Human PMNs mediated cytotoxicity of BsAb
[A77 × 520C9]-opsonized SK-BR-3 cells with similar efficacy as Tg
Mac-1+/ mouse PMNs (Figure 2B). Blocking Mac-1 by
anti-Mac-1 antibodies (M1/70, 44a) or NADG resulted in significantly
reduced FcR-mediated cytotoxicity (Figure 2B). PMN viability and FcR
expression were not altered on Mac-1 blocking, and isotype controls
(rat IgG2 and mouse IgG1) did not affect ADCC (data not shown). These
data implicate Mac-1 to be important for both mouse and human PMN
FcR-mediated cytotoxicity.
PMN binding and spreading on Ab-coated targets On observing an essential role for Mac-1 in FcR-mediated killing, we investigated the underlying mechanism. First, we aimed to analyze whether PMNs devoid of Mac-1 were defective in binding Ab-opsonized tumor cells. AIs were determined for Mac-1-expressing and Mac-1-deficient PMNs. Both Tg Mac-1+/ and Tg
Mac-1/ PMNs effectively bound Ab-coated tumor targets
within 30 minutes, albeit the AIs of Mac-1/ PMNs were
decreased compared with Mac-1-expressing PMNs (Figure 3A). Adherence of Ntg
Mac-1+/ PMNs to FcRI-directed BsAb
[A77 × 520C9]-coated tumor cells revealed hardly any binding
(< 50 PMNs/100 SK-BR-3 cells, n = 4). In the absence of Ab, PMNs
did not adhere to tumor targets (data not shown). Because the binding
of Mac-1/ PMNs to Ab-coated tumor cells was intact, we
next studied PMN interactions with Ab-coated targets in more detail.
Comparing Mac-1+/ PMNs with Mac-1/ PMNs
bound to Ab-coated surfaces (either IgA or IgG) revealed obvious
morphologic differences. Figure 3B displays confocal microscopic images
of the actin cytoskeleton of Mac-1+/ PMNs (left) and
Mac-1/ PMNs (right) adhered to IgA-coated glass slides.
Mac-1-expressing PMNs exhibited spreading (clearly visible after
analysis in x/z direction, inserts) after only 10 minutes,
in contrast to Mac-1-deficient PMNs. PMNs bound to BSA-coated glass
did not exhibit spreading (data not shown). Similarly, PMNs spreading
on Ab-coated tumor cells was dependent on Mac-1 (not shown). These
results show Mac-1/ PMNs able to bind but unable to
spread on Ab-coated targets.
PMN degranulation and respiratory burst activity We next investigated the role of Mac-1 in PMN degranulation and respiratory burst activity. First, exocytosis of azurophilic (primary) granules of mouse PMNs adhered to BsAb [A77 × 520C9]-coated tumor targets was analyzed. The-glucuronidase activities were measured in
supernatants after PMNs were detached from tumor cells. PMNs did not
release -glucuronidase after binding Ab-coated tumor cells, in
contrast to the PMNs stimulated with cytB/FMLP or PMA (Figure
4A). To rule out the possibility that
-glucuronidase interacts with tumor targets, precluding measurement
of its activity in supernatants, total -glucuronidase contents of
PMNs incubated with tumor cells in the absence or presence of Ab were
determined, again revealing no differences in -glucuronidase
activity. Extension of incubation periods to 4 hours did not result in
detectable -glucuronidase activity. Similar to these results, mouse
and human PMNs adhering to IgA- or IgG-coated surfaces did not release detectable -glucuronidase activity (Figure 4). To confirm the absence of PMN azurophilic granule mobilization after stimulation on
Ig-coated surfaces, an alternative detection method was used. Separation of different PMN fractions (cytosol, plasma membrane, and
granules) after PMN adherence to Ig-coated glass revealed -glucuronidase to be retained within the granule fraction (data not
shown). In contrast, -glucuronidase activity of PMNs stimulated with
cytB/FMLP was detectable in supernatants, consistent with the
results above.
Specific (secondary) granule mobilization was analyzed by studying
lactoferrin release and the translocation of p22-phox, a
component of PMN NADPH oxidase complexes.45 Human PMNs
released lactoferrin on adherence to IgA- or IgG-coated glass slides,
in contrast to PMNs bound to BSA-coated plates (Figure
5A). More importantly, PMNs bound to
Ab-opsonized tumor cells released lactoferrin within 30 minutes, in
contrast to PMNs incubated with uncoated tumor cells. This release was
detectable only after detaching PMN from Ab-coated tumor targets. Human
PMNs incubated with anti-Mac-1 mAb retained their ability to release
specific granules after activation (Figure 5A). As our lactoferrin
ELISA did not react with mouse lactoferrin, we developed another method
to study exocytosis of specific granules. Mouse PMNs (Tg
Mac-1+/
Next, respiratory burst activity of human and mouse PMNs bound to
Ab-coated tumor targets was analyzed. Oxygen radical production was
detected in time, using 2 types of substrates. Luminol, which is able
to pass cell membranes, detects total (intracellular and extracellular)
PMN oxygen radical production. On the contrary, isoluminol is membrane
impermeable and detects radicals outside membrane-enclosed compartments
only.44 PMA-stimulated Mac-1-expressing and
Mac-1-deficient PMNs exhibited similar respiratory burst activity (data not shown). Mac-1+/
Taken together, we observed effective mobilization of specific granules
and respiratory burst activity of Mac-1-deficient PMNs stimulated by
Ab-coated targets. Strikingly, however, oxygen radical production by
Mac-1 Immunologic synapse formation between PMNs and tumor cells Because of differences in spreading and respiratory burst activity by Mac-1-deficient PMNs, we hypothesized immunologic synapse formation between Mac-1/ PMNs and tumor targets to be defective.
Biotinylated tumor cells incubated with PMNs (Tg Mac-1+/,
Tg Mac-1/, and Ntg Mac-1+/) in the
absence or presence of FcRI-directed BsAb [A77 × 520C9] were
stained with streptavidin-FITC. Binding sites between PMNs and tumor
cells were analyzed for the presence of tumor cell membrane FITC
staining by "sectioning" at all levels through whole cells by
confocal microscopy. Quantification of absence (closed synapses) and
presence (open synapses) of membrane FITC staining showed Mac-1/ PMNs to exhibit significantly higher numbers of
open immunologic synapses than Mac-1+/ PMNs (Figure
7A). Ntg Mac-1+/ PMNs did
not adhere to Ab-coated tumor cells nor did Tg (Mac-1+/
and Mac-1/) PMNs bind tumor cells in the absence of Ab,
which was in all cases revealed by continuous membrane FITC staining
(data not shown).
These data were further supported by electron microscopic analysis of
PMN-tumor cell interactions. Both Tg Mac-1+/
Earlier studies documented Mac-1 to augment various FcR functions in myeloid cells. In the present work, we established a crucial role for Mac-1 in PMN-mediated ADCC. Mac-1 was found to be essential for PMN spreading and the formation of intact intercellular synapses. We propose these defective cytoskeleton-mediated processes to be responsible for defective FcR-mediated cytotoxicity by Mac-1-deficient PMNs. Various studies documented involvement of Mac-1 in phagocytosis of
Ab-coated targets.26,46-48 It is well known that antibody and complement can cooperate in the opsonization of pathogens for
phagocytosis. Complement-independent Mac-1 cooperation with Fc A number of earlier studies documented Mac-1 to be involved in
FcR-mediated extracellular cytotoxicity.33-38 Furthermore,
Mac-1 was documented to be critical in Fc To investigate the underlying mechanism of the requirement for Mac-1 in
PMN ADCC, we first analyzed the interaction between PMN and Ab-coated
targets. Mac-1 proved not to be crucial for mere FcR-mediated binding,
although AIs of Mac-1 The apparent discrepancy between impaired ADCC and intact activation of
Mac-1-deficient PMNs was resolved by investigating the initiation of
respiratory burst activity in more detail. Although total oxygen
radical production by Mac-1 Antibody therapies have been documented to be effective in the
eradication of different human tumors.2,3 The interaction of therapeutic antibodies with FcRs has been reported crucial for
therapeutic efficacy.6 PMNs, having potent cytolytic
capacity, gained recent interest as effector cells in Ab-mediated
antitumor immunity.14,39,73 Analyzing antibody treatment
of tumor-bearing Mac-1+/ In summary, we defined a crucial role for Mac-1 in FcR-mediated
extracellular PMN cytotoxicity and unraveled the underlying mechanism
of Mac-1 involvement in ADCC (Figure 8).
Mac-1 is not essential for the mere binding of PMNs to Ab-coated
targets and subsequent PMN activation (ie, degranulation and
respiratory burst activity). However, PMN spreading on Ab-coated
targets is fully dependent on Mac-1. Moreover, Mac-1 is required for
intact immunologic synapse formation between PMNs and Ab-coated tumor
cells. We postulate these impaired cytoskeleton-mediated processes to
be responsible for absent tumor cytolysis.
The authors thank Toon Hesp, Els Dorresteijn, Herma Boersma, and
Anja van de Sar for excellent assistance with the animal experiments;
Dr M. C. Dinauer for kindly providing
Submitted September 5, 2000; accepted December 21, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jan G. J. van de Winkel, Immunotherapy Laboratory, University Medical Center Utrecht, Rm KC02.085.2, Lundlaan 6, 3584 EA, Utrecht, The Netherlands; e-mail: j.vandewinkel{at}lab.azu.nl.
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Top
Abstract
Introduction
) and Tg Mac-1
). Results are expressed as percentage of PMNs that mediated
phagocytosis (mean ± SEM [n = 4]). (C) Fc
), Tg
Mac-1+/
), and Tg Mac-1
], and Tg Mac-1
]) were incubated with
BsAb-[A77 × 520C9]-coated SK-BR-3 cells, (
) represents blanco
(no cells). Oxygen radical production was measured in time by
chemiluminescence using either membrane-permeable luminol (A) or
impermeable isoluminol (B) as a substrate. Experiments were repeated 3 times, yielding similar results.
