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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3505-3511
Monoclonal Lym-1 Antibody-Dependent Cytolysis by Neutrophils
Exposed to Granulocyte-Macrophage Colony-Stimulating Factor:
Intervention of Fc RII (CD32), CD11b-CD18 Integrins,
and CD66b Glycoproteins
By
L. Ottonello,
A.L. Epstein,
P. Dapino,
P. Barbera,
P. Morone, and
F. Dallegri
From the Department of Internal Medicine, University of Genova
Medical School, Genova, Italy; and the Department of Pathology,
University of Southern California, Los Angeles, CA.
 |
ABSTRACT |
Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind
HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1
can synergize with granulocyte-macrophage colony-stimulating factor
(GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a
model of B-lymphoma targets. Here we provide evidence for the
intervention of certain neutrophil receptors or surface molecules in
this model of cell-mediated lysis. The lysis was completely inhibited
by the anti-Fc RII MoAb IV.3 and unaffected by the
anti-FcRIII MoAb 3G8. This suggests that neutrophil
cytolysis involves FcRII without cooperation of this
receptor with FcRIII. Moreover, the lysis was inhibited
by an anti-CD18 MoAb (MEM48) and by a MoAb specific for
carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol
(GPI)-linked glycoproteins (CD66b). Using an immunofluorescence
staining procedure, cross-linking of CD66b induced the redistribution
of CD11b on neutrophils with distinct areas of CD11b clustering via a
process susceptible of inhibition by D-mannose. This is
consistent with the ability of CD11b-CD18 and CD66b to undergo
lectin-like physical interactions on the neutrophil surface. Such a
type of interaction is presumably instrumental for neutrophil
cytolytic activity in that the lysis was inhibited by D-mannose and
enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b
and GPI-anchored molecules by specifically interacting with CD11b
lectin-like sites. Therefore, the present results prove the absolute
requirement for FcRII in neutrophil
GM-CSF/Lym-1-mediated cytolysis and, on the other hand, define the
crucial role of CD66b and CD11b/CD18 in the expression of the cell
lytic potential.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A PRELIMINARY CLINICAL trial with
intravenous infusion of the monoclonal antibody (MoAb) Lym-1 performed
in patients with refractory lymphoma has shown evident reduction of
lymph node size only in some cases.1 Although various
factors, including a relative inadequacy of the host immune effector
systems, are likely to be responsible for these partial
responses,1,2 there are some reasons for taking an interest
in the activity of this MoAb. Firstly, Lym-1 is a murine IgG2a MoAb
that recognizes a polymorphic variant of HLA-DR antigens present on the
membrane of B-lymphoma cells and incapable of shedding or undergoing
modulation after antibody binding.3 Secondly, in vivo
studies with radiolabeled Lym-1 have shown antibody localization at
sites of lymphomatous disease,4 the reactivity of normal
tissue cells including B lymphocytes being very low or
absent.3 Finally, using Raji cells as a model of B-lymphoma
targets, interleukin-2 and -interferon were found to increase Lym-1
antibody-dependent cytolysis (ADCC) by mononuclear
cells5,6 and by neutrophils,7,8 respectively, suggesting that the above-mentioned inadequacy of the immune effector systems in lymphoma patients might be susceptible to improvement.
Consistent with the attractive possibility of augmenting antitumor
immune effectors, we have recently shown that a variety of cytokines
and chemotaxins synergize with Lym-1 to greatly amplify the ADCC
activity of neutrophils towards Raji target cells.9 In this
context, granulocyte-macrophage colony-stimulating factor (GM-CSF) has
been shown to synergize with Lym-1 to induce ADCC by
macrophages,10 as well as neutrophils.9 These
findings, coupled with the capacity of GM-CSF to increase phagocyte
production and survival,11,12 make the cytokine an
attractive candidate to further develop Lym-1 antibody-based approaches
to the therapy of lymphomas. Despite these perspectives, no report is
presently available on the mechanisms of phagocyte-mediated,
GM-CSF/Lym-1 synergistic stimulation of cytolytic activity. Moreover,
few reports deal with the mechanism of neutrophil-mediated
MoAb-dependent ADCC in general. In this regard, using melanoma and
neuroblastoma cell lines and a murine antitarget monoclonal IgG3,
neutrophil-mediated tumor lysis was found to be strictly related to the
intervention of neutrophil Fc receptor type II
(FcRII), FcRIII, and adhesion molecules
CD11b-CD18.13
The present study shows that neutrophil-mediated GM-CSF/Lym-1 ADCC
absolutely requires FcRII without the contribution of FcRIII. Moreover, the cytolytic process involves
CD11b-CD18 integrins, which presumably undergo physical association
with CD66b molecules via lectin-like interactions.
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MATERIALS AND METHODS |
Culture medium and reagents.
The following culture medium was used: RPMI 1640 (Irvine Scientific,
Santa Ana, CA) supplemented with 10% heat-inactivated (56°C, 45 minutes) fetal calf serum (FCS, HyClone Eur, Ltd, Cramlington, NE), and
2 mmol/L glutamine (Irvine Scientific) (RPMI-FCS). Hanks' Balanced
Salt Solution (HBSS) was from Irvine Scientific. Ficoll-Hypaque was
purchased from Seromed, Berlin, Germany. Sodium chromate
51Cr was from the Radiochemical Center, Amersham, England.
Triton X-100, ethidium bromide, D-mannose, N-acetyl-D-glucosamine
(NADG), galactose, sodium azide, and fluorescein diacetate were
purchased from Sigma Chemical Co, St Louis, MO. Heparin was obtained
from Roche, Milano, Italy. Human recombinant GM-CSF was from Genzyme, Cambridge, MA.
MoAbs.
The previously described9 MoAb, Lym-1 (IgG2a), was used as
antitarget MoAb for the cytolytic assay. Moreover, the following MoAbs
were used: anti-CD32 IV.3 (Fab fragments, Medarex, West Lebanon, NH),
anti-CD16 3G8 [F(ab)2 fragments, Medarex], anti-CD18 MHM23 (IgG1, Dako AS, Glostrup, Denmark), anti-CD18 MEM48 (IgG1, kindly
provided by V. Horejsi, Praha, Institute of Molecular Genetics, Academy
of Science, Prague, Czech Republic), anti-CD18 60.3 (IgG2a, kindly
provided by J. Harlan, Department of Medicine, University of
Washington, Harborview Medical Center, Seattle, WA), anti-CD11a MEM25
(IgG1, kindly provided by V. Horejsi, Praha), anti-CD11b 2LPM19c (IgG1,
Dako AS), anti-CD11b 44 (IgG1, BioSource, Camarillo, CA), anti-CD11b
CBRM 1/5 (IgG1, kindly provided by T.A. Springer, Department of
Pathology, Harvard Medical School, Boston, MA), anti-CD11c 3.9 (IgG1,
BioSource), antiCD11c KB90 (IgG1, Dako AS), anti-intercellular adhesion
molecule (ICAM)-1 84H10 (IgG1, Immunotech, Marseille,
France), anti-CD11b VIM12 (IgG1, kindly provided by W. Knapp, Institute
for Immunology, The University of Vienna, Vienna, Austria), anti-CD66b
80H3 (IgG1, Serotec-Valter Occhiena, Torino, Italy). Fluorescein
isothiocyanate (FITC)-conjugated anti-CD11b (44, IgG1) was from
Biosource. FITC-conjugated anti-CD10 (ALB2, IgG2a), FITC-conjugated
anti-CD14 RMO52, FITC-conjugated anti-CD16 3G8, FITC-conjugated
anti-CD32 2E1, FITC-conjugated anti-CD64 22 were from Immunotech. The
appropriate control isotype-matched FITC-MoAbs were from Immunotech.
The goat antimouse (F[ab]2 fragments) was purchased from BioSource.
Neutrophil preparation.
Heparinized venous blood (heparin 10 U/mL) was obtained from healthy
volunteers (20 to 37 years old) after informed consent. No donor had an
infectious disease or was under medication at the time of and for 2 weeks before sampling. Neutrophils were prepared by dextran
sedimentation, followed by centrifugation (400g, 30 minutes) on
a Ficoll-Hypaque density gradient, as previously described.9 Contaminating erythrocytes were removed by
hypotonic lysis.9 Neutrophils resuspended in RPMI-FCS were > 97% pure and > 98% viable, as determined by assays previously
described.9 Morphologic and phenotypic
characteristics of cell preparations used are shown in
Table 1.
Target cells.
Lymphoblastoid Raji cells9 were used as targets in the
cytolytic assays. The Raji cell line was grown in RPMI-FCS and
subcultured every 3 days. The capacity of these cells to bind Lym-1
antibody was measured by indirect immunofluorescence with flow
cytometry using a rabbit antimouse IgG F(ab')2
polyclonal antibody conjugated with FITC (Dako).9 For
cytolytic assays, 4 × 106 Raji cells were labeled
with 100 to 200 µCi sodium chromate 51Cr by incubating
for 1 hour at 37°C (final volume, 0.5 mL; medium, RPMI 1640 plus
5% FCS). After washing, labeled cells were resuspended in RPMI-FCS.
Cytolytic assays.
Cytolytic activity of neutrophils was measured as described elsewhere
in detail.9 Briefly, target cells (2 × 104) were mixed with neutrophils at an effector:target
ratio of 20:1, with and without Lym-1 MoAb and/or GM-CSF appropriately
diluted in RPMI-FCS. The effector:target ratio of 20:1 was chosen on
the basis of preliminary experiments. Experiments were performed in the
absence or presence of the various MoAbs and reagents used to probe the
cytolytic process. The assays were performed in triplicate and in a
final volume of 150 µL using round-bottom microplates (Falcon,
Becton-Dickinson Italia, Milano, Italy). After a 14-hour incubation in
humidified atmosphere of 95% air and 5% CO2, the 51Cr-release was determined in the cell-free supernatants.
The percentage of cytolysis was calculated according to the formula 100 × (E-S)/(T-S), where E is the cpm released in the presence of
effector cells, T is the cpm released after lysing target cells with
5% triton X-100, and S is the cpm spontaneously released by target
cells incubated with medium alone (<18%).
Immunofluorescence neutrophil staining.
Purified neutrophils (1 × 106 cells in HBSS) were
incubated for 30 minutes at 4°C in the presence or absence of 4 µg/mL anti-CD66b MoAb 80H3. After washing twice with cold HBSS, the
cells were incubated (30 minutes, 4°C) with 20 µg/mL goat
antimouse F(ab')2. The cells were washed with HBSS
containing 0.1% sodium azide and then incubated (30 minutes, 37°C)
with FITC-conjugated anti-CD11b MoAb 44 or anti-CD10 MoAb ALB2. In some
experiments, the entire procedure was performed in the presence of 100 mmol/L D-mannose. The cells were examined using a fluorescence Nikon
Optiphot-2 microscope (Nikon, Melville, NY) and images were collected
by a Hamamatsu Color-chilled 3 CCD camera (Hamamatsu Italia, Arese, Italy).
Statistical analysis.
Each data point represents the mean of the results obtained by testing
each donor in triplicate. When present, the line graphs represent
single experiments performed in triplicate from different donors. The
standard deviation (SD) of triplicate samples was always
<10%. Results were expressed as mean ± 1 SD and/or as median with the 95% confidence interval. Statistical differences were analyzed by the nonparametric Mann-Whitney U test. Significance was accepted when P < .05.
| |
RESULTS |
Synergistic stimulation of neutrophil lytic activity by Lym-1 MoAb and
GM-CSF.
Human neutrophils, incubated with 51Cr-labeled Raji cells,
were incapable of inducing target cell lysis as detected by the release of the radiolabel (Fig 1). The addition of
1 ng/mL GM-CSF did not affect the phenomenon (Fig 1). As compared with
neutrophil-Raji cell coincubation in its absence, 10 µg/mL Lym-1 MoAb
caused low, but statistically significant (P < .01)
stimulation of neutrophil-mediated lysis, ie, Lym-1 triggers
antibody-dependent cellular cytotoxicity (ADCC) (Fig 1). Nevertheless,
only 12 of the 84 subjects studied were found to display an ADCC
activity higher than 10% (Fig 1). Finally, the simultaneous addition
of 10 µg/mL Lym-1 and 1 ng/mL GM-CSF resulted in a relevant
amplification of the neutrophil cytolytic activity (Fig 1). In fact,
the mean target cell lysis was 1.9% and 5.0% in the presence of
GM-CSF and Lym-1, respectively, but it increased to 29.1% when both
GM-CSF and Lym-1 were added simultaneously. Examples of effector:target
curves are shown in Fig 2. In conclusion,
it appears that Lym-1 and GM-CSF synergize to amplify neutrophil lytic
efficiency.
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| Fig 1.
Neutrophil-mediated cytolysis in the absence or
presence of 10 µg/mL Lym-1 and/or 1 ng/mL GM-CSF.
51Cr-labeled Raji cells were at 2 × 104. The
neutrophil:Raji cell ratio was 20:1. The incubation time was 14 hours.
In the absence of Lym-1 and GM-CSF (Nil), neutrophil-mediated lysis was
0.25 ± 0.77 (mean ± 1 SD, n = 21) with a median of 0.00 (confidence interval 95%,  0.09 to 0.60). In the presence of
GM-CSF, the lysis was 1.97 ± 2.28 (mean ± 1 SD, n = 31) with a
median of 0.80 (confidence interval 95%, 1.14 to 2.81). In the
presence of Lym-1, the lysis was 5.08 ± 7.5 (mean ± 1 SD, n = 82)
with a median of 2.2 (confidence interval 95%, 3.43 to 6.73). In the
presence of both GM-CSF and Lym-1, the lysis was 29.14 ± 16.29 (mean ± 1 SD, n = 82) with a median of 27.20 (confidence interval 95%,
25.55 to 32.73). Neutrophil-mediated lysis in the presence of Lym-1
versus that observed in the absence (Nil) was P < .01. The lysis in the presence of GM-CSF versus that observed in the
absence (Nil) was P > .05. Finally, the lysis in the presence
of both Lym-1 and GM-CSF versus that in the absence (Nil) or that in
the presence of Lym-1 or GM-CSF alone was always P < .001.
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| Fig 2.
Neutrophil-mediated cytolysis in the presence of 10 µg/mL Lym-1 and 1 ng/mL GM-CSF. 51Cr-labeled Raji cells
were at 2 × 104. Neutrophil:Raji cell ratios (E:T ratios)
are shown in abscissa. The incubation time was 14 hours. The two curves
show the results obtained with neutrophils from two donors.
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Inhibition of cytolysis by certain MoAbs against neutrophil antigens.
As shown in Fig 3, the anti-CD32
(FcRII) MoAb IV.3 efficiently and significantly
inhibited GM-CSF-amplified Lym-1 ADCC activity of neutrophils. In
contrast, the anti-CD16 (FcRIII) MoAb 3G8 did not affect
the lysis. These data are consistent with a crucial role of
FcRII without the intervention of FcRIII.
The role of CD11-CD18 integrins was then tested using a panel of MoAbs.
At the concentration of 4 µg/mL, the anti-CD11a MoAb MEM-25,
anti-CD11b MoAbs (3.9 and KB90) and anti-CD18 MoAbs (60.3 and MHM23)
had no inhibitory activity (data not shown). Nevertheless, although ineffective in some cases, the anti-CD18 MoAb MEM48 inhibited neutrophil-mediated lysis significantly
(Fig 4 and its legend). The MoAb inhibitory
activity was dose-dependent (Fig 5).
Moreover, the lysis was inhibited by MoAb 80H3
(Fig 6), able to recognize a member of the
carcinoembryonic antigen (CEA) family (CD66b) expressed on human
neutrophils.14,15
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| Fig 3.
Effect of the anti-FcRII MoAb IV.3 (Fab
fragments) and anti-FcRIII MoAb 3G8
(F[ab']2 fragments) on the GM-CSF-stimulated
neutrophil-mediated Lym-1 antibody-dependent cytolysis.
51Cr-labeled Raji cells were at 2 × 104. The
neutrophil-Raji cell ratio was 20:1. Lym-1 and GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation time was 14 hours.
(A) The lysis was 15.43 ± 6.17 (mean ± 1 SD, n = 4) with a median
of 12.90 (confidence interval 95%, 5.61 to 25.24) in the absence of
IV.3 and 0.02 ± 0.05 (mean ± 1 SD, n = 4) with a median of 0.00 (confidence interval 95%, 0.05 to 0.10) in the presence of 4 µg/mL IV.3. Cytolysis in the absence versus that in the presence of
IV.3 was P = .0286. (B) The lysis was 16.34 ± 7.10 (mean ± 1 SD, n = 5) with a median of 14.10 (confidence interval 95%,
7.53 to 25.15) in the absence of 3G8 and 19.92 ± 11.43 (mean ± 1 SD, n = 5) with a median of 20.00 (confidence interval 95%, 5.73 to
34.11) in the presence of 4 µg/mL 3G8. Cytolysis in the absence
versus that in the presence of 3G8 was P = 1.000.
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| Fig 4.
Effect of the anti-CD18 MoAb MEM48 on the
GM-CSF-stimulated neutrophil-mediated Lym-1 antibody-dependent
cytolysis. 51Cr-labeled Raji cells were at 2 × 104. The neutrophil-Raji cell ratio was 20:1. Lym-1 and
GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation
time was 14 hours. (A) The lysis was 21.94 ± 11.30 (mean ± 1 SD, n
= 10) with a median of 23.00 (confidence interval 95%, 13.86 to
30.02) in the absence of MEM48 and 7.30 ± 4.82 (mean ± 1 SD, n = 10) with a median of 7.60 (confidence interval 95%, 3.85 to 10.75) in
the presence of 1 µg/mL MEM48. Cytolysis in the absence versus that
in the presence of 1 µg/mL MEM48 was P = .0052. (B) The lysis was 23.58 ± 12.64 (mean ± 1 SD, n = 13)
with a median of 26.90 (confidence interval 95%, 15.94 to 31.22) in
the absence of MEM48 and 10.29 ± 6.96 (mean ± 1 SD, n = 13) with a median of 10.30 (confidence interval 95%, 6.08 to 14.50) in
the presence of 4 µg/mL MEM48. Cytolysis in the absence versus that
in the presence of 4 µg/mL MEM48 was P = .0061.
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| Fig 5.
Effect of different doses of the anti-CD18 MoAb MEM48 on
the GM-CSF-stimulated neutrophil-mediated Lym-1 antibody-dependent
cytolysis. 51Cr-labeled Raji cells were at 2 × 104. The neutrophil-Raji cell ratio was 20:1. Lym-1 and
GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation
time was 14 hours. The two curves show the results obtained with
neutrophils from two donors.
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| Fig 6.
Effect of the anti-CD66b MoAb 80H3 on the
GM-CSF-stimulated neutrophil-mediated Lym-1 antibody-dependent
cytolysis. 51Cr-labeled Raji cells were at 2 × 104. The neutrophil-Raji cell ratio was 20:1. Lym-1 and
GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation
time was 14 hours. The lysis was 24.5 ± 12.83 (mean ± 1 SD, n = 6) with a median of 21.45 (confidence interval 95%, 11.04 to 37.86) in
the absence of 80H3 and 5.92 ± 8.22 (mean ± 1 SD, n = 6) with a
median of 1.30 (confidence interval 95%, 2.71 to 14.55) in the
presence of 4 µg/mL 80H3. Cytolysis in the absence versus that in the
presence of 4 µg/mL 80H3 was P = .0087.
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Effect of certain saccharides on the neutrophil-mediated cytolysis.
The data presented above are consistent with the intervention of
glycophosphatidyl inositol (GPI)-anchored CD66b molecules and an
unknown CD11-CD18 integrin in the GM-CSF stimulated Lym-1 ADCC by
neutrophils. Owing to their heavily glycosylated structure, GPI-linked
molecules might undergo lectin-like interactions with integrins in a
manner similar to those occurring between CD11b/CD18 and other
GPI-linked glycoproteins such as CD16 and CD87.16,17 As
these glycoproteins have been found to interact with CD11b-CD18 through
a process inhibitable by NADG and D-mannose,16,18 the effect of these saccharides was tested in the present cytolytic system.
As shown in Fig 7, both the compounds
efficiently inhibited the lysis. In contrast, an equimolar amount of
galactose was completely ineffective (data not shown). These results
raise the possibility that saccharide-inhibitable, ie, lectin-like,
interactions between CD66b and CD11b-CD18 take place in neutrophil
ADCC. Consistent with this possibility, the MoAb VIM12, able to bind
CD11b and mimic the CD11b-CD18 interaction with GPI-linked
molecules,19 was found to significantly amplify
neutrophil-mediated Lym-1 ADCC (Fig 8).
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| Fig 7.
Effect of NADG and D-mannose on the GM-CSF-stimulated
neutrophil-mediated Lym-1 antibody-dependent cytolysis.
51Cr-labeled Raji cells were at 2 × 104. The
neutrophil-Raji cell ratio was 20:1. Lym-1 and GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation time was 14 hours.
(A) The lysis was 24.15 ± 7.72 (mean ± 1 SD, n = 6) with a median
of 24.75 (confidence interval 95%, 10.06 to 32.25) in the absence of
NADG and 6.08 ± 3.70 (mean ± 1 SD, n = 6) with a median of 6.20 (confidence interval 95%, 2.19 to 9.97) in the presence of 100 mmol/L
NADG. Cytolysis in the absence versus that in the presence of NADG was
P = .0022. (B) The lysis was 28.12 ± 4.81 (mean ± 1 SD, n
= 4) with a median of 29.70 (confidence interval 95%, 20.46 to
35.78) in the absence of D-mannose and 3.30 ± 1.31 (mean ± 1 SD, n
= 4) with a median of 3.15 (confidence interval 95%, 1.22 to 5.38)
in the presence of 100 mmol/L D-mannose. Cytolysis in the absence
versus that in the presence of D-mannose was P = .028.
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| Fig 8.
Effect of the anti-CD11b MoAb VIM12 on the
GM-CSF-stimulated neutrophil-mediated Lym-1 antibody-dependent
cytolysis. 51Cr-labeled Raji cells were at 2 × 104. The neutrophil-Raji cell ratio was 20:1. Lym-1 and
GM-CSF were at 10 µg/mL and 1 ng/mL, respectively. The incubation
time was 14 hours. The lysis was 26.71 ± 21.62 (mean ± 1 SD, n = 8) with a median of 19.15 (confidence interval 95%, 8.64 to 44.79) in
the absence of VIM12 and 49.3 ± 22.01 (mean ± 1 SD, n = 8) with a
median of 54.50 (confidence interval 95%, 30.89 to 67.70) in the
presence of VIM12. Cytolysis in the absence versus that in the presence
of VIM12 was P = .037.
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Cross-linking of CD66b affects membrane distribution of CD11b on
neutrophils.
When incubated with a FITC-conjugated anti-CD11b MoAb, purified
neutrophils displayed a uniform distribution of fluorescence (Fig 9). On the contrary, distinct areas of
CD11b clustering were observed when the cells, first exposed to an
anti-CD66b MoAb, were incubated with second step goat
F(ab')2 fragments against mouse MoAbs to cross-link
CD66b (Fig 9). This suggests that cross-linking of CD66b causes CD11b
redistribution on neutrophil membranes. As shown in Fig 9, the
phenomenon was prevented by D-mannose. Finally, cross-linking of CD-66b
did not induce clustering of control antigens, such as CD10 molecules,
on the neutrophil surface (Fig 9).
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| Fig 9.
Distribution of CD11b on the neutrophil surface after
cross-linking of CD66b. Upper left panel, neutrophil exposed to
FITC-conjugated anti-CD11b MoAb; upper right panel, neutrophil exposed
to anti-CD66b MoAb before incubation with a second step
goat-F(ab2') fragment against mouse MoAbs to cross-link
CD66b and labeled with FITC-conjugated anti-CD11b MoAb; lower left
panel, neutrophil treated as in the upper right panel, but in the
presence of 100 mmol/L D-mannose; lower right panel, neutrophil exposed
to anti-CD66b MoAb before incubation with a second step
goat-F(ab2') fragment against mouse MoAbs to
cross-link CD66b and labeled with FITC-conjugated anti-CD10 MoAb.
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DISCUSSION |
The present results confirm previous observations on the ability of
Lym-1 and GM-CSF to synergistically activate neutrophil ADCC activity
towards B-lymphoblastoid tumor target cells.9 Consistent
with these findings, GM-CSF was also shown to augment the ability of
normal neutrophils to lyse MoAb-sensitized melanoma, neuroblastoma, and
colorectal cells.13,20,21 The intersubject variability
herein reported confirms previous findings.9 Although no
definitive explanation is available, in our experience the existence of
high and low responders in Lym-1 ADCC is a well-established phenomenon.
Moreover, it seems unlikely that the relatively low level of cytolysis
observed in a subset of donors actually reflects a poor neutrophil
viability. In fact, neutrophils engaged in ADCC are known to display
good viability even after an 18-hour incubation.22 In
addition, GM-CSF is able to prolong neutrophil survival,23 also during FcR-mediated neutrophil activities (unpublished
observations). Finally, major findings of the present report are: (1)
neutrophil-mediated ADCC strictly requires the intervention of
FcRII without the contribution of FcRIII;
(2) neutrophil CD11b-CD18 integrins are presumably involved via
lectin-like interactions with CEA-related CD66b glycoproteins.
Previous studies dealing with the mechanisms of neutrophil-mediated
monoclonal antibody-dependent lysis of tumor cells have been performed
using melanoma and neuroblastoma cell lines.13,20 In these
systems, both FcRII and FcRIII have been
reported to be required for neutrophil lytic activity.13 In
the present setting, only FcRII was found to be critical
for neutrophil ADCC. The reasons underlying these discrepancies remain
to be clarified. Nevertheless, it is possible that inhibition of
neutrophil ADCC toward neuroectodermal tumor cells by the
anti-FcRIII MoAb 3G813,20 was related to the
ability of this MoAb to interfere with FcRII function.
This MoAb was indeed recently shown to bind with its Fc region to the
binding site of FcRII, thereby inhibiting
FcRII-dependent functions24 (and unpublished
observations). On the other hand, the selective intervention of
FcRII shown here is consistent with at least two major
and previously reported findings. First, neutrophils lyse murine
hybridoma cells bearing antibodies directed against
FcRII, but not those bearing antibodies against
FcRIII.25 Second, FcRII, but
not FcRIII, are capable of clustering at the
effector-target interface during neutrophil ADCC.26
It is well-known that CD11-CD18 integrins participate in various
neutrophil FcR-mediated responses, including ADCC.13,27 Under the present conditions, the requirement for CD11-CD18 molecules was suggested by the inhibitory activity of the anti-CD18 MoAb MEM48, a
component of the panel of anti-CD11/CD18 MoAbs used in an attempt to
prove the involvement of integrins. Moreover, using the anti-CD66b MoAb
80H3, evidence for the intervention of CEA-like glycoproteins (CD66b)
was obtained. In general, this is consistent with recent observations
showing that CEA-related antigens are involved in neutrophil adhesive
responses, including cell-to-cell interactions.28 Taking
into account that CD66b molecules are glycoproteins attached to the
neutrophil surface via a GPI anchor28 and that other
GPI-linked glycoproteins such as CD16 and CD87 can undergo physical and
sugar-inhibitable association with CD11b-CD18 on the neutrophil
membrane,18 we hypothesized the intervention of lectin-like
interactions between CD11b-CD18 and CD66b during neutrophil ADCC.
Various experimental findings are in agreement with this possibility.
First, both anti-CD18 (MEM48) and anti-CD66b (80H3) MoAbs inhibited
neutrophil ADCC efficiently. Second, appropriate concentrations of
D-mannose or NADG16 displayed potent inhibitory activity.
Third, the MoAb VIM-12, which binds CD11b at or near its lectin-like
site and mimics the cooperation between CD11b and GPI-anchored
molecules,19 was found to stimulate neutrophil ADCC.
Fourth, antibody-induced cross-linking of CD66b molecules caused a
mannose-preventable clustering of CD11b on the neutrophil surface. On
the other hand, although the occurrence of these phenomena in other
models of cell-mediated cytolysis has not been evaluated, it is
possible that interaction between CD11b-CD18 and CD66b plays a more
general role. Consistent with this possibility, adhesion-dependent and
CD11b-CD18-related neutrophil functions have been found to be
regulated by CD66b activation.15,28,29
The interpretation of our results in favor of a physical association of
CD11b-CD18 integrins with CD66b on the neutrophil surface during ADCC
does not per se exclude the intervention of CD11b-CD18 in
neutrophil-target adhesion. In fact, CD11b-CD18 molecules might promote
target cell adhesion to effectors by interacting with ICAM-1 on the
Raji cell surface. Nevertheless, if such a type of CD11b-CD18/ICAM-1
interaction occurs, it does not appear to be relevant to the neutrophil
cytolytic response. In fact, the lysis was unaffected by MoAb 80H10
specific for ICAM-1 and by MoAb CBRM1/5, able to recognize a CD11b
neo-epitope required for ICAM-1 binding.30 Consistent with
the possibility that CD11b-CD18 can intervene in neutrophil ADCC
without binding ICAM-1 on the target cell surface is also the observed
requirement of CD11b-CD18 for the lysis of melanoma and neuroblastoma
cells despite their low or absent ICAM-1 expression.13 The
binding of CD11b-CD18 to a presently unknown antigen on the target
cells cannot be ruled out. On the other hand, the ligand or ligands of
CD66b on the target cell surface remain to be identified.
In conclusion, the present data show that Lym-1 ADCC selectively
involves neutrophil FcRII and provides evidence for the intervention of CD11b-CD18 integrins interacting with CD66b
glycoproteins. Such a type of interaction between CD11b-CD18 and CD66b,
probably occurring via a lectin-like physical linkage on the cell
membrane, appears to be instrumental for the efficient expression of
the neutrophil cytolytic potential.
| |
FOOTNOTES |
Submitted July 13, 1998; accepted January 8, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Prof Franco Dallegri, MD,
Semeiotica Medica 2, Dipartimento di Medicina Interna, Viale Benedetto
XV, n.6, I-16132 Genova, Italy; e-mail: otto{at}csita.unige.it.
| |
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ABSTRACT
INTRODUCTION