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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3505-3511
By
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.
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
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 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 The present study shows that neutrophil-mediated GM-CSF/Lym-1 ADCC
absolutely requires Fc 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.
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.
Inhibition of cytolysis by certain MoAbs against neutrophil antigens.
As shown in Fig 3, the anti-CD32
(Fc
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).
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).
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 Fc 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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J Cell Biol
120:545, 1993 This article has been cited by other articles:
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RII (CD32), CD11b-CD18 Integrins,
and CD66b Glycoproteins
TOP
ABSTRACT
INTRODUCTION
RII MoAb IV.3 and unaffected by the
anti-Fc
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.
receptor III on human neutrophils.
J Immunol
150:3030, 1993
2(CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors.
J Lab Clin Med
129:492, 1997
-chain (CD11b) potentially involved in intramembrane complex formation with gly-cosylphospatidylinositol-anchored Fc
