RII-dependent cytolysis
* Department of Internal Medicine, University of Genova Medical School, Genova, Italy; and
Department of Pathology, University of Southern California, Los Angeles, California
Correspondence: Prof. Franco Dallegri, Dipartimento di Medicina Interna e Specialità Mediche, Viale Benedetto XV n.6, I-16132 Genova, Italy. E-mail: MACROBUTTON HtmlResAnchor otto{at}csita.unige.it
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RII, CD11b-CD18
integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis
by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was
inhibited by the anti-FcRII mAb IV.3, the anti-CD18 mAb MEM 48, and
the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and
N-acetyl-D-glucosamine. Furthermore, the PMA-
and GM-CSF-stimulated cytolysis was insensitive and sensitive to
inhibition by pertussis toxin, respectively. Thus, the use of PMA and
GM-CSF as neutrophil stimulants uncovers the existence of distinct
mechanisms of Lym-1 mAb-mediated cytolysis.
Key Words: human neutrophils • ADCC • Lym-1 • Fc receptors • GM-CSF • phorbol myristate acetate
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RII), with the concomitant and absolute requirement for
CD11b-CD18 integrins and carcinoembryonic antigen (CEA)-like CD66b
glycoproteins [14
]. In the present study, the
replacement of GM-CSF with phorbol 12-myristate 13-acetate (PMA)
uncovers the existence of alternative mechanisms of cytolysis, which is
CD66b- and pertussis toxin (PT)-independent. |
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mAbs
The previously described anti-B cell lymphoma mAb Lym-1 (IgG2a)
[1
] was used for the cytolytic assay. Lym-1 has been
shown to bind to a discontinuous epitope on the HLA-DR-10 ß
chain, overexpressed on human B cell lymphomas and leukemia
[15
]. Moreover, the following mAbs were used: anti-CD32
IV.3 (IgG2b, Fab fragments, Medarex, Annandale, NJ), anti-CD16 3G8
(IgG1, native mAb and F(ab')2 fragments, Medarex),
anti-CD18 MEM 48 (IgG1) and anti-CD11a MEM 25 (IgG; kindly provided by
V. Horejsi, Institute of Molecular Genetics, Academy of Science,
Prague, Czech Republic), anti-CD11b VIM12 (IgG1 kindly provided by W.
Knapp, Vienna, Austria), anti-CD11b 2LPM19c (IgG1, Dako A/S, Denmark),
and anti-CD66b 80H3 (IgG1, Serotec, Torino, Italy). Fluorescein
isothiocyanate (FITC)-conjugated anti-CD11b (44, IgG) was from
BioSource, Camarillo, CA. The appropriate control, isotype-matched,
FITC mAbs were from Immunotech, Marseille, France. FITC rabbit
anti-mouse IgG F(ab')2 fragment was from BioSource.
Neutrophil preparation
Heparinized venous blood (heparin 10 U/mL) was obtained from
healthy volunteers (20–45 years old) after informed consent. No donor
had an infectious disease or was under medication at the time of and
for two weeks before sampling. Blood was also obtained from a
previously described [16
] patient with chronic
granulomatous disease (CGD). Neutrophils were prepared by dextran
sedimentation, followed by centrifugation (400 g, 30 min) on
Ficoll-Hypaque density gradient, as previously described
[8
]. Contaminating erythrocytes were removed by
hypotonic lysis [8
]. Neutrophils resuspended in RPMI-FCS
were >97% pure, as determined by morphologic analysis of
Giemsa-stained cytopreps [8
] and greater than 98%
viable. Experiments were also carried out using neutrophils pretreated
with 10 ng/mL PMA. These cells were prepared as previously described
[17
]. Briefly, neutrophils were incubated (15 min,
4°C) with 10 ng/mL PMA, subsequently centrifuged at 4°C, and then
washed twice in 50 mL vol ice-cold RPMI [17
].
Target cells
Burkitt’s lymphoma Raji cells [8
] 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 anti-mouse IgG F(ab')2
polyclonal antibody conjugated with FITC (Dako A/S) [8
].
For the cytolytic assays, 4 x 106 Raji cells were
labeled with 100–200 µCi sodium chromate 51Cr by
incubating for 1 h at 37°C (final vol, 0.5 mL; medium, RPMI
1640+5% FCS). After washing, the labeled cells were resuspended in
RPMI-FCS.
Cytolytic assays
Cytolytic activity of neutrophils was measured as described
elsewhere in detail [8
, 18
]. Briefly,
target cells (2x104) were mixed with neutrophils at an
effector:target ratio of 20:1, with and without 10 µg/mL Lym-1 mAb
[1
] and/or 10 ng/mL PMA appropriately diluted in
RPMI-FCS. Experiments were carried out also using 1 ng/mL GM-CSF
instead of PMA. The effector:target ratio of 20:1 was chosen on the
basis of preliminary experiments, also taking into account previous
observations [8
]. Experiments were carried out in the
absence or presence of the various mAbs and reagents used to probe the
cytolytic process. The replacement of Lym-1 with 10 µg/mL mouse
IgG2a, in the presence of 10 ng/mL PMA, resulted in no lysis. The
assays were carried out in triplicate and in a final vol of 150 µL,
using round-bottom microplates (Falcon, Becton-Dickinson Italia,
Milano, Italy). After 14 h of incubation in a humidified
atmosphere of 95% air and 5% CO2, the
51Cr-release was determined by the formula 100 x
(E-S)/(T-S), where E is the cpm released in the presence of effector
cells, T is the cpm released after target cells with 5% Triton X-100,
and S is the cpm spontaneously released by target cells incubated with
medium alone (<18%).
Superoxide release assay
The release of superoxide anions (O2-)
was studied in duplicate by using a modification of the method of
Babior et al. [19
], as previously described
[20
]. Briefly, neutrophils (5x105 cells) in
a final vol of 0.5 mL HBSS were incubated (37°C, 20 min) with 10
ng/mL PMA. Incubations were carried out in the presence of 80 µM
ferricytochrome c. Parallel tubes, with the same components, included
150 U/mL SOD also. After incubation, the tubes were centrifuged (400
g, 10 min) at 4°C, and the optical density of the
supernatants was measured. The O2- production
was determined from the A550 of the samples without SOD,
minus the A550 of matched samples with SOD, using an
extinction coefficient of 2.1 x 104
M-1cm-1.
Immunofluorescence analysis
Purified neutrophils (1x106 cells in HBSS) were
preincubated (15 min, 4°C) with 10 ng/PMA and subsequently incubated
for 30 min at 4°C in the presence or absence of 4 µg/mL anti-CD66b
mAb 80H3 [14
]. After washing twice with cold HBSS, the
cells were incubated (30 min, 4°C) with 20 µg/mL goat anti-mouse
F(ab')2. The cells were washed with HBSS containing 0.1%
sodium azide and then incubated (30 min, 37°C) with FITC-conjugated,
anti-CD11b mAb 44. The cells were examined using a fluorescence Nikon
Optiphot-2 microscope and images were collected by a Hamamatsu
Color-chilled 3 CCD camera. The ability of neutrophils to bind mAb 80H3
(anti-CD66b) and the capacity of Raji cells to bind Lym-1 mAb were
measured by indirect immunofluorescence with flow cytometry (EPICS XL
flow cytometer, Coulter, Hialeah, FL), using a rabbit anti-mouse IgG
F(ab')2polyclonal antibody conjugated with
FITC.
Statistical analysis
Results were expressed as mean ± 1 SE and/or a
median with the 95% confidence interval. Statistical differences were
analyzed by the Mann-Whitney test. Significance was accepted when
P < 0.05.
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Figure 1. Neutrophil-mediated cytolysis in the absence or presence of 10 µg/mL
Lym-1 and/or 10 ng/mL PMA. 51Cr-labeled Raji target cells
were at 2 x 104. The neutrophil:Raji cell ratio was
20:1. The incubation time was 14 h. Results are expressed as
mean ± 1 SE (Nil, Lym-1, and Lym-1+PMA:
n=15; PMA: n=9). PMA vs. Nil, p < 0.05; Lym-1 vs. Nil, p < 0.01; Lym-1 + PMN vs.
PMA, p < 0.05; Lym-1 + PMA vs. Lym-1,
p < 0.01.
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Figure 2. Cytolysis mediated by neutrophils from a patient with CGD and by
neutrophils from two normal controls (N1 and N2).
51Cr-labeled Raji cells were at 2 x 104.
The neutrophil:target cell ratio was 20:1. The incubation time was
14 h. Lym-1 = 10 µg/mL; PMA = 10 ng/mL. Results are
mean ± 1 SE from one experiment performed in
triplicate.
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RII (CD32) and III (CD16), as well as mAbs
specific for CD11b-CD18 integrins, were tested in a
PMA/Lym-1-stimulated cytolytic system. As shown in Figure 3
, mAb IV.3 toward CD32 molecules inhibited significantly and
efficiently neutrophil-mediated cytolysis. On the contrary, the
anti-CD16 mAb 3G8 was ineffective (Fig. 3)
. These data are consistent
with the intervention of FcRII (CD32) without the involvement of
FcRIII. Moreover, the lysis was inhibited by mAb MEM 48 and mAb
2LPM19c, which, respectively, recognize CD18 and CD11b (Fig. 3)
.
Conversely, mAb MEM 25 (4 µg/mL) against CD11a [lymphocyte
function-associated antigen-1 (LFA-1)] had no effect (% cytolysis,
34.9±4.5 and 32.1±1.4 in the presence and absence of MEM 25; mean±1
SE, n=6). This suggests an absolute requirement
for CD11b-CD18 integrins in the process. In agreement with an active
role of these adhesion molecules in the present cytolytic system, the
lysis was enhanced significantly by the anti-CD11b mAb VIM-12
(Fig. 4
), a mAb known to activate these integrins [21
].
Using GM-CSF-stimulated neutrophils, Lym-1 mAb-mediated lysis was found
to be dependent strictly on the intervention of CEA-like CD66b
glycoproteins, which cooperate with CD11b-CD18 via
saccharide-inhibitable, i.e., lectin-like, interactions
[14
]. In the present PMA system, the lysis was found to
be unaffected by the addition of anti-CD66b mAb 80H3 (lysis, 33.17±3.2
and 35.10±0.7 in the absence or presence of 4 µg/mL mAb 80H3;
mean±1 SD, n=4). Moreover, the use of 8 instead
of 4 µg/mL mAb 80H3 did not produce an inhibitory effect (%
inhibition, 4.5±4.0, mean±1 SE, n=3). The
effect of the anti-CD66b mAb was tested simultaneously also in the
GM-CSF and PMA-stimulated system. As shown in Figure 5A
, the PMA system was confirmed to be insensitive to mAb 80H3,
whereas the GM-CSF system was inhibited efficiently. Consistent with
this conclusion, the GM-CSF but not the PMA system was inhibited by the
saccharide NADG (Fig. 5B)
. Conversely, neutrophil exposure to PMA
results in the CD66b surface-expression upregulation and allows the
CD66b/CD11b interaction (unpublished results). This suggests that
PMA-treated neutrophils express high amounts of functionally active
CD66b molecules, which, however, do not intervene in PMA-stimulated
cytolysis.
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Figure 3. Effect of the anti-CD32 (FcRII) mAb IV.3, anti-CD16 (FcRIII) mAb
3G8, anti-CD18 mAb MEM 48, and anti-CD11b mAb 2LPM19c on
PMA-stimulated, neutrophil-mediated, Lym-1, mAb-dependent lysis.
51Cr-labeled Raji cells were at 2 x 104.
The neutrophil:Raji cell ratio was 20:1. The incubation time was
14 h. (A) Lysis in the absence vs. presence of IV.3: p =
0.0286. (B) Lysis in the absence vs. presence of 3G8: p = 0.200. (C) Lysis in the absence vs. presence of MEM 48:
p = 0.0006. (D) Lysis in the absence vs. presence of
2LPM19c: p = 0.0286.
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Figure 4. Effect of the anti-CD11b mAb VIM12 on the PMA-stimulated,
neutrophil-mediated, Lym-1, mAb-dependent lysis.
51Cr-labeled Raji cells were at 2 x 104.
The neutrophil:target cell ratio was 20:1. The incubation time was
14 h. Lysis in the absence vs. presence of VIM12:
p = 0.0043.
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Figure 5. Effect of the anti-CD66b mAb 80H3 (A) and NADG (B) on the PMA- (solid
bars) and GM-CSF- (open bars) stimulated Lym-1, mAb-dependent lysis by
neutrophils from five (A) and four (B) donors. 51Cr-labeled
Raji cells were at 2 x 104. The neutrophil:target
cell ratio was 20:1. The incubation time was 14 h. Results are
shown as mean ± 1 SE. (A) PMA: lysis in the presence
vs. absence of 80H3: p = 0.1508. GM-CSF: lysis in the
presence vs. absence of 80H3: p = 0.0079. (B) PMA:
lysis in the presence vs. absence of NADG: p > 0.9999.
GM-CSF: lysis in the presence vs. absence of NADG: p =
0.0286.
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Figure 6. Effect of PT on the PMA- and GM-CSF-stimulated, neutrophil-mediated,
Lym-1, mAb-dependent lysis. 51Cr-labeled Raji cells were at
2 x 104. The neutrophil:target cell ratio was 20:1.
The incubation time was 14 h. Neutrophils (2x106/mL)
were incubated with PT or control medium for 2 h at 37°C and
washed twice before use. (A) PMA: lysis by PT-treated neutrophils vs.
control neutrophils: p = 0.222. (B) GM-CSF: lysis by
PT-treated neutrophils vs. control neutrophils: p =
0.0022.
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Figure 7. Effect of GST (A and D), H7 (B and E), and WMN (C and F) on
PMA-stimulated (left panels) and GM-CSF-stimulated (right panels),
neutrophil-mediated, Lym-1, mAb-dependent lysis.
51Cr-labeled Raji cells were at 2 x 104.
The neutrophil:target cell ratio was 20:1. The incubation time was
14 h. Concentrations in abscissa are expressed as µM. Results
are shown as mean ± 1 SE of four (A, C, E, and F),
five (B), or six (D) determinations for each point.
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Figure 8. Superoxide anion generation by neutrophils stimulated with PMA in the
absence or presence of GST, H7, or WMN. PMA = 10 ng/mL; GST =
25 µM; H7 = 100 µM; WMN = 0.1 µM. Results are expressed
as mean ± 1 SE, n = 3. PMA + H7
vs. PMA: p < 0.001.
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RII and CD11b-CD18
integrins, without the intervention of CD66b glycoproteins; and 3) the
activation of neutrophil-cytolytic potential proceeds through
PT-insensitive but kinase-dependent pathways. It is well-known that PMA is the most potent stimulant as far as the neutrophil-respiratory burst is concerned [25 , 26 ]. Nevertheless, in the present setting, different findings suggest that the stimulatory activity of PMA is independent completely on its ability to induce oxidant production in neutrophils. First, the effects of several inhibitors of the cytolytic system differed from those exerted on PMA-induced oxidative burst. Second, neutrophils from a patient with CGD-mediated cytolysis in spite of their incapacity to generate oxidants. Therefore, the PMA-dependent effect on neutrophil Lym-1 mAb-mediated cytolysis resembles that exerted on IgG-induced cell phagocytosis [27 , 28 ]. Conversely, the independence of the lysis from oxygen metabolites is in agreement with our previous observation carried out using Raji cells sensitized with rabbit antiserum as targets [18 ]. In this regard, defensins are the major candidates for the role of cytolytic mediators [29 , 30 ].
Previous studies dealing with the mechanisms of Lym-1, mAb-dependent
cytolysis by GM-CSF-stimulated neutrophils [14
] have
shown that the process involves FcRII without the intervention of
other FcRs and strictly requires CD11b-CD18 integrins and CEA-like
CD66b glycoproteins to physically interact in a lectin-like manner on
the neutrophil surface. Similar to these findings, PMA-stimulated
neutrophils exerted Lym-1, mAb-mediated lysis via FcRII and
CD11b-CD18 integrins. Nevertheless, we were unable to prove the
intervention of CD66b molecules. This is suggested by the incapacity of
the anti-CD66b mAb 80H3 and NAGD to block PMA-stimulated cytolysis.
Nevertheless, the ability of PMA-triggered neutrophils to exert
cytolysis independently of CD66b is not related to the PMA-induced
glycoprotein’s shedding or inactivation. Indeed, in agreement with
previous observations [31
], activated neutrophils
underwent upregulation of CD66b on their surface. In addition,
upregulated CD66b maintain the ability to undergo physical association
with CD11b-CD18 integrins. Therefore, PMA stimulation appears to bypass
the CD66b requirement observed for GM-CSF-exposed neutrophils. Finally,
it is known that neutrophil activation via FcRII proceeds through
two pathways, only one of which is regulated by PT-sensitive G proteins
[32
, 33
]. Because of the observed different
susceptibility to PT, the GM-CSF/Lym-1 and the PMA/Lym-1 cytolytic
systems appear to use the toxin-sensitive and -insensitive FcRII
signaling pathway, respectively. Indeed, the inhibitory activity of the
toxin cannot be attributed to interference with GM-CSF cell stimulation
or CD11b-CD18/CD66b-delivered signals [34
35
36
37
].
Cooperation among plasma membrane receptors, such as FcR and
CD11b-CD18 molecules, in activating phagocyte-effector functions is a
well-recognized phenomenon, particularly with neutrophil phagocytosis
and related cell responses [38
, 39
]. In
fact, such a type of cooperation has been investigated extensively,
primarily in terms of the ability of CD11b-CD18 integrins to serve as a
signaling partner for the glycosyl-phosphatidyl-inositol-anchored
FcRIII [39
, 40
]. The present study
suggests a collaboration of CD11b-CD18 with FcRII as well, in a
manner similar to that observed in certain models of FcRII-mediated
phagocytosis or cell activation [41
, 42
].
In this regard, the known association of these receptors with various
intracellular kinases, including tyrosine kinases [43
,
44
], is consistent with the observed susceptibility of
the cytolytic process to suppression by various inhibitors.
Nevertheless, as the specificity of these inhibitory compounds can be
questioned, the present findings do not allow a detailed identification
of the signal-transducing pathway responsible for activation of the
neutrophil-cytolytic potential. However, the ability of PMA to
stimulate neutrophil-cytolytic activity is consistent with the
intervention of protein kinase C, although the involvement of a phorbol
ester receptor lacking kinase activity cannot be excluded
[45
].
In conclusion, the present data, coupled with the findings obtained
using GM-CSF instead of PMA as stimulant [14
], suggest
the existence of at least two mechanisms underlying neutrophil,
mAb-dependent cytolysis. In fact, although the PMA and GM-CSF systems
share the requirement for FcRII and CD11b-CD18 integrins, only the
GM-CSF system involves CD66b glycoproteins and PT-sensitive signaling
pathways. These findings, enlightening the molecular mechanisms that
govern neutrophil-cytotoxic activities, may provide rational bases for
improving mAb-dependent antilymphoma therapies by pharmacological
manipulation of effector cell efficiency. In this context, the present
data obtained in the PMA system may inspire new attempts for developing
novel immunotoxins such as protein kinase activators conjugated with
antitarget mAb, including Lym-1 mAb.
Received December 16, 1999; revised April 14, 2000; accepted June 9, 2000.
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RI, CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy Blood 82,931-939RII (CD32), CD11b-CD18 integrins, and CD66b glycoproteins Blood 93,3505-3511
-chain (CD 11b) potentially involved in intramembrane complex formation with glycosylphospatidylinositol-achored FcRIIIB (CD16) molecules J. Immunol. 154,5452-5463[Abstract] receptor IIa and Fc receptor IIIb induces different proadhesive phenotypes on human neutrophils J. Immunol. 159,3940-3948[Abstract]Mß2, CD11b/CD18) and FcRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRII and tyrosine phosphorylation J. Cell Biol. 125,1407-1416RIIA Eur. J. Immunol. 26,207-212[Medline]
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