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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2329-2336
IMMUNOBIOLOGY
Interferon- -induced membrane PAF-receptor expression
confers tumor cell susceptibility to NK perforin-dependent lysis
Christian Berthou,
Jean-François Bourge,
Yuehe Zhang,
Annie Soulié,
Daniela Geromin,
Yves Denizot,
François Sigaux, and
Marilyne Sasportes
From INSERM U462, Hôpital St Louis, Paris, France; and EP CNRS
1I8 Faculté de Médicine, Limoges, France.
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Abstract |
Perforin is known to display a membranolytic activity on tumor
cells. Nevertheless, perforin release during natural killer (NK)-cell
activation is not sufficient to induce membrane target-cell damage. On
the basis of the ability of perforin to interact with phospholipids
containing a choline phosphate headgroup, we identify the
platelet-activating factor (PAF) and its membrane receptor as crucial
components in tumor cell killing activity of human resting NK cells. We
demonstrate for the first time that upon activation, naive NK cells
release the choline phosphate-containing lysolipid PAF, which binds to
perforin and acts as an agonist on perforin-induced membrane damage.
PAF is known to incorporate cell membranes using a specific receptor.
Here we show that interferon- (IFN-) secreted from activated NK
cells ends in PAF-receptor expression on perforin-sensitive K562 cells
but not on perforin-resistant Daudi cells. In order to prove the
capacity of PAF to interact simultaneously with its membrane PAF
receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional
activity of this complex was further examined. The aim was to determine
whether membrane PAF-receptor expression on tumor cells, driven to
express this receptor, could render them sensitive to the perforin
lytic pathway. The results confirmed that transfection of the
PAF-receptor complementary DNA into major histocompatibility complex
class I and Fas-receptor negative tumor cells restored susceptibility
to naive NK cells and perforin attack. Failure of IFN- to induce
membrane PAF receptor constitutes the first described mechanism for
tumor cells to resist the perforin lytic pathway.
(Blood. 2000;95:2329-2336)
© 2000 by The American Society of Hematology.
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Introduction |
Natural killer (NK) cells are a distinct subpopulation
of lymphocytes that play an important role in natural immunity to tumor cells. Resting or naive NK cells are capable of killing target cells
without requiring prior activation or sensitization. On the basis of
the ability of NK cells to kill tumors lacking major histocompatibility
complex (MHC) class I, the identification of killer cell inhibitory
receptors CD94-NKG2 of the C-type lectin-like family and those
belonging to the immunoglobulin superfamily and of a recently
identified novel set of phosphorylated polypeptides termed
killer-activating-receptor-associated proteins have considerably clarified the regulation of NK-cell activation.1-4 Upon
interaction with their targets, NK effector cells release their granule
protein content, essentially perforin and granzymes, into the
intercellular space. The perforin-dependent granule exocytosis pathway
proved to represent the main lytic pathway in tests that compared
perforin knocked-out mice with wild-type littermates.5-10
In addition, studies performed in perforin-deficient mice emphasized
the role of perforin in control of tumor growth through NK and T-cell
cytotoxicity.11,12 Perforin exerts its homeostatic role
either by induced target-cell necrosis13-16 or by
relocalization of granzyme B to its cytoplasmic and nuclear
substrates,17-21 a phenomenon responsible for induced target-cell apoptosis.22-23
Perforin is a phospholipid-binding protein that can disrupt the
membrane of mammalian cells. Perforin-induced membrane damage is
related to successful binding and insertion into the lipid bilayer,
ending in pore formation. Uellner and coworkers24 showed that the removal of carbohydrates from the C-terminus of perforin allowed its C2 phospholipid-binding domain to bind Ca++ and
to initiate interactions with the negatively charged target-cell surface. Since perforin is primarily hydrophilic in nature, increasing hydrophobic interactions between perforin and the membrane phospholipid bilayer is required for protein insertion.25-27 Several
studies on the lipid dependence of perforin interaction with
target-cell membranes have been reported in the
literature.27-31 It appears that both the lipid sidechain
composition, which is predicted to alter the membrane
fluidity,30,31 and the phospholipid headgroup identity
could influence perforin lytic activity.27-29 In the
presence of Ca++, perforin was indeed shown to be capable
of binding directly to various lipid molecules, provided a
phosphorylcholine headgroup was present.29 These results
were in agreement with 2 other studies,27,28 which
demonstrated that choline phosphate-containing lipids represented
powerful ligands for perforin. In contrast, cephaline-containing
phospholipids (phosphatidylethanolamine and phosphatidylserine) were
virtually ineffective in perforin binding.28
Here we show that, upon activation, resting human NK cells release not
only perforin but also choline phosphate-containing lysolipids, among
which is the platelet-activating factor (PAF). The
significance of this finding remained unknown. We suspected that PAF
could affect NK-cell activity as a direct ligand for the lytic protein
perforin. Moreover, it is well established that PAF molecules act
through a specific cell membrane choline receptor, the PAF receptor,
which has been cloned.32-34 In this study, we explored
whether PAF and its specific receptor, when expressed on target-cell
membranes, could enhance NK-cell perforin-mediated cytotoxicity. The
results clearly demonstrated that, in the presence of extracellular
Ca++ and PAF, perforin was able to initiate tumor-cell
membrane damage, provided that target PAF-receptor membrane expression
could be induced via IFN-, an essential cytokine released after
NK-cell activation.35 Our present results indicate that the
lipid mediator PAF and its receptor are crucially involved in the
cytolytic function of resting NK cells. They further suggest that
target failure to express membrane PAF receptor could constitute a
mechanism for tumor cells to resist the perforin lytic pathway.
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Materials and methods |
Assay for perforin membranolytic activity
Perforin cytotoxicity was followed by means of a 4-hour
51Cr-release assay. The targets we used, K562 and Daudi
cells, were negative for Fas receptor (CD95) expression by
fluorescence-activated cell sorting (FACS) analysis (data not shown).
These cells have previously been shown to undergo perforin-induced
acute necrosis with the use of NK cells as effectors.36 As
already described, 51Cr-release assays were
performed.37 The effector-target-cell suspensions were incubated at 37°C in 5% C02 in the
presence or absence of defined concentrations of PAF
(1-0-octadecyl-2-0-acetyl-sn-glycero-3-phosphocholine) C16:0 alkyl moieties (10 10 to
105 mol/L), supplied by
France Biochem. The PAF-receptor antagonist WEB 2086 or SR
27417 was added into the assays at a concentration of 2 µmol/L.38-40 In experiments
using transfected PAF-receptor-expressing Daudi cells as targets, the
cell suspensions were incubated with and without the PAF-receptor
antagonist WEB 2086 (2 µmol/L).
Preparation of human natural killer effector cells
Human NK cells were purified as shown previously.37
Adult peripheral blood mononuclear cells were prepared by standard
Ficoll-Hypaque procedures. After 1 hour of adherence to
plastic at 37°C in 5% CO2, nonadherent cells were
loaded on a discontinuous Percoll gradient (Pharmacia
Fine Chemicals, Uppsala, Sweden) and centrifuged for 30 minutes at
500g. Cells were recovered from the low-density fraction and
purified for NK cells by lysis in the presence of anti-CD3 and
anti-CD19 hybridomas at 1:100 dilution of an ascite fluid and rabbit
complement (Fillorga Laboratories, Paris, France). On purification, the
cell population was analyzed by flow cytometry using anti-CD16
(fluorescein isothiocyanate [FITC]-labeled), anti-CD56 (FITC-labeled), and anti-CD3 (FITC-labeled) monoclonal antibodies (mAbs) from Becton Dickinson Immunocytometry Systems, Inc (San José, CA). NK cells were isolated to greater than 93% purity. Freshly isolated, purified human NK cells were tested for cytolytic activity in 51Cr-release assays in cell culture medium in
which fetal calf serum (FCS) was replaced by bovine serum albumin (BSA)
(2 mg/mL). These cells were referred to as resting or
naive NK cells.
Release by natural killer cells of platelet-activating factor,
perforin, and interferon-
Naive NK cells were activated with the use of
2 × 106 K562 and Daudi hematopoietic tumoral cells,
in an E-to-T ratio of 50:1, in duplicate. PAF assay and
characterization were performed as already described.41
Briefly, PAF released from NK cells were ethanol-extracted from
supernatants at different points of NK/target-cell incubation. After
centrifugation (1500g, 20 minutes), the ethanolic supernatants
were dried, recovered in 50 µL of 60% ethanol, and stored at  20°C until assayed. PAF activity was measured by
platelet aggregation of washed rabbit platelets. Aspirin-treated washed rabbit platelets were stirred in 300 µL Tyrode buffer
containing 0.25% gelatin, 1 mmol/L creatine phosphate,
and 10 U/mL creatine phosphokinase (pH 7.4). Aggregating activity of
the samples was measured by means of a calibration curve obtained with
2.5-20 pg of synthetic C18:0 PAF. Results were expressed
in picograms of PAF as the mean of duplicate samples. The
NK-cell-secreted lipid material was further characterized as PAF on
the basis of studies on the aggregating activity in the presence of 0.1 mmol/L BN 52021 or CV 3988, 2 specific
PAF-receptor antagonists, and on the retention time during thin-layer
chromatography analysis as shown.42
The capacity of the target K562 and Daudi cells to induce from freshly
isolated human NK cells the release of granule protein content into the
intercellular space was examined as already described.43 At
determined times of NK/target-cell incubation, the supernatants were
harvested (50 µL), and the secreted BLT-esterase
activity was measured. Each sample (20 µL) was
incubated in enzyme-linked immunosorbent assay (ELISA) plates with 200 µL of BLT-esterase substrate: 1.5 mg BLT
(N-carbobenzoxy L-lysine-thiobenzyl ester) (Sigma) and
1.5 mg of 5.5'-dithio-bis-(2-nitrobenzoic acid)/20 mL (Tris)-HCl
0.2 mol/L, pH 8 (Sigma). After 1 hour of
incubation at 37°C, absorption was measured at 450 nm. The
spontaneous BLT release was determined by incubating NK cells in medium
alone. The maximum release was determined by adding 50 µL of 10% NP-40 to 100 µL of NK-cell suspension. The percentage of specific
BLT-esterase activity was calculated as follows: ([experimental spontaneous]/[maximum spontaneous]) × 100, where
experimental is experimental BLT-esterase release (A450);
spontaneous is spontaneous BLT-esterase release (A450); and
maximum is maximum BLT-esterase release (A450).
At relevant times of NK/target-cell incubation, IFN- release from
NK cells into the supernatants (50 µL) was also
determined. The high sensitive IFN- human ELISA system
(Biotrak, Amersham) was used for the IFN- assays.
Control tests received either targets or effectors only.
Target-cell interferon- activation
Target K562 and Daudi cells were first prepared for flow cytometry
analysis of membrane PAF-receptor expression before and after 10 and 30 minutes of NK-cell contact into the NK assays. K562 cells expressing
glycophorin A and Daudi cells expressing the CD19 antigen were selected
in a flow cytometry analysis (FASCan, Becton Dickinson) with the use of
the appropriate mAbs (FITC-labeled anti-glycophorin A [Immunotech,
Marseilles, France]; FITC-labeled anti-CD19 [Becton Dickinson
Immunocytometry Systems, Inc) and were then analyzed for PAF-receptor
expression. On the other hand, 1 × 106 K562 and Daudi
cells were incubated with different concentrations of IFN- (Protech
Inc, Rocky Hill, NJ) (50 to 500 U/mL) for various times (30 minutes and
1, 2, and 4 hours), washed, and analyzed for PAF-receptor membrane
expression by flow cytometry with the use of an anti-human
PAF-receptor mAb (Cayman Chemical Co, MI). The
myelomonocytic cell line U937 was used as a positive control of
PAF-receptor expression. The neutralizing anti-IFN- antibody named
MAS 290 was used for blocking at a concentration of 25 ng/mL and was
purchased from Valbiotech (Paris, France). Mouse normal pooled sera
were used in blocking experiments as isotype control.
Binding assays of [3H]platelet-activating factor to
perforin
Antiperforin 6.4 (immunoglobulin [Ig]M, antiperforin) mAb
(PharmaCell, Paris, France) coupling to Affi-Gel 10 gel (Affi-Gel 10 gel, Bio-Rad Laboratories) was first performed. The gel
was washed with 20 vol of cold 10 mmol/L
sodium acetate, pH 4.5. Then, 1 mL of the gel was mixed with 0.5 mL of
antiperforin mAb (0.1 mol/L MOPS, pH 7.5, containing 6.3 mg antiperforin) and was sufficiently agitated to give a uniform
suspension. The gel was gently shaken for 12 hours at 4°C. The
coupling reaction was halted by adding 0.1 mol/L ethanolamine-HCl (pH
8) for 4 hours at 4°C. Perforin was then purified by specific
immunoprecipitation from the human NK-cell line YT2C2. These cells
(3.6 × 106) were lysed with 1 mL cold lysis buffer
(10 mmol/L Hepes-NaOH, pH 7.4, containing 25 mmol/L KCl, 5 mmol/L EDTA, 1 mmol/L DTT, and 0.5% NP-40) at 4°C for
1 minute. The cytoplasmic cell lysate was mixed with the antiperforin
coupling gel in a coupling buffer: 10 mmol/L Tris-HCl, pH
8.0, containing 0.5 mol/L NaCl, 5 mmol/L EDTA, 0.2 mmol/L ABESF (4-2-aminoethyl-benzene sulfonyl fluoride), 0.5% NP-40, and 0.02% NaN3 which was gently agitated for
4 hours at 4°C. The gel was washed with phosphate-buffered saline
(PBS) (20 mmol/L NaH2PO4, pH 7.0, containing 150 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L
CaCl2, 6 mmol/L glucose, and 0.25% BSA). The
experiments were further carried out by the specific binding of
[3H] octadecyl-9,10-PAF C18:0 (141.6 Ci/mmol) (purchased
from Dupont de Nemours Division NEN) to 50 µL perforin-antibody gel in a total volume of 300 µL PBS containing 20 nmol/L
[3H]PAF. The mixture was incubated at 20°C for 30 minutes. The binding gel was then washed 5 times with the cold buffer
mentioned above, and the radioactivity was measured by liquid
scintillation counting after gel incubation with 0.1 mol/L NaOH.
Specific binding was determined as the total radioactivity bound minus
the radioactivity bound in the presence of unlabeled PAF C18:0 from
France Biochem. The perforin-negative Jurkat cell line was used as
control under the same conditions.
Coimmunoprecipitation of the platelet-activating factor receptor,
platelet-activating factor, and perforin as a ternary complex
Biotinylation of membrane cell surface proteins of IFN--induced
PAF-receptor-positive K562 cells was first carried out. Cells were
washed 3 times with ice-cold PBS (pH 8.0), resuspended at a
concentration of 25 × 106 cells/mL in PBS in which
0.5 mg of Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) was
added per mL of reaction volume, incubated during 2 hours and washed 3 times with ice-cold PBS. In order to purify cell membranes, the
biotinylated cells were resuspended at 107 cells/mL in
ice-cold PBS containing protease inhibitors; they were equilibrated at
0°C in a nitrogen cavitation bomb for 1 minute; nuclei were removed
by centrifugation at 800 rpm at 4°C for 8 minutes; and the membrane
fraction was obtained by centrifugation at 14 × 103
rpm at 4°C for 1 hour. The anti-PAF-receptor mAb (Cayman Chemical) was covalently cross-linked with Protein G Sepharose (Pharmacia), as
previously shown.44 Membrane proteins were lysed at 4°C
for 45 minutes with the following buffer: 20 mmol/L
Hepes, pH 7.5, containing 150 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L
CaCl2, 1 mmol/L DTT, 1 mmol/L
AEBSF, and 0.5% NP-40. The anti-PAF-receptor sepharose was mixed with
biotinylated K562 membrane proteins and incubated at 4°C for 12 hours. The beads were washed 3 times with the same ice-cold buffer
containing 1 mol/L NaCl. PAF-receptor immunoprecipitated beads were
then washed 2 times in binding buffer (Hepes 10 mmol/L,
pH 7.4, containing 137 mmol/L NaCl, 2.6 mmol/L KCl, 6 mmol/L glucose, 1.3 mmol/L CaCl2, 1 mmol/L
MgCl2, 0.02% NaN3 and 0.25% BSA).
Immunopurified PAF-receptor microspheres (100 µL) were
incubated with 20 nmol/L [3H]PAF (1.4 mL)
at 20°C for 1 hour. After this binding step, beads were washed 3 times with the same ice-cold buffer. They were then incubated with the
perforin-positive NK-cell YT2C2 cytoplasmic fraction in the same
binding buffer at 20°C for 1 hour and then washed 3 times with the
washing buffer (Hepes 10 mmol/L, pH 7.4, containing 137 mmol/L NaCl, 2.6 mmol/L KCl, 6 mmol/L glucose, 1.3 mmol/L
CaCl2, 1 mmol/L MgCl2, and 0.02%
NaN3). Proteins were eluted with the use of a buffer
of 20 mmol/L Tris, pH 6.8, containing 4%
sodium dodecyl sulfate (SDS) and 20% glycerol, at 95°C for 5 minutes. Then 30 µL of elution proteins were incubated
with 10 µL of Laemmli electrophoresis buffer at
95°C for 5 minutes to Western blot analysis. Western blotting was
performed by loading the proteins on 10% SDS-polyacrylamide gel
electrophoresis. The human antiperforin (antiperforin, perforin A2
2d4.2.a8.E11, kindly provided by G. M. Griffiths, Sir William
Dunn School of Pathology, South Parks Rd, Oxford, UK) was
added at 1/50 final dilution for 1 hour, and then washed and detected
with a sheep antimouse IgG peroxydase conjugate (1/2000 final dilution
for 1 hour; Amersham, Arlington Heights, IL), as previously
described.45 PAF-receptor proteins were tested by
streptavidin immunoblotting. The binding of [3H]PAF was
analyzed by radioactive controls. U937 PAF-receptor positive membrane
biotinylated fractions were used as positive controls whereas
PAF-receptor negative Jurkat cell line membrane biotinylated fractions
and perforin-negative cell lysates from the same cell line were used as
negative controls under the same conditions.
Daudi cell transfections
The EcoR1 insert of the plasmid pBluescript vector32
containing the total coding region of the human
PAF-receptor complementary DNA (cDNA) (1.8 Kb) was
subcloned into the EcoR1 site of the pCI-neo vector
(Promega), in both sense (pCI-neo PAF-receptor sense) and antisense (pCI-neo PAF-receptor antisense) orientations. Wild-type Daudi cells, purchased from ATCC, were maintained in RPMI
1640 with 10% FCS, 2 mmol/L glutamine,
penicillin (100 U/mL), and streptomycin (100 µg/mL). Daudi cells
(1 × 107) were electroporated (Gene Pulser,
Bio-Rad) at 250 V and a capacitance of 960 microfarads in the presence
of the above-mentioned expression vectors (10 µg) and with a plasmid
expressing no gene (pCI-neo). Membrane PAF-receptor expression was
analyzed by flow cytometry (FASCan, Becton Dickinson) with the use of
an anti-PAF-receptor mAb (Cayman Chemical). At 4 days after
electroporation, membrane PAF-receptor expression was maximal at the
surface of transfected Daudi cells (data not shown). Wild-type and
transfected Daudi cells were used as targets in short-term
51Cr-release assays with naive NK cells used as effectors.
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Results |
Natural-killer-cell lytic activity is enhanced by the phospholipid
platelet-activating factor
Addressing the question of tumor cell susceptibility to
perforin-mediated lysis in humans, we took advantage of a model in which K562 and Daudi cells, both of which are MHC class I and Fas-receptor (FasR) negative, were shown to induce
activation of resting human NK cells, degranulation, and perforin
release.43 The kinetic of degranulation was analyzed and
shown to take place 5 minutes after incubation of freshly isolated NK
effectors with K562 targets (Table 1). A
maximum release (90%), as measured by the percentage of BLT-esterase
activity, occurred at that time. With the use of Daudi cells as
targets, a similar ability to induce a granule protein content release
from NK cells was clearly observed, as shown in Table 1. However, if
NK-cell activation and degranulation ended up in 51Cr
release from K562 cells, it did not result in Daudi cell lysis (Table
1).
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Table 1.
Release by NK cells of PAF, perforin, and IFN-
compared with PAF receptor expression on targets and 51Cr
release
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The capacity of naive NK cells to secrete the phospholipid PAF during
the NK/target-cell contact was analyzed. NK effector cells released the
significant amount of 62.5 pg of PAF after 10 minutes of interaction
with Daudi cells, whereas a maximal value of 250 pg of PAF was found at
30 minutes (Table 1). Then the lipid mediator became undetectable. We
observed that the kinetic of PAF release from NK cells when incubated
with K562 targets was slightly delayed, compared with the use of Daudi
cells as activators, with a maximal value of 175 pg obtained at 30 minutes but with the same general kinetic profile of PAF metabolism
(Table 1).
We subsequently explored whether PAF addition could overcome Daudi cell
resistance to NK-cell-dependent lysis. To answer this question, we
used resting NK cells and targets in 51Cr-release assays to
which exogenous PAF (C16:0 alkyl moieties) was added. The concomitant
increase in extracellular concentration of C16:0 PAF completely failed
to affect Daudi cell lysis (Figure 1a). In
contrast, K562 cells displayed, after addition of C16:0 PAF, a
significantly higher susceptibility to membrane damage, as demonstrated
by an increase of 22 ± 2% of 51Cr target-cell release
(Figure 1A). These results demonstrated an agonistic effect of PAF in
the lytic pathway on NK perforin-sensitive target cells.
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| Fig 1.
PAF and perforin-mediated NK target-cell
lysis.
(A) The phospholipid PAF enhances perforin-mediated NK target-cell
lysis. Resting NK cells were used in standard cytotoxic assays against
the MHC class I and FasR-negative K562 and Daudi cells, both of which
were shown to be capable of inducing NK-cell activation and
degranulation. Perforin-induced cell lysis was measured by target-cell
release of sodium-51Cr-labeled cytoplasmic proteins via
membrane damage and perforin pores. Resting human NK cells were mixed
with labeled target cells at the various E-to-T ratios of 50:1, 25:1,
12:1, and 6:1, and target-cell lysis was measured at 4 hours. Naive NK
cells were very efficient in provoking 51Cr release from
K562 targets. Following addition of PAF to targets (PAF C16:0 alkyl
moieties, [1 µmol/L]), K562 cells displayed a higher
susceptibility to the lytic activity of perforin, as reflected by an
increase in the amount of 51Cr target-cell release. In
contrast, increasing extracellular concentrations of C16:0 PAF turned
out to be inefficient in overcoming the failure of naive NK cells to
induce Daudi cell lysis. (B) Inhibition of the PAF receptor. PAF
receptor activity on K562 target cells was inhibited either by blocking
its IFN--induced expression using a neutralizing anti-IFN-
antibody or by adding specific PAF receptor antagonists, such as WEB
2086 and SR 27417. Resting NK cells were used as effectors. These
processes led to a potent inhibitory activity on NK-cell lysis, as
shown by a significant decrease in the amount of NK/target-cell
51Cr release.
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Perforin purified by immunoprecipitation efficiently binds to the
phospholipid platelet-activating factor
These results led us to examine the capacity of human purified
perforin to bind to the phospholipid mediator PAF.
[3H]PAF C18:0 was exposed to immunopurified perforin, as
shown in "Materials and methods," and the protein was then
counted for [3H] incorporation. As shown in Figure
2, perforin efficiently bound to PAF in the
presence of Ca++ and Mg2+, as demonstrated by
the detection of a 20 × 103
disintegration-per-minute radioactivity from perforin after
[3H]PAF coincubation (n = 3). Unlabeled PAF
competitively inhibited the binding of [3H]PAF, with 50%
inhibition observed by mixing 1 vol C18:0 PAF with 1 vol C18:0
[3H]PAF at 20°C (data not shown). There was no
[3H]PAF binding in the control tests (the
perforin-negative Jurkat cell line cytoplasmic extracts, the
antiperforin mAb alone, the gel itself, and BSA) (Figure 2).
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| Fig 2.
Purified human perforin efficiently binds to PAF.
Perforin was immunopurified from the perforin-positive YT2C2 NK-cell
line using an appropriate binding gel (antiperforin 6.4-IgM,
antiperforin mAb, coupled with Affi-Gel 10 gel). (A) Successful
purification of perforin was controlled by a Western blot using the
specific antiperforin antibody 2d4-perf. In lane C, for YT cell line
extracts as positive control, the procedure revealed 2 bands at the
molecular weight of 66 and 30-kd as recently reported by
Uellner et al.24 Perforin was further purified by
ultracentrifugation to obtain the active form of 66-kd
(Uellner et al24), expressed in lane 1. No other band was
detected in lanes 2, 3, 4, and 5 using, respectively, the
perforin-negative Jurkat cell line lysate, the gel cross-linked to the
mAb, the gel alone, or the gel with an irrelevant protein BSA instead
of perforin. Active perforin was then exposed to [3H]PAF
C18:0 during 30 minutes at 20°C and washed 5 times with the cold
buffer as shown in "Materials and methods." The resulting
radioactivity was then measured for perforin. (B) In this assay, lane 1 shows the formation of perforin-[3H]PAF complexes
expressed in disintegrations per minute. In contrast, no significant
radioactivity was detected in lanes 2, 3, 4, and 5 using, respectively,
the perforin-negative Jurkat cell line lysate, the gel cross-linked to
the mAb, the gel alone, or the gel with an irrelevant protein BSA
instead of perforin as already mentioned for Western blot analysis.
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Interferon- induced membrane platelet-activating
factor-receptor expression on K562 cells but not on Daudi cells
PAF molecules bind and incorporate cell membranes using a specific
receptor that has been cloned.33,34 We determined whether PAF could enhance NK-cell lytic activity through efficient membrane binding of perforin/PAF complexes to the PAF receptor. We analyzed PAF-receptor expression on K562 and Daudi targets, but neither group of
cells had a detectable level of membrane PAF receptor (Figure
3).
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| Fig 3.
IFN- induces K562 cells to rapidly express membrane
PAF receptors.
K562 and Daudi cells were stimulated with 300 U/mL of human
IFN- for 30 minutes and for 1, 2, and 4 hours and were analyzed by
flow cytometry for PAF receptor membrane expression. U937 cells were
used as positive controls. The background signal is shown (dotted
lines). Inset numbers indicate the percentage of cells expressing the
protein and the mean fluoresence intensity at the indicated times.
Regarding the capacity of tumor cells to express membrane PAF receptor,
K562 cells were IFN- sensitive; Daudi cells, IFN- resistant.
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We subsequently explored whether membrane PAF-receptor expression could
be induced on targets by coincubated NK cells. The expression of PAF
receptors on K562 and Daudi target cells was measured at 3 times (0, 10, and 30 minutes), using flow cytometry analysis and appropriate
double-fluorescence labeling (PAF receptor and glycophorin A for K562
cells; CD19 for Daudi cells). However, owing to a rapid and expected
NK-mediated induced-target-cell lysis, only a limited number of
double-labeled and viable target cells could be analyzed (ie,
5.102). In such conditions, 33% of K562
target cells were clearly found to express membrane PAF receptors at 10 minutes (Table 1 and Figure 4). In
contrast, there were no Daudi PAF-receptor-positive cells in the NK
assays at the same time and thereafter (Table 1, Figure
4).
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| Fig 4.
Comparative analysis of membrane PAF-receptor expression
on K562 and Daudi cells.
A representative experiment is shown. K562 and Daudi cells were
analyzed before and after 10 and 30 minutes of NK cell attack, stained
with anti-PAF-receptor mAb detected with phosphatidylethanolamine
(PE)-goat antimouse antibody and with FITC-conjugated
antiglycophorin (K562) or anti-CD19 (Daudi). Data are displayed as dot
plots, and PAF-receptor expression was analyzed only on glycophorin
(K562) and CD19 (Daudi) positive cells. Only a limited number of
double-labeled and viable target cells could be analyzed (ie,
5.102), owing to the expected rapid
NK-mediated induced-target-cell lysis. Cells that are double-stained
with corresponding PE- and FITC-conjugated antibodies are represented
in the upper right quadrant.
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Among the cytokines produced by activated NK cells, we hypothesized
that IFN- could be a reliable candidate for the induction of
membrane PAF-receptor expression on targets. Following a 2-hour treatment by IFN- (300 U/mL), we observed a large increase in the
percentage of membrane positive PAF-receptor K562 cells
(73.3 ± 7%) (Figure 3). This increase persisted after 4 hours.
In contrast, IFN- turned out to be ineffective in inducing Daudi
cells to express the receptor (Figure 3), even when the cells were
further exposed to the cytokine for 16 hours (data not shown). The next step was to further confirm the secretion of IFN- by NK cells during
their interaction with targets. After a 5-minute interval of NK
interaction with both target-cell lines, 715 pg of IFN- were indeed
found. The amount of released IFN- remained virtually constant during the 4 hours of NK/target incubation, with a slight decrease at 10 minutes (Table 1).
In order to provide further insights into the role of PAF receptor on
NK-cell lytic activity, we studied the effect of a neutralizing anti-IFN- antibody (25 ng/mL) and of different PAF-receptor
antagonists (2 µmol/L) on lysis of K562 targets by
resting NK cells. Inhibition of the IFN- pathway led to a 50 ± 5% reduction of NK-cell killing activity. This effect was not seen
when an irrelevant antibody was used (Figure 1B). We also observed that
addition in the K562 assays of the PAF-receptor antagonists, WEB 2086 and SR 27417, was followed by a potent inhibitory activity on NK-cell
lysis as shown by a decrease of, respectively, 50 ± 7% and
72 ± 5% in the amount of NK target-cell 51Cr release
(Figure 1B). Such a reduced, but not totally abolished, NK-cell
cytotoxicity could be explained by either an incomplete membrane
PAF-receptor saturation or ligation. Through the induction of
PAF-receptor expression on target cells, IFN- is therefore involved
in the successful perforin lytic pathway induced by naive NK cells.
Perforin, platelet-activating factor, and
platelet-activating factor receptor form a ternary complex
We immunopurified the PAF receptor from biotinylated membranes of
IFN--treated K562 cells. This receptor was then exposed, in the
presence of Ca++ and Mg2+, to
[3H]PAF C18:0 and to perforin present in a YT2C2
NK-cell-line lysate. Radioactivity and perforin detection within the
ternary complex were then analyzed as appropriate, as shown in
"Materials and methods." The successful in vitro constitution of
a ternary complex was demonstrated by the presence of the 3 components,
PAF receptor, [3H]PAF, and perforin, in the same elution
fraction. The PAF-receptor protein was detected in the complex as a
42-kd band using stavidin immunoblotting (Figure
5A). Western blot analysis also revealed the presence of a 66-kd band equivalent to active
perforin, as reported by Uellner et al,24 using the same
specific antiperforin antibody 2d4-perf (Figure 5B). Finally, the
presence of [3H]PAF in the complex was demonstrated by
the concomitant detection of a 6 × 103 cpm
radioactivity, as shown in column 2 of Figure 5. In contrast, purified
PAF receptors failed to bind to perforin in the absence of bridging PAF
molecules (Figure 5). These data proved the capacity of PAF to interact
simultaneously, in these experimental conditions, with its receptor and
the perforin protein.
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| Fig 5.
Perforin, PAF, and PAF receptor form a ternary complex.
PAF receptors were immunopurified from the biotinylated membrane of
IFN--treated K562 cells with the use of the specific
anti-PAF-receptor mAb. Purified receptors were incubated with or
without C18:0 [3H]PAF in the presence of Ca++
and Mg2+ and then exposed to YT2C2 cell cytoplasmic
extracts for perforin-binding analysis. To confirm the presence of the
3 components (PAF receptor, PAF, perforin) in the same elution
fraction, Western blot analysis using, respectively, anti-PAF receptor
(A1), antiperforin (A2) mAbs, and [3H]PAF radioactivity
detection (B) were performed. This procedure allowed simultaneous
detection of PAF receptor at the molecular weight (mw) of
42-kd (A1, lane 2); perforin at the mw of 66 kd (A2, lane 2); and radioactivity due to the presence of
[3H]PAF in the complex (B, column 2). In the absence of
[3H]PAF (B, column 3), PAF receptor was still detected
(A1, lane 3) but perforin failed to bind to the receptor (A2, lane 3).
In the absence of PAF receptors (Jurkat cell membrane extracts, A1,
lane 4), [3H]PAF (B, column 4) and perforin (A2, lane 4)
failed to form the ternary complex. The YT2C2 cell line
was used as negative control for PAF receptor expression (A1, lane 1),
as positive control for perforin expression (A2, lane1), and as
negative control for [3H]PAF binding (B, column 1).
|
|
Platelet-activating-factor-receptor complementary DNA
transfection into Daudi cells restores susceptibility to naive
natural-killer-cell-induced lysis
The observed failure of NK cells to end in membrane PAF-receptor
expression on Daudi targets, via IFN- secretion, could explain the
tumor-cell resistance to the perforin lytic pathway. To establish that
the absence of PAF receptor on Daudi cells was responsible for their
resistance to the NK-cell perforin lytic activity, we transiently
transfected Daudi cells with the human PAF-receptor cDNA. This led to
PAF-receptor membrane expression on 30 ± 5% of cells, as
measured by flow cytometry analysis 4 days after transfection (Figure
6A). The transfected cells were used as
targets in 4-hour 51Cr-release assays using resting,
purified human NK cells as effectors. Five successive experiments
showed that an efficient NK-induced target-cell 51Cr
release (20 ± 3%) was repeatedly observed when
PAF-receptor-expressing Daudi cells were used as targets (Figure 6B).
Moreover, the ability of NK cells to induce a cytolytic activity in
PAF-receptor-positive Daudi cells was totally reversed by the addition
of the PAF-receptor antagonist WEB 2086 into the lytic assays (Figure
6B). Such an observed complete inhibition of perforin-mediated lysis
via WEB addition was likely due to the low level of PAF-receptor
expression obtained on transfected Daudi cells. We conclude that
PAF-receptor expression on Daudi cells was necessary and sufficient to
restore NK-cell susceptibility to the perforin-dependent lytic
pathway.
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| Fig 6.
(A) Daudi cells transfected with the PAF-receptor cDNA express
membrane PAF receptors. Wild-type Daudi cells were transfected by the
plasmid pCI-neo vectors expressing the sense PAF receptor cDNA gene
(pCI-neo.PAF-R s), the PAF receptor antisense cDNA gene (pCI-neo. PAF-R
as), and by a plasmid expressing no gene (pCI-neo) control vector. At 4 days later, PAF receptor membrane expression was quantified by flow
cytometry using an mAb anti-PAF receptor on nonpermeabilized Daudi
transfected cells (solid lines). Cells were stained with propidium
iodide to facilitate discrimination among live and dead cells. The
background signal is shown (dotted lines). Inset numbers
indicate the percentage of cells expressing the PAF receptor on
untransfected Daudi cells (wild-type), Daudi cells transfected with
pCI-neo PAF receptor sense gene (pCI-neo PAF-R s), Daudi cells
transfected with the pCI-neo PAF receptor antisense gene (pCI-neo PAF-R
as), and Daudi cells transfected with pCI-neo. (B) PAF-receptor cDNA
transfection in the MHC class I and FasR-negative NK-resistant Daudi
cells restores susceptibility to NK perforin-dependent lysis. Wild-type
Daudi cells and PAF-receptor-positive (pCI-neo PAF-R s),
PAF-receptor-negative (pCI-neo PAF-R as and pCI-neo plasmids)
transfected Daudi cells were used independently as targets in
short-term 51Cr-release assays using naive NK cells as
effectors. PAF-receptor-positive Daudi cell susceptibility to NK cells
was tested with and without a PAF-receptor antagonist, WEB 2086, added
at 2 µmol/L. Resting NK cells were mixed with labeled
target cells at the E-to-T ratios of 50:1, 25:1, 12:1, and 6:1, and
perforin-induced Daudi cell lysis was measured at 4 hours. Naive NK
cells were totally unable to lyse PAF-receptor-negative Daudi cells
(wild-type and PAF-receptor-negative transfectants) but were efficient
in killing PAF-receptor-positive Daudi cells. The susceptibility of
PAF-receptor-expressing Daudi cells to NK lysis was totally reversed
in the presence of the PAF receptor antagonist (pCI-neo PAF-R s + WEB).
|
|
| |
Discussion |
Perforin and granzymes, which belong to the granule exocytosis lytic
pathway, are used by cytotoxic T lymphocytes (CTLs) and NK cells in
acquired and innate immunity.14 However, CTLs as well as NK
cells can use Fas ligand-induced cytotoxicity, provided that targets
express the membrane Fas receptor.36,46 Granzyme B is
involved in target-cell death apoptosis, while perforin is responsible
for target-cell membrane damage as measured by 51Cr
release. Even if the mechanisms by which both proteins might interact
are still under investigation,17,18,20 the production of
perforin-deficient mice has established beyond any doubt the powerful
role of the protein perforin in tumor growth control.11,12 Moreover, a recent study performed in double granzyme A and B knocked-out mice has clearly indicated that target-cell membrane damage
leading to cell death was independent of granzymes and related solely
to perforin cytolytic activity.10
Naive NK cells primarily use Fas-independent and perforin-based
cytotoxicity.47 In a previous study, using naive human NK cells as effectors and Fas-receptor negative targets, we showed that
NK-cell lytic activity was totally abolished in the presence of 0.75 mmol/L EGTA, further confirming a
perforin-induced Ca++-dependent cytolysis.43 In
addition, dependent on the hematopoietic tumor target cell, NK-cell
activation and perforin release were insufficient to regularly
determine successful target-cell lysis.43 This observation
suggested that a structure present at the surface of some but not all
tumoral targets could be critical in facilitating perforin-mediated
target-cell lysis induced by resting NK cells.
Perforin lytic activity has been shown to be dependent on the presence
of phosphorylcholines acting as specific Ca++-dependent
receptors for the protein.29 We here demonstrated a
concomitant release by NK cells, upon activation by tumor cells, of
lytic proteins and of PAF choline phosphate-containing molecules. This
observation raised the question of the potential facilitating effect of
the NK-cell-secreted phospholipid mediator on perforin lytic activity.
Purified human perforin was indeed shown to efficiently bind to the
phospholipid PAF. Furthermore, our present results identified PAF and
its specific membrane receptor as a critical component in the lytic
activity of naive NK cells. The major argument comes from the
observation that transfection of the PAF-receptor cDNA into
NK-resistant MHC class I and FasR negative Daudi cells confers
susceptibility to NK-cell-induced perforin-mediated lysis.
It has been demonstrated that a larger amount of perforin binds
perforin-resistant cell membranes following treatment with tunicamycin,
an inhibitor for N-glycosylation.48 This observation suggests that the protective cell surface layer, which contains a large
carbohydrate content, could prevent perforin from efficient binding to
membrane targets. On the other hand, hydrophobic interactions between
perforin and phospholipids in target-cell membranes are known to
represent essential requirements for protein insertion and pore
formation.25-27 In this context, phosphorylcholines
represent suitable ligands for the protein.27-29 As
perforin is primarily a hydrophilic protein, the role of the
phosphocholine PAF as a facilitating partner in inducing a perforin
conformational switch from a hydrophilic state to a membrane-inserted
hydrophobic form remains to be demonstrated. Whatever the role of PAF
on perforin function, our present study favors the use by tumor cells
of PAF receptors to increase the concentration of phosphorylcholines at
membrane-binding sites, through PAF recruitment. This process seems to
enhance perforin-induced target-cell pore formation. The successful
constitution we obtained in vitro of a tri-molecular complex,
consisting of perforin, PAF, and its receptor, strongly supports such a
facilitating role of the bridging PAF molecules in perforin function.
We are aware that perforin can determine membrane damage without the
presence of PAF, as shown by the capacity of the protein to induce
lysis of sheep red blood cells. However, the matrix composition of
erythrocytes, which contain in their membranes 23% of
phosphorylcholines31 and lack a protective cell surface
layer,49 is likely to allow direct access and facile pore
formation by perforin. Finally, lack of PAF-receptor detection on
erythrocyte membranes could explain the observed inhibition of
perforin-induced hemolysis when choline-phosphate lysolipids are added
into the red blood cell assays.28
In addition to perforin and PAF, IFN- is also released by activated
NK cells. This cytokine has been implicated in tumor-cell cytotoxicity,
but the mechanism(s) of such a target-cell activity remained
undetermined. Here we showed that IFN- was capable of inducing
membrane PAF-receptor expression in NK-sensitive K562 cells, but not in
NK-resistant Daudi cells. The observed failure of NK cells to achieve
PAF-receptor membrane expression on Daudi targets, through IFN-
secretion, could explain tumor-cell resistance to the perforin lytic
pathway. Whatever the strategy used by Daudi cells to down-regulate
PAF-receptor membrane expression, it allowed tumor cells to escape the
immune response. PAF-receptor expression variability has already been
reported among hematopoietic tumoral cells,50,51 suggesting
that this process could constitute a more general phenomenon of NK-cell
resistance. A role of both IFN- and perforin in the suppression of
tumor growth has been described in mice52; this is in
agreement with our present in vitro data using human cells.
In conclusion, we showed that a tumor cell could escape perforin attack
by preventing efficient perforin insertion into its cell membrane. We
propose PAF and its receptor as facilitating partners in the perforin
lytic pathway as these elements confer target-cell susceptibility to
cytolytic activity induced by naive NK cells.
| |
Acknowledgments |
We are very grateful to Claude Gazin for constructing the expression
vectors with the PAF-receptor cDNA. We thank Eva Ninio for helpful
suggestions concerning the ligation of perforin to [3H]PAF and the use of PAF-receptor antagonists (WEB 2086 and SR 27417) that she kindly provided.
| |
Footnotes |
Submitted October 8, 1998; accepted December 3, 1999.
Supported by grants from ARC, La Ligue Nationale contre le Cancer
(Laboratoire associe n°10, Comite de Paris).
Reprints: Marilyne Sasportes, INSERM U462, Hôpital
Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France; e-mail: msasportes{at}chu-stlouis.fr.
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.
| |
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Abstract
Introduction