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*
Department of Experimental Medicine and Pathology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome "La Sapienza," Rome, Italy;
Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy;
Biotechnology Section, Institute for the Study and Cure of Tumors, Genoa, Italy; and
§
Mediterranean Institute of Neuroscience "Neuromed," Pozzilli, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 and 5ß1
fibronectin receptors, to modulate CD16-triggered phospholipase
activation in human NK cells. ß1 integrin simultaneous
cross-linking selectively inhibited CD16-induced phospholipase D (PLD)
activation, without affecting either phosphatidylinositol-phospholipase
C or cytosolic phospholipase A2 (PLA2)
enzymatic activity. CD16-induced secretory PLA2
(sPLA2) protein release as well as its enzymatic activity
in both cell-associated and soluble forms were also found to be
inhibited upon ß1 integrin coengagement. The similar
effects exerted by specific PLD pharmacological inhibitors
(2,3-diphosphoglycerate, ethanol) suggest that in our experimental
system, sPLA2 secretion and activation are under the
control of a PLD-dependent pathway. By using pharmacological inhibitors
(2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that
PLD activation is an important step in the CD16-triggered signaling
cascade that leads to NK cytotoxic granule exocytosis. Consistent with
these findings, fibronectin receptor engagement, by either mAbs or
natural ligands, resulted in a selective inhibition of CD16-triggered,
but not of PMA/ionomycin-induced, degranulation that was reversed by
the exogenous addition of purified PLD from Streptomyces
chromofuscus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimeric adhesion receptors involved
in both cell-cell and cell-extracellular matrix
(ECM)3 interactions. Members
belonging to the same family share a common ß subunit paired with
different -chains and are endowed with distinct, although
overlapping, ligand binding specificities. Cell differentiation and
activation tightly regulate integrin expression and avidity for their
ligands. On the other hand, integrin engagement by natural ligands or
specific mAbs transduces intracellular signals that are important for
regulating many different cell functions, such as migration, adhesion,
proliferation, differentiation, apoptosis, and specific gene
expression. In both outside/in and inside/out signaling, integrins
exhibit a functional cross-talk with diverse activation molecules, such
as growth factor or Ag receptors 1, 2 . Integrin-mediated costimulation of leukocyte activities has been widely studied at both molecular and functional levels 3 . Increasing evidence indicates that integrins are also capable of transducing signals that negatively affect several receptor-stimulated cell functions, such as proliferation, degranulation, cytokine production, and specific gene expression, in different cell types 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 . However, both the biochemical nature of integrin-elicited negative signals and the molecular levels at which they interfere with known activator pathways are poorly defined. Lipid signaling has been recently indicated as one possible point of interference between integrin and other receptor-initiated pathways. Inhibition of T cell proliferation induced by the anti-ß1 integrin mAb K20 is indeed accompanied by cAMP accumulation as well as by a decrease in the production of phosphatidic acid (PA) and diacylglycerol (DAG) 4, 5 , pointing to an interference with phospholipid turnover; in addition, cytoskeletal translocation of phosphatidylinositol 3-kinase (PI 3-kinase) is up-regulated upon disruption of GPIIb/IIIa-mediated aggregation in von Willebrand factor-stimulated platelets 16 .
NK cells are a discrete
CD3-CD16+CD56+ lymphocyte subset
capable of lysing a broad range of tumor, virus-infected, and immature
hemopoietic cells without prior sensitization in a non-MHC-restricted
or Ab-dependent fashion (Ab-dependent cellular cytotoxicity).
Functional responses, such as exocytosis of cytotoxic granule content
and cytokine production, are the final result of NK cell activation
elicited by different stimuli 17, 18 . CD16, the low affinity receptor
for the Fc fragment of IgG (Fc
RIIIA), is a major signaling structure
on NK cells capable of triggering multiple biochemical pathways, among
which the activation of several enzymes involved in phospholipid
turnover, including phosphatidylinositol-phospholipase C (PI-PLC) 19, 20 , PLD 21 , and both secretory and cytosolic PLA2 22 .
Moreover, phospholipase activation and lipid second messenger
production are thought to be involved in the regulation of several NK
cell functions 19, 20, 21, 22, 23 . Human NK cells express several members of the
ß1 family of integrins, which have been suggested to play
a major role in the regulation of their migration and functions
24, 25, 26, 27 . ß1 integrin ligation on human NK cells
transduces different biochemical signals, including calcium
mobilization, tyrosine phosphorylation of various substrates, among
which is proline-rich tyrosine kinase-2 (PYK-2), and activation of the
Ras/mitogen-activated protein kinase pathway, which leads to IFN-
production 28, 29, 30, 31 . Moreover, ß1 integrin cross-linking
by either mAbs or natural ligands costimulates NK cytotoxic functions
28 .
Here we provide evidence that ß1 integrin coengagement specifically interferes with CD16-elicited PLD activation, without affecting PI-PLC and cPLA2 activity. Both sPLA2 activity and granule exocytosis are also inhibited as a consequence of PLD inhibition, suggesting a role for this enzyme in CD16-triggered NK cell degranulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mouse mAbs were used: anti-CD3 (Leu 4),
anti-CD16 (Leu 11c), anti-CD56 (Leu 19), and anti-CD14 (Leu
M3; Becton Dickinson, Mountain View, CA); anti-CD16 (B73.1) and
anti-MHC class I (W6/32; provided by Dr. G. Trinchieri, Wistar
Institute, Philadelphia, PA); anti-CD56 (B159.5.2; provided by Dr.
B. Perussia, Thomas Jefferson University, Jefferson Cancer Center,
Philadelphia, PA); anti-ß1 integrin subunit (4B4,
purchased from Coulter Immunology (Hialeah, FL), and TS2/16, provided
by Dr. M. Hemler, Dana-Farber Cancer Institute (Boston, MA));
anti-4 (HP2/1) and anti-5 (SAM-1)
purchased from Immunotech (Marseille, France); and affinity-purified
F(ab')2 of goat anti-mouse Ig (GAM) purchased from
Cappel Laboratories (Malvern, PA).
The sPLA2 inhibitor p-bromophenacyl bromide (pBPB) and the PLD inhibitor 2,3-diphosphoglycerate (DPG) as well as purified bacterial PLD from Streptomyces chromofuscus (S. chromofuscus) were obtained from Sigma (St. Louis, MO).
Two differently radiolabeled phosphatidylcholine (PC) were used to
prepare PC vesicles for PLA2 assay; both were purchased
from DuPont-New England Nuclear (Boston, MA):
L--1-stearoyl-2-arachidonyl[arachidonyl-1-14C]PC
(sp. act., 56 mCi/mmol) and
L--dipalmitoyl-[choline-methyl-14C]PC (sp.
act., 56 mCi/mmol). Phosphatidylinositol bisphosphate
(PtdIns4, 5 P2; L--1-stearoyl-2-arachidonyl
[arachidonyl-1-14C]PtdIns; sp. act., 20–50 mCi/mmol) and
inositol-1,4,5-trisphosphate (Ins1, 4, 5 P3) 3H
radioreceptor assay kit were also obtained from DuPont-New
England Nuclear (Boston, MA).
RIA kits for leukotriene (LT) C4/D4/E4, LTB4, and platelet-activating factor (PAF) as well as [9,10-3H]oleic acid were obtained from Amersham (Aylesbury, U.K.).
Preparation of human NK cell cultures
NK cells were obtained by coculturing nylon nonadherent human PBMC from buffy coats (4 x 105/ml) with irradiated (3000 rad) RPMI 8866 cells (1 x 105/ml) at 37°C in a humidified 5% CO2 atmosphere for 10 days as previously described 26 . On day 10 the cell population was routinely 80–90% CD56+CD16+CD3-CD14- as assessed by immunofluorescence and cytofluorometric analysis. In some experiments contaminating T cells were eliminated by negative panning on plastic dishes. Anti-CD5-pretreated cells (10–20 x 106) were added to plastic petri dishes coated with GAM (10 µg/ml) and incubated at 4°C for 2 h. The nonadherent cells were gently poured off. The resulting NK cell population was >90% CD16+CD56+CD3-CD14- as assessed by immunofluorescence and cytofluorometric analysis. NK cell populations containing no more than 2% contaminating CD16-CD56-CD3+ cells were used for the present experiments.
PLD assay
Human NK cells (500 x 106) were preincubated with 1 mCi of [3H]oleic acid in serum-free medium for 2 h at 37°C and then washed three times with medium containing 0.01% BSA. Radiolabeled cells (10 x 106) were incubated with saturating concentrations of anti-CD16 (B73.1), anti-CD56 (B159.5.2), anti-MHC class I (W6/32), or anti-integrin (4B4, TS2/16, HP2/1, SAM-1) mAbs for 15 min at 37°C in RPMI 1640 medium, then washed and incubated with GAM (1 µg/106 cells) for 5 min at 37°C in medium containing 0.5% ethanol. Stimulation was stopped by the addition of 2 vol of ice-cold methanol. In some experiments cells were treated with pBPB (10–20 µM), DPG (2.5–10 mM), or wortmannin (0.3–300 nM) for 15 min at 37°C before stimulation. Lipids were extracted as described by Bligh and Dyer 32 and were separated by TLC with a mobile phase system consisting of chloroform/pyridine/formic acid (50/30/7). Standards were chromatographed in parallel. TLC plates were then autoradiographed, and the phospholipid bands of interest were scraped and counted in a beta counter. PLD activity was quantitated by measuring the production of phosphatidylethanol (PEt) and was expressed as the percent increase above the basal level of untreated samples.
PI-PLC and PLA2 assays
Human NK cells (10 x 106) were incubated with saturating concentrations of anti-CD16 (B73.1), anti-CD56 (B159.5.2), anti-MHC class I (W6/32), or anti-integrin (4B4, TS2/16, HP2/1, SAM-1) mAbs for 15 min at 37°C in RPMI 1640 medium. Cells were washed and incubated with GAM (1 µg/106 cells) for different time periods at 37°C in RPMI 1640 medium. Stimulation was stopped by the addition of ice-cold medium and centrifugation at 500 x g for 1 min at 4°C. In some experiments, cells were treated with pBPB (10–20 µM) or DPG (2.5–10 mM) for 15 min at 37°C before stimulation. Supernatants were collected and assayed for the presence of sPLA2 activity; sPLA2 protein release was assessed by ELISA, and the production of peptido-LT, LTB4, and PAF was tested by RIA. Cell pellets were resuspended in 50 mM Tris-HCl buffer, pH 8.5, containing 10 µM PMSF, 100 µM bacitracin, 1 mM benzamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml soybean trypsin inhibitor. Cells were lysed by sonication, and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Sixty to one hundred micrograms of the whole cell lysate (or an appropriate amount of supernatant) was added to 250 µl of reaction buffer (50 mM Tris-HCl (pH 8.5), 5 mM CaCl2, 5 mM MgCl2, and 0.1% fatty acid-free BSA) containing 1 µM radiolabeled PC vesicles (prepared by sonication in 50 mM Tris-HCl, pH 8.5, in an ice bath for 5 min at 5 W and 80% output) and incubated at 37°C for 1 h. The reaction was stopped by the addition of 250 µl of chloroform/methanol/acetic acid (4/2/1). Two hundred and fifty microliters of H2O, 250 µl of CHCl3, and 100 µl of 4 M KCl were added, and the mixture was centrifuged at 4000 rpm for 5 min to separate the organic from the aqueous phase. The former was dried under nitrogen, resuspended in 200 µl of chloroform, and applied to a silica gel TLC plate (Merck, Darmstadt, Germany) in duplicate with an automatic applicator (Linomat IV, Camag, Muttenz, Switzerland). Samples were chromatographed in chloroform/methanol/acetic acid/water (100/60/16/8) to separate the major products of PLA2 activity, i.e., arachidonic acid (AA) and lyso-PC. Standards were chromatographed in parallel. The radioactive spots were visualized by autoradiography, scraped from the plate, and counted in a beta counter. PLA2 activity was quantitated by the release of AA or lyso-PC from PC and was expressed as the percent increase above the basal level of untreated samples. In some experiments cell lysates were pretreated with the reducing agent DTT (Sigma) for 15 min at 4°C before performing the assay.
PLC activity was assayed using radiolabeled PC or PtdIns4, 5 P2 vesicles as substrate in 50 mM Tris-HCl buffer, pH 7.0, containing 10 µM PMSF, 100 µM bacitracin, 1 mM benzamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml soybean trypsin inhibitor. Assay conditions were the same as those described for PLA2 activity measurement. To separate the parent phospholipid from PLC activity products, such as DAG, the organic phase was chromatographed in petroleum ether/diethyl ether/acetic acid (70/30/1). PLC activity was quantitated by the release of DAG from PC or PtdIns4, 5 P2 and was expressed as the percent increase above the basal level of untreated samples. Ins1, 4, 5 P3 production was measured by competitive radiobinding assay kit.
Serine protease secretion assay
Purified anti-CD16 (B73.1), anti-CD56 (B159.5.2) or anti-ß1 (4B4) mAbs and ECM proteins (human plasma fibronectin (FN; Life Technologies, Grand Island, NY), and its 120- and 40-kDa proteolytic fragments (Chemicon, Temecula, CA) were coimmobilized in flat-bottom tissue culture 24-well plates (Costar, Cambridge, MA) by overnight incubation at 4°C. Plates were washed twice with cold PBS and blocked with PBS containing 1% BSA for 30 min at room temperature. Two hundred and fifty microliters of an NK cell suspension (8 x 106 cells/ml) in RPMI 1640, 10 mM HEPES, and 1 mg/ml BSA were then plated for 5 h at 37°C, and supernatants were recovered by centrifugation at 100 x g for 5 min. In some experiments, cells were treated with DPG, ethanol, or wortmannin for 15 min at 37°C before being plated. Inhibitors were also present throughout the test and did not affect cell viability, as tested by trypan blue exclusion. In other experiments purified bacterial PLD from S. chromofuscus (25–100 U/ml) was added to the cell suspension immediately before seeding.
Lymphocyte-specific serine protease activity in the supernatants was
measured using the
N--benzyloxycarbonyl-L-lysine thiobenzyl
ester (BLT; Calbiochem, La Jolla, CA) synthetic substrate as previously
described 22 . Results were expressed as a percentage of the total
cellular enzyme content after subtracting the spontaneous release,
which did not exceed 15%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent evidence has shown that ß1 integrin ligation
inhibits CD3-induced T cell proliferation by interfering with membrane
phospholipid metabolism, namely by decreasing PA and DAG production
5 . Since CD16 ligation stimulates a PLD activity that results in PA
production 21 , we investigated whether ß1 engagement
might modulate CD16-induced PLD activation in human NK cells. To this
purpose, [3H]oleic acid-radiolabeled human NK cells were
incubated with saturating doses of anti-CD16 (B73.1) and
anti-ß1 (4B4) mAb, alone or in combination, and then
stimulated by GAM cross-linking in the presence of ethanol. After
stimulation, radiolabeled PEt production was analyzed as a measure of
PLD activity in vivo. As shown in Fig. 1
,
CD16-induced PEt production was completely inhibited by
ß1 integrin simultaneous cross-linking, whereas
anti-CD56 (B159.5.2) control mAb did not exert any effect. Similar
results were observed using the TS2/16 mAb, recognizing a different,
adhesion-stimulating, ß1 epitope (data not shown).
Anti-5 (SAM-1) and, to a lesser extent,
anti-4 (HP2/1) mAb exerted a similar inhibiting
effect on CD16-stimulated PEt production (Fig. 1). PEt production in
cells stimulated with GAM cross-linked anti-integrin mAbs alone was
superimposable to that observed in cells stimulated with anti-CD56
control mAb alone (data not shown).
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ß1 integrin cross-linking inhibits CD16-induced sPLA2, but not cPLA2 nor PI-PLC, activity in human NK cells
Since CD16 engagement triggers the activation of other enzymatic
activities involved in phospholipid turnover, such as PI-PLC and both
cytosolic and secretory PLA2 19, 20, 22 , we then
investigated whether the ß1 integrin inhibitory effect
was selective for PLD or affected other phospholipases. Human NK cells
were stimulated with anti-CD16 mAb plus GAM in the presence or the
absence of simultaneous cross-linking with anti-ß1 or
anti-CD56 control mAb; PI-PLC and cPLA2 enzymatic
activities in the cell lysates were then tested by measuring DAG or AA
release from radiolabeled PtdIns 4, 5 P2 or arachidonyl-PC
vesicles, respectively; in addition, Ins 1, 4, 5 P3
production was measured by competitive radioreceptor binding assay. As
shown in Fig. 2, neither DAG release nor
Ins1, 4, 5 P3 production stimulated by CD16 engagement was
affected by simultaneous ß1 cross-linking, suggesting
that CD16-induced PI-PLC activation is not modulated by
ß1 integrins. Similarly, ß1 integrin
coengagement did not affect CD16-stimulated DTT-insensitive AA release,
indicating that CD16-mediated cPLA2 activation is not
influenced by ß1 integrin ligation (Fig. 3A). PI-PLC activity and
Ins1, 4, 5 P3 generation as well as cPLA2
activity in cells stimulated with GAM cross-linked
anti-ß1 mAb alone were superimposable to those
observed in cells stimulated with anti-CD56 control mAb alone (data
not shown).
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B, ß1 integrin cross-linking abolished both
extracellular and cell-associated sPLA2 enzymatic
activities induced by CD16 engagement, whereas simultaneous stimulation
with the anti-CD56 control mAb had no effect. Similar results were
obtained using a different anti-ß1 (TS2/16) mAb (data
not shown). CD16-induced sPLA2 inhibition was also observed
using anti-5 (SAM-1) or, to a lesser extent,
anti-4 (HP2/1) mAbs, suggesting a role for both FN
receptors (Fig. 3B). Secretory PLA2 activity in
cells stimulated with GAM cross-linked anti-integrin mAbs alone was
superimposable to that observed in cells stimulated with anti-CD56
control mAb alone (data not shown).
We then measured by ELISA the amount of sPLA2 protein
secreted in the extracellular medium of stimulated cells and found that
simultaneous cross-linking with anti-ß1 (4B4) mAb
profoundly inhibited CD16-stimulated sPLA2 release in the
supernatant (Fig. 3C), suggesting that the inhibition of
enzymatic activity might be due to the inhibition of protein secretion
in the extracellular compartment.
We have recently demonstrated that both cPLA2 and
sPLA2 contribute to CD16-induced PAF and AA metabolite
generation in human NK cells in the early (1–5 min) and the late
(5–40 min) phases, respectively 22 . Consistent with a selective
inhibition of the sPLA2 enzyme, ß1 integrin
cross-linking partially inhibited CD16-induced peptido-LT
production at 5 min and prevented further accumulation at later time
points (10 min) as assessed by RIA (Fig. 3D). A similar
degree of ß1-mediated inhibition was also observed for
CD16-stimulated PAF and LTB4 production (data not shown).
Taken together, these data indicate that ß1 integrin
engagement in human NK cells selectively inhibits CD16-stimulated PLD
activation as well as sPLA2 protein secretion and enzymatic
activity, leaving unaffected CD16-dependent PI-PLC or cPLA2
activation. Both 4ß1 and
5ß1 FN receptors expressed on NK cells are
involved in this process. Moreover, sPLA2 inhibition
results in the partial inhibition of CD16-stimulated peptido-LT
production.
CD16-stimulated sPLA2 secretion and enzymatic activity are dependent on PLD activation in human NK cells
We then investigated whether there was a relationship between PLD
and sPLA2 inhibition mediated by ß1
integrins. To this purpose, we stimulated human NK cells with
anti-CD16 (B73.1) mAb plus GAM in the presence or the absence of
selective pharmacological inhibitors of either sPLA2
(pBPB) 22 or PLD (DPG) 33, 34 . After
stimulation, both enzymatic activities were tested as described above.
Secretory PLA2 inhibition by pBPB had no
detectable effect on CD16-triggered PLD activation (data not shown).
Conversely, competitive inhibition of PLD activity by DPG inhibited
both sPLA2 protein release in the supernatant (Fig. 4A) and its enzymatic activity
(Fig. 4B), in either cell-associated or extracellular form,
in a dose-dependent manner, suggesting that CD16-mediated PLD
activation might be upstream of sPLA2 release and
activation. These results were further confirmed by the use of ethanol,
which diverts PA generation to the production of the inactive
metabolite PEt; as in the case of DPG, ethanol inhibited
sPLA2 protein secretion as well as its extracellular or
cell-associated enzymatic activity (data not shown).
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PLD involvement in CD16-induced NK cell granule exocytosis
PLD enzymatic activity has been implicated in membrane trafficking
and granule secretion in several cell systems 35 ; in addition, we
have observed that PLD inhibition results in the inhibition of
CD16-stimulated sPLA2 protein secretion in human NK cells.
Thus, we investigated whether PLD enzymatic activity might be involved
in CD16-stimulated NK cell degranulation. To this purpose, human NK
cells were incubated with either DPG, a competitive inhibitor of PLD,
or wortmannin, a fungal metabolite that inhibits PLD activation 36, 37 , and were stimulated with anti-CD16 mAb cross-linking. PLD
enzymatic activity and BLT-esterase release in the supernatant of
stimulated cells were then analyzed. In experiments dealing with
granule exocytosis, NK cells were stimulated by plastic-immobilized
anti-CD16 mAbs, since GAM cross-linked mAbs were unable to induce
detectable degranulation (data not shown). As shown in Fig. 5, DPG inhibited both PLD enzymatic
activity and BLT-esterase release in a dose-dependent fashion; PLD
enzymatic activity was completely inhibited at a DPG concentration of 5
mM, while BLT-esterase release was inhibited by 25% at a DPG
concentration of 5 mM and almost completely at 10 mM. Similarly,
wortmannin completely inhibited PLD activation at a concentration of 30
nM, while complete inhibition of BLT-esterase release required a higher
inhibitor concentration (300 nM). These data suggest that PLD
activation plays an important, although not exclusive, role in
CD16-triggered NK cell degranulation. These results were further
confirmed by the observation that ethanol, which inhibits the PLD
pathway by leading to the production of the "false product" PEt
instead of PA, also inhibited CD16-stimulated BLT-esterase release in a
dose-dependent fashion (data not shown).
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ß1 integrin cross-linking inhibits CD16-stimulated, but not PMA/ionomycin-stimulated, BLT-esterase release from human NK cells
Recent evidence has shown that ECM components are able to modulate
receptor-stimulated granule exocytosis in several cell systems 3 . The
above reported evidence of a ß1 integrin-mediated
inhibition of a biochemical event (PLD activation) involved in
CD16-stimulated NK cell degranulation prompted us to investigate
whether ß1 integrin ligation might modulate
CD16-dependent granule exocytosis. To this purpose, human NK cells were
stimulated by plastic-coimmobilized anti-CD16 (B73.1) and
anti-ß1 (4B4) mAbs. After stimulation, BLT-esterase
enzymatic activity in the supernatant was quantified as a measure of
granule content secretion. As shown in Fig. 6A, ß1 integrin
coengagement partially inhibited CD16-induced degranulation, whereas
anti-CD56 (B159.5.2) control mAb costimulation did not affect
CD16-dependent secretion. More importantly,
4ß1 and 5ß1
receptor engagement by plastic-immobilized natural ligands, either
human plasma FN or its proteolytic fragments (40 and 120 kDa), resulted
in a similar inhibition of CD16-induced NK cell degranulation (Fig. 6B). BLT esterase release from cells stimulated with
plastic-immobilized anti-ß1 mAb or FN alone was
superimposable to that obtained in cells stimulated with anti-CD56
control mAb or BSA alone (data not shown).
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C, no difference in PMA and/or ionomycin-stimulated
BLT-esterase release was observed in the presence of
plastic-immobilized anti-ß1 or anti-CD56 control
mAb.
To further strengthen the correlation between ß1
integrin-induced inhibition of both PLD and granule exocytosis, we
investigated whether exogenously added PLD might restore lytic granule
secretion in NK cells stimulated by CD16 and ß1
simultaneous cross-linking. To this purpose, human NK cells were
stimulated with plastic-coimmobilized anti-CD16 (B73.1) and
anti-ß1 (4B4) mAbs in the presence or the absence of
escalating doses of purified bacterial PLD from S.
chromofuscus. As shown in Fig. 6D, CD16-stimulated
BLT-esterase release was inhibited by 30% by ß1
simultaneous cross-linking and was returned to normal levels in the
presence of exogenously added purified PLD, suggesting that
ß1-mediated PLD inhibition may be responsible for the
observed decrease in granule exocytosis. Exogenous PLD addition to
unstimulated cells did not cause BLT esterase release above the basal
level (data not shown).
Taken together, these data indicate that ß1 integrin simultaneous cross-linking selectively inhibits CD16-mediated granule exocytosis in human NK cells without affecting PMA- and/or ionomycin-induced degranulation. Moreover, reversal of this effect by exogenous PLD suggests that the ß1 integrin-mediated decrease in granule exocytosis may be attributable to endogenous PLD inhibition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we report the novel finding that cross-linking of ß1
integrins, namely 4ß1 and
5ß1 FN receptors, specifically inhibits
CD16-stimulated PLD activation in human NK cells. Moreover, our data
indicate that PLD inhibition by either ß1 integrin
engagement or pharmacological agents functionally results in the
inhibition of CD16-elicited NK cell granule exocytosis. To our
knowledge this is the first report of a role for PLD in NK cell
degranulation. Bonnema et al. have previously demonstrated that
cytotoxic granule secretion triggered via CD16 is inhibited by
wortmannin, a fungal metabolite that inhibits PI 3-kinase by binding
irreversibly to its p110 catalytic subunit 38 . Wortmannin, however,
has been used extensively in other cell systems to inhibit PLD
activation induced by several stimuli 36, 37 , and we have
demonstrated that it also inhibits CD16-stimulated PLD activation in
human NK cells (Fig. 5C). On these bases, we can hypothesize
that PLD might function as a downstream effector of PI 3-kinase along
the signaling pathway that links CD16 engagement to the release of NK
cytotoxic granules. Consistent with a specific interference of
integrin-generated signals on a CD16-triggered biochemical pathway that
involves both PI 3-kinase and PLD, ß1 integrin
cross-linking did not affect PMA- and/or ionomycin-induced secretion,
which has been demonstrated to be protein kinase C dependent and PI
3-kinase independent 38 . Although the molecular mechanisms by which
PI 3-kinase may control PLD activity are presently unknown, the recent
observation that PtdIns3, 4, 5 P3 directly stimulates the
activity of cloned, purified PLDs provides a reasonable link between
these two pathways 39 .
Several lines of evidence imply that PLD activation plays a pivotal role in agonist-dependent secretion in different cell types. PLD activation and exocytosis share common requirements, and alterations in PLD regulation are linked to resistance of the secretory pathway to protein traffic-disrupting agents, such as brefeldin A 40 . More direct approaches, using primary alcohols to divert PA generation into PEt production and exogenous PLD to increase PA levels, have also provided evidence that PLD and its product PA are involved in transport vesicle assembly and secretion in several cell types 35, 41, 42, 43 .
CD16-induced sPLA2 activity was also inhibited upon
ß1 integrin simultaneous cross-linking, functionally
resulting in a decrease in peptido-LT, LTB4, and PAF
production. Since pharmacological inhibition of PLD activity also
resulted in sPLA2 inhibition, it is conceivable that in
CD16-stimulated NK cells ß1 integrin-mediated inhibition
of sPLA2 may be a consequence of PLD inhibition. Although
the molecular mechanisms by which PLD may control sPLA2
activation in human NK cells are presently unknown, several lines of
evidence indicate that a secretory event may play a pivotal role in
this process. PLD inhibition by either ß1 integrin
engagement or pharmacological agents results in the parallel inhibition
of both sPLA2 protein secretion and enzymatic activity;
since enzyme release in the extracellular medium, where it finds
millimolar concentrations of Ca2+, may be per se sufficient
for its activation 44 , the inhibition of sPLA2 release
may explain the observed inhibition of its enzymatic activity. In
addition, although sPLA2 storage in NK granules has not
been demonstrated, enzyme secretion parallels granule exocytosis 22, 45 , and enzymatic activity is inhibited by several agents that
interfere with NK cell degranulation with different mechanisms, such as
the PI 3-kinase inhibitor wortmannin, the mitogen-activated protein
kinase kinase inhibitor PD 098059, or PLD inhibitors 22 (M. Milella
et al., unpublished observations; Fig. 4).
Together, our data provide evidence that simultaneous cross-linking of ß1 integrins results in a decrease in CD16-triggered NK cell granule exocytosis due to the selective inhibition of a PLD-dependent pathway. It is worth noting that while granule exocytosis was only observed after stimulation with plastic-immobilized anti-CD16 mAbs, PLD activation occurred with either GAM cross-linked or plastic-immobilized mAbs. However, ß1 integrin coengagement inhibited PLD activation in both conditions (M. Milella, unpublished observations). In addition, inhibition of degranulation required the coimmobilization of both anti-CD16 and anti-ß1 mAbs on the same plastic surface, since pretreatment with soluble or GAM cross-linked anti-ß1 mAb did not modify the ability of plastic-immobilized CD16 to induce granule exocytosis (data not shown).
Interestingly, NK cell adhesion to FN or its 120- and 40-kDa fragments
exerted a similar inhibitory effect on degranulation (Fig. 6B), further adding to the physiological relevance of these
findings. Similar results have been obtained in human eosinophils,
whose adhesion to laminin- and FN-coated wells inhibits degranulation
in a concentration-dependent and secretagogue-specific manner 13 .
Conversely, FN has been shown to costimulate TCR-induced degranulation
in mouse CTL clones 46, 47, 48 . One possible explanation for these
discrepant findings is that in such an experimental system adhesion to
FN was mediated by the vß3 vitronectin
receptor, as suggested by the blocking effect of the RMV-7 mAb 46 .
In previous studies we demonstrated that ß1 integrin cross-linking on human NK cells enhances both natural and Ab-dependent cytotoxicity 28 . Although apparently discrepant, these findings might be explained by the existence of diverse cytotoxic mechanisms 49, 50 that can be differently regulated by integrins and suggest that integrins may interact with CD16 in several ways, contributing to the fine regulation of NK cell functions. An even more intriguing explanation is that the integrin-mediated attenuation of NK cell degranulation might be instrumental in the potentiation of their cytotoxicity. Indeed, after delivering the lethal hit to a susceptible target, an NK cell may recycle, become inactivated (due to depletion of critical effector molecules or postreceptor desensitization), or undergo activation-induced cell death 18 . Both inactivation and apoptosis decrease the ability of the affected NK cell to mediate subsequent killing of additional targets. Decreased degranulation might limit both effector molecule depletion and the release of granzymes, which are involved in the apoptotic process, thereby preserving NK cell recycling ability and potentiating their cytotoxicity. Preliminary data indicate that ß1 integrin cross-linking protects CD16-stimulated NK cells from apoptosis (S. Morrone, unpublished observations). Similarly, eosinophil adhesion to FN and laminin prolongs their in vitro survival while attenuating degranulation 13 .
The molecular mechanisms by which ß1 integrin
cross-linking might interfere with CD16-induced PLD activation remain
to be established. Although CD16-induced PLD activation in human NK
cells is strongly dependent upon extracellular Ca2+ and is
accompanied by polyphosphoinositide breakdown 21 , ß1
interference at these levels seems unlikely. Indeed, we have previously
demonstrated that ß1 integrins synergize with CD16 in the
induction of a Ca2+ influx from the extracellular
compartment 27 , and we provide here evidence that CD16-stimulated
PI-PLC enzymatic activity and Ins1, 4, 5 P3 generation are
not affected by ß1 simultaneous cross-linking (Fig. 2).
Several other biochemical events involved in the regulation of
mammalian PLD have been identified in different cell types, including
activation of protein kinase C isozymes, ADP-ribosylation factors, and
small G proteins, such as RhoA, Rac1, and Cdc42Hs 42, 43 . In
addition, both PtdIns4, 5 P2 and
PtdIns3, 4, 5 P3 strongly stimulate PLD by directly
activating the purified enzyme 39 . Most of these pathways share a
common molecular theme, which is the need for translocation to specific
membrane sites. Thus, a possible explanation for our findings is that
integrin-induced cytoskeletal reorganization may interfere with the
effective recruitment of specific signaling components involved in
CD16-triggered PLD activation. Consistent with a pivotal role of the
cytoskeleton in integrin-mediated negative signaling, Bathia et al.
have recently demonstrated that actin polymerization inhibitors, such
as cytochalasin D, completely reverse the anti-ß1
mAb-mediated inhibition of chronic myelogenous leukemia progenitor
proliferation 12 . In this regard we have recently demonstrated that
ß1 integrin engagement in human NK cells results in the
phosphorylation of both PYK-2 (belonging to the focal adhesion kinase
subfamily) and the PYK-2-associated cytoskeletal protein paxillin 30 .
The formation of multicomponent signaling complexes in parallel to the
structural complexes at the site of integrin adhesion may therefore be
relevant to ß1 integrin-mediated interference with
CD16-generated signals. This hypothesis is currently under
investigation.
In summary, our data indicate that in human NK cells ß1 integrins may interact with CD16-initiated pathways in different and complex ways depending on the specific function. We also present the first evidence of a role for PLD activation in the secretory pathway elicited by CD16 engagement in NK cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Angela Santoni, Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Viale Regina Elena 324, 00161 Rome, Italy. E-mail address:
3 Abbreviations used in this paper: ECM, extracellular matrix; PA, phosphatidic acid; DAG, diacylglycerol; PI 3-kinase, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLD, phospholipase D; PLA2, phospholipase A2; PYK-2, proline-rich tyrosine kinase-2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; GAM, F(ab')2 of goat anti-mouse Ig; pBPB, p-bromophenacyl bromide; DPG, 2,3-diphosphoglycerate; PC, phosphatidylcholine; PtdIns(4,5)P2, phosphatidylinositol bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol trisphosphate; Ins(1,4,5)P3, inositol trisphosphate; LT, leukotriene; PAF, platelet-activating factor; PEt, phosphatidylethanol; AA, arachidonic acid; FN, fibronectin; BLT, N--benzyloxycarbonyl-L-lysine thiobenzyl ester.
Received for publication August 7, 1998. Accepted for publication November 9, 1998.
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