β1 Integrin Cross-Linking Inhibits CD16-Induced Phospholipase D and Secretory Phospholipase A2 Activity and Granule Exocytosis in Human NK cells: Role of Phospholipase D in CD16-Triggered Degranulation

Michele Milella, Angela Gismondi, Paola Roncaioli, Gabriella Palmieri, Stefania Morrone, Mario Piccoli, Luigi Frati, Maria Grazia Cifone and Angela Santoni
J Immunol February 15, 1999, 162 (4) 2064-2072;

Abstract

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of β1 integrins, namely α4β1 and α5β1 fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells. β1 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 β1 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.

Integrins are a superfamily of αβ heterodimeric adhesion receptors involved in both cell-cell and cell-extracellular matrix (ECM)3 interactions. Members belonging to the same family share a common β subunit paired with different α-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-β1 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 (FcγRIIIA), 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 β1 family of integrins, which have been suggested to play a major role in the regulation of their migration and functions 24, 25, 26, 27 . β1 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-γ production 28, 29, 30, 31 . Moreover, β1 integrin cross-linking by either mAbs or natural ligands costimulates NK cytotoxic functions 28 .

Here we provide evidence that β1 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.

Materials and Methods

Abs and reagents

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-β1 integrin subunit (4B4, purchased from Coulter Immunology (Hialeah, FL), and TS2/16, provided by Dr. M. Hemler, Dana-Farber Cancer Institute (Boston, MA)); anti-α4 (HP2/1) and anti-α5 (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-α-1-stearoyl-2-arachidonyl[arachidonyl-1-14C]PC (sp. act., 56 mCi/mmol) and l-α-dipalmitoyl-[choline-methyl-14C]PC (sp. act., 56 mCi/mmol). Phosphatidylinositol bisphosphate (PtdIns4, 5 P2; l-α-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 × 105/ml) with irradiated (3000 rad) RPMI 8866 cells (1 × 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 × 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 × 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 × 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 × 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 × 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-β1 (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 × 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 × 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-α-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%.

Results

β1 integrin cross-linking inhibits CD16-induced PLD activation in human NK cells

Recent evidence has shown that β1 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 β1 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-β1 (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 β1 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, β1 epitope (data not shown). Anti-α5 (SAM-1) and, to a lesser extent, anti-α4 (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).

FIGURE 1.
FIGURE 1.

α4β1 and α5β1 integrin cross-linking inhibits CD16-induced PLD activation in human NK cells. [3H]oleic acid-radiolabeled NK cells were stimulated with saturating doses of anti-CD16, anti-CD56, anti-β1, anti-α4, and anti-α5 mAbs, either alone or in combination, followed by cross-linking with GAM for 5 min in the presence of 0.5% ethanol. Lipids were then separated by TLC and the percentage of counts per minute in PEt with respect to the total counts per minute in phospholipids was quantified by liquid scintillation counting as a measure of PLD activity. Results are expressed as the percent increase in PEt production above the basal level of untreated samples (0.4 pmol/mg protein). Results are representative of one of four independent experiments.

Overall, these data suggest that the engagement of both FN receptors expressed by NK cells down-modulates CD16-triggered PLD activity.

β1 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 β1 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-β1 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 β1 cross-linking, suggesting that CD16-induced PI-PLC activation is not modulated by β1 integrins. Similarly, β1 integrin coengagement did not affect CD16-stimulated DTT-insensitive AA release, indicating that CD16-mediated cPLA2 activation is not influenced by β1 integrin ligation (Fig. 3⇓A). PI-PLC activity and Ins1, 4, 5 P3 generation as well as cPLA2 activity in cells stimulated with GAM cross-linked anti-β1 mAb alone were superimposable to those observed in cells stimulated with anti-CD56 control mAb alone (data not shown).

FIGURE 2.
FIGURE 2.

β1 integrin cross-linking does not affect CD16-stimulated PI-PLC enzymatic activity or Ins(1,4,5)P3 generation in human NK cells. Cells were stimulated with saturating doses of anti-CD16, anti-β1, and anti-CD56 control mAb, either alone or in combination, followed by cross-linking with GAM for 1 min, and PI-PLC enzymatic activity and Ins(1,4,5)P3 generation were then tested. A, PI-PLC activity in the cell lysate, evaluated as DAG release from radiolabeled PtdIns(4,5)P2 vesicles. B, Ins(1,4,5)P3 production, measured by competitive radiobinding assay. Results are expressed as the percent increase in DAG release from PtdIns(4,5)P2 vesicles above the basal level of untreated samples (1.9 μmol/mg protein/h; A) or net Ins(1,4,5)P3 production ± SE (picomoles per 106 cells) after subtracting the basal level of untreated samples (0.8 pmol/106 cells; B). Results are representative of one of three independent experiments.

FIGURE 3.
FIGURE 3.

Effects of β1 integrin cross-linking on CD16-stimulated cPLA2 and sPLA2 enzymatic activity, sPLA2 protein release, and peptido-LT production. Human NK cells were stimulated with saturating doses of anti-CD16, anti-CD56, anti-β1, anti-α4, and anti-α5 mAbs, either alone or in combination, followed by cross-linking with GAM. Cell lysates were then tested for PLA2 enzymatic activity, and supernatants were assayed for sPLA2 protein release and enzymatic activity as well as for peptido-LT (LTC4, -D4, and -E4) production. A, cPLA2 activity in DTT-pretreated cell lysates after 1 min of stimulation, evaluated as AA release from radiolabeled arachidonyl-PC vesicles. B, sPLA2 activity in cell lysates (Pellet) and supernatants (SN) after 5 min of stimulation, evaluated as lyso-PC release from radiolabeled dipalmitoyl-PC vesicles. C, sPLA2 protein release in the supernatant, evaluated by ELISA. D, Peptido-LT accumulation in the supernatant, evaluated by RIA. Results are expressed as the percent increase in AA or lyso-PC release from PC vesicles above the basal level of untreated samples (2 and 4 pmol/mg protein/h for AA and lyso-PC, respectively; A and B), micrograms of sPLA2 protein per 100 μg of protein in the cell lysate after subtracting the basal release of untreated samples (1.5 μg/100 μg protein; C), and picograms of peptido-LTs per milligram of protein in the cell lysate (D). Results are representative of one of three independent experiments.

We then tested the effect of β1 integrin coengagement on CD16-induced sPLA2 activity. Human NK cells were incubated with anti-CD16 (B73.1) or anti-β1 (4B4) mAb, either alone or in combination, and then stimulated with GAM. Secretory PLA2 enzymatic activity was then tested in both the supernatant and the cell lysate by measuring DTT-sensitive lyso-PC release from radiolabeled arachidonyl-PC vesicles. As shown in Fig. 3⇑B, β1 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-β1 (TS2/16) mAb (data not shown). CD16-induced sPLA2 inhibition was also observed using anti-α5 (SAM-1) or, to a lesser extent, anti-α4 (HP2/1) mAbs, suggesting a role for both FN receptors (Fig. 3⇑B). 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-β1 (4B4) mAb profoundly inhibited CD16-stimulated sPLA2 release in the supernatant (Fig. 3⇑C), 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, β1 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. 3⇑D). A similar degree of β1-mediated inhibition was also observed for CD16-stimulated PAF and LTB4 production (data not shown).

Taken together, these data indicate that β1 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 α4β1 and α5β1 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 β1 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. 4⇓A) and its enzymatic activity (Fig. 4⇓B), 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).

FIGURE 4.
FIGURE 4.

PLD involvement in CD16-stimulated sPLA2 protein release and enzymatic activity in human NK cells. Cells were pretreated with DPG for 15 min and stimulated with saturating doses of anti-CD16 or anti-CD56 control mAb followed by cross-linking with GAM for 10 min, and both sPLA2 protein release and enzymatic activity were then tested. A, sPLA2 protein release in the supernatant, evaluated by ELISA. B, sPLA2 activity in cell lysates (Pellet) and supernatants (SN), evaluated as lyso-PC release from radiolabeled dipalmitoyl-PC vesicles. Results are expressed as micrograms of sPLA2 protein per 100 μg of protein in the cell lysate after subtracting the basal release of untreated samples (1.2 μg/100 μg protein; A) and the percent increase in lyso-PC release from PC vesicles above the basal level of untreated samples (3.5 pmol/mg protein/h; B). Results are representative of one of three independent experiments.

Together, these data indicate that CD16-induced sPLA2 release and activation in human NK cells are PLD dependent and suggest that β1 integrin-mediated inhibition of CD16-stimulated sPLA2 secretion and activity might be secondary to PLD inhibition.

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).

FIGURE 5.
FIGURE 5.

Effect of PLD inhibition on CD16-stimulated PLD activity and BLT-esterase release in human NK cells. Human NK cells, radiolabeled (A and C) or not (B and D) with [3H]oleic acid, were pretreated with the indicated dose of either DPG or wortmannin for 15 and 30 min, respectively. A and C, PLD activity, evaluated as radioactive PEt production after 5 min of stimulation with GAM cross-linked anti-CD16 (solid circles) or anti-CD56 control mAb (open circles); results are expressed as indicated in Fig. 1⇑. B and D, BLT-esterase activity released in the supernatant after 5 h of stimulation with plastic-immobilized anti-CD16 (solid circles) or anti-CD56 control mAb (open circles), assessed by colorimetric assay. Results are expressed as the percentage ± SE of total cellular BLT-esterase content released in the supernatant after subtracting the basal release of untreated samples. Results are representative of one of three independent experiments.

Taken together, these data indicate that a PLD-dependent pathway is involved in CD16-mediated NK cell granule exocytosis.

β1 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 β1 integrin-mediated inhibition of a biochemical event (PLD activation) involved in CD16-stimulated NK cell degranulation prompted us to investigate whether β1 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-β1 (4B4) mAbs. After stimulation, BLT-esterase enzymatic activity in the supernatant was quantified as a measure of granule content secretion. As shown in Fig. 6⇓A, β1 integrin coengagement partially inhibited CD16-induced degranulation, whereas anti-CD56 (B159.5.2) control mAb costimulation did not affect CD16-dependent secretion. More importantly, α4β1 and α5β1 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. 6⇓B). BLT esterase release from cells stimulated with plastic-immobilized anti-β1 mAb or FN alone was superimposable to that obtained in cells stimulated with anti-CD56 control mAb or BSA alone (data not shown).

FIGURE 6.
FIGURE 6.

β1 integrin-mediated inhibition of CD16-stimulated, but not of PMA/ionomycin-induced, NK cell degranulation and reversal by exogenous PLD addition. Human NK cells were stimulated with plastic-immobilized anti-CD16, anti-β1 or anti-CD56 control mAb, and ECM proteins (FN and its proteolytic fragments, 40 and 120 kDa), either alone or in combination (A, B, and D) or with PMA and ionomycin (Iono) at the indicated doses (C). After 5 h of incubation at 37°C, supernatants were tested for BLT esterase activity in a colorimetric assay. A, Inhibition of CD16-induced BLT esterase release by escalating doses of coimmobilized anti-β1 (solid circles) or anti-CD56 (open circles) control mAb. B, Inhibition of CD16-induced BLT esterase release by coimmobilized FN and its proteolytic fragments (40 and 120 kDa). C, Effect of anti-β1 (solid bars) or anti-CD56 (white hatched bars) control mAb on BLT esterase release from cells stimulated by either coimmobilized anti-CD16 or escalating doses of PMA and ionomycin. D, Anti-β1-mediated inhibition of BLT esterase release from cells stimulated by coimmobilized anti-CD16 in the presence of escalating doses of exogenously added purified PLD from S. chromofuscus; the indicated doses of PLD were added simultaneously to cell seeding on mAb-coated plates. Results are expressed as the percent ± SE of inhibition of CD16-stimulated BLT esterase release (A,B, and D) and the percentage ± SE of total cellular BLT esterase content released in the supernatant after subtracting the basal release of untreated samples (B). Results are representative of one of five independent experiments.

We then tested whether β1 integrin engagement selectively interfered with CD16-induced degranulation by using pharmacological stimuli, such as PMA and ionomycin, which stimulate NK cell secretion through pathways different from that elicited by CD16 38 . As shown in Fig. 6⇑C, no difference in PMA and/or ionomycin-stimulated BLT-esterase release was observed in the presence of plastic-immobilized anti-β1 or anti-CD56 control mAb.

To further strengthen the correlation between β1 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 β1 simultaneous cross-linking. To this purpose, human NK cells were stimulated with plastic-coimmobilized anti-CD16 (B73.1) and anti-β1 (4B4) mAbs in the presence or the absence of escalating doses of purified bacterial PLD from S. chromofuscus. As shown in Fig. 6⇑D, CD16-stimulated BLT-esterase release was inhibited by 30% by β1 simultaneous cross-linking and was returned to normal levels in the presence of exogenously added purified PLD, suggesting that β1-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 β1 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 β1 integrin-mediated decrease in granule exocytosis may be attributable to endogenous PLD inhibition.

Discussion

Although integrin-mediated negative regulation of specific cell functions has been reported in several cell systems 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , the molecular levels of interference between integrin- and other receptor-generated signals are not clearly established.

Here we report the novel finding that cross-linking of β1 integrins, namely α4β1 and α5β1 FN receptors, specifically inhibits CD16-stimulated PLD activation in human NK cells. Moreover, our data indicate that PLD inhibition by either β1 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. 5⇑C). 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, β1 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 β1 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 β1 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 β1 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 β1 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, β1 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-β1 mAbs on the same plastic surface, since pretreatment with soluble or GAM cross-linked anti-β1 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. 6⇑B), 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 αvβ3 vitronectin receptor, as suggested by the blocking effect of the RMV-7 mAb 46 .

In previous studies we demonstrated that β1 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 β1 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 β1 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 , β1 interference at these levels seems unlikely. Indeed, we have previously demonstrated that β1 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 β1 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-β1 mAb-mediated inhibition of chronic myelogenous leukemia progenitor proliferation 12 . In this regard we have recently demonstrated that β1 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 β1 integrin-mediated interference with CD16-generated signals. This hypothesis is currently under investigation.

In summary, our data indicate that in human NK cells β1 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.

Acknowledgments

We thank Dina Milana, Anna Maria Bressan, Patrizia Birarelli, Alessandro Procaccini, Antonio Sabatucci, and Gasperina De Nuntiis for their expert technical assistance.

Footnotes

  • ↵1 This work was supported by grants from the Italian Association for Cancer Research and the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (40% from MURST and 60% from Facoltà and Ateneo).

  • ↵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: asantoni{at}axcasp.caspur.it

  • ↵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-α-benzyloxycarbonyl-l-lysine thiobenzyl ester.

  • Received August 7, 1998.
  • Accepted November 9, 1998.

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The Journal of Immunology: 162 (4)
The Journal of Immunology
Vol. 162, Issue 4
15 Feb 1999
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