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*
Department of Experimental Medicine and Pathology, Istituto Pasteur-Fondazione Cenci Bolognetti, University "La Sapienza", Rome, Italy;
Biotechnology Section, Institute for the Study and Cure of Tumors, Genda, Italy;
Laboratory of Pathophysiology, Regina Elena Cancer Institute, Rome, Italy; and
§
Istituto Mediterraneo di Neuroscienze "Neuromed", Pozzilli, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII). In addition,
we demonstrate that both direct and Ab-mediated NK cell binding to
target cells result in the activation of nucleotide exchange on
endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide
treatment leads to an impairment of NK cytotoxicity, with
FcRIII-mediated killing being more sensitive to the abrogation of
Vav expression. These results provide new insight into the signaling
pathways leading to cytotoxic effector function and define a role for
Vav in the activation of NK cell-mediated
killing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTL- or NK cell-mediated cytotoxicity largely involves cytoskeleton redistribution, which consists of a polarized concentration of cytotoxic mediators and repositioning of the microtubule-organizing center 8, 9 . However, the signaling pathways leading to such cytoskeleton reorganization have not been elucidated yet.
Recently, Syk activation has been shown to be a crucial step in the NK cytotoxic response 10 . Intriguingly, Syk/Vav association and subsequent Vav tyrosine phosphorylation have been described in B cells in response to B cell receptor engagement 6 . Altogether, these data led us to hypothesize a functional involvement of Vav in the regulation of NK cell-mediated cytotoxicity.
We report here that stimulation of human NK cells with a panel of
sensitive targets or through the low-affinity Fc receptor for IgG
(FcRIII) (CD16) induces tyrosine phosphorylation of Vav that is
associated with Rac1 activation. The down-regulation of Vav protein
affects both natural and FcRIII-mediated cytotoxicity, suggesting
that Vav-regulated signals play a central role in the activation events
leading to the NK cell-mediated cytotoxic response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following human cell lines were used as targets: K562
(erythroleukemia), LOVO (colon carcinoma), and Mel 116 (breast cancer)
(kindly provided by Dr. M. Maio, Istituto Nazionale Tumori, Centro
Europeo, Aviano, Italy). The murine FcR+ P815
(mastocytoma) was used for reverse Ab-dependent cellular cytotoxicity
(ADCC).
Preparation of human NK cells
NK cell populations were obtained by a 10-day coculture of nylon nonadherent PBMCs (4 x 105 cells/ml) with irradiated (3000 rad) EBV+ RPMI 8866 lymphoblastoid cell line (105 cells/ml) as described previously 11 . On day 10, the cell population was routinely 80–95% CD56+, CD16+, CD3-, as assessed by immunofluorescence and cytofluorometric analysis. The experiments were performed on NK cell populations that were >90% pure.
Antibodies
Anti-human Vav and anti-phosphotyrosine (pTyr) (4G10) mAbs
were obtained from Upstate Biotechnology (Lake Placid, NY);
anti-human Syk (4D10) mAb and affinity-purified rabbit
anti-Rac1 Ab were supplied by Santa Cruz Biotechnology (Santa Cruz,
CA). B73.1 (anti-FcRIII) and W6/32 (anti-MHC class I) mAbs
were kindly provided by Dr. G. Trinchieri (Wistar Institute,
Philadelphia, PA).
Cytotoxicity assay
The 51Cr release assay was performed as described previously 12 . Maximal and spontaneous release were determined by incubating 51Cr-labeled target cells with 1 M HCl or medium alone, respectively. The percentage of specific lysis was determined as follows: ([mean cpm experimental release - mean cpm spontaneous release]/[mean cpm maximal release - mean cpm spontaneous release]) x 100.
[32P]orthophosphate labeling, cell stimulation, and immunoblotting
NK cells were starved for 4 h at 37°C in phosphate-free
RPMI 1640 medium (Life Technologies, Gaithersburg, MD) and
subsequently labeled (25 x 106 cells/ml) for 4 h
with [32P]orthophosphate (250 µCi/ml) (Amersham
International, Little Chalfont, U.K.) in phosphate-free RPMI 1640
medium. Cells were washed and resuspended (7 x 107
cells/sample) in prewarmed RPMI 1640 medium. Target cells were added
(E:T ratio of 2:1), pelleted at 5000 rpm for 5 s, and incubated at
37°C. For anti-FcRIII stimulation, cultured NK cells were
incubated at 4°C for 15 min with a saturating dose of B73.1 mAb,
washed, and used as described above. After stimulation, cells were
lysed with lysis buffer (1% v/v Triton X-100, 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, plus
protease and phosphatase inhibitors) and immunoprecipitated with
anti-Vav or W6/32 control mAb bound to protein G-Sepharose beads;
the precipitated proteins were separated by SDS-PAGE. Lysates from an
equivalent number of target and effector cells were combined before
immunoprecipitation and used as time 0 of the kinetics. In the
experiments using labeled NK cells, autoradiography was performed on
dried gels. The rehydrated gels were then transferred to nitrocellulose
membranes and probed with anti-Vav mAb. For experiments with
unlabeled NK cells, proteins separated by SDS-PAGE were transferred to
nitrocellulose membranes, probed with anti-pTyr mAb, and, after
stripping, probed with the anti-Syk mAb. Immunoreactivity was
detected using an enhanced chemiluminescence kit (Amersham
International).
In vivo nucleotide labeling of Rac1
NK cells were labeled for 3 h with [32P]orthophosphate and stimulated with target cells as described above. Stimulation was stopped by lysis in 50 mM HEPES (pH 7.4), 1% v/v Triton X-100, 100 mM NaCl, and 5 mM MgCl2, plus protease and phosphatase inhibitors. Postnuclear lysates were adjusted to a final concentration of 500 mM NaCl, 0.5% sodium deoxycholate, and 0.05% SDS and immunoprecipitated with anti-Rac1 Ab precoated on protein A-Sepharose beads for 45 min at 4°C. Immunocomplexes were washed and eluted in 5 mM DTT, 5 mM EDTA, O.2% SDS, 0.5 mM GTP, and 0.5 mM GDP at 68°C for 20 min. The nucleotides were separated on polyethyleneimine-cellulose F plates (Merck, Darmstadt, Germany) with 0.75 M KH2PO4 (pH 3.5). TLC plates were analyzed by autoradiography and quantitated by direct scanning for ß radiation using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Oligodeoxynucleotides (ODNs) and cell treatment
Antisense (AS) and complementary sense (S) ODNs that targeted the translation initiation region of the protooncogene vav were synthesized in fully phosphorothioated form. The sequence was as follows: AS-vav (5'-CATTGGCGCCACAGCTCCAT-3'), S-vav (5'-ATGGAGCTGTGGCGCCAATG-3'). Cultured NK cells (2.5 x 105 cells/ml) were exposed to ODNs (100 µg/ml) in heat-inactivated (65°C for 20 min) conditioned medium, derived from primary culture (see preparation of human NK cells), for 56 h. To analyze Vav expression, equivalent amounts of cell lysates from ODN-treated or untreated cells were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-Vav mAb. The same membrane was probed with anti-Syk mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII-mediated killing induce tyrosine
phosphorylation of Vav in human NK cellsAs PTK activity has been demonstrated to provide early and requisite signals for the development of NK cell-mediated cytotoxic functions 13 , we investigated whether Vav could be one of the substrates of PTK(s) triggered by the interaction of NK cells with the cell line K562, the prototype NK-sensitive target.
To analyze NK cell- but not target cell-derived Vav, cultured NK cells
were labeled with [32P]orthophosphate. In
target-stimulated NK cells, Vav immunoprecipitates show increased
32P-incorporation in a 95-kDa band corresponding to Vav, as
revealed by anti-Vav immunoblot (Fig. 1
A). The increase in Vav
phosphorylation peaks at 5 min and almost declines to baseline levels
by 20 min.
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RIII-mediated
cytotoxicity would also result in the stimulation of Vav
phosphorylation. 32P-labeled NK cells were challenged with
NK-resistant FcR+ murine mastocytoma P815 cells in the
presence of anti-FcRIII B73.1 mAb (reverse ADCC). A rapid
phosphorylation of Vav was observed when NK cells were allowed to
contact Ab-coated P815 cells; however, unlike natural killing,
FcRIII-induced Vav phosphorylation was more persistent (still
present at 20 min of stimulation). Low levels of Vav phosphorylation
were also induced by direct contact with P815 cells, which are barely
lysed by cultured NK cells (Fig. 1
B). No bands corresponding
to Vav were observed in samples immunoprecipitated with isotype-matched
control mAb (data not shown).
Recent reports have shown that the nucleotide exchange activity of Vav
is regulated by PTK-dependent tyrosine phosphorylation 4 . Thus, we
further determined whether NK cell Vav was undergoing phosphorylation
on tyrosine residues upon target cell binding. For this purpose, NK
cells were stimulated with LOVO or Mel 116 Vav-negative tumor cell
lines, and Vav was subsequently immunoprecipitated and immunoblotted
with anti-pTyr mAb. Upon target stimulation, NK cells show a marked
tyrosine phosphorylation of Vav (Fig. 2,
upper panels). Interestingly, a 70-kDa
tyrosine-phosphorylated protein was consistently coprecipitated with
Vav from target-stimulated but not unstimulated NK cell lysates (Fig. 2, upper panels). On the basis of the described Vav/Syk
association 6 and the involvement of Syk in the NK cytotoxicity
signaling pathway 10 , the same blot was reprobed with anti-Syk
mAb after stripping of the bound Abs. Fig. 2 (middle panels)
shows that Syk was present in anti-Vav immunoprecipitates from
target-stimulated NK cells. The anti-Vav blot (Fig. 2, lower
panels) shows the absence of Vav in the LOVO and Mel 116 cell
lines and an equivalent amount of Vav immunoprecipitated from
unstimulated and target-stimulated NK cells.
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Vav tyrosine phosphorylation results in a dramatic enhancement of
its guanine nucleotide exchange activity toward Rac1 4 . Therefore, we
analyzed whether Rac1 was activated upon NK/target cell contact. The
high intrinsic GTPase activity of Rac1 has prevented the detection of
its GTP-bound form in living cells. Thus for these experiments, we took
advantage of a recently described technique that evaluates Rac1
exchange activity by determining the levels of 32P-labeled
GDP bound to Rac1 after a brief exposure to
[32P]orthophosphate-containing medium 4, 14 .
Stimulating NK cells by direct contact with K562 target cells or
through FcRIII consistently increased the levels of radioactive
GDP bound to Rac1 (three- to eightfold). Under these experimental
conditions, low levels of labeled nucleotides were observed in
unstimulated NK cells (Fig. 3).
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The above data indicate that Vav undergoes tyrosine
phosphorylation upon NK cell stimulation by sensitive targets or
through FcRIII; the activation of Rac1 is also observed under the
same conditions.
To provide direct evidence of the functional requirement for Vav in the
generation of NK cytotoxicity, we attempted to perturb Vav expression
using AS-ODNs. NK cells were incubated with AS-ODNs, S-ODNs, or control
medium for 56 h, and Vav expression was analyzed by
immunoblotting. The viability of cells cultured in the presence of
AS-ODNs, S-ODNs, or medium was >90% as assessed by trypan blue
exclusion (data not shown). Fig. 4g shows that Vav was almost
undetectable in cells exposed to AS-ODNs compared with cells exposed to
S-ODNs or to untreated controls. The specificity of the AS-ODN effect
is shown by blotting the same membrane with anti-Syk mAb (Fig. 4h). Vav AS-ODN treatment markedly reduced
FcRIII-mediated cytotoxic activity (Fig. 4, a,
c, and e), whereas natural killing was less
sensitive to the effect of Vav down-regulation (Fig. 4, b,
d, and f). S-ODN-treated cells exhibit a level of
cytotoxicity that is comparable with that of untreated NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII by
the Fc portion of IgG bound to cell-associated Ags. "Natural
cytotoxicity" refers to the capacity of NK cells to directly interact
with tumor or virus infected cells by means of incompletely defined
receptors 15 . Thus far, the signaling events leading to NK
cytotoxic functions have been only partially characterized.
In this report we have addressed the role of the specific Rac1
activator, Vav, in the generation of NK cell cytotoxic activity. Our
findings show that the activation of NK cells through FcRIII or by
interaction with sensitive target cells results in the rapid
stimulation of Vav tyrosine phosphorylation. The Vav phosphorylation
stimulated by FcRIII triggering exhibited a more sustained kinetic
(still persisting at 20 min) with respect to that observed upon direct
NK/target cell contact. This difference can be explained by the ability
of the D3 phosphoinositide products of phosphatidylinositol-3 kinase to
regulate Vav phosphorylation 16 . In this regard, recent evidence
indicates a requirement for phosphatidylinositol-3 kinase in
FcRIII-mediated NK cytotoxicity but not in natural killing 17 .
Vav/Syk association leading to Vav tyrosine phosphorylation has been
described in B lymphocytes upon Ag receptor stimulation 6 . A recent
study has identified Syk as a tyrosine kinase that is essential for NK
cell cytotoxic activity 10 . Our results show that NK/target cell
interaction induces the formation of Vav/Syk complexes, suggesting that
Syk could be the PTK responsible for Vav phosphorylation and control
the recruitment of activated Vav in the area of NK/target cell contact.
However, we cannot rule out the possibility that PTKs other than Syk
may be involved in Vav tyrosine phosphorylation in response to
FcRIII or target stimulation.
Our study also provides evidence that Vav is required for NK
cell-mediated cytotoxic function. Vav AS-ODN-treated NK cells exhibited
a marked decrease in FcRIII-mediated killing, whereas natural
killing was affected to a much lesser extent. This observation could be
explained by discrete signaling pathways regulating these different
modalities of cytotoxicity 15 ; in this context, Rac1 exchange factors
other than Vav (Dbl, Ost, and Tiam1) may be involved 18 .
The dynamic organization of the actin cytoskeleton is an essential
requirement for cytotoxic activity 8, 9 ; recent reports
demonstrate that Vav functions by regulating the actin cytoskeleton 2, 3 . Vav-mediated regulation of the cytoskeleton may be achieved via its
guanine nucleotide exchange activity on the small G protein Rac1 4 .
Rac1-mediated control of macrophage-dependent phagocytosis, actin
polymerization in platelets, and mast cell degranulation has been
demonstrated recently 19, 20, 21, 22 . We show here that both
FcRIII-mediated and direct NK/target cell contacts induce GDP/GTP
exchange activity on endogenous Rac1.
In agreement with our data, a recent report describes the induction of Vav phosphorylation upon NK/target cell contact. Moreover, it provides genetic evidence that the Vav/Rac1 pathway regulates effector/target cell binding and cytotoxic granule polarization during cellular cytotoxicity 23 . The downstream effectors of Rac1 that are involved in the Vav-mediated control of cytotoxic activity still need to be defined. Rho family GTPases may control actin polymerization by binding and activating the enzyme phosphatidylinositol-4 phosphate 5-kinase (PI-5K), thus leading to phosphatidylinositol 4,5 bisphosphate generation 21, 24 . Interestingly, Vav-mediated PI-5K activation upon CD19 coreceptor engagement on B lymphocytes 25 has been described recently. Moreover, the action of phospholipase C on phosphatidylinositol 4,5 bisphosphate generates phosphatidylinositol 3,4,5 trisphosphate and diacylglycerol, which lead to protein kinase C activation and intracellular calcium mobilization, two signaling intermediates that play a central role in the development of NK cytotoxicity 15 . The involvement of PI-5K in the Vav-mediated regulation of cytotoxic activity is under investigation.
Our results provide insight into the molecular events that regulate cytotoxic function and identify a critical role for Vav in NK-mediated killing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ricciarda Galandrini, Department of Experimental Medicine and Pathology, University "La Sapienza", Viale Regina Elena 324, 00161 Rome, Italy. E-mail address:
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; ADCC, Ab-dependent cellular cytotoxicity; FcRIII, low-affinity Fc receptor for IgG; pTyr, phosphotyrosine; ODN, oligodeoxynucleotide; AS, antisense; S, complementary sense; PI-5K, phosphatidylinositol-4 phosphate 5-kinase.
Received for publication September 28, 1998. Accepted for publication December 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI to the Rac1-JNK pathway. J. Biol. Chem. 272:10751.RI-activated rat basophilic leukemia (RBL-2H3) cells. J. Cell Sci. 110:2215.[Abstract]
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), S-ODN-treated (
), or untreated (solid gray line) NK cells
were incubated for 4 h with 51Cr-labeled K562 cells
(b, d, and f) or
51Cr-labeled P815 cells in the presence of 1 µg/ml of the
anti-Fc