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*
Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Milan, Italy;
Laboratory of Clinical Pathology, National Institute for Cancer Research, Genoa, Italy; and
Laboratory of Immunopathology, National Institute for Cancer Research and Advanced Biotechnology Center, Genoa, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII (CD16)-initiated Ab-dependent cellular cytotoxicity
(ADCC),3 i.e., killing of
opsonized targets, and natural killing, i.e., the ability to lyse tumor
or virus-infected targets in the absence of Ab (5, 6, 7, 8, 9). Both mechanisms
are highly dependent on intracellular calcium rise. Indeed, one of the
earliest detectable events following CD16 ligation is an increase in
intracellular free calcium concentration
([Ca2+]i) (5, 7, 9). In addition, natural
cytotoxicity is initiated by adhesion receptors that drive NK cells and
their targets to close contact and trigger NK cell activation (5, 6, 7, 8, 9);
these receptors include the 
Lß2 integrin
(CD11a/CD18), which is known to elicit a
[Ca2+]i elevation, mainly due to
extracellular calcium influx (9, 10, 11). The rise in
[Ca2+]i is needed for the granule release
involved in the delivery of a lethal hit (7, 12).
The molecular structures mediating extracellular calcium influxes in NK
cells are still poorly characterized. Recently, the presence of
functional L-type calcium channels, whereby extracellular calcium can
enter the cell, has been observed in B lymphocytes (13) and dendritic
cells (14), raising the possibility that similar structures are present
also in NK cells. L-type calcium channels are composed of three
transmembrane subunits (1C, , and the 2
complex) and one cytoplasmic chain (the ß1 chain) (15, 16). These structures can be identified using compounds such as the
phenylalkylamine (PAA) derivatives, which specifically bind with high
affinity to the pore-forming 1C chains, regulating their
functional state from blocking to opening (15, 16).
Interestingly, we have recently observed that exogenous HIV-1 Tat inhibits some dendritic cell functions by competing with L-type calcium channels expressed by these cells (14). Several lines of evidence indicate that extracellular Tat plays important roles in the development of immunodeficiency. Indeed, the HIV-1 transactivating factor Tat can be released by infected cells and exert many biologic effects on bystander cells (17, 18, 19, 20), including inhibition of several calcium-dependent immune cell functions (14, 21, 22, 23, 24, 25).
In this paper, we demonstrate that L-type calcium channels are expressed by NK cells and regulate the killing of tumor cells. Furthermore, we show that L-type calcium channels are the molecular target of HIV-1 Tat on NK cells: blocking of these channels by either Tat of PAA drugs results in lack of degranulation, which eventually leads to impairment of NK cell-mediated cytotoxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified CD3-CD16+ NK cells were obtained from PBL after depletion of monocytes by plastic adherence and of CD3+, CD4+, and CD8+ cells upon incubation with anti-CD3 (Leu4a), anti-CD4 (Leu3a), and anti-CD8 (Leu2a) mAbs (Becton Dickinson, Mountain View, CA) followed by immunomagnetic beads (Unipath, Milan, Italy), as described (10). NK cells were stimulated with 10 µg/ml PHA (Sigma Chemicals, St. Louis, MO) and then cultured in 96-well U-bottom microplates (Greiner Labortechnik, Nurtingen, Germany) with RPMI 1640 medium supplemented with 10% FCS, 1 mM glutamine, 100 U/ml penicillin-streptomycin (all from Biochrom, Berlin, Germany) and 100 U/ml of rIL-2 (Cetus, Emeryville, CA) in a final volume of 200 µl/well.
Indirect immunofluorescence and cytofluorometric analysis
Immunofluorescence staining was performed as described (10). Briefly, aliquots of 105 cells were stained with the anti-CD11a 70H12 mAb (10) or the anti-CD16 KD1 mAb (IgG1 (26)) followed by phycoerythrin (PE)-conjugated anti-isotype-specific goat anti-mouse serum (GAM; Southern Biotechnology, Birmingham, AL). Control aliquots were stained with the fluorescent reagent alone. Samples were analyzed on a flow cytometer (FACSort, Becton Dickinson) equipped with an argon ion laser exciting FITC and PE at 488 nm, gated to exclude nonviable cells and debris. Data were analyzed using the Lysis II computer program (version 1.1, Becton Dickinson).
Analysis of calcium fluxes by spectrofluorometry
NK cells were loaded with the acetoxymethyl-ester of fura 2 (fura 2-AM, 1 µM, Sigma) for 1 h at 37°C, placed in a quartz 2-ml cuvette and maintained at 37°C by a thermostatically controlled water bath. Fura 2-AM was excited at 334 and 380 nm; emitted light was filtered at 510 nm; and fluorescence was monitored with an LS-50B spectrofluorometer (Perkin-Elmer, Pomona, CA) (10). [Ca2+]i was calculated according to Grynkiewicz et al. (27). [Ca2+]i increases were measured upon cross-linking of CD11a or CD16 molecules, obtained by incubating NK cells with the F(ab')2 (5 µg/ml) of the specific anti-CD11a 70H12 mAb (10) or anti-CD16 KD1 mAb (20 min at 4°C) followed by adding 10 µg/ml of F(ab')2 GAM-Ig(H+L) (Zymed, San Francisco, CA) during the test at 37°C as described (10). In some experiments, NK cells were pretreated with the L-type Ca2+ channel antagonist verapamil (VPM; Calbiochem-Inalco, Milan, Italy; 10 or 1 µM) or chemically synthesized, >96% pure, HIV-1 Tat (Tecnogen, Piana di Monteverna, Italy, 10 or 100 nM) for 20 min (14), then washed before challenge with the various mAbs. Alternatively, [Ca2+]i increases were elicited with 10 µM (±) Bay K 8644 (Calbiochem; the net functional effect of the racemic mixture is that of the negative enantiomer, which is an L-type Ca2+ channel agonist), as described (14, 15). When indicated, experiments were performed in the presence of 2 mM EGTA (Sigma).
Calcium channel detection by fluorescence or Western blot. NK
cells (106/sample), untreated or preincubated 20 min with
10 µM VPM or 0.5 µM HIV-1 Tat or 1 µM recombinant fibronectin
type III repeat (Fn-III, from amino acid 1086–1172, kindly provided by
Dr. L. Zardi, IST-CBA, Genoa, Italy) as a control, were stained with 3
nM fluorescein DMBODIPYR PAA or
fluorescein-
-conotoxin GVIA (Molecular Probes Europe, Leiden, The
Netherlands) and run on a FACSort (14, 28). Alternatively, NK cells,
untreated or pretreated for 20 min with either 0.5 µM HIV-1 Tat or 10
µM VPM or 1 µM Fn-III, were stained with 100 nM biotinylated HIV-1
Tat (Tecnogen (14)) followed by PE-streptavidin and analyzed by
FACSort. Western blot was performed as described (14). Lysates from NK
or A431 cells (positive control, Transduction Laboratories, Lexington,
KY) were separated by SDS-PAGE (12% gel) under reducing conditions and
electrotransferred onto nitrocellulose filters (Hybond ECL; Amersham
Italia, Milan, Italy). In other experiments, biotinylated Tat or
biotinylated transferrin (Sigma) were cross-linked to avidin-Sepharose
(Boehringer Mannheim Italia, Monza, Italy) and incubated with NK cell
lysates at 4°C overnight. After elution, samples were run and
transferred as above. Membranes were then incubated for 1 h with
the anti-calcium channel ß1 subunit mAb (clone 44,
Transduction Laboratories) at 1:250 dilution, followed by horseradish
peroxidase-conjugated GAM Ig (Dako, Milan, Italy; 1:10,000 dilution).
The immunoreactive bands were revealed by luminol reaction (ECL;
Amersham).
Cytolytic assay and serine esterase measurement. Cytolytic
activity of CD3-CD16+ resting or activated NK
cells against the human erythroleukemia K562 cell line, the murine
mastocytoma cell line P815, or the human lung adenocarcinoma cell line
P71 was tested in a 4-h 51Cr release assay as described
(29). NK cells were used as effectors at different E:T ratios, from
40:1 to 1:1, in a final volume of 200 µl of RPMI 1640. NK cells were
also tested in a redirected killing assay using FcR+
P815 target cells, by adding saturating amounts (1 µg/106
cells) of the anti-CD16 mAb at the onset of the cytolytic assay. In
some experiments, NK cells were pretreated for 20 min with 10 or 1 µM
VPM, 100 nM Tat alone, or 100 nM Tat followed by 10 µM Bay K 8644 or
10 µM Bay K 8644 followed by 100 nM Tat. Drug concentrations were
chosen on the basis of titration experiments (14). Results are
expressed as percentage of cytotoxicity calculated as described (29).
Measurement of serine-esterases in the supernatants or in cell lysates
of NK cells, challenged as described above, was conducted according to
Shiver et al. (30). Briefly, 100 µl of supernatants (SN) or cell
lysates (CL, 0.5% Triton X-100) obtained from 106 NK
cells, activated upon CD16 or CD11a cross-linking before or after
treatment with 10 µM VPM or 100 nM Tat, were added to 50 µl of 1 mM
dithionitrobenzoic acid (DTNB, Sigma) and 50 µl of 1 mM
N-benzyloxycarbonyl-L-lysine
thiobenzylester (BLT; Calbiochem, La Jolla, CA), and the reaction was
performed at room temperature for 30 min. ODs were measured at 414 nm,
and results expressed as percentage of esterase release were calculated
as follows: (OD414 SN/OD414 CL +
OD414 SN) x 100.
Binding assay
Highly purified fresh or cultured NK cells were labeled with 51Cr (NEN, Boston, MA), washed, and added (105/well) to confluent monolayers of either the ICAM-1 + P71 tumor target cells or ICAM-1-transfected NIH/3T3 murine fibroblasts (31) and incubated at 37°C for 1 h. In some experiments, NK cells were preincubated for 20 min at 4°C with saturating amounts (5 µg/ml) of the anti-CD11a (70H12) F(ab')2, with 10 µM VPM, or with 100 nM Tat and washed twice before the onset of the adhesion assay. Nonadherent cells were removed by washing, and adherent cells were lysed with 1% Triton X-100 in 100 mM Tris buffer. The radioactivity of the samples was measured in a gamma counter (Packard, Sterling, VA). Results are expressed as percentage of adherent cells, calculated as previously described (31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood resting or rIL-2-cultured
CD3-CD4-CD8- cells
were stained with DM-BODIPYR PAA, a fluorescent
PAA derivative that binds to the 1C chain of
L-type calcium channels (28), and FACS analysis was performed. As shown
in Figure 1
(one representative
experiment of six) freshly purified
CD3-CD4-CD8- cells (60%
CD16+) are stained by DM-BODIPYR PAA (Fig. 1B); likewise, cultured NK cell populations (>96%
CD16+) express surface structures recognized by this
fluorescent compound (Fig. 1C). Conversely, the fluorescent
-conotoxin GVIA failed to stain both cultured (Fig. 1D)
and resting (not shown) NK cells, thus ruling out the surface
expression of N-type calcium channels. The presence of L-type calcium
channels on NK cells was confirmed by the finding of a specific 58-kDa
band corresponding to the ß1 calcium channel subunit in
NK cell lysates (Fig. 1E). Moreover, treatment of cultured
NK cells with Bay K 8644, a drug known to bind the open state of L-type
calcium channels and prolong their open time (15), elicits a sustained
[Ca2+]i rise (Fig. 1F). Unlike in
excitable tissues, these channels are voltage independent, since
exposure of NK cells to 50 mM of KCl failed to induce a calcium influx
(not shown).
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shows that VPM inhibits, in a
dose dependent manner, the lysis of K562 cell line by fresh NK cells
(Fig. 2A) and of the human lung adenocarcinoma cell line P71
(Fig. 2B) by cultured NK cells. A similar impairment of NK
cell-mediated cytotoxicity by VPM was also observed using the murine
mastocytoma P815 (Fig. 2C). Since CD16 represents an
important pathway of NK cell activation, which can trigger the
cytolytic activity (5), we asked whether VPM could also block
CD16-induced cytotoxicity in a redirected killing assay with P815
target cells. Interestingly, VPM significantly decreases the
CD16-mediated lysis of P815 targets by cultured (Fig. 2D)
and resting (not shown) NK cells.
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, exposure of cultured NK cells to VPM
completely prevented the [Ca2+]i rise
detected upon CD11a (Fig. 3A) and partially prevented
calcium mobilization induced by CD16 cross-linking (Fig. 3B). When CD16 triggering was performed in the presence of 2
mM EGTA to chelate extracellular calcium, a slight and quick
[Ca2+]i increase was observed, conceivably
due to mobilization from internal stores (Fig. 3C). This
calcium mobilization was not inhibited by VPM, supporting the
conjecture that this drug blocks the entry of extracellular
calcium (Fig. 3C).
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As HIV-1 Tat can inhibit several calcium-dependent immune cell
functions (21, 22, 23, 24, 25), we addressed the question of whether Tat can block
NK cell-mediated cytotoxicity. Exogenous Tat proved to inhibit both the
lysis of the K562 cell line by fresh NK cells (from 44 ± 12% to
15 ± 4% of specific lysis) and the killing of P815 targets by
cultured NK cells (Fig. 4A); a
similar inhibitory effect was observed on CD16-triggered cytotoxicity
(Fig. 4B). In both cases, inhibition was prevented by
exposing NK cells to the calcium channel agonist Bay K 8644 before
adding Tat (Fig. 4, A and B). However, Bay K
8644, when added after the binding of this protein to NK cells, was not
able to revert the effect of Tat (Fig. 4 A and
B).
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A)
or CD16 (Fig. 5B) molecules is strongly decreased by
pretreatment of NK cells with HIV-1 Tat. In contrast, Tat does not
affect the calcium channel-independent calcium rise that follows NK
cell exposure to the ionophore ionomycin (not shown). Interestingly,
Tat was not able to inhibit the calcium rise elicited upon CD16
cross-linking in the presence of 2 mM EGTA (Fig. 5C),
supporting the concept that Tat, similarly to VPM (Fig. 3
C), blocks the entry of extracellular calcium. Moreover,
Tat did not compete with the anti-CD11a or anti-CD16 mAbs for
binding to NK cells (Fig. 6), thus ruling
out the possibility that the inhibition of calcium mobilization
elicited via CD11a or CD16 was due to the inaccessibility of these
molecules.
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A); in turn, Tat antagonizes
the binding of DM-BODIPYR PAA to NK cells (Fig. 7B), whereas the unrelated peptide Fn-III, displaying the
same length of HIV-1 Tat, has no effect (not shown). Furthermore, we
found that the ß1 subunit of calcium channels is eluted,
after chromatography of NK cell lysates, from an avidin column
immobilizing biotinylated Tat (Fig. 7C). These data
indicate that the inhibitory effect of HIV-1 Tat on NK cell function is
mediated by its interaction with surface structures related to L-type
calcium channels.
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In NK cell-mediated cytotoxicity, three steps can be identified:
binding to target cells, degranulation, and killing (6, 7, 9). In our
experiments, adhesion of NK cells to the ICAM-1 + P71 tumor cell line
or to ICAM-1-transfected cells was unaffected by HIV-1 Tat (Fig. 8A), indicating that this
protein does not interfere with the first step of the cytotoxic
process. Similarly, VPM does not impair NK-target cell binding, ruling
out the involvement of L-type calcium channel in this step. To
understand whether secretion of cytolytic enzymes is affected by HIV-1
Tat, we evaluated the percent of release of serine esterases by
cultured NK cells pretreated with 100 nM Tat and triggered via CD11a or
CD16 molecules. Our results show that Tat inhibits the secretion of
serine esterases by NK cells upon engagement of CD16 or CD11a molecules
(Fig. 5B). Interestingly, VPM (10 µM) exerted the same
inhibitory effect (Fig. 8B), indicating that the release of
granule contents is dependent on calcium channel function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We show that rises in [Ca2+]i levels
occurring upon CD11a or CD16 engagement in NK cells, mainly due to
extracellular calcium entry (5, 10, 11), are mediated by L-type calcium
channels. The presence of functional L-type Ca2+ channels
in NK cells is supported by three lines of evidences: 1)
Ca2+ entry upon CD11a or CD16 cross-linking and cytolytic
activity is inhibited by L-type Ca2+ channel blockers; 2)
fluorescent PAA drugs, specific for the 1C chain of
L-type channels, bind to NK cell surface; and 3) the calcium channel
ß1 chain is detectable in NK cell lysates. At variance
with L-type calcium channels in excitable tissues, which are voltage
sensitive (34), PAA-sensitive Ca2+ channels in NK cells are
voltage independent, as described in B lymphocytes and dendritic cells
(13, 14). This points to a common mechanism responsible for
Ca2+ entry in immune cells, based on specialized
transmembrane structures.
L-type calcium channels appear to be the molecular target of HIV-1 Tat on NK cells: indeed, the inhibitory effect of Tat on NK cell activity is prevented by the activator Bay K 8644, which selectively binds to L-type calcium channels, prolonging their open time (15). Along this line, the ß1 calcium channel subunit is eluted by avidin-Sepharose columns linked to biotinylated Tat, and the binding of PAA derivatives to these channels is cross-inhibited by Tat. In turn, PAA drugs are able to strongly decrease the binding of biotinylated Tat to NK cells, indicating that PAA-sensitive calcium channels represent one major ligand for Tat on NK cells. The finding that Bay K 8644 is unable to restore cytotoxicity when NK cells are pretreated with Tat is in agreement with the observations that calcium channel antagonists can be displaced from purified receptors only by very high (1000-fold) doses of agonists (15, 16); we could not use these concentrations (>10 µM), as they lead to NK cell dysfunction in our system (not shown).
The physiologic relevance of our findings is supported by two previous observations: 1) nanomolar concentrations of HIV-1 Tat are detectable in the sera of AIDS patients (24), and 2) a serum factor contributes to the suppression of NK cell function (35). It is tempting to speculate that this factor is HIV-1 Tat: the local amount of Tat in the mucosal and lymphoid tissues should be conceivably higher, due to the concentrating effect of heparan sulfates (36), thus contributing to decreased NK cell function at the site of infection.
We cannot rule out that other mechanisms, operating synergistically, may contribute to NK cell deficiency in AIDS (3). Nevertheless, our data provide evidence for a molecular mechanism, based on the blocking of L-type calcium channels, possibly leading to NK cell impairment during HIV-1 infection. As L-type calcium channels are expressed by other immune cells (13, 14), they could represent a common target for HIV-1 Tat. Interaction with Tat would impair several immune functions that are dependent on extracellular calcium entry, thus contributing to a generalized immune dysfunction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria Raffaella Zocchi, Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Via Olgettina 60, I-20132 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; BLT, N-benzyloxy-carbonyl-L-lysine thiobenzylester; [Ca2+]i, intracellular free calcium concentration; Fn-III, recombinant fibronectin type-III repeat; fura 2-AM, acetoxymethyl-ester of fura 2; GAM, goat anti-mouse serum; PAA, phenylalkylamines; PE, phycoerythrin; VPM, verapamil.
Received for publication February 17, 1998. Accepted for publication May 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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