mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity

Mistletoe viscotoxins increase natural killer cell-mediatedcytotoxicity

Julie Tabiasco1, Frederic Pont2, Jean-Jacques Fournie1 and Alain Vercellone1

1Institut National de la Sante et de la Recherche Medicale U563 and 2Service de spectrometrie de masse de l¢ IFR 30,

CHU Purpan, BP3028, Toulouse, France

Mistletoe extracts have immunomodulatory activity. Weshow that nontoxic concentrations ofViscum album extractsincrease natural killer (NK) cell-mediated killing of tumorcells but spare nontarget cells from NK lysis. The com-pounds responsible for this bioactivity were isolated frommistletoe and characterized. They have low molecular massand are thermostable and protease-resistant. After completepurification byHPLC, theywere identified by tandemMSasviscotoxins A1, A2 and A3 (VTA1, VTA2 and VTA3,respectively). Whereas micromolar concentrations of these

viscotoxins are cytotoxic to the targets, the bioactivity withrespect toNK lysis is within the nanomolar range and differsbetween viscotoxin isoforms: VTA1 (85 nM), VTA2 (18 nM)and VTA3 (8 nM). Microphysiometry and assays of cellkilling indicate that, within such nontoxic concentrations,viscotoxins do not activate NK cells, but act on cell conju-gates to increase the resulting lysis.

Keywords: mistletoe; natural killer cells; tumor cells; visco-toxin; Viscum album.

Mistletoe preparations have been used for pharmacologicalpurposes since ancient times [1]. These days, industriallyproduced extracts from mistletoe are used in treatments ofsolid tumors [2–5]. It is thought that the molecular basis ofthe antitumoral activity of mistletoe lies in several distinctbioactivities. First, its lectin content is responsible for directtoxicity to tumor cells [6–9]. Secondly, the Viscum albumrhamnogalacturonan oligosaccharide favors bridging ofnatural killer (NK)–tumor cell conjugates, enhancing effi-ciency of killing [10–15]. Thirdly, it has been found that theantitumoral human cytotoxic T lymphocytes with cd T cellreceptor are selectively activated by mistletoe ligands ofphosphoantigen structure [16,17].NK cells play an important role in antitumoral immunity

as they directly kill tumor cells and regulate the adaptativeimmunity [18]. Activation of cytolytic functions of NK cellsrelies mainly on selective interactions of NK receptors withmajor histocompatibility complex (MHC) class I-relatedmolecules on the tumoral cells. Distinct ligands, however,may also induce NK cell stimulation [19–23]. Hence,immunotherapeutic modulation of NK cell activation isan important issue in current antitumoral approaches.We analysed the bioactivity of V. album compounds in

the in vitro killing of tumor cells by human NK cells andfound that the components that mediate this bioactivity areviscotoxins.

M A T E R I A L S A N D M E T H O D S

Preparation of V. album (Va) extract

The green and white parts of mistletoe (V. album L.,Viscaceae; 1 kg) freshly collected on Robinia pseudacacia L.were crushed and extracted twice with 5 L methanol/water(1 : 1, v/v). After filtration and volume reduction to600 mL, the aqueous phase was successively partitionedwith cyclohexane, dichloromethane and ethyl acetate. Thisaqueous phase was positive in tests of enhancement ofNK-mediated killing of tumor cells, but was directly toxicfor the tumor target cell line. Ethanol was added to theconcentrated aqueous phase to achieve 85% (v/v) concen-tration. A precipitate was obtained and separated from thesupernatant by centrifugation (2000 g; 10 min). The super-natant was concentrated and ethanol was added to 85%(v/v). After centrifugation, the precipitate was dissolved inwater, pooled with the former precipitate, and constitutedthe Va extract. The final yield of Va extract was 50 g from1 kg plant extracted. The stock solution of Va extract(237 mgÆmL)1) was stored at )20 �C.

Viscotoxins

The protocol for purifying viscotoxins was modified from aprevious method [24,25]. Briefly, to fractionate Va extract,we replaced ion-exchange and exclusion chromatographywith C18 reverse-phase open column chromatography toavoid the use of salt solutions. A 4-g portion of Va extractwas applied to a column (2.5 · 20 cm) of LichroprepRP-18(Merck, Darmstadt, Germany), which was irrigated with300 mL 20%, 40% and 100% acetonitrile in 0.1% aqueousacetic acid. The former 40% acetonitrile eluate was freeze-dried, dissolved in 20% acetonitrile in 0.1% trifluoroaceticacid and chromatographed by C18 reverse-phase HPLC(Nucleosil 5 lm; 300 A pore size; Bischoff, Leonberg,Germany). The column (250 · 4.6 mm) was eluted at

Correspondence to: A. Vercellone, INSERM U395, CHU Purpan,

BP 3028, 31024 Toulouse, France.

Fax: 335 6274 8386, Tel.: 335 6274 8364,

E-mail: [email protected]

Abbreviations: MHC, major histocompatibility complex; MSn,

multiple-stage MS; NK, natural killer; VTA, viscotoxin A; TNF-a,tumor necrosis factor a.(Received 28 November 2001, revised 27 March 2002,

accepted 15 April 2002)

Eur. J. Biochem. 269, 2591–2600 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02932.x

1 mLÆmin)1 by a linear gradient from 20% to 50% ofacetonitrile in 0.1% trifluoroacetic acid over 30 min. Forfinal purification of VTA2 and VTA3, the elution wascarried out with the following gradient: 25% solvent B(acetonitrile in 0.1% trifluoroacetic acid) and 75% solventA (0.1% aqueous trifluoroacetic acid) during the first10 min, 1 min up to 30% solvent B, 9 minwith 30% solventB, and from 30% up to 40% over 5 min. Cation-exchangechromatographywas conduced on a polypore SP 10micron(100 · 2.1 mm) column (Applied Biosystems) using lineargradient elution at 0.3 mLÆmin)1 from 100% solvent A(50 mM phosphate buffer, pH 7) to 100% solvent B (1 M

NaCl in 50 mM phosphate buffer, pH 9) in 30 min. TheVTA3 standard was kindly provided by K. Urech andpurified as described by Schaller et al. [24].

Chemical and enzymatic treatments

Dilution in organic solvent was achieved by addition of pureacetonitrile to 80% final volume. After 2 h at roomtemperature, organic solvent was removed by evaporationwith a Speed-vac centrifuge before further bioassays. Forchemical treatments, 1 vol. Va extract or purified viscotoxinwas mixed with 1 vol. 4 M NaOH or 4 M HCl andincubated for 2 h at 37 �C. After neutralization with HClor NaOH, the samples were immediately diluted in RPMImedium supplemented with 10% human serum, and pHwas adjusted to 7.0 before further bioassays. For periodateoxidation, sodium periodate (Sigma) was added (finalconcentration 5 mM) to the Va extract or to purifiedviscotoxin, and samples were left for 2 h at room tempera-ture. Unchanged sodium periodate was further neutralizedby the addition of a few drops of glycerol to the sample.Reduction and alkylation were performed as describedpreviously [26]. Excess reagent was removed by purificationon a tC18 September–Pak� cartridge (Walters, Milford,MA, USA) eluted successively with increasing percentagesof acetonitrile.Enzymatic treatments consisted of incubating Va extract

for 2 hat 37 �Cwithproteinase K (1.5 UÆmL)1; Boehringer,Mannheim, Germany), calf alkaline phosphatase(1 UÆmL)1; Boehringer), or sulfatase (16.8 UÆmL)1

Aeromonas aerogenes sulfohydrolase; Sigma) in 10 mM

Tris/HCl, pH 7.2. Heating at 75 �C for 10 min stoppedenzymatic reactions before further bioassay.

SDS/PAGE

SDS/PAGE analysis of Va extract and viscotoxins wasperformed using 7 · 10 cm gels of 20%acrylamide (37.5 : 1ratio of acrylamide to bisacrylamide) for the resolving geland 5% acrylamide for the stacking gel. Samples wereelectrophoresed at 75 V, and further stained by CoomassieBlue or silver nitrate.

Mass spectrometry

MS was performed using the LCQ Ion-trap mass spectro-meter (Thermo-Finnigan, San Jose, CA, USA) by liquidchromatography/MS and nanospray as described [27]. Theviscotoxin masses were obtained by spectral deconvolutionwith Bioworks software from the Xcalibur 1.2 suite(Thermo-Finnigan). Multiple stage MS (MSn) sequencing

of viscotoxins was carried out on the underivatized sample.The peak at m/z 966.5 (i.e. z ¼ + 5 for VTA2 for whichthe mass is ¼ 4827 Da) was selected in full-scan MS andfragmented. In its MS2 spectrum, Y ions corresponding tothe disulfide-free C-terminal moiety of VTA2 were selectedand fragmented by MS3. Fragmentation of the Y6 ion (m/z706.3) led to overlapping sets of Y and b ions identifying thesix C-terminal amino acids of VTA2. The precision of thisexperiment did not allow lysine to be distinguished fromglutamine.

Cell lines and cultures

Peripheral blood lymphocytes were obtained from hepari-nized venous blood of healthy volunteers using Ficoll-Paque(Amersham Pharmacia Biotech AB, Uppsala, Sweden).Fresh NK cell populations were obtained by peripheralblood lymphocyte depletion of non-NK cells using the NKCell Isolation Kit (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). The phenotype of isolated cells wasanalyzed by flow cytometry with CD16 mAb and CD56mAb (clone 3G8 and N901-NKH1, respectively; Immuno-tech-Beckman-Coulter, Marseilles, France). The resultingNK population typically comprised more than 85% ofCD56 cells and less than 0.5% CD3 cells. The differenthuman cell targets (K562, an MHC class I-deficient mono-myelocytic tumor [28]; Daudi, a b2m– Burkitt’s lymphomacell line [29]; Val, a non-Hodgkin B cell lymphoma [30];C1R, a lymphoblastoid tumor expressing only the HLAclass I allele Cw 0401 [31]; and C1R-B27 [32]) weremaintained in culture in complete RPMI 1640 mediumwith Glutamax-I supplemented with 10% fetal calf serum(Gibco–BRL, Life Technologies, Cergy Pontoise, France).The murine FccR+ mastocytoma P815 cell line wasmaintained in complete Dulbecco’s modified Eagle’s culturemedium (Sigma Aldrich, St Louis, MO, USA) supplemen-ted with 5% human serum. The human interleukin-2-dependent NK cell lines NKL [33] and NK-92 [34] (kindlyprovided by E. Vivier, CIML, Marseilles, France) weremaintained in RPMI 1640 medium with Glutamax-I(Gibco–BRL) supplemented, respectively, with 10% pooledhuman AB serum plus recombinant interleukin-2(100 UÆmL)1; Chiron) and 10% fetal calf serum plusrecombinant interleukin-2 (200 UÆmL)1). The cd cell linehas been described previously [27].All the culture media were complemented with

100 UÆmL)1 penicillin, 100 UÆmL)1 streptomycin and1 mM sodium pyruvate (all from Gibco–BRL).

Bioactivity assays

The cytolytic activity was assessed in a 4-h 51Cr-release assayin which effector cells (NKL,NK-92, freshly isolatedNKorcd cytotoxic T lymphocytes) were mixed with differenttarget cells. Lysis assays were carried out with or without Vafractions added at different concentrations. Briefly, 106

target cells were labeled with 100 lCi sodium [51Cr]bichro-mate (10 mCiÆmL)1; ICN) at 37 �C for 1 h. The cells werethen washed three times with RPMI and added to theeffector cells at 3 · 103 cells/well in 96-well round-bottommicroplates, resulting in an effector to target cell (E/T) ratioranging from 30 : 1 to 3 : 1 in a final volume of 0.2 mL ineach well. For the redirected killing assay, target cells were

2592 J. Tabiasco et al. (Eur. J. Biochem. 269) � FEBS 2002

the (FccR+) P815 cell line, and effector cells the CD16+

NKL cell line, using an E/T ratio of 3 : 1. The assays werecarried out with or without 2 lgÆmL)1 anti-CD16 IgG1(clone 3G8). After 4 h of incubation at 37 �C, 100 lLsupernatant was harvested and counted in the gammacounter. The percentage specific 51Cr release was deter-mined from the relation:[(experimental c.p.m. ) spontaneous c.p.m.)/(total c.p.m.

incorporated ) spontaneous c.p.m.)] · 100All determinations were performed in triplicate in each

assay, and results shown are the mean ± SEM fromtriplicate determinations of a representative experiment outof five independent experiments (on average). For clarity,SEMs are not represented on most of the figures becausethey were below 3% of specific lysis.Release of tumor necrosis factor a (TNF-a) was meas-

ured by a bioassay using TNF-a-sensitive cells (WEHI-13VAR, ATCC CRL-2148). Briefly, 5 · 104 NKL cells perwell were incubated for 24 h at 37 �C with or withoutvarious concentrations of each viscotoxin in 100 lL culturemedium. A 50-lL portion of supernatant was then added to50 lLWEHI cells plated at 3 · 104 cells per well in culturemedium containing actinomycin D (2 lgÆmL)1) and LiCl(40 mM). WEHI cells were incubated for 20 h at 37 �C.Viability of WEHI cells was then measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(Sigma) assay. Levels of TNF-a release were then calculatedfrom a standard curve obtained using purified human

recombinant TNF-a (PeproTech, Inc., Rocky Hill, NJ,USA). Typically, unstimulated NKL cells release5 ± 2 ngÆmL)1 TNF-a compared with 60 ± 5 ngÆmL)1

when induced by phorbolmyristate acetate (4 ngÆmL)1) andionomycin (500 ngÆmL)1).To probe for soluble cytolytic factors released by NK

cells during the lysis assay, 9 · 104 NK cells were mixedwith 3000 K562 cells and incubated with viscotoxins for 4 hat 37 �C. A 100-lL portion of supernatant was then addedto 3000 51Cr-labeled K562 cells plated in a 96-well round-bottom microplate. After 4 h incubation at 37 �C, 100 lLsupernatant was collected and counted in a gamma counter.The percentage of specific 51Cr release was determined as forthe cytotoxicity assay.

R E S U L T S

Nontoxic concentrations of Va extract enhance NKcell-mediated and antibody-redirected lysis

A crude hydrosoluble extract was prepared from crushedmistletoe leaves, and added to in vitro lysis assays withK562tumor targets and variousNK effector cells. As indicated bythe spontaneous release of 51Cr from target cells alone, Vaextract diluted 1 : 5000 was not directly toxic for thesetargets. On the other hand, Va extract increased K562 lysisby freshly isolated NK cells or by a strongly cytolytic cd Tcell line [35]. NK cell lysis in the presence of Va extract was

Fig. 1. V. album extract (VaE) enhances

cytolytic activity of different killer cells for

various target cells. (A) Lysis of K562 target

cells by the following effector cells: fresh NK

cells, cd cytotoxic T lymphocytes, NKL, and

NK-92 titrated at different E/T ratios, in the

presence (d) or absence (s) of Va extract

diluted 1 : 5000 in all assays. Shown on the

right of each lysis assay are the c.p.m. of

spontaneous 51Cr release by the target cells

alone (target spontaneous release) incubated

under the same conditions in the presence

(filled bars) or absence (empty bars) of Va

extract diluted 1 : 5000 in all assays. Each

result represents the mean of triplicate experi-

ments. (B) NKL effectors were tested for kill-

ing of the target cells lines, C1R, C1R-B27,

Daudi and Val, at different E/T ratios, in the

presence (d) or absence (s) of Va extract

diluted 1 : 5000. Shown on the left of each

cytotoxic experiment are the c.p.m. of spon-

taneous 51Cr release by the target cells alone as

above. (C) NKL cells were tested in a redi-

recting killing assay against the FccR+ P815

target cells. These assays were carried out in

the absence or presence of CD16 mAb

(2 lgÆmL)1) and with or without Va extract

(VaE) diluted 1 : 5000. Each result represents

the mean of triplicate experiments at an E/T

ratio of 3 : 1.

� FEBS 2002 Viscotoxins enhance NK cell-mediated killing (Eur. J. Biochem. 269) 2593

equivalent to four times the number of killer cells whenpresent alone (Fig. 1A). Similar results were found with twoother NK cell lines, NK-92 and NKL. Thus nontoxicconcentrations of Va extract increased NK lysis.NK cells eliminate tumor cells in the absence of

appropriate interactions between their inhibitory receptorsandMHC class I molecules on the surface of the target cells.Thus we investigated whether Va extract would alter thelysis of target cells other thanK562 by the cell line NKL. Vaextract also enhanced killing of the HLA class I-deficienttargets C1R and Daudi (Fig. 1B). However, Va extract didnot trigger killing of theHLA class I cells Val or C1R-B27, aC1R cell line transfectedwith theHLA-B27 allele protectingagainst lysis by NKL cells [33].NK cells may also lyse IgG-bound tumor cells [36], so we

investigated whether Va extract was also bioactive in thiscase. Lysis of the FccRIII+ cells P815 by NKL cells in thepresence of CD16mAb [37] wasmeasured with andwithoutVa extract. Again, Va extract was not directly toxic to P815cells but enhanced its antibody-redirected killing (Fig. 1C).Therefore, the bioactivity of this Va extract appears to beindependent of the NK-lysis activation pathway.

Bioactivity-guided isolation of thermostableand low-molecular-mass proteins from Va extract

Bioactivity of Va extract in NKL cells could be titrated inlysis assays by varying the E/T ratio and Va extractconcentrations (Fig. 2A). This helped to guide the isolationof the molecule(s) responsible. After various treatments of

Va extract, titration of the remaining bioactivity indicatedthat the bioactive component(s) were resistant to heating,acid, protease, phosphatase and sodium periodate (Ta-ble 1). This suggested that the molecule was thermostable,although presumably neither a protein nor a carbohydrate,despite their relative abundance (carbohydrate content10 mgÆmL)1; data not shown) in Va extract revealed bysilver nitrate and Coomassie Blue staining of SDS/poly-acrylamide gels (Fig. 2B). However, bioactivity in the Vaextract was degraded by alkali or a reduction–alkylationtreatment that targeted disulfide bridges (Table 1). Furtherisolation of the NK-bioactive compound(s) was based onHPLC with macroporous reverse-phase support (Fig. 2C).Bioassay-guided fractionation of the Va extract indicatedthat the bioactivity was due to distinct UV-absorbing peakssplit into two consecutive HPLC fractions. The HPLCfractions 14–17 min and 17–20 min increased the NKL-mediated lysis of K562 cells (Fig. 2C). With regard to thespecific NK lysis, no other bioactive fraction could berecovered by this procedure. Both absorbance characteris-tics of the active fractions (not shown) and their appearanceon SDS/PAGE (Fig. 2B) revealed that they comprisedsmall protein(s) with estimated molecular mass of 5 kDa.

The only Va extract molecules bioactive in NK lysisare VTA1-3

On the one hand, proteins were unexpected for the unusualthermostability and protease-insensitivity of the bioactiveextract, but, on the other hand, they could account for the

Fig. 2. NK cell lysis bioassay-guided isolation

of viscotoxins. (A) Cytolytic activity of NKL

cells against K562 target cells with Va extract

diluted 1 : 5000 (j), 1 : 15 000 (m), 1 : 45 000

(e) or without Va extract (s). (B) SDS/

PAGE of Va extract and fraction 17–20 min

(VT) stained with either AgNO3 or Coomassie

Blue. (C) RP-HPLC separation of bioactive

fractions from the Va extract monitored for

total ion current and for bioactivity on NKL

cells. Fractions were collected every 3 min,

lyophilized, and tested in the NKL/K562

killing assay as above. Online coupling of

HPLC to ion trap electrospray ionization MS

was used to identify viscotoxins at the specified

peaks.


sensitivity to disulfide-specific reaction (Table 1). To unam-biguously identify these molecules, we used online couplingof the above HPLC separation with ion-trap MS detection(liquid chromatography/MS). Using positive-mode electro-spray, analysis of the HPLC peaks gave a series ofmulticharged ion species (not shown) that were furtherdeconvoluted in the 2000–6000 mass range. The deconvo-lution spectrum of the major peak from bioactive fraction17–20 min gave two molecular species of 4827.0 and4925.0 Da (Fig. 3A). They correspond, respectively, to the

expected molecular mass of VTA2 (theoretical mass 4828.4)and to a noncovalent phosphate adduct of VTA2 [25].Similar analysis of the other bioactive HPLC peaksidentified VTA1 (mass 4884.0) and VTA3 (mass 4830.0)(Fig. 2C and [25]). This conclusion was checked by partialpeptide sequencing usingMSn. As the N-terminal moiety ofnative viscotoxins involves a disulfide bridge [25], sequentialfragmentation of each C-terminal moiety was performed toconfirm these assignments. From native VTA2, we selectedby MS2 two ions belonging to the Y series (m/z 407, 706.3)(data not shown). Its fragmentation into the expectedY-type and b-type fragments characterized the C-terminalPSDYPK hexapeptide sequence (Fig. 3B). Similar experi-ments established the identity of VTA1 and VTA3 (data notshown).The HPLC fraction eluted at 17–20 min was dried and

weighed, and, although its bioactivity in NK-mediatedkilling was clear-cut, it still comprised a mixture of distinctviscotoxins. The same reverse-phase HPLC procedure wasrepeated, but using a slower elution sequence. This resultedin resolution into three peaks corresponding to each of thethree distinct viscotoxins. Each of the separated viscotoxinswas then individually controlled chromatographically byreverse-phase (Fig. 4A) and cation-exchange (Fig. 4B)chromatography. Parallel bioactivity monitoring of theseruns revealed that NK lysis activity was recovered with eachof the purified viscotoxin peaks. A molecular mass of4884 Da identified VTA1 in the first bioactive fraction (notshown), a molecular mass of 4827 Da identified VTA2 inthe second bioactive fraction (Fig. 4A), and a molecularmass of 4830 Da (and 4928 Da for the phosphate adduct)identified VTA3 in the third bioactive fraction (Fig. 4A).The bioactivities of VTA1–3 were destroyed by alkali and

reduction–alkylation of disulfide bridges, but not by heat orperiodate, as described above for Va extract (Table 1). So,this profile establishes the involvement of VTA1–3 as theonly components of Va extract that enhance NK lysis.Moreover, the fact that polymyxin B (10 lgÆmL)1) did notaffect the bioactivity of the purified viscotoxins ruled outany contribution of traces of lipopolysaccharide to theobserved effect (not shown).

Table 1. NK bioactivity of Va extract and purified viscotoxins after

chemical and enzymatic treatments. The NK lysis-increasing bioactivity

was normalized to 100% using the largest increase in lytic response in

the presence of untreated Va extract (here + 22% of specific K562

lysis), VTA1 (here + 16%) or VTA2 (here + 20%). For comparison,

the NK lysis-increasing bioactivity of chemically treated extracts is

expressed as a percentage of the reference bioactivity. Va extract,

VTA1 and VTA2 were used at 1 : 5000 dilution, 100 nM and 40 nM,

respectively, which had no direct toxicity for K562 cells. NT, Not

tested; DTT, dithiothreitol; IEt, iodoethanol.

Treatment

NK bioactivity (%)

Va extract VTA1 VTA2

None 100 100 100

Heat (75 �C, 2 h) 91 74 88

Dilution in organic solvent

(80% acetonitrile)

73 100 100

Acid

(2 M HCl, 2 h, 37 �C)67 NT NT

Alkali

(2 M NaOH, 2 h, 37 �C)13 0 0

Periodate oxidation

(5 mM NaIO4, 2 h, 20 �C)100 91 120

Reduction + alkylation

(DTT, IEt)

0 0 0

Proteinase K 90 NT NT

Alkaline phosphatase 77 NT NT

Sulfatase 100 NT NT

Fig. 3. Biochemical identification of the visco-

toxin A2. (A) Deconvoluted spectrum from

the electrospray ionization MS analysis of the

VTA2 peak corresponds to the expected mass

of VTA2. The amino-acid sequence with

disulfide bridges of VTA2 is shown on the

right [25]. (B) MS3 spectrum of the ion at m/z

706.3 generated from pentacharged VTA2

(m/z 966.6). The sequence of the C-terminal

moiety and assignment of the fragmentation

series are indicated.


Using SDS/PAGE examination of serial Va extractdilutions, we estimated the total viscotoxin concentration inthe Va extract to be � 3 mM. This estimate is in concor-dance with the respective bioactivities measured above forpurified viscotoxin and Va extract.

Each purified viscotoxin species was quantified byanalytical HPLC, and its bioactivity in the NKL/K562assay titrated by serial dilutions. As each of the viscotoxinsamples produced similar increases in NK lysis, wecompared the three viscotoxins by determining theirrespective EC50 values, i.e. the VTA concentration produ-cing half-maximal increase in NK-mediated lysis. TheseEC50 values are significantly different: 85 ± 6.4 nM forVTA1, 18 ± 4.2 nM for VTA2 and 8 ± 3.0 nM for VTA3(two-by-two comparison, highest two-tailed P value forstatistical difference equalled 0.0284). These findingsmatched that of an authentic VTA3 standard [24], whichenhanced NK-mediated lysis with EC50 ¼ 6.5 ± 3.5 nMwithout direct cytotoxicity within the 1–100 nM range(Fig. 5). This reference VTA3 EC50 value was not signifi-cantly different from that of the VTA3 (P ¼ 0.6031)purified in this study. Together these data confirm thatviscotoxins are the Va extract compounds responsible forthe increase in NK cell-mediated cytotoxicity.

VTA2 acts on NK–target cell conjugates

The interaction between NK and target cells successivelyinvolves the formation of conjugates, the specific recogni-tion phase, and the lethal hit delivery. Afterwards, the killercells are recycled and may serially kill several targets [38].Because the action of viscotoxin was found to be independ-ent of the NK activation pathway (Fig. 1), we investigated

Fig. 5. Effect of a VTA3 standard on NK-mediated lysis in the absence

of direct cytotoxicity.Lysis ofK562 target cells byNK cells at E/T ratio

of 1 : 1 in the absence (0) or presence of pure VTA3 standard (kindly

provided byK.Urech [24]) added in the concentration range 0–100 nM

to the culture wells (up). Toxicity of VTA3 for the target cells alone

was titrated over the same concentration range as above by measure-

ment of the direct 51Cr release from pulsed K562 cells (down). Each

result represents the mean of triplicate experiments.

Fig. 4. Biochemical characterization of the

purified viscotoxins A1, A2 and A3. (A)

RP-HPLC and (B) cation-exchange HPLC of

purified viscotoxins A2 and A3 monitored for

absorbance at 220 nm and for bioactivity on

NKL cells. Fractions delimited by tick marks

were collected, lyophilized, and tested in the

NKL/K562 killing assay. The deconvoluted

mass spectrum of each purified viscotoxin is

shown in the inset.


whether a viscotoxin pulse of either NKL effector or K562target cells before the assay could also lead to thisbioactivity. Neither VTA2-pulsed killers nor VTA2-pulsedtargets alone reproduced the bioactivity of viscotoxins in theusual lytic assay (Fig. 6A). Therefore, to exert its bioactiv-ity, viscotoxin must be present during the killing assay.Moreover, viscotoxins did not induce production of solubletoxic factors by NKL cells during the 4-h incubation withtarget cells (data not shown; see Materials and methods).These results indicate that VTA2 acts on NK–target cellconjugates.From cell mixing to final cell harvest, these cytotoxicity

assays span 4 h, so we determined viscotoxin bioactivitywhen added at various time points. The optimum for VTA2bioactivity was when it was added � 30–45 min after cellcontact (Fig. 6B). When compared with other drugstargeting NK activation [39], this time-course suggests thatVTA2 affected an event subsequent to conjugate formation,in agreement with the former conclusion.

VTA2 does not activate NK cells

NK–target cell conjugation precedes NK cell activation,which follows a complex array of activatory and inhibitory

receptor–ligand interactions. In hematopoietic cells, intra-cellular transduction of activation signals involves changesin metabolic rates, which may be detected by micro-physiometry [27]. As Va extract acts on both redirected andnatural lysis by NK cells (Fig. 1), its effects appear to beindependent of the NK activation pathway. Therefore,viscotoxins were not expected to directly activate NK cells.To address this issue, we testedmicrophysiometric responsesof NKL cells to VTA2. Although a short pulse with themitogen combination of phorbol ester and ionomycinincreased the metabolic rate of NKL cells (Fig. 6C), nosuch change was detected in parallel assays with NK cellsexposed to various concentrations of VTA2 alone (shownfor 50 nM in Fig. 6C) or to phorbol and VTA2 together(data not shown). So VTA2 does not constitute an activatorsignal to NK cells nor bring them a cosignal additional tophorbol esters. This conclusion was also supported by thefinding that NKL cells incubated with VTA2 alone did notproduce any more TNF-a than unstimulated ones (data notshown).After killer–target cell binding and NK cell activation,

early intracellular events involved in NK-mediated lysiscomprise polarization and delivery of cytotoxic granulesat the target cell synapse. We investigated whether the

Fig. 6. VTA2 acts on NK–target cell conjugates without a change in extracellular acidification rate of NK cells and killing of bystander cells. (A)

Enhancement of NK cytotoxicity cannot be reproduced by a VTA2 pulse of effector or target cells before the lysis assay. The effector cells or target

cells were separately preincubated with 50 nM VTA2 for 4 h and washed before running the usual cytotoxic assay without VTA2. For comparison,

the same assaywas runwithout preincubation andwith or without VTA2 during the killing assay. (B) Time course of VTA2 bioactivity in the killing

assay. At t ¼ 0 min, effector and target cells were mixed. At the times indicated, 50 nM VTA2 (final concentration) was added, and 4 h after

mixing of the cells, specific 51Cr release was determined. For each time point, viscotoxin bioactivity was calculated as follows: (% of specific lysis

with VT) ) (% of specific lysis without VTA2). For each time point, the viscotoxin bioactivity was normalized using the largest increase found with

viscotoxin in the assay (+15% at t ¼ 30 min; s). For comparison, the time course of bioactivity of ion channel blockers was plotted (d) as

described by Sidell et al. [39]. (C) NKL cells in microphysiometer chambers were drained with complete medium only (n), with complete medium

containing phorbol myristate acetate + ionomycin (m) or with complete medium containing 50 nM VTA2 (s). For 80 min, extracellular

acidification was monitored every 90 s in each chamber, and the response was normalized to the rate value before sample introduction. (D)

Bioactivity of VTA2 was tested in a three-cell lysis assay involving NKL killer, K562 target and C1R-B27 bystander cells. While the presence of

bystander cells did not interfere with viscotoxin bioactivity in the killing assay (left), VTA2 did not induce any detectable killing of 51Cr-labeled

bystander cells (right).


killing-enhancing bioactivity of VTA2 resulted from alteredpolarization of lytic granules in such a way as to killbystander cells in addition to bound targets. NKL cells weresimultaneously exposed to K562 target cells and C1R-B27bystander cells (one target for one bystander mixedtogether). We then measured the effect of VTA2 on eitherlysis of 51Cr-labeled K562 target cells in the presence ofbystanders, or reciprocally, on lysis of 51Cr-labeled C1R-B27 bystanders in the presence of K562 target cells. VTA2was devoid of direct toxicity to the bystander cells whilesimultaneously being bioactive against the NK targets(Fig. 6D).So, whereas VTA2 enhances killing of K562 target cells in

the presence of bystanders, it does not induce furthercollateral lysis and hence does not affect the polarization ofthe cytolytic activity of NK cells.

D I S C U S S I O N

The lytic activity of human NK cells is enhanced byV. album compounds [10,40,41]. Previous studies haveinvestigated this effect using fermented Va extracts,peripheral blood lymphocytes, and cytokine-dependentand lymphokine-dependent contexts [13,15,42]. Theresults presented here indicate that the lysis of K562cells by either freshly isolated NK cells or the tumoralNK cell lines NK-92 and NKL is increased by thepresence of Va extract molecules in the lytic assay. Thelytic potential of killer cells in the presence of the extractwas almost the same as that of four times as many killercells alone. This effect of Va extract is not restricted toNK cells, as other types of cytotoxic T lymphocyte, suchas cd T cells, that exert strong NK-like activity againstthe same targets are also enhanced by these molecules.So we investigated the V. album-mediated enhancementof killing by NK cells independently of exogenouscytokine supply.In previous studies, the V. album molecules bioactive in

tumor cell killing by NK cells and monocyte/macrophageswere identified as oligosaccharides (arabinogalactan [11] orrhamnogalacturonan [12,43]), which link killer and targetcells [43]. This report describes the isolation and identifica-tion of VTA1, VTA2 and VTA3 as the only Va extractcomponent of NK-cell-mediated lysis. Mistletoe producestwo protein families with related biological properties:lectins and viscotoxins [44,45]. Viscotoxins are a group ofhighly basic cysteine-rich small proteins related to the familyof thionins [46] and notoriously resistant to protein-denaturing agents [45,47]. Table 1 shows that the startingbioactivity of the Va extract has such characteristics. Otherworkers had already reported an uncharacterized Va-derived peptide that activated NK cells in vivo [48]. Thioninsare cytotoxic for a large variety of eukaryote and proka-ryote cells [49]. This toxicity relies on positive charges ofthionins interacting unspecifically with phospholipids. Thus,by passive insertion into the cell membranes, thioninspermeabilize and kill tumoral cells [49,50]. Few studies havein fact elucidated the basis of viscotoxin toxicity. Using themost viscotoxin-sensitive cell line [51], micromolar concen-trations of viscotoxins are toxic for the rat renal sarcomaYoshida [24]. More recently, VTA3 has been found toinduce cell membrane stiffening and destabilization [52].Here, the action on NK-cell-mediated killing of tumor

targets was analysed using nontoxic nanomolar concentra-tions of viscotoxin. The data presented lead to clearerconclusions than earlier studies of the effect of V. album ontumor cell lysis by monocytes and NK cells. The synergisticenhancement of lysis by mistletoe arabinogalactan ismediated by cytokines secreted from killer cells after itsbinding to NK surface receptors [13,15]. These cells pulsedwith high concentrations of arabinogalactans killed moretarget cells [13,15].Our study shows a different spectrum of effects of

purified viscotoxins which appear to be focused on estab-lished killer–target cell conjugates.NK cell lysis results from intracellular integration of a

wide array of receptor-mediated activatory and inhibitorysignals. As the absence of a lytic response could result fromdominance of negative signaling by cell targets, the actualactivity of viscotoxins on positive signaling pathwaysneeded to be investigated in NK cells more directly.Activation of hematopoietic cells involves increased glyco-lysis, Na+/H+ antiport, and lactate and carbonic acidexcretion, which together cause medium acidification [53].Microphysiometry monitors extracellular acidification as areal-time follow-up of metabolic rate, and detected NKLresponse to a mitogen combination. VTA2 alone does notalter themetabolic rate of NKL cells, so they do not providea self-sufficient positive signal for activating NK cells.Furthermore, the action of VTA2 on killer–target cellcouples spares the �unproductive� killer-bystander ones.Therefore VTA2 does not alter the polarization of cytolyticactivity against the activating target, nor does it weaken thelatter in such a way as to increase their lysis in the assay.Besides the lack of viscotoxin toxicity for the various targetscells in this study, this conclusion can be drawn from theseveral lines of evidence discussed below. First, a pulse andrinse of targets with viscotoxins did not increase theirsubsequent lysis by NK cells. Secondly, viscotoxins do notcause the lysis of bystander cells exposed to viscotoxins andactivated NK.Investigation of the molecular basis of viscotoxin action

on NK–target cell conjugates was not within the scope ofthis study. However, the high bioactivity of viscotoxins (i.e.in the nanomolar range) in this assay, and the differential(8–80 nM) of bioactivity between the structurally relatedVTA1–3 proteins strongly suggest an interaction with anunidentified receptor involved in NK lysis. Viscotoxinsare structurally related to thionins [46], which behaveas selective ion channels in nanomolar concentrationranges [50]. As functional ion channels are involved inNK-mediated lysis [54], a related role for viscotoxins ishypothesized.Long-standing medicinal virtues have been imputed to

mistletoe extracts, used in the treatment of some solidtumors. Although mistletoe was thought to owe much ofits bioactivity to the direct toxicity of several componentsto tumoral cells, this report shows that highly purifiedviscotoxins alone enhance the efficiency of tumor celllysis by NK cells in the absence of any direct cytotoxicityto the target. Although the mode of viscotoxin actionhas not been defined, we show that viscotoxins areinvolved in immunologically mediated tumour celldestruction. Future studies will aim to identify theviscotoxin receptors involved in this therapeuticallyinteresting bioactivity.


A C K N O W L E D G E M E N T S

We wish to thank E. Vivier for helpful discussions and provision of cell

lines, and E. Espinosa for the cd cell line. We also thank Dr K. Urech

for the gift of the highly purified viscotoxin A3. This work was

supported by institutional grants from INSERM and Programme

Eureka from ARC.

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mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity

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