Biomed& Plurrmacother( 1991)45.279-288 8 Elsevier, Paris

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Platelet-mediated cytotoxicity and its enhancement by platelet activating factor SN Bykovskaya, AV Bolvacheva, MV Kiselevsky, VA Khaylenko, AF Bykovslcy Laboratory of Celiulor Immunity. All-Unian Cancer Research Cower of the Academy of Medical Sciences of rhe USSR, Kashirskoye Shosse 24, 115476, Moscow; Gamaleya institute of Epidemiology and Microbiology of the Academy of Medicul Sciences of the USSR, Moscow, USSR

(Received 15 April 1991; accepted 25 June 1991)

Summary - Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ACL cell line displays the highest sensitivity to platelet cytotoxicity. Using the ACL cells, we discovered that platelets from oncological patients and normal donors display comparable cytotoxicity. The level of platelet lytic activity is irrelevant to tumor localisation; however, it appears to be dependent on the stage of tumor growth. Incubation of platelets, both from donors and patients, with PAF (concentration range 10 pM to 10 nM) results in a significant rise of the killing activity of platelets. PAF induces greater cytotoxicity enhancement for platelets with lower initial activity, this pattern appearing to be the specific feature of the PAF mediated effect. Hence, platelets can be considered as effector cells relevant to antitumor immunity; PAF-mediated enhancement of platelet cytotoxicity can appear to be useful in the search for new immunotherapeutic drugs. platelet I platelet activating factor ! cytotoxleity R&urn& - Cytotoxicit~ induite par Ies plaquettes et son renf~eement par fe facteur aetivateut de pl~quette. La cytotoxicitt! des plaquettes a Et& &ok&e chez 70 makdes atteints de cancer avec des localisatio~ tmorales vuriries, et c&e-z 30 donneurs sains. Les rt%ultats pr&ent& ici rPvPlent que la 1ignPe cellulaire ACL se montre la plus sensible ci lu ryrotoxicit6 des plaqueties. En se servant des cellules ACL, les auteurs ont constat qua les plaqttettes provenant de maludes atteints de cancer excercent la mbte cyroroxicit& que celles qui proviennent de donneurs sains. Le degrt! d’activit& lytique des plaquettes est indPpendant de la localisation tumorale; il apparart nkanmoins comme dPpendont du stade de dPveloppement de la tumeur. Le fait de mettre en incubation les plaquettes, celles des donneurs comme celles des malades, avec du PAF (ri des concentrations allant de 10 pM b 10 nM) a pour r&dtat une au~mentution significotive de I’activitP rtueuseu des plaquettes. Le PAF provoque un ren*forcement plus important de la cytotoxicit6 pour les plaquettes ayont une activit6 initiale plus foible. Cette caract&istique apparait come spkifque des effets ayant pour midiateur le PAF. Ainsi les piaquetles peuvent&es Ptre consid&Jes comme des cellules effectrices relevant de I’immunite celluiaire. Ce renforcement induit par le PAF de la cytotoxicitC: plaquettaire prt%entera sans doute un it&r& &ns la recherche de nouveaux mtiicaments en immunothtirapie.

plaquette I PAF I cytotoxlcit6

Introduction Recent studies have demonstrated that the role of platelets is not confined to their participation in thrombus formation. Although formally these cells do not constitute the immunocompetent system, they can be activated in various immunoiogiAbb~vialions: PAF, platelet activating factor; ACL, lung adenoearcinoma cancer cell; Mel-l, melanoma

cal reactions and involved as effector cells in infectious processes [6. 131. Platelets are also involved in the host response to malignant tumor growth [4, 8, 9, 14-161. In particular, in cancer patients with various tumor localizations a characteristic reaction of the megacaryocyte-platelet system was reported, manifested in increased granularity of these cells [14]. Besides, cancer patients, as a rule, display platelet deficiency which is accompanied by a tendency to increased

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platelet aggregation [ 14-161. It is also known that platelets facilitate the metastasis and endovascular invasion of malignant cells 19. 161. As demonstrated in a number of studies, platelet activity is stimulated by the platelet activation factor (PAF), which causes liberation of biologically active substances from the secretory granules [2, 31. Furthermore, PAF can also activate the elements of the immunocompetent system during tumor growth [ 1, 171. Incubation of monocytes with PAF was shown to increase their cytotoxicity [17]. PAF and its analogs were shown to display antitumor effect in trials in laboratory animals; the authors assigned their action to the direct effect of these phospholipids on the tumor cells as well as to the possible activation of the immunocompetent system [ 10-12. 171. Hence, it appears quite probable that PAF is capable of enhancing the antitumor activity of platelets. Similar to T-lymphocytes and monocytes, platelets display killing activity against malignant cells. Grethen and colleagues [S] demonstrated that donor platelets can lyse melanoma cells. We have earlier reported that platelets from lung cancer patients kill freshly isolated autologous tumor cells and the allogeneic transplantable lung adenocarcinoma (ACL) cell line [4]. The aim of this study was to compare platelet activities against ACL in normal donors and in cancer patients with various tumors at different stages of the disease; a panel of other tumor cell lines was also examined as well as the influence of PAF on platelet cytotoxicity.

the laboratory for experimental tumor models; AllUnion Cancer Research Center of the Academy of Medical Sciences of the USSR, Head - Dr Sci (Med)

Ye S Revazova). HeLa and Molt-4. Prior to assay tumor target cells were labeled with 25-50 mCi/ml of Naz “CrG4 (Medradiopreparat. USSR) in a water bath at 37’C. After 45-60 rnin incubation the cells were washed three times with RPM1 1640 and plated in microtiter wells (2 x lo4 cells per well) of a 96-well Falcon plate. Platelets were added in 100: I, 5O:l. 25:1, 12: 1 effector tp target ratios, the total added volume being 0.2 ml per well. The cell mixtures were incubated for 18 h at 37°C and 5% CO2 in RPM1 1640 containing PAF (Sigma, USA) in concentrations from 10 nM to IO pM. Supernatant was carefully sampled into plastic tubes to measure the radioactivity with the I&spectrometer. Specific chromium release was calculated using the formula: experimental cpm - spontaneous cpm x loo % % lysis= maximal cpm - spontaneous cpm where experimental cpm gives the mdan isotope on platelet addition; spontaneous cpm refers to the isotope release with intact target cells: maximal cpm (complete lysis) corresponds to the total amount of isotope incoiporated by the target cell. Each assay was performed in triplicate. The values in the tables are the mean of triplicates. The suspension containing platelet-target cell conjugates was incubated at 37°C for 5, 15, 30 and 60 min and fixed in 1% glutaraldehyde with phosphate buffeg at pH 7.2, centrifuged for 15 min at 250 g, the pellets were treated with 1% 0~04 using Dalton’s method [7], and embedded in the mixture of Epon and araldite. UItrathin sections were cut on an LKB-4800 ultratome. stained with 1% water solution of uranyl acetate and then with lead citrate as described in [l8], or with 15% solution of uranil acetate in methanol. The preparations were examined in an JEM-IOOB electron microscope (x 5000).

Materials and methods Platelet cytotoxicity was assessed in 85 patients (30-70 years old) with various tumors: lung cancer, 31; breast cancer, 18; gastric cancer, 14; Kaposi’s sarcoma, 22. Platelet cytotoxicity of 39 normal donors was also examined. IO ml of peripheral venous blood was collected into tubes containing heparin (15 U/ml). Platelet-rich plasma was Qlbtained by centrifugation of heparinized blood first at 200 p (IO min. 4”C), and then at 1600 g (20 min, 4’C). The platelet precipitate was then resuspended in 1 ml RPM1 1640 (Gibco. Flow) containing 5-10% of inactivated human serum, 2 mM L-glutamine (Gibco), 5 mM Hepes (Gibco), 3 x 10” M mercaptoethanol (Merck), and 100 U penicillin, and 100 mg streptomycin per 1 ml. The platelet suspension thus obtained contained no contaminating mononuclear cells. The following tumor cell lines were used as targets: ACL monolayer culture of lung adenocarcinoma cancer Cells (Timofeevskaya et al, 1983). Mel-l (produced in

Results In the studies of cytotoxicity of platelets from tumor patients on ACL, HeLa, K-562, and Mel-l cell lines it was discovered that ACL cells are the most sensitive. Platelets from patients with breast cancer, lung and gastric cancer demonstrated higher cytotoxicity for ACL cells (percent lysis 15.2 f 4.4%) as compared with HeLa (9.2 f 2.3%). Mel-l (4.0 f 0.9%). and Molt-4 (0.3 f 0.1%) (fig 1). Hence, taking into account the high sensitivity of ACL line to platelet cytotoxicity, it is this line that was used in further studies. The cytotoxic effects of platelets from normal donors and cancer patients were found to lack significant difference. Cytotoxicity for cancer

Platelet cytotoxicityin cancer patients patients was on the average 17.7 jI 4.8%, whereas in normal subjects it was 13.0 f 5.2%. In both cases the killing activity was subject to vary ranging from 0 to 38.8% in normal donors and to 47.9% in oncologicat patients (fig 2). % aytotoxicrity

8

$& oe

I) b

Mel-1

)o

E”ii 1. Lung adcn~carcjnoma cetl line sensitivity to plarelei cytotoxic elfect. Pla~etets from patients and normai dcmrs were incubsied for I8 h with Naz skrU.+ tabekd target cells; effector to target ratios were I#:I, J&I, 25:5, f2:i. Platelets from breast cancer, lung cancer, and gastric cancer patients were more toxic for ACL cells (percent lysis 15.2 f 4.4%) than for HeLa (9.2 f 2.3%), Mel-i (4.0 f 0.9%). and MOB-4 (0.3% f 0. I Sbf lines.

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Platelet cytotoxicity for malignant cells was actually indepertdent of tumor localization (fig 3); however, platelets from patients suffering of breast cancer were sI~ghtly more cyt~t~~i~ (21.1 f 4.5%) than from lung cancer (16.2 f 3.3%) Kaposi’s sarcoma (16.2 f 5.3%) and gastric cancer (15.0 f 2.2%) patients. The lytic activity of platelets was found to be essentially dependent on the tumor growth stage (fig 4). At the initial stage platelet activity (10.3 f 2.7%) was the same as in normal donors (13.1 f 5.2%). At the second stage platelet cytotoxicity increased approximately two-foEd (24.3 f 4.2%). At the third stage the pfatelets cytotoxicity was comparable to that of the initial stage (10.5 f 3.4%). whereas at the fourth stage of the disease platelet cytotoxi~ity decreased to 6.1 SC2.3%. Incubation of platelets both from normal donors and cancer patients with PAF (concentration range from 10 pM to 10 nM) resulted in a significant enhancement of the killing activity of platelets. In this series of experiments it was established that initial cytotoxicity increased from 12.3 f 2.4% to 21 S f 3.5% in normal donors and from 12.2 i 1.9% to 21.8 f 2.7% in patients (fig S), PAF was found to induce greater cytotoxicity enhancement for platelets with the lower initial activity, this appearing to be a specific feature of the PAF mediated effect. Indeed, the killing activity of platelets with initial cytotoxicity Wow % cytotcmlcrity

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25

40 15

20

9 0 -10

Fig 2. Ptatelet cytotoxicity in normai donors and cancer patients. Platelet cytotoxicity was assessed in 70 cancer patients with various tumor vocalizations aad 50 normal donors. The difference in platelet cytoto%icity for ACL cells in patients (17.7 f 4.8%) and donors { 13.0 f 5.2%) is not statjstjcally signi~can~

0

Fig 3. P&At cytotoxicitg in normaI donors and in CSEW patients. Cytotoxic effects of platelets from 30 donors (I); gastric cancer, 10 (2); Iung cancer, 24 (4); breast cancer, 16 (5); and Kaposi sarcoma, 20 (3).

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10% was increased several times on incubation with PAF, whereas the platelets with high intrinsic lytic activity were unaffected by PAF (fig 6). % cytotorlclty

40

1

30

20

10

0

donore lonors

I

II

Fig 4. Platelet cylotoxicity in cancer patients: dependence on the stage of the disease. The lytic activity was assessed in groups: 1. IO patients with TINOMOor TI-zNeMo stage; II. 20 padents with TI-~NIMo. TI-zNo_lMo or TI-nNo_zMostage: Ill. 20 patients with TI-IN-2hlo. TI-~Nz-~Mo or Tz-~No-zMo stage; and IV. 20 patients with TLINO-~MI or TI-~N-0-2Ml stage. Cytotoxicily was the highest in patients of the II group (24.3 f 4.2%): in patients of the I (10.3 f 2.7%) and the III (10.5 f 3.4%) groups cytoloxicity was not different from that in normal donors (13.1 f 5.2%); in the IV group cytotoxicity decreased to 6.1 f 2.3%.

0 cptotoxiclty

patiente

Fig 6. The level of PAF-mediated increase of platelet cytotoxicity (A) depends on the initial cytotoxicity level ( causes significant increase of platelet cylotoxicity in patients and normal donors with low initial platelet activity: it produces only a slight increase of the lytic effect of platelets with high initial acidvity.

As demonstrated in electron microscopic studies, platelets are absorbed on the target cells followed by the changes in platelet ultrastructure, which are characteristic of secretory cells with discontinuous secretive type. These changes involve hypertrophy of the Golgi apparatus, growth of secretory granules, and their orientation towards the contact zone (figs 7-10).

Discussion

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Fig 5. Platelet cytotoxicity in 15 patients ( (0) in the presence of PAF. Platelets fromsgatients and normal donors were incubated for 18 h wilh Naa CrO4-labeled large1 cells and PAF added at doses from IO nM to IO pM; effector to target ratios were 5O:l. Maximal cytotoxicity enhancement was observed with PAF concemrations ranging from 500 pM to I nM.

The data presented demonstrate that the ACL cell culture exhibits the highest sensitivity to plateletmediated cytotoxicity; platelet lytic activity for HeLa, Mel-l, Molt-4, K-562 cells are significantly lower (P c 0.01). Hence, the ACL line can be suggested as the standard target in investigations of platelet cytotoxicity. The reasons for the observed differences in sensitivity of the target cells to the lytic effect of platelets are still unclear. Further investigations should be undertaken to clarify the phenomenon. Using the ACL cells we discovered that platelets from both cancer patients and normal donors

Platelet

cytotoxicity

in cancer patients

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F&J 7. a. ~~5u~~~~n of pfrtelrts art the surface of target cetls. Section across the equatorial angle of ptateletswA~ITWS Sb+~vv mieretubule~ at the outlying area of platelet. x 2~0~.

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Fig 8. a. Adsorption of platelet on the cell. Arrows show the bundles of peripheral microtubules. x 20.000. b. a fragment of figure S a. MT microtubules (transverse section); SVS, surface vacuolar system; DB, dense body. x I15 000.

Platelet cytotoxicity

in cancer patients

Fig 9. Orientation of Go&i complex near the zone of platelet-terget cell contact. Arrow points to the Golgi complex vacuole and plasma membrane of platelet. x 3500~.

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area of fusion between

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Fig 10. a. Accumulation StrucIures

in the cytoplasm

of Igranules of platelet.

in the cytoplasm x 100000.

of platelet,

SVS.

surface

vacuolar

system.

x 120000.

b. Crystal-like

Platelet cytotoxicity display comparable cytotoxicity. We want to stress that the cytolytic activity of platelets was found in all cases of cancer, whereas it was not

detectable in 15% of healthy donors. The level of platelet lytic activity is irrelevant to tumor localization: however, it appears to be dependent on the stage of the tumor growth. The cytolytic effect of platelets observed at the first stage of disease is the same as in control. At the second stage platelet cytotoxicity is almost two-fold higher. At the third stage platelet cytotoxicity is comparable to that of normal donors. At the terminal (IV) stage cytolytic activity of the platelets from patients decreases by two-fold as compared to donors. The observed connection between cytotoxicity level W-MS tumor growth stage leads to the assumption that the platelet response to tumor cells involves immune mechanisms. This assumption is in accordance with the data of Capron and colleagues [ 1I, who reported on the immune character of the protective action displayed by platelets in inflammatory and infectious diseases. As demonstrated

in electron microscopic studies, platelets are adsorbed on the target cells and this is followed by changes in platelet ultrastructure, which are characteristic of secretory cells with discontinuous secretive type. These changes involve hypertrophy of the Golgi apparatus, enlargement of secretory granules, and their orientation towards the contact zone; these are very similar to those observed in T-lymphocytes upon contact with tumor cells [5]. Hence, it can be suggested that platelet cytotoxicity is due to their ability to produce and liberate a mediator capable of inducing the lytic effect via transmembrane route or by permeating into the cell via endocytosis. The increase of the killing activity of platelets on incubation with PAF supports the secretory hypothesis of platelet cytotoxicity since PAF csn stimulate the evolution of the contents of platelet secretory granules [2, 31. Different mechanisms of plate!ct cytotoxicity can be suggested: in particular, specialized enzymatic systems at the platelet membrane or cytophilic antibodies could be involved. Like T-killers, platelets might have been also capable of binding the antigens of tumor cells with complementary receptors. By activating

phosphoinositide

metabolism, to contract and, thus, affects the shape of the platelets. Consequently, receptor structures can become exposed at the cellular membrane, these being PAF causes the proteins of cytoskeleton

in cancer patients

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capable of interacting with antigenic determinants of tumor cells. Hence, relying on the presented experimental data one may consider platelets to constitute one of the elements of the immunocompetent system, like lymphocytes and monocytes, displaying specific antitumor activity. The platelet cytotoxicity enhancing effect of PAE as well as the reported in viva antitumor action of the analogs of this endogenous mediator, give grounds to suggest that, along with interleukins, this class of phospholipids may appear to provide a fruitful area for the search of new immunotherapeutic antitumor drugs.

References Urcotte S, Rola-Pleszczynski N (1988) Platelet activating factor enhances the production of

1 Bosse J.

cytotoxic cytokines during natural cell-mediated cytotoxicity. Am Sot Exp Biol J 2, 691 2 Braquet P, Shen TY, Touqui L, Vargaflig BB (1987) Perspectives in platelet-activating factor research. Pharmacol

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3 Brindley LL, Swet JM. Goetzl EJ (1983) Stimulation of histamine release from human basophils by platelet factor. J Clin Itzst 72. 1218 4 Bykovskaya SN, Blochina NG. Speransky DL, Kuprijanova TA (1988) Spectfic antitumor cytotoxity of platelets from patients with lung cancer. Bull Exp Biol Med 6, 708

5 Bykovskaja SN, Rytenko AN, Raushenbach MO, Bykovsky AF (I 979) Ultrastructural alteration of cytolytic T-lymphocytes following their interaction with target cells. Cell Immunol 42, 197 6 Capron A, Joseph M, Ameisen JC, Capron M, Pancre V, Auriault C (1987) Platelet as effecters in immune and hypersensitivity reactions. Int Arch Allergy Appl Immunol 82, 307 7 Dalton AT (1955) A chrome-osmium fixative for electron microscopy. Anat Ret 121, 281 8 Grethen MI, Kay NE, Jonson KJ, Jacol G (1985) Human platelets exert cytotoxic effects on tumor cells. Blood 65. 1252 9 Hilgard P, Heller H, Schmidt CG (1976) The influence of platelet inhibitors on metastasis formation in mice. Z Krebsforsch 86. 243 10 Hoffman DK, Hajdu J, Snyder E (1984) Cytotoxicity of platelet activating factor and related alkyl-phospholipid analogs in human leukemia cells, potymorphonuclear neutrophils and skin fibroblasts. Blood, 63, 545 I1 lchiro K, Shoahichi N, Wook CH, Ryohei Y. Hidetoshi H, Eri K, Hiroaki H, Keizo I (1987) Antitumor

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activity of synthetic alkylphospholipi~ with or without PAF activity. Lipids 22, 862 12 Koetting J, Fleer EAM. Unger C, Eibf H (1988) Synthetic alkyllysophosphoiipids as antitumor drugs structural relation to platelet activating factor. Ferr Wiss Technol 90%345

Panre V, Auriault C, Joseph M, Cesbron JY. Kusnien JP, Capron A (1986) Suppressive lymphokine of platelet cytotoxic functions J lmmunol 137, 585 14 Prijivot GN (1964) Platelet and megacaryocyte contents in tat intmnspiantabletumors. Vopr Onkoi 3, 46 I3

15 Scheider W, Scharf RE. Au1 C (1984) Trombozyten und Tumork~n~eit. 2 Gtrsrroenzerol 22, 80 16 Skoinik G, Ericson LE. Bagge UJ (1983) The effect of thrombocytopenia and antiserotonin treatment on the lodgment of circulating tumor cells. Res C/in Oncol 105, 30 17 Valone FH, Philip K, Dess RJ (1988) Enhanced human monocyte cytotoxicity by platelet activating factor (PAF). Immunology 64, 715 18 Vanable JH, Hoggeshall RJ (1965) A simplified lead citrate strain for use in electron microscopy. J Cell &of 25, 407

Platelet-mediated cytotoxicity and its enhancement by platelet activating factor.

Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ...
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