The role of reactive oxygen metabolites in lymphocyte-mediated
cytolysis
C. J. BISHOP*, C. M. RZEPCZYK, D. STENZEL and K. ANDERSON
Queensland Institute of Medical Research, Bramston Terrace, Herston, Queensland 4006, Australia
* Author for correspondence
Summary
To examine the possible role of reactive oxygenmetabolites in lymphocyte-mediated cytolysis,the morphology of cell death following the ex-posure of cells to reactive oxygen metabolites invitro was compared with the morphology of cell-mediated killing in vitro of tumour cells by natu-ral killer (NK) cells. Ultrastructural examinationof human tumour cells that were dying followingincubation for 60 min with the oxygen metabolitegenerating systems, xanthine-xanthine oxidaseor f-butylhydroperoxide, showed that cell deathin both instances was exclusively by necrosis. Itwas unclear which oxygen metabolites were in-volved in killing. Cell death was not decreasedby the addition of superoxide dismutase, a scav-enger of the superoxide anion, to the xanthine-xanthine oxidase mixture. Although the cells
were not killed by incubation with 1 mM-hydro-gen peroxide, the addition of catalase, a scaven-ger of hydrogen peroxide, to the xanthine-xanthine oxidase mixture significantly reducedcell death. The addition of scavengers for thehydroxyl radical to either the xanthine-xanthineoxidase mixture or t-butylhydroperoxide gaveinconsistent protection.
In contrast, tumour cell killing mediated bynatural killer cells was by apoptosis, a morpho-logically distinct mode of cell death with adifferent basic mechanism, indicating that reac-tive oxygen metabolites are not directly involvedin lymphocyte-mediated cytolysis.
It has clearly been shown that reactive oxygen metab-olites are involved in phagocyte-mediated antimi-crobial activity (Babior, 1978; Segal, 1984) and thereis considerable evidence that antibody-dependent cell-mediated cytotoxicity (ADCC) against parasites byphagocytes also depends on the generation of reactiveoxygen metabolites (Klebanoff, 1980; Klebanoff etal. 1983). However, the role of reactive oxygenmetabolites in lymphocyte-mediated cytolysis againsttumour cells is unclear as many published findingsare contradictory. Studies have shown that T-cell-mediated cytolysis does (Devlin et al. 1981; Nathan etal. 1982) or does not (MacDonald & Koch, 1977)depend on oxygen and is (Thorne et al. 1980; Thorne& Franks, 1983) or is not (Nathan et al. 1982)mediated by reactive oxygen metabolites. Similarly, ithas been suggested that natural killer (NK) cell-Journal of Cell Science 87, 473-481 (1987)
mediated killing does (Roder et al. 1982; Babior &Parkinson, 1982; Duwe & Roder, 1984; Suthanthiranet al. 1984) or does not (Kay et al. 1983; El-Hag &Clarke, 1984; Ramstedt et al. 1984) utilize reactiveoxygen metabolites. Moreover, antibody-dependentlymphocytotoxicity mediated by killer (K) cells hasbeen shown to involve (Bowman & Shoeb, 1984) ornot involve (Katz et al. 1980) reactive oxygen metab-olites.
It has been recently recognized that there are twodistinct morphologically recognizable types of celldeath, necrosis and apoptosis (sometimes referred toas zeiosis), which have different basic mechanisms(Wyllie et al. 1980; Searle et al. 1982; Kerr etal. 1984). Morphological studies of complement-me-diated lysis (Goldberg & Green, 1959) and phagocyte-mediated antimicrobial or antiparasitic activity(Thorne & Blackwell, 1983; Rzepczyk & Bishop,1984), show cell death by necrosis. However, studies
473
of lymphocyte-mediated cytolysis by either T cells(Don et al. 1977; Liepins et al. 1977; Sanderson &Glauert, 1977; Matter, 1979; Kerr et al. 1984),human K cells (Stacey et al. 1985) or murine NK cells(Bishop & Whiting, 1983) have shown that target cellsdie by apoptosis.
To examine the possible role of reactive oxygenmetabolites in lymphocyte-mediated cytolysis, themorphology of cell death following the exposure ofcells to reactive oxygen metabolites in vitro wascompared with the morphology of NK cell-mediatedkilling of tumour cells. Cells were exposed to either axanthine-xanthine oxidase mixture, /-butylhydroper-oxide or hydrogen peroxide (H2O2). The xanthine-xanthine oxidase mixture generates the superoxideanion (O2~) and to a lesser extent H2O2 (Fridovich,1970). The further reduction of enzymically producedH2O2, in the presence of ferrous or cuprous ions,results in the formation of the hydroxyl radical ('OH)(refer to Halliwell & Gutteridge, 1984). i-butylhydro-peroxide is homolytically split, in the presence ofcellular haemoproteins, into its alkoxy radical and'OH (Cadenas & Sies, 1982). In this paper we showthat the morphology of cell death following exposureto xanthine-xanthine oxidase or f-butylhydroperoxidediffers from that involved in the killing of tumour cellsby NK cells.
Materials and methods
Culture medium
Cells were cultured in RPMI 1640 (Commonwealth SerumLaboratories, Vic.) supplemented with 10% heat-inacti-vated foetal calf serum (FCS), penicillin (lOOi.u. ml"1) andstreptomycin '
Cell lines
Experiments involving the addition of reactive oxygenmetabolites were carried out on the human myeloid leu-kaemic line K562 (Lozzio & Lozzio, 1975), the humanBurkitt's lymphoma cell line BL36 (Rooney et al. 1984) andthe human lymphoblastic leukaemic line HSB2 (Krishan &Raychaudhuri, 1970).
Lymphocyte-mediated cytolysis
Unfractionated human peripheral blood mononuclear cellswere isolated from heparinized blood by isopycnic centri-fugation on Ficoll/Paque (Pharmacia) as described (Boyum,1968). Monocytes were removed by filtration through cottonwool as described (Stacey et al. 1985). The lymphocyteswere resuspended in culture medium at 106ml~' beforebeing mixed with K562 or HSB2 cells in a ratio of 4:1 inculture medium, centrifuged at 250£ for 3 min to facilitatecell to cell contact and incubated at 37°C. At various timesthe mixtures were centrifuged at 10000£ for 1 min (in abench microfuge) and the pellets processed for electronmicroscopy.
Exposure to reactive oxygen metabolitesCells were exposed to either a xanthine (Sigma, USA)-xanthine oxidase (Calbiochem, Australia) mixture, /-butyl-hydroperoxide (Sigma) or H2O2. All reagents except xan-thine were made up as required, in RPMI 1640. Xanthinewas made up in 0-015 M-NaOH. Cells were incubatedat 37CC at 105ml~' in xanthine (1 mM)-xanthine oxidase(125munitsmr' or 50munitsml~') mixture, H2O2 (1 mMor 10 mM) or f-butylhydroperoxide (1 mM) in 1 ml microfugetubes in triplicate. Various scavengers of reactive oxygenmetabolites were added to some cell cultures 10 min beforethe addition of xanthine-xanthine oxidase or f-butylhydro-peroxide as above. Superoxide dismutase (SOD, frombovine erythrocytes, Calbiochem) was used at 350 unitsml"1 as a scavenger for O2 ', catalase (from bovine liver,Calbiochem) was used at 1000 units ml"1 as a scavenger forH2O2; L-histidine (Sigma) was used at 100 mM as a scavengerfor the singlet oxygen radical (O2); dimethylsulphoxide(DMSO), ethanol, methanol and mannitol (Sigma) wereused at 100 mM, 80 mM, 300 mM and 100 mM, respectively, asscavengers for 'OH. After incubation for 60 min, the cellswere centrifuged and resuspended in cold RPMI 1640.Some cells were processed for electron microscopy. Percent-age cell death was estimated as below.
Morphological markers of cell deathCertain histological changes associated with cell death,classically designated as nuclear pyknosis and karyorrhexis,occur in both necrosis and apoptosis (Wyllie et al. 1980;Searle et al. 1982). Necrotic cells also show karyolysis andswelling of the cytoplasm, which eventually loses its baso-philia, and cell boundaries become indistinct (Trump &Arstila, 1975; Wyllie et al. 1980; Searle et al. 1982).Apoptotic cells appear to be condensed, typically withintensely eosinophilic cytoplasm, which frequently showssurface protrusion. These protuberances separate to formroughly spherical bodies sometimes containing basophilicnuclear fragments (Wyllie et al. 1980; Searle et al. 1982).
By electron microscopy, necrotic cells exhibit aggregationof chromatin and extensive nuclear and cytoplasmic swell-ing. Mitochondria show gross swelling and develop floccu-lent and sometimes granular densities. Nuclear, organelleand plasma membranes rupture, and cellular organellesdisintegrate and disperse (Trump & Arstila, 1975). Typicalfeatures of apoptosis are aggregation and margination ofchromatin with nuclear and cytoplasmic condensation andloss of microvilli. Generally this is followed by nuclearfragmentation and the budding off of surface protuberancesto form membrane-bound bodies that contain intact cyto-plasmic organelles and/or nuclear fragments (Wyllie et al.1980; Searle et al. 1982).
Estimation of cell death
The percentage of cell death by necrosis was determined bythe failure of necrotic cells to exclude Trypan Blue dye andwas estimated by light microscopy from counts, in haema-cytometer chambers, of 300 cells from each of duplicatewells from each sample. The percentage of cell death byapoptosis was estimated by electron microscopy from countsof 300 cells in each of duplicate thin sections from each
474 C. jf. Bishop et al.
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Fig. 1. HSB2 cells, following incubation in xanthine—xanthine oxidase (125 munitsml ) for 1 h, showing mitochondrialand cellular swelling, membrane rupture and dissipation of cellular contents. X5500.
sample. Where apoptotic bodies were in a compact cluster,the cluster was scored as one.
Electron microscopy
Cells were fixed in 3 % glutaraldehyde in 0-1 M-cacodylatebuffer (adjusted to 300mmolkg~' H2O real osmolality withsucrose) for 1 h at room temperature, washed, post-fixedwith 1 % OsC>4 for 1 h at room temperature, en bloc stainedwith uranyl acetate, dehydrated in ethanol and embedded inSpurr's low viscosity embedding media (Polyscience, PA).Centrifugation was used to compact the specimen after eachtreatment. Thin sections were stained with uranyl acetateand lead citrate and examined in a Philips EM 4€0.
Results
Ultrastructure of cell death by reactive oxygenmetabolites
When K562, BL36 or HSB2 cells were incubated witha xanthine-xanthine oxidase mixture or (-butylhydro-peroxide, a significant proportion of the cells died bynecrosis within 60min. Cells incubated with eitherxanthine-xanthine oxidase or f-butylhydroperoxideinitially showed a decrease in the number of microvilli,marked condensation of the mitochondria and somedilation of the rough endoplasmic reticulum (ER).Typically, with further incubation there was aggre-gation of chromatin, gross mitochondrial swelling,
increased dilation of the ER, and the cells becameswollen with a resulting decrease in the density of thematrix of both the cytoplasm and nucleus and, eventu-ally, membrane rupture (Fig. 1). Although a system-atic study was not carried out, the speed of the abovesequence of events, including mitochondrial swelling,varied according to the target cell line and the concen-tration of xanthine oxidase or /-butylhydroperoxide.For example, while mitochondrial swelling alwaysoccurred sequentially to mitochondrial condensation,both condensed and swollen mitochondria couldsometimes be observed in otherwise normal BL36 cellsafter incubation with 1 mM-Z-butylhydroperoxide forlh (Fig. 2). In some K562 cells incubated withxanthine-xanthine oxidase (125 munitsmP1) for 1 h,mitochondria remained condensed although the cyto-plasm was severely disrupted (Fig. 3). In some K562cells, there was also a marked increase in the numberof cytoplasmic multivesicular bodies following incu-bation (Fig. 4). In all cell lines, there was, finally,membrane rupture and dissipation of the cellularcontents (Fig. 1). Cells incubated for l h with 1 mM-H2O2 generally showed condensation of mitochondriaand some dilation of the ER (not shown), although asmall proportion of cells (<5 %) were observed withswollen mitochondria as described above. Incubationof cells for l h with 10mM-H2O2 resulted in rapiddeath by necrosis as described above (not shown). Inall instances, apoptosis was rarely observed.
Oxygen metabolites in lymphocyte-mediated cytolysis 475
Percentage cell death induced by reactive oxygenmetabolites
The percentage cell death by necrosis, as measured bythe dye-exclusion assay, in cultures of K562 and BL36cells following exposure for 60 min to xanthine-xanthine oxidase, /-butylhydroperoxide or H2O2 isshown in Table 1. There was a significant increase innecrosis by xanthine-xanthine oxidase and by t-butylhydroperoxide. The percentage necrosis in cul-tures incubated with 1 mM-H2O2 was low or notsignificant. Incubation of cells with IO1T1M-H2O2 for60min resulted in 100% necrosis (not shown). There
was no significant increase in the percentage cell deathby apoptosis, as determined by counting in the elec-tron microscope, in any of the cultures (not shown).
The percentage necrosis, as measured by the dye-exclusion assay, in cultures of K562, BL36 and HSB2cells after incubation for 60 min with xanthine-xanthine oxidase in the presence of various scavengersof reactive oxygen metabolites is shown in Table 2.The percentage necrosis was not decreased by theaddition of superoxide dismutase (SOD), a scavengerfor C>2~, or by histidine, a scavenger for "02, but wassignificantly decreased by the addition of catalase, a
Fig. 2. BL36 cell, following incubation with 1 mM-<-butylhydroperoxide for 1 h, showing both condensed and swollenmitochondria. X 15 000.
Fig. 3. KS62 cell, following incubation in xanthine-xanthine oxidase (125munitsml ') for 1 h, showing condensedmitochondria associated with severe cytoplasmic disruption. X 11 500.
476 C. Jf. Bishop et al.
Fig. 4. Numerous multivesicular bodies in a K562 cell, following incubation in xanthine-xanthine oxidase(125 munitsmr1) for 1 h. X21 000.
Table 1. Percentage cell death in cultures of tumourcells following incubation with xanthine-xanthineoxidase (125munitsmr'), t-butylhydroperoxide
scavenger for H2O2. Most scavengers for "OH thatwere tested, i.e. mannitol, DMSO or methanol, didnot consistently decrease the percentage necrosisalthough DMSO and methanol markedly reducednecrosis in some experiments, resulting in the largestandard deviations shown in Table 2. However,ethanol, another scavenger for 'OH, significantlyreduced necrosis. There was no significant increase innecrosis in cultures incubated with scavengers alone(not shown). Similarly, the percentage cell death bynecrosis in cultures of K562 and BL36 cells afterincubation for 60min with /-butylhydroperoxidecould be reduced, sometimes markedly but incon-sistently, by the addition of mannitol or ethanol(Table 3).
Ultrastructure of lymphocyte-mediated cytolysisWhen tumour cells were incubated with peripheralblood lymphocytes, there was attachment of some
Oxygen metabolites in lymphocyte-mediated cytolysis 477
Fig. 5. K562 cell, showing the morphological changes associated with cell death by apoptosis, following incubation for 3 hwith human peripheral blood lymphocytes. X 12 500.
lymphocytes to some of the tumour cells. Followinglymphocyte attachment, the tumour cells showed earlychanges associated with cell death by apoptosis, suchas fragmentation of the nucleus into membrane-boundfragments, loss of microvilli and budding of thecytoplasm (Fig. 5). Once the process of apoptosisbegan, it appeared that the lymphocytes detachedfrom the dying cells and the latter budded into anumber of membrane-bound fragments termed apop-totic bodies (Fig. 6). Eventually apoptotic bodiesunderwent secondary disintegration, with mitochon-drial swelling and membrane disruption (not shown).
Discussion
A significant percentage of cells die following incu-bation for 60 min with xanthine—xanthine oxidase or t-butylhydroperoxide. Ultrastructural examination ofdying cells showed that cell death in both instanceswas exclusively by necrosis. Cells initially showed theearly reversible changes associated with cell injury,such as condensation of mitochondria, but within60 min showed the irreversible changes associated withnecrosis, such as high amplitude swelling of mitochon-dria and membrane rupture (Trump & Arstila, 1975).
It was unclear which oxygen metabolites wereinvolved in killing, f-butylhydroperoxide is homolyti-cally split, in the presence of cellular haemoproteins,into its alkoxy radical and 'OH (Cadenas & Sies,1982). These radicals can induce lipid peroxidation
and the subsequent production of lipid oxyl andperoxyl radicals (Halliwell, 1984; Halliwell & Gutter-idge, 1984). The aerobic oxidation of xanthine byxanthine oxidase generates C>2~ and to a lesser extentH2O2 (Fridovich, 1970). C>2~ is only poorly reactive inaqueous solution and it has been suggested that it isnot reactive enough to be directly toxic in mostinstances (Klebanoff et al. 1983). Moreover, in theexperiments described here, cell death was not de-creased by the addition of SOD, a scavenger of O2~, tothe xanthine-xanthine oxidase mixture, indicatingthat O2~ was not directly involved in the killing.
It has been shown that different mammalian cellsexhibit different sensitivities to H2O2 and that thiscorrelates with their susceptibility to killing by acti-vated phagocytes (Nathan et al. 1979a). The tumourcells used as targets in this study, particularly K562and HSB2 cells, are commonly used as sensitivetargets in assays of NK cell-like cytotoxicity (e.g. seeRickinson et al. 1981) yet were relatively insensitive toH2O2. Although the cells rapidly underwent necrosisin high concentrations of H2O2, they were not killedby incubation with 1 mM-H2O2- As the xanthine-xanthine oxidase concentrations used in the aboveexperiments were unlikely to generate concentrationsof H2O2 greater than 1 mM within 60 min, it appearedthat in this instance H2O2 was not killing directly.Nevertheless, H2O2 appeared to mediate killing in-directly as the addition of catalase, a scavenger of
478 C. J. Bishop et al.
Fig. 6. Apoptotic bodies containing nuclear fragments formed after incubation of K562 cells for 3 h with human peripheralblood lymphocytes. X9000.
H2O2, to the xanthine-xanthine oxidase mixture sig-nificantly reduced cell death.
Recent studies suggest that erythrocyte membranedamage resulting from incubation in xanthine-xanthine oxidase is due to OH (Girotti & Thomas,1984). H2O2 can be reduced in the presence of ferrousor cuprous ions, resulting in the formation of 'OH(Halliwell & Gutteridge, 1984). It has been suggestedthat ferrous ions may be generated in the xanthine-xanthine oxidase mixture by the reduction of ferricions by O2~ (Halliwell & Gutteridge, 1984). How-ever, in the experiments described here, the additionof SOD to xanthine—xanthine oxidase did not preventkilling. The addition of scavengers for 'OH to thexanthine—xanthine oxidase mixture and to ?-butylhy-droperoxide gave inconsistent results. Of the scaven-gers for -OH added to xanthine-xanthine oxidase,only ethanol significantly reduced cell death, althoughcell death was sometimes markedly reduced by theaddition of DMSO or methanol. Similarly, the ad-dition of mannitol or ethanol to i-butylhydroperoxidesometimes markedly reduced cell death. It has beensuggested that the failure of some 'OH scavengers toprevent membrane damage induced by xanthine-xan-thine oxidase is due to 'OH being produced onmembranes at iron-binding sites and subsequentlyreacting so rapidly with target molecules that scaven-gers cannot compete (Girotti & Thomas, 1984).
It has been suggested that membrane damage byoxygen metabolite-generating systems is mediated inpart by singlet oxygen, '02, derived from the spon-taneous dismutation of 02~ or its reduction by H2O2(Lynch & Fridovich, 1978). However, there is littleevidence to support this (Halliwell, 1984) and in the
studies described here the addition of scavengers for02~ and '02 did not reduce killing.
Irrespective of the reactive oxygen metabolite in-volved, the killing associated with H2O2, xanthine-xanthine oxidase and /-butylhydroperoxide was bynecrosis. However, killing mediated by NK cells wasby apoptosis, a morphologically distinct mode of celldeath with a different basic mechanism, indicatingthat reactive oxygen metabolites were unlikely to bedirectly involved in NK cell killing. As killing me-diated by other lymphocytes (i.e. T and K cells) is alsoby apoptosis, reactive oxygen metabolites are unlikelyto be directly involved in lymphocyte-mediated cytoly-sis.
Some of the confusion in the literature may beexplained by the important observation that a res-piratory burst can be detected in cytotoxic T-cell-mediated killing of tumour cells only in the presence ofmycoplasma (Koppel et al. 1984). Recent studiessuggest that lymphocytes mediate cell killing by pro-ducing other cytotoxic substances, such as lympho-toxin (refer to Ruddle, 1985) and/or pore-formingmolecules (refer to Henkart, 1985; Podack, 1985), butagain there are contradictions. For example, the poresinduced in target cell plasma membranes by pore-forming molecules isolated from cytotoxic T cells andNK cells are said to be morphologically and bio-chemically related to those pores produced duringcomplement-mediated lysis (e.g. see Dennert &Podack, 1983; Henkart et al. 1984). However, asdescribed above, cell death resulting from lympho-cyte-mediated killing and from complement-mediatedlysis are morphologically quite distinct. It is unclearwhy lymphocytes induce apoptosis while complement
Oxygen metabolites in lymphocyte-mediated cytolysis 479
induces necrosis, if lymphocytes and complementproduce similar lesions.
The mechanism of phagocyte-mediated killing ofnucleated, non-erythroid mammalian cells is alsounclear. For example, it has been suggested that thephagocyte-mediated killing of tumour cells does(Nathan et al. 197%) or does not (Weinberg & Haney,1983) involve reactive oxygen metabolites, while otherstudies have implicated the involvement of cytotoxins(Mannel et al. 1980; Matthews, 1981) and pore-forming molecules (Young et al. 1986), depending onthe effector cell. Moreover, it has been shown thatmacrophages can kill tumour cells either in the pres-ence or absence of antibody, the resulting.target celldeath having the morphological appearance of necrosisor apoptosis, respectively (Dingemans et al. 1983). Itis possible that there may be a number of mechanismsthat can be used by different cell types and/or indifferent circumstances. However, one of these mech-anisms, the production of reactive oxygen metabolites,does not appear to be utilized by cytotoxic lympho-cytes.
References
BABIOR, B. M. (1978). Oxygen-dependent microbial killingby phagocytes. New Engl. J. Med. 298, 659-688 &751-775 (two parts).
BABIOR, B. M. & PARKINSON, D. W. (1982). The NK cell:a phagocyte in lymphocyte's clothing. Nature, Land.298, 511.
BISHOP, C. J. & WHITING, V. A. (1983). The role ofnatural killer cells in the intravascular death ofintravenously injected murine tumour cells. Br. J.Cancer 48, 441-444.
BOWMAN, B. U. & SHOEB, H. A. (1984). Effect of oxygenradical scavengers on K-cell cytolysis. Infect. Immun. 43,942-946.
BOYUM, A. (1968). Isolation of mononuclear cells andgranulocytes from human blood. Scand.J. din. Lab.Invest. 21(Suppl. 97), 77-89.
CADENAS, E. & SIES, H. (1982). Low levelchemiluminescence of liver microsomal fractions initiatedby tert-butyl hydroperoxide. Relation to microsomalhemoproteins, oxygen dependence, and lipidperoxidation. Eur.J. Biochem. 124, 349-356.
DENNERT, G. & PODACK, E. R. (1983). Cytolysis by H-2-specific T killer cells. Assembly of tubular complexes ontarget membranes. J . exp. Med. 157, 1483-1495.
DEVLIN, R. G., LIN, C. S., PERPER, R. J. & DOUGHERTY,
H. (1981). Evaluation of free radical scavengers instudies of lymphocyte-mediated cytolysis.Immunophannac. 3, 147—159.
DINGEMANS, K. P., PELS, E., VAN DONGEN, G. & DEN
OTTER, W. (1983). Destruction of murine lymphomacells by allogeneic peritoneal macrophages in vitro:Influence of antiserum. jf. natn. Cancer Inst. 70,181-192.
DON, M. M., ABLETT, G., BISHOP, C. J., BUNDERSEN, P.
G., DONALD, K. J., SEARLE, J. & KERR, J. F. R.
(1977). Death of cells by apoptosis following attachmentof specifically allergized lymphocytes in vitro. Aust. J.exp. Biol. med. Set. 55, 407-417.
DUWE, A. R. & RODER, J. C. (1984). Involvement ofhydroxyl free radical, but not superoxide, in the cytolyticpathway of natural killer cells. Med. Biol. 62, 95-100.
EL-HAG, A. & CLARK, R. A. (1984). Intact natural killeractivity in Chronic Granulomatous Disease: evidenceagainst an oxygen-dependent cytotoxic mechanism. .7.Immun. 132, 569-570.
FRIDOVICH, I. (1970). Quantitative aspects of theproduction of superoxide anion radical by milk xanthineoxidase. J. biol. Chem. 215, 4053-4057.
GIROTTI, A. W. & THOMAS, J. P. (1984). Damagingeffects of oxygen radicals on resealed erythrocyte ghosts.J. biol. Chem. 259, 1744-1752.
GOLDBERG, B. & GREEN, H. (1959). The cytotoxic actionof immune gamma globulin and complement on KrebsAscites tumour cells. I. Ultrastructural studies, jf. exp.Med. 109, 505-510.
HALLIWELL, B. (1984). Oxygen radicals: a commonsenselook at their nature and medical importance. Med. Biol.62, 71-77.
HALLIWELL, B. & GUTTERIDGE, J. M. C. (1984). Oxygentoxicity, oxygen radicals, transition metals and disease.Biochem. J. 219, 1-14.
HENKART, P. A. (1985). Mechanism of lymphocyte-mediated cytotoxicity. A. Rev. Immun. 3, 31-58.
HENKART, P. A., MILLARD, P. J., REYNOLDS, C. W. &
HENKART, M. P. (1984). Cytolytic activity of purifiedcytoplasmic granules from cytotoxic rat large granularlymphocyte tumours. .7. exp. Med. 160, 75-93.
KATZ, P., SIMONE, C. B., HENKART, P. A. & FAUCI, A. S.
(1980). Mechanisms of antibody-dependent cellularcytotoxicity. The use of effector cells from chronicgranulomatous disease patients as investigative probes. J.din. Invest. 65, 55-63.
KAY, D., SMITH, D. L., SULLIVAN, G., MANDELL, G. L.
& DONOWTTZ, G. R. (1983). Evidence for a non-oxidative mechanism of natural killer (NK) cellcytotoxicity by using mononuclear effector cells fromhealthy donors and from patients with granulomatousdisease. J. Immun. 131, 1784-1788.
KERR, J. F. R., BISHOP, C. J. & SEARLE, J. (1984).
Apoptosis. In Recent Advances in Histopathology, vol.12 (ed. P. P. Anthony & R. N. M. MacSween), pp.1—15. Edinburgh: Churchill Livingstone.
KLEBANOFF, S. J. (1980). Oxygen metabolism and thetoxic properties of phagocytes. Ann. intern. Med. 93,480-489.
KLEBANOFF, S. J., LOCKSLEY, R. M., JONG, E. C. &
ROSEN, H. (1983). Oxidative response of phagocytes toparasite invasion. In Cytopathology of Parasitic Disease.Ciba Foundation Symposium 99 (ed. D. Evered & G.M. Collins), pp. 92-111. London: Pitman Books.
KOPPEL, P., PETERHANS E., BERTONI, G., KEIST, R.,
GROSCURTH, P., WYLER, P. & KELLER, R. (1984).
Induction of chemiluminescence during interaction oftumoricidal effector cell populations and tumour cells is
480 C. jf. Bishop et al.
dependent on the presence of mycoplasma. J. Immun.132, 2021-2029.
KRISHAN, A. & RAYCHAUDHURI, R. (1970). Chromosomestudies of cell lines and tumours derived from a singlespecimen of human leukemic blood by cell culture andheterotransplantation. Cancer Res. 30, 2012-2016.
LIEPINS, A., FAANES, R. B., LIFTER, J., CHOI, Y. S. &
D E HARVEN, E. (1977). Ultrastructural changes duringT-lymphocyte-mediated cytolysis. Cell Immun. 28,239-257.
Lozzio, C. B. & Lozzio, B. B. (1975). Human chronicmyelogenous leukaemia cell line with positivePhiladelphia chromosome. Blood 45, 321-324.
LYNCH, R. E. & FRIDOVICH, I. (1978). Permeation of theerythrocyte stroma by superoxide radical..?, biol. Chem.253, 4697-4699.
MACDONALD, H. R. & KOCH, C. J. (1977). Energymetabolism and T-cell-mediated cytolysis. I. Synergismbetween inhibitors of respiration and glycolysis. J. exp.Med. 146, 698-709.
MANNEL, D. N., MELTZER, M. S. & MERGENHAGEN, S. E.
(1980). Generation and characterization of alipopolysaccharide-induced and serum-derived cytotoxicfactor for tumor cells. Infect. Immun. 28, 204—211.
MATTER, A. (1970). Microcinematographic and electronmicroscopic analysis of target cell lysis induced bycytotoxic T lymphocytes. Immunology 36, 179-190.
MATTHEWS, N. (1981). Production of an anti-tumourcytotoxin by human monocytes. Immunology 44,135-142.
NATHAN, C. F., BRUKNER, L. H., SILVERSTEIN, S. C. &
COHEN, Z. A. (1979a). Extracellular cytolysis byactivated macrophages and granulocytes. I.Pharmacologic triggering of effector cells and release ofhydrogen peroxide. J . exp. Med. 149, 84-99.
NATHAN, C. F., MERCER-SMITH, J. A., DESANTIS, N. M.
& PALLADINO, M. A. (1982). Role of oxygen in T cell-mediated cytolysis. J . Immun. 129, 2164-2171.
NATHAN, C. F., SILVERSTEIN, S. C , BRUKNER, L. H. &
COHN, Z. A. (19796). Extracellular cytolysis by activatedmacrophages and granulocytes. II. Hydrogen peroxide asa mediator of cytotoxicity. J. exp. Med. 149, 100-113.
PODACK, E. R. (1985). The molecular mechanism oflymphocyte-mediated tumor cell lysis. Immun. Today 6,21-27.
RAMSTEDT, U., ROSSI, P., KULLMAN, C , WARREN, E.,
PALMBLAD, J. & JONDAL, M. (1984). Free oxygenradicals are not detectable by chemiluminescence duringhuman natural killer cell cytotoxicity. Scan. J. Immun.19, 457-464.
RICKINSON, A. B., Moss, D. J., WALLACE, L. E., ROWE,
M., MISKO, I. S., EPSTEIN, M. A. & POPE, J. H.
(1981). Long-term T-cell-mediated immunity to Epstein-Barr virus. Cancer Res. 41, 4216-4221.
RODER, J. C , HELFAND, S. L., WERKMEISTER, J.,
MCGARRY, R., BEAUMONT, T. J. & DUWE, A. (1982).
Oxygen intermediates are triggered early in the cytolyticpathway of human NK cells. Nature, Land. 298,569-572.
ROONEY, C. M., RICKINSON, A. B., Moss, D. J., LENOIR,
G. M. & EPSTEIN, M. A. (1984). Paired Epstein-Barr
virus-carrying lymphoma and lymphoblastoid cell linesfrom Burkitt's lymphoma patients: Comparativesensitivity to non-specific and allo-specific cytotoxicresponses in vitro. Int.J. Cancer 34, 339-348.
RUDDLE, N. H. (1985). Lymphotoxin redux. Immun.Today 6, 156-159.
RZEPCZYK, C. M. & BISHOP, C. ] . (1984). Immunological
and ultrastructural aspects of the cell-mediated killing ofDirofilaria immitis microfilariae. Parasit. Imimni. 6,443-457.
SANDERSON, C. J. & GLAUERT, A. M. (1977). The
mechanism of T cell mediated cytotoxicity. V.Morphological studies by electron microscopy. Proc. R.Soc.Lond. B, 198, 315-323.
SEARLE, J., KERR, J. F. R. & BISHOP, C. J. (1982).
Apoptosis. Path. Ann. 17(2), 229-259.
SEGAL, A. W. (1984). How do phagocytic cells killbacteria? Med. Biol. 62, 81-84.
STACEY, N. H., BISHOP, C. J., HALLIDAY, W. J.,
COOKSLEY, W. G. E., POWELL, L. W. & KERR, J. F. R.
(1985). Apoptosis as the mode of cell death in antibody-dependent lymphocytotoxicity. Evidence that killing doesnot depend on the production of plasma membranelesions. J. CellSci. 74, 169-179.
SUTHANTHIRAN, M . , SOLOMON, S . D . , WILLIAMS, P. S . ,
mediated killing of protozoa. Adv. Parasit. 22, 43-151.THORNE, K. J. I. & FRANKS, D. (1983). Importance of
oxidative metabolism in T cell cytotoxicity: a comparisonof cloned T cells and spleen cells. Immunology 50,645-650.
THORNE, K. J. I., SWENNSEN, R. J. & FRANKS, D.
(1980). Role of hydrogen peroxide in the cytotoxicreaction of T lymphocytes. Clin. exp. Immun. 39,486-495.
TRUMP, B. F. & AKSTILA, A. U. (1975). Cellular reactionto injury. In Principles of Pathobiology, 2nd edn (ed. M.F. Lavia & R. B. Hill), pp. 9-96. New York: UniversityPress.
WEINBERG, J. B. & HANEY, A. F. (1983). Spontaneous
tumor cell killing by human blood monocytes and humanperitoneal macrophages: Lack of alteration by endotoxinor quenchers of reactive oxygen species. J. natn. CancerInst. 70, 1005-1010.
WYLLIE, A. H., KERR, J. F. R. & CURRIE, A. R. (1980).
Cell death: The significance of apoptosis. Int. Rev.Cytol. 68, 251-306.
YOUNG, J. D., PETERSON, C. G. B., VENGE, P. & COHN,
Z. A. (1986). Mechanisms of membrane damagemediated by human eosinophil cationic protein. Nature,Land. 321, 613-616.
(Received 9 September 1986 -Accepted, in revised form, IIDecember 1986)
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