A view to a kill: cytotoxic mechanisms of human polymorphonuclear leukocytes compared with monocytes and natural killer cells.

Cytotoxic Mechanisms © 1990 S. Kargcr AG. Basel 1015-2008/90/0585-024952.75/0

Pathobiology 1990;58:249-264

A View to a Kill: Cytotoxic Mechanisms of Human Polymorphonuclear Leukocytes Compared with Monocytes and Natural Killer Cells Kok P.M. van Kessel. Jan Verhoef Eijkman-Winkler Laboratory of Medical Microbiology, University Hospital, Utrecht. The Netherlands

Key Words. Cytotoxicity • Polymorphonuclear leukocytes • Antibody-dependent, cell-mediated cytotoxicity • Oxygen Abstract. Polymorphonuclear leukocytes (PMN) are able to exert cell-mediated cytotoxic reactions in order to eliminate tumor cells and virus-infected cells. Appropriate stimulation is needed to activate the potential cytotoxic arsenal of the PMN in contrast to the spontaneous cytotoxicity mediated by natural killer cells. Stimulation with phorbol esters induces an oxygen-dependent killing mechanism which results in highly efficient lysis of red blood cell targets. Tumor target cells are more resistant to oxygen-dependent killing mechanisms due to effective antioxidant capacities. Antibody-coated tumor target cells are easily recognized and bound by PMN via a cooperative action of Fc receptors and adhesion molecules. This firm contact and receptor occupation result in efficient killing of the tumor cells which does not require production of oxygen radicals. The mechanisms of PMN-mediated cytotoxicity are discussed and compared with data known from natural killer cells and monocytes.

Directed cell killing is an important defense mecha nism in the control and elimination of tumor cells and virus-infected cells. Cytotoxicity or lysis of target cells can be mediated by various systems involving several leukocyte populations which possess cytotoxic capacities under different conditions with a variety of killing mech anisms. Some of these cytotoxic cells are highly special ized for target cell killing, while others exert multiple functions. In vitro studies of cell-mediated cytotoxicity are important in order to understand the role of leuko cytes in vivo against malignancies and in the control of the spread of viral infections. Insight into the working mechanisms and regulation of cytotoxicity may contrib ute to effective therapeutic treatments. Both phagocytic cells, polymorphonuclear leukocytes (PMN) and mono cytes, as well as natural killer (NK) cells play a role in the defense against tumor cells or virus-infected cells [124], Cytologic studies have shown substantial numbers of

PMN surrounding tumor cells in ascites fluid from can cer patients suggesting an active role in tumor clearance [1, 65]. The phagocytic cells possess powerful toxic mechanisms against ingested bacteria which are also effective against extracellular targets. The killing mecha nisms of PMN, monocytes, and NK cells share some common characteristics as well as major differences. In this review, the cytotoxic capacity and killing mecha nism of PMN are discussed and compared with that of monocytes and NK cells.

Characteristics of PMN, Monocytes, and NK Cells PMN and monocytes derive from separate progeni tors in the bone marrow. Promonocyte precursors enter the circulation where they remain as monocytes or mi grate into the tissues and become macrophages, while PMN may leave the circulation to an inflammatory site but do not return. PMN, monocytes, and macrophages

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Introduction

are phagocytic cells, while NK cells are of lymphoid ori gin and are not able to phagocytize. Although Abo et al. [2] and Kang et al. [64] showed that a subpopulation of NK cells is capable of ingesting gram-positive bacteria, this is not recognized as a general property of NK cells. NK cells are defined as effector cells with spontaneous cytotoxicity against various target cells without any re striction related to the major histocompatibility complex [58, 124], Monocytes. PMN, and NK cells each have a charac teristic phenotype as defined by monoclonal antibodies to surface antigens. Although defined as a separate cell, NK cells are a heterogeneous population with respect to surface antigens and functional activity [98]. PMN have also been shown to be heterogeneous by several parame ters such as receptor expression, cell density, and surface determinants [46]. Granules Phagocytes, PMN and monocytes, as well as NK cells contain cytoplasmic granules which are apparent in Giemsa-stained cell preparations. Morphologically, the NK cell activity is closely related to that of large granular lymphocytes (LGL) which contain azurophilic cytoplas mic granules [123]. Electron microscopic analysis re veals that LGL have round or indented nuclei, and the cytoplasm is characterized by an extended Golgi appara tus and numerous vesicles with a characteristic ‘tubular array’ structure [54], PMN have two major types of gran ules which contain a number of products and enzymes. The different granules can be separated by sucrose and Percoll gradient centrifugation procedures and are char acterized by their ultrastructural morphology and cyto chemistry [8,51, 103]. Specific (secondary) granules con tain lactoferrin, collagenase, vitamin B12 binding pro tein, alkaline phosphatase, and (i-glucuronidase, while the azurophilic (primary) granules contain neutral pro teases such as elastase and myeloperoxidase (MPO). Ly sozyme is found in both types of granules. Monocytes and macrophages secrete many substances with variable biologic activities, including lysozyme, cationic proteins, antibacterial toxins, and immuno-modulators such as tumor necrosis factor and interleukin-I [95] Degranula tion of lysosomal contents occurs either into phagocytic vacuoles within the cell or into the cell’s environment. Surface Antigens Recognition of antibody-coated particles occurs via Fc receptors (FcR) which bind to the Fc region of the IgG molecule. Complement receptors (CR) recognize parti

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cles which bear activated complement factor C3. There are three types of FcR identified on the basis of their molecular weight and reactivity with monoclonal anti bodies: FcRI, FcRlI, and FcRIlI. These receptors exhibit different binding properties [3]. The FcRI is expressed only on mononuclear phagocytes and binds free mono meric IgG in contrast to the other FcR which only recog nize surface-bound or aggregated IgG. Although freshlyisolated PMN express no detectable quantities of FcRI. incubation of the cells for 16 h with recombinant inter feron gamma results in expression of moderate amounts of FcRI [113]. FcRII is expressed on PMN and mononu clear phagocytes and has affinity for all subclasses of human IgG [3], FcRIII is present on both PMN and NK cells and preferentially binds human IgGl and IgG3 and mouse IgG2a and IgG2b [101]. Two different receptors for activated complement C3 are present on PMN: CR1 which binds C3b-coated particles and CR3 which binds C3bi/C3d-coated particles [146], A number of related cell surface molecules present on all leukocytes associated with cell-cell interactions are defined by specific monoclonal antibodies. This is a family of related glycoproteins which share an identical (3-subunit and a distinguishable a-subunit: leukocyte function associated antigen 1 (LFA-I), p i 50/95, and Mo-l/Mac-1 (or CR3) [120]. Patients with inherited deficiency in expression of these adhesion molecules suf fer from severe recurrent bacterial infections and dimin ished in vitro leukocyte functions [4, 91]. These cell adhesion molecules play an important role in cell-cell interactions of the immune system and are involved in the adhesion properties of monocytes and PMN [78] and in cytotoxicity mediated by NK cells, K cells, and PMN [74, 90, 108], The PMN adhesion molecules per se are apparently not sufficient in promoting adequate (non antibody-coated) target cell binding, either because this binding is weak in comparison to FcR-mediated binding, or additional triggering is needed which switches these adhesion molecules into ‘active’ molecules or enhanced surface expression. Adhesion molecules could stabilize the interaction or accelerate and facilitate speed and effi ciency of target cell binding, especially in the situation of limited amounts of target cell bound immunoglobulins [128], Inflammatory mediators stimulate the expression of several surface receptors from latent intracellular pools [121], The surface expression of both CR1 and CR3 on PMN can be upregulated from different intracellular compartments, while some stimuli also induce internali zation ofCRI [99, 100].


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Oxidative Burst Stimulation of phagocytes by a variety of stimuli, such as opsonized particles or soluble mediators, leads to an increase in oxygen consumption, activation of the hexose monophosphate shunt, and an increased glucose uptake. These changes in oxidative metabolism are known as the ‘respiratory burst’ and result in the produc tion of reduced toxic oxygen species [6, 72]. Toxic oxy gen metabolites are generated from oxygen via a mem brane-bound flavoprotein that uses NADPH as a reduc ing substance. A b-type cytochrome is related to this oxi dase, probably as part of a chain of electron carriers. The NADPH-oxidase system catalyzes the one-electron re duction of O2 to superoxide anion (05). Via oxidation and reduction steps, O 2 is converted to several other toxic oxygen species such as hydrogen peroxide (H 2O 2). hydroxyl radicals ( ’OH), and singlet oxygen. In combi nation with an enzyme from the azurophilic granules, MPO, H 2O 2, and a halide form a powerful oxidation sys tem resulting in the generation of oxidized halogens such as hypochlorous acid (HOC1). A substantial portion reacts with amines, particularly taurine, to yield chlo ramines. Disorders in the NADPH-oxidase system are known as chronic granulomatous disease, and the phago cytes from such patients are unable to produce toxic oxy gen species [106]. Phagocyte-derived reactive oxygen metabolites are important mediators in bacterial and tumor cell killing and play a role in inflammatory reac tions leading to tissue injury [7, 43, 72]. Many cytokines (such as tumor necrosis factor and colony stimulating factors) enhance the ability of PMN and monocytes to respond to a second stimulus, result ing in increased production of oxygen radicals, release of

lysosomal enzymes, phagocytic and cytotoxic capacity, and ability for localization and adherence at sites of infection [121].

Cytotoxicity

The interaction between effector cell and target cell which leads to extracellular killing of the target cell can be divided into at least two distinct steps. The target cell must be recognized and bound to bring the effector cell in close proximity, and, secondly, the lytic machinery of the effector cell must be triggered. After activation of the effector cell, the various lytic mediators are released and attack different target sites, leading ultimately to target cell death. Recognition of specific target cell structures itself is a possible stimulus for cytotoxicity (spontaneous cytotoxicity). Target cells coated with specific antibodies are recognized and bound by FcR-bearing cytotoxic ef fector cells and subsequently killed; this process is known as antibody-dependent, cell-mediated cytotoxic ity (ADCC). Stimulation of cytotoxic effector cells with agents that activate potential lytic machineries provides an alternative cytotoxic response which does not require specific target cell binding, but only proximal contact. Spontaneous Cytotoxicity NK cells are defined as spontaneous cytotoxic cells which kill their targets without any major histocompati bility complex restriction. NK cell sensitive target cells in vitro include several tumor cell lines (such as the cell line K562 which is used to define the population of NK cells), freshly isolated tumor cells, and virus-infected cells [58, 124, 143], The cytotoxic process of NK cells involves three separate phases: binding to the target cell, activation of lytic mediators, and killer cell independent lysis [59], After binding and triggering or activation of intracellular processes in the NK cell, release of granule content takes place at or in close proximity to the bound target cell. PMN do not spontaneously kill target cells, but need an additional stimulus. However, some reports de scribed spontaneous cytostatic and cytotoxic activities of PMN from cancer patients [13, 49, 76] and spontaneous tumor cell lysis by murine inflammatory peritoneal PMN [81]. The potential spontaneous cytotoxic properties of monocytes are more controversial. Research on mono cyte spontaneous cytotoxic reactions to tumor cells are hampered by the isolation procedure. A conventional


The adhesion molecules Mac-1 and p i50/95 are stored in specific granules in PMN [9] which are trans ferred to the cell surface after stimulation. The FcRIl and FcRIII expression on PMN is not upregulated after stimulation. Both monocytes and PMN are able to eliminate mi croorganisms by engulfment and subsequent ingestion into a phagosome. The granules fuse with this phago some and degranulate, upon which the bacteria are killed and degraded. Bacteria are easily taken up by phagocytes when they are loaded with opsonins (com plement factors and immunoglobulins) which are recog nized by specific receptors on the phagocyte cell surface [131, 132]. However, PMN are also able to ingest unop sonized bacteria when they are trapped against a surface [129, 130].

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method is to use the adherent properties of monocytes to separate them from the nonadherent lymphocytes on plastic [44], However, attachment of the cells to a surface and the unintended exposure to trace levels of endotoxin may act as an activation or maturation signal. Moreover, contamination of the adherent monocytes by NK cells makes it difficult to discriminate between spontaneous monocyte cytotoxicity and NK cell activity, especially when NK cell susceptible targets are used [73], Potential activation of monocytes is minimized by using autolo gous serum-coated plastic, a short incubation period, and detachment by an EDTA medium without cell scraping, resulting in a highly monocyte-enriched cell population with good viability. The initially observed spontaneous killing of K562 tumor cells by adherence-isolated cells has been shown to be due to contamination with NK cells. Monocytes were not triggered to kill K562 target cells spontaneously, unless the cells were activated by phorbol myristate acetate (PMA) to lyse the tumor cells via an oxygen-dependent mechanism. Killing of K562 tumor cells by PMA-activated monocytes was only mea surable after a prolonged incubation period of 18 h in contrast to the rapid (2 h) cytotoxicity mediated by NK cells [125], This may be due to the difference in lytic mechanisms: non-oxygen-derived NK cell products and oxygen radicals may cause different patterns of cell death. On the other hand, NK cells deliver their products directly in close proximity to the target cell, while PMA stimulates the monocytes randomly. As a consequence of this nonselective monocyte stimulation by PMA, NK cells in the close proximity to activated monocytes are also affected by the oxygen radicals produced. In the pres ence of PMA the spontaneous killing of the K562 target cells, mediated by contaminating NK cells, is abolished either because the NK cells are killed or because their cytotoxic machinery is (temporally?) paralyzed by the monocyte-released oxygen radicals [125]. Oxygen species and proteases released by activated phagocytes (mono cytes, macrophages, and PMN) have been shown to mod ulate other leukocyte activities such as NK and K cell function and lymphocyte mitogenic response. Not only down-regulation by monocytes and PMN is observed, but also upregulation of NK cell activity is reported, indicat ing a potential role for phagocytes to interfere with the functional activity of lymphocytes [68], An alternative way to test monocyte cytotoxicity is to use drug-treated target cells which are insensitive to NK cells, usually actinomycin D treated WEHI-164 murine sarcoma cells [24], These drug-treated tumor cells are rapidly killed by monocytes. However, it is not clear

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whether killing of drug-treated targets represents sponta neous monocyte cytotoxicity, or whether it should be considered an alternative cytotoxic mechanism. Antibody-Dependent Cell-Mediated Cytotoxicity Antibody-coated target cells are recognized by FcR on monocytes, PMN, and NK cells. The recognition of a target cell loaded with specific antitarget antibodies en ables the effector cell to kill selected target cells. The killing efficiency is defined by the immunoglobulin class and the density bound to the target cell and by the FcR type involved on the effector cell [69, 80], Variations in the efficiency of ADCC are not solely the result of a dif ference in the antibody subclass, but spatial orientation and organization of the antibodies on the target cell membrane may also contribute to a sufficient trigger for a lytic process [ 16], The specific FcR interaction initiates the various killing mechanisms which are probably local ized at the site of contact with the target cell because non-antibody-coated innocent bystander cells are not lysed. In addition, non-antibody-coated target cells bound to monocytes via concanavalin A or protein Aimmunoglobulin bridges were also not killed, while the specific interaction of antibody-coated red blood cells resulted in lysis of these targets [ 136], The existence of a narrow-sealed compartment between the target cell and the effector cell has been proposed which is inaccessible to extracellular proteins and provides a microenviron ment where lytic molecules are released [145], PMA Cytotoxicity Induction of non-receptor-mediated cytotoxicity in monocytes and PMN is achieved with various soluble and particulate mediators. The most potent activator is the phorbol ester PMA, but also zymosan-activated se rum [28], aggregated IgG [29], and lectins such as conca navalin A [21, 30, 53, 115, 116] are all capable of stim ulating PMN-mediated target cell lysis. Most of these agents stimulate the oxidative burst of monocytes and PMN indicative of the highly oxygen-dependent charac ter of their induced cytotoxicity. Although target cell lysis is demonstrated with various types of target cells (red blood cells, mouse and human tumor cell lines), quantitative and qualitative differences in the cytotoxic mechanisms and their effectiveness are evident. These differences are not only due to the assay conditions, but may be due to the heterogeneous nature of the target cells or may reflect the target cell sensitivity to particular lytic processes and its escape mechanism against damage (see below).


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Factors Involved in Cytotoxicity

Oxygen Radicals Oxygen radicals are highly reactive agents which con tribute to cell killing. Stimulation of monocytes and PMN with PMA results in a strong activation of the oxi dative burst and cytotoxicity against a variety of target cells. The oxygen-dependent cytotoxicity mediated by monocytes or PMN involves either O2 and H2O 2, alone or in combination with MPO and halide, depending on the effector cell and the target cell used (table 1). Al though O2 by itself induces damage, the major part of this molecule is rapidly converted to H2O 2 in the pres ence of a hydrogen donor. Furthermore, the combina tion of O 2, H 2O 2, and Fe3+ results in the formation of highly reactive hydroxyl radicals and subsequent second ary radicals. PMN from CGD patients are ineffective in cell lysis after PMA stimulation. Oxygen radicals are the major lytic agents in PMA-stimulated, PMN-mediated cytotoxicity. High concentrations of PMA (60,000 ng/ml in contrast to a commonly used amount of 10-100 ng/ml) were shown to increase PMN-mediated binding

and killing of K.562 tumor cells by a nonoxidative path way, as indicated by scavengers and normal cytotoxicity with PMN from a patient with CGD [85). However, the normally used PMA concentrations predominantly pro voke activation of the oxidative burst. Spontaneous cy totoxicity by inflammatory PMN also involves an oxy gen-dependent process [82], Monocyte-mediated sponta neous cytotoxicity and killing of actinomycin-D-treated target cells does not primarily depend on reactive oxygen metabolites (15, 25, 136). Binding of antibody-coated target cells by the FcR results in activation of the metabolic burst and production of oxygen radicals which contribute to target cell death. The role of the metabolic burst in monocyte and PMNmediated ADCC is still somewhat controversial (table 1). Participation of oxygen products in monocyte- and PMNmediated ADCC is suggested by several parameters of the oxidative burst such as oxygen consumption, superoxide release, and l4C-glucose oxidation, while ADCC per formed under anaerobic conditions is diminished [20, 55). However, scavengers of oxygen products are not always inhibitory, and phagocytes from patients with a

Target

Effector

Stimulus

Mechanism

References

RBC (H, O) RBC(H) RBC (C, H) RBC (C, H, O) RBC(O) RBC (C) RBC (C)

PMN PMN, monocyte PMN PMN PMN PMN PMN

PMA PMA ConA aggregated lgG ZAS ConA ConA

MPO Oi, non-MPO active metabolism MPO (catalase) H 2O 2 Oj, MPO non-MPO, O dependent

27,32 137-139 115. 116 29, 33, 34 28 30 53

Tumors Tumors (M) Tumors Tumors (M) Tumors (M) Tumors (H. M) Tumors (H) Tumors (H) Endothelium

PMN macrophage monocyte PMN PMN PMN PMN monocyte, macrophage PMN

PMA PMA PMA ConA zymosan none none none PMA

MPO H2 O 2 non-MPO, O dependent MPO MPO, cationic pr. defensins more cytostasis O independent h 2o 2

22, 26. 141 5, 93, 94 89 21 17. 18 82, 83 13, 49. 76 136 141

RBC(H) RBC(H) Tumors (M) Tumors (rat) Tumors (H)

PMN, monocyte PMN, monocyte PMN PMN PMN

ADCC ADCC ADCC ADCC ADCC

O independent partly O dependent O dependent, non-MPO Oj partly O dependent

45, 66, 75 12 20 55 31, 35

RBC = Red blood cells (C - chicken, H = human, M = mouse, O - ox); ConA = concanavalin A, ZAS - zymosan- = activated serum. For explanation of the other abbreviations see text.


Table 1. Studies of phagocyte-mediated cytotoxicity toward red blood and tumor target cells

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PMA induced

Ratio PMN: K562

ADCC

defect in their oxidative burst (CGD cells) displayed a normal ADCC against antibody-coated red blood cells [45, 66] and Raji tumor cells [31], Inhibition of the gluta thione content of Raji cells increased their susceptibility to lysis by PMN from normal donors, but not to lysis by PMN from CGD patients, indicating that both oxidative and nonoxidative mechanisms are involved. The contribution of oxygen radicals in PMN-mediated ADCC is suggested by the induction of a chemi luminescence response in PMN after FcR stimulation by antibody-coated K562 membrane fragments as a conve nient, reproducible substitute for intact cells [127], The amount of chemiluminescence initiated by these antibody-coated fragments (but also by whole intact anti body-coated K.562 target cells) is far less in comparison with the response induced by PMA under the same assay conditions (fig. 1). Despite this small oxidative burst triggered via the FcR. killing of antibody-coated K562 target cells is much more efficient (after 2 h with low effectontarget ratios) in comparison with the same non antibody-coated targets and PMA-stimulated PMN (fig- 2). Efficient contact may be part of the explanation. Antibody-coated K562 target cells are efficiently bound by PMN. as indicated by flow cytometric analysis of con

Ratio PMN: K562

jugate formation [128]. A tight binding might provide a perfectly sealed microenvironment where locally re leased and produced toxic mediators attack only target cell structures in contrast to the randomly released prod ucts from PMA-stimulated PMN which do not form such a close, extended contact area with target cells. Addition of PMA to ongoing ADCC against K562 tumor cells where conjugation is already established is shown to increase the cytotoxicity due to generation of oxygen radicals (HiCE) [62]. Preincubation with PMA decreased the ADCC without affecting the FcR-binding capacity. Alternatively, Fc and complement receptors are suscep tible to the MPO-H2O 2 system, resulting in a reduced attachment of opsonized yeast particles and conse quently a reduced phagocytotic capacity [122], The involvement of oxygen species has been sug gested for NK cell mediated cytotoxicity [104], How ever, several other studies have indicated that scavengers have no effect on cytotoxicity, while NK cells from chronic granulomatous disease patients display normal cytotoxic capacities [67], and K562 target cells are un able to stimulate purified NK cells to an oxidative burst as measured by chemiluminescence [125]. Alternative evidence for the oxygen-independent character of NK cell mediated killing is presented by the fact that impair


Fig. 1. PMN (1 X 105 cells) were stimulated with buffer, 25 ng/ml PMA, 3 X 10s K562 tumor cells (K562/-), or K562 tumor cells sensitized with 10% rabbit antiserum (K562/Ab), and peak chemiluminescence values in the presence of 3.5 \iM luminol were measured (mean ± SEM). Fig. 2. Difference in efficiency of PMA-stimulatcd cytotoxicity and ADCC of PMN towards K562 tumor cells. PMN were mixed at various ratios with chromium-labeled K562 targets, and lysis was measured after 2 h at 37 °C. For PMA (25 ng/ml) induced cytotox icity, control K562 cells were used, and for ADCC targets were sen sitized with 10% rabbit antiserum.

ment of K562 target cell antioxidants greatly enhances their susceptibility to oxygen radical induced damage as shown with PMA-stimulated PMN. However, no en hanced NK cell mediated killing of the antioxidantdepleted targets was observed, supporting the nonoxygen radical character of the NK cell killing process [126], Degranulation Exocytosis of granular content at the interphase be tween effector and target cell provides the delivery of possible lytic molecules which play a role in target cell killing. Lysosomal enzymes have a more specific reac tion pattern and act over a longer period of time in com parison to oxygen radicals. Several products in PMN granules have been identified which display either spe cific antibacterial or antiviral activities such as cationic proteins or possess catalytic activities such as lysozyme, elastase, and collagenase [41, 119]. Although most data available concern the antimicrobial activities of these PMN granular products, purified cationic proteins are also cytotoxic for mouse tumor cell lines [ 18]. Defensins, a group of small cysteine-rich cationic peptides isolated from the azurophilic granules of human and rabbit PMN and characterized by their activities against bacteria and herpes viruses [47], also lyse human and murine target cells and act synergistically with H 2O 2, resulting in en hanced cytolysis [83]. A predominant role for granule-derived lytic mole cules in PMN-mediated ADCC is suggested. Degranula tion of PMN is evident when they are allowed to adhere to surface-bound IgG or stimulated by soluble agents [57]. PMN stimulation causes the differential discharge of small organelles (which contain among others cathepsin and gelatinase) and specific and azurophil granules [8, 51]. Azurophilic (secondary) granules have a com plete enzymatic armamentarium including MPO, lyso zyme, and neutral serine proteases and several other effector molecules which possess cytotoxic properties. Also defensins are partly discharged upon stimulation with PMA and opsonized zymosan which correlates with the small release of azurophil granule markers [48], A small and selective exocytosis of specific granules from PMN into the extracellular medium is evident when they are exposed to PMA [8, 144], Exocytosis of primary granules is related to PMN-mediated ADCC, indicating that granular products play a role in the PMN cytolytic process [35], There is no evidence for stable soluble cyto toxic mediators of PMN-mediated ADCC. The release of soluble cytotoxic or cytostatic factors by monocytes plays an important role in the killing

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repertoire of these cells. A number of different soluble factors are known, including toxic oxygen species, cyto toxic factor, neutral proteases, and tumor necrosis factor [86, 95]. Monocyte cytotoxic factor(s) are also involved in the killing of antibody-coated target cells [42] and in the killing of drug-treated cells [10]. Directed exocytosis is an important feature of the NK cell killing process [14, 133], and several cytolytic media tors have been identified. Stimulation of NK cells re leases a soluble NK cell cytotoxic factor (NKCF) which binds to susceptible cell lines. The NKCF-mediated cy totoxic mechanism does not involve lymphotoxin (a T lymphocyte produced factor) and is partly mediated bytumor necrosis factor-like activity [11, 59], Isolation and purification of cytoplasmic granules from rat LGL tu mor cells revealed another cytotoxic factor, cytolysin or perforin, which acts through the insertion of pores into the target cell membrane. This either results in direct cell lysis or provides a channel for insertion of other lytic factors [59]. Although purified NK cell derived NKCF possess in vitro cytolytic activities against tumor cell lines, long incubation periods are required to give chromium release [11] which is also described for PMN granule derived defensins [84], However, the situation in a sealed contact area as observed in PMN ADCC and in NK-target cell interaction may provide more physiologi cal conditions in combination with additional factors. Alternative candidates may be involved as mediators or cofactors in the killing mechanism. Cell membrane asso ciated proteases (serine proteases and chymotrypsin-like molecules) are possibly involved in the sequence of events after PMN activation of O 2 generation, granule enzyme release, and PMN-mediated ADCC against tu mor cells at some step after target cell recognition [36, 71], A membrane-bound neutral serine protease from PMN has been shown to enhance the lytic action of H 2O 2 against red blood cells [102], Platelets are also capable of recognition and lysis of sensitized red blood cells. Their cytotoxic system has been shown to reside in the plasma membrane. The molecules actually operating in platelet-mediated target cell killing have not been identified [118]. Cell Surface Structures Although experimentally documented, the precise na ture of the tumor cell recognition structures and the NK cell receptor are not fully defined. Binding to virusinfected target cells involves the recognition of viral gly coproteins expressed on the membrane of infected cells or of upregulated normal cell structures or membrane


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alterations as a consequence of the virus infection [143], Studies with monoclonal antibodies against surface structures on NK cells have revealed that besides the tar get cell recognition structures, other target cell antigens are involved in triggering lysis [59], In addition to the target cell specific binding struc tures, cell adhesion molecules (LFA) are involved in establishing an effective binding between effector and target cell [ 120]. Patients with a genetic deficiency in the LFA glycoprotein family have a decreased NK cell and PMN mediated lysis of antibody-coated herpes simplex virus infected target cells due to a decreased formation of effector-target conjugates [74],

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lytic process). A target cell programmed self-destruction is suggested as a possible mechanism of lymphocytemediated cytotoxicity [52, 107], Target cell enzymes (en donucleases and proteases) are activated by effector cell products which introduce single-strand breaks, resulting in DNA fragmentation [149], Tumor necrosis factor and lymphotoxin also induce target cell DNA fragmentation and cytolysis which is enhanced by interferon gamma [37], Activated PMN produce sister chromatid exchange in cell lines by an oxygen-dependent mechanism, possi bly via hydroxyl radicals. These target cells are protected by supplementation of intracellular antioxidants [142], PMA-stimulated PMN can damage each of the four bases in calf thymus DNA which is likely mediated via hydroxyl radicals [63],

Target Cell Damage

Morphology The morphological and biochemical aspects of nu cleated cell death can be divided into two distinct path ways: necrosis and apoptosis [40], Necrosis occurs after complement attack and after exposure to oxygen radicals or hyperoxia. The plasma membrane permeability in creases, resulting in ion and water redistribution. The cell alters the shape with blebbing of the surface, and the nuclear chromatin flocculates. The morphological changes are observed before lysis of the target cell occurs and are initially reversible. Cell-mediated killing by cyto toxic T lymphocytes (CTL) and NK cells or isolated lymphotoxin induces apoptosis. This process gives a bub bling appearance on electron microscopy, the cytoplasm condenses, the chromatin forms dense aggregates, and the nucleus fragments. The plasma membrane invaginates, and clusters of membrane bound vesicles are formed. Damage of target cell nuclear membrane with con densation of chromatin is an early event in CTL and NK cell mediated lysis. DNA fragmentation is observed in mouse and human target cells lysed by mouse or human effector cells, CTL, NK cells, and K cells, but not in complement-mediated lysis (or only very late during the

Cytolysins Cell killing mediated by NK cell and CTL-released cytolysins share immunological and morphological char


Once the effector cell is properly stimulated to exert its cytotoxic potential, the lytic mediators attack the tar get cell, resulting in cell damage and ultimately in cell death. The various lytic molecules all lead to the same result: lysis of a target cell. Their mechanisms and point of attack, however, are quite diverse. However, despite the great variety of lytic molecules, some aspects of cell killing have some mechanisms in common.

Oxygen Radicals The major physiological target for oxygen radicals is the cell membrane where unsaturated lipids undergo peroxidation reactions and structural and functional molecules may be affected. The oxidants or their prod ucts of interaction may be responsible for changes in ion fluxes and disturbance of the membrane integrity. Dif ferent forms of membrane damage are responsible for cell death which is not solely determined by lipid perox idation [50], Oxygen products can pass through mem branes either passively (such as H 2O 2) or via anion chan nels (O 2), thereby affecting vital intracellular systems [87], Early parameters during H 2O 2 injury of nucleated cells include disturbance of intracellular Ca2+ [61], mor phological changes evident as surface blebs, and an increase in F actin content [60]. These processes precede the loss of membrane permeability and the subsequent cell death. Arrangement of F actin is also altered in L cells attacked by CTL or lymphotoxin. The decrease in F actin organization is prominent within microvilli and beneath the plasma membrane [79]. Polyadenosine diphosphate ribose polymerase activa tion is associated with conditions that cause DNA dam age and results in depletion of cellular nicotinamide-ade nine dinucleotide (NAD) and adenosine triphosphate (ATP). Irreversible depletion of cellular NAD and ATP is caused by H 2O 2. The loss of NAD and ATP is partly explained by the excessive activation of polyadenosine diphosphate ribose polymerase [109, 110].

acteristics which resemble the membrane damage pro duced by the membrane attack complex of the comple ment system [88, 92, 147, 149]. Examination of the membranes of target cells lysed by complement, NK cells, and CTL or of their isolated granules exhibits cir cular lesions on the surface. These proteins are secreted into the intercellular space between effector and target cell which form membrane lesions after assembly and polymerization of the monomer into functional chan nels. These granular molecules are, therefore, called pore-forming protein or perforins [59, 147], The assem bly mechanism of pore-forming protein is under the con trol of Ca and pH. Ring-shaped membrane lesions on target cell mem branes are observed in lymphocyte-mediated ADCC [39]. The formation of pores in target cell membranes which are responsible for lethal damage is also demon strated in PMN-mediated ADCC with erythrocyte ghosts [117], Eosinophil cationic protein which is shown to damage Schistosoma mansoni also forms stable transmembrane pores in nucleated target cells and lipid vesi cles. These pores resemble the pores formed by C9 and pore-forming protein from CTL/NK [148]. Human PMN peptide defensin mediated cytotoxicity requires initial binding of the molecules to target cells and subse quent translocation of internalization. The kinetics of defensin-mediated lysis are slow as measured by chrom ium release, and lysis is not due to rapid creation of plasma membrane lesions [84], Lysis by NKCF does not involve pore formation. The release of NKCF is triggered by a second signal different from the target cell binding structure. Following release. NKCF binds to specific glycoprotein or glycolipid-binding sites on the target cell which may be processed, lead ing ultimately to cell death. The precise mechanism is not yet known [11]. The cytotoxic mechanisms of PMN and monocytes/ macrophages have been suggested to involve production of oxygen metabolites, release of cytotoxic factors and serine proteases, translocation of lysosomal enzymes, and phagocytosis. Time-lapse fluorescence-intensified microscopy studies suggest that PMN can internalize tumor cell membrane and cytosol from antibody-coated YAC tumor cells in a piecemeal fashion [150]. Target Cell Determinants The efficiency of target cell killing is not solely deter mined by the effector cell lytic arsenal. The target cell itself influences the outcome of the cytotoxic process. Different target cell types may react differently to the

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same lytic molecules, as shown for complement-me diated lysis of red blood cells and nucleated cells. Assem bly of a single pore in the red blood cell membrane is sufficient to cause colloid osmotic imbalance and lysis, while nucleated cells require multiple and large mem brane pores before complement-mediated lysis occurs [70, 77]. The composition of the plasma membrane and the rapid removal of complement channels influence the ability of complement to mediate cytolysis [114]. In addition, nucleated cells are relatively resistant because they possess mechanisms of defense against complement attack. Lipid synthesis and active cell metabolism are related to resistance to complement, while endocytosis establishes elimination of terminal complexes from the plasma membrane [97], The cytocidal activity of tumor necrosis factor and monocyte-released cytotoxic fac tors) also involves active protein and RNA synthesis in susceptible targets and is controlled by microtubule for mation [96]. Tumor cell lysis by NKCF and PMN defensins requires energy and cytoskeleton-dependent pro cesses of the target cell for expression of cytolysis [38, 84], Although oxygen radical induced damage is not mediated through channel formation as demonstrated for the complement membrane attack complex and NK cell derived perforins, red blood cells and K562 tumor cells show a differential susceptibility for oxygen radicals (fig. 3). Most cells produce small amounts of oxygen radicals themselves as a result of normal metabolic processes. These small quantities are easily detoxified by the cell’s own protective antioxidant mechanisms such as vitamin E, superoxide dismutase, catalase, and the glutathione oxidation-reduction cycle. These endogenous natural an tioxidants contribute to the elimination of intra- and extracellularly produced oxygen radicals and provide a defense against oxygen-dependent injury by activated phagocytes. A simplified liposome model target system illustrates the role of membrane-associated target de fense properties against MPO-dependent cytotoxicity [111, 112]. Modification of the antioxidant status of a target cell renders the cell more susceptible to damage by exogenously produced oxygen products. This mecha nism is described for glutathione depletion in murine and human cell lines [5, 26, 94] and endothelial cells [56] and for catalase depletion in red blood cells [33, 34], Both glutathione and catalase which detoxify HjCF and SOD which eliminates Oi are involved in K562 tumor cell protection against oxygen radicals produced by PMA-stimulated PMN [126], The nature of the gener ated oxygen radicals in combination with additional


A View to a Kill

Fig. 3. Difference in target cell susceptibility for PMA-induced PMN cytotoxicity. Human red blood cells (RBC) or K562 tumor cells were mixed with PMN at various ratios, and the cytotoxicity was measured with 25 ng/ml PM A after 2 h at 37 °C.

van Kessel/Verhoef

granule products also affects the outcome of the cyto toxic reaction. PMA-stimulated cytoplasts, which have lost their granules but retain the ability to produce oxy gen radicals, lyse red blood cell targets, but not K.562 tumor cells. Inhibition of K.562 antioxidants to improve their susceptibility to oxygen radicals did not result in killing by PMA-stimulated cytoplasts, even not a high effectontarget ratios or long incubation periods (fig. 4). The resistance of K562 tumor cells to PMA-stimulated cytoplasts indicates that additional granule products are required for nucleated cell killing. A possible important granule product is myeloperoxidase which is known to be involved in PMA-stimulated killing of mouse and human tumor cell lines. Granule-free cytoplasts. there fore, lack an important component of this potent cyto lytic combination against tumor cell targets. Exogenous MPO and lactoferrin did not improve cytoplast-mediated lysis of red blood cells, nor did they initiate K.562 tumor cell killing. The fact that red blood cells are highly susceptible to oxygen radicals and that they, in contrast to tumor cells, are lysed by PMA-activated cytoplasts suggests that a non-MPO, oxygen-dependent mechanism is sufficient to lyse red blood cells, but is ineffective in killing of K.562 tumor cells. Generation of oxygen radicals by activated phago cytes is not only effective towards tumor and red blood cell target cells. The producers themselves are also af fected due to direct autocytotoxic effects [19, 105], mod ification of cell surface receptors [ 122], or inactivation of secretory products [23, 134], thereby modulating the phagocyte bactericidal and tumoricidal functions.

Fig. 4. Comparison of intact PMN and granule-de pleted neutroplasts (NPL) for their PMA-induced cyto toxic capacity against red blood cells (RBC). K562 tumor cells (K562/c), and K562 tumor cells depleted for gluta thione (K562/GSH). The effectontarget ratio was 1:2 for RBC targets and 100:1 for K562 targets, and lysis was measured after 2 h at 37 °C.


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Summary and Model for PM N Cytotoxicity

Fig. 5. PMN-mediated ADCC. I = Binding mediated through interaction of target cell bound IgG and the PMN FcyR in conjunc tion with leukocyte adhesion molecules (LFA-I) and target cell counter-structures (ICAM-1; intercellular adhesion molecule); 2 = signal transduction; 3 = degranulation; 4 = NADPG-oxidase activa tion and generation of reactive oxygen intermediates (ROI): 5 = target cell damage by granule constituents; 6 = target cell damage by ROI; 7 = inactivation of granule products by ROI, and 8 = target cell detoxification of ROI.

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PMA directly activates PKC and the subsequent gen eration of oxygen radicals, favoring a predominantly oxygen-dependent cytotoxic mechanism. There is no specificity in target cell recognition or binding. The oxy gen radicals are randomly released in the direct environ ment where they attack any potential target, either a scavenger, other leukocytes, or a ‘target’ cell. The target cell ability to escape oxygen radical damage determines severity and speed of cell lysis. A model of PMNmediated cytotoxicity is shown in figure 5. IgG-loaded target cells are efficiently bound via the PMN surface FcR, and an extensive, tight contact area is formed. Speed and efficiency of this target cell binding is accelerated and facilitated via PMN adhesion molecules. They provide additional ‘catching’ arms on the PMN surface in addition to the FcR. This is specially true under conditions where limited amounts of target cell bound immunoglobulins exist. The PMN adhesion mol ecules per se are apparently not sufficient to promote adequate target cell binding, either because this binding is weak (in comparison to FcR-mediated binding) or because additional triggering is needed to activate these adhesion molecules or enhance their surface expression. The PMN projects its surface along the target cell mem brane over a large surface area, resulting in close contact without junction formation. A tight cleft is formed be tween PMN and target cell linked via FcRs and adhesion molecules. Adhesion molecules do not activate a meta bolic response or degranulation, but the FcR occupation is linked to the generation of intracellular ‘second mes sengers’ which translate the receptor-ligand interaction into a cellular response. Both activation of the oxidative burst and degranulation are triggered. Production of oxygen radicals, however, is not absolutely necessary for ADCC, but is certainly initiated. Because the target cell is bound via a sealed area, local generation of small amounts of oxygen radicals together with degranulation into the compartment between PMN and target cell is sufficient to participate in target cell lysis. The small amount of oxygen radicals produced is by itself not suf ficient to kill the target cell directly (as measured by chromium release), but does produce damage and may facilitate subsequent killing by other lytic mediators. On the other hand, the generation of oxygen radicals in the microenvironment between PMN and target may be del eterious for the released granular products (inactivation) or may modulate initially established FcR and adhesion molecule bridges.

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Received: February 25, 1990 Accepted: April 8, 1990 Dr. Kok P.M. van Kessel Eijkman-Winkler Laboratory of Medical Microbiology University Hospital G04.614 Heidelberglaan 100 NL-3584 CX Utrecht (The Netherlands)


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van Kessel/Verhoef

Natural killer (NK) cells augment atherosclerosis by cytotoxic-dependent mechanisms.

Phagocytosis of Mycoplasma salivarium by human polymorphonuclear leukocytes and monocytes.

Cytotoxic activity against HIV-infected monocytes by recombinant interleukin 2-activated natural killer cells.

Thyrotropin-specific binding to human peripheral blood monocytes and polymorphonuclear leukocytes.

How cells kill a "killer" messenger.

Human polymorphonuclear neutrophils specifically recognize and kill cancerous cells.

Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes.

Influence of lactoferrin on the function of human polymorphonuclear leukocytes and monocytes.

Lysis of myotubes by alloreactive cytotoxic T cells and natural killer cells. Relevance to myoblast transplantation.

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells.

Enhanced cytotoxic activity of ex vivo-differentiated human natural killer cells in the presence of HOXB4.

Quantitative iodination of human blood polymorphonuclear leukocytes.

Programmed differentiated natural killer cells kill leukemia cells by engaging SLAM family receptors.

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells.

In vitro generation of activated natural killer cells and cytotoxic macrophages with lentinan.

Phosphorylase kinase from human polymorphonuclear leukocytes.

Creatine phosphokinase activity in human polymorphonuclear leukocytes.

Human Adaptive Natural Killer Cells: Beyond NKG2C.

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor.

Cytotoxic T lymphocytes and natural killer cells display impaired cytotoxic functions and reduced activation in patients with alcoholic hepatitis.

Development of natural killer cytotoxicity during childhood: marked increases in number of natural killer cells with adequate cytotoxic abilities during infancy to early childhood.

Cells with natural killer activity in human rabies.

Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate.

Natural killer cells: a TACTILE restraint.