Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function?

Freshly plated 3T3 cells send out radial projections or filopodia. We observed cells which happended to settle on glass near the borderline of a gold-plated area. When some of the filopodia contacted the gold-plated area and others the glass substratum and remained attached for a few minutes, lamellipodia then extended preferentially toward the gold-plated area. 1-2 h later, most of the cells were found in the gold-plated area. When the filopodia of a spreading 3T3 cell contacted another already spread 3T3 cell and also the glass substratum, the first lamellipodia extended preferentially towards the glass. These observations suggest a directionally differentiated extension of lamellipodia after the filopodia of a spreading 3T3 cell have contacted different substrates in their environment. Before filopodia contact a substrate, they perform a rapid "scanning" motion. Therefore, we suggest that the filopodia of a spreading 3T3 cell serve as organs which explore the nonfluid environment and react to a certain quality of the substrate that is presently unknown. Subsequently, they mediate the extension of lamellipodia into the direction in which this quality is found. The described phenomena are reversibly inhibited by Cytochalasin B at concentrations above 5 mug/ml although filopodia are produced.     

Granzyme B activity in target cells detects attack by cytotoxic lymphocytes

Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.     

Proximity oscillations of complement type 4 (alphaX beta2) and urokinase receptors on migrating neutrophils.

Migrating neutrophils utilize beta2 integrins for substrate attachment and urokinase receptors (uPAR) to focus pericellular proteolysis. Our studies show that CR3 associates with uPAR on resting cells, whereas uPAR associates with CR4 at lamellipodia of migrating cells. Using resonance energy transfer (RET) microscopy, we show that the molecular proximity between CR4 and uPAR oscillates on migrating cells, thus suggesting that CR4 molecules periodically bind/release uPAR. Cell contact with fibrinogen, endothelial cells, chemotactic factors and indomethacin, and treatment with sub-optimal doses of signal transduction inhibitors, affect the oscillations' period, amplitude, and/or waveform. The oscillations were indistinguishable in period and 180 degrees out-of-phase with cytosolic NAD(P)H autofluorescence oscillations. Thus, CR4 and CR3 identify a neutrophil's axis of migration and CR4 may restrain uPAR at lamellipodia. Oscillations in signal transduction and energy metabolism may coordinate cell adherence, local proteolysis, oxidant release, actin assembly, and cell extension.     

Tether-guided lamellipodia enable rapid wound healing

Adhesion between animal cells and the underlying extracellular matrix is challenged during wounding, cell division, and a variety of pathological processes. How cells recover adhesion in the immediate aftermath of detachment from the extracellular matrix remains incompletely understood, due in part to technical limitations. Here, we used acute chemical and mechanical perturbations to examine how epithelial cells respond to partial delamination events. In both cases, we found that cells extended lamellipodia to establish readhesion within seconds of detachment. These lamellipodia were guided by sparse membrane tethers whose tips remained attached to their original points of adhesion, yielding lamellipodia that appear to be qualitatively distinct from those observed during cell migration. In vivo measurements in the context of a zebrafish wound assay showed a similar behavior, in which membrane tethers guided rapidly extending lamellipodia. In the case of mechanical wounding events, cells selectively extended tether-guided lamellipodia in the direction opposite of the pulling force, resulting in the rapid reestablishment of contact with the substrate. We suggest that membrane tether-guided lamellipodial respreading may represent a general mechanism to reestablish tissue integrity in the face of acute disruption.     

Cytostasis of tumor cell lines by human granulocytes

Purified peripheral blood granulocytes from normal adult donors were tested for cytolytic and cytostatic activity against a variety of tumor-derived, virus-transformed, and normal cell lines. Altogether, 45 donors and 16 cell lines were tested. Although granulocytes mediated antibody-dependent cell-mediated cytolysis, no spontaneous cytolysis, as measured by chromium-51 (⁵¹Cr) or [³H]thymidine ([³H]TdR) release could be detected in assays performed for up to 12 hr, even at an effector:target (E:T) cell ratio of 100:1. In contrast, granulocytes exhibited substantial growth-inhibitory activity (GIA) against most target cells, as measured by uptake of [³H]TdR by the target cells. These results were confirmed by visual counting of target cells. The degree of cytostasis was dependent on the E:T ratio, with a plateau of 80–95% inhibition usually reached at a ratio of 40:1. Inhibition of growth of adherent tumor target cells was accompanied by cell detachment, with both effects apparent by 5 hr and reaching a peak after 15 hr of incubation. With nonadherent targets, the onset and the peak of cytostasis were delayed, being observed after 8 and 24 hr, respectively. Growth of target cells remained inhibited for up to 4 days of culture. A wide variety of target cells were sensitive to granulocyte-mediated cytostasis, including tumor-derived human and mouse cell lines, lymphoblastoid cell lines from normal donors, and embryo fibroblasts. Normal human fibroblasts were inhibited only at high E:T ratios (40:1). PHA-induced lymphoblasts were the only target cells tested that were completely resistant to the cytostatic effects of granulocytes and in fact, their growth was slightly stimulated. There appeared to be two somewhat different mechanisms of growth inhibition by granulocytes, which varied with the target cell. Trypsinization of granulocytes markedly reduced their reactivity against adherent target cells but had little effect on GIA against suspension target cells. Also, the activity against F-265, but not against other target cells, was almost completely abrogated in the presence of catalase, suggesting an important role of hydrogen peroxide in one mechanism of granulocytemediated cytostasis.     

Estimation of cell surface associated protease activity and its application to lymphocytes

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.     

Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells

By immunofluorescence observations with cell couples of cloned murine cytotoxic T lymphocytes (CTL) and target cells, evidence is presented for a rapid reorientation of the microtubule-organizing center (MTOC) and the Golgi apparatus (GA) in the effector cell (but not in the target cell) toward the contact area with the target. The reorientation of the MTOC/GA and the cytotoxic activity of the CTL were inhibited reversibly by nocodazole, a microtubule-disrupting agent. In lectin-formed cell couples of CTL and neuraminidase-treated target cells, the MTOC in essentially all of the CTL was oriented toward the effector-target contact area of a lysable target cell, but was left randomly oriented with a nonlysable target cell. A similar random orientation of the effector-MTOC was also observed in cell couples of cloned natural killer cells and nonlysable targets. These findings indicate that the repositioning of the MTOC and the GA, which is shared by CTL and natural killer cells, is an essential and early event in the onset of the cytolytic mechanism. It is suggested that this reorientation serves the purpose of directing to the bound target cell secretory vesicles derived from the GA that contain cytotoxic substances.     

Possible mechanism for target cell lysis by cytotoxic T-cells

The mechanism of the lysis of target cells by cytotoxic T-cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti-viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus-infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates 'suicide capsule' lysosomes.     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

In vitro natural cell-mediated cytotoxicity against Candida albicans: macrophage precursors as effector cells

Bone marrow cells, cultured in L-929 CSF, consist of cells of granulocyte and macrophage lineages. Cells of the granulocyte lineage are known to be cytotoxic for Candida albicans. In this paper we report that macrophage precursor cells also display strong cell-mediated cytotoxicity against the yeast form of the dimorphic fungus C. albicans. The macrophage precursors responsible for this activity are nylon wool-nonadherent, nonphagocytic cells and lack asialo GM1 surface antigen. A purified population of macrophage precursors (greater than 95%) was obtained by means of Percoll density centrifugation. The interaction of these purified effectors with the target yeast cells was analyzed at a single cell level, and their activity was compared with that displayed by cells of the granulocytic series derived from the same bone marrow culture. Macrophage precursor cells proved to be more effective in binding the target cells and showed the same killing ability as the granulocytes: macrophage precursors were not damaged by contact with the target, in contrast to that which happened with granulocytes. In a long-term colony-forming unit assay, in fact, granulocytic cells showed a decrease over time in their ability to inhibit the growth of C. albicans, probably due to cell damage and death after the interaction with the target. In contrast, no loss of activity was observed with the macrophage precursor fraction. The same macrophage precursor cells also proved able to exert good natural killer activity against YAC-1 lymphoma cells, but not against P815 mastocytoma cells, as reported previously. The macrophage precursor cells, when cultivated in vitro to mature macrophages, lost completely their natural cytotoxicity against C. albicans and YAC-1 cells. The implications of these findings, as well as the possible role in vivo of such a precursor cell population during an infection, are discussed.     

Change in activity of nuclear poly(ADP-ribose) glycohydrolase during the HeLa S3 cell cycle

The change in activity of nuclear poly(ADP-ribose) glycohydrolase during the cell cycle of HeLa S3 cells was investigated. The poly(ADP-ribose) glycohydrolase activity was solubilized from HeLa S3 cell nuclei and chromosomes only by sonication at high ionic strength. The enzyme hydrolyzed poly(ADP-ribose) exoglycosidically, producing ADP-ribose. After release from mitosis, the activity of the solubilized nuclear poly(ADP-ribose) glycohydrolase per nucleus or per unit protein, assayed with [3H]poly(ADP-ribose) (average chain length, n = 15) as substrate, was lowest in the early G1 phase and highest in the late G1 phase. The specific activity in the late G1 phase was about two times that in the early G1 phase. The high activity remained constant during the S-G2-M phase. A similar change during the cell cycle was observed after release from hydroxyurea block. These results suggest that the activity of poly(ADP-ribose) glycohydrolase doubled during the G1 phase of the cell cycle of HeLa S3 cells.     

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.     

Spontaneous cytotoxic activity of eosinophilic granule cells separated from the normal peritoneal cavity of Dicentrarchus labrax

In this study the spontaneous in vitro cytotoxic activity to tumour cell lines, (K562), by unstimulated sea bass (Dicentrarchus labrax) leukocytes was examined by trypan blue exclusion test and lactate dehydrogenase release assay. A high anti-tumour cell line activity of resident peritoneal leukocytes was found at an effector to target ratio (E:T) of 25:1 after incubation for 2 h at 18 degrees C. Rabbit and sheep erythrocytes were not lysed. A low activity was displayed by head kidney and spleen cell populations whereas blood leukocytes revealed no significant activity. The effect of E:T ratio on cytotoxicity as well as microscopy observations suggested that the cytotoxic reaction required effector-target cell contact. Eosinophilic granule cells, isolated on a Percoll density gradient from a peritoneal wash, appeared to be responsible for the in vitro cytotoxic activity.     

Compound exocytosis and cumulative degranulation by eosinophils and their role in parasite killing

The killing of metazoan parasitic larvae by eosinophils occurs following cell adhesion and the secretion o f their cytotoxic proteins onto the surface o f these targets. In eosinophils, as in mast cells and neutrophils, stimulus-secretion coupling is mediated by GTP-binding proteins. In this article, Susanne Scepek Redwon Moqbel and Manfred Lindou summarize recent results indicating that the granule-fusion events activated by GTP-binding proteins lead to compound exocytosis and cumulative fusion. They propose that these exocytotic processes, following contact with opsonized larvae, may direct secretion to a restricted space defined by the site o f contact Such a focused release may be essential for effective targeting in parasite killing, thus preventing uncontrolled random diffusion of the secreted cytotoxic proteins with the possible undesirable consequences of damage to intact host tissue.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.