K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.     

Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.     

The effect of RU 41.740 (Biostim) on the production of interleukin-1 by monocytes and enriched large granular lymphocytes in normals and patients with hepatocellular carcinoma

Summary The effect of RU 41.740 on natural killer (NK) cell cytotoxicity from normals and patients with advanced hepatocellular carcinoma (HCC) was studied. RU 41.740-treated normal enriched large granular lymphocytes (LGLs) showed increased cytotoxic activity against ⁵¹Cr-labelled K562 cells. Although the drug increased the cytotoxic activity of LGLs from HCC patients, these results were not statistically significant. As LGLs have been shown to release interleukin-1 (IL-1) after suitable stimulation the effects of RU 41.740 on production of this cytokine from both monocytes and LGLs was studied. The drug stimulated IL-1 release from normal cells but monocyte and LGL IL-1 production from HCC patients was not influenced by RU 41.740 by RU 41.740. It is concluded, therefore, that although RU 41.740 can stimulate natural cytotoxicity and IL-1 production by normal LGLs, it is unable to correct the defect which exists in these functions in patients with large mass tumours.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of target cell recognition by CD3- LGL. I. Development of a monoclonal antibody to a K562-associated target cell antigen.

In an attempt to identify the target recognition molecule(s) involved in the interaction between CD3- large granular lymphocyte (LGL) and a tumor cell target, monoclonal antibodies (mAb) to NK-susceptible K562 tumor cell membrane glycoproteins were developed. After screening by ELISA for reactivity to K562 membrane glycoproteins, two monoclonal antibodies were identified (mAb 35 and mAb 36). One of the monoclonal antibodies (mAb 36) was found to inhibit conjugation between LGL and K562 target cells and also to inhibit lysis of K562 by LGL. Upon further testing, mAb 36 also inhibited the binding between LGL and other NK-susceptible target cells, e.g., Daudi and Molt 4. In contrast, mAb 35, even though binding to K562, did not inhibit the binding of LGL to tumor targets and therefore was used as an isotype control. When mAb 36 was utilized as an affinity matrix, bound proteins specifically inhibited CD3- LGL-K562 conjugation. Experiments involving tunicamycin treatment of tumor target cells demonstrated that mAb 36 recognized a carbohydrate moiety rather than the protein core. Therefore, these data suggested that the target cell recognition molecule which is recognized by mAb 36 appears to be a membrane carbohydrate-associated molecule.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Macrophage cytotoxicity: interleukin 1 as a mediator of tumor cytostasis

Purified macrophage interleukin 1 (IL 1) induced a concentration-dependent inhibition of the proliferation of two commonly used tumor cell target lines, the human myeloid K562 and the murine T lymphoma Eb. In contrast, mastocytoma-derived P815 cells were not inhibited. The cytostatic action of IL 1 was not associated with direct cytotoxicity and was only partially reversible. PGE or interferon did not appear to mediate these effects. IL 1 treatment of the multipotential K562 cells revealed no morphologic evidence for the induction of specific differentiation. FACS analysis of IL 1-treated K562 cells showed a rapid decrease in transferrin receptor density, and a more delayed, but highly significant, increase in HLA-A,B,C antigen density. These findings provide one explanation for the frequently reported macrophage cytostatic actions against tumor cells, and indicate as well that IL 1, like interferon, may enhance the expression of Class I MHC antigens. These observations further extend the range of IL 1 actions and underscore the fundamental and direct role of this monokine in macrophage antitumor activity.     

Effects of natural and recombinant IL 2 on regulation of IFN gamma production and natural killer activity: lack of involvement of the Tac antigen for these immunoregulatory effects

Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histamine-induced suppressor factor inhibition of NK cells: reversal with interferon and interleukin 2

Culture supernatants of lymphocytes stimulated with 10(-3) to 10(-8) M histamine contain histamine-induced soluble suppressor factor (HISSF) that significantly inhibits the natural killer (NK) cell functions of allogeneic lymphocytes. Lymphocytes precultured with increasing concentrations of HISSF showed a dose-dependent suppressive effect on their NK activity. HISSF was not cytotoxic itself and produced suppressive effects on PBL, NK-enriched large granular lymphocytes (LGL), and isolated T cells. Suppression was evident throughout a range of effector:target cell ratios. Production of HISSF was specifically blocked by the H2 antagonist cimetidine, but not by the H1 antagonist clemastine fumarate. Furthermore, H1 and H2 antagonists themselves do not induce production of HISSF. Although HISSF could inhibit the cytotoxicity of LGL, LGL themselves do not produce HISSF. HISSF inhibition of NK activity could be completely reversed by treating effector lymphocytes with recombinant interferon-alpha (IFN) for 1 or 2 hr or culturing them with purified interleukin 2(IL 2) for 36 hr. Our data suggest that exogenous IFN and IL 2 may have therapeutic potential in the treatment of immunological diseases associated with histamine-induced suppressor cell activity.     

Evidence for an early calcium-independent event in the activation of the human natural killer cell cytolytic mechanism

In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.     

Role of lipoxygenation in human natural killer cell activation

Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC.     

Defective interleukin-1 production by natural killer cells of patients with cancer

Summary Large granular lymphocytes (LGLs) from patients with malignant disease and from controls were activated by endotoxin or K562 cells, and the supernatants assayed for interleukin-1 (IL-1) activity. Normal LGLs produced significant amounts of IL-1, the activity of which could be neutralized by anti-human IL-1 antiserum. In patients with advanced cancer depressed IL-1 production was observed, which generally correlated with the degree of cytotoxicity produced by the LGLs. Prior treatment of the LGLs with interferon increased production of IL-1 by both control and patient cells. It is suggested that LGLs coming into contact with K562 cells produce IL-1, which is important in the effector-target cell interaction. The decreased cytotoxic activity of LGLs from cancer patients could be related to a defect in IL-1 production, an effect which can be partially corrected by in vitro interferon treatment.Abbreviations IL-1 Interleukin-1 - LGLs large granular lymphocytes - NK cells natural killer cells - IFN interferon - IL-2 interleukin-2 - HCC hepatocellular carcinoma - MN mononuclear cells - LPS lipopolysaccharide Supported in part by grants from the South African Medical Research Council, the National Cancer Association of South Africa, and the South African Chamber of Mines     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.