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.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.     

Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

The "lazy" NK cells of Chediak-Higashi syndrome

Natural killer (NK) function, measured in a short-term (4-hr) 51Cr-release assay, is profoundly depressed in circulating PBL of donors with Chediak-Higashi syndrome (CHS). In this study, we demonstrate that CHS NK cells can express relatively normal lytic function after prolonged exposure in vitro to high levels of activating as well as cytotoxic stimuli. After activation with the human cloned interferon (B1) for 24 hr, CHS NK cells have lytic activity comparable to unactivated normals in a 4-hr 51Cr-release assay. In addition, after 5 days of activation with mitomycin C-treated B cell lines, CHS NK cells have levels of activity similar to those of activated normals but are defective in generating cytotoxic cells capable of lysing the stimulator B cell. Even though CHS NK cells are defective in a 4-hr 51Cr-release assay, after 16 hr they enhance their killing capability 200 to 400-fold. In fact, after 16 hr of interaction with K562 target cells, CHS NK cells are capable of releasing NK soluble cytotoxic factors. These results are consistent with the hypothesis that CHS NK cells have all the necessary cellular structures and molecules required for them to function as lytic effector cells, but their lack of cytotoxic function is due to a relative refractoriness in initiating the post-binding lytic mechanism.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

The functional loss of human natural killer cell activity induced by K562 is reversible via an interleukin-2-dependent mechanism

In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells.     

Sensitivity to lysis by natural killers depends on the integrity of lipid rafts in plasma membrane of transformed cells

The present work focused on the role of cholesterol-rich membrane microdomains (rafts) in cellular mechanisms of innate immunity and anticancer defence. The lytic effect of natural killers (NK) was examined in dependence on cholesterol content in transformed target cells. In the current study, K562 human erythroleukaemia cells were the targets. K562 cells were treated with methyl-beta-cyclodextrin (MbCD) to deplete membrane cholesterol that was verified by enzymatic method. With the use of 3H-uridine test, NK (mouse splenocytes) cytotoxity was estimated under various conditions, specifically, after incubation of K562 cells with MbCD or inactive analog alpha-cyclodextrin. The data obtained show that cholesterol-depleting treatment (2.5 or 5 mM MbCD) of target cells results in full loss of their sensitivity to NK lysis. The effect is likely to be due to disintegrity of lipid rafts that is critically dependent on the level of membrane cholesterol. Visualization of cell surface changes by fluorescent labeling of ganglioside GM1 confirmed our conclusions.     

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Mechanism of defective natural killer cell activity in patients with AIDS is associated with defective distribution of tubulin

Previous studies have demonstrated the importance of some cytoskeleton components in killing mechanisms. In fact, a microtubule and microfilament (MF) rearrangement in the lytic sequence of CTL and NK cells has been observed. In particular, MF seem to be related to the binding phase, because MF inhibitors suppress the binding of NK cells to the target, whereas microtubule inhibitors suppress only the killing phase. In this paper, the distribution of two cytoskeleton components, actin and alpha- and beta-tubulin, has been studied in PBL from AIDS patients, who maintain the capacity to bind to the target cell line K562 but are not able to kill it. PBL were labeled with mAb to these two cytoskeleton components, and then indirect immunofluorescence was used to visualize their distribution in the conjugates. A normal polarization of actin in the effector PBL was found, whereas no tubulin rearrangement was evident in the effector and target cells. On the contrary, in conjugates of PBL or large granular lymphocytes from normal donors and K562, a polarization of actin in the effector cell and a polarization of tubulin both in the effector and in the target cells, at the site of the attachment, was evident. These data suggest that a deficiency of tubulin rearrangement may underlie the inability of the NK cells from AIDS patients to kill their target.     

Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p15E

It has been shown previously that the retroviral envelope protein p15E suppresses certain monocyte and lymphocyte functions. In this paper, we describe the effects on natural killer (NK) activity of a synthetic peptide (CKS-17) with homology to a region of p15E conserved among numerous retroviruses. Enriched human NK cells were assayed against K562 tumor target cells in a 51Cr-release cytotoxicity assay. Pretreatment of NK cells with CKS-17 at concentrations as low as 1.5 microM, but not with equivalent concentrations of control materials, markedly and reproducibly suppressed NK lytic activity. Prior exposure of NK cells to interferon-alpha (IFN-alpha) at 1000 U/ml did not alter their sensitivity to CKS-17-induced inhibition. Pretreating NK cells with CKS-17 almost entirely diminished their responsiveness to IFN-alpha and IFN-gamma, but not to interleukin 2 (IL 2). Kinetics experiments demonstrated that CKS-17-mediated suppression of both endogenous and activated NK cells was reversible after 18 hr at 37 degrees C. Experiments designed to examine the CKS-17 mechanism of action revealed that the peptide bound to all Leu-11+ lymphocytes, as shown by two-color flow cytometry. CKS-17 did not, however, inhibit effector cell/target cell conjugate formation. These data suggest a new mechanism for immune suppression mediated by retroviruses; inhibition of NK function. They moreover imply that the CKS-17 peptide interferes with the lytic phase of NK cytolysis.     

Modulation of natural cytotoxicity by alloantibodies. V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab)₂ fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR⁺ target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.     

Sensitivity to attack of natural killers depends on integrity of lipid rafts in plasma membrane of transformed cells

This work deals with an analysis of the role of cholesterol-rich lipid microdomains (rafts) in cellular mechanisms of natural immunity and antitumor defense. The lytic action of natural killer (NK) cells was studied depending on the cholesterol content and the state of lipid rafts in the plasma membrane of transformed cells. In this work, the targets were human leukemia K562 cells. For the partial extraction of cholesterol, methyl-beta-cyclodextrin (MbCD), cyclic oligosaccharide selectively binding sterols, was used. A decrease in the cholesterol level after the incubation of cells with MbCD was confirmed by the enzymatic method. Using the ³H-uridine test, the activity of NK (mouse splenocytes) towards the cultivated K562 cells was estimated under different conditions, including those after the cell incubation with MbCD or alpha-cyclodextrin (aCD), a structural MbCD analog that does not bind sterols. The results obtained indicate that a decrease in the cholesterol content in K562 cells (after treatment with MbCD at a concentration of 2.5 or 5 mM) leads to the complete loss of their sensitivity to the NK lytic action. Most likely, this is caused by a disturbance of the structure of the lipid rafts whose integrity critically depends on the membrane cholesterol level. These conclusions agree with the data on the visualization of the cellular surface changes obtained at the fluorescent labeling of the ganglioside GM1, a marker of the cholesterol-rich lipid microdomains.     

Altered production and renewal of natural killer cells in B-lymphocyte-deficient CBA/N mice

The present study was designed to measure by quantitative and kinetic methods the production and renewal of natural killer (NK) cells in congenitally B-lymphocyte-deficient (CBA/N) mice. The total NK activity (percent specific lysis corrected for changes in whole organ cellularity) of the bone marrow and spleen of immunologically normal (CBA/CaJ) and CBA/N mice was assayed prior to and immediately after 48 h treatment (2 X/day, i.p.) with the cell cycle poison hydroxyurea (HU) and at various intervals throughout the subsequent post-HU recovery period. The total NK activity (TNKA) of untreated CBA/N bone marrow was 154% of that of CBA/CaJ bone marrow while the TNKA of CBA/N spleen was not significantly different (112%) from that of CBA/CaJ spleen. At the conclusion of 48 h HU, bone marrow TNKA of CBA/N and CBA/CaJ mice fell to 60 and 49%, respectively, of their saline-injected (2 X/day, i.p.) control levels, while spleen TNKA fell to 42 and 61%, respectively, of their saline-injected control levels. In the bone marrow, NK cell depletion in response to HU was more rapid in CBA/N mice (day 0.5 after HU) than in CBA/CaJ mice (day 2 after HU). TNKA of the spleen also decreased more rapidly in CBA/N mice (day 2 after HU) than in CBA/CaJ mice (day 3 after HU). The data indicate an enhanced production and turnover of NK cells in CBA/N mice relative to CBA/CaJ mice. Moreover, increased production and renewal of NK cells in CBA/N mice together with virtually unchanged levels of NK activity (112% of CBA/CaJ mice) in CBA/N mouse spleens indicate that mature lytic NK cells in CBA/N spleen but not bone marrow have a significantly shorter post-mitotic life span than do NK cells in the spleens of immunologically normal (CBA/CaJ) mice.     

Definition of a secondary target cell trigger during natural killer cell cytotoxicity: possible role of phospholipase A2

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.     

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.