Evidence of a dynamic role of the target cell membrane during the early stages of the natural killer cell lethal hit

The role of membrane movement during the stages of human NK cytolysis has been examined by using the bifunctional protein cross-linking reagent, glutaraldehyde. The binding stage was inhibited when K562 target cells or NK cells were pretreated with glutaraldehyde. When added post-binding, after initiation of calcium pulse, glutaraldehyde did not dissociate conjugates, but inhibited NK cytolysis. In contrast to the early stages of NK cytolysis, glutaraldehyde enhanced lysis during the terminal stage, killer cell independent lysis ( KCIL ). Lysis of the preprogrammed target cells, however, was enhanced only when glutaraldehyde was added immediately after dispersion of the conjugates, before target cell lysis. The mechanism of enhancement of lysis during the terminal stages of cytolysis was further explored in assays for NK cell-derived cytolytic factor (NKCF). L929 target cells prebound with NKCF were lysed more readily in the presence of glutaraldehyde, but as in KCIL , maximum enhancement of lysis occurred when glutaraldehyde was added immediately after NKCF was bound to the target cell. These results suggest that the target cell membrane may play a dynamic role during the terminal stages of the NK lethal hit.     

Studies on the mechanism of the human natural killer cell lethal hit: a new method for rapid physical isolation of lethally hit K562 targets and inhibition of cytolysis by reduced temperature or trypsin

A new method was developed which allows for rapid (2 min) physical isolation of viable K562 target cells after being programmed to lyse (lethally hit) by purified human natural killer (NK) cells (LGL). To achieve this K562 cells which were obtained from the 34-36% interface of discontinuous Percoll gradients and purified human NK cells (LGL) which were obtained from the (43-45% Percoll) interface were employed. Using a Ca2+ pulse method and the separation of NK-K562 conjugates with EDTA and rapid centrifugation on Percoll gradients at 4 degrees C we could physically isolate the lethally hit K562 cells from the LGL allowing the study of the events leading to their subsequent lysis. Lysis of "purified" lethally hit K562 cells occurred in the absence of Ca2+ or Mg2+ and was blocked by reduced temperature (4 degrees C), or by the protease enzyme trypsin. When lethally hit targets were held at 4 degrees C (to block lysis) then rewarmed to 37 degrees C lysis ensued but with a rate slower than that of control cells not held at 4 degrees C. These data support the concept that transfer of protease-sensitive and possibly temperature-dependent structures from the NK cell to the target is a requisite step in NK cytolysis.     

T-lymphocyte mediated cytolysis: Temperature dependence of killer cell dependent and independent phases and lack of recovery from the lethal hit at low temperatures

Using alloantiserum and complement to inactivate cytolytic T-lymphocytes after they had administered the “lethal hit” to target cells, the rate of killer-cell independent lysis (KCIL) as measured by radiochromium release was followed at various temperatures. Under usual conditions, KCIL was half-completed on the average after 1.7 hr at 37 °C. The average Q₁₀ of KCIL is about 1.6 during the first few hours after cooling, but near 0 °, lysis slows down at later times. Thus, the extent of KCIL after 6–8 hr at 0 ° is frequently less than one-tenth of that at 37 °C. The Q₁₀ of the whole killing process is 2.5 near 37 °C but exceeds 6 near 22 °C.Evidence has been presented elsewhere suggesting that recovery from complement mediated damage may occur under appropriate conditions. Since KCIL can largely be arrested at low temperatures, we tested for possible recovery from or repair of the T-cell administered “lethal hit” during incubations at low temperature following (i) inactivation of killer cells by antiserum and complement or (ii) detachment of killer cells with EDTA and prevention of subsequent killer-target cell contact with dextran. No evidence for recovery from the “lethal hit” was found during incubations from 0.3 to 5 hr at 20 °, 15 °, or 0 °C. The temperature dependence of KCIL raises the possibility that metabolic events are of importance during KCIL. However, the previous finding that lysis following damage mediated by antiserum and complement is equally temperature sensitive leaves no basis for postulating such metabolic events. Hence, although unequivocal direct evidence has been difficult to obtain, colloid osmotic lysis is at present the simplest and most plausible explanation of killer-cell independent lysis.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane phospholipids

Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.     

Evidence for involvement of serine proteases in the late stages of the natural killer cell lytic reaction

An irreversible inhibitor (L-1-tosylamide-2-phenylethyl-chloromethylketone) and substrate (N-acetyl-L-tyrosineethylester) of the neutral serine protease chymotrypsin were evaluated for their effects on the natural killer cell lytic reaction sequence. During direct cell-mediated cytolysis these inhibitors had no effect on natural killer cell binding to target cells but were able to inhibit the "trigger" mechanism which initiates killing. In addition, they inhibited later calcium-dependent events in the lytic reaction and killer cell-independent lysis. These findings suggest that serine proteases may be required during several stages of natural killer cell lysis, including calcium-dependent programming as well as the actual lethal hit.     

Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.     

Further characterization of PNK-E: a monoclonal antibody enhancing porcine natural killer cell activity

Monoclonal antibody PNK-E binds to approximately 15% of porcine peripheral blood lymphocytes (PBL) which are PT4 negative and PT8 positive. When cells from tissues of adult pigs are treated with PNK-E, enhancement of natural killer (NK) cell activity is observed from PBL and spleen cells, and a dramatic induction of NK activity is observed from bone marrow cells. With cells derived from tissues of neonatal piglets, PNK-E induces NK activity from PBL and bone marrow cells. To investigate the mechanism of PNK-E-mediated enhancement of NK, proliferation assays, calcium-pulse assays, single-cell assays, and kinetic analyses were performed. PNK-E did not induce proliferation of PBL. PNK-E could be added as late as 30 min prior to termination of Ca(2+)-pulse assays and still enhance NK activity. Using kinetic analysis PNK-E was found to increase the rate of NK lysis (Vmax) and rate of lytic programming per NK cell (k2). In addition, results from single-cell assays indicate that PNK-E activates a population of normally inactive effector cells. These results indicate that PNK-E enhances the lytic capacity of mature NK cells and induces a population of nonlytic cells to become highly cytolytic cells. Furthermore, the enhancing effects are immediate and do not require an induction period. Thus, PNK-E recognizes and activates a unique triggering molecule that is present on NK cells.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Target-effector interaction in the natural killer cell system. IV. Modulation by cyclic nucleotides.

Dibutyryl cAMP (dB-cAMP) and the cAMP elevating agents, prostaglandin E1, theophylline, and histamine markedly suppressed NK cytolytic function in a dose- and rate-dependent manner. The inhibition was rapidly induced and persisted in the presence of the drugs. Separate pretreatment of targets and highly purified NK cells, isolated by a target binding and velocity sedimentation technique, revealed that PGE1 and dB-cAMP acted at the level of the effector cell in a short-term cytolytic assay. In contrast to the inhibitory effects of cAMP elevating agents, dB-cGMP and carbamylcholine caused a small but significant acceleration in the rate of lysis and could compete with inhibitory doses of dB-cAMP to reduce the level of suppression thereby suggesting that the cAMP-cGMP ratio might be important in NK-mediated lysis. Insulin had no effect on NK activity, whereas T cell-mediated cytolysis was augmented by insulin and cGMP if the effector cells were taken early after alloimmunization but not later. Neither cAMP- nor cGMP-elevating agents affected the frequency of NK-target cell conjugates. These results are compatible with the hypothesis that cyclic nucleotides may be involved in triggering the lytic event within NK cells.     

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.     

Mechanism of cell contact-mediated inhibition of natural killer activity

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.     

Augmentation of human natural cytotoxic cell activity by leukotriene B4 mediated by enhanced effector-target cell binding and increased lytic efficiency

The addition of leukotriene B4 (LTB4) to cytotoxicity assays measuring natural killer (NK) or natural cytotoxic (NC) cell activities resulted in significantly augmented killing of K562 or herpes simplex virus (HSV)-infected target cells, respectively. Since the mechanism of cytotoxicity implies several steps, including the binding of effectors to targets which is Mg2+-dependent and the programming of lysis of the target which is Ca2+-dependent, we undertook to define the step(s) at which LTB4 acted in augmenting cytotoxicity. Our results showed that LTB4 significantly increased the percentage of effector-target conjugates when K562- or HSV-infected targets were incubated with lymphocytes. Maximal binding occurred at a concentration of LTB4 of 1 X 10(-10) M. Preincubation of lymphocytes and not target cells with LTB4 was sufficient to observe the increased binding. PBML binding to and killing of the NK-resistant target clone I, derived from K562, was not enhanced by LTB4. In the absence of Ca2+, cytotoxicity was impaired and LTB4 could not restore it. Use of a single cell lytic assay demonstrated augmented efficiency of lysis of both K562 and HSV-infected targets in the presence of LTB4. These findings suggest that LTB4 may augment natural cytotoxicity by enhancing target cell recognition by cytotoxic effector cells and subsequently by augmenting their lytic efficiency.     

Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes.

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37 degress C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0 degrees thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di- and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75 degrees C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10-second intervals. This method was more convenient than standard procedures and gave yields of di- and triphosphoinositide equivalent to that obtained by the method of Folch.     

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.     

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer: target cell interactions

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.     

Characterization of antibody-mediated inhibition of natural killer (NK) cytotoxicity: Evidence for blocking of both recognition and lethal hit stages of cytolysis

Rat antisera prepared against murine, periodate-activated alloimmune cytotoxic lymphocytes (termed RAT¹) have previously been shown to effectively block T-cell-mediated cytotoxicity (CMC) at the “lethal hit” stage of cytolysis (J. C. Hiserodt and B. Bonavida, J. Immunol.126, 256, 1981). Both natural killer (NK) and cytotoxic T lymphocytes (CTL) have been shown to mediate lysis by the same pathway, namely binding of effector to target cells, programming for lysis, and killer cell-independent target cell lysis. This result suggested that the molecular mechanism of NKCMC and CTLCMC may also be similar. In this context, RAT¹-mediated blocking of CTL was examined for its ability to block NKCMC. The results show that (1) addition of RAT¹ serum or IgG fractions blocked NKCMC in the absence of complement in a 4-hr ⁵¹Cr-release assay, and blocking was directed at the effector cell; (2) at the single-cell level, RAT¹ serum blocked the formation of conjugates between effector and target cells; (3) in a Ca²⁺-pulse experiment, in which the effectors and targets were first allowed to bind in the absence of Ca²⁺ for 1 hr at 37 °C, followed by the addition of Ca²⁺ to initiate the lytic event, RAT¹ was capable of blocking cytotoxicity after conjugate formation at the Ca²⁺-dependent lethal hit stage of cytolysis. The similarity of results in RAT¹ blocking experiments of both the CTL and NK systems suggests a common molecular mechanism of cytolysis.     

Inhibition of human natural killer (NK) activity by calcium channel modulators and a calmodulin antagonist

The presence of calcium (Ca2+) in the culture medium is a requirement for the NK cytotoxic reaction. To further explore the role of Ca2+ and calmodulin (a cytoplasmic protein that mediates most of the biological effects of Ca2+) in this process, we evaluated the effects of nifedipine (a Ca2+ channel antagonist), BAY-K-8644 (a Ca2+ channel agonist), and haloperidol (an inhibitor of calmodulin) on the NK activity of human peripheral blood mononuclear cells (PBMC), and the augmentation of this activity by recombinant interleukin 2 (r-IL 2) and interferon-gamma (r-gamma-IFN). We found that all of these drugs inhibit NK activity in a dose-dependent fashion. This appears to result from interference with the programming for lysis stage of the lytic process. In contrast, the presence of these agents during the incubation of PBMC with r-IL 2 or r-gamma-IFN did not induce any change in the enhancement of NK activity. These data suggest that Ca2+ exerts its effect at the intracellular level during the NK cytotoxic process, and that the augmentation of NK activity by lymphokines is independent of the calcium-calmodulin system.     

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.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.