Human Natural Killer Activity Research Articles

Relationship between expression of herpes simplex virus glycoproteins and susceptibility of target cells to human natural killer activity.

Cells normally insensitive to human natural killer (NK) activity were rendered susceptible by infection with HSV-1. The cytotoxic effector cell was a nonadherent, non-T, non-B lymphocyte. Antibody plus complement treatment, using a monoclonal antibody that recognizes an antigen present on NK cells, removed much of the cytotoxic activity, and a density gradient fraction enriched for NK cells yielded cells of increased virus-specific cytotoxicity. It was concluded that the effector cell active against infected targets possessed characteristics of an NK cell. Blockage of viral protein synthesis during infection inhibited development of increased susceptibility of infected targets to NK activity. When targets were infected with a mutant virus unable to produce viral glycoprotein C (gC), NK activity against these targets was reduced approximately 30% compared with activity against targets infected with wild-type virus. Similarly, activity against targets infected in the presence of 2-deoxyglucose (2dG), which prevents cell surface expression of viral glycoprotein B (gB), was also reduced approximately 30%. An approximately 60% reduction in activity was seen against targets infected with mutant virus in the presence of 2dG; these targets express gD, but neither gB nor gC. When cells expressing various combinations of HSV-1 glycoproteins were used as both labeled targets and cold target competitors, it was found that the susceptibility of a particular target to NK activity was paralleled by its ability to act as a cold target competitor. This indicates that targets with decreased sensitivity to NK cells were less able to bind NK effectors. Further, the amount of interferon produced in co-cultures of NK effectors and infected target cells did not directly correlate with the amount of NK activity generated, and interferon pretreatment of effectors did not decrease virus-specific cytotoxicity. The present results suggest that HSV-1 glycoproteins expressed at the surface of infected targets may act as recognition structures for NK cells.

Ultraviolet radiation inhibits human natural killer activity and lymphocyte proliferation.

Exposure to ultraviolet radiation (UVR) has been implicated in the predisposition to certain neoplasms and leads to viral reactivation. Natural killer (NK) activity may play a role in immunosurveillance and response to certain viral infections. We have evaluated the sensitivity to UVR of human NK activity, a nonproliferative function, and the proliferative response to the mitogen phytohemagglutinin (PHA). In vitro exposure to UVR resulted in a dose-dependent inhibition of NK activity and response to PHA. The wavelength dependence for UVR inhibition of NK activity and of the PHA response of lymphocytes were virtually superimposable at wavelengths at or above 300 nm, but NK activity was less sensitive to UVR at 260 and 280 nm. Maximal sensitivity for both functions was found at 260 nm, consistent with a nucleic acid chromophore mediating UVR inhibition of both activities. The DNA-directed drugs mitomycin C, acridine orange, and adriamycin at concentrations that inhibit proliferation are poor inhibitors of NK activity. These results suggest that UVR inhibition of NK activity as well as proliferation may be mediated by a nucleic acid chromophore. NK activity, however, is less sensitive to direct damage of DNA by alkylation, distortion, or oxidation. At 300 nm, the amount of radiation required to inhibit NK activity and proliferation is within the range penetrating to the dermis and capillaries during environmental exposure to sunlight.

Cyclic AMP as a mediator of prostaglandin E-induced suppression of human natural killer cell activity.

The role of cyclic AMP in mediating the prostaglandin (PG) E2-induced suppression of natural killer (NK) cell function was studied. Highly purified preparations of human large granular lymphocytes (LGL; which have been shown to be closely associated with human NK activity) and T lymphocytes were obtained by using Percoll gradients. Basal cyclic AMP was similar in both cell populations. With LGL, PGE2 (but not PGF2 alpha) suppressed NK activity (75%) and enhanced cellular cycle AMP (600%). In contrast, in small, high density T lymphocytes, PGE2 caused very small increases in cyclic AMP (20%). Phosphodiesterase inhibitors (PDEI) such as isobutyl methylxanthine and theophylline also increased cyclic AMP and suppressed NK activity in LGL. The effect of PGE2 and PDEI, in combination on cellular cyclic AMP and NK activity, was greater than the effect of each agent alone. Finally, exogenous derivatives of cyclic AMP also suppressed NK activity. These results indicate that in LGL, increased cellular cyclic AMP mediates the action of PGE2 on the suppression of NK activity.

Involvement of transferrin and transferrin receptors in human natural killer effector:Target interaction

In this current study one of the determinants of natural killer cell specificity in immunosurveillance against cancer, may be the recognition of transferrin receptors on neoplastic cells by the NK effectors. Human transferrin, when saturated with iron (FeTf), was found to inhibit human natural killer (NK) activity against K562 tumor cells, if included in assay mixtures at physiologically relevant levels. Whereas both FeTf and iron-free transferrin (ApoTf) inhibited initial conjugate formation at the level of the target cell, only FeTf inhibited NK cytolytic activity, as judged by release of chromium from the targets. This suggested a functional role for FeTf on either NK or tumor cells. When either targets or effector lymphocytes were pre-incubated with FeTf, inhibition of killing was strongest when the targets were first exposed to FeTf. The evidence suggested that NK-associated transferrin mediated the interaction with target cells through free target-associated transferrin receptors. The finding that rabbit anti-human transferrin antibody (RaHTf) inhibited killing, when reacted with effector lymphocytes but not with target cells, supported this hypothesis.

The differential effects of human leukocyte pyrogen/lymphocyte-activating factor, T cell growth factor, and interferon on human natural killer activity.

The differential effects of human leukocyte pyrogen/lymphocyte-activating factor, T cell growth factor, and interferon on human natural killer activity.

Contrasting effects of lactoferrin on human lymphocyte and monocyte natural killer activity and antibody-dependent cell-mediated cytotoxicity.

Iron and the iron-binding protein lactoferrin (LF) have significant effects on natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC). Human adherent and nonadherent blood mononuclear cells were incubated with K562 cells and antibody-sensitized Chang cells in short-term chromium-release assays. Ferric citrate (10(-3) M) inhibited both adherent and nonadherent NK cell activity, but had no effect upon ADCC. LF markedly affected adherent monocyte cytotoxicity, but had no effect on nonadherent lymphocytes. LF enhanced NK in a dose-dependent manner, but similar concentrations paradoxically inhibited ADCC. LF acted directly upon the adherent effector cell because pretreatment of cells for 30 min was sufficient for enhancement. Fe-saturated LF as well as the unsaturated protein enhanced adherent cell NK. Transferrin in all concentrations tested did not alter NK activity. These studies show inhibitory effects of iron on immune function in addition to those previously described and reveal a new regulatory role for LF. The selective effect of LF on adherent cells provides further evidence that monocytes, at least in the adherent state, can have potent NK activity. The opposite effects of LF on NK and ADCC are unexplained and may serve as a probe to define the mechanisms involved.

Changes in human natural killer activity early and late ater renal transplantation using conventional immunosuppression.

The natural killer (NK) cell activity of human peripheral blood lymphocytes falls following major surgical procedures including renal transplantation but in non-immunosuppressed individuals returns to normal levels within the first 72 hr after operation. In renal allograft recipients, if this early postoperative fall is excluded from the analysis, NK cell function appears to follow changes in allograft function, suggesting that in vivo, as has been reported in vitro, NK activity is generated during activation of the alloreactive process. In an additional group of patients whose grafts were functioning for between 3 and 102 months after cadaveric renal transplantation using conventional immunosuppression, NK function was depressed in comparison with that of control subjects. However, some patients who were more than 48 months post-transplant had normal NK cell activity. Collectively, these results suggest that NK cell function may recover despite the continued administration of conventional immunosuppressive agents.

Trimethoprim-induced augmentation of human natural killer activity in vitro

Trimethoprim-sulfamethoxazole enhanced human natural killer activity in vitro at concentrations that did not alter lymphocyte mitogenesis and that were within protein-free therapeutic levels. Trimethoprim is the active component.

Human natural killer activity: Effects of leukocytic pyrogen/lymphocyte activating factor (LP/LAF), T-cell growth factor (TCGF), and interferon (IFN)

Human natural killer activity: Effects of leukocytic pyrogen/lymphocyte activating factor (LP/LAF), T-cell growth factor (TCGF), and interferon (IFN)

In vitro augmentation of human natural killer (NK) cell activity by a streptococcal preparation OK-432 and its extracts, protein M and polysaccharides.

Effect of a streptococcal preparation OK-432 and its derivatives on the augmentation of human natural killer cell activity in vitro was examined. The strong augmentation was found by the OK-432 stimulation. Cell separation procedures revealed that cytotoxic activity augmented by the stimulant was considered to be mediated mainly by nonadherent, nonphagocytic, IgG-Fc receptor positive, non-T cells, i.e. NK cells. In addition, supernates cultured with OK-432 augmented NK activity, suggesting some kinds of soluble factors released in the supernates may be participating on the augmentation of human NK activity by OK-432.

Human natural killer-like activity against mouse spleen cells.

Unstimulated human peripheral lymphocytes were cytotoxic for normal mouse spleen cells and suppressed the in vitro antibody plaque-forming cell (PFC) response of these cells to sheep red blood cells and dinitrophenylated Ficoll. The cells in the lymphocyte population that were responsible for the immunosuppression had properties of natural killer (NK) or NK-like cells in that they were: (a) non-E-rosetting, (b) nonadherent, (c) unaffected by treatment with anti-human immunoglobulin plus complement, (d) cytotoxic against an established human NK target, K562 leukemic cells, and (e) partially inactivated by mitomycin C. Addition of the human NK-like cells to mouse spleen cell cultures at the time of antigen addition and at an effector cell to target cell ratio as low as 0.67:1 resulted in 85 to 96% suppression of the PFC response. Addition of NK-like cells to cultures 18 h before harvesting in 5-day cultures required higher concentrations and ratios (2.7:1) of effector to target cells to significantly suppress the PFC response. The data suggest that human NK-like activity in suppression of the mouse PFC response is due to killing of the targets. The mouse spleen cell PFC system represents a potential model for assessment of human NK activity that is quite dramatic in its effect and can be used in addition to the well known 51Cr-release assay. Also, since the mouse spleen cell is a normal cell, it provides a model in the PFC system for studying the mechanism of NK regulation of normal cellular function. An additional finding of this study was the observation that E-rosetting T cells significantly enhanced the mouse PFC response. Thus, human peripheral lymphocytes contain discrete cellular population that either enhance or suppress the mouse PFC response.

Regulation of human natural killing. I. The role of monocytes, interferon, and prostaglandins.

We have found that human endogenous natural killer activity as measured in a rapid 51Cr release assay is unaffected by the presence of monocytes. Moreover, stimulation of natural killer cells by poly I:C is independent of monocytes. In contrast, the presence of monocytes in a mixed population of mononuclear cells that have been stimulated by poly I:C suppresses NK activity. The suppression is shown to be partially reversible if indomethacin (10(-6) M) is added to the cultures during stimulation. Culture supernatants of monocytes stimulated with poly I:C are shown to contain PGE1 in a radioimmunoassay, and have NK inhibitory activity comparable to that obtained with exogenous PGE1 added to NK assays at a concentration range of 10(-7) to 10(-9) M. Culture supernatants from poly I:C-stimulated monocytes do not have detectable levels of antiviral activity. In contrast, plastic nonadherent cells stimulated with poly I:C produce significant levels of interferon (100 to 200 U/ml/2 x 10(6) cells) but almost undetectable amounts of PGE. Supernatants of nonadherent cells incubated with indomethacin (10(-6) M) alone augment NK activity. Taken together, the results suggest that stimulation of mononuclear cells with poly I:C is dependent on and regulated by the relative levels of interferon produced by lymphocytes and PGE produced by monocytes.

Natural killer (NK) cell activity in the rat. I. Isolation and characterization of the effector cells.

This study has demonstrated the presence of large granular lymphocytes (LGL) in the rat and implicated a central role for these cells in mediating natural killer (NK) cell activity in this species. The LGL frequency from various organs was shown to be: peripheral blood = lung greater than spleen greater than peritoneal exudate greater than lymph node. Little or no LGL were found in the thymus or bone marrow. This pattern was similar to the distribution of NK activity, except for the lungs, which had no detectable activity. These studies have also shown that discontinuous Percoll density gradients can be used to highly enrich or deplete LGL from both spleen and peripheral blood. Enriched LGL populations were shown to bind selectively to NK-susceptible target cells and to demonstrate very high NK cell cytolysis. Since human NK activity has also been found to be clearly associated with LGL, the present findings indicate that rats may provide a very useful animal model for detailed studies of the ontogeny, regulation, and in vivo relevance of NK cells.

Phospholipid methylation and phospholipase A2 activation in cytotoxicity by human natural killer cells.

The role of phospholipid methylation and phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) in natural killer (NK) function by human peripheral blood mononuclear cells was studied. Pretreatment of effector cells with a methyltransferase inhibitor, 3-deazaadenosine, in the presence of homocysteine thiolactone, reduced cytotoxicity in a dose-dependent fashion. This effect was closely associated with inhibition of methylation of lipids but not of nucleic acids or proteins. The suggestion for a role of phospholipid methylation was supported by the observation that the interaction between NK-susceptible tumor targets and peripheral blood mononuclear cells caused increased phospholipid methylation only when susceptible target cells were used. Phospholipase A2 was also implicated in human NK activity. Inhibitors of the enzyme such as tetracaine, mepacrine, Rosenthal's inhibitor, and corticosteroids impaired NK function. Rosenthal's inhibitor was also shown to exert an inhibitory effect on a purified NK-cell population obtained by the isolation of large granular lymphocytes on Percoll gradients. Peripheral blood mononuclear cells were also directly shown to display phospholipase A2-like activity, as measured by the decrease in radioactive arachidonate from prelabeled phospholipids, specifically phosphatidylcholine, in effector cells. These data suggest that enhanced phospholipid methylation occurs during the recognition function of NK cells. Consequent activation of phospholipase A2 might be involved in the mechanisms leading to lytic events within the target cell.

Inhibition of human natural killer activity by cyclosporin A.

The effect of in vitro exposure to cyclosporin A on human natural killer (NK) activity was investigated using peripheral blood lymphocytes as effectors and 51Cr-labeled K562 cells as targets. After preincubation of the effectors with the drug for 20 hr, a dose-dependent reduction of NK activity was observed at concentrations ranging from 0.1 to 10 micrograms/ml. Exposure to the drug during the 20-hr preincubation and the 4-hr 51Cr-release assay resulted in greater impairment of NK cytotoxicity than preincubation alone, whereas the drug, present only during the assay, had no effect on cytolytic activity. Inhibition of NK activity required a minimal exposure time to the drug of 8 hr. The inhibitory effect of cyclosporin A on NK activity was rapidly reversible as, after a 20-hr incubation with 19 micrograms/ml, complete recovery of cytotoxicity was already observed after a further 4-hr culture in medium. Partially purified human fibroblast interferon augmented NK activity even in the presence of high concentrations (10 micrograms/ml) of cyclosporin A.

Regulation of natural killer activity in vivo. II. The effect of alcohol consumption on human peripheral blood natural killer activity.

Natural killer (NK) activity of human peripheral blood lymphocytes (HPBL) was studied in 32 alcoholics and 15 control subjects in a 4-h chromium release assay using K562 tumor target cells. The natural killer activity of human peripheral blood lymphocytes in alcoholics was significantly higher than the level found in the control group. NK activity of PBL from alcoholics was still higher than that of similarly treated PBL from control subjects after carbonyl iron and magnet treatment, or after passage through a nylon wool column or after removal of cells rosetting with sheep erythrocytes. Changes in T cell, B cell or macrophage populations, therefore, are not the mechanism for increased NK activity in alcoholics.

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.

Morphological and Functional Characterization of Isolated Effector Cells Responsible for Human Natural Killer Activity to Fetal Fibroblasts and to Cultured Cell Line Targets

Morphological and Functional Characterization of Isolated Effector Cells Responsible for Human Natural Killer Activity to Fetal Fibroblasts and to Cultured Cell Line Targets

Evaluation of the Role of IgG Antibodies in Human Natural Cell-Mediated Cytotoxicity Against the Myeloid Cell Line K562

Abstract The possible role of anti-target cell antibodies in human natural cell-mediated cytotoxicity against the K562 myeloid leukemia cell line was investigated in a 4-hr 51chromium release assay (CRA) by using, as effector cells, peripheral blood lymphocytes from normal, healthy donors. We investigated whether anti-target cell antibodies (IgG) were involved in the mechanism of natural cytotoxicity, either 1) as “arming” antibodies bound to the Fc receptor of the natural killer (NK) cell in vivo, or 2) by synthesis during the in vitro incubation period of the CRA. NK activity was not affected by procedures that were expected to remove cytophilic IgG from effector lymphocytes, such as washing whole, unseparated blood at 37°C three times before exposure to Ficoll-Hypaque; extensive washing (up to ten times) of purified lymphocytes at 37°C; or incubation of lymphocytes at 37°C for as long as 48 hr. After exposure of effector cells to trypsin for 30 min at 37°C, NK activity was always significantly reduced, and was not spontaneously recovered, even after incubation in culture medium for up to 48 hr. NK activity was not restored to trypsinized lymphocytes by incubation with normal autologous or allogeneic sera at 4°C, 24°C, or 37°C for times ranging from 0.5 to 18 hr before the CRA. Further, K562 was not sensitized for ADCC by these same normal sera, in contrast to the strong ADCC, which resulted when the target cells were preincubated with rabbit anti-K562 sera. Only when normal sera were left in the assay for 4 hr did we very occasionally observe boosting of cytotoxicity of untreated or trypsinized lymphocytes. More frequently, sera left in the assay caused some inhibition of cytotoxicity. When effector cell suspensions were incubated before the CRA, or for the duration of a 4 hr or 18 hr CRA, with various concentrations of potent Fab or F(ab′)2 fragments of goat anti-human F(ab′)2, NK activity was not specifically inhibited. Further, protein A from Staphylococcus aureus, which binds to the Fc portion of most subclasses of human IgG, did not inhibit NK when left in the assay at concentrations up to 100 µg/ml, but significantly inhibited, in a dose-dependent manner, K cell activity to Change cells coated with human anti-HLA serum. Thus, no clear evidence in support of a role for IgG in human NK activity against K562 cells could be demonstrated.

Interferon enhanced human natural killer and antibody-dependent cell-mediated cytotoxic activity.

Preincubation of normal human lymphocytes with human interferon for 18-24 h at 37 degrees C resulted in an increase of the activity of both natural killer (NK) cells and antibody-mediated cytotoxic cells (ADCC). The human myeloid line, K-562, which is highly susceptible to NK cells, was employed. ADCC was assessed with antibody-coated chick erythrocytes as targets. NK cells and ADCC were detected in a 4-hour 51Cr release assay. The magnitude of the enhancement was proportionate to the amount of interferon used in preincubation of the effector cells. Preincubation of tumor-target cells with interferon does not increase their susceptibility or resistance to lysis. The major effect of interferon on the cellular metabolism of the tumor-target cell is inhibition of DNA synthesis, but no direct cytotoxic effect was detected. Our findings may be important in understanding the mode of action of interferon in increasing host resistance to a variety of pathogens and tumors. This may be accomplished by inhibiting the growth of the tumor while simultaneously enhancing the natural killing mechanism for immunosurveillance.