Human Natural Killer Cell Activity Research Articles

Effects of isoniazid treatment on human lymphocyte proliferative response, lymphocyte subsets and natural killer cell activity

The effect of isoniazid on proliferative response, natural killer (NK) cell activity and lymphocyte subset distribution of blood mononuclear cells (BMNC) was investigated. To evaluate the effect of treatment with isoniazid in pharmacologic concentrations, twenty healthy HIV-seronegative volunteers were randomized into two groups: one group received isoniazid tablets plus pyridoxin tablets once a day for 30 days, the other group received pyridoxin only. Blood samples were collected on day 0 and day 30. Inhibition of the PHA-induced proliferative response was demonstrated in lymphocyte cultures from isoniazid-treated volunteers (p< 0.001). However, no effect was seen on the IL-2- or antigen (PPD)-induced proliferative response or the NK cell activity of isolated BMNC. Inhibition of the PHA-induced proliferative response could not be related to changes in the distribution of CD3 + , CD4 + , CD8 + , CD14, or CD19 + lymphocyte subsets. The effects, in vitro, were investigated by addition of isoniazid to cultures of BMNC isolated from either HIV-seroposive or HIV-seronegalive donors who did not receive any treatment. We found that isoniazid did not influence the mitogen- or antigen-stimulated proliferative response or the NK cell activity.

The role of natural killer cells in the treatment of chronic myeloid leukemia.

The success of chemotherapy in patients with leukemia whose marrow appears to be replaced by leukemia cells must be due to the persistence of normal stem cells. In this normal population are the progenitors of the cells of the immune system. Natural killer (NK) cells originate in the bone marrow. On maturation and activation with interleukin 2 (IL-2) or other cytokines, NK cells develop cytotoxic activity against a variety of leukemic blasts, including those from patients with chronic myeloid leukemia (CML). In the past few years, bone marrow transplantation (BMT) and alpha-interferon (IFN-alpha) have proved to be the most promising therapies for the treatment of CML. In both these therapies, NK cells may play a prominent role. In this article, we discuss the antitumor/antileukemia activity of human NK cells, the presence of benign NK cell precursors in the different stages of CML, the role of NK cells in BMT and IFN-alpha treatment, and the potential therapeutic applications of NK cells in patients with hematologic malignancies.

Human natural killer cytotoxic activity is not affected by in vitro exposure to 50-Hz sinusoidal magnetic fields.

Epidemiological studies have suggested, but not demonstrated, a role of exposure to 50/60-Hz magnetic fields in increasing cancer risk in man (workers and the general population). A possible target of magnetic fields is the immune system. In particular, it is known that an important defence against cancer is represented by natural killer (NK) cells capable of killing cancer cell targets. To test this hypothesis, human NK cells, stimulated or not with phytohaemagglutinin or interleukin 2, were exposed to 50-Hz sinusoidal magnetic fields before or during the cytotoxicity test, and then mixed with a variety of target cancer cell lines (Daudi, Raji, U937, H14, IGROV, SW626, K562, HL60). The experiments were performed in two laboratories (Rome and Modena) by means of two different exposure systems. The results of both laboratories suggest that 50-Hz sinusoidal magnetic fields with flux densities up to 10 mT do not affect the cytotoxic activity of human NK cells.

Effects of ethanol on human natural killer cell activity: in vitro and acute, low-dose in vivo studies.

Chronic use of ethanol may cause a variety of immunological abnormalities in humans. In this study, we have determined the effects of an acute, low dose of ethanol (0.5 g/kg), administered either intravenously or orally, to normal, nonalcoholic male volunteers, on natural killer cell (NK) activity. We have also examined the effects of a 4-hr incubation with ethanol, in concentrations ranging from 0 to 320 mg/dl, on human NK activity in vitro. NK activity was measured by the 51Cr release assay technique in all of these studies, using peripheral blood mononuclear cells prepared from blood obtained from healthy, nonalcoholic volunteers. Eight subjects received ethanol in vivo; cells from nine subjects were used for the in vitro studies. Blood ethanol concentrations were determined at multiple time points before and after ethanol administration for the in vivo studies; for the in vitro studies, ethanol concentrations were measured from each assay sample both before and after the incubation period. Gas chromatography was used for determinations of both blood alcohol and medium ethanol concentrations. Results of the in vivo studies showed that a single dose of ethanol (0.5 g/kg), administered either intravenously (with resultant peak blood levels transiently up to 89 mg/dl) or orally (with resultant peak blood levels transiently up to 40 mg/dl at the time of the NK assay), did not alter NK activity. However, results of the in vitro studies showed a significant dose-dependent decrease (p < 0.001) in NK activity when ethanol exposure was sustained for 4 hr at concentrations of 80 mg/dl and above.(ABSTRACT TRUNCATED AT 250 WORDS)

Delta-9-tetrahydrocannabinol treatment results in a suppression of interleukin-2-induced cellular activities in human and murine lymphocytes

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been shownto suppress a variety of interleukin-2-(IL-2)-dependent cellular functions in both murine and human lymphocytes. These effects were examined in both human peripheral blood lymphocytes (hPBL) and the IL-2-dependent murine cytotoxic T-cell line CTLL-2. Interleukin-2-induced thymidine uptake and uridine uptake were suppressed in a dose related manner when cells were co-incubated for 48 h with 100 U rhIL-2/ml and 1 – 10 μg THC/ml. Interleukin-2-induced protein synthesis was also suppressed in a dose related manner over this THC concentration range, with the hPBL being more susceptible to the suppressive effect of THC than the CTLL-2 cells. Autoradiographic analysis of the synthesized proteins from hPBL cell lysates reveals a generalized suppression of all nascent proteins in THC-treated cultures. Human natural killer cell activity is only affected at the highest concentration tested (10 μg THC/ml) while lymphokine-(IL-2)-activated natural killer cell activity is affected throughout the range of 1 – 10 μg THC/ml. Together these results suggest that THC interferes with the IL-2 : IL-2 receptor signaling cascade at one or possibly many points causing a decrease in IL-2-induced metabolic activity and cytolytic function.

The expanding field of hypothalamic-pituitary-adrenal modulation of human natural killer cell activity.

The expanding field of hypothalamic-pituitary-adrenal modulation of human natural killer cell activity.

Trans-urocanic acid, a natural epidermal constituent, inhibits human natural killer cell activity in vitro.

UV irradiation has been reported to influence NK cell function both in vitro and in vivo. Since urocanic acid may mediate UV-induced immune modulation we tested the effect of trans- and cis-urocanic acid (UCA) on the cytotoxic activity of human peripheral blood lymphocytes against the erythroleukemic target cell line K562 in vitro. Trans-UCA was found to be a strong inhibitor of NK cell activity whereas cis-UCA had no effect. Trans-UCA also partially inhibited cytotoxic function of IL-2-activated NK cells and reduced IL-2-induced activation of NK cells. This is the first report describing trans-UCA to be active, and cis-UCA inactive, in regulating an immune function. In the skin, a decrease in epidermal trans-urocanic acid concentration by UV radiation could produce a favorable milieu for NK cell activity, and thus counteract the impairment of antigen-specific immune surveillance, induced by increased cis-urocanic acid concentrations.

Cell Death Protection by 3-Aminobenzamide and Other Poly(ADP-Ribose)polymerase Inhibitors: Different Effects on Human Natural Killer and Lymphokine-Activated Killer Cell Activities

The death of target cells by cytotoxic effector cells is a relevant biological phenomenon, where cells are activated and a very quick apoptotic program occurs. In order to test the hypothesis that the nuclear enzyme poly(ADP-ribose)polymerase (PADPRP) plays a role in such a process, a variety of PADPRP inhibitors such as 3-aminobenzamide, nicotinamide, 4-aminobenzamide and luminol were used. All of them were able to strongly inhibit K562 target cell killing by human effector natural killer cells (NK) in a 4hr 51Cr release assay. PADPRP inhibitors were much less effective in protecting target cells when lymphokine activated killer cells (LAK) were used as effectors. These substances were active only when both target and effector cells were mixed, being ineffective on target or effector cells alone. On the whole, these data indicate that PADPRP is involved in the death of target cells. Moreover, the different sensitivity of NK and LAK activities to PADPRP inhibitors suggests that the molecular mechanisms underlying these two types of cytotoxicity are at least partially different.

Inhibitory effect of somatostatin-14 and some analogues on human natural killer cell activity

The effect of somatostatin-14 (SST) and the SST analogues SMS and RC160 on human natural killer (NK) activity mediated by large granular lymphocytes (LGL), as well as on IL-2-and/or anti-CD16 monoclonal antibody (mAb)-induced activation of these cells, was investigated. The NK activity of LGL was studied by the release of 51Cr by the erythroleukemia-derived cell line K562, whereas 51Cr release by the P815 murine mastocytoma-derived cell line, for which lysis was redirected by the use of an anti-CD16 mAb, was used to study the cytolytic potential of these cells. IL-2 was used at the final concentration of 100 IU and was incubated overnight with LGL. SST and the analogues, added to these systems at final doses ranging from 10 −12 to 10 −5 M, were inhibitory of the NK cell activity to K562, with a dose-response curve starting from 10 −8 M and reaching a significant level at 10 −6 M. On the contrary, no effect was observed on the redirected killing assay to P815 and on the IL-2-induced activation of NK cells. These results provide additional evidence for the immunomodulatory action of somatostatin.

Immunomodulatory properties of diazepam-binding inhibitor: Effect on human interleukin-6 secretion, lymphocyte proliferation and natural killer cell activity in vitro

We have examined the influence of diazepam binding inhibitor (octadecaneuropeptide, DBI 33–50) on cell mediated immune responses including LPS-stimulated monocyte IL-6 secretion, PHA induced lymphocyte proliferation and NK cell function in humans. All studies were performed in vitro on isolated human peripheral blood mononuclear cells in the absence or presence of synthetic DBI 33–50. It has been shown that DBI 33–50, in concentration between 10 −6–10 −8 M, enhances the LPS-induced secretion of IL-6, as determined by specific bioassay for this monokine. On the other hand DBI 33–50 (10 −6–10 −12 M), had no significant effect on either PHA-induced lymphocyte proliferation or NK cell function. This data suggests a possible immunomodulatory role for DBI 33–50 as an endogenous neuropeptide, which stimulates IL-6 secretion by human monocytes.

Enzyme release assay of human NK cell activity using β-galactosidase-expressing K562 target cell line

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli β-galactosidase (β-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 X 10 4) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of β-gal activity depending on the incubation time and the number of effector cells. Released β-gal activity was assayed sensitively by using 4-methylumbelliferyl-β-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.

In vivo effect of ascorbic acid on enhancement of human natural killer cell activity

The in vivo effect of ascorbic acid on human natural killer (NK) cell activity was determined. Twenty control healthy subjects were given ascorbic acid at a concentration of 60 mg/kg, and blood was drawn at 0,1,2,4,8, 24 and 48 hours after treatment with ascorbic acid. Peripheral Blood Lymphocyte-NK activity was measured by a 4-hr.- 51Cr-release assay using K562 tumor cells as targets. Treatment with ascorbic acid was shown to have a biphasic effect on NK activity: a transient slight suppression between 1 to 2 hrs. (20% of control) was followed by a significant enhancement (an over-shoot) at 8 hrs. that was further increased at 24 hrs., then the activity returned to the normal level by 48 hrs. Changes in the activity of ascorbate treated NK cells were inversely related to the E:T ratio; namely 231%, 189%, 141% and 127% at 6:1, 12:1, 25:1 and 50:1 E:T ratio respectively. Flow cytometry analysis indicated no quantitative changes in the NK cell sub-populations post treatment with ascorbic acid in the experimental subjects as compared with control untreated subjects. Simultaneous to measurement of NK count and activity, ascorbic acid and its uptake by PBL was measured in the plasma. The uptake of the vitamin was maximized at 2–4 hours and maintained at a high level up to 24 hours. We conclude that ascorbic acid is a potent immunomodulator and its effect in enhancement of NK cytotoxicity may explain one mechanism by which ascorbic acid exerts its probable anti cancer activity.

Modified procedure for labelling target cells in a europium release assay of natural killer cell activity

Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuCl 3. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium.

Interplay in Vitro Between ACTH, β-Endorphin, and Glucocorticoids in the Modulation of Spontaneous and Lymphokine-Inducible Human Natural Killer (NK) Cell Activity

Release of pro-opiomelanocortin (POMC)-derived peptides and glucocorticoids characterizes the activation of the hypothalamic–pituitary–adrenal (HPA) axis and represents a major adaptive response to stress. Both glucocorticoids and POMC-derived hormones are known to be crucial modifiers of the immune response. Natural killer (NK) cells are a lymphocyte subset deeply involved in immunosurveillance. Cortisol, the most important glucocorticoid hormone in humans, is a well-established inhibitor, whereas the two lymphokines, immune interferon (IFN-γ) and interleukin-2 (IL-2), are important physiological stimulators. In the present study, physiological as well as superphysiological concentrations of two POMC-derived peptides, ACTH and β-endorphin, were shown not only to affect in vitro spontaneous and lymphokine-inducible NK activity of peripheral blood mononuclear (PBM) cells, but also to modify cortisol-mediated inhibition. NK activity was measured in a 4-h cytotoxic assay using the cell line K562 as a target, after prior incubation with ACTH (10−8–l0−12M) and β-endorphin (10−8–l0−14M) in the presence or absence of cortisol(10−6MAl), IFN-γ (325 IU/ml), and IL-2 (25 IU/ml.). ACTH was ineffective in changing spontaneous NK activity at all concentrations, whereas β-endorphin enhanced NK cytotoxicity (p < .02). The concomitant exposure of PBM cells to the two POMC-derived peptides and IFN-γ or IL-2 significantly enhanced the lymphokine-induced boosting of NK activity. Moreover, ACTH and β-endorphin were able to significantly reduce the cortisol-dependent inhibition (p < .05). These data are compatible with the hypothesis that POMC-derived peptides have a role in the modulation of NK cell activity. It seems likely that in cases of activation of the HPA axis, ACTH and β-endorphin may effectively counteract the negative effects of glucocorticoids on NK cell activity, and prevent, at least in some instances, any overshooting of the glucocorticoid-dependent effect on immune cells.

Augmentation of human natural killer cells by splenopentin analogs.

Splenopentin, Arg-Lys-Glu-Val-Tyr (SP-5) and its synthetic analogs; Arg-D-Lys-Glu-Val-Tyr (pentapeptide 1), Lys-Lys-Glu-Val-Tyr (2), D-Lys-Lys-Glu-Val-Tyr (3), Arg-Lys-Gly-Val-Tyr (4), and Arg-Lys-Gln-Val-Tyr (5) have been examined for augmentation of human natural killer (NK) cell activity and human T-cell transformation response. Pentapeptides 2 and 3 were found to significantly augment the in vitro human NK cell activity. However, none of them had any effect on lymphocyte proliferative responses.

Effects of CCK-8 and CCK-33 on human natural killer cell activity: studies on intestinal lamina propria and peripheral blood mononuclear cells

Two different molecular forms of cholecystokinin (CCK) were studied with respect to their modulatory effect on non-MHC restricted or natural killer (NK) cell cytotoxicity. NK activity of peripheral blood mononuclear cells was found to be hardly affected by co-incubation with either CCK-8 or CCK-33 within a physiological concentration range against K-562 or Caco-2 tumour target cells. NK activity by lamina propria mononuclear cells isolated from histologically normal intestinal mucosa was found to be enhanced dose-dependently by incubation with CCK-8 in the 4-h assay against K-562, but not in the prolonged 18-h assay or against Caco-2 targets. In contrast, CCK-8 showed a tendency to inhibit NK activity in the prolonged 18-h assay against K-562; however, these alterations were not found to be statistically significant. CCK-33 was not found to modulate the NK activity of lamina propria mononuclear cells. It is suggested that NK activity by lamina propria mononuclear cells may be stimulated preferentially by CCK-8 because this molecular form of CCK in particular predominates in the nervous tissue of the intestinal mucosa.

Psychobiological factors related to human natural killer cell activity and hormonal modulation of NK cells in vitro

The present report investigated whether percentages of circulating natural killer (NK) cells and NK cell activity (NKCA) are associated with psychological variables. Subjects (n=95) were selected, based on a combination of low or high scores on questionnaires on daily hassles and self-reported symptoms, to create four extreme groups. NK cell percentages were different between two of the four groups, only when the analysis was not controlled for gender, life style and endocrine parameters. No evidence was found for a relationship between group membership and NKCA. NKCA, however, was found to differ between men and women and to be associated with percentages of NK cells and intracellular levels of cAMP. Furthermore, the hypothesis was tested, that hormone-induced changes in NKCA in vitro are dependent on the individual's current stress profile. To investigate this issue, NKCA was measured after cells had been incubated with hydrocortisone (10 −6 or 10 −7 M) or the β-adrenergic agonist isoprenaline (10 −5 or 10 −7 M) in vitro. Changes in NKCA were found to be related to plasma adrenaline levels, but no evidence was found for involvement of psychological variables. It is concluded that, in the current setting, there is no association between the combination of scores on the two psychological questionnaires, and NKCA or hormone-induced changes therein.

Inhibition of interleukin-2-stimulated enhancement of human natural killer (NK) cell activity by carbaryl, an anticholinesterase insecticide

The potency of the anticholinesterase (antiCHE) insecticides as serine hydrolase inhibitors, and evidence for serine hydrolase activity in interleukin-2 (IL2) signalling suggest that the natural killer (NK) cell may be a target for dysregulation by antiCHE insecticides. NK cells are large granular lymphocytes (LGL) that respond to IL2 by proliferating and increasing their cytolytic efficiency. In the present study, we assessed the effects of carbaryl (CA, an antiCHE insecticide) and α-naphthol (NA, the major metabolite of CA) on both target cell killing per se and IL2 enhancement of target cell killing by human NK cells. Human LGL, collected from the peripheral blood of normal donors, were cultured for 4 days with human recombinant IL2 (HRIL2), then assayed by a 51Chromium ( 51Cr) release assay for lytic activity against human K562 cells. When added at the beginning of the culture period, CA inhibited enhancement of cytolytic efficiency in a concentration-dependent manner; at concentrations (0.5 and 5.0 μM) compatible with no cholinergic toxicity. Reduction of the effector/target cell (E/T) ratio in the 51Cr release assay markedly enhanced the observed inhibition by CA. In one experiment, inhibition increased from 6% to 20%, 17% to 35%, and 53% to 73% at 0.5, 5.0, and 50 μM CA, respectively, when E/T was reduced from 10:1 to 2.5:1. This result is consistent with reduced cytolytic efficiency of individual NK cells exposed to CA. NA had no effect at 0.5 or 5.0 μM but caused some inhibition at 50 μM. Neither CA nor NA produced LGL death. When CA or NA was added directly to the 51Cr release assay, inhibition was not observed. The mechanism of inhibition of IL2-stimulated enhancement of target cell killing is not yet known, however, the results are consistent with impairment of IL2 signalling, by CA.

Negative modulation of human NK cell activity by purinoceptors: 2. Age-associated, gender-specific partial loss of sensitivity to ATP

Aging is known to modulate the affinity and sensitivity of receptors for hormones and regulatory molecules. We have shown previously that exogenous adenosine triphosphate (ATP), perhaps acting as a purinoceptor agonist, can down-regulate the cell-mediated anti-tumor natural cytotoxic activity of human peripheral blood natural killer (NK) cells. We have extended these studies to investigate whether this effect is modulated during immunosenescence, and if so, whether it is gender-restricted or NK subset-associated. While the inhibitory effect is demonstrable in most individuals, there is a gender-restricted, age-associated transition in the sensitivity of NK cell activity to inhibition by ATP at 2.5 × 10 −5to 80 × 10 −5 M in vitro. Data from both suboptimal (100 μ M) and optimal (800 μ M) inhibitory doses of ATP support this conclusion. The ID 20ATP were 10.2 × 10 −5 and 17.8 × 10 −5 M for the young (<40 years) and elderly (>70 years) females, respectively ( P = 0.02). The frequency distribution curve of ATP sensitivity shifts to the left in the elderly, i.e., the sensitivity to be inhibited at 50% or more by ATP was expressed by one-half of young and one-fifth of elderly female donors. Linear regression analysis suggests an inverse relationship between percentage CD57 + and percentage CD16 +57 + (but not percentage CD16 +) NK subsets and sensitivity to down-regulation by ATP. The mean percentage of the above NK cell phenotypes among lymphocytes from young and old female donors differ significantly (<0.0001). The data suggest that the presence of CD57 antigen-positive cells may render NK cells relatively more resistant to the action of purinoceptor agonists such as ATP. Thus in females, immunosenscence results in a diminished ability of NK cells to transduce those signals that may normally mediate ATP-induced suppression of NK cytolytic activity. Such a diminished ability may be an immunobiological advantage to aging NK cells since they can be kept at a higher steady-state level of (anti-tumor cytotoxic) activity through a protection from negative modulators. These findings have an implication on the lower rate of mortality due to cancer seen in older women compared to that in older men. It is suggested that the ATP-NK cell interaction through the P 2 purinoceptor may serve as a potentially useful model to study immunosenescence, ontogenic, or gender-specific changes in NK cells at the cell surface level.

Plasma beta-endorphin and natural killer cell activity in major depression: a preliminary study.

Low concentrations of beta-endorphin have been found to enhance human natural killer (NK) cell activity in vitro. Both beta-endorphin and NK activity are changed by clinical depression. To evaluate whether circulating concentrations of beta-endorphin have a role in the in vivo modulation of cellular immunity in humans, we measured plasma beta-endorphin and NK cell activity in 14 depressed patients and 14 age-matched control subjects. In the depressed patients, both plasma beta-endorphin and NK cell activity were reduced to 76% and 57%, respectively, of the mean levels in the control subjects. In addition, beta-endorphin showed a significant positive correlation with lytic units of NK cell activity in the combined group of all subjects and in the patient group (p = 0.04), but not in the control group. The study supports the hypothesis that circulating endorphin is correlated with NK cell activity in vivo. This correlation may be higher in the depressed patient group.