Calcium-dependent natural killer and calcium-independent natural cytotoxic activities in an IL-2-dependent killer cell line

Using a cloned murine cell line, NKB61A2, that concomitantly exhibits both NK and natural cytotoxic (NC) activities, we investigated the biochemical mechanisms involved in natural cell mediated cytotoxicity against NK-sensitive YAC-1 tumor cells and against the NC-sensitive WEHI-164 tumor cells. Recent reports have suggested that target cell lysis by cytotoxic lymphocytes occurs by either a calcium dependent and/or a calcium-independent mechanism(s). To determine the role of calcium in NK and NC activities of the NKB61A2 cell line, we evaluated the effect of: 1) extracellular Ca2+ depletion by the divalent cation chelator, EGTA, 2) Ca2+ influx blockade by the Ca2+ channel blocker verapamil, and 3) blocking of intracellular Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). We found that EGTA, verapamil, and TMB-8 were all capable of inhibiting NK activity, but they had little effect on NC activity of the NKB61A2 cells. Using 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide which are inhibitors of protein kinase C and calmodulin respectively, we determined that protein kinase C and calmodulin do play a role in the NK activity of NKB61A2 cells. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalanesulfonamide, similar to Verapamil and TMB-8, had no effect on NC activity. Thus, the data indicate that the NK activity of NKB61A2 cells is calcium dependent whereas NC activity is not. These results may explain the disparate reports seen in the literature of calcium-dependent and -independent lysis of tumor cells.     

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.     

Analysis of natural killer activity and natural killer cell subsets in patients with bladder cancer

Summary In order to analyze the state of the natural resistance system of bladder cancer patients in vivo, we measured natural killer (NK) activity and NK cell subsets of peripheral blood lymphocytes (PBL) from 46 patients with bladder cancer and 25 age- and sex-matched healthy volunteers. The mean NK activity in patients with lowstage bladder cancer was similar to that in the controls, while NK activity in patients with high-stage bladder cancer was significantly depressed. The mean proportions of Leu7⁺ cells in patients with both low-stage and highstage bladder cancer were significantly higher than that in the controls. The mean proportion of Leu11a⁺ cells in patients with low-stage bladder cancer was similar to that in the controls, while in patients with high-stage bladder cancer it was significantly higher. This study demonstrates the abnormal immunological state of bladder cancer patients; namely, abnormalities exist not only in NK activity but also in the proportions of circulating NK cell subsets.     

Immunomodulatory effects of corticosteroids on natural killer and antibody-dependent cellular cytotoxic activities of human lymphocytes

The in vitro effect of prednisolone (PRD) on NK and ADCC activities of human lymphocytes was investigated. PRD at concentrations ranging from 7.5 X 10(-3) to 1 X 10(-5) M significantly inhibited NK activity, while concentrations of 7.5 X 10(-3) to 1 X 10(-4) M inhibited ADCC activities of PBL when added directly to the mixture of effector and target cells. Lymphocytes pre-cultured for 24 hr with PRD at concentrations ranging from 1 X 10(-4) M to 1 X 10(-6) M showed significant suppression of their NK activity. Inhibition was proportional to the concentration of the drug, and was observed at as early as 1 hr of incubation at various effector to target cell ratios with several targets. PRD also inhibited NK and ADCC activities of purified T cells, non-T cells, and NK-enriched effector cells. In target-binding assays, PRD decreased the target-binding capacity of effector lymphocytes in a dose-dependent manner. PRD-induced inhibition could be reversed by incubating lymphocytes for 1 hr with interferon or IL 2. Pretreatment of targets with PRD for 4 hr did not affect cytotoxic activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells because lymphocytes treated with PRD showed normal spontaneous 51Cr release, and their viability after 24 hr of pre-culture with PRD was comparable to that of untreated control cells. These results demonstrate that PRD has significant immunomodulatory effects on human NK and ADCC activities that may be of clinical relevance.     

Immunomodulation of human natural killer cell cytotoxic function by triazine and carbamate pesticides

Triazine (atrazine) and carbamates (maneb, metiram, and ziram) are used as pesticides on a variety of crops around the world. To our knowledge, there have been no studies dealing with the effects of these compounds on human natural killer (NK) cells cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of atrazine, maneb, metiram, zineb, and ziram on the ability of human NK cells to lyse tumor cells. The compounds were tested in both purified NK cells as well as a cell preparation that contained both T and NK lymphocytes (T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 h to 6 days. Exposure of highly purified NK cells to 10 microM atrazine, maneb, or metiram inhibited K562 tumor cell lysis by 63+/-25, 95+/-4, and 50+/-6%, respectively, after a 24 h exposure and by 83+/-21, 70+/-39, and 48+/-41% after a 6-day exposure. Exposure to 2.5 microM ziram for 24 h caused a 99+/-2% decrease in lytic function and at 1 microM for 6 days caused a 96+/-4% decrease. However, when T/NK cells were exposed to atrazine, maneb, or metiram for 24 h only 10 microM atrazine and maneb caused a significant decreases in lytic function (61+/-13 and 38+/-18%) and after 6 days only atrazine was inhibitory (54+/-12%). A 24-h exposure to 2.5-microM ziram caused a 41+/-51% decrease in function, but a 6-day exposure to 1 microM ziram caused no inhibition of lytic function. The results provide evidence of relative toxic potential for the five compounds and the immunomodulatory effects on both T and NK lymphocyte function.     

The cytotoxic activity of human natural killer cells requires an intact secretory apparatus

We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations.     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Phenotypic studies of natural killer cell subsets in human transporter associated with antigen processing deficiency

Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56(bright) cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, (51)Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright) NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56(dim)CD16(-) NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56(bright) NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro

To study cell-to-cell interactions in microgravity we examined the functional activity of natural killer cells on board of the ISS. NK cells are the effector cells with direct cytotoxic activity to oncogenic, virus-infected cells and cells with modified differentiation. Ground-based experiments have shown that the examination of target cell lysis after incubation with NK cells is a simple and informative model for studying the influence of microgravity. NK cytotoxicity was measured as the value of non-degradeted labeled myeloblasts (K-562) after 24 hrs exposure with human lymphocytes in suspension. A special device was developed for space flight experiments. Human cultured lymphocytes and labeled K-562 cells were loaded into special syringes and delivered to the Russian segment of the ISS. Cosmonauts prepared co-cultured suspensions during the first day of microgravity, exposed them at 37 degrees C for 24 hrs and then separated H3-labeled cells on special filters. The results of ISS-8 mission showed that human NK cells in vitro remain lysis activity toward target cells in microgravity. The basal level of NK cytotoxicity was low and we did not found significant differences between "control" and "flight" values. Interferon production during the interaction between immune and target cells (ratio 10:1) in microgravity did not differ compared with ground-based control experiments. Ground exposure of the same lymphocyte samples with K-562 cells to 24 hrs clinorotation also did not lead to significant differences. These experiments paved the way for understanding the cell interaction mechanisms in space flight and the obtained results suggest that microgravity does not disrupt the interaction of NK cells with tumor cells.     

Human natural killer cells and cytotoxic T lymphocytes require cell surface carbohydrate determinants for lytic function

Cloned and uncloned populations of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) were treated with tunicamycin, an antibiotic that inhibits N-linked glycosylation, in order to study the potential role of cell surface carbohydrate determinants in lytic function. It is shown that tunicamycin-treated NK and CTL effector cells lose killer function in a dose-dependent manner. This effect is reversible; cells washed free of tunicamycin begin to recover their killer activity within 2 to 3 days after initial treatment. Conjugate experiments indicate that killer-target cell binding is not affected by tunicamycin treatment of the NK cells. It is also shown that tunicamycin treatment of target cells does not significantly affect their ability to be lysed by NK or CTL effector cells. These studies provide evidence that carbohydrate determinants are important in the lytic mechanism of both CTL and NK cells, rather than in specific effector-target cell binding.     

Age-associated alterations in human natural killer cells. 2. Increased frequency of selective NK subsets

We have analyzed the peripheral blood lymphocytes from healthy volunteers (20 to 94 years) for the expression of natural killer (NK) cell surface markers, NK activity, and B-cell proliferative response. An increase (2- to 3.5-fold) in relative percentage and absolute number of lymphocytes expressing Leu-7 (HNK-1) or Leu-11a (CD 16) antigen was found in the elderly group (greater than 80 years) as compared to young adults (less than 40 years). A two-color immunofluorescence analysis revealed that the age-associated increment was both progressive and selective; the actual increase occurred in Leu-7+11a+ and Leu-7+11a- populations (subsets with variable and weak NK activity) but not in the Leu-7-11a+ (most active) subset. There is a corresponding decrease in the 7-11a- cells. The ratios of 7+11a+/7-11a+ and 7+11a-/7-11a+ cells doubled with advancing age. Linear regression analysis suggests that the 7-11a+ cells are highly preserved through human senescence and the ratio of 7+11a- cells to the most stable subset, 7-11a+, could expand nearly 100-fold from birth to old age. Further analysis of Leu-7+ cells for the coexpression of Leu-11c (an epitope of Leu-11a) confirmed a similar pattern of changes in 7+11c+ and 7+11c- NK subsets with advancing age. The frequency of Leu-11+ (epitopes 11a+ or 11c+), but not of the subsets of 7+ phenotype (7+11a- or 7+11c-), correlates well with the NK activity (spontaneous killing of K562 tumor cell line). The 7+11c+ cells may directly or indirectly be responsible for the increase in NK activity observed with a majority of aged donors. The inverse relationship observed between the mitogenic response of lymphocytes to pokeweed mitogen (PWM) and the initial frequency of 7+11a-, but not other phenotypes, raises a potential functional significance for the expansion of the 7+11a-(7+11c-) subset. These age-associated NK phenotypic changes provide a cellular basis for our observations on age-associated increase in NK activity and decrease in mitogenic response to PWM.     

Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.     

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

Suicide behavior of target cells after binding with natural killer cells

Human natural killer (NK) cell activity seems to be related to the integrity and function of the cytoskeletal apparatus. It has been hypothesized that microfilaments and microtubules play a pivotal role. In particular, the binding of the NK cell to the target cell requires microfilament integrity, and the lysis of bound targets seems to depend on microtubule assembly. We focused on the changes occurring in cytoskeletal elements and surface structures of NK cells and of target cells highly sensitive to NK activity (K562). Our observations, performed by fluorescence and scanning electron microscopy, besides confirming a rearrangement of the cytoskeletal apparatus in the effector cell, provide evidence that target cell cytoskeletal elements are involved in NK cell function. In K562 cells, after binding with NK cells, there is marginal rearrangement of actin and polarization of tubulin and vimentin in the contact regions, accompanied by modification of surface structures. These findings suggest that the target cell plays an active role in its own death by participating in the formation of an extended area of intimate contact with the killer cell. In addition, they lend credence to the surprising proposal that NK cells may induce a suicide mechanism in target cells.