Histone deacetylase inhibitors suppress natural killer cell cytolytic activity

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Effects of endogenous and exogenous opioids on splenic natural killer cell activity

Previous work from our own and other laboratories has shown that electroshock-induced neurohormonal changes in rodents could modify host-tumor interactions by both increasing the frequency and growth rate of transplanted tumors and decreasing the elimination rate of a radiolabelled natural killer (NK) cell sensitive tumor. To test whether such neurohormonal changes could affect NK activity we subjected mice to tail electrode shock (TES) and examined in vitro splenic NK activity. We found that between 30 and 60 min after TES there is a significant but transient suppression of their splenic NK activity. To determine whether TES-induced endogenous opioids might be involved in this suppression mice were given intraperitoneal injections of the opioid antagonists naloxone or naltrexone before or at the end of the TES session. These drugs prevented NK suppression. In a further test of the hypothesis that opioids alter NK activity mice were given a single intraperitoneal injection of morphine or [D-Ala2-Met5]-beta-endorphin, a relatively stable analogue of beta-endorphin, an endogenous opioid. Contrary to expectations these opioids enhanced splenic NK activity. Our interpretation of these results is that shock-induced NK suppression may not be mediated by endogenous opioids and that the effects of naloxone and naltrexone on NK activity may not be related to their opioid antagonist properties. On the contrary, opioids may participate in a homeostatic rebound from suppression mediated by other neurohormonal mechanisms activated during TES.     

Evaluation of natural killer cell activity

Natural killer (NK) cells are being appreciated not only for their ability to recognize and lyse tumor cells and virus-infected cells but also for their immunoregulatory properties. NK cells provide a first line of defense against invading pathogens with a two pronged attack, lysis of infected cells and secretion of cytokines and chemokines with potent antipathogen effects. This article describes the standard chromium release assay, which measures the ability of NK cells derived from the peripheral blood to lyse appropriate target cells.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

Recombinant interleukin 2 rapidly augments human natural killer cell activity

Recombinant human interleukin 2 (r-IL-2) rapidly stimulated human natural killer cell activity in vitro. Augmentation of NK activity occurred within 1 hr of preincubation with r-IL-2. Responsive killer cells were typical NK cells as shown by cell fractionation procedures. These included Percoll density gradient separation and depletion of OKT3+ T cells by an indirect rosetting method. Analysis with a panel of polyclonal and monoclonal antibodies against alpha and gamma interferon revealed that this early enhancement of NK activity by r-IL-2 was independent of the production of both types of interferon.     

The influence of natural mineral water on aquaporin water permeability and human natural killer cell activity

Aquaporins are the intrinsic membrane proteins functioning as water channel to transport water and/or mineral nutrients across the biological membrane systems. In this research, we aimed to clarify if the selected mineral water can affect aquaporin functions in vitro and the assumption of the mineral water can modify aquaporin expression and activate natural killer cell activity in human body. First, we expressed six human and eight plant aquaporin genes in oocytes and compared the effect of different kinds of natural mineral water on aquaporin activity. The oocyte assay data show that Hita tenryosui water could promote water permeability of almost all human and plant aquaporins in varying degrees, and freeze-dry and organic solvent extraction could reduce AQP2 activity but pH change and boiling could not. Second, each volunteer in two groups (10 in one group) received an oral Hita tenryosui or tap water load of 1000 ml/day for total four weeks. We found that these two kinds of water did not directly affect the relative expression levels of AQP1 and AQP9 in the blood cells, but intriguingly, the natural killer cell activities of the volunteers drinking Hita tenryosui water were significantly improved, suggesting that Hita tenryosui water has obvious health function, which opens a new and interesting field of investigation related to the link between mineral water consumption and human health and the therapies for some chronic diseases.     

Histamine H2-receptor-mediated regulation of human natural killer cell activity

We have investigated effects of histamine on the spontaneous cytotoxic activity of human natural killer (NK) cells in vitro. Addition of histamine (10(-3) to 10(-7) M) to assay cultures of Percoll-fractionated mononuclear cells (MNC) and erythroleukemic K 562 target cells resulted in a strong enhancement of the cytotoxicity of low-density MNC, enriched for NK cell cytotoxicity (NKCC). No enhancing or suppressing effects of histamine could be detected after removal of monocytes/adherent cells from the effector cell suspensions. When unfractionated MNC were used as NK effectors, similar results were obtained, i.e., dose-dependent enhancement of NKCC by histamine in the presence of monocytes and lack of effect in nonadherent effector cells. Freshly isolated monocytes displayed low spontaneous cytotoxicity against K 562 targets and were not induced by histamine. The histamine-induced enhancement was mimicked by dimaprit, a specific histamine H2-receptor agonist, but not by N-methyldimaprit, a chemical control for H2-receptor agonist activity of dimaprit. Furthermore, the enhancement was completely antagonized by the specific histamine H2-receptor antagonists cimetidine and ranitidine. The effect of histamine could not be ascribed to endogenous interferon (IFN) production, since no IFN activity could be detected in histamine-treated MNC effectors. Also, the enhancing effects of histamine and human leukocyte IFN-alpha were clearly additive. On the basis of these findings, we suggest that histamine, via specific activation of H2 receptors, may be an important regulator of human NK cell activity.     

Cytotoxic T-lymphocyte and spontaneous killer cell activity against human T- and B-lymphoid cell lines.

We have investigated cell-mediated immune responses to cultured human T- and B-cell lines. Two effector mechanisms were explored and found to have different capabilities for mediating cytotoxic reactions. Cytotoxic T lymphocytes were generated by stimulation with irradiated B-cell lines and demonstrated cross-reactive cytotoxicity against these lines but not against T-cell lines. Unseparated mononuclear cells showed spontaneous cytotoxicity for both T- and B-cell lines; however, T-cell lines appeared more susceptible. Cell separation procedures were employed to determine functional differences in effector cells. In contrast to cytotoxic T lymphocytes induced in vitro, spontaneous killer cells (SKC) were shown to be nylon wool adherent, non-T lymphocytes with receptors for IgG-coated sheep erythrocytes.     

Mechanism of action of phorbol myristate acetate on human natural killer cell activity

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that “regenerated EC” are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.     

Inhibition of human natural killer cell activity by platelet-derived growth factor

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.     

Immunomodulatory activity of commonly used drugs on Fc-receptor-mediated human natural killer cell activation

Natural killer (NK) cells mediate defense against neoplastic as well as infected cells. Yet, how their effector functions are affected by the large variety of pharmacological compounds commonly in use has not been investigated systematically. Here, we screened 1,200 in-use or previously approved drugs for their biological effect on freshly isolated human peripheral blood-derived NK cells. Mimicking antibody-dependent cellular cytotoxicity (ADCC), known to be important in antibody-based immunotherapies against, e.g., human malignancies, the cells were stimulated by Fc-receptor (CD16) engagement. Cellular responses were assessed by flow cytometry. Fifty-six compounds that significantly inhibited and twelve that enhanced one or more of the readouts of adhesion, exocytosis, and chemokine production were identified and confirmed as hits. Among the confirmed inhibitors, 80 % could be assigned to one of seven major pharmacological classes. These classes were β2-adrenergic agonists, prostaglandins, phosphodiesterase-4 inhibitors, Ca²⁺-channel blockers, histamine H1-receptor antagonists, serotonin/dopamine receptor antagonists, and topoisomerase inhibitors that displayed distinct inhibitory patterns on NK cell responses. Among observed enhancers, interestingly, two ergosterol synthesis inhibitors were identified that specifically promoted exocytosis. In summary, these results provide a comprehensive knowledge base of the effect known drugs have on NK cells. More specifically, they provide an overview of drugs that may modulate NK cell-mediated ADCC in the context of clinical immunotherapies.