Blockade of physiologically secreted IFN-gamma inhibits human T lymphocyte and natural killer cell activation

The role of physiologically secreted human IFN-gamma in T lymphocyte and NK cell activation has been probed with a panel of mouse mAb directed against various epitopes of the human IFN-gamma molecule, or human IFN-gamma R. Addition to the culture medium of those mAb that neutralize the antiviral activity of IFN-gamma or interact with its receptor inhibited proliferative and cytotoxic responses elicited in PBL by HLA alloantigens, anti-CD3 mAb, and IL-2, but not the proliferative response to PHA. The IFN-gamma blockade also inhibited IFN-gamma, IL-2, and TNF-alpha release during MLC. Kinetic experiments showed that reduction of proliferative and cytotoxic responses to HLA alloantigens is maximal when IFN-gamma is blocked within the first 48 h. Exogenous rIFN-gamma restored the proliferative response only when added at the beginning. Moreover, when IFN-gamma was blocked, T lymphocytes recovered from 6-day MLC displayed a profound decrease in their expression of p55 and p75 chains of the IL-2R, as well as in the number of high-affinity IL-2 binding sites. These findings strongly suggest that IFN-gamma is required in the early phases of induction of the oligo- and polyclonal proliferative and cytotoxic responses of lymphocytes.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Role of lipoxygenation in human natural killer cell activation

Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC.     

Effect of millimeter waves on natural killer cell activation

Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions.     

NK细胞和NKT细胞发育的转录调控

自然杀伤（natural killer,NK）细胞和自然杀伤T（natural killer T,NKT）细胞是参与机体抗病毒免疫和肿瘤免疫的两群淋巴细胞亚群,是介导先天性免疫（innate immunity）应答和调节适应性免疫（adaptive immunity）应答的重要效应细胞。近年来,随着对NK细胞和NKT细胞及其转录调控因子研究的不断深入,NK细胞和NKT细胞的发育机制逐步被阐明,这将为提高NK细胞和NKT细胞的抗病毒和肿瘤免疫疗效提供新的策略。     

自然杀伤细胞和自然杀伤T细胞在动脉粥样硬化形成中的作用

动脉粥样硬化发生发展与免疫细胞参与的免疫反应密切相关,其中自然杀伤细胞主要是通过释放IFN-γ、穿孔素和颗粒酶等方式发挥生物学作用,自然杀伤T细胞通过释放多种细胞因子影响动脉粥样硬化形成,但其具体机制未明。本文就自然杀伤细胞和自然杀伤T细胞对动脉粥样硬化的影响做一综述,为动脉粥样硬化及其相关疾病的防治研究提供新的思路。     

Alternative mechanisms of natural killer cell activation during herpes simplex virus infection

Although NK cells can kill both malignant cells and virus-infected cells without prior sensitization, it has remained unclear whether the mechanism by which an NK cell is activated in the presence of a tumor cell is similar to that induced by the presence of a virus-infected cell. In our experimental system using homogeneous populations of cloned human CD16+ NK cells, we found that HSV-infected target cells do not induce in the NK cells the same pharmacologically-active second messengers elicited by NK-sensitive tumor cells. Although phosphoinositide turnover and calcium signaling were generated in NK cells exposed to NK-sensitive tumor cells, the recognition of HSV-infected cells by NK cells did not result in similar transmembrane signaling. Furthermore, depending on the cell type infected by HSV, alternative mechanisms of cytotoxicity were employed. HSV-infected foreskin fibroblasts were rapidly and selectively killed by cloned NK cells without a requirement for IFN or accessory cells. In contrast to this direct cytotoxicity against HSV-infected foreskin fibroblasts, NK cell-mediated cytotoxicity against an HSV-infected fibrosarcoma cell line (1591) was dependent on IFN-alpha production by accessory cells. Importantly, in both systems of cytotoxicity, IFN-alpha activation of NK cells resulted in augmented killing against both infected and uninfected targets. These results suggest that NK cell activation induced during antiviral immunity is distinct from activation elicited during an antitumor response. These differences include the utilization of alternative forms of signal transduction and alternative mechanisms of cytotoxicity.     

Regulation of natural killer cell activation: implementation for the control of tumor metastasis

The cellular interactions that regulate natural killer (NK) cell-mediated cytotoxicity and antimetastatic activity following treatment with biological response modifiers (BRM) were studied. The transient activation of NK cells by a single injection of Corynebacterium parvum is followed by a refractory period during which a second injection of BRM fails to stimulate the already depressed NK cell activity. During this hyporesponsive period, the in vivo NK cell-dependent BRM-induced antimetastatic activity is markedly reduced and cannot be enhanced by multiple injections of BRM. A correlation exists between the generation of hyporesponsiveness to NK cell activation and the activation of suppressor macrophages by C. parvum. BRM that selectively activate NK cells without subsequently activating suppressor macrophages do not induce hyporesponsiveness to further activation of NK cells in vivo and, when given in multiple injections, retain the ability to inhibit hematogenous tumor metastasis.     

Molecular mechanisms of natural killer cell activation in response to cellular stress

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.     

Avian influenza virus directly infects human natural killer cells and inhibits cell activity

Natural killer (NK) cell is a key component of innate immunity and plays an important role in host defense against virus infection by directly destroying infected cells. Influenza is a respiratory disease transmitted in the early phase of virus infection. Evasion of host innate immunity including NK cells is critical for the virus to expand and establish a successful acute infection. Previously, we showed that human influenza H1N1 virus infects NK cells and induces cell apoptosis, as well as inhibits NK cell activity. In this study, we further demonstrated that avian influenza virus also directly targeted NK cells as an immunoevasion strategy. The avian virus infected human NK cells and induced cell apoptosis. In addition, avian influenza virion and HA protein inhibited NK cell cytotoxicity. This novel strategy has obvious advantages for avian influenza virus, allowing the virus sufficient time to expand and subsequent spread before the onset of the specific immune response. Our findings provide an important clue for the immunopathogenesis of avian influenza, and also suggest that direct targeting NK cells may be a common strategy used by both human and avian influenza viruses to evade NK cell immunity.

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Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

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.     

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.     

Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle

Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target Cell Cycle.     

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)     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Toward an optimal oligosaccharide ligand for rat natural killer cell activation receptor NKR-P1

Aminosugars have a good affinity for the NKR-P1A protein, the major activating receptor at the surface of rat natural killer cells. We have systematically investigated the structural requirements of the recombinant soluble dimeric form of the receptor for its optimal carbohydrate ligands. While N-acetylD-mannosamine was the best neutral monosaccharide ligand, its participation in the context of an extended oligosaccharide sequence was equally important. The IC(50) value for the GalNAcbeta1 --> ManNAc disaccharide was nearly 10(-10) M with a further possible increase depending on the type of the glycosidic linkage and the aglycon nature. From the point of view of its availability, stability, and affinity for the receptor and a potential in vivo use, these studies are pivotal for the design of an oligosaccharide or glycomimetics suitable for further clustering into the multivalent glycodendrimers.     

Evidence for an early calcium-independent event in the activation of the human natural killer cell cytolytic mechanism

In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium.