A test for increased lethality in land snails exposed to 60 Hz magnetic fields.

A recent report suggested that exposures of land snails to 0.1 mT, 60 Hz magnetic fields for periods of 48-120 h increase mortality levels by 2-10 times. In direct experimental tests, we were unable to confirm this effect.     

Human natural killer cell lysis of virus-infected cells. Relationship to expression of the transferrin receptor

Natural killer (NK) cells lyse tumor and virus-infected cells yet the nature of the target structure they recognize is unknown. A normal host cell glycoprotein, the transferrin receptor (TfR), has been proposed as a target structure on tumor cells. We therefore investigated whether changes in the number or physiological recycling of the TfR, consequent on virus infection, were related to the differential susceptibility of virus-infected cells to NK lysis. There was a direct correlation between TfR expression, susceptibility to NK lysis and ability to act as cold target competitors, for human fibroblasts infected with RNA and DNA viruses (cytomegalovirus, herpes simplex, polio, vaccinia and Semliki Forest virus). The NK lysis of human cytomegalovirus-infected fibroblasts was studied in more detail. NK lysis was increased coincident with human cytomegalovirus early antigen expression and this susceptibility to lysis was associated with increased total and recycling TfR but only a slight increase in surface TfR expression. In addition, susceptibility of uninfected human fibroblasts to NK lysis directly correlated with TfR number. However, we were unable to inhibit NK lysis by either excess iron-saturated Tf or affinity-purified TfR. We conclude that there is a direct correlation between total TfR expression and susceptibility to NK lysis of human virus-infected cells; however, the NK target structure on virus-infected cells is probably not the TfR itself.     

Human adipose-derived stem cells impair natural killer cell function and exhibit low susceptibility to natural killer-mediated lysis

Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared.     

Human natural killer cells: Molecular mechanisms controlling NK cell activation and tumor cell lysis

Natural killer cells represent a highly specialized lymphoid population with a potent cytolytic activity against virus-infected or tumor cells. Their function is regulated by a series of inhibiting or activating signals. The mechanisms by which NK cells kill susceptible target cells was thought to be elucidated after the discovery of inhibitory receptors specific for MHC-class I molecules: NK cells would kill those target cells that lack MHC-class I molecules. However, the actual scenario revealed more complex with the discovery of activating receptors and their ligands. Thus, in certain pathological conditions, corticosteroid treatment or exposure to TGFbeta, NK cells may under-express activating receptors. In addition, target cells may lack ligands for activating receptors and thus fail to activate NK cells upon cell-to-cell contact. This clearly implies that activation of NK cells and of their potent effector mechanism are under the control of different checkpoints.     

Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction.

When T effector cells meet antigen-bearing target cells, there is a specific accumulation of T-cell receptors, co-receptors and structural proteins at the point of cell-cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer-(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell-cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK-16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell-target cell contact when the target cells expressed H-2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR-P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell-cell contact which may be necessary for them to mediate their full inhibitory effect.     

Transferrin receptors on tumor and bone marrow cells: Lack of involvement as target structure for natural killer cells

Summary Two different experimental approaches based on the specificity of monoclonal antibodies (mAbs) have been taken to verify the hypothesis that the transferrin receptor (TfR) on proliferating cells serves as a common target structure for natural killer (NK) cells. Thus, by the lysostrip technique the TfR was removed from the surface of K562 and Molt4 tumor cells by incubation with two different anti-TfR mAbs. The effect of removal of the TfR was controlled by uptake of radiolabeled transferrin, and by binding of non-cross-reacting monoclonal anti-TfR receptor antibodies. Though the modulation of TfR on the membrane of viable cells was nearly complete, the cells remained fully susceptible to NK lysis. The second approach consisted in removal of TfR-bearing cells from bone marrow cell suspensions by an indirect rosetting technique. Using mAbs bound to ox erythrocytes the rosetted TfR-bearing cells could be removed from bone marrow cell suspension by density centrifugation with an efficiency of >99%. It could be shown that both fractions, TfR⁺ and TfR^– cells, could be lysed to the same degree by NK cells. Thus, the evidence obtained speaks against a role of TfR as a recognition structure for NK cells.Abbreviations BM Bone marrow - mAb Monoclonal antibody - NK cell Natural killer cell - PBL Peripheral blood mononuclear cell - TfR Transferrin receptor This work is dedicated to Professor Hans Dierk Waller on the occasion of his 60th birthdayThis work was supported in part by the Deutsche Forschungsgemeinschaft, Bonn, SFB 217     

Rat natural killer cell and cytotoxic T cell lysis of H-2-negative murine embryonal carcinoma cells

H-2-lacking murine embryonal carcinoma (EC) cells have been proposed as universal targets for natural killer (NK) effectors from different species because their killing appeared to be uncomplicated by potential T cell effector mechanisms (Stern, P. L. et al., Int. J. Cancer 1981. 27:679). While some previous studies had shown that murine cytotoxic T cells were unable to lyse EC cells, rat T killers are shown here to be active against these targets and to be distinguishable from NK cells. Percoll density fractionation of rat peripheral blood lymphocytes enriches in parallel for NK-mediated lysis of both EC or YAC target cells. These NK cells unlike T cells, do not mediate lectin-dependent and cell-mediated cytotoxicity (LDCC) of NK-insensitive target cells. This procedure is thought to reveal the total cytolytic potential of stimulated T cell populations, regardless of specificity. In contrast to previous results with mice, we found that allogeneically primed rat cytotoxic T cells can kill murine EC cells in LDCC and, further, that rat cytotoxic T cells, generated by stimulation with mouse spleen cells in vitro, can lyse murine EC cells directly. This demonstration of T cell lysis of EC cells suggests that either there is a novel mechanism of lysis operating without requirement for major histocompatibility complex (MHC) structures, or EC cells express some hitherto unidentified MHC-like structures on their cell surface.     

Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity.

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

Conserved and variable natural killer cell receptors: diverse approaches to viral infections

Natural killer (NK) cells are lymphocytes of the innate immune system with essential roles during viral infections. NK cell functions are mediated through a repertoire of non-rearranging inhibitory and activating receptors that interact with major histocompatibility complex (MHC)–peptide complexes on the surface of infected cells. Recent work studying the conserved CD94–NKG2A and variable killer cell immunoglobulin-like receptor–MHC systems suggest that these two receptor families may have subtly different properties in terms of interactions with MHC class I bound peptides, and in recognition of down-regulation of MHC class I. In this review, we discuss how these properties generate diversity in the NK cell response to viruses.     

Proliferative and cytotoxic response of human natural killer cells exposed to transporter associated with antigen-processing-deficient cells

In transporter associated with antigen-processing (TAP)-deficient patients affected by a severe downmodulation of human leucocyte antigen class I (HLA-I) molecules, natural killer (NK) cells have an increased expression of the inhibitory receptor CD94/NKG2A. Focusing our attention on NK cells, we have investigated the phenotype, function and proliferative response of peripheral blood lymphocytes (PBLs) derived from healthy donors after coculturing with TAP (T2)- or HLA-I-deficient (721.221) cell lines and their related HLA-I-expressing transfectants (T3 and DT360, respectively). After 4 days, NK cells cocultured with T2 cells had a threefold increased CD94 expression compared to NK cells cocultured with T3. This increase was due to proliferation of the CD56brightCD94bright subset. In contrast, expression of other inhibitory receptors [killer cell immunoglobulin (Ig)-like receptors] was variable during time and was not related to HLA-I molecules expressed by stimulating cells. Similar results were obtained using HLA-I-deficient cells (721.221). The PBLs cocultured for 4 days with T2 cells displayed enhanced cytotoxic responses. The results suggest that CD56brightCD94bright NK cells are induced to proliferate and kill in response to a TAP-deficient environment. The changes seen in the NK-cell compartment were partially contributed by T lymphocytes present in the coculture. These data could explain the increased CD94 expression and autoimmune manifestations observed in TAP-deficient patients.     

Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities.     

A functional comparison of tumor cell killing by activated macrophages and natural killer cells.

This report compares the sensitivity of 17 tumor cell lines to cytolysis mediated by natural killer (NK) cells or by activated, bone marrow-derived macrophages (AM) from 15 inbred mouse strains. Some tumor cell lines, notably P815, were highly sensitive to AM-mediated lysis but almost completely insensitive to NK cells, whereas other cell lines were lysed by NK cells but not AM. In a genotype survey, some low-responder strains in the NK system, such as A/Sn, were high responders in the AM system, and conversely, one intermediate to high-responder strain (C3H/HeJ) in the NK system was a low responder in AM-mediated cytolysis. In addition, macrophage cytotoxicity factor was necessary to activate macrophages, but this lymphokine did not augment NK activity. Furthermore, the NK population did not contain pre-activated macrophages since pre-activated cells were removed on glass bead columns or by iron carbonyl and a magnet; treatments which have been previously shown not to affect NK cells. These results suggest that NK cells are distinct from AM in physical characteristics, target selectivity, genotype distribution and the mechanism of cytolysis.     

Induction of erythroid differentiation in K562 cells and natural killer cell-mediated lysis: distinct effects at the level of recognition and lysis in relation to target cell proliferation

The erythroleukemic K562 cell line was induced to erythroid differentiation by a variety of agents, including hemin, bleomycin, and cytosine arabinoside. The sensitivity of induced cells to binding and lysis by non-sensitized peripheral blood mononuclear cells (MNC) in agarose was studied in relation to the target cell division rate. Differentiated K562 cells formed a lower proportion of conjugates with MNC, when compared with non-induced controls. The reduction correlated significantly with the level of differentiation, irrespective of the inducer and the proliferative status. The differentiation-induced alterations of lysis, however, were strongly influenced by the modification of target cell growth rate which was caused by the differentiating agent. These data suggest that target cell differentiation has distinct effects upon the steps of recognition and lysis by natural killer cells.     

Interferon-alpha-dependent and -independent participation of accessory cells in natural killer cell-mediated lysis of HSV-1-infected fibroblasts.

Human natural killer (NK) cells require an HLA-DR+ accessory cell (AC) population to lyse herpes simplex virus-type 1 (HSV-1)-infected fibroblasts (HSV-Fs) but not K562 target cells. It has been postulated that ACs may function by producing interferon-alpha (IFN-alpha), which stimulates NK cells. Using a sequential enrichment protocol, ACs were found to coenrich with the interferon-producing cells (IPCs). Treatment of the ACs with a protein synthesis inhibitor, emetine, inhibited both their IFN production and AC function, results that support a central role for IFN in AC activity. In contrast, when the arginine analogue canavanine was added to NK assays, no IFN-alpha was produced and NK(HSV-Fs) activity was only partially inhibited. Consistent with IFN-independent AC function, treatment with either polyclonal sheep or bovine anti-IFN-alpha neutralized all the IFN-alpha produced during the NK assays but caused either no or partial reduction of NK(HSV-Fs) activity, respectively. However, when limiting numbers of ACs were used, the bovine antiserum neutralized both IFN-alpha and NK(HSV-Fs) activity. We further found that HLA-DR+ cells are required for cell clustering, suggesting a role for cell contact. Finally, fixation of activated ACs prevented the accessory function. Together, these results demonstrate that ACs can provide help to NK cells in both IFN-alpha-dependent and -independent manners.     

Homogenous expression of killer cell immunoglobulin-like receptors (KIR) on polyclonal natural killer cells detected by a monoclonal antibody to KIR2D

The activity of human natural killer (NK) cells is in part regulated by the expression of killer cell immunoglobulin (Ig)-like receptors (KIR) that recognize major histocompatibility complex (MHC) class I and can inhibit NK cell cytotoxicity. A monoclonal anti-KIR antibody was established and designated Lig1. Lig1 was shown to be specific for KIR in cell-surface staining and to react with all KIR2D, except KIR2DL4 which lacks a D1 domain, but not with KIR3D molecules in an enzyme-linked immunoadsorbent assay (ELISA) and Western blotting. Unlike other anti-KIR antibodies, Lig1 did not inhibit binding of KIR-Ig-fusion proteins to MHC-class I expressing cells nor did it interfere with KIR-mediated inhibition of NK cell cytotoxicity in a functional assay. Lig1 reacted with all NK cells in polyclonal NK populations from different donors, demonstrating that all NK cells express at least one KIR2D receptor.     

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

A study of heart rate and heart rate variability in human subjects exposed to occupational levels of 50 Hz circularly polarised magnetic fields

The effects of power-frequency magnetic fields on heart rate and heart rate variability (HRV) were studied in groups of adult volunteers. Exposure consisted of 28 microT (280 mG) at 50 Hz (circularly polarized) for 100 or 150 seconds either following or prior to a similar period of sham-exposure. A small but significant slowing of heart rate of the order of 2% was observed in two separate studies in which the fields were generated by continuous sinusoidal currents. Magnetic fields generated by square-wave currents or by currents turned alternatively on and off at 15 second intervals during the exposure period produced inconsistent effects on heart rate. Analysis of the HRV spectra in relation to continuous sinusoidal exposure showed a consistent reduction in the ratio of power in the Low Band (0.02-0.15 Hz) to the High Band (0.16-1.0 Hz). This reduction in ratio was significant for experiments in which respiration was controlled at 0.2 Hz (12 breaths/minute) where the order was actual exposure followed by sham exposure (On-->Off). The spectral power in the Low Band was significantly reduced for both orders, but the High Band power was significantly raised only for the On-->Off order. Although there are some inconsistencies, these data indicate that short exposures to magnetic fields at occupational levels may influence heart rate control mechanisms.