Characterization of natural killer (NK) cells and killer (K) cells in human blood: discrimination between NK and K cell activities.

Cell suspensions enriched and depleted for rosette-forming cells with sheep red blood cells (E-RFC) and depleted for RFC with antibody complexes were prepared. The isolated fractions were characterized by cell surface marker analysis and tested for their natural killer (NK) and killer (K) cell activity against K-562 cells and IgG-coated P-815 cells, growing in suspension, and against a number of monolayer tumor cell lines. It was found that the NK cells most likely belong to the T cell lymphocyte subpopulation. Furthermore, this study indicates that several subpopulations exist, e.g. NK cells that have no IgG Fc receptor (FcR) on their surface and NK cells that bear IgG FcR, indicating that for a proportion of NK cells the IgG FcR is not involved in the NK lytic process and hence antibody-independent. Moreover, monocytes and B lymphocytes appear not to be directly involved in the NK cell lytic process. Furthermore, cell separation procedures were used to obtain cell suspensions either bearing IgG FcR or lacking IgG FcR. Cells bearing IgG FcR were isolated in such a way that they lost their IgG FcR by shedding, as a result of the separation procedure. Again, all fractions were simultaneously characterized by cell surface marker analysis and tested for their NK and K cell lytic activity. The effect of immune complexes on the NK and K cell lytic activities was investigated. The data indicate that the IgG FcR is not involved in the NK lytic mechanism, although this receptor may be present on the NK cell. Moreover, prolonged culturing of lymphocytes increases and/or induces NK cell lytic activity.     

Inhibition of NK and K cell function by phenothiazines

Several phenothiazines have been shown to inhibit NK cell-mediated cytotoxicity (NKC) and antibody-dependent cell-mediated cytotoxicity (ADCC) effected by human peripheral blood lymphocytes. Of those tested, chlorpromazine was found to inhibit at the lowest concentration (5 microM) with a 90% inhibition at 10 microM for NKC and 70% inhibition for ADCC. Pre-incubation of either effector or target cells with chlorpromazine did not provide any evidence of inhibition in subsequent cytotoxicity assays. The data for chlorpromazine in particular may be of clinical significance. In vivo inhibition would presumably require the presence of the drug since preincubation did not produce inhibition of NK or K cell function.     

Interactions between human natural killer (NK) lymphocytes and yeast cells: human NK cells do not kill Candida albicans, although C. albicans blocks NK lysis of K562 cells.

Rodent natural killer (NK) lymphocytes are cytotoxic to certain fungi. We investigated whether human NK cells are cytotoxic to the yeast Candida albicans. We found that human peripheral blood lymphocytes possessing NK cell activity had little or no effect on the viability of the yeast. Unopsonized C. albicans, however, were able to block NK cell-mediated cytotoxicity at a ratio of 100 yeast to one K562 erythroleukemia cell. C. albicans was not toxic to the lymphocytes nor did it take up isotope released by the K562 cells. Furthermore, C. albicans that was pretreated with human serum blocked NK cell activity more than did untreated C. albicans. Binding of the yeasts to NK cells could account for the blocking effect of serum-treated yeasts, but not for that of the untreated yeasts. Flow cytometry indicated that there was preferential binding of C. albicans to NK lymphocytes but not to T cells when the yeasts were pretreated with human serum. In this report we affirm the results of the study by Vecchiarelli et al. (A. Vecchiarelli, F. Bistoni, E. Cenci, S. Perito, and A. Cassone, Sabouraudia 23:377-387, 1985), that the first report of rodent NK cell activity against the yeast Cryptococcus neoformans (J. W. Murphy and D. O. McDaniel, J. Immunol. 128:1577-1583, 1982) cannot be extrapolated to a general phenomenon of unprimed lymphocyte-mediated destruction of all species of yeast. Our data extend the observations to humans and also suggest that in vivo interactions between NK lymphocytes and opportunistic fungal pathogens may affect NK cell function.     

Immunoultrastructural studies of human NK cells: II. Effector-target cell binding and phagocytosis

The binding of NK cells to a target cell appears to be a necessary step for NK cell-mediated cytolysis. In this report, we demonstrated effector-target binding by immunoelectron microscopy by using monoclonal antibodies against NK cells (Leu-7, Leu-11a) and T-cell subsets (Leu-2a/T8, Leu-3a/T4). The surfaces of NK and K562 cells were characterized by antitransferrin receptor antibody and various lectins. In addition, the controversial phagocytic activity of NK cells was studied by incubation of peripheral blood mononuclear cells with opsonized Staphylococcus aureus and labeling with anti-Leu-7 or anti-Leu-11a antibody. Results showed that only Leu-11a+ cells displayed a broad cell-to-cell contact with the target by a shallow intercellular interdigitation of cytoplasmic projections, while Leu-7+, Leu-2a+, or Leu3a+ cells showed only a partial contact with target without interdigitation. The Leu-11a+ cells were frequently observed in small clusters and in close association with monocytes. Cluster formation and association with monocytes were not observed in other NK and T-cell immunophenotypes. In Leu-11a+ cells conjugated with target cells, membrane-bound granules, small vesicles, parallel tubular arrays, Golgi apparatus, endoplasmic reticulum, and small vacuoles were evident and concentrated toward the target. The surface of NK cells was intensely stained for glycoprotein by chromic acid-phosphotungstic acid, whereas target cells were not stained. Transferrin receptors were stained only on the surface of target cells. Only the lectins RCA and UEA labeled the surfaces of both NK and target cells. Phagocytic vacuoles containing cell debris or fragments and ingested bacteria were found in the cytoplasm of Leu-11a+ cells but not in Leu-7+ cells. NK cells were also found within the cytoplasm of K562 target cells. All these findings suggest that Leu-11a+ cells are the true functional NK cells involved in NK cell-mediated cytolysis, phagocytosis, and emperipolesis. Therefore, the NK cell is probably "a phagocyte in lymphocyte's clothing." The presence of peroxidase in the small vesicles of NK cells and endocytotic vesicles of target cells at the effector-target contact area indicates that cytolytic enzymes or factors derived from NK cells may be transported into the target by endocytosis.     

Inhibition of SK cell activity in frogs by certain drugs and sugars

We present similarities between mammalian natural killer (NK) cells and (anuran amphibian) frog spontaneous killer (SK) cells. A cytotoxic assay utilizing allogeneic erythrocytes as target cells was used and lysis assessed by measuring release of hemoglobin. SK effector cells, just as mammalian NK cells, are not sensitive to cycloheximide nor most simple sugars (50 mM glucose, glucose-6-phosphate, galactose, fucose, mannose). However, SK activity is inhibited by chloroquine, colchicine and mannose-6-phosphate. When SK cells were co-incubated with mammalian tumor cells, they were able to lyse only the NK-sensitive target YAC-1, but not other mammalian tumor cell targets including K562, Molt-4, Raji, P815 and EL4. Lysis of YAC-1 cells was also inhibited by colchicine and chloroquine. These results allow speculation on the evolution of cell mediated cytotoxicity since natural cytotoxic cells are present in ectothermic vertebrates.     

The 'T-cell-ness' of NK cells: unexpected similarities between NK cells and T cells

NK cells are considered as prototypical innate immune cells. However, recent discoveries have tended to refine the dogmatic concepts of innate and adaptive immunity. In many ways, NK cells are highly related to T cells and represent the closest innate immune cell lineage to adaptive immune cell populations. Here, we review the relationships between NK cells and T cells and discuss the recently described cell-intrinsic-adaptive features of NK cells.     

Stimulation of human NK cell activity by cultured cells. II Ingestion of aspirin by blood donors suppresses induced NK activity.

Incubation of mononuclear cells with a melanoma tumour cell. M4, induces a rapid increase in NK cell activity in both normal and tumour patient cells. A marked individual variation among the patient population can be attributed to the prior ingestion of aspirin. Single therapeutic doses of aspirin (two tablets, 660 mg) taken 12 hr prior to donation of the blood sample cause an 80-100% reduction in the NK cell activity induced by M4. Since many patients coming to cancer clinics take aspirin regularly, it is essential that they be questioned about their recent usage of this drug if their cells are to be used in assays for NK activity.     

Inhibition by sugars of Candida albicans adherence to human buccal mucosal cells and corneocytes in vitro.

The adherence and inhibition of adherence of Candida albicans to epithelial cells was studied for human cells obtained from skin (corneocytes) and buccal mucosa. The yeast adhered to both kinds of cells, although in somewhat greater numbers to buccal mucosal cells. Adherence to the cells of different individuals was variable, but the ratios of values for the two kinds of cells from a single subject were quite constant. Inhibition of adherence was produced by several sugars, including the aminosugars mannosamine, glucosamine, and galactosamine. The pattern of inhibition produced by the sugars was similar for the two types of cells. Pretreatment of the yeast with mannosamine, followed by dilution to a subinhibitory concentration, produced some inhibition of yeast-buccal mucosal cell attachment, indicating some direct interaction between the sugar and the fungal cell. These data suggest that the mechanisms whereby C. albicans attaches to corneocytes and to buccal mucosal cells are probably similar.     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

Recognition of autologous dendritic cells by human NK cells

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK-target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40-transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co-stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL-2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF-alpha of the cultures stimulated the development of a more mature DC phenotype, while addition of IL-10 resulted in a less mature phenotype, with lower expression of CD40 and other co-stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.     

Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride

Ammonium-chloride-containing solutions (AC) are routinely used to lyse red blood cells during preparation of PBMC. Although exposure to AC has been described to affect the ultrastructural appearance of large granular lymphocytes and to temporarily inhibit cytolytic activity of PBMC preparations, the cellular basis of this phenomenon has not been studied. Here, the inhibitory effect of AC on human CTL and NK-mediated cytotoxicity has been analyzed in 4-h 51Cr-release assays. The results show that NK killing of K562 leukemia cells and xenogeneic endothelial cells is inhibited by AC exposure. The effect is dose-dependent and reversible, because recovery of cytotoxicity is observed within 15 h of re-culturing. AC does not reduce the viability of NK cells and the inhibitory effect is not mediated by the exhaustive release of granzymes upon AC treatment. In contrast, antigen-specific CTL killing of EBV-transformed B-lymphoblastoid cell lines and xenogeneic PHA lymphoblasts was less sensitive to AC and data are presented suggesting that FasL-induced apoptosis is not inhibited by AC. In conclusion, perforin-mediated NK killing is AC-sensitive whereas CTL killing and FasL-mediated killing appear to be AC-resistant. Therefore, AC represents a powerful tool to study different mechanisms of cell-mediated cytotoxicity and may be helpful in assessing antigen-specific CTL cytotoxicity without the influence of NK cell-mediated background killing.     

Modulation of in vitro natural cell-mediated activity against enteropathogenic bacteria by simple sugars.

Lymphoid cells from mouse Peyer's patches and spleens were tested in a 2-h in vitro assay for their natural activity against the enteropathogenic bacteria Salmonella typhimurium, Salmonella enteritidis, Salmonella tel aviv, and Shigella sp. X16. The antibacterial activity expressed by normal cells was detected against all the bacterial strains tested with the exception of Peyer's patch lymphocytes against S. tel aviv and splenocytes against Shigella sp. X16. To determine whether the different expression of natural antibacterial activity might be due to lectin-like proteins interacting with the saccharidic moieties of the bacterial wall, 11 simple sugars were preincubated with the effector cells before the in vitro assays. We found that some of them could block the natural antibacterial activity as well as induce antibacterial activity when this was not spontaneously expressed. Interestingly, a different panel of sugars among those employed was observed to affect the antibacterial activities for each of the above-mentioned bacterial targets and each effector cell. However, the same panel of sugars was able to block or stimulate the lymphocyte activity when bacteria with the same somatic antigens as two substrains of S. typhimurium and one strain of Salmonella schottmuelleri were employed. To further investigate the interaction between effector cells and bacteria, effector cells or Shigella sp. X16 targets were treated with proteolytic, glycolytic, and lipolytic enzymes before the in vitro assays. Furthermore, EDTA was used to analyze the role of divalent cations in this experimental system. The results obtained suggest that lectin-like proteins playing a role in this interaction are present not only on lymphocytes but also on bacteria and that divalent cations are essential for the expression of in vitro antibacterial activity.     

Enhancement of NK, but not K cell activity by different interferons

Two different interferons derived either from a human lymphoblastoid cell line (Namalva) or from human fibroblasts were tested for their ability to modulate natural killer (NK) or killer (K) cell activity. The lymphoblastoid interferon was purified by ion-exchange chromatography on SP-Sephadex C-25 and gel filtration on Sephadex G-100, the fibroblast-derived interferon was purified by chromatography on porous glass beads. Evidence is presented that NK cell activity is enhanced by both of these interferons being active to a similar extent. When tissue culture cells are employed as targets for measurement of K cell activity, the augmentation of cytotoxicity by interferons has to be attributed to the inherent NK cell activity. With the use of the autologous hapten-coated target cells and of affinity chromatography purified antibodies, the cytotoxicity is displayed solely by K cells and this activity is not enhanced by either interferons tested.     

Inhibition of blood clearance and hepatic tissue binding of Escherichia coli by liver lectin-specific sugars and glycoproteins.

The effects of sugars and glycoproteins that are known to bind to lectins of liver tissue on the clearance of cells of Escherichia coli from mouse blood was investigated. The administration of 100 mg per mouse of methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, or methyl-alpha-D-fucoside, but not of methyl-alpha-D-galactoside or L-rhamnose, markedly inhibited the blood clearance of cells of E. coli 346. Clearance was similarly inhibited by 0.1 and 1.0 mg per mouse of asialofetuin or ovalbumin, respectively, whereas fetuin had no effect. The inhibitory effects of the sugars on blood clearance was abolished by pretreating the E. coli cells with antibodies against whole organisms. All of these effects were equal for fimbriated and nonfimbriated phenotypes of E. coli 346. Homogenates of mouse liver tissue coaggregated with nonfimbriated cells of E. coli. The aggregation was blocked by 100 mM solutions of methyl-alpha-D-mannoside, or methyl-alpha-D-glucoside, 1 mg of bacterial lipopolysaccharide per ml, or 10 mM EDTA but not by L-rhamnose. These results suggest that the mannose-N-acetylglucosamine hepatic lectin recognizes specific sugars on the surface of E. coli and may be centrally involved in the nonimmune clearance of nonfimbriated E. coli from the blood of the infected host.     

Arousal and inhibition of human NK cells

Summary: NK cells express receptors for polymorphic MHC class I molecules that inhibit killing of potential target cells bearing appropriate class I allotypes. Here we review the membrane receptors on human NK cells that are known to initiate cell-mediated cytotoxicity and demonstrate regulation of these responses by the killer cell inhibitory (KIR) receptors.     

Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation

We previously reported that intracellular oxidation-reduction (redox) regulates NK cell functions and that IL-2-activated NK cells undergo apoptosis upon contact with NK-sensitive target cells. We now report that apoptosis in activated human NK cells is also regulated by redox. Thiol deprivation increased apoptosis in NK cells induced by anti-Fas mAb or Fas ligand-transfected cells, and pretreatment of cells with N- acetyl cysteine, which increased intracellular glutathione, partially inhibited the apoptosis and reversed the effect of thiol-deficient medium, suggesting that Fas-induced apoptosis in NK cells is also redox sensitive. Thiol deprivation did not alter cell surface Fas expression, but did increase ceramide generation following Fas engagement. Although exogenous ceramides induced apoptosis of NK cells, thiol depletion had no effect on this apoptosis. Thiol deprivation increased CPP32 activation induced by Fas engagement, but not by ceramides. These findings suggest that, if ceramide is required for Fas-induced apoptosis, thiol deprivation affects the Fas-mediated signaling pathway at the generation of ceramide and/or upstream thereof. Though tyrosine phosphorylation following Fas engagement was not significantly affected by thiol deprivation, tyrosine dephosphorylation was delayed, suggesting that tyrosine phosphatases may also be redox sensitive. The notion that dephosphorylation is important in the Fas signaling pathway is supported by the finding that tyrosine phosphatase inhibitors significantly enhanced both CPP32 activity and apoptosis following Fas ligation. We conclude that events downstream of tyrosine phosphorylation and upstream of CPP32 activation, including tyrosine dephosphorylation and possibly ceramide generation, are sensitive to regulation by redox in human NK cells, requiring a reducing environment for optimal protection from apoptosis induced by Fas ligation.      

Modulation of human NK cells by interferon and prostaglandin E2

Our studies have shown that stimulation of human natural killer (NK) cells by poly I:C does not depend on or require monocytes. In contrast, the presence of monocytes in a mixed population of mononuclear cells stimulated by poly I:C suppresses NK activity. The suppression can be partially overcome if indomethacin (10(-6) M) is added to the culture during stimulation. Culture supernatants from poly I:C stimulated monocytes do not have detectable levels of anti-viral activity but contained appreciable amounts of PGE2. Our results offer an explanation as to how NK cells may protect themselves from suppression by PGE2. We have demonstrated that IFN-activated NK cells become resistant to PGE2-mediated suppression; moreover the suppression does not require cells other than the large granular lymphocytes, the major effector cell type for NK. Taken together, the data suggest that stimulation of NK cells is dependent on and regulated by the relative levels of interferon produced by lymphocytes and PGE2 produced by monocytes.     

Catecholamines induce alterations of distribution and activity of human natural killer (NK) cells

Catecholamines have been suggested to be responsible for altered cellular immunity after stress. This study was performed to determine the effects of adrenaline and noradrenaline on lymphocyte subpopulations and NK cell functions. Subjects were given a subcutaneous injection of either NaCl, adrenaline (5 µg/kg), or noradrenaline (10 µg/kg). Catecholamine concentrations, subsets of peripheral blood lymphocytes, NK activity, and antibody-dependent cellular cytotoxicity (ADCC) were analyzed before (baseline) and 5, 15, 30, 60, and 120 min after injection. There were no differences between groups in the distribution of CD2⁺ and CD8⁺ lymphocytes over time. However, CD3⁺ and CD4⁺ T cells decreased significantly 5 to 60 min after injection of adrenaline. In contrast, NK cell numbers (CD16⁺, CD56⁺) increased significantly 5 min after injection of adrenaline and noradrenaline, reached the highest values 15 to 30 min postinjection, and subsequently declined to baseline values 60 (noradrenaline) and 120 (adrenaline) min, respectively, after injection. Similar alterations for NK activity and ADCC were observed after administration of both catecholamines. These data suggest that both sympatheticadrenal hormones are similarly potent modulators of natural immunity and provide further evidence that catecholamines might be responsible for the observed alterations in immune functions after phases of acute stress.