Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. II. Increased Sensitivity to Natural Killer Cytotoxic Factor (NKCF)

Abstract

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.     

Effects of X-ray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumor cells and lytic function of NK cells

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

Abstract

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Enhancement by X-ray irradiation of target cell susceptibility to natural killer cells

The in vitro effect of X-ray irradiation on tumor cell susceptibility to lysis by natural killer (NK) cells was studied in human systems. When relatively NK-resistant T24 bladder transitional carcinoma cells were irradiated with X-rays and cultured for 18 h, they showed increased sensitivity to lysis by blood lymphocytes in a 4-h 51Cr release assay. No enhancement was seen when irradiated target cells were tested immediately after exposure to X-rays. The X-ray-induced augmentation was observed with as little as 1 Gy of irradiation, the level of which was comparable to that observed at higher doses. The doses of X-rays did not influence the viability and spontaneous release of the target cells. Treatment with mitomycin C of target cells did not change their NK sensitivity. On the other hand, irradiation with X-rays of blood lymphocytes resulted in a transient increase in NK activity at 3 h, and then the activity declined and was completely lost by 24 h. However, when irradiated lymphocytes were stimulated with interferon (IFN), they maintained the high activity against untreated T24 cells. These results suggest the possible use of relatively low doses of X-ray irradiation in combination with IFN for treatment of human cancer.     

Suppressed natural killer cell activity in patients with euthyroid Graves' ophthalmopathy

The purpose of the present study was to determine whether patients with euthyroid Graves' exophthalmopathy have an impaired NK cell function compared to patients with Graves' hyperthyroidism and healthy controls. The NK cell activity measured against K562 target cells was significantly suppressed (p less than 0.01) in patients with euthyroid Graves' ophthalmopathy, whereas the NK cell activity of patients with Graves' hyperthyroidism was not. Although interferon-alpha, interleukin-2 and indomethacin significantly enhanced (p less than 0.01) the NK cell activity in all three groups, none of these agents fully restored the defective NK cell activity in euthyroid Graves' ophthalmopathy. The concentrations in the blood of large granular lymphocytes and CD16 positive cells did not differ between the three groups, furthermore an immunosuppressive serum factor was not detected. The number of effector/target cell conjugates did not differ between patients and controls, whereas the interferon-alpha induced production of a soluble natural killer cytotoxic factor (NKCF) with specificity for NK sensitive target cells was suppressed in patients with Graves' euthyroid ophthalmopathy. We conclude that one of the mechanisms underlying the defective NK cell activity in patients with euthyroid ophthalmopathy may be an impairment of the release of NKCF from the NK cells.     

Lysis of natural killer-sensitive and -resistant tumor cells by natural killer cytotoxic factors (NKCF)-containing liposomes

The lethal hit stage in NK cell-mediated lysis requires a complex series of events involving the release of NKCF, subsequent binding of these factors to the target cell, and susceptibility of the target cell to lysis by NKCF. Binding of NKCF alone is not sufficient because a number of tumor cells are able to bind NKCF without being lysed, suggesting the need for an additional processing step active on susceptible target cells. In the present study, we show that the interaction with liposome-incorporated NKCF renders NK resistant target cells sensitive to NKCF-mediated lysis. These results suggest that NKCF may mediate their cytotoxic effects through internalization of these factors into the cytosol.     

The effect of differentiation inducers on the sensitivity of two myeloid cell lines to natural killer (NK) cell-mediated lysis

The effects of differentiation inducers on the sensitivity of two human myeloid cell lines, K562 and HL-60, to natural killer (NK) cell lysis were examined. K562 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and HL-60 cells treated with either TPA or dimethylsulfoxide (DMSO) not only became less sensitive NK targets, but also became less competitive in cold target inhibition experiments. Target binding cell assays revealed that TPA-treated target cells bound fewer NK effector cells than did untreated targets. TPA treatment renders K562 and HL-60 target cells more susceptible to hypotonic lysis, indicating that the decreased NK sensitivity is not due to altered osmotic fragility. The observed reduction in sensitivity to NK lysis was not apparently mediated by interferon (IFN) released by the target cells, as neither alpha nor gamma IFN were detected in culture supernatants. Furthermore, the effects of TPA were additive to the known protective effect of IFN on NK target cells. In contrast to the parent line, a subclone of HL-60 resistant to chemically induced differentiation did not become a less sensitive target after exposure to TPA. These observations with myeloid and erythroid target cells imply that the reduced killing of the TPA-treated cells was secondary to impaired recognition of the target by the effector, and that the state of cellular differentiation is a major determinant of susceptibility to NK cell-mediated lysis.     

Impaired release of natural killer cytotoxic factor in patients with primary Sjögren''s syndrome.

The impaired natural killer (NK) cell activity against K562 target cells of patients with primary Sjögren''s syndrome (primary SS) was re-examined in a 2-year follow-up study of 10 patients and 10 normal controls. The ability of blood mononuclear cells (BMNC) to form effector/target cell conjugates and to release NK cytotoxic factor (NKCF) were studied. NK cell activity of the patients was unchanged low (P less than 0.01) compared with the controls. The number of effector/target cell conjugates did not differ between patients and controls, whereas NKCF-release from interferon-stimulated BMNC was significantly (P less than 0.01) reduced in the patients with primary SS and positively correlated to the reduced NK cell activity (r = 0.85, P = 0.0002). The permanently low NK cell activity of patients with primary SS appears therefore, at least in part, to be due to an impaired release of NKCF and not to a defective ability of effector cells to recognize and/or adhere to target cells.     

Target structure for natural killer cells: evidence against a unique role for transferrin receptor

The transferrin receptor (TfR) was recently proposed as putative natural killer (NK) cell target structure. Here data are presented against this hypothesis and it is shown that low TfR expression and high NK sensitivity can occur concommitantly . K562 cells were studied at various stages of cell proliferation. No change in NK sensitivity could be observed between exponential growth and the plateau phase, whereas TfR expression completely disappeared during the latter. Protein synthesis inhibitors such as cycloheximide (1 microgram/ml, 48 h) and actinomycin D (50 micrograms/ml, 48 h), that abolished the TfR expression at the K562 cell surface, had no effect on NK sensitivity. Similarly, hemin induction (0.1 mM, 5 days) did not change NK susceptibility of K562 cells but considerably diminished TfR expression. Moreover, attempts to block NK sensitivity with anti-TfR monoclonal antibodies were unsuccessful, even when the 42.6 antibody, which is known to bind to the active site of TfR, was used. Finally, no blocking of NK sensitivity could be achieved when K562 cells were preincubated with saturating concentrations of transferrin or when transferrin was added during the NK assay. It therefore seems doubtful that TfR is the unique target structure for NK cells. It remains possible that TfR and NK target structures are often coexpressed on actively dividing cells.     

Pathogenesis of the natural killer cell deficiency in AIDS

Deficiency in natural killer (NK) cell activity is a common feature of acquired immune deficiency syndrome (AIDS). This is part of a general immune dysfunction in AIDS and may lead to progression of the disease, since NK cells are known to be involved in protection against tumors and against viral infections. The lack of immunological surveillance by NK cells of the growth of pathogens that activate the HIV-1 tat infectivity gene may also favor progression to AIDS. The pathogenesis of NK cell deficiency in AIDS is not known. Previous studies have shown that NK cells from AIDS patients are able to bind but not to lyse the target cell line K562. This results from an inability to rearrange the cytoskeleton microtubular (MT) system and to release the natural killer cytotoxic factor (NKCF). This report by Maria Caterina Sirianni and colleagues evaluates the possible mechanisms leading to this NK cell deficiency.     

Interaction of natural killer cells with MHC class II: reversal of HLA-DR1-mediated protection of K562 transfectant from natural killer cell-mediated cytolysis by brefeldin-A

Major histocompatibility complex (MHC) class I antigens on tumour cell surfaces have been shown to modulate target susceptibility to natural killer (NK) cell-mediated lysis in some, although not all, systems investigated. MHC class II expression may also affect NK cell function, but the mechanism by which MHC class II antigen regulates NK cell activity has not been fully examined. In this study we induced HLA-DR1 expression by gene transfection into the classic NK-sensitive K562 cell line to study the interaction of NK cells with MHC class II molecules and the effect of brefeldin-A (BFA), an endogenous antigen-processing pathway blocker, on NK–target cell interaction. We demonstrated that the expression of HLA-DR1 on the cell surface reduced K562 cell susceptibility to NK lysis by peripheral blood monuclear cells and a NK cell line. The effect was demonstrable in prolonged (8 hr) cytotoxicity assays and was blocked by pretreatment of target cells with anti-HLA-DR antibody. Treatment of K562 DR transfectant with BFA abrogated the resistance of K562 transfectant to NK-mediated cytolysis. These findings indicate that HLA class II molecules regulate NK cell function and target recognition, and suggest that endogenous peptides presented through MHC molecules are responsible for regulating NK cytolysis.     

The chemiluminescence response of human natural killer cells. II. Association of a decreased response with low natural killer activity

Low natural killer (NK) responders selected from a panel of 600 normal, healthy volunteers exhibited 5- to 10-fold less cytotoxicity against the human erythroleukemic cell line K562, compared with high NK responders. Antibody-dependent cellular cytotoxicity against tumor cells, which is mediated by similar or identical effectors, was also depressed in low NK donors whereas lectin-dependent T cell killing and monocyte-mediated cytolysis of tumor cells was normal. Low NK donors exhibited normal frequencies of cells expressing the HNK-1 marker of human NK cells and highly enriched NK fractions were not impaired in their ability to recognize and bind to NK-sensitive target cells. Interferon partially activated low responder NK cells but did not restore the response to normal levels. The burst of chemiluminescence that is generated by NK cells within seconds of target cell contact was markedly impaired in low NK responder donors. We have previously shown that chemiluminescence detects reactive oxygen intermediates which are necessary but not sufficient for the activation of the NK cytolytic pathway.     

Phospholipase C activation in the cytotoxic response of human natural killer cells requires protein-tyrosine kinase activity.

Treatment of highly purified natural killer (NK) cells with the protein-tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was not affected by PTK inhibition. However, activation of phospholipase C (PLC) in response to K562 cell binding (as measured by inositol phosphate turnover) was decreased by as much as 75% when PTK activity was inhibited. Furthermore, there was an increase in tyrosine phosphorylation of NK cell PLC gamma 2 after exposure to K562 target cells. These data indicate that a PTK is involved in the activation of NK PLC by tumour target cells in the cytotoxic response.     

Activation of human blood lymphocytes and monocytes by the streptococcal preparation OK432: enhanced generation of soluble cytotoxic factors

The streptococcal preparation OK432 augments natural cytotoxicity of human blood lymphocytes and monocytes. It also enhanced the production of natural killer soluble cytotoxic factors (NKCF) when the effector cells interact with K562 cells. There was a good correlation between the OK432-induced enhancement of NK cell-mediated cytotoxicity and the released NKCF activity. OK432-pretreated monocytes secreted higher amounts of monocyte cytotoxic factors (MCF) than the untreated monocytes. With the monocytes the enhanced generation of MCF was not always accompanied by the increase in direct cell-mediated lysis of K562. OK432 treatment alone did not induce NKCF release from lymphocytes, and the presence of K562 in the culture was necessary. In contrast, monocytes generated MCF when exposed to OK432. In the supernatants of cocultures of OK432-activated effectors and K562 the NKCF and MCF activity was elevated two- to ten-fold. The OK432-induced augmentation of natural cytotoxicity exerted by lymphocytes and monocytes may be mediated through an increase in the synthesis, activation and/or release of NKCF and MCF.     

Selective expansion of human natural killer cells leads to enhanced alloreactivity

In allogeneic stem cell transplantation （SCT）, natural killer （NK） cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease （GVHD）. This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors （KIRs） within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 （C1） or Cw4 （C2） transfected K562 stimulator cells. Coculture of KIR＋ NK cells with C1 or C2 positive K562 cells, in the presence of IL-2＋ IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR＋ NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107~ cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.     

Effects of pertussis toxin treatment on human natural killer cell function.

Membranes from highly purified natural killer (NK) cells were ADP ribosylated by treatment with pertussis toxin (PTX). PTX treatment resulted in a single band of 32P incorporation at M(r) 41,600. PTX treatment of NK cells diminished their ability to lyse K562 tumour cells by about 50%. However PTX treatment had no measurable effect on cAMP levels in NK cells. PTX pretreatment also had no effect on the ability of target cells to induce phosphoinositide turnover or on the ability of the NK cells to conjugate with the K562 tumour cells. Movement toward the chemoattractants interleukin-2 (IL-2) and formylmethionylleucylphenylalanine (FMLP) was significantly inhibited indicating that a PTX substrate in NK cells may be involved in the transduction of signals which are involved in cell motility.     

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.     

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and visual estimation of the frequency of fresh or IL-2-activated human lymphocytes that form conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E:T ratio, with extrapolated maximum conjugate frequencies (alpha max) of 40-50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.