Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.     

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)     

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.     

Analysis of natural killer activity and natural killer cell subsets in patients with bladder cancer

Summary In order to analyze the state of the natural resistance system of bladder cancer patients in vivo, we measured natural killer (NK) activity and NK cell subsets of peripheral blood lymphocytes (PBL) from 46 patients with bladder cancer and 25 age- and sex-matched healthy volunteers. The mean NK activity in patients with lowstage bladder cancer was similar to that in the controls, while NK activity in patients with high-stage bladder cancer was significantly depressed. The mean proportions of Leu7⁺ cells in patients with both low-stage and highstage bladder cancer were significantly higher than that in the controls. The mean proportion of Leu11a⁺ cells in patients with low-stage bladder cancer was similar to that in the controls, while in patients with high-stage bladder cancer it was significantly higher. This study demonstrates the abnormal immunological state of bladder cancer patients; namely, abnormalities exist not only in NK activity but also in the proportions of circulating NK cell subsets.     

Lysosomal enzymes and natural killer activity

The natural killer activity (NKA) of human mononuclear cells and the activity of the lysosomal enzymes of these cells (arylsulfatase, acid phosphatase and beta-glucuronidase) has been studied in norm and under human lung cancer. The mononuclear cells were isolated from peripheral blood of 10 healthy donors and 20 patients with lung cancer of II-III stages. Under the action of mononuclear cells on the target cells (human erythroleukosis cells K-562 labeled with 3H-uridine) the NKA of mononuclear cells of patients was seen to decrease (cytotoxic index = 54.8 +/- 6.4%), in comparison with that of healthy donors (cytotoxic index = 65.1 +/- 4.5%). Simultaneously a decrease in arylsulfatase activity (0.05 +/- 0.01 nmoles/10(6) cells/min) was found in comparison with the control value (0.11 +/- 0.01 nmoles/10(6) cells/min). In 2-3 weeks after the operation the NKA value (cytotoxic index = 50.2 +/- 5.8%) was restored and arylsulfatase activity (0.09 +/- 0.02 nmoles/10(6) cells/min) was increased. There was no correlation between the NKA value and the activities of acid phosphatase and beta-glucuronidase. The parallelism observed between changes in NKA value and arylsulfatase activity may suggest a possible participation of this enzyme in the killing mechanism at the stage of cerebroside sulfate ester degradation of the target cell membrane to initiate the lytic events.     

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

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

Recombinant gamma interferon enhances natural killer cell activity similar to natural gamma interferon

The enhancement of human natural killing activity by recombinant human gamma interferon (IFNγ) and natural human IFNγ were similar over a wide concentration range. Enhancement of natural killing activity by both interferons was neutralizable by antibody to natural IFNγ, as well as by antibody to a synthetic peptide representing the first 20 N-terminal amino acids of IFNγ provide conclusive evidence that IFNγ is responsible for the enhanced natural killing activity seen in IFNγ preparations.     

Increase in liver-associated natural killer activity by polyribonucleotides

Several polyribonucleotides are currently in clinical trials for the treatment of cancer or viral diseases. The present report in mice demonstrates that polyinosinic-polycytidylic acid and poly-L-lysine which has been stabilized in carboxymethylcellulose (poly (ICLC) as well as polyadenosinic-polyuridylic acid (poly AU), both potently augment natural killer (NK) activity in the liver, which is often a target organ for the formation of metastases during the progression of human cancer. Following the administration of poly ICLC (10 micrograms/mouse), greater NK activity as measured by lytic units (LU), was observed in the liver (445 LU) than in blood (63 LU) or spleen (20 LU). The high level of NK activity in the liver was in contrast to the low levels observed in untreated mice, and was maintained for at least 9 days post injection. NK activity in the blood and spleen returned to normal levels by day 6. Similar results were obtained with poly AU except that approximately 10-fold more poly AU (100 micrograms/mouse) was required to induce optimal augmentation of NK activity. Further studies demonstrated that the increase in liver-associated NK activity induced by poly ICLC was associated with a 10- to 20-fold increase in liver-associated leukocytes, termed nonparenchymal cells (NPC). Fractionation of the NPC on discontinuous density gradients of Percoll demonstrated that the NK activity mediated by NPC was associated with cells morphologically characterized as large granular lymphocytes (LGL). Further studies demonstrated that the repeated administration of poly ICLC resulted in significantly higher levels of liver-associated NK activity and total liver-associated LGL as compared to a single injection.     

Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lytic activity of natural killer cells

The goal of the present work was to find out whether the actin reorganization of 3T3-SV40 cells affects their sensitivity to the activity of natural killer (NK) cells. The effects of N-acetylcysteine (NAC) and the inhibitor of actin depolymerization, latrunculin B on both cellular parameters were studied. Experiments with NAC demonstrated that the sensitivity of 3T3-SV40 cells to the NK cell activity remained unchanged with disordered microfilaments, but decreased upon the appearance of structured stress-fibers. With respect to latrunculin B action, the opposite conclusion has been drawn, which is that the more microfilaments disorganization in the presence of latrunculin B, the lower the 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent, which confirms our previous conclusion (Gamaley et al., 2006). A decrease in the 3T3-SV40 sensitivity to the NK cell activity accompanied by actin reorganization resulting from the influence of both latrunculin B and NAC suggests changes in the cellular surface that ultimately lead to the inactivation (or loss) of molecules that are activating signals to NK cells.     

Characterization of natural killer cell activity in Macaca nemestrina

Natural killer (NK) cell activity was evaluated in three groups of Macaca nemestrina that varied with respect to SAIDS D retrovirus serotype 2 (SRV-2/W) and viremic status. Target cells used were Raji and K562 cells. No significant differences (ANOVA) in mean NK activity were detected among the three groups of animals studied. Using Raji targets, mean LU₃₀/10⁶ ± SEM was 6.3 ± 1.6 for seronegative (V-Ab−) animals, 7.3 ± 1.5 for seropositive (V-Ab+) animals, and 10.2 ± 3.5 for persistently viremic (V + Ab−) animals. Using K562 targets, mean LU₃₀/10⁶ was 7.6 ± 1.7 for seronegative (V-Ab−) animals, 6.5 ± 2.5 for seropositive (V-Ab+) animals, and 5.1 ± 1.9 for persistently viremic (V+Ab−) animals. Percentage blood CD16⁺ and CD8⁺cells also were not different in the three groups of animals. NK activity did not always correlate with percentage of CD16⁺ or CD8⁺ cells in peripheral blood at the time the assays were done. In persistently viremic animals, there was a strong positive correlation between percent CD16⁺ and CD8⁺ cells and NK activity using K562 cells but not Raji cells. Depletion experiments indicated that lysis was mediated by both CD8⁺ and CD16⁺ cells with both Raji and K562 cells. However, Raji targets were a better indicator of killing mediated by CD16⁺ cells. Our studies indicate that M. nemestrina may be classified as high or low responders with regard to NK activity, and there was no correlation with SRV-2/W viral or antibody status. Additionally, our results suggested that group housing of M. nemestrina was usually associated with increased NK activity. In conclusion, studies of NK activity in M. nemestrina should consider target cells used, phenotype of effectors, endogenous (high or low) levels of NK activity in individual animals, and housing conditions. © 1996 Wiley-Liss, Inc.     

Characterization of natural killer activity in sponge matrix allografts

NK cell activity, defined by the ability of infiltrating host cells to lyse the YAC-1 tumor target, can be detected in sponge matrix allografts across all genetic barriers tested. Nonspecific tumor cell killing cannot be detected either within bulk populations of host-infiltrating cells or in populations enriched for non-adherent lymphocytes. NK activity is also detected in cells infiltrating a syngeneic sponge matrix graft although to a much lesser extent than an allogeneic graft. NK cell functional activity at the graft site precedes the appearance of alloimmune CTL by several days. The surface phenotype of the NK cell is Thy-1.2+ and L3T4- as determined by depleting the various subpopulations with antibody and C. Systemic treatment of sponge-bearing animals with repeated injections of anti-asialo GM1 (AGM1) results in inhibition of both NK activity and CTL activity recovered from the graft on days 5 to 9 after grafting, but on days 11 to 13 after grafting both NK activity and CTL activity are found within the sponge graft. Treatment of sponge-associated cells with anti-AGM1 in vitro or intrasponge injection of anti-AGM1 at various times after grafting eliminates NK activity more readily than alloimmune CTL activity. The intimate association observed between NK cells and alloimmune CTL at the graft site prompts further investigation into the role of NK cells in the allograft response.     

Inhibition of natural killer cell activity by IgA

The in vitro effect of IgA on natural killer (NK) activities of human peripheral blood mononuclear cells was investigated. Purified myeloma polymeric IgA2 (pIgA2) and secretory IgA (S-IgA) from human colostrum inhibited NK activity, while myeloma polymeric IgA1 (pIgA1), monomeric IgA1 (mIgA1), IgG, and IgM were ineffective. Inhibition was proportional to the concentration of pIgA2 (0.125-1 mg/ml) and was observed after as little as 1 hr of incubation at various effector to K562 target cell ratios. pIgA2 and S-IgA also inhibited NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells, but did not interfere with effector-target cell binding. The inhibitory effect was slightly diminished after 24 hr culture in pIgA2-free medium. Inhibition of cytotoxicity was not due to direct toxicity on lymphoid cells by IgA because PBL treated with pokeweed mitogen in the presence of pIgA2 or S-IgA differentiated into immunoglobulin-producing cells. Viability after 24 hr of preculture with pIgA2 and S-IgA was comparable to that of untreated control cells. Morphological examination of effector cells cultured with pIgA2 or S-IgA showed a decrease in the number of granules, and the formation of cytoplasmic vacuoles. These morphological changes appeared to coincide with the depressed cytotoxicity of NK cells. The results demonstrate that purified pIgA2 and S-IgA have significant immunomodulatory effects on human NK activity.     

Increased sensitivity of cholera toxin B treated K562 cells to natural killer cells

Cholera toxin B-subunit (CTB) treatment of K562 erythroleukemia cells increased their sensitivity to be killed by NK-92 cells with more than 10%, compared to untreated cells. A similar treatment of non-T, non-B acute lymphoblastic REH leukemia cells, known to be unsensitive to NK cell mediated cytotoxicity, did not have any impact at all. Visualization of the cross-linked ganglioside_M1 (GM₁) using fluorescent labeled CTB, indicated accumulation of the fluorescence to one cap and a few smaller patches in both type of cells. Additional cross-linking using anti-CTB antibodies further accentuated capping and increased lysis in the case of K562 cells. Blocking experiments performed with anti-MICA/B, ULBP-2 and/or CD59 antibodies could not inhibit the increased sensitivity mediated by CTB.     

Increased natural killer cell activity in experimental American trypanosomiasis

When purified murine plasminogen was added to cultures of mouse spleen B cells, active plasmin progressively appeared in the supernatants. This reaction, resulting from the specific cleavage of the plasminogen by lymphocyte plasminogen activator (LPA), was measured in a fibrinolysis assay using 125I-fibrinogen. T cells were totally ineffective; under certain conditions, they could even antagonize the B cell action. Of the various B populations studied, i.e., obtained from spleen, lymph nodes, or blood of various mouse strains, all expressed the same property of plasminogen activation, which concerned mainly medium-sized B cells. Since only slight activities were detected in extracellular or intracellular compartments, a membrane-associated proteolytic enzyme may be responsible for plasminogen activation. Submitted to a series of group-specific antiproteases, the lymphocyte plasminogen activator essentially behaved as a serine-protease, with sensitivity to diisopropyl fluorophosphate, phenyl methyl sulfonyl fluoride, and nitro phenyl guanidino benzoate. The fast renewal of the enzyme in the membrane was also demonstrated by different techniques, using modifiers of cell physiology, like cycloheximide and dexamethasone, or following the reexpression of the enzyme by the cell kinetically.