Enhancement Of Natural Killer Activity Research Articles

Inhibitory effect of sera from patients with liver cirrhosis on natural killer activity

ABSTRACTThe effect of sera from patients with liver cirrhosis (LC) and from healthy donors on natural killer (NK) activity of normal peripheral blood mononuclear cells (PBMC) was investigated. PBMC from a healthy donor were pre‐incubated for 3 h at 37°C in media containing 50% test sera and washed twice with phosphate‐buffered saline (PBS). Cytotoxic activity was determined using 51Cr‐labelled K‐562 cells as target. It was demonstrated that sera from both healthy donors and patients with LC suppressed the NK activity of normal PBMC compared with a PBS control. But the level of inhibition caused by patients’ sera (n= 30) was significantly higher than that caused by normal sera (n= 30; P < 0.001, Wilcoxon rank sum test). Sera from a patient with LC and a healthy subject were fractionated by a Sephacryl S‐300 column and the effect of five fractions on NK activity of normal MNC was assessed. Fractions 3 and 4 from both sera had inhibitory effects although Fraction 5 revealed an enhancing effect. These results suggest that human sera contain factors which both inhibit and enhance NK activity and that their net effects are inhibition. Since it is known that IgG is eluted in Fraction 4, IgG was separated from sera with a DEAE‐Sephacel column and its effect on NK activity was examined. IgG from patients’ sera was observed to suppress NK activity more often than those from healthy donors at the same concentrations.

Immunoregulatory dysfunctions in type I diabetes: Natural and antibody-dependent cellular cytotoxic activities

Peripheral blood lymphocytes from 13 patients with established insulin-dependent diabetes mellitus (IDDM) and 2 prediabetic patients were examined for natural killer (NK) and antibody-dependent cellular cytotoxic activities (ADCC), lectin-dependent cellular cytotoxicity (LDCC), interferon- and interleukin-2-induced cytotoxicity, and concanavalin A-induced suppressor-cell activities in comparison with age-matched normal controls. IDDM patients demonstrated normal levels of NK and ADCC activities against K562 and antibody-coated SB target cells, respectively, compared to controls. IDDM patients showed normal levels of LDCC activity. Notable deviations from control values were, however, observed with diabetic lymphocytes in the following systems. Interferon- and interleukin-2-induced NK activities were significantly higher with IDDM lymphocytes than with control cells. IDDM lymphocytes precultured with concanavalin A demonstrated lower NK and ADCC activities than control cells and manifested decreased suppressor effects on the NK activity of normal allogeneic lymphocytes. Lymphocytes from one of two prediabetic patients showed increased NK, ADCC, and LDCC activities in comparison to controls. The increased interferon- and interleukin-2-induced enhancement of NK activity and reduced suppressor activity of lymphocytes from IDDM patients may be involved in the pathogenesis of the disease.

Suppression of natural killer cell activity by surgical stress in cancer patients and the underlying mechanisms.

The influence of surgical stress on the natural killer (NK) activity of peripheral blood lymphocytes in patients with carcinoma of the lung or gastrointestinal system was studied. The peripheral blood lymphocytes of the patients showed a marked decrease in NK activity against K-562 cells as target cells 1-2 days after surgery. The activity remained lowered for 2 weeks after thoractomy and for 1 week after laparotomy. No appreciable suppression of NK activity was observed with normal human peripheral blood lymphocytes preincubated with postoperative patient sera. Peripheral blood mononuclear cells obtained postoperatively from patients lost NK activity after ultraviolet irradiation, without any detectable loss of viability. Such irradiated mononuclear cells showed inhibition of NK activity after a 24-hour preincubation with peripheral blood lymphocytes from normal subjects. Similar suppressive activity was demonstrable in a fraction of mononuclear cells with adhesiveness to plastic petri dishes, while non-adherent cells had no such activity. When added immediately to the cytotoxicity assay system without the 24-hour preincubation, patient mononuclear cells caused no inhibition of NK activity, whereas adherent cells from normal subjects enhanced NK activity. The findings seems to indicate that, following surgical stress, plastic dish-adherent peripheral blood mononuclear cells become deprived of NK helper activity and exert suppression, thus causing postoperative depression of NK activity.

Beta-Endorphin augments the cytolytic activity and interferon production of natural killer cells.

We have investigated the in vitro effects of the neurohormone beta-endorphin (b-end) on natural killer (NK) activity and interferon (IFN) production mediated by large granular lymphocytes (LGL). LGL-enriched fractions from peripheral blood mononuclear cells (PBMC) from normal human volunteers were obtained by fractionation over discontinuous Percoll gradients. LGL were preincubated with or without various concentrations of b-end or the closely related peptides alpha-endorphin (a-end), gamma-endorphin (g-end), or D-ALA2-beta-endorphin (D-ALA2-b-end), a synthetic b-end analogue. NK activity was assayed on 51Cr-labeled K562 target cells. Preincubation of LGL effectors (but not K562 targets) for 2 to 18 hr with concentrations of b-end between 10(-7) M and 10(-10) M produced significant augmentation of NK cytolytic activity (mean percentage increase: 63%). The classic opiate antagonist naloxone blocked the enhancing effect when used at a 100-fold molar excess relative to b-end. Neither a-end nor g-end could augment NK activity, whereas D-ALA2-b-end produced an enhancement comparable with that produced by b-end. In addition, incubation of LGL with b-end in the presence of phytohemagglutinin or poly I:C significantly augmented IFN production. These findings demonstrate that b-end enhances NK activity and IFN production of purified LGL, and suggests that b-end might bind to an opioid receptor on LGL that can be blocked by naloxone. These results lend support to the concepts of regulation of the immune response by neurohormones and the functional relationship between the nervous and immune systems.

Enhancement of lymphocyte proliferation, interleukin-2 production and NK activity by inmunoferon (AM-3), a fungal immunomodulator: Variations in normal and immunosuppressed mice

The in vivo effect of Inmunoferon (AM-3), a glycophosphopeptide of fungal origin, has been studied on T and B lymphocyte mitogenesis, Interleukin 2 (IL-2) synthesis and natural killer (NK) activity. Inmunoferon (30 mg/kg/day) was administered to several groups of mice 2, 3 or 7 days/week for 2 weeks, and its effect assessed on day 15 of treatment. Every treatment assayed enhanced IL-2 and NK activity in the spleen. The greater effect was produced by daily administration of the immunomodulator. No enhancement was found in mitogen-induced proliferation of T and B lymphocytes. Similar treatments with Inmunoferon enhanced NK activity in the spleens of mice treated with 250 mg/kg cyclophosphamide. In addition, mitogenic responses of T lymphocytes, but not IL-2 production, were also increased in immunosuppressed mice after treatment with the immunomodulator.

Natural killer cell activity in multiple sclerosis patients treated with recombinant interferon- α2

Natural killer (NK) cell activity was evaluated in multiple sclerosis (MS) patients during a phase II trial of recombinant interferon- α 2 (IFN). Spontaneous NK activity against the K562 myeloid target cell increased significantly during the first week of treatment in the IFN treatment group. However, NK activity was also increased in the placebo treatment group. Long-term administration of IFN caused a decline of NK activity, below the pretreatment values. This decline was not paralleled by a decline in the percentage of Leu-7 cells in the peripheral blood. The ability of IFN to enhance NK activity during treatment was also evaluated. Enhancement of NK activity by IFN was depressed for the duration of the study in the IFN treatment group. After treatment was stopped, IFN enhancement of NK activity returned to the pre-study value. These studies demonstrate that spontaneous and IFN enhanced NK activity are profoundly affected by the administration of recombinant IFN- α 2 in MS patients.

Augmentation of Natural Killer Cell Activity by Anti‐Host Delayed‐Type Hypersensitivity during the Graft‐versus‐Host Reaction in Mice

During a graft-versus-host (GVH) reaction in unirradiated F1 hybrid mice there is a generalized activation of natural killer (NK) cells. We have examined whether the enhanced NK activity is due to an F1 resistance mechanism directed at the parental cells used to induce the GVH reaction. Spleen cells of C57BL/10 origin induce much more NK cell activation in B10F1 hybrids than the opposite parental type, despite a similar intensity of systemic GVH reactions. However, this does not correlate with in vivo resistance of mice with GVH reaction to a local challenge dose of B10 cells. NK cell activation in (CBA X BALB/c)F1 mice with GVH reaction involves both host and donor cells and is preceded by an anti-host delayed-type hypersensitivity (DTH) response. B10 cells have a greater ability to induce DTH in B10F1 mice than cells from the opposite parent. We propose that NK cells are one group of non-specific effector cells recruited by DTH in a GVH reaction and may contribute to the tissue pathology.

The effect of recombinant alpha 2-interferon on defective natural killer cell activity in multiple sclerosis.

Natural killer (NK) cell activity was evaluated in exacerbating/remitting MS patients. Peripheral blood mononuclear cells (MNC) from MS patients had impaired NK-cell cytotoxicity against the K562 myeloid target cell. NK activity was depressed, irrespective of the interval (2 to 13 months) after the last exacerbation. Recombinant alpha 2-interferon (100 U/ml) enhanced NK activity of both MS and control MNC. Cytotoxicity mediated by interferon-treated MS MNC was increased to the level of untreated control MNC. These studies show that MNC from exacerbating/remitting MS patients possess a defect in NK-cell activity that can be corrected in vitro by treatment with interferon.

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.

Recombinant interleukin 2 enhanced natural killer cell-mediated cytotoxicity in human lymphocyte subpopulations expressing the Leu 7 and Leu 11 antigens.

Highly purified recombinant human interleukin 2 (rIL 2) markedly augments the natural killer (NK) cell-mediated cytotoxicity of peripheral blood lymphocytes. In this study, we examined the cellular and metabolic basis of rIL 2-mediated activation of human lymphocyte subpopulations expressing the NK cell-associated surface antigens Leu 7 and Leu 11. All rIL 2-responsive cytotoxic NK cells were found within the subset of lymphocytes expressing the Leu 11 marker, an antigen associated with the Fc-IgG receptor on human NK cells. Cells lacking the Leu 11 antigen, including cells expressing another NK cell-associated marker, Leu 7, did not express NK cell-mediated cytotoxicity either before or after rIL 2 treatment. By contrast, rIL 2 augmented the NK activity of both Leu7-,11+ and Leu 7+,11+ subpopulations. Activation of Leu 11+ NK cells resulted from a direct effect of rIL 2 on these cells and neither required nor was amplified by the presence of T lymphocytes. Enhanced NK cell-mediated cytotoxicity occurred within 4 hr after exposure to rIL 2, and was blocked by the protein synthesis inhibitor cyclohexamide, but not by the DNA synthesis inhibitor mitomycin C or 1500 rad of x-irradiation. Neither Tac antigen, a high-affinity receptor for IL 2, nor other activation markers, such as transferrin receptor or HLA-DR antigen, were detectable on a significant proportion of Leu 11+ cells, either before or after incubation with rIL 2 for 48 hr. In addition, saturating concentrations of antibodies to each of these markers had no effect on the enhancement of NK activity by rIL 2. Finally, preliminary experiments with neutralizing antibodies to gamma- and alpha-interferons also failed to prevent rIL 2 enhancement of NK cell-mediated cytotoxicity, suggesting that rIL 2 does not mediate its effect via release of these cytokines.

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augumenting human natural killer (NK) cell activity. A dosedependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.

Natural killer activity in patients with acute viral hepatitis

Using the natural killer (NK) sensitive K562 cell line, enhanced NK cell cytotoxicity was demonstrable early in the course of acute hepatitis B while normal values were obtained in patients studied during convalescence. No evidence of enhanced NK activity was instead obtained in the course of acute non-A, non-B hepatitis. Serum levels of alpha-interferon, as determined by radioimmunoassay (RIA), were significantly increased in patients with acute hepatitis B showing enhanced NK cell activity but not in those with acute non-A, non-B hepatitis and normal NK cell activity. These results suggest that natural cytotoxicity may play a role early in the course of acute hepatitis type B, before antigen-specific T lymphocytes become fully operative.

Association of decreased T-cell-mediated natural cytotoxicity and interferon production in Down's Syndrome

Total peripheral blood lymphocytes (PBL) and isolated subpopulations from children with Down's Syndrome (DS) and age-matched healthy controls were investigated for their (1) natural killer (NK) and antibody-dependent cellular cytotoxic activities, (2) interleukin 2 (IL-2)-induced augmentation of NK activity, (3) lectin-dependent cellular cytotoxicity (LDCC), (4) ability of serum- and culture-derived soluble suppressor factor(s) to inhibit NK activity of normal lymphocytes, and (5) capacity to produce interferon (IFN) against tumor targets in vitro. T lymphocytes from DS patients demonstrated significantly decreased NK activity against K562 target cells compared to controls. DS lymphocytes also demonstrated a significant reduction in LDCC activity and IL-2-induced enhancement of NK activity. Furthermore, the ability of DS lymphocytes to produce IFN in vitro against K562 target cells was also significantly lower than that for normal PBL. Although sera from DS patients showed a significantly greater inhibitory effect on the NK activity of allogeneic normal PBL than normal sera, culture supernates from DS lymphocytes demonstrated suppressive effects comparable to culture supernates from normal PBL. These studies suggest an association between the decreased NK activity of T-cell subpopulations and lower IFN production by PBL from patients with DS.

Natural killer (NK) cell activity in murine muscular dystrophy: II. Age-related tissue distribution and enhanced NK activity in the thymus of dystrophic mice

The age-related tissue distribution of natural killer (NK) cell activity in murine muscular dystrophy was investigated. Lymphoid tissues including the spleen, thymus, mediastinal (or bronchial) lymph nodes (BLN), mesenteric lymph nodes (MLN), inguinal/popliteal lymph nodes (PLN1), and axillary/brachial lymph nodes (PLN2) were obtained from various aged normal (+/+) and dystrophic (dy 2J/dy 2J) C57BL/6J mice. Cell suspensions were incubated with 51Cr-labeled YAC-1 lymphoma target cells in a 4-hr 51Cr-release assay. The data indicated that dystrophic mice, at all ages studied, had elevated levels of NK activity in the spleen, BLN, MLN, PLN1, and PLN2 as compared with the normal age- and sex-matched control group. The NK activity in the thymocyte population from dystrophic mice at 2 weeks of age was found to be negligible, while at 8 weeks of age it was two-fold higher than that for the normal control group. In addition, dystrophic mice had an age-related decline in NK activity in all tissues after 10 weeks of age but the activity was still elevated at 40 weeks of age as compared with the normal control group. Target cell binding studies revealed that the number of conjugate-forming cells in thymocytes from 8-week-old dystrophic mice were found to be significantly higher than that found in normal mouse thymocytes using NK-sensitive YAC-1 tumor target cells. The number of cells bound per YAC-1 target cell ranged from 1 to 3 for dystrophic mouse thymocytes as compared with 1 to 2 for the normal control group. Thus, the data indicate an elevated NK activity in all lymphoid tissues studied from dystrophic mice of different ages. In addition, the thymus from dystrophic mice at 8 weeks of age contains an enhanced number of conjugate-forming NK cells and NK activity.

Natural killer cells are present in the normal human lung but are functionally impotent.

The lung is affected by disorders in which natural killer (NK) cells are thought to play an important defensive role. This study, however, demonstrated that normal lung lymphocytes actually express very little NK cell activity (P less than 0.001 compared with blood lymphocytes). This was true independent of the NK-sensitive target used (K562, U937, MOLT-3, or Daudi). This lack of lung lymphocyte NK activity occurred even though the proportions of lymphocytes in the normal lower respiratory tract with the morphology (large granular lymphocytes) and surface antigen markers of NK cells were similar to that of blood (P greater than 0.5). Although normal lung lymphocytes bound to known NK-sensitive targets, they did not lyse these cells (P less than 0.001 compared with blood), which suggested that the lack of lung NK cell activity resulted from a relative inability of lung NK cells to destroy their targets. While the mechanisms of this functional impotence of lung NK cells are not clear, normal human alveolar macrophages and lower respiratory tract epithelial lining fluid exerted a profound suppressive effect on blood NK cell activity (P less than 0.001 for both) by inhibiting their ability to lyse target cells after binding (P less than 0.001). Though impotent initially, when incubated for 24 h in medium alone, normal lung lymphocytes demonstrated markedly enhanced NK activity (P less than 0.02), which suggested that lung NK cells do have the potential to express NK activity. Interleukin-2 (IL-2) further augmented this effect (P less than 0.05), but gamma interferon did not (P greater than 0.2). Consistent with this observation, lung lymphocytes from patients with active sarcoidosis, a disease in which lung lymphocytes are spontaneously releasing IL-2, did express NK cell activity (P less than 0.01). These studies suggest that although NK cells are present in the normal lung, they are functionally inactive, due, at least in part, to local inhibitory influences. In the presence of IL-2, however, lung NK cell activity is expressed, which suggests that lung NK cell activity can be modulated.

Plasmodium berghei sporozoites are mitogenic for murine T cells, induce interferon, and activate natural killer cells.

Enhanced natural killer (NK) activity was detected in the spleens of mice as early as 24 hr after single i.v. inoculation with gamma-irradiated Plasmodium berghei sporozoites. The activity peaked at 48 hr post-injection, and declined below baseline level by day 8. Reinoculation of mice with irradiated sporozoites produced an increased NK activity significantly smaller than the original activity. Spleen cells sensitized in vivo as well as nonsensitized spleen cells stimulated in vitro with sporozoites produced high levels of interferon (IFN) and displayed enhanced NK activity. Characterization of the IFN through the use of specific antibodies revealed that it was mainly IFN-gamma. The cellular basis for IFN-gamma induction was linked to the mitogenicity of P. berghei sporozoites for T cells. The possibility exists that IFN-gamma may have a regulatory effect on antibody production against P. berghei sporozoites.

Enhancement of human natural killer cell activity by subcellular components of Toxoplasma gondii

The ability of sonicates and subcellular fractions of the intracellular parasite Toxoplasma gondii to enhance in vitro human natural killer (NK) cell activity was examined. Incubation of nylon-wool-non-adherent human peripheral blood lymphocytes (PBL) with sonicates of T. gondii for 18–72 hr resulted in increased NK activity against an NK-sensitive, as well as an insensitive, target cell. Single-cell assays revealed that augmentation of NK activity was not due to an increased binding of K562 target cells to effector cells. Differential centrifugation studies indicated that NK-augmenting activity was distributed in membrane-enriched and cytoplasmic fractions. This activity was found to be resistant to treatment with ribonuclease (RNase) and deoxyribonuclease (DNase), but susceptible to proteolysis. Antibodies present in the serum of humans infected with Toxoplasma blocked the NK cell-augmenting effect of the membrane-enriched fractions. Enhancement of NK activity by PBL incubated with Toxoplasma sonicate was accompanied by a concomitant increase in interferon (IFN), but not of interleukin 2 (IL-2), levels in supernatants of the cell cultures.

Neuroimmunomodulation with enkephalins: Enhancement of human natural killer (NK) cell activity in vitro

To further define the effects of enkephalins on immune function, the effect of methionine-enkephalin and leucine-enkephalin on natural killer cell (NK) activity in isolated human peripheral blood lymphocytes was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in natural killer cell activity. At effector:target cell ratios of 11:1 methionine-enkephalin significantly ( P < 0.05) enhanced NK activity at dilutions of 10 −6, 10 −8, 10 −10, and 10 −14 mg/ml, while leucine-enkephalin significantly ( P < 0.05) enhanced NK activity at dilutions of 10 −4, 10 −6, 10 −8, 10 −10, and 10 −14 mg/ml. Cells from individuals with low NK activity showed greater percentage increases in NK activity following enkephalin than did cells from individuals with high NK activity.

Modulation of natural killer activity in mice following infection with Listeria monocytogenes

Mice that received a sublethal, intraperitoneal dose of viable Listeria monocytogenes, virulent strain 10403, exhibited a systemic increase in natural killer (NK) activity. The kinetics of the response differed with respect to the various effector cell populations analyzed. Resident peritoneal cells and peripheral blood leukocytes demonstrated high NK activity on Days 3, 7, and 10. Peak spleen and bone marrow NK activity was observed on Day 3, returning to normal levels by Day 7. In contrast, peritoneal exudate cells, elicited with proteose peptone, expressed enhanced NK activity for 60 days following infection with viable Listeria. Augmented NK activity was detected with all cell types as early as 12 hr after infection. The intraperitoneal injection of nonviable antigenic preparations derived from L. monocytogenes, strain 10403, resulted in the enhancement of peritoneal and splenic NK activity. In contrast, mice that received an intraperitoneal injection of avirulent Listeria, strain 19113, failed to express enhanced levels of NK activity. The genetic trait of anti-listerial resistance which is associated with non-H-2 linked genes was of no importance with respect to enhanced NK activity. Listeria-resistant C57BL/6J and Listeria-susceptible DBA/2J mice both produced systemic augmentation of NK activity following infection. NK activity was not abrogated by macrophage depletion or by treatment with anti-Thy 1.2 serum plus complement. These results confirm the potent immunostimulatory capacity of virulent Listeria for NK activity and provide further insight into the kinetics of this response in various lymphoid compartments. The protracted augmentation of NK activity of elicited peritoneal exudate cells as compared to nonelicited peritoneal cells in Listeria-primed mice suggests that the influx of inflammatory cells may provide NK-enriched and/or accessory populations for immunopotentiation of NK activity in inflammatory sites.

Effect of low dosage of interferon on natural killer activity in patients with HBsAg-positive chronic active hepatitis.

The effect of human leukocyte interferon (IFN) on natural killer (NK) activity in 7 patients with HBsAg-positive chronic active hepatitis was investigated. Human leukocyte IFN was intramuscularly given once a week for 4 consecutive weeks (10 X 10(5), 5 X 10(5), 2 X 10(5) and 1 X 10(5) U). 1 week after the initial injection, NK activity showed a significant rise compared with the preinjection levels (p less than 0.001) and remained elevated during the 1-month treatment, while it fell after the cessation of IFN therapy. 4 of 5 patients showed a marked decrease in Dane particle-associated DNA-polymerase activity. Serum HBeAg disappeared in 3 of 6 patients and serum transaminase levels markedly improved in all patients. Only a transient reduction in HBsAg was observed in 2 patients after the treatment period. The findings indicate that a low dosage of human leukocyte IFN such as 18 X 10(5) U, administered in decreasing doses, enhances NK activity in patients with HBsAg-positive chronic active hepatitis, and that the effect of IFN is involved in the immune mechanisms associated with natural cytotoxicity.