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.     

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 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.     

Inhibition of human natural killer cell activity by platelet-derived growth factor

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.     

Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid

The effect of eicosapentaenoic acid (EPA) on natural killer (NK) cell activity of human lymphocytes was examined. The addition of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG) emulsified with purified phosphatidylcholine from krill to a cytotoxicity assay system resulted in a marked depression of NK activity. The inhibition was proportional to the concentration of EPA-TG emulsion, and was observed as early as the first one hour of incubation at various effector to target cell ratios. Pretreatment of effector cells with EPA-TG emulsion resulted in significant suppression of their NK activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells or decreased target cell binding. These results indicate that EPA is a potent inhibitor of NK activity in vitro.     

Inhibition of human natural killer activity by monolayers of primary cell cultures

Some primary and continuous cell cultures were tested for their capacity to regulate human natural killer (NK) activity. Primary cultures of endothelial cells, fetal fibroblasts, adult fibroblasts, amnion epithelial cells, renal parenchymal cells, and ovarian carcinoma cells inhibited NK activity when peripheral blood lymphocytes were preincubated on target cell monolayers for 18 h before testing the cytotoxicity against K-562. The supernatants of the inhibiting cell cultures were not suppressive. Prostaglandins or suppressive lymphocytes were not involved in the phenomenon. The binding capacity of the effector cells was not changed, suggesting that the suppressive signal was targeted at the cytolytic machinery of NK cells. The down-regulating capacity of the cell cultures weakened significantly during subculturing in vitro, and continuous cell lines were not inhibitory. The inactivation of NK cells may be one of the mechanisms by which target cells are protected from NK activity.     

Inhibition by cortisol of human natural killer (NK) cell activity

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10(-8) to 1 X 10(-5) M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-gamma). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 (PgE2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.     

Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

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.     

Inhibition of normal human natural killer cell activity by human immunodeficiency virus synthetic transmembrane peptides

The inhibitory effect on normal natural killer (NK) cell activity of two synthetic peptides corresponding to amino acid sequences 735-752 and 846-860, respectively, as deduced from the amino acid sequences of HTLV-IIIB gp160, was assessed. Sequences 735-752 and 846-860 correspond to regions located within the HIV transmembrane gp41, the carboxy terminus of HIV gp160. These two synthetic peptides have been shown previously to suppress the mitogen- and alloantigen-induced normal human lymphocyte blastogenic responses. Peptides 735-752 and 846-860 conjugated to protein carriers exerted a significant inhibition on the normal NK cell activity assayed against K562 tumor target cells in an in vitro 51Cr-release cytoltoxicity assay. At variance, control peptides similarly conjugated had no effect on NK activity. Addition of exogenous recombinant human interleukin-2 (IL-2) resulted in a partial restoration of the suppression of NK cell activity exerted by both peptides. Binding experiments indicated that peptides 735-752 and 846-860 did not affect the formation of effector cell-target cell conjugates, suggesting inhibitory effect(s) subsequent to the formation of the lytic complex as one potential mechanism of the observed NK suppression. These results suggest that peptides 735-752 and 846-860 homologous to sequences within the HIV transmembrane gp41 may play an important role in the pathogenesis of the defective NK cell activity observed in patients with acquired immunodeficiency syndrome (AIDS).     

Suppression by cannabinoids of a cloned cell line with natural killer cell activity

Preincubation of a cloned cell line with natural killer (NK) cell activity, as well as splenic mononuclear cells with either delta 9-tetrahydrocannabinol (THC) or 11-hydroxy-delta 9-tetrahydrocannabinol (11-OH-THC) suppressed NK cytolytic activity against YAC-1 target cells in a dose-dependent manner. THC was more inhibitory for cloned cells than 11-OH-THC and suppressed the lytic activity of these cells without reducing cell viability in the concentration range of 5 micrograms/ml (16 microM) to 10 micrograms/ml (32 microM). THC also inhibited proliferation of cloned NK cells, but this inhibitory effect was reversible in that extensive washing of cells following cannabinoid pretreatment eliminated the suppressive effect. Single-cell analysis revealed that THC did not inhibit the binding of cloned NK cells to target cells and further showed that NK cells freshly isolated from mouse spleen were restricted in killing capacity following binding to target cells. Therefore, THC and 11-OH-THC appear to directly inhibit NK cell cytolytic activity in a postbinding stage.     

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.     

Cytolytic activity of human natural killer cell subpopulations isolated by four-color immunofluorescence flow cytometric cell sorting

The natural killer activity and phenotypic properties of six different subpopulations of normal human peripheral blood lymphocytes obtained by four-color immunofluorescence cell sorting were examined. Phycoerythrin-conjugated CD16 and CD56 were used simultaneously to identify (CD16 + CD56+) NK cells. The cells most effective in mediating NK cytolysis against K562 target cells were CD3-(16 + 56+)57 -8+. Although most of the K562 killing was found in the CD3-(16 + 56 +) groups of cells, a substantial degree of NK activity was detected in the CD3+(16 + 56 +) subpopulations of some individuals. The level of expression of CD57 and CD8 was significantly higher on CD3+(16 + 56 +) than on CD3-(16 + 56 +) cells.     

Recombinant interleukin 2 rapidly augments human natural killer cell activity

Recombinant human interleukin 2 (r-IL-2) rapidly stimulated human natural killer cell activity in vitro. Augmentation of NK activity occurred within 1 hr of preincubation with r-IL-2. Responsive killer cells were typical NK cells as shown by cell fractionation procedures. These included Percoll density gradient separation and depletion of OKT3+ T cells by an indirect rosetting method. Analysis with a panel of polyclonal and monoclonal antibodies against alpha and gamma interferon revealed that this early enhancement of NK activity by r-IL-2 was independent of the production of both types of interferon.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.