大豆异黄酮体外对大鼠淋巴细胞激活和天然杀伤细胞活性的作用(英文)

目的 :研究大豆异黄酮对大鼠免疫细胞的体外作用。方法 :取大鼠抗凝血 ,密度梯度离心法分离外周血单个核细胞 ,以伴刀豆球蛋白A(ConA)和体内所能达到的不同浓度的金雀异黄素或大豆苷元共同孵育 6 8h后 ,四唑蓝还原法 (MTT)测定体外ConA诱导的淋巴细胞增殖 ;另取外周血单核细胞为效应细胞 ,YAC 1细胞为靶细胞 ,以乳酸脱氢酶法测定天然杀伤细胞 (NK )活性。结果 :在 0 .0 1～ 1μmol·L- 1浓度范围内 ,大豆苷元可剂量依赖性的增强体外ConA诱导的淋巴细胞增殖 (P <0 .0 1) ,1μmol·L- 1的大豆苷元可明显增强NK细胞活性 (P<0 .0 1)。而同样浓度范围内的金雀异黄素对淋巴细胞增殖及NK细胞活性均无明显作用。结论 :大豆苷元体外能剂量依赖性的刺激ConA诱导的淋巴细胞转化 ,增强NK细胞活性 ,这种增强的免疫功能可能是大豆制品抗肿瘤作用的机制之一     

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.     

Inhibition of natural killer cell function by marijuana components

The extent of modulation of host resistance mechanisms by marijuana components is not fully understood. Natural killer (NK) cells are a subpopulation of lymphoid cells and are important in host resistance mechanisms against malignant cells, virus-infected cells, and possibly pathogenic bacteria and fungi. We report that the marijuana component delta-9-tetrahydrocannabinol (THC) injected into mice results in a suppression of splenic NK activity. Furthermore, THC and the hydroxylated metabolite 11-hydroxy-delta-9-tetrahydrocannabinol (11-hydroxy-THC) suppress the NK activity of cultured murine splenocytes in a dose-dependent manner (range 1 X 10(-5) to 3.2 X 10(-5) M) without diminishing NK cell viability. The hydroxylated derivative appears to possess a more potent suppressive effect, in that it suppresses at lower concentrations than THC does and requires a shorter incubation time with the effector cells for its suppressive action. Purification of NK cells by Percoll density-gradient centrifugation suggests that both cannabinoids act directly on the natural killer cell population, resulting in suppression. Studies involving target binding analysis and calcium ionophore experiments suggest that cannabinoids do not suppress NK cell killing by the inhibition of effector/target binding or by disruption of calcium ion flux. These results suggest that two principal psychoactive cannabinoids can suppress natural killer cell function by interacting directly with the killer cells and disrupting cellular events postbinding and during the programming for lysis. Furthermore, the data suggest different modes of action for THC and the hydroxylated metabolite.     

Ethanol activation of human natural cytotoxicity

Human lymphocytes cultured with ethanol and subsequently assayed for natural killer (NK) activity to K562 cells have enhanced NK activity compared to lymphocytes cultured without exposure to ethanol. Optimal enhancement occurred at 0.64% (v/v) ethanol, and required several hours of culture. Lymphocytes retained their enhanced cytolytic ability for several hours after removal from the ethanol-containing medium. The enhancement correlated with a faster rate of cytolysis by ethanol-treated lymphocytes, rather than recruitment of an increased number of killer cells, as measured with single cell assays. Inclusion of ethanol directly in the NK assays was inhibitory. Cells that had been cultured with ethanol were less sensitive to inhibition of NK activity by the proteinase substrate acetyl tyrosine ethyl ester than were control cells cultured without ethanol. Although this observation and the increased rate of cytolysis in the single cell assays are consistent with increased production of a chymotrypsin-like proteinase involved in cell-mediated cytotoxicity, no alteration in protein synthesis was detected concomitant with ethanol treatment. This report demonstrates that even without hepatic metabolism, ethanol can produce effects on lymphocyte function which remain after exposure to the reagent is discontinued.     

Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes by the streptococcal preparation OK432

In vitro overnight exposure to the streptococcal preparation OK432 of blood and tumor-associated lymphocytes enhanced their lytic activity against autologous, freshly isolated tumor cells from malignant pleural effusions of cancer patients. This enhancement was mediated through factors that were distinct from interferon (IFN) and interleukin 2 (IL 2). Treatment with IFN-alpha, -beta, or -gamma, or IL 2 failed to augment autologous tumor killing (ATK) activity, although the treatment enhanced natural killer (NK) activity. Intrapleural (i.pl.) injection of OK432 induced or enhanced ATK and NK activities of effusion large granular lymphocytes (LGL) in patients who showed a reduction or disappearance of effusion tumor cells. No such increase in ATK and NK activities was seen with effusion LGL of patients who had no clinical benefit from i.pl. OK432 therapy. Blood ATK and NK activities were not consistently modified by the therapy. These results indicate that i.pl. administration of OK432 induces an augmentation of ATK activity of effusion LGL, which may be involved in the elimination of effusion tumor cells. The data also suggest that the host immune defense against tumor may be better represented by tumor-associated lymphocytes than by blood lymphocytes, and that it is important to examine the effect of biological response modifiers on ATK activity of tumor-associated effector cells.     

Marijuana components suppress induction and cytolytic function of murine cytotoxic T cells in vitro and in vivo.

Killer lymphocytes play a major role in host defense against tumors and infectious diseases. Previously, we reported that delta-9-tetrahydrocannabinol (THC) and II-hydroxy-delta-9-tetrahydrocannabinol (II-hydroxy-THC) suppressed the cytolytic activity of cultured natural killer (NK) cells. Also, we showed that the drugs appeared to be affecting a stage in the killing process subsequent to the binding of the killer cell to the target cell. In the present report, we have extended these studies to an examination of the effect of cannabinoids on the activity of cytotoxic T lymphocytes (CTLs). The cytolytic activity of CTLs generated by cocultivation with either allospecific stimulators or TNP-modified-self stimulators were suppressed by both THC and II-hydroxy-THC treatment. Allospecific CTLs generated in vivo were also inhibited by an in vitro exposure to either THC or II-hydroxy-THC, and the sensitivity of these cells to drug effects appeared to be greater than the sensitivity of the in vitro generated CTLs. Suppression of cytolytic function by THC and II-hydroxy-THC was maximal after a 4-h drug treatment, suggesting that the drug effects were inducible and therefore required a finite period of time to develop maximally. As seen in previous studies involving NK cells, drug treatment of mature CTLs appears to have little effect on the binding capacity of these cells for the target. However, the maximal killing capacity of the cells and the frequency of CTLs were significantly reduced by drug treatment. In addition to suppressing the cytolytic activity of mature effector CTLs, we also show that drug treatment inhibits both the proliferation of lymphocytes responding to an allogeneic stimulus and the maturation of these lymphocytes to mature CTLs. Similarly, CTL activity developing in vivo could be inhibited by THC injection. These results suggest that CTLs are inhibited by cannabinoids by at least two mechanisms. First, the cytolytic activity of mature killers is suppressed at some point beyond the binding to the target cell. Second, the cannabinoids appear to suppress the normal development of these mature effector cells from less mature precursor cells.     

Augmentation of murine natural killer cell activity by manganese chloride

Natural killer (NK) cell activity of spleen cells from male CBA/J mice was augmented by a single parenteral injection of MnCl2 administered 1 day prior to testing by in vitro and in vivo isotope release assays. Increased cytotoxic activity was observed in vitro against both NK-sensitive (YAC-1) and NK-resistant (EL-4) target cells. NK activity was also enhanced in normally low (A/J) and intermediately (C57Bl/6J) reactive strains of mice. Augmentation of NK activity by MnCl2 was accompanied by an increase in circulating interferon (IFN) levels.     

Species-specific effect of proteins secreted by cultured pre-pubertal rat Sertoli cells on natural killer cell activity.

The effect of proteins secreted by cultured pre-pubertal rat Sertoli cells (pSCP) on natural killer (NK) cell activity of rat, mice and guinea pig splenocytes and human peripheral blood lymphocytes was estimated. The results indicate that pSCP inhibited, in a dose-dependent manner, NK cell activity of mice, guinea pig and human lymphocytes but did not suppress lysis of YAC-1 target cells by rat NK cells. Species-specific differences in the effect of pSCP on NK cell activity probably result from differences in the binding of proteins within the effector cells. These data indicate that in the pre-pubertal period of gonadal development immature Sertoli cells synthesize a factor/s which might contribute to the maintenance of specific testis immunological environment.     

Immunoregulation of natural and lymphokine-activated killer cells by selenium

The effect of selenium (Se) on natural killer (NK) and lymphokine-activated killer (LAK) cell activities and proliferative responses of human lymphocytes was studied in vitro. Direct addition of Se at 1.0 microgram/ml final concentration to the mixture of target and effector cells during a 4 h cytotoxicity assay significantly suppressed the NK activity of normal lymphocytes. When lymphocytes were preincubated with Se at concentrations as low as 0.2 microgram/ml for a period of 48 h, a significant inhibitory effect on NK activity was observed. In the LAK cell assay, direct addition of Se at concentrations of 0.2-1.0 microgram/ml to a mixture of target and effector cells did not show any effects on LAK cell activity, whereas LAK cells generated in the presence of Se at 0.8 microgram/ml showed significant inhibition of their functions. Lymphocyte proliferative responses to T cell mitogens such as phytohemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed by direct addition of Se at 0.5-1.0 microgram/ml. The inhibitory effect of Se was not due to nonspecific toxicity of effector cells as demonstrated by viability nor was the effect directed against target cells. These studies suggest that although Se is an essential micronutrient for various immune mechanisms, an excess of Se may have a deleterious effect on certain immunological functions. As these activities are considered to be important defense mechanisms against tumors and virus infections, a nutritional imbalance of Se could result in an increased risk of these disorders.     

In vitro immunosuppressive potency of deflazacort, a new bone-sparing corticosteroid on T lymphocytes, NK and K cells.

The in vitro immunosuppressive effect of deflazacort, a new bone-sparing glucocorticoid, and its biologically active metabolite, 21-deacetyl-deflazacort, was examined on phytohaemagglutinin (PHA) stimulated human peripheral blood lymphocytes (PBL) as well as on natural killer (NK) and killer (K) cell activity. Deflazacort and the 21-deacetyl metabolite were as potent as prednisolone and hydrocortisone in suppressing PHA stimulated lymphocytes in a dose dependent way, but all were less potent than methylprednisolone. The physiological metabolites of hydrocortisone, dihydrocortisol and tetrahydrocortisol were without any immunosuppressive effects in vitro. Deflazacort, 21-deacetyl-deflazacort, and methylprednisolone suppressed NK cell activity, while hydrocortisone and aldosterone had no effect on NK cells. K cell activity was resistent to all tested glucocorticoids except methylprednisolone at high concentrations. The present results indicate that deflazacort and 21-deacetyl-deflazacort are potent immunosuppressive drugs in vitro and, on a molar basis, equally as potent as prednisolone.     

Swainsonine,a glycosylation inhibitor,enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity

Swainsonine (SW) inhibits the formation of N-linked complex oligosaccharides and has previously been shown to inhibit experimental metastasis in nude mice models. The present studies with human effector cells have shown that SW enhanced both lymphokine activated killer cell (LAK) and natural killer (NK) cytotoxicity in standard ⁵¹Cr-release assays. SW also increased the susceptibility of human K562 and Colo 320 target cells to NK and LAK cytotoxicity. The peak response of both LAK effectors and targets to SW occurred at 1–2 μg/ml SW. A novel finding was that SW enhanced the interleukin 2 (IL-2) β chain receptor subunit expression on both LAK and NK cells to a greater extent than its enhancement of the IL-2Rα (CD25 or TAC) receptor expression on LAK effectors. In addition, increases in both these receptors occurred at the doses of SW which augmented LAK cytotoxicity. We conclude that the anti-metastatic effects of SW have an immunological component which is maximal at 1–2 μg/ml SW. This suggests that dosage may be an important consideration to obtain optimal potential of SW in any future human cancer therapy.     

New cancer immunotherapy using autologous lymphocytes activated with trastuzumab

It is well known that trastuzumab (TTZ) is molecular target drug for breast cancer overexpressing human epidermal growth factor receptor 2 (HER2). Novel immunotherapy by human peripheral blood mononuclear cells (PBMCs) activated with TTZ were examined. Proliferation of lymphocytes after adding of TTZ was obtained. Furthermore, lymphocytes activated with TTZ inhibited growth of breast cancer cells in vitro. It is noteworthy that remarkably high cellular cytotoxicity in lymphocytes activated with TTZ compared with that of CD3- and lymphokine (interleukin (IL)2)-activated killer (CD3-LAK) cells commonly used in immunotherapy were revealed.     

Inhibition of cytotoxicity by sulfasalazine. II. Sulfasalazine and sulfapyridine inhibit different stages of the NK and NKCF lytic processes

Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibit spontaneous cytotoxicity mediated by human natural killer (NK) cells. The aim of this study was to determine which stage(s) of the NK cytotoxic reaction is inhibited by these compounds. Effector/target cell binding studies performed in parallel with cytotoxicity assays using purified large granular lymphocytes indicated that inhibition is a post-binding event. The kinetic profile of inhibition in a calcium pulse assay showed that inhibition continues long after the effector cell triggering stage and that although sulfasalazine may have some inhibitory effect on the calcium-dependent events of the programming phase, sulfapyridine continues to inhibit during the calcium-independent or lethal hit phase of the cytotoxic sequence. The NK soluble cytotoxic factor (NKCF) assay was used as a measure of the lethal hit since the time course of this assay permits study of the various substages of this terminal event in the lytic sequence. Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibited NKCF-mediated target cell lysis. Different substages of the NKCF-induced lytic reaction were affected by these agents. Sulfasalazine appears to inhibit binding of NKCF to the target cell whereas sulfapyridine predominantly inhibits early post-binding events.     

Effects of cadmium on cytotoxic functions of human natural killer cells

The effect of cadmium (Cd²⁺) was studied on natural killer (NK) cell-mediated cytotoxicity as well as on antibody-dependent cellular cytotoxicity (ADCC) of human peripheral blood lymphocytes. The results show that cadmium chloride inhibits human NK and ADCC activities against K562 and Ab-coated P815 target cells in a dose- and time-dependent manner. Maximal inhibition (80–90%) was observed in the presence of 100 μ -cadmium chloride, and it does not appear to be due to toxic effects on effector cells. Purification of large granular lymphocytes by Percoll density gradient centrifugation suggests that Cd²⁺ acts directly on this cell population. No effects of Cd²⁺ could be found after pre-exposure of either effector or target cells separately. The inhibitory effect of Cd²⁺ was manifested only when effector and target cells were present simultaneously. The greatest inhibition of NK and ADCC activities occurred when cadmium chloride was added to the assay within the first 60 min, suggesting that an early event was affected by Cd²⁺. However, Cd²⁺ did not block effector-target conjugate formation or affect leucocyte function associated Ag-1 expression on effector cells. This indicates that an initial triggering of effector cells by target cells was required before Cd²⁺ could exert its effect. Cd²⁺-induced inhibition of cytotoxic functions of NK cells could be only partially prevented by increasing the external Ca²⁺ concentration or by adding Zn²⁺ to the culture medium.     

The effects of triodothyronine and thymulin on avian NK cytolytic activity

The effects of triiodothyronine (T3), thymulin, and recombinant chicken interferon-y (ChIFN-gamma) on natural killer (NK) cell cytotoxicity was investigated using the euthyroid control K and the T3-deficient sex-linked dwarf (SLD) chicken strains. Factorial design experiments were used to investigate the effects of T3 treatments where animals of both strains received either 0 or 0.1 ppm T3 supplementation to the standard chick starter diet. The ChIFN-gamma treatments were administered in vitro by incubation with effector cells overnight prior to the addition of the RP9 lymphoblastoma target cell line. All cytotoxicity assays were run at 50:1 and 25:1 effector/target (E/T) ratios. Treatments were begun at hatching and continued through 7 weeks. NK cells for these assays were enriched by separation of splenocytes over ficoll. Splenocyte preparations from untreated K strain consistently had significantly higher NK-mediated cytolysis than did samples from the untreated SLD at both E/T ratios. T3 treatment alone had no effect on NK activity in cell preparations from the K strain but did significantly enhance that activity in the T3-deficient SLD whereas IFN treatment alone enhanced NK activity in both strains. The combined T3 and IFN treatments resulted in a greater enhancement of NK cytolytic activity in both strains than any separate treatment and resulted in an elimination of differences in NK cell responsiveness between the K and SLD strains. Similar results were obtained when NK cell cultures were incubated in vitro with thymulin prior to assessing cytotoxicity. In vitro thymulin treatments alone significantly enhanced cytolytic activity for NK cells for both K and SLD strains. The greatest effect of in vitro thymulin exposure was to increase the responsiveness to NK cells to ChIFN-gamma stimulation.     

Natural killer cells and nitric oxide

Natural killer (NK) cells and nitric oxide (NO) are both important components of the natural or innate immune response. NK cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by MHC antigens. They are abundant in blood, spleen, liver and lungs and are distinct from both T and B lymphocytes in their circulation patterns, profile of surface antigens, receptor repertoire and the way in which they discriminate between self and non-self. Uniquely, NK cells express receptors that can recognize and discriminate between normal and altered MHC class I determinants. NK cell cytotoxic activity is strongly induced by cytokines such as IL-2 and IL-12, and this activation is associated with synthesis of NO. Inhibitors of NO synthesis impair NK cell-mediated target cell killing, demonstrating a role for NO in NK cell function. Furthermore, NO itself can regulate NK cell activation. In this article, evidence that NO is a mediator of NK cell-mediated target cell killing, and that NO is a regulator of NK cell activation will be reviewed. Results of NO synthase gene deletion studies will be discussed, and rodent and human NK cells will be compared.     

Effect of cianidanol on natural killer cell activity in patients with chronic B virus hepatitis

The hepatoprotective antioxidant bioflavonoid cianidanol has beneficial therapeutic and immunomodulatory effects in chronic hepatitis. Its action on natural killer (NK) cell activity has not yet been studied in hepatitis B virus (HBV) infection. In the present study, the in vitro and in vivo effects of the drug on NK cell activity have been determined in six patients with chronic HBV hepatitis and in ten healthy control subjects. Two methods were used: an enzyme release assay and a cytotoxicity test based on the assessment of endogenous alkaline phosphatase activity of the target cells. The in vitro effect of the drug was assessed using cianidanol at 10(-6), 10(-5) and 10(-4) M concentrations. For in vivo studies, HBV hepatitis patients were treated with cianidanol at a daily dose of 3.0 g cianidanol for seven days and were investigated before and after the treatment. Chronic HBV hepatitis patients showed a moderate decrease in NK cell activity compared to the controls, but after the cianidanol therapy their NK cell activity significantly rose to 68.0% +/- 9.5% (p less than 0.01). Cianidanol in vitro inhibited the NK cell activity both in hepatitis and healthy groups when using K-562 target cells and the lactic acid dehydrogenase enzyme release assay, but did not influence or even slightly enhance the NK activity when human embryonic fibroblast cells and alkaline phosphatase assay were used for the test. After the 7-day in vivo treatment, the in vitro inhibitory action of the drug was diminished or absent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations induced in vitro by ochratoxin A in rat lymphoid cells

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by species of the genus Penicillium and Aspergillus that is present in food and feed as a natural contaminant. It modifies the immune function in animals and inhibits the proliferative response of lymphocytes in vitro. The toxic effect of OTA (0.5, 2, 20 microM) in lympho-proliferative response, natural killer (NK) cell activity, cytotoxic T lymphocytes (CTL) activity and macrophages' bacteriolytic capability was studied in vitro after 1 hour of treatment. The proliferative response of lymphocytes to concanavalin A and lipopolysaccharide was not affected by OTA; the cytotoxic activity of NK cells was dose-dependent decreased; the CTL activity was significantly decreased at the lowest concentration; the bacteriolytic activity of macrophages varied only slightly. These in vitro results reproduced, at least in part, some effects detected previously in vivo. The protein synthesis inhibition and the oxidative metabolism of OTA coupled to the prostaglandin synthesis are probably implicated in NK cells' toxicity, because the effects were reverted by the addition of phenylalanine or piroxicam to the culture medium. The induction of apoptosis seems to be the principal mechanism of action in the CTL effect. The intracellular concentration of OTA after 1 hour was analysed by HPLC and was found to be proportional to the quantity of OTA added to the culture medium for the three cell types; the presence of phenylalanine and piroxicam on the culture medium did not change the intracellular OTA concentration.