Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides

Organochlorine pesticides are used worldwide. To our knowledge there have been no studies dealing with the effects of these agents under in vitro conditions on human natural killer (NK) cell cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of organochlorine pesticides and some of their breakdown products on the ability of human NK cells to lyse tumor cells. A total of 11 compounds were tested. The compounds were tested in both purified NK cells as well as a cell preparation that contained other mononuclear cells (predominantly T cells) and NK lymphocytes (referred to as T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 hour to 6 days. Exposure of highly purified NK cells to 5 microM alpha-chlordane, gamma-chlordane, 4,4'-DDT, heptachlor, oxychlordane, or pentachlorophenol (PCP) inhibited their ability to destroy K562 tumor-cells by 88+/-5, 92+/-8, 61+/-13%, 64+/-10%, 69+/-11%, 76+/-12%, respectively, after a 24 h exposure. The loss of cytotoxic function seen with alpha-and gamma-chlordane remained essentially constant out to 6 days, while that seen with 4,4'-DDT, oxychordane and PCP increased with longer exposures (6 d). PCP was the most effective of the compounds tested at decreasing NK function. Of the compounds that caused decreased lytic function when tested in purified NK cells, only PCP and oxychordane decreased the lytic function of the T/NK cell preparation after any exposure. The results provide evidence of relative toxic potential for the 11 compounds and their immunomodulatory effects on other mononuclear cells (such as T-cells, B-cells, and monocytes) as well as NK lymphocyte function.     

Effects of D-Fraction,a polysaccharide from Grifola frondosa on tumor growth involve activation of NK cells

Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we examined tumor volume and levels of IFN-gamma and TNF-alpha in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases in TNF-alpha and IFN-gamma released from spleen cells and a significant increase in TNF-alpha expressed in NK cells. This suggests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction increased macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from macrophages.     

Effects of manganese, calcium, magnesium, and zinc on nickel-induced suppression of murine natural killer cell activity

The effects that divalent metals have on nickel-induced suppression of natural killer (NK) cell activity were studied in mice. Male CBA/J mice were given a single intramuscular injection of metal salt on a body weight basis. The metal doses used were the following: nickel chloride, 4.5-36 micrograms/g; manganese chloride, 20-80 micrograms/g. Twenty-four hours after metal injection, splenic NK cell activity was assessed using a 51Cr-release assay. Ni significantly (p less than 0.01) suppressed NK activity, while Mn significantly (p less than 0.01) enhanced NK activity. No alteration in NK activity was observed in mice injected with Mg, Ca, or Zn. Since these divalent metals have been shown to have antagonistic effects on Ni-induced carcinogenicity and toxicity, they were used in combination with Ni to determine if such antagonisms exist for NK cell activity. The injection of Ni and Mn in combination at a single site resulted in the enhancement of NK activity, although this enhancement was at a level below that observed following the injection of Mn alone. Injection of Mg, Zn, or Ca in combination with Ni did not affect NK activity compared to saline controls. In contrast, the injection of Ni in one thigh followed immediately by Mn, Mg, Ca, or Zn into the other thigh resulted in significant suppression of NK activity for all metals compared with saline controls. An interesting finding was that the injection of Ni followed immediately by Mn into the opposite thigh resulted in even greater reductions in NK activity than Ni alone. Suppression of NK activity by Ni and Mn injected at separate sites was not seen when Mn injection preceded Ni injection by 1 h. These data indicate that both the divalent metal and the timing of its injection relative to Ni injection are critical for altering Ni-induced suppression of NK cell activity.     

Effects of nickel sulfate on pulmonary natural immunity in Wistar rats

This study was designed to investigate the in vivo effect of nickel sulfate on the pulmonary non-specific immune defences. Groups of four male Wistar rats were treated with a single intratracheal instillation of NiSO(4) at different doses: 1, 2, 4 and 8 micromol of NiSO(4) per rat. Control rats received a corresponding instillation of the saline vehicle. The effect of NiSO(4) on the cytotoxic activity of the pulmonary natural killer (NK) cells and alveolar macrophages (AM), as well as the pulmonary production of cytokines such as alpha-tumor necrosis factor (TNF-alpha) and gamma-interferon (IFN-gamma), were examined 1, 2 and 7 days later. Spontaneous NK-cytotoxicity towards mouse-derived tumor cell line, Yac-1 was suppressed 1 day after treatment at doses of 2 micromol/rat and above with only one result significant (P<0.05); 2 days after treatment the suppression was increased with all results significant at the same doses; 1 week after treatment NK activity restoration was observed except for the highest dose, 8 micromol/rat. AM-mediated cytotoxicity towards mouse-derived tumor cell line, 3T12, did not show any significant difference in treated and untreated animals. In contrast, whereas moderate levels of TNF-alpha were detected in the broncho-alveolar lavage (BAL) fluid supernatants of controls, the NiSO(4) treatment highly suppressed TNF-alpha production with a maximum observed after 2 days. TNF-alpha suppression was found to be transient, at least with the lowest NiSO(4) dose, with levels returning to normal after 7 days. A non-significant increase in IFN-gamma was observed in the BAL fluids of treated animals at each time of examination. Taken together, these results indicate that NK cell activity and TNF-alpha secretion are sensitive targets for instilled NiSO(4) in Wistar rats.     

Correlation of suppressed natural killer cell activity with altered host resistance models in B6C3F1 mice.

A number of methods have been developed to assess the impact of a xenobiotic on the various components of the immune system. For risk analysis, it is necessary to determine what degree of chemically induced immune perturbation translates into altered host resistance. Natural killer (NK) cells play a pivotal role in the innate immune system with the ability to lyse cells infected with intracellular pathogens and certain tumors without previous exposure to the antigen. Spontaneous NK activity in B6C3F1 mice could be incrementally and consistently decreased by 20 to > or =80% by the intravenous administration of a range of dilutions of anti-asialo GM1 (AAGM1) antibody. The decrease in spontaneous NK activity following a single iv administration of AAGM1 antibody persisted for up to approximately 3 weeks when the initial suppression (e.g., 24 h after AAGM1 antibody injection) was almost 100%. Treatment with AAGM1, however, did not appear to perturb the function of other immune cells, based on results of the plaque assay, the mixed lymphocyte response, the cytotoxic T lymphocyte assay, the reticuloendothelial system clearance of sRBC assay, and the Streptococcus pneumoniae host resistance assay. Following a > or =80% decrease in spontaneous NK activity in mice, challenge with > or =1 x 10(3) B16F10 melanoma cells resulted in an increase in tumor burden based on the number of lung nodules. However, following challenge with 1 x 10(5) melanoma cells, a significant increase in tumor burden in mice was not observed until spontaneous NK activity had been decreased by > or =50-60%. Altered host resistance is a function not only of the magnitude of the decrease in NK activity but also of the magnitude of the challenge to the host.     

Disparate effects of representative dithiocarbamates on selected immunological parameters in vivo and cell survival in vitro in female B6C3F1 mice.

Recent studies indicate that sodium methyldithiocarbamate is immunotoxic. Major effects of this compound in female B6C3F1 mice include decreased thymus weight, increased spleen weight, and decreased natural killer (NK) cell activity. The effects of other dithiocarbamates on these parameters are not known, and the immunotoxic potential of this important class of compounds is uncertain. In the present study, the effects of sodium methyldithiocarbamate (SMD), sodium diethyldithiocarbamate (DEDTC), and disodium ethylene-bis(dithiocarbamate)(EBD) on thymus weight, spleen weight, and NK cell activity were compared in female B6C3F1 mice. SMD caused significant loss of thymic weight following oral administration at 200, 225, or 300 mg/kg/d for 7 d and caused significant suppression of splenic NK cell activity at doses of 150, 225, or 300 mg/kg/d for 7 d. In contrast, a dose of 1000 mg/kg/d of DEDTC was required to decrease significantly thymus weight or increase spleen weight, and the only significant change produced by EBD was a slight increase in spleen weight at a dose of 675 mg/kg/d. EBD and DEDTC did not affect NK cell activity at any dose tested. Dithiocarbamates are known to be cytotoxic for a variety of cell types, and it seemed possible that SMD might be more potent in vivo than EBD or DEDTC because it was more cytotoxic than these compounds. However, direct measurement of the cytotoxicity of all three compounds toward splenocytes and thymocytes in vitro demonstrated that SMD and EBD are approximately equally potent (EC50 from 6.1 to 10.5 microM), whereas DEDTC is much more potent (EC50 from 0.13 to 0.15 microM). Of the three compounds examined in this study, only SMD affected thymus weight, spleen weight, and splenic NK cell activity in vivo. Thus, this pattern of immunological effects is not produced by all dithiocarbamates. In addition, the data demonstrate that differences in the potency of SMD, DEDTC, and EBD in vivo do not correlate with their relative cytotoxic potencies in vitro.     

Dendritic cell-tumor coculturing vaccine can induce antitumor immunity through both NK and CTL interaction

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.     

Effect of bacterial purified antigenic fractions on natural defence mechanisms

The in vitro ability of bacterial purified antigenic fractions to interfere with the immune system has been investigated on human mononuclear cells from peripheral blood. Exposure of purified monocytes to the drug at concentrations from 1 to 1000 micrograms/ml, for different periods from 0 to 18 h, significantly increased cell-mediated cytotoxicity against TU5 target cells. Moreover, monocytes exposed for 1 to 18 h to drug concentrations from 0.1 to 1000 micrograms/ml released significant amounts of tumor necrosis factor alpha in a dose-dependent manner in the culture supernatants. The drug was also tested on natural killer (NK) cell activity; mononuclear cells exposed to antigenic fractions for different periods showed a significant increase of NK cytotoxic activity against K562 target cells after 3 and 6, but not 0 and 18 h. Active concentrations were from 1 to 100 micrograms/ml, higher and lower doses being ineffective. Bacterial purified antigenic fractions thus have some ability to interfere in vitro with mechanisms of cytolysis mediated by cells and soluble factors.     

Formaldehyde exposure impairs the function and differentiation of NK cells

We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation.     

Pulmonary Host Defenses and Resistance to Infection Following Subchronic Exposure to Phosgene

Abstract

Acute exposure to phosgene, a toxic gas widely used in industrial processes, decreases resistance to bacteria in mice and rats and enhances susceptibility to B16 tumor cell challenge in mice. These effects appear to be due to impaired alveolar macrophage and natural killer (NK) cell activity, respectively. In this study effects of repeated phosgene exposures on bacterial infection and NK activity were determined. Rats were exposed for 4 or 12 wk, 6 h/day, 5 days/wk, to 0.1 or 0.2 ppm phosgene or 2 days/wk to 0.5 ppm and infected by aerosol with Streptococcus zooepidemicus immediately after the last exposure. An additional group was also infected after 4 wk of recovery following the 12-wk exposure regimens. Bronchoalveolar lavage (BAL) fluid was assessed 0, 6, and 24 h postinfection for bacteria and inflammatory cells. Differential cell counts in BAL and pulmonary NK activity were also determined in uninfected rats 18 Is after the last exposure. All phosgene exposures impaired clearance of bacteria from the lungs and caused an increase in polymorphonuclear leukocytes (PMNs) in BAL of infected rats. Effects in the 0.5 ppm exposure group were greatest, and were significantly different from those in the 0.2 ppm exposure group, although the product of concentration × time was the same. BAL cell counts and bacterial clearance were normal in rats assessed 4 wk after the 12-wk phosgene exposures. Bacterial clearance and the PMN response to infection following repeated exposure were similar to those observed after a single exposure; that is, for these endpoints, effects due to repetitive exposure were neither additive nor attenuated. In contrast, NK activity was suppressed only at the 0.5 ppm level, and the magnitude of suppression was much less than that following acute exposure, suggesting that attenuation of this effect did occur with repeated exposure. The data indicate that susceptibility to streptococcal infection is a sensitive endpoint for phosgene toxicity following subchronic exposure.     

The effects of nickel on immune function in the rat

The immunotoxic potential of NiCl2 was evaluated in Fischer 344 rats following a single intramuscular injection at doses ranging from 10 to 20 mg/kg. Twenty-four hours following treatment, selected cellular and humoral immune function parameters were examined. Significant (P less than 0.05) decreases in body weights were observed in rats injected with 15 and 20 mg/kg NiCl2 as were decreases in spleen weights of rats receiving 20 mg/kg. The lymphoproliferative responses of splenocytes to the T cell mitogens concanavalin A (Con A), phytohemagglutinin (PHA), the T and B cell mitogen pokeweed mitogen (PWM) and the B cell mitogen Salmonella typhimurium mitogen (STM) were not significantly different from controls. No significant differences were observed between control and Ni-treated rats in the primary antibody response to sheep red blood cells (SRBC). On the other hand, natural killer (NK) cell activity was significantly (P less than 0.05) suppressed in rats injected with 10, 15, or 20 mg/kg NiCl2. NK cell suppression was observed in both male and female rats and for both allogeneic W/Fu-G1 target cells as well as xenogeneic YAC-1 target cells. Ni-induced suppression of NK activity was transient, with levels returning to control values within three days following treatment. Ni-induced suppression of NK activity was also manifested by an increase in mortality of rats injected with MADB106 tumor cells. These results extend to a second species our earlier findings that Ni suppresses NK activity.     

Methylcholanthrene: a possible pseudosubstrate for adrenocortical 17 alpha-hydroxylase and aryl hydrocarbon hydroxylase

In cultured bovine adrenocortical cells, loss of 17 alpha-hydroxylase activity was observed after incubation with 3-methylcholanthrene (3-MC). The suppression of 17 alpha-hydroxylase by 3-MC was rapid (50% loss of activity in 10 hr at 1 microM 3-MC), did not exhibit a lag period, and was not affected by cycloheximide. Direct effects of 3-MC on 17 alpha-hydroxylase were observed only at high concentrations, but the concentration for 50% loss of activity was 0.3 microM when 3-MC was added for 24 hr prior to assay of 17 alpha-hydroxylase. High concentrations (to 40 microM) of substrate (progesterone), did not affect the loss of activity due to 3-MC. Loss of 17 alpha-hydroxylase activity was specific; 11 beta-hydroxylase was unaffected and cell growth was unaltered. However, 22-amino-23,24-bisnorchol-5-en-3 beta-ol, an inhibitor of 17 alpha-hydroxylase, partially prevented the loss of 17 alpha-hydroxylase at 1-30 nM. 3-MC is thought to induce cytochrome P-450s via a receptor with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD was without effect on 17 alpha-hydroxylase over the range of 10 nM to 10 microM. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, chrysene, and methylphenanthrenes suppressed 17 alpha-hydroxylase at high concentrations (10-50 microM for 50% loss of activity). Some steroids that lack a substituent at position 17 also caused loss of 17 alpha-hydroxylase. Like 17 alpha-hydroxylase, bovine adreno-cortical cell AHH was found to be suppressed by exposure to 3-MC. Compounds that caused loss of 17 alpha-hydroxylase caused loss of AHH, with a similar order of potency and at similar concentrations. Suppression of AHH by 3-MC did not require protein synthesis and was prevented by an inhibitor of enzymatic activity, alpha-naphthoflavone. This implies a degree of similarity of the cytochrome P-450s for 17 alpha-hydroxylase and adrenal AHH, but the activities were shown to be likely due to different enzymes. The suppression of 17 alpha-hydroxylase and AHH by 3-MC appears not to occur by a receptor-mediated mechanism but to be similar to the suppression of 11 beta-hydroxylase and 21-hydroxylase by steroid pseudosubstrates previously observed.     

Antitumor activity of the Korean Mistletoe Lectin is attributed to activation of macrophages and NK cells

Inhibitory effect of the lectins (KML-C) isolated from Korean mistletoe (KM; Viscum album coloratum) on tumor metastases produced by murine tumor cells (B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 lymphoma cells) was investigated in syngeneic mice. An intravenous (i.v.) administration of KML-C (20-50 ng/mouse) 2 days before tumor inoculation significantly inhibited lung metastases of both B16-BL6 and colon 26-M3.1 cells. The prophylactic effect of 50 ng/mouse of KML-C on lung metastasis was almost the same with that of 100 microg/mouse of KM. Treatment with KML-C 1 day after tumor inoculation induced a significant inhibition of not only the experimental lung metastasis induced by B16-BL6 and colon 26-M3.1 cells but also the liver and spleen metastasis of L5178Y-ML25 cells. Furthermore, multiple administration of KML-C given at 3 day-intervals after tumor inoculation led to a significant reduction of lung metastasis and suppression of the growth of B16-BL6 melanoma cells in a spontaneous metastasis model. In an assay for natural killer (NK) cell activity, i.v. administration of KML-C (50 ng/mouse) significantly augmented NK cytotoxicity against Yac-1 tumor cells 2 days after KML-C treatment. In addition, treatment with KML-C (50 ng/mouse) induced tumoricidal activity of peritoneal macrophages against B16-BL6 and 3LL cells. These results suggest that KML-C has an immunomodulating activity to enhance the host defense system against tumors, and that its prophylactic and therapeutic effect on tumor metastasis is associated with the activation of NK cells and macrophages.     

PHARMACOKINETIC DISPOSITION AND TISSUE DISTRIBUTION OF BISPHENOL A IN RATS AFTER INTRAVENOUS ADMINISTRATION

Among the different chemicals present in tobacco and tobacco smoke, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen. In the present study the immunosuppressive effect of NNK was investigated in laboratory animals by analyzing the antitumor immune responses. Mice of B6C3F1 strain were treated with different doses of NNK by IP and assayed for natural killer cell activity by the lysis of 51Cr-labeled YAC-1 lymphoma cells. The control mice received physiological saline. The results showed a significant inhibition of natural killer cell activity in the spleen cells of mice treated with 100 or 250 mg/ kg NNK. In contrast to the high-dose NNK group, treatment of mice with lower doses of NNK like 10 or 50 mg/kg had no significant effect on the natural killer cell activity. In addition to spleen, the natural killer cell activity was also suppressed in the hilar lymph nodes and lung cells of NNK-treated mice. The clearance of 125I labeled YAC-1 tumor cells was also reduced from the lungs of mice injected with NNK. Further, the metastatic potential of B16F10 melanoma cells was significantly higher, as evidenced by the increased lung tumor nodules in the high-dose NNK-treated mice. The decreased antitumor immune response in the carcinogen-treated mice was not due to a decrease of NK cells, because flow cytometric analysis indicated no change in the frequency of NK 1.1+ cells between control and treated animals. However, there was an increased plasma cortisone levels in the carcinogen-treated mice compared to control animals. Injection of mice with poly I:C or interleukin-12 was able to restore natural killer cell activity in the tobacco carcinogen-treated mice.     

Activation of natural killer cell cytotoxicity of human blood monocytes by a low molecular weight component from viscum album extract

Viscum album extracts (Helixor) were investigated for their potency to influence natural killer cell (NK) cytotoxicity of human peripheral blood mononuclear cells (PBMC) in vitro. The samples investigated were unable to enhance NK cytotoxicity in PBMC/tumor cell co-cultures by direct short-term mediation but NK cytotoxicity of human PBMC was strongly stimulated when PBMC were pre-incubated for 72 h with a partly purified fraction (HM-BP) derived from extracts of V. album mali. Stimulation of NK cytotoxicity was not dependent from age and sex of PBMC donors and was directed against highly as well as moderately NK-sensitive human tumor cells. The responding effector cells were identified as monocytes/macrophages and stimulation of the NK cytotoxicity of these cells was not based on increased proliferation. The active component in the HM-BP fraction has a molecular weight of about 1000 Da or smaller and correlates with the structural criteria of an oligosaccharide. The activity was completely abrogated when the active fraction was treated with endoglycosidase F or alpha-glucosidase. Partial inactivation was observed after treatment with endoglycosidase D or hemicellulase. Moreover, the active fraction induced a reduction in tumor take incidence and tumor development in mice when applied before and after tumor challenge.     

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.     

Immunomodulation by 9-amino-1,2,3,4-tetrahydroacridine (THA): 1. Down-regulation of natural cell-mediated cytotoxicity in vitro.

THA (Tacrine), a drug used in the experimental therapy of dementia of Alzheimer's disease type, and whose biochemical site of action is believed to be the neural cholinesterase, is shown, for the first time, to be an immunosuppressant in vitro on normal human peripheral blood lymphocytes in microgram quantities. THA down-regulates non-MHC restricted natural killer (NK) cell activity without affecting the general viability of cells. This down-regulation can be demonstrated at all effector and target (K562) concentrations, in purified resting NK cells as well as in lymphokine (interleukin 2) activated killer cells in 3- or 16-h NK assays and in all the blood samples tested. Kinetic analysis shows that the Vmax (maximal cytotoxic potential) and Km of NK cell-mediated cytolysis are also attenuated. Single cell assays using agarose matrix reveal that THA moderately interferes with tumor target binding/recognition events and strongly abrogates the delivery of lethal hit, thus lowering the frequency of active killer cells among THA-treated lymphocytes. THA down-regulates NK cells upon direct interaction and does not require the help of non-NK cells. The THA sensitive site(s) on NK cells does not appear to be perturbed significantly either by their proliferative status or by membrane modulations that may be normally induced by interleukin 2. The in vitro immunomodulatory pharmacological properties of THA reveal that the biological site of action of THA extends to non-neural cells also. Such non-neural models may be helpful in exploring the pathophysiological neuroimmunomodulatory properties of THA at cellular and molecular levels.     

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.     

Enhancement of natural killer cell activity of aged mice by modified arabinoxylan rice bran (MGN-3/Biobran)

The present study is aimed to examine the possibility of enhancement of natural killer (NK) cell activity in aged C57BL/6 and C3H mice using MGN-3, a modified arabinoxylan from rice bran. Intraperitoneal injection of MGN-3 (10 mg kg(-1) per day) caused a remarkable increase in the peritoneal NK activity as early as 2 days (35.2 lytic units), and the level remained elevated through day 14. The control aged mice had a level of 5.8 lytic units. Enhancement in NK activity was associated with an increase in both the binding capacity of NK cells to tumour targets and in the granular content as measured by BLT-esterase activity. Treatment did not alter the percentage of peritoneal NK cells. Data showed that peritoneal macrophages inhibit NK activity. In conclusion, MGN-3 enhances murine NK activity of aged mice and may be useful for enhancing NK function in aged humans.     

Correlation between chemical suppression of natural killer cell activity in mice and susceptibility to cytomegalovirus: rationale for applying murine cytomegalovirus as a host resistance model and for interpreting immunotoxicity testing in terms of risk of disease.

The purpose of this study was to determine the relationship between chemical suppression of natural killer (NK) cell activity in mice and chemical effects on susceptibility to murine cytomegalovirus (MCMV) infection. The goal was to provide a rational basis for applying MCMV as a host resistance model for immunotoxicity testing and to provide risk assessors some guidance in relating suppression of NK cell activity to enhanced risk of disease. Data from studies with eight chemicals administered in various doses and by various routes were evaluated, and a significant correlation was observed between chemical suppression of virus-augmented NK cell activity and increased mortality due to MCMV infection. In contrast, effects of the same chemical treatments on spontaneous NK cell activity (i.e., basal activity in uninfected mice) did not correlate with effects of these chemicals on mortality due to MCMV. Although chemicals that suppressed spontaneous NK cell activity enhanced infection, the converse was not always true--that is, increased susceptibility to infection and suppression of virus-augmented NK cell activity were observed on three occasions when spontaneous NK cell activity was unaffected. This latter phenomenon plus the fact that for two chemicals spontaneous NK was suppressed at concentrations twofold below that which affected mortality appear to account for the poor statistical correlation. Nevertheless, the data indicate that MCMV is a useful host resistance model to be applied in immunotoxicity testing when suppression of NK cell activity has been demonstrated. However, virus-augmented activity may be a better indicator than spontaneous activity. The data also indicated that suppression of NK cell activity is predictive of increased susceptibility to infection and hence provides qualitative guidance (hazard identification) to risk assessors.