NK ACTIVITY OF LYMPHOCYTE SUBSETS AND THE EFFECTS OF LOW DOSE RADIATION

This work was supported by the National Natural Science Foundation of China (No. 3870902). Objective: To determine the NK activity of lymphocyte subsets and the effects of low dose radiation. Methods: Lymphocyte subsets were separated by monoclonal antibodies. The NK activity of each subset on tumor cells was detected by radioactive release method. Results: The results showed that besides NK cells, CD 4, CD 8 and B cells alone can kill tumor cells. But the cellkilling activity of NK cells appeared to be strongest. There was synergistic effect between CD 4 and NK cells. The activity of mixed lymphocytes was more than that of only one subset. The effect of low dose radiation (LDR) on NK activity of panlymphocytes or NK cells was different. Conclusion: This paper demonstrated that NK activity of mononuclear cells was called “NK activity of lymphocytes”, but it is not true. Only when NK cells were separated by monoclonal antibodies, its killer activity can be called “activity of NK cells”.

IgE PRODUCTION IS INVOLVED IN BUTYRATE- ENHANCED NK CELL ACTIVITY IN VIVO

It has been demonstrated that patients with asthma have a large number of NK cells and show a stronger NK activity. These results indicate that NK cell activity may be related to total IgE level in serum in healthy subjects. Previously,we have found that sodium butyrate (NaBu) markedly enhanced the IL- 4- induced IgE production in the LPS- stimulated murine splenocytes in vitro, and inductive rat IgE production in vivo, and enhanced the NK cell activity ex vivo .We hypothesized that the IgE production might be involved in butyrate- enhanced NK cell activity in vivo. Mice were intraperitoneally treated/immunized with NaBu or/and Ascaris suum extract (ASC),and the spleen NK cell activity was evaluated. Furthermore, the effect of serum (NAS) on IL- 2- or IFN-γ- induced spleen NK cell activity was determined. The spleen NK cell activity and IL- 2- or IFN-γ- induced spleen NK cell activity of mice treated/immunized with NaBu or/and ASC were stronger than those of untreated/unimmunized mice. Although IL- 4 blocked IL- 2 (100 U/ml)- or IFN-γ (100 U/ml)- induced increase in NK cell activity,these NK cell activities in mice treated/immunized with NaBu/ASC were not inhibited. IgE production showed a tendency to rise in NaBu- treated mice serum, and a synergistic effect was observed with treatment of NaBu and ASC. Moreover, the NAS significantly increased IL- 2(25 U/ml)- or IFN-γ (25 U/ml)- induced NK cell activity, and its effect was inhibited by anti- mouse IgE mAb. These data show that IgE plays an important role in NAS- enhanced IL- 2/IFN-γ- induced NK cell activity, and IL- 4 does not inhibit IgE and IL- 2/IFN-γ- induced NK cell activity in mice.

A Sensitive and Simple Method to Assess NK Cell Activity by RT-qPCR for Granzyme B Using Spleen and Blood

A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.

Im m unoregulatory Effects of Triptergium W ilfordii Hook at Antifertility Dose on Splenic NK Cells Activityin Male Mice

Effects of Tripterypium Wilfordii Hook f (TWH) on sperm atozoa in the epi- didym is and splenic NK cells activity in m ale m ice w ere observed using MTT assay and silver impregnation m ethods. The results show ed that the density, viability and m otility of the epididym alsperm atozoa in the experim entalgroupstreated w ith TWH w ere m ore significantly reduced than those in the controlgroup (P< 0.01). The head sw elling, head separation from tailin the groups treated w ith TWH w ere observed. The inhibition of splenicNK cellsactivity in m iceby TWH w asdose-dependent. Inhi- bition by TⅡand TWH athigh dose on the NK cells activity w as significant (P< 0.01 and P< 0.05), w hileinhibitory effectsof TWH atinterm ediateand low doseson the NK cells activity w ere notobserved (P> 0.05). Itw as concluded thatTWH at low er antifertility dose did not significantly inhibit the splenic NK cells activity. It m ightbe usefulforevaluating thetherapeuticeffectsof TWH in futureclinicalprac- tice.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C18 column/time-of-flight mass spectrometry system

Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell activators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside IV,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory components of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Assistant radiotherapeutic effect of Jiaqi Mixture on tumor-bearing mice

Objective:The aim was to study the assistant radiotherapeutic effect of Jiaqi Mixture (JQM) on Ehrlich's ascites carcinoma (EAC) mice.Methods:The EAC-cancer model was made up with Kunming mice.The tumor-bearing mice were treated with whole body exposure (8 Gy) and intragastric administration of JQM,and the changes of tumor weight,the total number of white blood cells (WBC) and immune system were observed.Results:The average tumor weight,WBC,spleen coefficient,the stimulation index (SI) of Con A and LPS and the natural killing (NK) cell activity of mice decreased in some degree after radiotherapy,but the average tumor weight decreased more obviously in radiotherapy + medicine groups (compared with tumor control group,P < 0.05);and the other above indexes were much higher in radiotherapy + medicine groups than those in radiotherapy groups (P < 0.05-0.01).Conclusion:It was suggested that JQM can enhance the effect of radiation therapy and protect the normal immune system caused by radiation therapy.

Plasma membrane lipid scrambling causing phosphatidylserine exposure negatively regulates NK cell activation

One of the hallmarks of live cells is the asymmetric distribution of lipids across their plasma membrane.Changes in this asymmetry due to lipid"scrambling"result in phosphatidylserine exposure at the cell surface that is detected by annexin V staining.This alteration is observed during cell death processes such as apoptosis,and during physiological responses such as platelet degranulation and membrane repair.Previous studies have shown that activation of NK cells is accompanied by exposure of phosphatidylserine at the cell surface.While this response was thought to be indicative of ongoing NK cell death,it may also reflect the regulation of NK cell activation in the absence of cell death.Herein,we found that NK cell activation was accompanied by rapid phosphatidylserine exposure to an extent proportional to the degree of NK cell activation.Through enforced expression of a lipid scramblase,we provided evidence that activation-induced lipid scrambling in NK cells is reversible and does not lead to cell death.In contrast,lipid scrambling attenuates NKcell activation.This response was accompanied by reduced cell surface expression of activating receptors such as 2B4,and by loss of binding of Src family protein tyrosine kinases Fyn and Lck to the inner leaflet of the plasma membrane.Hence,lipid scrambling during NK cell activation is,at least in part,a physiological response that reduces the NK cell activation level.This effect is due to the ability of lipid scrambling to alter the distribution of membrane-associated receptors and kinases required for NK cell activation.