AIMTo investigate the effect of acetylcholine (ACh) on the cytotoxicity of natural killer (NK) cells and to explore the receptor mechanisms involved in the effect.

METHODSThe effector cells (i. e. NK cells) from the spleens of rats were collected and cultured with the target cells (Yac-1 cells). The various concentrations of ACh, cholinergic receptor agonists or antagonists were added to the cultures, respectively according to distinct experimental purposes. Lactate dehydrogenase (LDH) release assay was used to evaluate NK cell cytotoxicity.

RESULTSNK-cell-mediated lysis of Yac-1 lymphoma cells was reduced by 10(-10) - 10(-6) mol/L ACh. The inhibitory effect of ACh on NK cell cytotoxicity was mimicked by pilocarpine, an agonist of muscarinic receptor, and by nicotine, an agonist of nicotinic receptor, at all applied concentrations (10(-10) - 10(-6) mol/L). Muscarinic receptor antagonist atropine blocked the inhibitory effect of ACh on the cytotoxicity of NK cells. Nevertheless, tubocurarine, an antagonist of nicotinic receptor, had no blocking effect on the suppression of NK cell cytotoxicity by ACh.

CONCLUSIONACh results in an inhibition of the cytotoxicity of NK cells, and this inhibition is realized mainly through M and N1 cholinergic receptor.

Acetylcholine ; pharmacology ; Animals ; Cells, Cultured ; Female ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Male ; Rats ; Receptors, Natural Killer Cell ; drug effects

A depressed level of natural killer (NK) activity is one of the various immunologic abnormalities in human immunodeficiency virus (HIV) infection. Interleukin-15 (IL-15), an immunotherapeutic candidate in HIV infection, increases NK activity and induces the excretion of CC-chemokines from divergent immune cells, but the mechanisms of NK activity enhancement by IL-15 stimulation is not clearly established in HIV infection. This study examined whether CC-chemokines, which are known to increase NK activity, are secreted adequately in HIV-infected individuals, and also investigated whether P-glycoprotein is involved in NK activity enhancement after IL-15 administration. NK activity increased with IL-15 stimulation in NK cells of HIV-infected individuals, as it does in normal NK cells. IL-15 stimulates NK cells to secrete CC-chemokines, such as, macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage chemotactic protein-1alpha (MCP-1alpha) and regulated upon activation, normal T cells expressed and secreted (RANTES) in both HIV-infected individuals and controls with no significant difference. P-glycoprotein expression and function is decreased in HIV-infected individuals and restored only in NK cells of HIV-infected individuals after IL-15 stimulation. P-glycoprotein may play a role in the mechanism of increased NK cell activity in HIV-infected individuals after IL-15 stimulation.
HIV Infections/physiopathology* ; HIV Infections/pathology ; Human ; Interleukin-15/pharmacology* ; Killer Cells, Natural/physiology* ; Killer Cells, Natural/drug effects* ; P-Glycoprotein/physiology* ; Recombinant Proteins/pharmacology

The aim of this study was to explore the cytotoxicity of fresh cord blood(CB) NK cells and the influence of IL-12 and IL-15 on activity of the NK cells killing K562 and Jurkat cells lines. The NK cells were isolated from cord blood by depleting CD3(+) cells and then enriching CD56(+) cells using sorting with immunomagnetic beads. The experiment was divided into 3 groups: group A (fresh CB-NK cells without cytokines), group B (CB-NK cells cultured by IL-2) and group C (CB-NK cells cultured by IL-2 and IL-15). The purity of NK cells was determined by flow cytometry; the cytotoxity of fresh and different cytokine-treated CB-NK cells on K562 and Jurkat cell lines was detected by LDH release test. The results showed that the purity of NK cells before and after sorting was 14.88 ± 9.2% and 92.39 ± 0.8% respectively. After culture for 3 days, NK-forming colony amounts in group B and group C were 148.60 ± 13.0 and 831.80 ± 23.0 respectively, the comparison between group B and group C showed the significant difference (p < 0.05). The cytotoxicities of NK cells in group A, B and C on K562 and Jurkat cell lines were 27.76 ± 8.8%, 61.90 ± 9.1% and 87.62 ± 3.7%; 29.32 ± 2.5%, 69.43 ± 4.4% and 92.95 ± 3.2% respectively, the difference was significant (p < 0.05). It is concluded that the fresh isolated CB-NK cells show low cytotoxic activity. After stimulated with IL-2 or IL-2 plus IL15, cytotoxicity of CB-NK cells increases obviously, the effect of IL-2 plus IL-15 is much better than IL-2 alone for promoting the growth and enhancing the cytotoxicity of CB-NK cells.
Cytotoxicity, Immunologic ; drug effects ; immunology ; Fetal Blood ; drug effects ; immunology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Jurkat Cells ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

OBJECTIVETo study the protective effect of Sarcandra glabra extract (SGE) on immune system in restrained mice.

METHODThe male C57BL/6 mice were randomly divided into normal control group, stress control group, 125, 500 mg x kg(-1) SGE group. The spleen lymphocyte suspensions of each group were prepared. The parameters of spleen T cells subsets, NK cell and NKT cell proportion and number was detected by Flow cytometry.

RESULTSGE regulated the balance of T cell subsets, increased the percent of NK cells and NKT cell proportion and number in restrained mice.

CONCLUSIONSGE has immunologic protective effect in restrained mice probably via the amelioration of immune cells proportion and number.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Killer Cells, Natural ; drug effects ; immunology ; Magnoliopsida ; chemistry ; Male ; Mice ; Mice, Inbred C57BL ; Natural Killer T-Cells ; drug effects ; immunology ; Restraint, Physical ; Stress, Psychological ; immunology ; T-Lymphocyte Subsets ; drug effects ; immunology

The objective of this study was to explore the effect of astragalus polysaccharide (APS) on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism. The cytotoxicities of NK cells against HL-60 cells were analyzed by LDH releasing assay at different effect-to-target cell ratios (E:T) before and after treated with APS. The gene expression of MHC class I chain-related (MICA) in HL-60 cells before and after APS treatment was assayed with RT-PCR. Protein expression of MICA in HL-60 cells was assayed by flow cytometry before and after treated by APS. The results showed that after treated with APS 15 mg/ml for 48 h, the cytotoxicities of NK cells against HL-60 cells enhanced at different effect-to-target (P < 0.05), and the gene and protein expressions in MICA of HL-60 cells were up-regulated (P < 0.05). It is concluded that the APS can obviously up-regulate the expression of MICA in HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.
Astragalus Plant ; Cytotoxicity, Immunologic ; drug effects ; HL-60 Cells ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Killer Cells, Natural ; Polysaccharides ; pharmacology

This study was purposed to investigate the amplification of CD3(-)CD56(+)NK cells in umbilical cord blood and their change of immunophenotype and cytotoxicity after stimulation with IL-2 and IL-15. Mononuclear cells were isolated from umbilical cord blood and cultured in serum-free medium supplemented with IL-2 or (and) IL-15 for 14 d. The subset level of CD3(-)CD56(+)NK cells and expression of CD16, CD62L, NKG2A, NKG2D, NCR44, NCR46, granzyme B and perforin were analyzed by flow cytometry. The cytotoxicity of NK cells to K562 was detected by WST-1 method. The results showed that NK cells stimulated with IL-2, IL-15 and IL-2/IL-15 were amplified by 10.78 ± 2.51, 10.42 ± 3.72, and 10.54 ± 6.24 times respectively after 14 d, there was no statistically significant difference between these three groups. The expression of CD16 decreased obviously in NK cells after amplification; there was significant difference between IL-2 and IL-15 groups. The expression of CD62L was not changed statistically after stimulation with cytokines, the IL-2 down-regulated the expressions of NKG2A and NCR46, while IL-15 showed the opposite effect. IL-2 or IL-15 displayed upregulation effect on the expression of NKG2D, perforin and NCR44, but there was statistically significant difference between effects of these two cytokines. IL-15 up-regulated the expression of granzyme B on NK cells. The cytotoxicity of NK cells stimulated and amplified by cytokines significantly increased, but there was no statistically significant difference between IL-2 and IL-15. It is concluded that IL-2 or IL-15 can effectively amplify umbilical cord blood NK cells under serum-free conditions. Although the immunophenotype associated with NK cells function showed different characteristics between them, however, cytotoxicity of NK cells increased obviously after amplification and there is no statistically significant difference between effect of these two cytokines, their synergistic effect is not obvious. The cytotoxicity of NK cells is the result from combined effect of all active molecules.
Cells, Cultured ; Cytotoxicity, Immunologic ; drug effects ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunophenotyping ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects

OBJECTIVETo investigate whether bortezomib can enhance the sensitivity of human prostate cancer (PCa) cells to natural killer (NK) cell-mediated cytotoxicity, and whether it produces the same effect on different PCa cell lines.

METHODSWe treated androgen-dependent PCa LNCaP cells and androgen-independent PCa DU145 cells with bortezomib at the concentrations of 0, 5, 10, 15, 20 and 25 nmol/L for 24, 48 and 72 hours, and then detected the proliferation and apoptosis of the tumor cells by CCK-8 and Annexin V/PI, respectively.

RESULTSThe proliferation rates of the DU145 cells treated with 15, 20 and 25 nmol/L bortezomib were (82.79 +/-2.04)%, (73.59+/- 2.95)% and (74.16+/- 6. 16)% at 48 hours and (71.24+/- 5.30)%, (51.20+/- 2.91)% and (38.02+/- 2.67)% at 72 hours, and those of the LNCaP cells were (77.04+/- 7.74)% , (42.61 +/- 6.62)% and (23.85 +/-6.04)% at 48 hours and (36.45 +/-7.02)%, (14.94 +/-5.76)% and (11.65 +/-5. 87)% at 72 hours, both significantly inhibited as compared with the control group (P <0.05). At 24 hours, the apoptosis rates of the DU145 cells treated with 15, 20 and 25 nmol/L bortezomib were (14.41 +/- 1.32)% , (16.13 +/- 1.55)% and (14.48 +/- 1.42)% , and those of the LNCaP cells treated with 20 and 25 nmol/L bortezomib were (12.77 +/- 1.28)% and (14. 84 +/- 1.65)% , significantly higher than those of the control group (P <0.05) , and the DU145 cells showed an even higher sensitivity to bortezomib than the LNCaP cells. Bortezomib failed to sensitize these two cell lines to NK cell-mediated cytotoxicity in short-term assay, while long-term assay manifested that the apoptosis rates of DU145 and LNCaP cells after treated with 20 nmol/L bortezomib + NK cells were (41.83 +/- 5.06)% and (30.31 +/- 3.62)% , respectively, significantly higher

CONCLUSIONBortezomib enhances the sensitivity of than those after treated with either bortezomib or NK cells alone (P <0.05). PCa cells to NK cell-mediated cytotoxicity and adds to the effect of current cancer therapies, and it is more efficacious for androgen-independent prostate cancer.

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; drug effects ; Humans ; Killer Cells, Natural ; drug effects ; Male ; Prostatic Neoplasms ; pathology ; Pyrazines ; pharmacology

AIM AND METHODSThe effect of intrahippocampal microinjection of noradrenaline (NA) and its receptors antagonists and agonists on cellular immune functions were investigated in normal and adrenalectomy rat by determine the proliferative activity of Con A-stimulated splenic lymphocytes in MTT method and natural killer (NK) cell activity.

RESULTS(1) In normal group, the proliferative activity of Con A-Stimulated splenic lymphocytes were inhibited and the activity of NK cell were reduced with microinjection NA and beta1-, beta2-adrenergic receptor agonists Dobutamine (Dob, 4 microl, 6.0 x 10(-3) moL/L), Metaproterenol (Met, 4 microl, 8.0 x 10(-3) mol/L), compared with their intensity of effect, NA > Met > Dob; the immunosuppression effect induced by NA was partly hindered by alpha- and beta-receptor antagonists, phentolamine (Phen, 2 microl, 1.6 x 10(-2) mol/L) and propranolol (Prop, 2 microl, 1.6 x 10(-3) mol/L), and the action of Prop was more evident. (2) In adrenalectomy group, immunosuppression effect induced by NA was unconspicuous.

CONCLUSIONThe results suggested that NA in hippocampus could inhibit distinctly cellular immune functions, which was predominantly mediated by beta2- adrenergic receptor with a minor contribution of beta1- and alpha- adrenergic receptors. Moreover, keeping intact construction and function of adrenal gland have an important role in the effect of NA on cellular immune function.

Adrenergic Agonists ; pharmacology ; Animals ; Hippocampus ; drug effects ; Immunity, Cellular ; drug effects ; Killer Cells, Natural ; immunology ; Lymphocytes ; immunology ; Microinjections ; Norepinephrine ; pharmacology ; Rats ; Rats, Wistar ; Spleen ; cytology ; immunology

Astragalus Plant ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Genes, MHC Class I ; genetics ; HL-60 Cells ; Humans ; K562 Cells ; Killer Cells, Natural ; drug effects ; Leukemia ; genetics ; metabolism ; Polysaccharides ; pharmacology

This study was purposed to explore the changes in biological functions of human peripheral blood derived NK Cells after ex vivo expansion with different combinations of interleukin IL2 and/or IL12, IL15. According to different combination of cytokines, cultured NK cells were divided into 4 groups: group IL2, group IL2 + IL12, group IL2 + IL15 and group IL2 + IL15 + IL12. The group in which NK cells were cultured without cytokines was used as control. The cytotoxicity of cultured NK cells to target K562 cells was determined by using cell counting kit-8; the level of IFN-gamma in supernatants of NK cell culture was detected by ELISA; the perforin and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR. The results showed that the cytotoxicity of expanded NK cells in groups cultured with cytokines at different E:T ratio was significantly higher than that in group without cytokines (p < 0.01), although the cytotoxicity of NK cells in IL2 + IL15 + IL12 group seem to be slightly higher than that in IL2 + IL15 group, but there was no statistic difference (p > 0.05). The IFN-gamma levels in the supernatants of NK cell culture in the presence of cytokines significantly increased, and the IFN-gamma levels in IL2 + IL15 + IL12 group and IL2 + IL12 group were significantly higher than that in others (p < 0.01). The expressions of perforin and granzyme B mRNA of expanded NK cells in groups cultured with cytokines was significantly higher than that in control group (p < 0.01), and was consistent with cytotoxicity of NK cells. It is concluded that there are differences in the functions of NK cells cultured with different cytokines. IL2 and IL15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. However, the main function of IL12 promotes NK cells to secrete IFN-gamma, which plays a role in immunoregulation.
Humans ; Interferon-gamma ; secretion ; Interleukin-12 ; administration & dosage ; pharmacology ; Interleukin-15 ; administration & dosage ; pharmacology ; Interleukin-2 ; administration & dosage ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology