This study was aimed to explore the killing effect of PBMNC induced by IL-23 alone or combined with IL-2 on K562 cells and its mechanism. The PBMNC were induced in vitro by IL-23 (50 ng/ml) alone or IL-23 combined with IL-2 (100 U/ml) for 72 h, and then were co-cultured with leukemia cell line K562. The CCK-8 method was used to detect the effect of PBMNC induced at different times on K562 cells, the ELISA was performed for detecting IFN-γ level in culture supernatant, and the perforin and granzymes B were detected by RQ-PCR. The results showed that the killing effect of PBMNC induced by IL-23 alone or IL-23 combined with IL-2 on K562 cells was observed, and obviously enhanced with prolonging of time, moreover, there was statistical difference among different time points (P < 0.05). The IFN-γ level in supernatant of PBMNC cultured with cytokines significantly increased, and the IFN-γ levels in group of IL-23 combined with IL-2 were higher than that in other groups (P < 0.05). The mRNA expressions level of perforin and granzymes B of the expanded PBMNC in groups cultured with cytokines were higher than that in control group (P < 0.05), and the mRNA expressions of perforin and granzymes B in group of IL-23 combined with IL-2 were significantly higher than that in others (P < 0.05). It is concluded that IL-23 can promote the killing effect of PBMNC on K562 cells. The combination of IL-2 with IL-23 displays synergic effect and a time-dependent manner. IL-23 also enhances the expression of IFN-γ, perforin and granzyme B in PBMNC. Its combination with IL-2 displays synergistic effect, suggesting that the anti-leukemic activity of IL-23 may be realized through inducing PBMNC to express IFN-γ, perforin and granzyme B.
Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-23 ; pharmacology ; K562 Cells ; Monocytes ; drug effects ; metabolism ; Perforin ; metabolism

The aim of this study was to compare cell proliferation and function of the T cells acquired under various culture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells (DC) and EBV-transformed B lymphoblastoid cell line (BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3(+)CD8(+) population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
Cell Line, Transformed ; Cell Proliferation ; Cell Separation ; Dendritic Cells ; cytology ; metabolism ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; metabolism

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

In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.
Animals ; Chick Embryo ; Chickens ; Fowlpox virus ; genetics ; Interleukin-2 ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology

OBJECTIVETo study the effect of Bushen Huayu Composite (BSHY) on interleukin-2 (IL-2) secretion and IL-2 receptor expression in the aged.

METHODSIL-2 was examined by methyl thiazolyl tetrazolium (MTT) method and IL-2 dependent cellular stains, IL-2 receptor expression was examined by FACS-indirect immunofluorescence.

RESULTSAging could result in the decrease in lymphocytic IL-2 secretion and IL-2 receptor expression (P < 0.01, P < 0.05), which could be improved by BSHY (P < 0.01, P < 0.05).

CONCLUSIONBSHY has significant up-regulatory effect on immune function of lymphocytes in the aged.

Adjuvants, Immunologic ; pharmacology ; Aged ; Aging ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Interleukin-2 ; blood ; Lymphocytes ; immunology ; Male ; Middle Aged ; Phytotherapy ; Receptors, Interleukin-2 ; blood

Killer cell Ig-like receptor (KIR) binds to HLA class I molecules on the surface of target cells, and it confers inhibitory signals to NK cells. Although NK cytotoxicity can be affected by the change of the surface expression of KIR on NK cells, the effect of cytokines on the regulation of KIR expression has not been thoroughly investigated. Here in our study, we investigated the effect of several cytokines, including IL-2, TGF-beta, IFN-gamma, IL-12 and IL-18, on the surface expression of CD158 KIR, which binds to HLA-C, by the use of FACS analysis. In the isolated NK cells, IL-2 obviously increased the surface expression of CD158 KIR after 72 hr in vitro culture, and this was evidenced by the increased percentage of CD158+ NK cells and the increased mean fluorescence intensity of CD158 in CD158+ NK cells. In contrast, TGF-beta decreased the surface expression of CD158 KIR after 72 hr culture. However, IFN-gamma, IL-12 and IL-18 did not change the expression of CD158 KIR. The modulated expression of KIR by IL-2 and TGF-beta can be associated with the changed NK-cytotoxic target-discriminating ability of NK cells upon their exposure to IL-2 and TGF-beta.
Antineoplastic Agents/*pharmacology ; Cells, Cultured ; Human ; Interferon Type II/pharmacology ; Interleukin-12/pharmacology ; Interleukin-18/pharmacology ; Interleukin-2/*pharmacology ; Killer Cells/cytology/*drug effects/*metabolism ; Receptors, Immunologic/*metabolism ; Support, Non-U.S. Gov't ; Transforming Growth Factor beta/*pharmacology

To explore the efficacy of the different combinations of stem cell factor (SCF), FLT3 ligand (FL) and interleukin (IL) 2, 7, 15 on the induction and expansion of cord blood (CB) derived CIK/ NK cells in vitro, according to the different combinations of cytokines, combinations of cytokines were divided into 3 groups: group A (SCF + IL2 + IL7 + IL15), group B (SCF + FL + IL2 + IL7 + IL15) and group C (IL2 + IL7 + IL15 as control group). At 21 days of culture, the proportion and the expansion rate of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells were measured. The results showed that at 21 days in culture, the percentages of CD3(+)CD56(+) CIK cells derived from CB-MNC in group A, B and C were (19.84 +/- 2.93)%, (26.20 +/- 4.05)%, (24.03 +/- 4.99%) and that of NK cells were (49.60 +/- 1.40)%, (51.16 +/- 6.45)% and (30.85 +/- 8.12)%, respectively. The proliferation of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells was the most effective in group B (SCF + FL + IL2 + IL7 + IL15). Expansion multiple of CIK cell number increased from 575.81 +/- 221.72 (control) to 796.09 +/- 278.47 with peak value of 1 313, and that of NK cells increased from 30.39 +/- 14.47 (control) to 65.85 +/- 30.83 with peak value of 121.06. In conclusion, a proper cytokines (SCF + FL + IL2 + IL7 + IL15) culture system can effectively induce and expand CB CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells simultaneously.
CD3 Complex ; analysis ; CD56 Antigen ; analysis ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; Killer Cells, Natural ; drug effects ; Membrane Proteins ; pharmacology ; Stem Cell Factor ; pharmacology

OBJECTIVETo investigate the influence of the LPS of Bacteroides fragilis on the secretion of IL-2 and IL-4 from the peripheral blood mononuclear cells of normal individuals, so as to elucidate the mechanism of the infection by Bacteroides fragilis.

METHODSLPS was obtained from both the strains isolated from patients and from standard NCTC9343. Peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of LPS thus obtained. The supernatants from the cell culture of the PBMCs were harvested at 24 PBHs and were subjected to the determination of the IL-2 and IL-4 contents by ELISA method. RESULTS The IL-2 secretion from the PBMCs of normal volunteers was obviously inhibited by the LPS from Bacteroides fragilis (P < 0.01), and the inhibitory effect was dose-dependent. Nevertheless, the IL-4 secretion from the PBMCs of normal volunteers was significantly stimulated by the LPS from Bacteroides Fragilis (P < 0.05), and it was not concentration dependent. There was no difference between the effects of the LPSs from patients and standard strains (P < 0.05).

CONCLUSIONThe LPS from Bacteroides fragilis was inhibitory to the secretion of IL-2 from PBMCs and was stimulative to that of IL-4 from PBMCs of normal human persons.

Bacteroides fragilis ; metabolism ; Cells, Cultured ; Humans ; Interleukin-2 ; immunology ; secretion ; Interleukin-4 ; immunology ; secretion ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; immunology

OBJECTIVE: To investigate the effects of inflammation cytokines, (FK506) and cyclosporine (CSA) on albumin secretion, and the effects of FK506 and CSA on the IL-6 induced suppression of albumin synthesis in cultured human hepatocytes. METHODS: Human hepatoma cell lines (HepG2 cells) were separately cultured with IL-6, IL-2 and IL-10 (0 approximately 10 microg/L) and FK506, CSA (0 approximately 100 microg/L) for 48 h. In another experiment, HepG2 cells were stimulated with different doses of FK506 and CSA (0 approximately 10 microg/L) in the presence of IL-6 (5 microg/L) for 48 h. Albumin levels in the supernatant of all groups were measured by radioimmunoassay (RIA). The concentration of LDH secreted by cells stimulated with FK506 and CSA were detected with spectrophotometry. RESULTS: For cultured HepG2 cells, IL-6 significantly decreased albumin levels in a dose-dependent manner (P <0.01), and the maximal inhibition occurred at 5 microg/L. CSA mildly decreased albumin levels and a significant reduction in albumin production was first visible at 10 microg/ L (P <0.05). In contrast, IL-2, IL-10 and FK506 did not significantly influence albumin pro- duction (P > 0.05). FK506 obviously decreased LDH levels in the supernatant (P < 0.05) and attenuated IL-6 induced suppression of albumin synthesis (P < 0.01). But CSA slightly increased LDH concentration and could not block the IL-6 induced decrease of albumin synthesis (P > 0.05). CONCLUSION IL-6 but not IL-2 and IL-10 suppressed the production of hepatic albumin in vitro. FK506 protected against the suppression of hepatic albumin synthesis caused by IL-6, suggesting its potential role in improving hypoalbuminaemia in immune glomerulonephritis.
Albumins ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cyclosporine ; pharmacology ; Hepatocytes ; physiology ; Humans ; Interleukin-10 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-6 ; pharmacology ; Liver Neoplasms ; metabolism ; pathology ; Tacrolimus ; pharmacology ; Tumor Cells, Cultured

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.
Cell Separation ; methods ; Humans ; Interleukin-2 ; analysis ; Lymphocytes ; cytology ; drug effects ; metabolism ; Phytohemagglutinins ; pharmacology