Chuankezhi (CKZ), a new Chinese medicine, plays an important role in immunoregulation. Cytokine-induced killer (CIK) cells have been commonly used for immunotherapy in recent years. In this study, we aimed to investigate the immunoregulatory effect of CKZ on CIK cells. Peripheral blood monocytes were isolated from healthy donors, and CIK cells were generated by culturing monocytes with interferon-gamma (IFN-γ) and interleukin 2. Different concentrations of CKZ were added on day 2. After incubation for 14 days in culture, the antitumor effects of CIK cells were measured by cytotoxicity assay. Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype, intracellular cytokine production, and apoptosis. The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated. CKZ increased the percentage of CD3+CD56+ CIK cells but did not significantly change the percentage of CD4+, CD8+, or CD4+CD25+ CIK cells. CKZ-conditioned CIK cells showed a greater ability to kill tumor cells, as well as a higher frequency of IFN-γ and TNF-α production, compared with the CIK cells in the control group. CKZ also suppressed the apoptosis of CIK cells in vitro. Furthermore, CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK, CKZ, or normal saline control groups. Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.
Animals ; Apoptosis ; drug effects ; CD3 Complex ; metabolism ; CD56 Antigen ; metabolism ; Cell Line, Tumor ; drug effects ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; immunology ; Cytotoxicity, Immunologic ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Epimedium ; chemistry ; Female ; Humans ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morinda ; chemistry ; Neoplasm Transplantation ; Plants, Medicinal ; chemistry ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism

This study was aimed to investigate the effects of different stimulatory factors on proliferation and function of cytokine induced killer (CIK) cells. Peripheral blood mononuclear cells (PBMNC) were separated by Ficoll-Hypacue gradient. According to supplement of different stimulatory factors (CD28 mAb, IL-15 and IL-21), the experiment was divided into five groups:control group (CIK), CB28+IL-15+IL-21 group, IL-15+IL-21 group, CD28+IL-15 group and CD28+IL-21 group. Effects of different stimulatory factors on the proliferation of CIK cells were assayed by an automated hematology analyzer. Changes of granzyme B,perforin and CD107a were detected by flow cytometry. IL-10, IL-12, INF-γ and TNF-α were quantified by ELISA. Cytotoxicities on lung cancer cell line A549, breast adenocarcinoma cell line MFC-7 and human melanoma cell line HME1 were examined by lactate dehydrogenase release method. The results showed that there were significant differences among different groups. The highest proliferation index on days 10 was observed in group CD28mAb, IL-15 and IL-21(255.3 ± 6.3), which was higher than control group, IL-21+IL-15 group and CD28 mAb+IL-21 group (166.6 ± 13.5, 199.4 ± 15.0 and 228.8 ± 16.6) (P < 0.05). The expression of perforin in CD28 mAb+IL-15 group was higher than the other groups. The expression of perforin,GranB and CD107a of costimulatory groups was higher than control group. The cytotoxicities of CD28 mAb+IL-15 group on A549, MFC-7 and HME1 cells (82.2%, 59.3% and 70.6%) were much higher than that of control group (60.9%, 49.6% and 48.4%) (P < 0.05). The highest IFN-γsecretion was found in CD28 mAb, IL-15 and IL-21 groups. It is concluded that there are significant difference of proliferative capacity, cytokine secretion and cytotoxicity after being activated by different stimulatory factors. Adding corresponding stimulatory factors into the culture system displays a great value for target cells culture.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-15 ; pharmacology ; Interleukins ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).

METHODSGingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.

RESULTSArginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.

CONCLUSIONS1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.

Adhesins, Bacterial ; pharmacology ; Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Bcl-2-Like Protein 11 ; Cell Line ; Cysteine Endopeptidases ; pharmacology ; Humans ; MCF-7 Cells ; Membrane Proteins ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Tosyllysine Chloromethyl Ketone ; pharmacology ; bcl-2 Homologous Antagonist-Killer Protein ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.

METHODSMononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.

RESULTSAfter IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-α increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-α increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.

CONCLUSIONHumanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.

Cells, Cultured ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; Fas Ligand Protein ; metabolism ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukins ; pharmacology ; K562 Cells ; Perforin ; metabolism ; Receptors, Interleukin-21 ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDActivation in vitro of natural killer T (NKT) cells in systemic lupus erythematosus (SLE) with α-galactosylceramide (α-GalCer) and dendritic cells (DC) may affect the immunoregulatory role of NKT cells. This study was designed to compare the number of NKT cells in patients with SLE to the number in healthy volunteers and measure the cytokines secreted from these NKT cells in vitro.

METHODSThree sets of culture conditions using (i) α-GalCer, (ii) DC, or (iii) both α-GalCer and DC (α-GalCer+DC) were adopted to expand NKT cells from peripheral blood mononuclear cells (PBMC) of patients with SLE and healthy volunteers. Flow cytometry was used to assess the levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by the Vα24(+)Vβ11(+) NKT cells.

RESULTSAfter 14 days in culture, the total cell count and percentage of Vα24(+)Vβ11(+) NKT cells were increased under all conditions but were highest in the α-GalCer+DC group. The level of IL-4 and IL-10 secreted by Vα24(+)Vβ11(+) NKT cells from patients with active SLE was found to be higher than that of inactive patients and the control group (P < 0.05), while the levels of IFN-γ and TNF-α were lower than those found in the inactive and control groups (P < 0.05).

CONCLUSIONSVα24(+)Vβ11(+) NKT cells showed the greatest expansion in vitro with α-GalCer and DC. Th2-type cytokines from Vα24(+)Vβ11(+) NKT cells are the predominant type in patients with SLE, while Th1 cytokines predominate in the control group. This evolution of NKT cell function during the progression of the disease may have important implications in understanding the mechanism of SLE and for the development of possible therapies using NKT cell agonists.

Adolescent ; Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dendritic Cells ; metabolism ; Female ; Flow Cytometry ; Galactosylceramides ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-4 ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Male ; Middle Aged ; Natural Killer T-Cells ; cytology ; drug effects ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Young Adult

Although nickel hypersensitivity is known as a delayed-type hypersensitivity mediated by nickel-specific T cells, it is greatly influenced by other immune cells. Here we show that splenic natural killer cells (NK cells) directly or indirectly respond to nickel by secretion of IFN-gamma. Using enzyme-linked immunosorbent spot (ELISPOT) assays, we found that nickel-reactive cells readily secreted IFN-gamma when splenocytes were cultured in the presence of varying concentrations of nickel sulfate (NiSO4) for 24 h. However, nickel-reactive IL-2- or IL- 4-secreting cells were infrequent during the 24-h culture with NiSO4. Immune responses to nickel were innate, not adaptive, in nature since the frequency of nickel-reactive IFN-gamma-secreting cells did not increase upon previous exposure to NiSO4 and recombination activating gene (RAG)-1-deficient mice contained nickel-reactive IFN-gamma-secreting cells. The involvement of NK cells in the innate response to NiSO4 was confirmed since we could observe a significant reduction of the frequency of nickel-reactive cells in NK cell-depleted mice. Furthermore, the number of IFN-gamma secreting cells was significantly reduced in the ELISPOT assays when NKG2D was blocked by anti-NKG2D antibody. These results suggest that there is an early and rapid innate immune response to nickel, which is mediated by NK cells and the NKG2D receptor. The significance of the innate response to nickel is that it may contribute to development of the late T cell-mediated delayed type hypersensitivity against nickel.
Animals ; Homeodomain Proteins/genetics/metabolism ; Humans ; Immunity, Innate/immunology ; Interferon-gamma/*secretion ; *Irritants/immunology/pharmacology ; *Killer Cells, Natural/drug effects/immunology/secretion ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism ; *Nickel/immunology/pharmacology ; Spleen/*cytology/immunology

OBJECTIVETo explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.

METHODSNK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.

RESULTSCD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).

CONCLUSIONHigh purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.

Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukins ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; metabolism ; Perforin ; metabolism

OBJECTIVETo investigate the effects of interferon (IFN) on the natural killer (NK) cytotoxicity and the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and other apoptosis-inducing genes in peripheral blood mononuclear cells (PBMNC) from polycythemia rubra vera (PV).

METHODSPBMNC were collected from 12 PV patients. The NK cytotoxicity was assessed by lactate dehydrogenase release assay and lytic units (LU) were calculated based on specific lysis. The TRAIL mRNA and other apoptosis-inducing genes were determined by RNase protection assay.

RESULTSThe NK cytotoxicity of untreated PBMNC from PV was (152.0 +/- 146.6) LU. And of IFNalpha1b- and IFNalpha2b-treated PBMNC were up to (250.9 +/- 197.4) LU and (355.9 +/- 249.9) LU, respectively (P < 0.05 and P < 0.01, respectively). The expression of TRAIL mRNA was upregulated in IFNalpha1b and IFNalpha2b stimulated-PBMNC. Up-regulation of mRNA levels of FLICE, DR3, DR4 and TNFRp55 genes was observed in PBMNC after stimulated with IFNalpha, which also induced the mRNA expressions of FasL, Fas, TRADD and RIP. To explore the role of TRAIL in IFNalpha-augmented NK cytotoxicity, neutralizing assay was used and the results showed that IFNalpha-augmented NK activity could be blocked partially by peptides DR4-Fc and DR5-Fc.

CONCLUSIONIFNalpha induced/upregulated the expression of TRAIL and other apoptosis-inducing genes in PBMNC from PV patients, which partially contribute to the IFNalpha-augmented NK cytotoxicity augmentation. This may be one of the mechanisms of IFNalpha therapy for PV.

Adult ; Aged ; Cells, Cultured ; Female ; Humans ; Interferon-alpha ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Male ; Middle Aged ; Polycythemia Vera ; blood ; immunology ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; Up-Regulation ; drug effects ; genetics

OBJECTIVETo study the anticancerous effect of Fuganchun 6 (FGC-6) and its immunoregulatory effect on tumor-bearing mice.

METHODThe mice inoculated by H22 cells were divided into 5 groups: model group, 5-Fu group and FGC-6 in high dose, medium dose, and low dose groups. The normal mice were also observed. These mice were treated for 10 days. The weight of tumor mass and mouse were examined. The target-cell-killing activity of NK cells. The proliferation activity of lymphocyte and the production of IL-2 of murine splenocytes were detected respectively. The serum containing FGC-6 was prepared and its inhibition effect on H22 cells was examined by MTT assay and growth curve in vitro.

RESULTGrowth of tumor was inhibited markedly by FGC-6 high dose. The inhibition of serum containing FGC-6 on the proliferation of H22 cells in vitro was observerd in a dose and time-dependent manner. The target-cell-killing activity of NK cells and the production of IL-2 of murine splenocytes of model group were lower than those of normal group (P < 0.05). When compared with model group, FGC-6 in high dose elevated the two indexes above-mentioned, and also enhanced the proliferation activity of lymphocyte markedly (P < 0.05). The production of IL-2 of murine splenocytes was also improved when treated by FGC-6 in medium dose (P < 0.05).

CONCLUSIONFGC-6 can inhibite the growth of H22 cells markedly and also can strengthen the immunity of H22 transplanted mouse.

Animals ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Interleukin-2 ; metabolism ; Killer Cells, Natural ; immunology ; Liver Neoplasms, Experimental ; immunology ; pathology ; Lymphocytes ; pathology ; Male ; Mice ; Plants, Medicinal ; chemistry ; Spleen ; cytology ; metabolism

OBJECTIVETo prepare human interferon-k (hIFN-kappa) and study its biological activities.

METHODSWhole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities.

RESULTSThe purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity.

CONCLUSIONAntiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.

Animals ; Antiviral Agents ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Interferon Type I ; genetics ; isolation & purification ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Microbial Sensitivity Tests ; Plasmids ; genetics ; Recombinant Proteins ; isolation & purification ; metabolism ; pharmacology ; Vero Cells