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

OBJECTIVETo explore the putative effects of Vitamin C (Vit C) on inhibition of human leucocyte antigen class I (HLA-I) expression of human peripheral blood mononuclear cells (HPBMCs) induced by deoxynivalenol (DON) in vitro.

METHODSThe effects of Vit C on the changes of HLA-I expression of HPBMCs induced by DON in vitro were evaluated with cell culture, flow cytometry (FCM), Western blotting and immunocytochemical methods.

RESULTSFCM analysis showed that HLA-I expression of HPBMCs in DON treated cells was significantly lower than that in controls (FI 0.88 +/- 0.02 vs 1.00 +/- 0.03, P < 0.05). As compared with DON group, the HLA-I expressions of HPBMCs in the two Vit C (25 micromol/L and 100 micromol/L) pretreatment groups were all significantly increased (1.15 +/- 0.06 and 1.10 +/- 0.02 vs 0.88 +/- 0.02, P < 0.05). Exposure to different dosage of Vit C alone could dramatically increase the expression of HLA-I of HPBMCs in vitro as compared with that in the normal control (FI for 25 micromol/L and 100 micromol/L Vit C treatment group was 1.28 +/- 0.03 and 1.25 +/- 0.05 respectively, P < 0.05). Immunocytochemical results showed that the percentages of HLA-I positive expression of HPBMCs in Vit C pretreatment groups at different dosages were significantly higher than those in DON group (70.10 +/- 6.90)%, (64.50 +/- 5.50)% vs (42.20 +/- 4.30)%, P < 0.05. Western blotting confirmed the results of FCM and immunocytochemistry.

CONCLUSIONSVitamin C pretreatment at different dosages could reverse at some extent the inhibitive effects of DON on HLA-I expression of HPBMCs.

Ascorbic Acid ; pharmacology ; Cells, Cultured ; Flow Cytometry ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Monocytes ; drug effects ; metabolism ; Trichothecenes ; pharmacology

OBJECTIVETo investigate the antitumor effect of recombinant IFN-alpha-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms.

METHODSMB49 cells were co-cultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining.

RESULTSThe hIFN-alpha-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoechst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9%, respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group.

CONCLUSIONThe recombinant hIFN-alpha-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.

Adjuvants, Immunologic ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; BCG Vaccine ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytotoxicity, Immunologic ; Histocompatibility Antigens Class I ; metabolism ; Interferon-alpha ; pharmacology ; Mice ; Recombinant Proteins ; pharmacology ; Urinary Bladder Neoplasms ; metabolism ; pathology

Adenocarcinoma ; chemically induced ; metabolism ; Animals ; Apoptosis ; drug effects ; Carcinogens ; toxicity ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; metabolism ; Lung Neoplasms ; chemically induced ; metabolism ; Lymphocytes ; cytology ; metabolism ; Pulmonary Surfactant-Associated Protein C ; metabolism ; Sterigmatocystin ; toxicity

A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.
Animal ; Antibodies/pharmacology ; Chymotrypsin/metabolism ; Chymotrypsin/chemistry ; Comparative Study ; Concanavalin A/pharmacology ; Heat ; Histocompatibility Antigens Class I/metabolism* ; Histocompatibility Antigens Class I/drug effects ; Interferon Type II/pharmacology ; Interferon Type II/metabolism ; Interferon Type II/immunology ; Lymphocytes/physiology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Proteins/pharmacology* ; Proteins/metabolism ; Proteins/isolation & purification* ; Spleen/cytology ; Trypsin/metabolism ; Trypsin/chemistry ; Tumor Cells, Cultured/immunology ; Tumor Cells, Cultured/drug effects

OBJECTIVETo investigate the hepatic expression of immunological markers relevant to a cytotoxic response in relation to viral genotype.

METHODSThe frozen liver biopsies were obtained from 28 HF genotyped patients and made the sections stained. The morphometry was used to analyze the major histocompatibility complex class I (MHC-I), CD8, beta(2)-microglobulin (beta(2) -mG), HFE and CD68 in the stained sections. Biopsy data of response to therapy with interferon were available in 18 cases.

RESULTSCD8+ was usually clustered together and localized in portal tracts and sinusoids, and seen to interact with MHC I positive lining cells. MHC-I and beta(2) -mG were expressed mainly in endothelial and Kupffer cells. HFE was expressed in most round and dendritic CD68+ cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better sustained response to interferon (IFN) therapy than patients without.

CONCLUSIONThe MHC-I expression in the liver of patient with chronic hepatitis C virus infection seems to relate to viral-genotype. The hepatic MHC-I and HFE expression are higher in patients with virus genotype 3a than that in patients with non-3a genotype.

Adult ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Antiviral Agents ; therapeutic use ; Blotting, Western ; CD8 Antigens ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Genotype ; Hemochromatosis Protein ; Hepacivirus ; drug effects ; genetics ; Hepatitis C, Chronic ; genetics ; metabolism ; virology ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Interferons ; therapeutic use ; Liver ; immunology ; metabolism ; virology ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo analyze the expression of NKG2D ligands on human nasopharyngeal carcinoma cell line CNE2 and the multidrug-resistant lin CNE2/DDP and investigate its impact on cytotoxicity of natural killer (NK) cells.

METHODSExpression of NKG2D ligands on the surface of CNE2 and CNE2/DDP cells was analyzed by flow cytometry, and their HLA genotypes, along with inhibitory killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells from 5 healthy donors, were determined by PCR with sequence specific primers. Cytotoxicity of NK cells against CNE2 and CNE2/DDP cells was evaluated by LDH-releasing assay at different effector-to-target ratios (E:T). In blocking experiments, different monoclonal antibodies (mAb) were added to the target cells at the E:T of 20:1 ratio.

RESULTSThe HLA genotypes of CNE2 and CNE2/DDP cells were A2, 24, B18, 35, Cw4, 7, and the inhibitory KIR genotypes of the 5 healthy donors were KIR2DL1, KIR2DL3, KIR3DL1, and KIR3DL2, mismatched with the HLA -class I molecules expressed by the CNE2 and CNE2/DDP cells. The expression of MHC class I chain-related proteins A and B (MICA and MICB) on CNE2 cells was higher than that on CNE2/DDP cells (P<0.01), and ULBP1 and ULBP3 were not detectable. NK cells displayed highly in vitro cytotoxicity against CNE2 and CNE2/DDP cells with a mean cell lysis rate of (10.50-/+2.17)%, (4.98-/+0.95)%; (27.68-/+1.47) %, (15.48-/+2.10) %; (36.99-/+3.13) %, (28.46-/+4.30) %; (55.00-/+2.20) %, (40.95-/+2.21) %, respectively, corresponding to the E:T ratios of 5:1, 10:1, 20:1, and 30:1 (P<0.01). Blocking experiments confirmed that killing of CNE2 and CNE2/DDP cells by NK cells was efficiently inhibited by anti-MICA, anti-MICB, and anti-ULBP2 mAbs, whereas anti-ULBP1 and anti-ULBP3 mAbs had no effects on the cytotoxicity of the NK cells.

CONCLUSIONExpression of NKG2D ligands on CNE2 and CNE2/DDP cells is correlated with NK cell-mediated lysis, and NK cells display higher cytotoxicity against parental CNE2 cells than the multidrug-resistant CNE2/DDP cells.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Drug Resistance, Multiple ; Flow Cytometry ; Genotype ; HLA Antigens ; genetics ; Histocompatibility Antigens Class I ; immunology ; metabolism ; Humans ; Killer Cells, Natural ; immunology ; NK Cell Lectin-Like Receptor Subfamily K ; immunology ; metabolism ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Polymerase Chain Reaction ; Receptors, KIR ; genetics

OBJECTIVETo investigate the regulatory effect of IFN-gamma on recognition of target cells by human natural killer (NK) cells.

METHODSThe cytotoxic activity of human NK cell lines (NK92, NKL) was detected by MTT method. Expression of NK cell receptors (NKG2D, NKG2A/B, KIR2DL1 and KIR2DS1) and MICA on target cells (the ligand of NKG2D) was measured by RT-PCR.

RESULTSBoth NK92 and NKL cells exerted higher cytotoxicity to tumor cells with MICA expression, while tumors without MICA expression could resist NK cell lysis. IFN-gamma (> 1000 U/ml) inhibited NK lysis of tumor cells with MICA expression through down-regulating the expression of NKG2D, but up-regulating the expression of NKG2A/B and KIR2DL1.

CONCLUSIONIFN-gamma has a negative effect on activation and cytotoxicity of human NK cells by altering the balance between the expression of activating and inhibitory receptors on NK cells in favor of inhibition. This may serve to limit NK cell over-activation in vivo.

Cell Division ; drug effects ; Cytotoxicity, Immunologic ; drug effects ; Histocompatibility Antigens Class I ; analysis ; physiology ; Humans ; Interferon-gamma ; pharmacology ; Killer Cells, Natural ; immunology ; NK Cell Lectin-Like Receptor Subfamily C ; NK Cell Lectin-Like Receptor Subfamily K ; Receptors, Immunologic ; metabolism ; Receptors, KIR2DL1 ; Receptors, Natural Killer Cell ; Recombinant Proteins ; Tumor Cells, Cultured

OBJECTIVETo study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells.

METHODSAfter establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching.

RESULTSProteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT.

CONCLUSIONThe present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calbindins ; Chloride Channels ; analysis ; DNA-Binding Proteins ; analysis ; Electrophoresis, Gel, Two-Dimensional ; methods ; HL-60 Cells ; Harringtonines ; pharmacology ; Histocompatibility Antigens Class I ; analysis ; Humans ; Ikaros Transcription Factor ; Inhibitor of Apoptosis Proteins ; Microtubule Proteins ; Phosphoproteins ; analysis ; Proteins ; analysis ; Proteome ; analysis ; S100 Calcium Binding Protein G ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; Transcription Factors ; analysis

OBJECTIVETo investigate the mechanism underlying the effects of matrine in enhancing the cytotoxic sensitivity of CNE2/DDP cells highly expressing ATP-binding cassette superfamily G member 2 (ABCG(2)(High)) to allogenic natural killer (Allo-NK) cells.

METHODSABCG(2)(High) CNE2/DDP cells and Allo-NK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of the isolated cells and the expression of NKG2D ligands on the target cells before and after incubation with matrine. The cytotoxic sensitivity of the treated and non-treated ABCG(2)(High) CNE2/DDP cells to Allo-NK cells was measured by LDH releasing assay.

RESULTSThe expression rate of ABCG2 was (91.40-/+2.32)% in ABCG(2)(High) CNE2/DDP cells. More than 90% of the isolated NK cells were identified to be CD3(-)CD16(+)CD56(+) cells. The expression rates of MICA, MICB, ULBP1, ULBP2, and ULBP3 on the target cells incubated with matrine increased from (2.92-/+0.33)%, (4.27-/+0.33)%, (5.80-/+0.62)%, (11.10-/+3.15)%, and (7.75-/+1.14)% to (11.30-/+0.89)%, (14.29-/+2.61)%, (12.56-/+1.06)%, (43.24-/+4.43)%, and (12.77-/+1.06)%, respectively. At the E: T ratio of 10:1 and 20:1, the cytotoxic sensitivity of ABCG(2)(High) cells to Allo-NK cells increased from (15.32-/+1.34)% and (27.26-/+6.81)% in un-treated cells to (28.53-/+1.37)% and (42.72-/+2.80)% in matrine-treated cells, respectively, showing significant differences in the cytotoxic sensitivity of the target cells in both groups produced by matrine treatment (F=29.05, P=0.000).

CONCLUSIONMatrine can up-regulate the expressions of NKG2D ligands (MICA/B and ULBP1-3) in ABCG(2)(High) nasopharyngeal carcinoma cells, which results in increased cytotoxic sensitivity of the cells to Allo-NK cells.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Alkaloids ; pharmacology ; Carcinoma ; Cell Line, Tumor ; Flow Cytometry ; GPI-Linked Proteins ; metabolism ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Killer Cells, Natural ; drug effects ; metabolism ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K ; metabolism ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Proteins ; metabolism ; Quinolizines ; pharmacology