Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Inhibitory effects of gossypol on the activation of human T-lymphocytes stimulated with polyclonal activators

AIM: Peripheral blood mononuclear cells (PBMC) were cultured in vitro to study the effect of gossypol, a polyphenolic antifertility agent, on the activation of normal human T cells. METHODS: Double fluorescent staining together with flow cytometry was adopted to analyze the influence of gossypol on expression of the early activation antigen CD69 on T-lymphocytes under stimulation of mitogen or phorbol ester. RESULTS:Analysis of T cell activation in vitro revealed that preincubation of PBMC with 100 μmol/L gossypol could completely inhibit the expression of early activation marker CD69 on CD3⁺ T cells in response to 10mg/L PHA, and block T cell activation by 10^-7 mol/L PDB as well. The suppression of CD69 expression was dose-dependent and IC₅₀ of gossypol on PDB and PHA were (35.7±2.9) μmol/L and (32.8±1.5) μmol/L(x ±s), respectively. Besides, gossypol had similar inhibitory effect on CD69 expression of CD3^- lymphocytes. However, it did not have any significant effect on T cell surface molecule CD3 down-regulation. CONCLUSION: Gossypol could inhibit T cell activation in vitro in response to polyclonal activators, both PHA and PDB, suggesting that its action site may be at PKC or its downstream and that gossypol possessed potential immuno-regulatory effect.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Early apoptotic T lymphocytes induce DCs from bone marrow to tolerogenic DCs

AIM: To study the immunosuppressive effects of early apoptotic T lymphocytes.METHODS: Early apoptotic spleen T cells were induced by ultraviolet irradiation for 5 min.After irradiation,spleen T cells were incubated at 37 ℃ with 5% CO₂ for 2 h and thus early apoptotic T lymphocytes were obtained.Three to four freeze thaw cycles resulted in disruption of the spleen T cells into fragments.imdendribic cells(DCs) were prepared from red cells and T cells depleted bone marrow cells.The imDCs were divided into five groups: group A: necrotic spleen T cells were added to imDCs;group B: early apoptotic spleen T cells were added to imDCs;group C: supernatants from early apoptotic spleen T cells alone with necrotic spleen T cells were added to imDCs;group D: TGFβ₁ neutralizing antibody along with early apoptotic T lymphocytes were added to imDCs;group E: immature dendridic cells culture in RPMI-1640 for 5 days were used as negative control.Flow cytometry was employed to analyze the expression of MHCⅡ,CD40,CD80 and CD86 on DCs in each group.ELISA was employed to assay the IL-12 p70 produced by DCs in different groups.The amounts of TGFβ₁ released by early apoptotic T lymphocytes were also determined by ELISA.T cells proliferation assay was employed to study DCs T cells stimulatory capacity.RESULTS: The DCs expressed high level of MHCⅡ,CD40,CD80 and CD86 when exposed to necrotic cells while early apoptotic cells did not.The supernatants from early apoptotic spleen T cells suppressed the expression of MHCⅡ,CD40,CD80 and CD86 on DCs exposed to necrotic spleen T cells.When TGFβ₁ neutralizing antibody along with early apoptotic spleen T were added to imDCs,the expression of MHCⅡ,CD40,CD80 and CD86 was increased significantly.The necrotic spleen T cells increased IL-12 p70 production by DCs,while apoptotic spleen T cells at early stage did not (P<0.01,group B vs group A or B;P>0.05,group B vs group E).Only the DCs that exposed to necrotic spleen T cells gained significant T lymphocytes stimulatory capacity,while DCs exposed to apoptotic cells at early stage did not.The amounts of TGFβ₁ released by early apoptotic spleen T cells were much higher than those released by viable spleen T cells.CONCLUSION: Apoptotic spleen T cells at early stage have the capacity to induce the generation of tolerogenic DCs.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Determination of T cell cycle and the expression of bcl- 2 in asthmatic mice and its significance

AIM: To investigate the changes of T cell cycle, the expression of bcl- 2 in allergic asthmatic mice and the effects of dexamethasone on them. METHODS: An animal model with asthma was established by means of ovalbumin sensitizing-challenging. CD3 expression in spleen and lymphocytes in bronchoalveolar lavage fluid (BALF), T cell cycle and Bcl-2 expression in spleen were detected by flow cytometry. RESULTS: In BALF lymphocytes and spleen lymphocytes, CD3 expression rate in the asthmatic group was significantly higher than that of control group. In BALF lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly lower than that of the asthmatic group. However, in spleen lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly higher than that of the asthmatic group. In spleen lymphocytes, the cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmatic group were significantly higher than that from the control group. Cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmaplus dexamethasone group was significantly lower than that from the asthmatic group. The Bcl-2 expression rate of T cell from the asthmatic group was significantly higher than that from the control group. CONCLUSIONS: In the allergic asthmatic mice model, T cell count, proliferation and activation of T cells, apoptosis rate of T cells in spleen lymphocytes increase, meanwhile bcl- 2 expression also increases significantly. There was no significant effect of dexamethasone on the bcl- 2 expression. The therapeutic effects of dexamethasone on asthma may be not due to the inhibition of the bcl- 2 expression in T cells.     

Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Valproic acid exerts differential effects on cytokine synthesis in human peripheral lymphocytes

AIM: Valproic acid (VPA) is a histone deacetylase inhibitor and is believed to have anti-tumor activity. The present study aims to investigate the effect of VPA on the, apoptosis and cytokine synthesis of human peripheral lymphocytes.METHODS: The activation and cytokine synthesis in lymphocytes in whole blood stimulated with phorbol dibutyrate (PDB) and ionomycin were evaluated with flow cytometry after fluorescent staining. The mitochondrial membrane potential was examined using 3, 3-dihexyloxacarbocyanine iodide staining.RESULTS: VPA at low doses (1 and 5 mmol/L) promoted CD69 expression in activated lymphocytes, whereas it turned to inhibit the expression of CD69 at a high dose (25 mmol/L). Meanwhile, VPA at low doses increased the mitochondrial membrane potential, while a high dose of VPA decreased it in activated lymphocytes. Furthermore, interleukin-2 (IL-2) synthesis was enhanced by low doses of VPA but inhibited by a high dose. However, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) synthesis were dose-dependently enhanced by VPA as compared with those of PDB plus ionomycin-treated cells.CONCLUSION: VPA exerts biphasic effect on the further activation and apoptosis of human peripheral lymphocytes stimulated with mitogens and exhibits differential activity on the synthesis of several important cytokines in human lymphocytes.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Effect of lactacystin and β-lactacystin on the activation and proliferation of T-lymphocytes

AIM: To evaluate the effects of lactacystin (LAC) and β-lactacystin (β-LAC), proteasome inhibitor, on the proliferation and activation of T lymphocytes. METHODS: Flow cytometry was used to analyse the proliferation and the expression of CD69, CD25 and CD3 in PHA activated T-lymphocytes. Furthermore, the expression of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. RESULTS: (1) LAC and β-LAC significantly decreased the incorporation in PHA activated T-lymphocytes. (2) Although LAC and β-LAC did not affect the expression of CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P＜0.05). (3) In comparison with control, LAC and β-LAC significantly down-regulated the expression of PA28 and IL-2 mRNA (48 h, 72 h, P＜0.05).CONCLUSIONS: LAC and β-LAC significantly inhibit the proliferation and activation of T lymphocytes. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNA expression.     

Effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro

AIM: To observe the effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro. METHODS: The lymphocytes from the lymphoid nodes of BALB/c mice were isolated and primarily cultured. The viability of T cells was assessed by MTT assay. Fluorescence-conjugated monoclonal antibody and flow cytometry (FCM) were used to analyze the expression of T-cell activation marker CD69 in response to concanavalin A (Con A) in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to detect the proliferation of T cells in vitro. FCM analysis was used to determine the production of reactive oxygen species (ROS) in the T cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). The mean fluorescence intensity of DiOC₆(3) staining in the T cells was detected by FCM in order to analyze the effects of salidroside on the activity of the mitochondrial and the mitochondrial membrane potential in the T cells induced by dexamethasone (DEX). The thymus T cells from BALB/c mice were isolated and primarily cultured, and then FCM was also used to analyze the apoptosis of the thymus T cells treated with DEX. RESULTS: Salidroside increased the expression of T-cell activation marker CD69 at the final concentration of 80, 160 and 320 μmol/L (P<0.05). Salidroside promoted the proliferation of T cells induced by Con A for 72 h in vitro (P<0.01). Salidroside reduced the production of ROS (P<0.05) and protected the mitochondrial membrane potential of T cells from the injury of DEX (P<0.01). Salidroside also decreased the apoptosis rate of the thymus T cells induced by DEX in vitro (P<0.01). CONCLUSION: Salidroside promotes the activation and proliferation of T cells induced by Con A, reduces the production of ROS, maintains the mitochondrial membrane potential and protects thymus T cells against apoptosis induced by DEX in vitro.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy persons

AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69⁺CD3⁺/CD3⁺%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.