The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

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.     

Relationship between invasion of tumor-associated macrophages and phenotype and immune efficacy of tumor-infiltrating lymphocytes in advanced ovarian carcinoma

AIM:To explore the relationship between the invasion of tumor-associated macrophages(TAM) and the phenotype and immune efficacy of tumor-infiltrating lymphocytes(TIL) in advanced ovarian carcinoma. METHODS:Immunohistochemical analysis of TAM density in 175 cases of poorly-differentiated ovarian cancer tissue biopsy was performed. The cases were divided into TAM high-density(TAM^High) group and TAM low-density(TAM^Low) group according to the median of TAM density. The control group included 32 cases of benign ovarian lesions. The changes of CD8⁺ and CD25⁺ phenotypes of TIL were detected by flow cytometry analysis. TIL in the 2 groups were cultured in vitro and the conditioned-medium was collected for detecting the expression of IL-2, IL-10, TGF-β and IFN-γ by ELISA. RESULTS:The average TAM infiltration density was 62.8/high-power field(HP, ×400) in 175 cases of poorly-differentiated ovarian carcinoma, and the median was 53.3/HP. TAM^High group was 87 cases and TAM^Low group was 88 cases. A significant difference between malignant ovarian carcinoma group and control group(10.5/HP) was observed. The mean expression of CD8⁺ TIL in TAM^High group was 24%, and CD8⁺ TIL in TAM^Low group was 52%(P<0.05). The mean expression of CD25⁺ TIL in TAM^High was 48%, and CD25⁺ TIL in TAM^Low was 25%(P<0.05). The average infiltration density of CD8⁺ and CD25⁺ TIL in control group was 7%. The average infiltration density of CD8⁺ and CD25⁺ TIL in TAM^High and TAM^Low groups was significantly higher than that in control group(P<0.05). Compared with TAM^Low group, TIL destruction cytokines IL-2 and IFN-γ were significantly decreased in TAM^High group(P<0.05), while the inhibitory cytokines IL-10 and TGF-β were significantly increased(P<0.05). CONCLUSION:In high-density TAM infiltration of ovarian cancer tissues, CD25⁺ TIL type and inhibitory cytokines IL-10 and TGF-β increase, while CD8⁺ TIL type and destruction cytokines IL-2/IFN-γ decrease, suggesting that the high-density TAM has relationship with the phenotype and immune efficacy of TIL.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

The effect and mechanism of H. polygyrus infection in T-cell-mediated inflammatory bowel disease in mice

AIM: To investigate the effect and mechanism of Heligmosomoides polygyrus (H. polygyrus) infection in mouse inflammatory bowel disease (IBD) mediated by CD4⁺ helper T-cells. METHODS: Ovalbumin (OVA) -specific CD4⁺ helper T-cells were transferred into SCID (severe combined immunodeficiency) mice to establish an IBD model. The IBD mice were infected by H. polygyrus and sacrificed 14 days later. The histological changes of the colon were observed, and the expression of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA and flow cytometry. Additionally, IL-4 monoclonal antibody was intraperitoneally injected into the H. polygyrus-infected IBD mice to block the secretion of IL-4. The IL-4-blocking IBD mice were sacrificed 9 days later and the above indexes were also determined.RESULTS: Compared with the non-infection group, the H. polygyrus-infected IBD mice had more severe colonic lesions, higher level of IL-4 and lower level of IFN-γ in mesenteric lymph nodes (all P<0.05). Compared with the non-blocking group, the H. polygyrus-infected IBD mice with IL-4 blockage had less colonic lesions, lower IL-4 level and higher IFN-γ level (all P<0.05).CONCLUSION: H. polygyrus infection in CD4⁺ T-cell-mediated IBD model promotes inflammation in the early stage probably by inducing the secretion of Th2 cytokine and inhibiting the secretion of Th1 cytokine. The finding suggests that using worms for treatment of IBD needs to be cautious.     

Immunological mechanism of long-term stimulation by LPS in macrophages

AIM: To investigate the molecular mechanism and the immunosuppressive phenotype of macrophages under long-term exposure to lipopolysaccharide (LPS). METHODS: We used Ficoll-Hypaque density gradient centri-fugation combined with MicroBeads Separation Kits to separate peripheral blood mononuclear cells from human blood, and then induced the monocytes into macrophages. We observed the morphology of the macrophages by treating the cells with LPS for 48 h, in comparison with a negative control and IFN-γ treatment. ELISA was used to detect the levels of cytokines, such as IL-10, IL-12, IL-6 and TNF-α, and flow cytometry was used to detect the expression of the surface molecules (HLA-DR, CD14, CCR7, HLA-ABC and CD40). To observe the effect of macrophage on T cell proliferation, co-culture experiment was carried out for 6 d. Real-time PCR was used to validate the expression levels of molecules related to MyD88-independent pathway in Toll-like receptor 4 (TLR4) signal pathway. RESULTS: The antigen-presenting ability of the macrophages was reduced and the IL-10 expression level was increased after the cells were treated with LPS for 48 h. We observed a poor proliferative capacity of CD8⁺ T cells after co-culturing of LPS-induced macrophages with CD3⁺ T cells for 6 d. The results of real-time PCR indicated that TRIF, IRF3 and CIITA were down-regulated in LPS-induced macrophages.CONCLUSION: We successfully established a macrophage model in vitro and observed that LPS-induced macrophages into an immunosuppressive phenotype with poor CD8⁺ T cell proliferative capacity, in which MyD88-independent TLR4 signaling pathway was impaired.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

The biological characteristics of cytokine-induced killer cells

AIM:To investigate the biological characteristics of cytokine-induced killer (CIK) cellsin vitro.METHODS:The non-adhere peripheral blood monoclear cells from healthy donors were induced into CIK cells in the presence of IFN-γ, IL-1β, IL-2 and anti-CD3 antibody. LAK (lymphokine activated killer) cells were prepared as a control. The cellular phenotype were detected by FCM and immunocytochemistry and the cytotoxicity was measured by LDH release assay.RESULTS:After 2 weeks of induction, the proliferation rate of CIK cells reached a peak and the proportion of CD3⁺ population was above 95%, and then the cells growth entered to plateau phase at week 3. The proportion of CD3⁺CD56⁺ NKT subset cells was 16.5% on day 15 and it had no obvious variety between 2 and 4 weeks. Correspondingly, LAK cells grew slowly and had lower proliferation rate compared with the CIK cells (P<0.01). CIK cells showed higher specific lysis rates to BeL-7402 hepatoma cells than those of LAK cells at different effector to target ratio (P<0.01). Immunocytochemical staining showed the CIK cells highly co-expressed HLA-DR and CD54 antigens. The NKT cells were slightly bigger than CD3⁺CD56^- cells and a large quantity of pseudopodia were observed on their surface.CONCLUSION:The CIK cells have higher proliferation potency and stronger cytotoxicity to lyse tumor cells than LAK cellsin vitro.Within the span of time from 14 to 21 days, the proliferation rate and the proportion of CD3⁺CD56⁺ subset of CIK cells all reach peaks. Therefore, CIK cells in this period are suitable for clinical application.     

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.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats

AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4⁺ was increased in rabbit corneal stroma (P＜0.05), CD4⁺, CD4⁺ CD71⁺ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).     

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.     

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34⁺ cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34⁺ cells simultaneously expressed mRNA for IL-1(α,β),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGFβ₁ ,TNFα and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGFβ 1 , TNFα and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.