OBJECTIVETo investigate the effects of letherally total body irradiation (TBI) on distribution of T-lymphocyte subtypes and their cytokine expression.

METHODSThe BALB/c mice were divided randomly into ⁶⁰Co gamma rays TBI group and control group. Mice were sacrificed 7 days after irradiation. The lymphocytes in spleens, mesenteric lymphonodes, livers and bone marrow were collected and counted. Changes of CD4(+) T and CD8(+) T cell subsets as well as the expressions of IFN-γ and IL-17 were analyzed by flow cytometry.

RESULTS(1)Compared with control group, the total number of lymphocytes in marrow, spleen, lymph node and liver distinctively decreased in TBI group [(5.34±1.14)×10⁵ vs (3.08±1.13)×10⁷, (2.10±0.54)×10⁵ vs (2.71±0.83)×10⁷, (5.89±1.07)×10⁵ vs (7.92±1.15)×10⁷ and (3.45±1.01)×10⁵ vs (7.44±0.79)×10⁶, respectively, and the significant differences were observed between two groups in each organ (P<0.05)]. (2)The level of IFN-&gamma; produced by CD4(+) T in spleen, lymph node and liver elevated in TBI group compared to control group, which were (20.77±2.03)% vs (3.69±3.13)%, (6.28±0.46)% vs (1.11±0.17)%, (27.24±5.79)% vs (9.01±1.24)% respectively, the differences between two groups in each organ were significant (P<0.05). (3)Percentages of IFN-&gamma;(+)CD8(+) T in spleen, lymph node and liver in TBI group increased compared to control group [(52.40±9.26)% vs (43.06±1.04)%, (33.56±5.02)% vs (21.83±4.22)%, and (44.27±8.97)% vs (19.32±3.11)%, respectively, and the differences between two groups in each tissue were significant (P<0.05)]. (4)However, IL-17A expressions in CD4(+) T and CD8(+) T cells from spleen and liver were lower than those in control group.

CONCLUSIONTBI induced the reduction of lymphocytes and the expansion of IFN-&gamma; producing Th1 and Tc1 effector cells in mice.

Animals ; Interferon-gamma ; secretion ; Interleukin-17 ; secretion ; Mice ; Mice, Inbred BALB C ; T-Lymphocyte Subsets ; immunology ; secretion ; Whole-Body Irradiation

In order to explore a new way to study allogeneic reactive T lymphocytes, detection of cytokine-secreting T lymphocytes after allogeneic peripheral blood mononuclear cells (PBMNCs) stimulation and investigation of its clinical significance were performed. A novel cytokine secretion assay (CKSA) was first applied to detect T lymphocytes secreting cytokine including IFN-gamma, IL-4 and IL-10 at single cell level in human mixed lymphocytes reaction. IFN-gamma-secreting T cells from PBMNCs were then evaluated in 2 patients with acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation. The results showed that compared with IL-4 and IL-10 (which were 0.12 +/- 0.03% and 0.10 +/- 0.03% respectively), a sizable proportion of IFN-gamma-secreting T lymphocytes could be detected (1.12 +/- 0.13)% after allogeneic PBMNCs stimulation. Preliminary results indicated that frequency of IFN-gamma-secreting T lymphocytes correlated with the onset and severity of clinical aGVHD. In conclusion, it is feasible to detect IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation and to apply the CKSA technique for clinical identification of aGVHD.
Acute Disease ; Cytokines ; secretion ; Graft vs Host Disease ; etiology ; Humans ; Interferon-gamma ; secretion ; Interleukin-10 ; secretion ; Interleukin-4 ; secretion ; T-Lymphocytes ; immunology ; Transplantation, Homologous ; immunology

ISG15 is a well-known intracellular ubiquitin-like molecule involved in ISGylation. However, a recent study has revived the notion first put forward two decades ago that ISG15 is also a secreted molecule. Human neutrophils, monocytes and lymphocytes can release ISG15, even though this protein has no detectable signal peptide sequence. ISG15 has also been found in the secretory granules of granulocytes. The mechanism underlying ISG15 secretion is unknown. Secreted ISG15 acts on at least T and natural killer (NK) lymphocytes, in which it induces interferon (IFN)-gamma production. However, the mechanism by which ISG15 stimulates these cells also remains unclear. ISG15 and IFN-gamma seem to define an innate circuit that operates preferentially, but not exclusively, between granulocytes and NK cells. Inherited ISG15 deficiency is associated with severe mycobacterial disease in both mice and humans. This infectious phenotype probably results from the lack of secreted ISG15, because patients and mice with other inborn errors of IFN-gamma immunity also display mycobacterial diseases. In addition to raising mechanistic issues, the studies described here pave the way for clinical studies of various aspects, ranging from the use of recombinant ISG15 in patients with infectious diseases to the use of ISG15-blocking agents in patients with inflammatory diseases.
Amino Acid Sequence ; Animals ; Cytokines/chemistry/*secretion ; Humans ; Interferon-gamma/secretion ; Metabolism, Inborn Errors/metabolism ; Models, Biological ; Molecular Sequence Data

OBJECTIVETo investigate the antitumor effects of cytotoxic T lymphocytes (CTLs) induced by autologous dendritic cells that were inspired by autologous tumor lysates (ATLs-mDCs).

METHODSPrimary gastric cancer cells prepared by short-term culture were used as targets. ATLs-mDCs were subjected to activate autologous T cells to generate CTLs. The immunological functions of DCs were evaluated by flow cytometry and by mixed leukocyte response (MLR) assay. The antitumor outcome of tumor antigen specific CTLs was tested by cytotoxicity assay. Concentrations of IL-12 in cultured DCs and INF-gamma in CTLs were measured by ELISA.

RESULTSThe expressions of MHC-II, CD80, CD83 and CD86 were significantly up-regulated in ATLs-mDCs, moreover, the ATLs-mDCs obtained the capability of stimulating the proliferation of autologous T cells with high efficiency. The secretion of IL-12 in ATLs-mDCs was significantly higher than that in pure mature DCs (t = 15.47, P < 0.01) and in immature DCs (t = 28.44, P < 0.01). The secretion of INF-gamma in CTLs activated by ATLs-mDCs was significantly higher than that in CTLs by pure mature DCs (t = 4.84, P < 0.05) and in CTLs by immature DCs (t = 13.74, P < 0. 01). The antigen specific cytotoxicity of CTLs induced by ATLs-mDCs was significantly higher against autologous tumor cells [(84 +/- 11)%] than that against two allogeneic tumor cell lines [(19 +/- 7)% and (19 +/- 11)%; t = 54.18 and 56.46, P < 0.01, respectively].

CONCLUSIONSATLs-mDCs might mediate the antigen specific CTLs against autologous gastric cancer cells ex vivo with high efficiency.

Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Humans ; Immunotherapy, Adoptive ; methods ; In Vitro Techniques ; Interferon-gamma ; secretion ; Interleukin-12 ; secretion ; Stomach Neoplasms ; therapy ; T-Lymphocytes, Cytotoxic ; immunology

T lymphocytes are an integral part of the effective immune response against various tumors, but they are frequently functionally unresponsive in tumor-bearing patients. This study was aimed to investigate the T lymphocyte function of patients with acute myeloid leukemia (AML) in different status through analyzing intracellular cytokine characteristics of T lymphocytes in AML patients. The T lymphocytes from 18 de nuevo AML patients in different status and 10 healthy controls were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin, and were stained with fluorescent McAbs CD4-PITC, CD8-FITC and IFNgamma-PE, ILA-PE, then their T lymphocytes were analyzed by flow cytometry. The results showed that IFN-gamma level in CD4+ and CD8+ T cells of de novo AML patients was significantly lower as compared with healthy controls (p<0.05), while IL-4 level in CD4+ and CD8+ T cells was low too, there was no significant difference between de novo AML patients and healthy controls. In AML patients in clinical remission, IFNgamma level in CD8+ T cells was significantly higher than that in de novo AML patients (p<0.05), but there was no significant difference as compared with healthy controls (p>0.05). In relapsed AML patients, IFNgamma level in CD4+ and CD8+ T cells was significantly lower than that in healthy controls and in AML patients with CR (p<0.05), while IL-4 level was significantly higher than that in healthy controls and de nuevo AML patients (p<0.05). It is concluded that the cytokine secretion in T cell subsets of AML patients in different status is changed. Correspondingly, the Th1/Tc1 level is low after stimulating CD4+ and CD8+ T cells in peripheral blood of de novo AML patients, but not different from healthy controls. Though the Th1 level in T cells of AML patients in complete remission is low, but Tc1 response is even enhanced as high as the healthy controls. The Th2/Tc2-like response of CD4+ and CD8+ T cells in relapsed AML patients obviously increases when compared with Th1/Tc1-like response.
CD4-Positive T-Lymphocytes ; metabolism ; CD8-Positive T-Lymphocytes ; metabolism ; Humans ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; Leukemia, Myeloid, Acute ; immunology

In order to investigate the effect of vitamin A (VA) on the secretion of IFN-gamma and IL-4 in Mycoplasma Pneumoniae (MP)-induced A549 cells, A549 cells were co-cultured with MP for different time lengths and then the levels of IFN-gamma and IL-4 in the cell culture supernatants were detected before and after treatment with different concentrations of VA by using the enzyme-linked immunosorbent assay (ELISA). The results showed that the level of IFN-gamma and IL-4 in the supernatants of MP-induced A549 cells was much higher than that in non-induced cells (P<0.01). After application of VA, IL-4 level was not increased until the concentration of VA was up to 0.5x10(-5) mol/L (P<0.01). However, with concentration of VA increased up to 1x10(-4) mol/L, IL-4 was significantly suppressed (P<0.01). It was concluded that MP could induce the secretion of IFN-gamma and IL-4 in A549 cells. VA could inhibit the secretion of IFN-gamma and increase the IL-4 level in MP-induced A549 cells. However, high concentration of VA had an inhibitory effect on the secretion of IL-4 as well as on the IFN-gamma. These data provided a theoretical basis for the application of VA in MP pneumonia in the clinical practice.
Cell Line, Tumor ; Coculture Techniques ; Culture Techniques ; Interferon-gamma/*secretion ; Interleukin-4/*secretion ; Lung Neoplasms/pathology ; Mycoplasma pneumoniae/growth & development ; Mycoplasma pneumoniae/*physiology ; Vitamin A/*pharmacology

This study was aimed to investigate the expression and clinical significance of IL-18, IL-18 binding protein (IL-18BP), IFN-&gamma; and IL-4 secreted from splenocytes of patients with idiopathic thrombocytopenic purpura (ITP) in vitro. Spleen mononuclear cells (MNC) were prepared by using routine sterile method, and were cultured in RPMI 1640 complete medium containing 10 µg/ml PHA, 10% fetal calf serum at 37°C and 5% CO2. The levels of IFN-&gamma;, IL-4, IL-18 and IL-18BP secreted from MNC of ITP patients and normal controls were determined after culture for 48 hours. The results showed that after culture of spleen MNC for 48 hours, the levels of IL-18 and IFN-&gamma; were significantly higher in patients with ITP than that in controls, but the levels of IL-18BP was not significantly elevated in ITP patients. The level of IL-4 was below the detectable limit of the assay used. It is concluded that imbalance between IL-18 and IL-18BP may play an important role in pathogenesis of ITP, and regulation of balance between IL-18 and IL-18BP may be a therapeutic approach against ITP.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cells, Cultured ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; secretion ; Interferon-gamma ; secretion ; Interleukin-18 ; secretion ; Interleukin-4 ; secretion ; Lymphocytes ; cytology ; metabolism ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; metabolism ; Spleen ; cytology ; metabolism ; Young Adult

OBJECTIVETo investigate the effect of FK506 on cytokine secretions in whole blood from healthy individuals.

METHODSBlood samples collected from healthy volunteers were co-cultured with different concentrations of FK506 and stimulated with PMA and IONO. The concentrations of 8 cytokines including IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF were detected by Bio-Plex suspension system.

RESULTSCompared with the control group, high-concentration FK506 (20 ng/ml) significantly inhibited the secretions of IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF. At a moderate concentration (5 ng/ml), FK506 inhibited the secretion of GM-CSF significantly.

CONCLUSIONFK506 effectively inhibits the secretion of proinflammatory cytokines including IL-6, IFN-gamma and TNF-alpha and also the secretion of IL-2, IL-12, IL-17, GM-CSF and G-CSF. FK506 might play the role of immunosuppression by inhibiting the production of these cytokines by the immune cells. Monitoring the levels of these cytokines might be a potential method for evaluating the adequacy of FK506 doses administered.

Adult ; Cytokines ; blood ; secretion ; Down-Regulation ; drug effects ; Female ; Humans ; Immunosuppressive Agents ; pharmacology ; Interferon-gamma ; blood ; secretion ; Interleukin-6 ; blood ; secretion ; Male ; Tacrolimus ; pharmacology ; Tumor Necrosis Factor-alpha ; blood ; secretion ; Young Adult

OBJECTIVETo investigate the effects of apoptotic lymphocytes on the secretion of cytokines by hepatic sinusoidal endothelial cells (HSEC).

METHODSHuman HSEC cells were co-cultured for 16 h with allogenetic apoptotic lymphocytes induced by UVB irradiation. The supernatants were collected and the levels of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha were detected by Luminex technique.

RESULTSAll the cytokines were down-regulated by about 50% in HSECs after co-culture with the apoptotic lymphocytes as compared with those in the control group (P<0.05).

CONCLUSIONSCo-culture with apoptotic lymphocytes can down-regulate the secretion of pro-inflammatory cytokines in HSECs, which may contribute to tolerogenic microenvironment in the liver.

Apoptosis ; physiology ; Cells, Cultured ; Coculture Techniques ; Cytokines ; secretion ; Down-Regulation ; Endothelial Cells ; cytology ; metabolism ; Humans ; Immune Tolerance ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Liver ; cytology ; Lymphocytes ; cytology ; Tumor Necrosis Factor-alpha ; secretion

AIMTo study the effect of prothymosin alpha (Pro T alpha) as a fusion protein on secretion of IFN-gamma, IFN-alpha and TNF-alpha in vitro.

METHODSThe in vitro study was carried out on the culture of splenocytes, splenic and peritoneal macrophages isolated from Balb/c mice. Splenocytes were incubated with various concentrations of Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) with or without Con A (5 micrograms.mL-1) for 72 h. Splenic and peritoneal macrophages were respectively treated with Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) in the presence of LPS (10 micrograms.mL-1) for 24 h. Then IFN-gamma, IFN-alpha and TNF-alpha levels in the supernatant were detected by ELISA.

RESULTSPro T alpha (1 x 10(-7) mol.L-1) was found to obviously increase IFN-gamma level (P < 0.05) in the supernatant of splenocytes compared with the control group. Moreover, Pro T alpha (1 x 10(-7) mol.L-1) significantly induced the secretion of IFN-alpha (P < 0.01) and TNF-alpha (P < 0.01) in splenic and peritoneal macrophages.

CONCLUSIONIn vitro, Pro T alpha could increase the secretion of IFN-gamma, IFN-alpha and TNF-alpha.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cell Separation ; Female ; Glutathione Transferase ; pharmacology ; Interferon-alpha ; secretion ; Interferon-gamma ; secretion ; Lymphocytes ; secretion ; Macrophages ; drug effects ; secretion ; Macrophages, Peritoneal ; secretion ; Mice ; Mice, Inbred BALB C ; Protein Precursors ; pharmacology ; Recombinant Fusion Proteins ; pharmacology ; Spleen ; cytology ; Thymosin ; analogs & derivatives ; pharmacology ; Tumor Necrosis Factor-alpha ; secretion