AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepa...AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepaticlymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestionpercentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h ^51Cr release assay.RESULTS: NK1.1^+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ^+(68%) or TNF-α^+(15%) NK1.1^+ cells from poly (I:C)treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells.CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1^+ cell.展开更多
We have previously shown a critical role of prolactin (PRL) during maturation and anti-tumor effects of murine natural killer (NK) cells in vitro and in vivo. We extended that study by exploring the ability of human N...We have previously shown a critical role of prolactin (PRL) during maturation and anti-tumor effects of murine natural killer (NK) cells in vitro and in vivo. We extended that study by exploring the ability of human NK cell lines (NK-92 and YT cell) to express PRL receptor (PRL-R) and to respond to PRL stimulation in vitro. Both human NK cell lines constitutively expressed PRL-R on membrane and mRNA transcripts,NK-92 cells contained higher level of PRL-R than YT cells,which correlated to the enhanced capacity of the cells to proliferate and to lyse target cells in response to PRL stimulation in the presence of trace amount of IL-2 or IL-15 in vitro. Two differences between IL-2 and IL-15 in functioning on human NK cells were for the first time observed. PRL synergized with IL-15 to improve proliferation of NK cells in a dose-dependent manner without double peak manifesting like IL-2. Although PRL enhanced the cytotoxicity of IL-2 or IL- 15 activated NK cells,it exerted the function through up-regulating gene expression of perforin without influence of FasL in IL-2-stimulated NK cells,while in IL-15-stimulated NK cells,PRL did the function through up-regulating gene expression of both perforin and FasL but not IFNγ. PRL increased expressions of IL-2Rα on membrane and of IL-2 mRNA in cells,indicating that PRL up-regulated NK cell function by improving positive feedback between IL-2 and IL-2R. The similar results were also observed in network between IL-15 and IL-15R. These data indicate a potential role of PRL in human NK cell modulation.展开更多
Type 2 cytokines are usually predominant in tumor patients and associated with tumor progression.To explore whether reversing of type 2 predominance could be a promising strategy in tumor immunotherapy,PBMCs of 35 lun...Type 2 cytokines are usually predominant in tumor patients and associated with tumor progression.To explore whether reversing of type 2 predominance could be a promising strategy in tumor immunotherapy,PBMCs of 35 lung cancer patients and 19 healthy subjects were prepared and subjected to be examined for cytokine secretion and gene expression.Tetra-Methylpyrazine (TTMP),extracted from a traditional Chinese medicinal herb which has been used in clinic to reverse the Th2 status of cancer patients in China,was added to PBMC culture. Determined by RT-PCR,the positive percentages of mRNA expression of type 1 cytokines (8.6% for IFN-γ and 11.4% for IL-2) were lower than those of type 2 cytokines (71.4% for IL-4,60% for IL-6 and 80% for 1L-10) in patients' PBMCs.The potential of gene expressing (measured as relative intensity to the ratio of β-actin) in the patients for type 1 cytokines was also in a low level (0.111 for IFN-γ,0.119 for IL-2) in comparison with a relative high level for type 2 cytokines (0.319 for IL-4,0.303 for IL-6 and 0.377 for IL-10).Meanwhile,both positive percentage and relative intensity of gene expression were lower for a type 1 cytokine-related transcription factor T-bet (31.4% and 0.142,respectively) than those for type 2 cytokine-related GATA3 (85.7% and 0.378, respectively).The blood serum levels of IFN-γ and IL-2 in the patients were slightly lower but not significantly when compared with healthy control.In contrast,the levels IL-4 and IL-6 in patients were significantly higher than those in healthy subjects by ELISA analysis.TTMP could enhance supernatant concentration and gene expression levels of IFN-γ,IL-2 and T-bet,but reduced those of type 2 cytokines.These results demonstrate that the lung cancer patients had a predominant expression of type 2 cytokines and TTMP could reverse the type 2 dominant status,which might offer an alternative therapeutic regime for lung cancer patients.Cellular & Molecular Immunology.2004;1(1):63-70.展开更多
Recombinant human prolactin(rhPRL)was administered to huPBL-SCID mice to determine its effects on human immunologic reconstitution and function.The huPBL-SCID mice were given 10 μg i.p.injection of rhPRL every other ...Recombinant human prolactin(rhPRL)was administered to huPBL-SCID mice to determine its effects on human immunologic reconstitution and function.The huPBL-SCID mice were given 10 μg i.p.injection of rhPRL every other day for a total of 10 injections after huPBL were transfered.The results demonstrated that rhPRL improved the engraftment of lymphocytes into thymus,lymph nodes and spleens,showing that the cellularities of these organs increased although the cellularities tended to vary depending on the donor.The amounts of human T cells(HLA-ABC^+/CD3^+)increased greatly in thymus(14.2 folds),spleen(4.16 folds)and lymph nodes(40.18 folds)after rhPRL injections.The amounts of human B cells(HLA-ABC^+/CD19^+)also increased greatly in lymph nodes(42.5 folds)and spleen(5.78 folds).The lymph node cells from the rhPRL-treated huPBL-SCID mice were more sensitive to PHA stimulation([~3H]thymidine incorporation).The supernatant of PHA-stimulated PBL from rhPRL-treated huPBL/SCID chimerism contained more cytokines (IFN-γ and IL-2).The natural cytotoxicity against human sensitive target cells,K562 cells,from spleen and bone marrow of hPBL/SCID chimerism was significantly enhanced by rhPRL administration.The lymph node cells were stimulated with LPS in vitro for 3 days and the lymphocytes from the rhPRL-treated huPBL-SCID mice were more sensitive to mitogen stimulation.Both serum total IgG level and IgM level of rhPRL-treated huPBL/SCID chimerism were increased,and even without DT-rechallenge the base line of DT-specific IgG was elevated after rhPRL treatment in huPBL-SCID mice.Thus,rhPRL stimulation promotes reconstitution of human immune system in huPBL-SCID mice.Cellular & Molecular Immunology.2004;1(2):129-136.展开更多
基金Supported by the National Science Fund for Distinguished Young Scholars,No.30125038the Key Project of National Natural Science Foundation of China,No.30230340the National High Technology Research and Development Program of China(863 Program),No.2002A
文摘AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepaticlymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestionpercentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h ^51Cr release assay.RESULTS: NK1.1^+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ^+(68%) or TNF-α^+(15%) NK1.1^+ cells from poly (I:C)treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells.CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1^+ cell.
基金supported partly by Outstanding Young Scientist Award and Key Project by Natural Science Foundation of China(No.30125038,No.30230340)The Major Sate Basic research Development program of China(No.2001CB510009)+1 种基金The National high technology research and Development program of China(No.2002AA216151)by Ministry of Science and Technology of ChinaKey Project by Chinese Academy of Science(No.KSCX2-2-08).
文摘We have previously shown a critical role of prolactin (PRL) during maturation and anti-tumor effects of murine natural killer (NK) cells in vitro and in vivo. We extended that study by exploring the ability of human NK cell lines (NK-92 and YT cell) to express PRL receptor (PRL-R) and to respond to PRL stimulation in vitro. Both human NK cell lines constitutively expressed PRL-R on membrane and mRNA transcripts,NK-92 cells contained higher level of PRL-R than YT cells,which correlated to the enhanced capacity of the cells to proliferate and to lyse target cells in response to PRL stimulation in the presence of trace amount of IL-2 or IL-15 in vitro. Two differences between IL-2 and IL-15 in functioning on human NK cells were for the first time observed. PRL synergized with IL-15 to improve proliferation of NK cells in a dose-dependent manner without double peak manifesting like IL-2. Although PRL enhanced the cytotoxicity of IL-2 or IL- 15 activated NK cells,it exerted the function through up-regulating gene expression of perforin without influence of FasL in IL-2-stimulated NK cells,while in IL-15-stimulated NK cells,PRL did the function through up-regulating gene expression of both perforin and FasL but not IFNγ. PRL increased expressions of IL-2Rα on membrane and of IL-2 mRNA in cells,indicating that PRL up-regulated NK cell function by improving positive feedback between IL-2 and IL-2R. The similar results were also observed in network between IL-15 and IL-15R. These data indicate a potential role of PRL in human NK cell modulation.
基金supported partly by 0utstanding Young Scientist Award(#30125038)Key Project(#30230340)by Natural Science Foundation of China+2 种基金Chinese Key Basic Science Program by Ministry of Science and Technology of China(#2001CB510009)Foundation of Chinese Academy of Science(#KSCX2-2-08)UICC Yamagiwa-Yoshida Memorial International Cancer Study Grant.
文摘Type 2 cytokines are usually predominant in tumor patients and associated with tumor progression.To explore whether reversing of type 2 predominance could be a promising strategy in tumor immunotherapy,PBMCs of 35 lung cancer patients and 19 healthy subjects were prepared and subjected to be examined for cytokine secretion and gene expression.Tetra-Methylpyrazine (TTMP),extracted from a traditional Chinese medicinal herb which has been used in clinic to reverse the Th2 status of cancer patients in China,was added to PBMC culture. Determined by RT-PCR,the positive percentages of mRNA expression of type 1 cytokines (8.6% for IFN-γ and 11.4% for IL-2) were lower than those of type 2 cytokines (71.4% for IL-4,60% for IL-6 and 80% for 1L-10) in patients' PBMCs.The potential of gene expressing (measured as relative intensity to the ratio of β-actin) in the patients for type 1 cytokines was also in a low level (0.111 for IFN-γ,0.119 for IL-2) in comparison with a relative high level for type 2 cytokines (0.319 for IL-4,0.303 for IL-6 and 0.377 for IL-10).Meanwhile,both positive percentage and relative intensity of gene expression were lower for a type 1 cytokine-related transcription factor T-bet (31.4% and 0.142,respectively) than those for type 2 cytokine-related GATA3 (85.7% and 0.378, respectively).The blood serum levels of IFN-γ and IL-2 in the patients were slightly lower but not significantly when compared with healthy control.In contrast,the levels IL-4 and IL-6 in patients were significantly higher than those in healthy subjects by ELISA analysis.TTMP could enhance supernatant concentration and gene expression levels of IFN-γ,IL-2 and T-bet,but reduced those of type 2 cytokines.These results demonstrate that the lung cancer patients had a predominant expression of type 2 cytokines and TTMP could reverse the type 2 dominant status,which might offer an alternative therapeutic regime for lung cancer patients.Cellular & Molecular Immunology.2004;1(1):63-70.
基金supported by 0utstanding Young Scientist Award and Key Project by Natural Science Foundation of China(#30125038,#30230340)Key Basic Science Program by Ministry of Science and Technology of China(#2001CB510009)Foundation of Chinese Academy of Science(#KSCX2-2-08).
文摘Recombinant human prolactin(rhPRL)was administered to huPBL-SCID mice to determine its effects on human immunologic reconstitution and function.The huPBL-SCID mice were given 10 μg i.p.injection of rhPRL every other day for a total of 10 injections after huPBL were transfered.The results demonstrated that rhPRL improved the engraftment of lymphocytes into thymus,lymph nodes and spleens,showing that the cellularities of these organs increased although the cellularities tended to vary depending on the donor.The amounts of human T cells(HLA-ABC^+/CD3^+)increased greatly in thymus(14.2 folds),spleen(4.16 folds)and lymph nodes(40.18 folds)after rhPRL injections.The amounts of human B cells(HLA-ABC^+/CD19^+)also increased greatly in lymph nodes(42.5 folds)and spleen(5.78 folds).The lymph node cells from the rhPRL-treated huPBL-SCID mice were more sensitive to PHA stimulation([~3H]thymidine incorporation).The supernatant of PHA-stimulated PBL from rhPRL-treated huPBL/SCID chimerism contained more cytokines (IFN-γ and IL-2).The natural cytotoxicity against human sensitive target cells,K562 cells,from spleen and bone marrow of hPBL/SCID chimerism was significantly enhanced by rhPRL administration.The lymph node cells were stimulated with LPS in vitro for 3 days and the lymphocytes from the rhPRL-treated huPBL-SCID mice were more sensitive to mitogen stimulation.Both serum total IgG level and IgM level of rhPRL-treated huPBL/SCID chimerism were increased,and even without DT-rechallenge the base line of DT-specific IgG was elevated after rhPRL treatment in huPBL-SCID mice.Thus,rhPRL stimulation promotes reconstitution of human immune system in huPBL-SCID mice.Cellular & Molecular Immunology.2004;1(2):129-136.
