Expression of prolactin receptor and response to prolactin stimulation of human NK cell lines

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.

Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A 4 (LXA 4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β, with or without preincubation with LXA 4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzyme-linked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2(SH 2) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results: IL-1β-stimulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA 4 in a dose-dependent manner. LXA 4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β. Conclusion: LXA 4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cells through the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.

INTERLEUKIN-6 PROTECTS ANNULUS FIBROSUS CELL FROM APOPTOSIS INDUCED BY INTERLEUKIN-1β IN VITRO

Objective To investigate the effect of interleukin-6 （ IL-6 ） on the apoptosis of annulus fibrosus （AF） cell induced by intedeukin-1β （IL-1β）. Methods Cultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1β, 10 ng/mL IL-1β and Z-VAD-FMK （ a caspase-9 inhibitor）, 10 ng/mL IL-1β and 10 ng/mL IL-6, 10 ng/mL IL-1β and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry. Results The apoptosis rates of cells in group 1 to 6 were 2.67% ± 1.08%, 2.71% ± 0.53%, 20. 37% ± 1.57 %, 11.34% ± 0.67 %, 18.17 % ± 0.74%, and 9.42 % ± 1.08 %, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 （ P = 0. 001, P =0. 172, and P =0. 001, respectively）. Positive rates of caspase-3 in group 5 （ 12. 35% ±0.64% ） and 6 （9.26% ±0. 36% ） were significantly lower than group 3 （ 17.14% ±0. 72% ; P =0. 001 and P 〈0.001, respectively）. And positive rates of caspase-9 in group 5 （ 15.13% ± 1.45% ） and 6 （ 10.17% ± 2.50% ） were significantly lower than group 3 （ 19.4% ±0.98% ; P =0. 014 and P =0. 004, respectively）. But there was not obvious change of caspase-8 activity after IL-6 was added. Conclusion IL-6 is capable of protecting AF cells from IL-1β induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Modulation of cellular and humoral immune responses to anHIV-1 DNA vaccine by interleukin-12 and interleukin-18 DNA immunization

Objective: To investigate the effect of interleukin-12 (IL-12) and interleukin-18 (IL-18)DNA immunization on immune response induced by HIV-1 DNA vaccine and to explore new strategies for therapeutic HIV DNA vaccine. Methods: The recombinant expression vector pCI-neoGAG was constructed by inserting HIV Gag gene into the eukaryotic expression vector pCI-neo. Balb/c mice were immunized with pCI-neoGAG alone or co-immunized with the DNA encoding for IL-12 or IL-18.Anti-HIV antibody and IFN-γ were tested by ELISA,and splenocytes were isolated for detecting antigen-specific lymphoproliferative responses and specific CTL response by MTT assay and LDH assay respectively. Results: The anti-HIV antibody titers of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were lower than that of mice immunized with pCI-neoGAG alone(P<0.01). In contrast, the IFN-γ level of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 was higher than that of mice immunized with pCI-neoGAG alone (P<0.01).Furthermore, compared with mice injected with pCI-neoGAG alone, the specific CTL cytotoxity activity and antigen-specific lymphoproliferative responses of mice immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were significantly enhanced respectively (P<0.01). Conclusion: The DNA encoding for IL-12 or IL-18 together with HIV DNA vaccine may enhance specific Th-1 responses and cellular immune response elicited in mice. Hence, the DNA encoding for IL-12 or IL-18 are promising immune adjuvants for HIV-1 DNA vaccine.