Effect of gamma radiation on cytokine expression and cytokine-receptor mediated STAT activation.

PURPOSE: The expression of cytokine mRNA and their related transcription factors was examined in order to assess the effects of gamma radiation on the immune function of murine splenocytes. MATERIALS AND METHODS: Splenocytes were collected from seven-week-old female Balb/c mice, and then irradiated at a dose of 5 Gy of 60Co gamma-ray at a dose rate of 1.394 Gy/min. Total RNA was extracted from both irradiated and non-irradiated splenocytes at 1/2, 1, 3, 6, and 24 h and analysed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mRNA level of interferon (IFN)-gamma, which is a Th1-type (T helper cell type 1) cytokine, was reduced after 3 h post-irradiation, whereas the interleukin (IL)-2 mRNA in the naïve splenocytes had no significant changes within the 24 h after irradiation. Moreover, IFN-gamma and IL-2 mRNA expression in concanavalin A (Con A, 2.5 mug/ml) activated-splenocytes was significantly reduced by gamma irradiation. On the other hand, the mRNA level of the Th2 type (T helper cell type 2) cytokines, such as IL-4, IL-5 and IL-10, was increased both in naïve and activated splenocytes, and pro-inflammatory cytokines were also rapidly induced in response to irradiation in naïve splenocytes. Interestingly, gamma irradiation had no effect on transforming growth factor (TGF)-beta mRNA expression. Moreover, the mRNA levels of the leucine zipper trqnscription factor c-Maf and GATA binding protein-3 (GATA-3), which regulate IL-4 and IL-5 transcription, were found to have been up-regulated. However, the mRNA coding for interferon regulatory factor (IRF)-1, which is involved in IFN-gamma production, was reduced 6 h post-irradiation. The level of signal transducers and activators of transcription (Stat)-1 and Stat-4 phosphorylation, which are activated by IFN-gamma and IL-12, respectively, was significantly reduced by gamma irradiation, but IL-4 receptor mediated Stat-6 activation remained unchanged. CONCLUSIONS: These results suggest that gamma irradiation may play a role in Th1 and Th2 cytokine expression, via regulation of the level of cytokine-mediators through transcriptional modulation and Stat signaling. These results are helpful to understand general profile of cytokine expression in response to gamma irradiation.

Enhancement of anti-inflammatory tendency by SB203580, p38alpha specific inhibitor, in human fibroblast-like synoviocyte cell line, MH7A.

Interleukin-1 beta (IL-1beta) is an abundant cytokine, which, together with TNF-alpha, mediates inflammatory events in rheumatoid arthritis (RA). IL-1beta is known to induce the induction of inflammatory cytokines and metalloproteinases (MMPs) in rheumatoid synovial cells. Here, we assessed these inflammatory events by measuring IL-1beta levels in the human synovial cell line, MH7A. We observed that the activation of p38 MAP kinase by IL-1beta was involved in the induction of inflammatory cytokines, as well as several genes, including MMP-1 and MMP-3. SB203580, a specific p38 MAP kinase inhibitor, inhibited the production of IL-1beta-induced cytokines and MMPs, while the levels of the tissue inhibitor of metalloproteinase (TIMPs) were unchanged by treatment with SB203580. Moreover, the induction of suppressor of cytokine signaling 3 (SOCS3) and interferon regulatory factor 1 (IRF-1) were both found to be induced by the inhibition of p38 MAP kinase. Therefore, we suggested that the inhibition of p38 MAP kinase might enhance anti-inflammatory tendencies in the MH7A cells.

Ginsan improved Th1 immune response inhibited by gamma radiation.

Gamma radiation causes suppression of the immune function, and immune properties are related to cytokine production. In the present study, the polysaccharide, Ginsan, purified from an ethanol-insoluble fraction of Ginseng (Panax ginseng C.A. Meyer, Araliaceae) water extract was studied to assess its effects on the immunosuppressive activities of gamma radiation. Ginsan was found to stimulate murine normal splenocytes by inducing the mRNA expressions of Th1 and Th2 type cytokines, and also restore the mRNA expression of IFN-gamma, Th1 cytokine, after its inhibition by whole-body gamma irradiation. Therefore, Ginsan was found to restore the T lymphocytes function that had been suppressed by gamma irradiation in allogenic MLR (mixed lymphocyte reactions). However, Ginsan exhibited no excessive stimulatory effects on the control group. The above results indicated that Ginsan may constitute a new noble agent for the improvement of gamma radiation-induced immunosuppression.

Radioprotective effects of ginsan, an immunomodulator.

We previously reported that ginsan, a purified polysaccharide isolated from Panax ginseng, had a mitogenic activity, induced LAK cells, and increased levels of several cytokines. In an effort to identify other immunostimulatory effects, we evaluated the protective effects of ginsan injected in vivo against radiation by measuring its effects on the CFU-S bone marrow cells and spleen cells. Ginsan was found to significantly increase the number of bone marrow cells, spleen cells, granulocyte-macrophage colony-forming cells (GM-CFC), and circulating neutrophils, lymphocytes and platelets in irradiated mice. In addition, ginsan induced the endogenous production of cytokines such as Il1, Il6, Ifng and Il12, which are required for hematopoietic recovery, and was able to enhance Th1 function while interfering with the Th2 response in irradiated mice. We demonstrated that pretreatment with ginsan protected mice from the lethal effects of ionizing radiation more effectively than when it was given immediately after or at various times after irradiation. A significant increase in the LD(50/30) from 7.54 Gy for PBS injection to 10.93 Gy for mice pretreated with 100 mg/kg ginsan was observed. These findings indicate that ginsan may be a useful agent to reduce the time necessary for reconstituting hematopoietic cells after irradiation.

Gamma irradiation-reduced IFN-gamma expression, STAT1 signals, and cell-mediated immunity.

The signal transducer and activator of transcription (STAT)1 is a cytoplasmic-transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon gamma(IFN-gamma). The phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFN-gamma-inducible genes, such as the interferon regulatory factor (IRF)-1. We show here that gamma irradiation reduces the IFN-gamma mRNA expression. The inhibition of the STAT1 phosphorylation and the IRF-1 expression by gamma irradiation was also observed. In contrast, the mRNA levels of IL-5 and transcription factor GATA-3 were slightly induced by gamma irradiation when compared to the non-irradiated sample. Furthermore, we detected the inhibition of cell-mediated immunity by gamma irradiation in the allogenic-mixed lymphocytes' reaction (MLR). These results postulate that gamma irradiation induces the polarized-Th2 response and interferes with STAT1 signals, thereby causing the immunosuppression of the Th1 response.

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan.

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1)beta, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-alpha mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1beta and IL-12 mRNA were highly induced at 24 h. IL-1beta and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 microg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-alpha activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 microg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-gamma. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 microg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.