| Objective: To study the effects of two antioxidants (N-acetyl-cysteine; NAC, Tetra-methylthiourea; TMTU)on immune responses, to provide experimental evidence for using antioxidants to treat reactive oxygen species (Reactive Oxygen Species; ROS) -induced diseases.Methods: 1 Spleen cell part: (1) Using [3H]-TdR incorporation method, we studied the effect of antioxidants on murine spleen cell proliferation in vitro. After treating spleen cells with anti-Thy1 antibody or anti-Ig antibody and complement, we studied their proliferative response to antioxidants. Furthermore, after T cell and macrohage depletion with Sephadex G-10, B cell proliferative response was investigated. (2) Using flow-cytometry method, we studied the effect of antioxidants on B cell and T cell population in spleen cells. (3) IL-2, IL-4 production in spleen cells was detected by ELISA. (4) IgG production in spleen cell or in purified B cells was also detected by ELISA.2 In vivo part: To investigate the effect of NAC on antibody production in vivo, mice were immunized with Ovalbumine and administered with NAC orally, then anti-Ovalbumine IgG production in sera was assayed by ELISA.3 Macrophage part: (1) Using ELISA, we detected IL-6, IL-12p40, IL-12p70 productions after antioxidants treatment. (2) Effect of antioxidants on TNF-α production was studied by L929 bioassay. (3) mRNA expression of IL-12p40, p35 was studied by RT-PCR. (4) To investigate the effect of antioxidants on IL-12p70 bioactivity, spleen cells were cultured with supernatant from macrophages pre-cultured with LPS and antioxidants, in the presence or absence of IL-12p70, then IFN-γ production of spleen cells was determined by ELISA.Results: 1 T cell mitogen, ConA and B cell mitogen, LPS, stimulated proliferative response of spleen cells. NAC markedly enhanced this ConA- and LPS-induced proliferative response dose dependently. NAC didn't stimulate spleen cells without ConA or LPS. The effect of TMTU was not so clear.2 In order to study which cell types are enhanced by NAC-treatment, the fractionation of spleen cells was performed by the treatment with anti-Thy1 antibody or anti-Ig antibody and complement, then the T cell depletion and B cell depletion kept their proliferative response by NAC-treamtment. These results suggest that NAC stimulates both T cell and B cell responses. The proliferative response of T cell- and macrophage- depleted spleen cells was reduced about 50%. This result suggests that the stimulation effect of NAC on B cells requires macrophages.3 Flow-cytometry data showed that NAC and LPS treatment increased B cell population in spleen cell, but T cell population was decreased. NAC and ConA stimulation didn't change the T cell and B cell populations. 4 NAC and TMTU enhanced the Th1 cytokine IL-2 and Th2 cytokine IL-4 production which was induced by ConA in spleen cells, suggesting that antioxidants have stimulation effect on both T cells and B cells. 5 NAC enhanced LPS-induced IgG production of spleen cells and B cells in a dose-dependent manner. And in vivo data also showed that NAC enhanced anti-Ovalbumine IgG production. TMTU didn't change antibody production.6 Antioxidants suppressed LPS- and SAC- induced proinflammatory cytokine, IL-6 and TNF-α,production in macrophages.7 NAC and TMTU enhanced IL-12p40 subunit production, but suppressed IL-12p70 heterodimer production in macrophages, and this effect is neither due to a dose difference nor a time course difference. This result suggests the suppressive effect of antioxidants on IL-12p35 subunit. RT-PCR showed that the effects of NAC and TMTU on IL-12p40 and p35 productions were at mRNA level.8 Spleen cells were cultured with the supernatant from macrophages pre-cultured with LPS and NAC or TMTU, in the presence or absence of IL-12p70, then IFN-γ in the culture supernatant was determined. The macrophage supernatant pre-cultured with antioxidants suppressed IFN-γ production in spleen cells, which indicated the suppressive effects of antioxidants on IL-12p70 production; whi... |
PDF Full Text Request