An investigation of the effects of the antioxidants, ebselen or N-acetyl cysteine on human peripheral blood mononuclear cells and T cells

Abstract

BackgroundThe research literature has documented age-related increases in genetic damage, including oxidative DNA damage, in human T lymphocytes, in vitro and ex vivo. Such damage has the potential to interfere with the ability of the T cells to proliferate at times when they need to, such as when antigen challenged. The consequence of this could be a sub-optimal immune response in vivo.Context and purposeThe purpose of the research reported in this paper was to investigate the impact of two antioxidants, which can be administered in vivo, Ebselen and N-acetyl L-cysteine, on the age-related increase in genetic damage, and on T cell proliferation and lifespan. In vitro human T cell clones, ex vivo peripheral blood mononuclear cells or T cells were supplemented with different concentrations of antioxidants, under standard conditions and for different periods of time. A range of assays were then applied in order to determine any impact of the antioxidants.Results30 μM ebselen or 7.5 mM N-acetyl L-cysteine supplementation resulted in a significantly higher intracellular GSH: GSSG ratio. This increased ratio was accompanied by reduced levels of oxidative DNA damage in established CD4+ human T cell clones, from a young or a middle-aged donor. Additionally, cultures of primary human peripheral blood mononuclear cells and CD4+ T cells from donors aged 25–30 or 55–60 years were also supplemented with these agents. Cells from all sources exhibited increased proliferation, and in the case of the T cell clones, an increase in cumulative population doublings. Neither ebselen nor N-acetyl L-cysteine had such effects on clones supplemented from the midpoint of their in vitro lifespan.ConclusionsEbselen and N-acetyl L-cysteine, under certain conditions, may have anti-immunosenescent potential in T cells in in vitro clonal and ex vivo polyclonal culture models.

SENIEUR status of the originating cell donor negates certain ‘anti-immunosenescence’ effects of ebselen and N-acetyl cysteine in human T cell clone cultures

“…A viable cell count was performed on harvested cells using a Neubauer Counting Chamber, and a new culture cycle was set up with fresh medium and RJK853 feeder cells on day 7 [ 3 , 8 ]. The proliferative capacity and lifespan were determined similar to the protocol described previously [ 3 , 8 , 10 ]. The TCCs used in this study were kindly provided by the group of Professor Graham Pawelec.…”

“…Further to our previous study [ 10 ] we examined the impact of titrated concentrations of Ebselen (0, 10, 30, 60, 100 μM) or NAC (0, 1.25, 5, 7.5, 10 mM) in three pooled 399-37 (80 year old) TCCs, 385-7 (45 year old) TCCs and 400-23 (26 year old) TCCs respectively. n = 3 in each case.…”

“…Previous work from our group has provided evidence of an increase in the level of ROS-induced DNA damage with age in CD4 + TCCs cultured at 20% O 2 [ 3 , 8 - 10 ] and an increase in DNA damage and mutation with age in human lymphocytes [ 11 ]. A more recent study demonstrated anti-immunosenescence effects of two antioxidants, 2-phenyl-1,2-benzisoselenazol-3 (2H)-one (Ebselen; [ 12 ]) or N-acetyl cysteine (NAC; [ 13 ]) on CD4 + TCCs derived from healthy 26 year old and 45 year old donors [ 10 ]. In this paper we now detail the impact of each of these two antioxidants on CD4 + TCCs derived from a healthy 80 year old donor (conforming to the SENIEUR protocol for healthily aged individuals; [ 14 ]).…”

“…In these cases individuals have been shown to be able to raise an effective immune response, contributed to by adequate T cell function [ 17 - 19 ]. We were interested to examine whether the anti-immunosenescent effects of Ebselen or NAC, which we have previously reported, in TCCs from donors of 26 or 45 years of age [ 10 ] were also present when TCCs from a SENIEUR selected healthy aged donor were tested.…”

“…The antioxidant potential of Ebselen has been previously demonstrated in a number of other cell lines; HepG 2 cells [ 20 ], human HL-60 [ 22 ] and PC-12 cells [ 23 ]. Their ability to scavenge intracellular ROS resulting in reduction of hydroxyl radical formation may have contributed to the antioxidant potential in TCCs derived from healthy 26 and 45 year old donors displayed by their impact on certain markers of T cell integrity and function [ 10 ].…”