No abstract

2-Mercaptoethanol (2-ME) is widely used in rodent lymphoid cell cultures as an enhancer of multiple cellular functions. We have confirmed that the action of 2-ME must be on a serum component(s), rather than a direct action on the cells. The serum component(s) is contained within the dialyzable fraction of fetal calf serum (FCS) since: (a) dialysis of FCS diminished the ability of FCS to support an antibody response even in the presence of 2-ME; and (b) FCS dialysate, pulsed with 2-ME, restored the ability of dialyzed FCS to support an antibody response. Diminution of the reduced glutathione content of FCS by heating reduced the capacity of FCS to support an antibody response, whereas addition of 2-ME-pulsed glutathione restored the supportive capacity of heated FCS. Conversely, oxidized glutathione inhibited the antibody response in the absence of 2-ME, but that inhibition was not seen in the presence of 2-ME. We have concluded that reduced glutathione is an essential component in FCS in order for 2-ME to produce its enhancing effect. The most plausible explanation for the enhancement of antibody responses, in vitro, by 2-ME, is the concomitant reversal of the inhibitory effect of oxidized glutathione and the increased availability of reduced glutathione which can scavenge oxygen-derived radicals, thus protecting macrophages and lymphocytes from the deleterious effects of oxygen-derived free radicals.

A broad spectrum of agents known to block various steps in the lipid peroxidation process were tested for their ability to protect mouse spleen cells and thereby enhance their activities, in vitro, in either the primary antibody response or the lipopolysaccharide-stimulated proliferation response. Each agent (superoxide dismutase, butylated hydroxyanisole/butylated hydroxytoluene/n-propyl gallate, lucigenin, and alpha-tocopherol) was able to enhance the cellular response in both assay systems. The degree of enhancement of these immune functions was in proportion to the efficacy of each agent in blocking the overall process in lipid peroxidation. Previous work in this laboratory has shown that the enhancement of the primary antibody response by 2-mercaptoethanol (2-ME) is mediated by the enhanced availability of reduced glutathione in the culture medium. Suboptimal doses of each lipid antioxidant agent were able to enhance the antibody response in the additive manner with a suboptimal dose of 2-ME up to a maximum response equal to that achieved with an optimal dose of 2-ME alone. These data support the hypothesis that the enhancement of cellular responses in the presence of 2-ME is mediated by the lipid antioxidant activity of reduced glutathione.