Enhancement of In vitro antibody production of murine splenocytes by ascorbic acid 2-O-α-glucoside

Abstract

Using an antigen-specific plaque forming cell (PFC) assay, we have studied the effect of ascorbic acid 2-O-α-glucoside (AA-2G), a new stable derivative of ascorbic acid (AsA), on antibody production in cultured murine splenocytes in comparison with that of AsA. A single addition of AA-2G (0.0625–1.0 mM) remarkably stimulated both anti-SRBC (sheep red blood cell, TD antigen) and anti-TNP (trinitrophenyl, TI antigen) PFC responses in a dose-dependent manner, although AsA failed to stimulate their responses. However, repeated additions of AsA at 12-h intervals during the cultivation resulted in enhancement of the anti-SRBC PFC response, and the magnitude of stimulation was comparable to that obtained by a single addition of AA-2G. AA-2G's effect was abrogated in the presence of castanospermine (α-glucosidase inhibitor) in the medium, indicating that the immunostimulation brought about by AA-2G is attributed to AsA released from the glucoside by α-glucosidase of cultured cells. In fact, the cells maintained a certain amount of AsA for a relatively long time after exposure to AA-2G. In contrast, AsA levels in the cells treated with AsA quickly decreased. AA-2G markedly enhanced lipopolysaccharide(LPS)-induced proliferative response, and could affect the concanavalin A (Con A) response weakly. These results suggests that AsA effectively potentiates B-cell functions in the humoral immune system. Thus, we conclude that AA-2G is capable of enhancing antibody production in the cultured splenocytes via a continuous supplementation of AsA and that AA-2G is available for long-term cell cultures as a useful tool for analyzing biological function of AsA.