Impaired immunoglobulin M production by incubation of hybridoma cells with ethanol

Abstract 

Several reports have presented results that demonstrate suppression of the immune system by ethanol. Using a hybridoma cell model, we studied the effects of ethanol on cell proliferation and on the production of immunoglobulin M (IgM) antibodies. The number of cells decreased while incubated with as little as 25 mM ethanol but not in a clonal subline incapable of IgM production, indicative of an increased vulnerability associated with the antibody-producing machinery. Levels of antibodies in cell culture supernatants were monitored by μ-heavy-chain-specific and κ-light-chain-specific enzyme-linked immunosorbent assays. We found a significant decrease in antibody concentration at 200 mM ethanol compared with findings for nonexposed cells. In addition, lower κ-chain compared with μ-chain values were monitored at ethanol concentrations of 50 mM and higher. This difference suggests irregular composition of the antibodies in the supernatant. Determination of IgM levels within the hybridoma cells revealed a linear increase in antibody concentrations by as much as three times the control levels with increasing ethanol concentrations when correlated with cell numbers. Analysis of the mRNA levels of two ethanol-inducible stress proteins, the 78-kilodalton glucose-regulated protein (GRP78) and the 70-kilodalton heat-shock protein (HSC70), by quantitative Northern hybridization yielded increased mRNA in a nonlinear fashion. The results demonstrate that ethanol impairs IgM composition, whereas antibody production within hybridoma cells is increased and the assembling machinery is activated, indicating compensating processes.

Keywords:  Immunoglobulin IgM, Hybridoma cells, Ethanol, ELISA, Cytotoxicity, Glucose-regulated stress protein 78 kDa (GRP78), Heat-shock cognate protein 70 kDa (HSC70)