The effects of cell number, concentrations of mitogen and glutamine and time of culture on [ 3H]thymidine incorporation into cervical lymph node lymphocytes stimulated by Concanavalin-A

Abstract

The amount of [ 3H]thymidine incorporated into DNA in lymphocytes stimulated with Concanavalin-A increases exponentially with time at different concentrations of glutamine, reaches a peak value, then gradually decreases. When the value (log 10 thymidine incorporation glutamine present−log 10 thymidine incorporation glutamine absent) obtained from the exponential phase is plotted against time, a linear plot is obtained for each glutamine concentration. When these linear rates of incorporation are plotted against glutamine concentration, hyperbolic curves are obtained for different times of culture. The peak value of incorporation (which reflects the final number of cells which entered the cell cycle) is determined by the concentration of mitogen and occurs at an earlier time as the number of cells in culture is increased and as the concentration of glutamine is increased. These findings suggest that increasing the plasma glutamine concentration above the normal physiological level may be of value in increasing the proliferation of lymphocytes in conditions of lymphopenia. Adenosine, a fuel of purine nucleotide synthesis, which may affect the lymphoproliferative response also via specific adenosine receptors, increases the rate of incorporation of [ 3H]thymidine but this effect depends upon the concentration of glutamine; at low concentrations of glutamine, the stimulation by adenosine is apparent whereas at high concentrations of glutamine adenosine appeared to inhibit proliferation. Our data presented in this paper suggest that not only the number of cells and the concentration of mitogen, but also the concentration of glutamine, may play a role in determining the proliferative response of mitogenic stimulated peripheral lymphocytes. This emphasises that care must be taken to carry out a full kinetic study, including varying the concentration of glutamine to assess in these types of experiments whether a particular compound or condition causes immunostimulation or immunoinhibition.