The effect of phytohaemagglutinin and other lymphocyte mitogens on immunoglobulin synthesis by human peripheral blood lymphocytes in vitro

Abstract

Amino acid incorporation techniques and immunofluorescence have been used to investigate the effect of mitogenic substances on immunoglobulin synthesis by human peripheral blood lymphocytes in vitro. Radioelectrophoresis, radioimmunoelectrophoresis and controlled immunological precipitation methods suggest that only a small amount of immunoglobulin is synthesized in the culture system used. Immunofluorescent staining of fixed cell preparations showed that during the first 24 hr in culture only a small percentage of cells reacted positively for immunoglobulin; after 24 hr these cells were no longer demonstrable. This suggests that the small amount of immunoglobulin detected was synthesized during the first few hours in culture by these cells, having the morphological appearance of medium lymphocytes. The slight enhancement of immunoglobulin synthesis obtained in one experiment with phytohaemagglutinin (PHA) probably occurred within this same cell type since after 24 hr in vitro no cells in the transformed cultures could be stained by the fluorescent anti-immunoglobulin. Fixed preparations of blast cells obtained by stimulation with anti-lymphocytic serum and staphylococcal filtrate also gave negative reactions. However, using a staining technique with suspensions of viable cells, it was possible to demonstrate positive staining for immunoglobulins with PHA stimulated cells as previously described by Ripps & Hirschhorn (1967). A number of controls suggest that this reaction depends upon the presence of exposed immunoglobulin groups or markers on the cell surface and that intracytoplasmic staining is the result of endocytosis of conjugate.

In contrast with the negative results obtained with PHA-transformed blasts, a small percentage of lymphocytes from cultures stimulated by pokeweed or tuberculin reacted positively when fixed preparations were stained with conjugated anti-immunoglobulin.