The effect of splenic extract on the reaction of cells to phytohaemagglutinin.

Abstract

Bain, Vas and Lowenstein (1963, 1964) reported a reaction between peripheral blood leucocytes from different healthy donors, grown together in mixed culture. Cells were mixed in equal numbers in their own plasma following separation from red cells by centrifugation. Control cultures of the cells from each donor were grown separately. Cultures were examined at 5 days. Smears from the mixed cultures showed that some of the cells had undergone blastic transformation and occasional mitoses were observed. Autoradiographs after exposure to 3H‐thymidine revealed a striking increase in cells synthesizing desoxyribose nucleic acid (DNA) compared with the controls. Other elements of the blood, red cells, platelets, and plasma did not have this effect on homologous leucocytes. Hirschhorn, Bach, Kolodny, Firschein and Hashem (1963) observed a similar reaction with both viable and frozen‐thawed lymphocytes. Skin extracts either from a biopsy specimen, or long‐term cultures of human skin fibroblasts, also produced mitosis when added to lymphocytes of an unrelated individual. Hashem and Rosen (1964) fractionated extracts from human lymphocytes and found that fractions containing ribose nucleic acid (RNA) were mitogenic to homologous lymphocytes. This action was inhibited by RNA‐ase.Craddock (1960) and his co‐workers had noticed that intact or homogenized human granulocytes inhibited DNA synthesis by dog thoracic duct lymphocytes. A later paper from one of his collaborators and a colleague (Perry and Marsh, 1964) indicated that this same effect could be observed with human leucocytes in cultures. When cells from a patient with chronic myeloid leukaemia were grown for a few hours in vitro, normal granulocytes or homogenates of normal or leukaemic cells inhibited DNA synthesis. In contrast to this, ‘incubation supernates’ from chronic myeloid leukaemic cells increased DNA synthesis.These findings with cellular material suggested it would be interesting to observe the effect of extracts from the reticulo‐endothelial system. Extracts of healthy human spleen were obtained and the effect on cells growing in tissue culture observed. Normal peripheral blood leucocytes were stimulated with phytohaemagglutinin and then 24 hours later splenic extract added. In some cases, the extract was added first and the phytohaemagglutinin after 24 hours. Activity in the cultures was assessed by the number of cells taking up 3H‐thymidine.