Experimental Studies Upon Lymphocytes

A simple method is presented by which, with the diffusion of trypan blue into the nucleus as a criterion of cell injury, it is possible to study quantitatively the effect of various agencies upon the small thymus cells and upon the tissue lymphocytes. Preliminary studies with this method have led us to the following conclusions, which, however, unless otherwise stated, may be taken as applying only to the lymphocytes of the rat thymus. 1. The small thymus cells, when suspended in balanced phosphate solutions, show no distinct reaction to variations in hydrogen ion concentrations ranging between P(h) 7.0 and P(H) 7.8. Beyond P(H) 7.0 there is a sudden increase in the permeability of the cells to the dye; plasmolysis of the cells occurs when the alkalinity exceeds P(H) 8.0. 2. Heating to 49 degrees or 50 degrees C. is accompanied by a critical increase in the permeability of the cells to the dye. 3. The injury caused by lack of oxygen can be demonstrated by the increase in the number of stained cells. 4. The addition of serum to suspensions of thymus cells or tonsil lymphocytes greatly inhibits the diffusion of the trypan into the cells. The protection afforded is roughly proportionate to the amount of serum added. Gelatin also exerts a marked protective influence; egg albumin affords a partial protection; starch and gum arabic are inert. Hemoglobin and cholesterol do not modify the stainability of the cells. Arsenious sulfide in weak concentrations partially inhibits the diffusion of the dye. Colloidal iron is without effect, and is precipitated about the cells. 5. The toxicity of the photodynamic substance, hematoporphyrin, and of an impure chlorophyll solution in the presence of sunlight could be strikingly demonstrated by the greatly increased permeability of the cells to the stain. 6. Acute and chronic inanition produces an increased fragility of the cells. The protective power of the serum in acute starvation appears to be increased. 7. The small thymus cells of old animals are more readily injured than are those of young ones, as indicated by the increased proportion of stained cells. 8. The method has been applied to the demonstration of the action of cytotoxic immune sera for rat thymus cells and for human tonsil lymphocytes in vitro. Further experiments dealing with the question of specificity are in progress. The cytotoxins are inactivated by the addition of complement. Thermostabile cytagglutinins have also been produced.