In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.