In vitro studies of delayed-type hypersensitivity: The time course of the primary immunological reaction in individual rats determined by macrophage spreading inhibition

The aim of this work was to develop a method for the continuous follow-up of the onset and evolution of delayed-type hypersensitivity in individual rats. This hypersensitivity was determined by an in vitro macrophage spreading inhibition test in Wistar rats sensitive to tuberculin and diphtheria toxoid. Circulating antibodies to diphtheria toxoid were evaluated by passive haemagglutination. Samples of peritoneal cells and blood were taken 2 days and a few hours before sensitization, and on days 3, 4, 5, 6, 7, 11, 20, 27 and 40 after sensitization. It was found that: (1) Multiple consecutive washings of the peritoneal cavity, repeated bleedings and sensitization alone produced no change in the percentage of peritoneal macrophages spreading in medium alone. (2) Similarly, the percentage of spread macrophages of non-sensitized rats in the presence of tuberculin remained unaltered during the course of daily peritoneal washings. (3) In sensitized rats, sensitization was followed by a significant inhibition in macrophage spreading in the presence of sensitizing antigens. Thereafter, it was possible to trace individual curves reflecting the onset and evolution of delayed-type hypersensitivity to tuberculin and diphtheria toxoid. (4) The mean macrophage spreading inhibition corresponding to the delayed-type hypersensitivity was found to be maximal for both antigens on day 7 following sensitization, and decreased thereafter. (5) On the other hand, high titres of circulating antibodies to diphtheria toxoid did not appear earlier than 20 days after sensitization and continued to increase towards the end of the observation period.